Bioactive Molecules in Food

Reference Series in Phytochemistry
Series Editors:
J.-M. Mérillon · K. G. Ramawat
Jean-Michel Mérillon
Kishan Gopal Ramawat Editors
Bioactive
Molecules
in Food
Reference Series in Phytochemistry
Series Editors
Faculty of Pharmaceutical Sciences
Institute of Vine and Wine Sciences
University of Bordeaux
Villenave d’Ornon, France
Kishan Gopal Ramawat
Department of Botany, University College of Science, M. L. Sukhadia University
Udaipur, Rajasthan, India
This reference works series provides a platform for all information on plant metab-
olites and phytochemicals, their chemistry, properties, applications, and methods. By
the strictest definition, phytochemicals are chemicals derived from plants. However,
the term is often used to describe the large number of secondary metabolic com-
pounds found in and derived from plants. These metabolites exhibit a number of
nutritional and protective functions for human welfare such as colorants, fragrances
and flavorings, amino acids, pharmaceuticals, hormones, vitamins and agrochemi-
cals. Besides food, fibers, fuel, cloth and shelter, a vast number of wild plants can
hence provide important sources for medicines, especially in developing countries
for their traditional health systems. Natural products have inspired and provided the
foundation to the bulk of FDA-approved compounds and there is tremendous
increase in natural products and natural products derived compounds that have
been registered against many prevailing diseases. Natural product industry has
shown tremendous growth and is expected to continue to do so in the near future.
The present series compiles reference information on various topics and aspects
about phytochemicals, including their potential as natural medicine, their role as
chemo-preventers, in plant defense, their ecological role, their role in plants as well
as for pathogen adaptation, and disease resistance. Volumes in the series also contain
information on methods such as metabolomics, genetic engineering of pathways,
molecular farming, and obtaining metabolites from lower organisms and marine
organisms besides higher plants. The books in the series are hence of relevance in
various fields, from chemistry, biology, biotechnology, to pharmacognosy, pharma-
cology, botany, or medicine. Each volume is edited by leading experts and contains
authoritative contributions by renowned authors.
More information about this series at http://www.springer.com/series/13872

Kishan Gopal Ramawat
Editors
Bioactive Molecules in
Food
With 238 Figures and 206 Tables

Editors
Jean-Michel Mérillon Kishan Gopal Ramawat
Faculty of Pharmaceutical Sciences Department of Botany
Institute of Vine and Wine Sciences University College of Science
University of Bordeaux M. L. Sukhadia University
Villenave d’Ornon, France Udaipur, Rajasthan, India
ISSN 2511-834X ISSN 2511-8358 (electronic)

ISBN 978-3-319-78029-0 ISBN 978-3-319-78030-6 (eBook)
ISBN 978-3-319-78031-3 (print and electronic bundle)
https://doi.org/10.1007/978-3-319-78030-6
Library of Congress Control Number: 2018964906
© Springer Nature Switzerland AG 2019

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Preface
We are pleased to present a three-volume treatise on Bioactive Molecules in Food.

Bioactive molecules are present in all the food consumed, and we come to know
more about them with the availability of more and more sophisticated tools and
techniques. As we know more about benefits of food and their molecules, we intend
to improve food quality and fortify the food for human welfare and disease preven-
tion. The aim of the book is to present the state of information about bioactive
molecules present in various foods we consume daily and their effect on our physical
and mental state of body. The concept of functional foods is of recent origin, but in
old civilizations like that of China and India, there are sayings about consumption of
selected food and their effect on well-being. Hence history is being revisited.
Selection, cultivation, and domestication of plants for human welfare is almost
9,000 years old which is reconfirmed by a recent report from the remains of
domesticated crop plants at an archaeological site in southwest Amazonia. Plants
are easy to catch as compared to animals, and hunting evolved with evolution of
tools and techniques with civilization of men. Thus, fruits, tubers, bulbs, and seeds
were easy to gather and consumed by early men. Some chapters deal with cultivation
and improvement of nutrients in food plants.
The debate about food consumption and vegetarian and nonvegetarian diet or
consumption of various fatty acids in oils (PUMA, MUFA, omega-3 FA) may
continue for another decade. With the new facts coming out day by day, this scene
changes for a while and then resets to a new concept. Whatever the diet regimen, a
cereal, a pulse, and a fruit make an essential part of all diets. With the advent of new
and sophisticated technology, more information available about bioactive molecules
present in the food, best combinations of food components to meet nutritional
requirement, and fate of ingested molecules as well as their beneficial effects are
known. Major part of the book is devoted to this aspect.
Consumers have become very attentive to quality, safety, and health benefits of
food. This has direct repercussion on food industry to develop such foods, especially
processed and packaged foods and particularly in terms of calorie, quality, nutri-
tional value, and bioactive molecules present in them. Market for nutraceuticals and
food supplements is growing at rapid pace with increasing middle class in develop-
ing countries and elderly population in developed countries. Therefore, this is a very
v
vi Preface
timely compilation of information about bioactive molecules present in our

daily food.
Longevity of life is associated with preferential consumption of selected food
which can prevent diseases and keep overall health in good condition. Society is
keen to know the benefits of organic food, bioactive molecules present in their food,
and possibility of food fortified with beneficial molecules. This has direct impact on
research related to this field by all those involved in food cultivation, processing, and
production including meat quality. Certain chapters are devoted to this aspect in
the book.
The book is divided into ten parts (I. Favorable Regimen for Good Health and
Epidemiological Studies, II. Proteins and Their Biological Activity, III. Lipids and
Their Biological Activity, IV. Food Carbohydrates and Derived Compounds,
V. Food Carotenoids, VI. Food Polyphenols, VII. Functional Foods, VIII. Agricul-
tural Practices and Food Quality, IX. Methods of Food Analysis and Quality Control,
X. Miscellaneous Case Studies) covering entire gamut of bioactive molecules
present in daily foods like cereals (carbohydrates), pulses, and other sources (pep-
tides and proteins); oils (lipids, fatty acids); mushrooms, wine, and fruits and
vegetables (carotenoids, polyphenols, etc.); and fibers and methods of analysis of
some foods. Concepts like French paradox, Mediterranean diet, healthy diet of
eating fruits and vegetables, vegan and vegetarian diet, and functional foods are
also described with suitable case studies.
Well-recognized international specialists in their respective fields of research
contributed these chapters. This book will be useful to all those working in the
field of botany, phytochemistry, pharmacy, drug delivery, molecular biology, for-
estry, biotechnology, industrial food, and medical products. This work is arranged in
78 well-illustrated chapters.
Because of the voluminous work for the treatise, this project was spread over
almost 2 years, from concept to print. We would like to acknowledge the coopera-
tion, patience, and support of our contributors who have put their serious efforts to
ensure the high scientific quality of this book with up-to-date information. We are
thankful to the staff at Springer, namely, Drs. S. Costa, S. Blago, and N. Clifford, for
their professional support in this project.
Prof. Jean-Michel Mérillon

Prof. Kishan Gopal Ramawat
Contents
Volume 1
Part I Favorable Regimen for Good Health and

Epidemiological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 Health Benefits of Dietary Phenolic Compounds and

Biogenic Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Hector Alonzo Gomez-Gomez, Cristine Vanz Borges, Igor Otavio
Minatel, Aline Carbonera Luvizon, and Giuseppina Pace Pereira Lima
2 Intake of Mediterranean Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Charalampos Siotos, Marco Vinceti, and Androniki Naska
3 Flavonoids – Food Sources, Health Benefits, and Mechanisms

Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Aleksandra Kozłowska and Dorota Szostak-Węgierek
4 Bioactive Molecules, Nutraceuticals, and Functional Foods in

Indian Vegetarian Diet and During Postpartum Healthcare . . . . . 79
Jaya Arora and Kishan Gopal Ramawat
5 Challenges in Optimal Utilization of Bioactive

Molecules Clinically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Kotamballi N. Chidambara Murthy, M. Shivapriya, P. Monika, and
B. Tejashree
6 Bioactive Food Components in the Prevention of Cardiovascular

Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Arti Parihar and Mordhwaj S. Parihar
7 Capsicum annuum Bioactive Compounds: Health Promotion

Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Muhammad Imran, Masood Sadiq Butt, and Hafiz Ansar Rasul Suleria
vii
viii Contents
8 Sesame: Bioactive Compounds and Health Benefits . . . . . . . . . . . . 181

Niti Pathak, Asani Bhaduri, and Ashwani K. Rai
9 Nutritional Quality of Mangifera Species . . . . . . . . . . . . . . . . . . . . 201
Luis M. Anaya-Esparza and Efigenia Montalvo-González
Part II Proteins and Their Biological Activity . . . . . . . . . . . . . . . . . 221
10 Plant Proteins from Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Catherine Bennetau-Pelissero
11 Soybean Bioactive Molecules: Current Trend and
Future Prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Brij Pal Singh, Deepika Yadav, and Shilpa Vij
12 Wheat Bran Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
René R. Balandrán-Quintana and Ana María Mendoza-Wilson
13 Antihypertensive Peptides from Animal Proteins . . . . . . . . . . . . . . 319
Z. F. Bhat, Susan Mason, James D. Morton, Alaa El-Din A. Bekhit,
and Hina F. Bhat
14 Bioactive Peptides from Fish Protein By-Products . . . . . . . . . . . . . 355
Aurélien V. Le Gouic, Pádraigín A. Harnedy, and
Richard J. FitzGerald
15 Edible Insects as Source of Proteins . . . . . . . . . . . . . . . . . . . . . . . . 389
Ewelina Zielińska, Monika Karaś, Anna Jakubczyk, Damian
Zieliński, and Barbara Baraniak
16 Plant Proteases in Food Processing . . . . . . . . . . . . . . . . . . . . . . . . . 443
Manzoor Ahmad Shah and Shabir Ahmad Mir
Part III Lipids and Their Biological Activity . . . . . . . . . . . . . . . . . . . 465
17 Bioactive Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

Luis Vázquez, Marta Corzo-Martínez, Pablo Arranz-Martínez,
Elvira Barroso, Guillermo Reglero, and Carlos Torres
18 Oxidative Stability of Edible Plant Oils . . . . . . . . . . . . . . . . . . . . . 529
Terrence Madhujith and Subajiny Sivakanthan
19 The Anti-inflammatory Properties of Food Polar Lipids . . . . . . . . 553
Ronan Lordan, Constantina Nasopoulou, Alexandros Tsoupras, and
Ioannis Zabetakis
20 Neuroprotective and Antiaging Essential Oils and Lipids
in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Mamali Das and Kasi Pandima Devi
Contents ix
21 Protective Effect of Omega 3 Fatty Acids EPA and DHA in

the Neurodegenerative Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Edwin E. Martínez Leo, Rafael A. Rojas Herrera, and
Maira R. Segura Campos
22 Application of Lipid Nanocarriers for the Food Industry . . . . . . . 623
Zahra Rafiee and Seid Mahdi Jafari
23 CLAs in Animal Source Foods: Healthy Benefits for Consumers . . . 667
Paolo Polidori, Silvia Vincenzetti, Stefania Pucciarelli, and
Valeria Polzonetti
Part IV Food Carbohydrates and Derived Compounds . . . . . . . . . 699
24 Total Dietary Fiber Intake, Whole Grain Consumption, and

Their Biological Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
Semih Otles and Emine Nakilcioglu-Tas
25 Inulin-Type Fructans Application in Gluten-Free Products:
Functionality and Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . 723
Natalia Drabińska, Cristina M. Rosell, and Urszula Krupa-Kozak
26 Development of Dietary Fiber-Rich Meat Products:
Technological Advancements and Functional Significance . . . . . . . 763
Nitin Mehta, Manish Kumar Chatli, Pavan Kumar, Om Prakash
Malav, Akhilesh Kumar Verma, Yogesh Kumar, and Dinesh Kumar
27 Chemistry, Biological, and Pharmacological Properties of
Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Hassan Hussein Musa, Abdelkareem Abdall Ahmed, and
Taha Hussein Musa
28 Resistant Starch in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
Pinky Raigond, Som Dutt, and Brajesh Singh
29 Gluten-Free Cereals and Pseudocereals: Nutrition and Health . . . 847
Mario Fernández de Frutos, Bartosz Fotschki, Ricardo Fernández
Musoles, and José Moisés Laparra Llopis
Volume 2
Part V Food Carotenoids ................................. 865
30 Natural Food Pigments and Colorants . . . . . . . . . . . . . . . . . . . . . . 867

Delia B. Rodriguez-Amaya
31 Lycopene: Metabolism and Functional Aspects . . . . . . . . . . . . . . . 903
Soma Srivastava
x Contents
32 Sweet Potato: Bioactive Compounds and Health Benefits . . . . . . . 919

Remya Mohanraj
33 Anticancer Properties of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . 935
Kazim Sahin, Cemal Orhan, Nurhan Sahin, and Omer Kucuk
Part VI Food Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
34 Bound Phenolics in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973

Liliana Santos-Zea, Javier Villela-Castrejón, and
Janet A. Gutiérrez-Uribe
35 Tea, Coffee and Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
Sumio Hayakawa, Yumiko Oishi, Hiroki Tanabe, Mamoru Isemura,
and Yasuo Suzuki
36 Theobroma cacao and Theobroma grandiflorum: Bioactive
Compounds and Associated Health Benefits . . . . . . . . . . . . . . . . . . 1049
Maria Inés Genovese and Helena Rudge de Moraes Barros
37 Olive Oil and Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
Álvaro Hernáez, Julieta Valussi, Alejandra Pérez-Vega,
Olga Castañer, and Montserrat Fitó
38 Anthocyanins: Nutrition and Health . . . . . . . . . . . . . . . . . . . . . . . 1097
Iva Fernandes, Cláudia Marques, Ana Évora, Ana Faria,
Conceição Calhau, Nuno Mateus, and Victor de Freitas
39 Wine Polyphenols and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135
Giovanna Giovinazzo, Maria A. Carluccio, and Francesco Grieco
40 Natural Estrogenic Substances, Origins, and Effects . . . . . . . . . . . 1157
41 Jaboticaba: Chemistry and Bioactivity . . . . . . . . . . . . . . . . . . . . . . 1225
Natália Crialeison Balbo Vall Ribeiro, Andressa Mara Baseggio, and
Vicki Schlegel
42 Pomegranate Bioactive Molecules and Health Benefits . . . . . . . . . 1253
Saeed Akhtar, Tariq Ismail, and Anam Layla
Part VII Functional Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281
43 Antidiabetic Functional Foods with Antiglycation Properties . . . . 1283

Mutiu Idowu Kazeem, Habeeb Adebodun Bankole, Azeez Ayomide
Fatai, Abiola Fatimah Adenowo, and Theophilus Clavell Davies
44 Nutraceutical Potential of Apiaceae . . . . . . . . . . . . . . . . . . . . . . . . 1311
Milica G. Aćimović
Contents xi
45 Functional Components and Medicinal Properties of Food . . . . . . 1343

Christian Izuchukwu Abuajah
46 Development of Functional Dairy Foods . . . . . . . . . . . . . . . . . . . . 1377

Natália Martins, Maria Beatriz P. P. Oliveira, and Isabel C. F. R.
Ferreira
47 Nutrients and Nutraceuticals from Seafood . . . . . . . . . . . . . . . . . . 1397

V. Venugopal
48 Roots and Tubers as Functional Foods . . . . . . . . . . . . . . . . . . . . . . 1441

Anoma Chandrasekara
49 Betalains: Application in Functional Foods . . . . . . . . . . . . . . . . . . 1471

Wee Sim Choo
50 Nutraceutical Potential of Guava . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

Moni Gupta, Anshu Wali, Anjali, Sachin Gupta, and
Sudheer K. Annepu
51 Cereal-Based Fermented Foods of Africa as Functional Foods . . . 1527

Ome Kalu Achi and Naomi U. Asamudo
52 A Novel Delivering Agent for Bioactive Compounds:

Chewing Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559
Ibrahim Palabiyik, Haniyeh Rasouli Pirouzian, Nevzat Konar, and
Omer Said Toker
53 Bioactive Molecules in Edible and Medicinal Mushrooms for

Human Wellness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1597
Chia-Wei Phan, Elson Yi-Yong Tan, and Vikineswary Sabaratnam
54 Bioactive Molecules of Spirulina: A Food Supplement . . . . . . . . . . 1621

Meeta Mathur
55 Bioactive Compounds from Garcinia Fruits of High Economic

Value for Food and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1643
Hosakatte Niranjana Murthy, Vijayalaxmi S. Dandin,
Dayanand Dalawai, So-Young Park, and Kee-Yoeup Paek
Volume 3
Part VIII Agricultural Practices and Food Quality . . . . . . . . . . . . . . 1671
56 Factors Influencing Sweet Taste in Apple . . . . . . . . . . . . . . . . . . . . 1673

Mathilde Charles, Eugenio Aprea, and Flavia Gasperi
xii Contents
57 Advances in Pseudocereals: Crop Cultivation, Food

Application, and Consumer Perception . . . . . . . . . . . . . . . . . . . . . 1695
Natalia Manzatti Machado Alencar and Ludmilla de Carvalho
Oliveira
58 Pesticide Residues in Fruits and Vegetables . . . . . . . . . . . . . . . . . . 1715

Samira Mebdoua
59 Banana and Plantains: Improvement, Nutrition, and Health . . . . 1755

Siddhesh B. Ghag and Thumballi R. Ganapathi
60 Veterinary Antibiotics in Animal Diet: Effects on

Waste/Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1775
Lizbeth E. Robles Jimenez, Juan C. Angeles Hernandez, Jorge Osorio
Avalos, Xunde Li, Edward Rob Atwill, Octavio Castelan Ortega, and
Manuel Gonzalez Ronquillo
61 Insecticide Residues in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1793

Ramesh Kumar Sharma
62 Edible Mushrooms: Cultivation, Bioactive Molecules,

and Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1815
Sachin Gupta, Baby Summuna, Moni Gupta, and Sudheer K. Annepu
63 Plants Probiotics as a Tool to Produce Highly Functional Fruits . . . 1849

Alejandro Jiménez-Gómez, Paula García-Fraile, José David
Flores-Félix, and Raúl Rivas
64 Bioactive Compounds of the Wonder Medicinal

Mushroom “Ganoderma lucidum” . . . . . . . . . . . . . . . . . . . . . . . . . 1863
Surya Sudheer, Ibrahim Alzorqi, Sivakumar Manickam, and Asgar Ali
65 Minas Frescal Cheese as a Probiotic Carrier . . . . . . . . . . . . . . . . . 1895

Tatiana Colombo Pimentel, Vanessa Aparecida Marcolino, Carlos
Eduardo Barão, Suellen Jensen Klososki, and Michele Rosset
Part IX Methods of Food Analysis and Quality Control . . . . . . . . . 1927
66 Dietary Phenolic Compounds in Biological Samples:

Current Challenges in Analytical Chemistry . . . . . . . . . . . . . . . . . 1929
Maike Passon
67 Rheology of Food Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

Seyed Mohammad Ali Razavi and Mahdi Irani
68 GLC/HPLC Methods for Saffron (Crocus sativus L.) . . . . . . . . . . . 1987

Asghar Amanpour, Hasim Kelebek, and Serkan Selli
Contents xiii
69 Use of Nanotechnology for Immobilization and

Entrapment of Food Applicable Enzymes . . . . . . . . . . . . . . . . . . . 2037
Milad Fathi, Mehri Karim, Soroush Rahimi Khoigani, and
Vahid Mosayebi
70 Methods for Seafood Authenticity Testing in Europe . . . . . . . . . . . 2063
Véronique Verrez-Bagnis, Carmen G. Sotelo, Rogério Mendes,
Helena Silva, Kristina Kappel, and Ute Schröder
71 Some Statistical Considerations Regarding the Occurrence
and Analysis of Bioactive Materials in Foods . . . . . . . . . . . . . . . . . 2119
Basil Jarvis
72 LC-MS/MS Determination of Pesticide Residues in Fruits and
Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2137
Anna Stachniuk
73 Encapsulation to Protect Different Bioactives to Be Used as
Nutraceuticals and Food Ingredients . . . . . . . . . . . . . . . . . . . . . . . 2163
Jacqueline Ruiz Canizales, Gustavo R. Velderrain Rodríguez,
J. Abraham Domínguez Avila, Alejandra M. Preciado Saldaña, Emilio
Alvarez Parrilla, Mónica A. Villegas Ochoa, and Gustavo A. González
Aguilar
Part X Miscellaneous Case Studies ......................... 2183
74 Are Myristica fragrans L. (Myristicaceae) and Its

Phytochemicals Useful for Human Health? . . . . . . . . . . . . . . . . . . 2185
Monica Rosa Loizzo, Vincenzo Sicari, Jianbo Xiao, and Rosa Tundis
75 Coriander (Coriandrum sativum L.): Bioactive Molecules
and Health Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2199
Muhammad Jawad Iqbal, Masood Sadiq Butt, and Hafiz Ansar
Rasul Suleria
76 Jackfruit (Artocarpus heterophyllus): Biodiversity,
Nutritional Contents, and Health . . . . . . . . . . . . . . . . . . . . . . . . . . 2237
Shrikant Baslingappa Swami and Sandeep Baban Kalse
77 Sambucus nigra Berries and Flowers Health Benefits:
From Lab Testing to Human Consumption . . . . . . . . . . . . . . . . . . 2261
Ângelo C. Salvador, Ricardo J. R. Guilherme,
Armando J. D. Silvestre, and Sílvia M. Rocha
78 Bioactive Compounds and Health Benefits of Jamun
(Syzygium cumini) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2297
Shalini S. Arya, Kakoli Pegu, and Prajakta D. Sadawarte
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2317
About the Editors
Prof. Dr. Jean-Michel Mérillon received his

M.Pharma. (1979) and Ph.D. (1984) from the University
of Tours, France. He joined the university of Tours as
assistant professor in 1981, became associate professor
in 1987. In 1993 he moved to the faculty of Pharmacy,
University of Bordeaux, France, accepting a position
as full professor. He has been the director of the research
group on biologically active plant substances for over
15 years, at the Institute of Vine and Wine Sciences
(University of Bordeaux, France), which comprises
25 scientists and research students. The group has been
working on phenolic compounds from vine and wine
for many years, mainly complex stilbenes and their
involvement in health. He is involved in developing
teaching on plant biology, natural bioactive compounds
and biotechnology. Prof. Mérillon has published more
than 160 research papers in internationally recognized
journals, and has coedited books and reference works
on secondary metabolites and biotechnology. In 2004,
he founded the technology transfer unit “Polyphenols
Biotech”, providing support for R&D programs for SMEs
and major groups from the cosmetic, pharmaceutical,
agricultural and health-nutrition sectors. He is currently
the manager of this unit.
xv
xvi About the Editors
Prof. Dr. Kishan Gopal Ramawat is former professor

and head of the Botany Department, M.L. Sukhadia
University, Udaipur, India, and has a long-standing
research experience. He received his Ph.D. in Plant
Biotechnology in 1978 from the University of Jodhpur,
India, and afterward joined the university as a faculty
member. In 1991, he moved to the M.L. Sukhadia
University in Udaipur as associate professor and became
professor in 2001. He served as the head of the
Department of Botany (2001–2004, 2010–2012), was
in charge of the Department of Biotechnology
(2003–2004), was a member of the task force on medic-
inal and aromatic plants of the Department of Biotech-
nology, Government of India, New Delhi (2002–2005),
and coordinated UGC-DRS and DST-FIST programs
(2002–2012).
Professor Ramawat had done his postdoctoral studies
at the University of Tours, France, from 1983 to 1985 and
later returned to Tours as visiting professor (1991). He
also visited the University of Bordeaux 2, France, several
times as visiting professor (1995, 1999, 2003, 2006,
2010) and, in 2005, in Poland in an academic exchange
program (2005). Through these visits in France, Prof.
Ramawat and Prof. Mérillon established a strong con-
nection, which has resulted in productive collaborations
and several book and reference work publications.
Professor Ramawat has published more than
170 well-cited peer-reviewed papers and articles and
edited several books and reference works on topics
such as the biotechnology of medicinal plants, second-
ary metabolites, bioactive molecules, herbal drugs, and
many other topics. His research was funded by several
funding agencies.
In his research group, Prof. Ramawat has supervised
doctoral thesis of 25 students. He is an active member of
several academic bodies, associations, and editorial
boards of journals. Botany Department, M.L. Sukhadia
University, Udaipur, India.
Contributors
Christian Izuchukwu Abuajah Department of Food Science and Technology,

University of Uyo, Uyo, Nigeria
Ome Kalu Achi Department of Microbiology, Michael Okpara University of
Agriculture, Umudike, Nigeria
Milica G. Aćimović Department of Alternative Crops and Organic Agriculture,
Institute of Field and Vegetable Crops, Novi Sad, Serbia
Abiola Fatimah Adenowo Department of Biochemistry and Microbiology, Uni-
versity of Zululand, Kwadlangezwa, South Africa
Abdelkareem Abdall Ahmed Department of Physiology and Biochemistry, Fac-
ulty of Veterinary Sciences, University of Nyala, Nyala, Sudan
Saeed Akhtar Institute of Food Science and Nutrition, Faculty of Agricultural
Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan
Natalia Manzatti Machado Alencar Department of Food and Nutrition, School of
Food Engineering, University of Campinas, Campinas, São Paulo, Brazil
Asgar Ali Centre of Excellence for Postharvest Biotechnology (CEPB), School of
Biosciences, University of Nottingham, Semenyih, Malaysia
Emilio Alvarez Parrilla Instituto de Ciencias Biomédicas, Universidad Autónoma
de Ciudad Juárez. Anillo Envolvente del PRONAF y Estocolmo s/n. Cd. Juárez,
Chihuahua, Mexico
Ibrahim Alzorqi Department of Chemical and Environmental Engineering, Fac-
ulty of Engineering, University of Nottingham, Semenyih, Malaysia
Asghar Amanpour Department of Food Engineering, Faculty of Agriculture,
Cukurova University, Adana, Turkey
Luis M. Anaya-Esparza Laboratorio de Microbiología de Alimentos,
Departamento de Ciencias Pecuarias y Agrícolas, Universidad de Guadalajara,
Centro Universitario de los Altos, Tepatitlán de Morelos, Jalisco, Mexico
Laboratorio Integral de Investigación en Alimentos, Instituto Tecnológico de Tepic,
Tepic, Nayarit, Mexico
xvii
xviii Contributors
Juan C. Angeles Hernandez Departamento de Nutricion Animal, Facultad de

Medicina Veterinaria y Zootecnia, Universidad Autonoma del Estado de Mexico,
Toluca, Estado de Mexico, Mexico
Anjali Division of Biochemistry, Faculty of Basic Sciences, Sher-e-Kashmir Uni-
versity of Agricultural Sciences and Technology, Chatha, Jammu, Jammu and
Kashmir, India
Sudheer K. Annepu Divison of Crop Improvement, ICAR-Directorate of Mush-
room Research, Solan, Himachal Pradesh, India
Vanessa Aparecida Marcolino Federal Institute of Paraná (IFPR), Paranavaí,
Parana, Brazil
Eugenio Aprea Department of Food Quality and Nutrition, Research and Innova-
tion Centre, Fondazione Edmund Mach (FEM), San Michele all’Adige, Trento, Italy
Jaya Arora Department of Botany, University College of Science, M. L. Sukhadia
University, Udaipur, India
Pablo Arranz-Martínez Department of Production and Characterization of Novel
Foods, Institute of Food Science Research (CIAL,CSIC-UAM), Madrid, Spain
Shalini S. Arya Food Engineering and Technology Department, Institute of Chem-
ical Technology, Mumbai, India
Naomi U. Asamudo Department of Microbiology, University of Uyo, Uyo, Nigeria
Edward Rob Atwill Western Institute for Food Safety and Security, School of
Veterinary Medicine, University of California Davis, Davis, CA, USA
René R. Balandrán-Quintana Centro de Investigación en Alimentación y
Desarrollo, A.C., Coordinación de Tecnología de Alimentos de Origen Vegetal,
Hermosillo, Son., México
Habeeb Adebodun Bankole Department of Biochemistry, Faculty of Science,
Lagos State University, Ojo, Lagos, Nigeria
Barbara Baraniak Department of Biochemistry and Food Chemistry, University
of Life Sciences in Lublin, Lublin, Poland
Helena Rudge de Moraes Barros Department of Food Science and Experimental
Nutrition, University of São Paulo, São Paulo, Brazil
Elvira Barroso Department of Production and Characterization of Novel Foods,
Institute of Food Science Research (CIAL,CSIC-UAM), Madrid, Spain
Andressa Mara Baseggio Department of Food Science, Faculty of Food Engi-
neering, University of Campinas, Campinas, Brazil
Alaa El-Din A. Bekhit Department of Food Sciences, University of Otago, Dun-
edin, New Zealand
Contributors xix
Catherine Bennetau-Pelissero Department of Life Science and Health, University

of Bordeaux, Bordeaux, France
Department Feed and Food, Bordeaux Sciences Agro, Gradignan, France
Asani Bhaduri Cluster Innovation Centre, University of Delhi, Delhi, India
Hina F. Bhat Division of Biotechnology, Sher-e-Kashmir University of Agricul-
tural Sciences and Technology of Kashmir, Shuhama, Alusteng, Srinagar, Jammu
and Kashmir, India
Z. F. Bhat Division of Livestock Products Technology, Faculty of Veterinary
Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sci-
ences and Technology of Jammu, Jammu, Jammu and Kashmir, India
Cristine Vanz Borges Department of Chemistry and Biochemistry, Institute of
Biosciences, Sao Paulo State University – UNESP, Botucatu, São Paulo, Brazil
Masood Sadiq Butt National Institute of Food Science and Technology, Faculty of
Food, Nutrition and Home Sciences, University of Agriculture, Faisalabad, Pakistan
Conceição Calhau Nutrição e Metabolismo, NOVA Medical School, Faculdade de
Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal
ProNutri - Clinical Nutrition and Disease Programming, CINTESIS - Center for
Research in Health Technologies and Information Systems, Porto, Portugal
Maria A. Carluccio Institute of Clinical Physiology (IFC), National Research
Council, Lecce, Italy
Olga Castañer Cardiovascular Risk and Nutrition Research Group (CARIN),
Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
CIBER of the Physiopathology of Obesity and Nutrition (CIBEROBN), Institut
Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain
Octavio Castelan Ortega Departamento de Nutricion Animal, Facultad de
Anoma Chandrasekara Department of Applied Nutrition, Faculty of Livestock
Fisheries and Nutrition, Wayamba University of Sri Lanka, Gonawila, Sri Lanka
Mathilde Charles BIOFORTIS Sensory and Consumer, Saint Herblain, France
Manish Kumar Chatli Department of Livestock Products Technology, College of
Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University,
Ludhiana, Punjab, India
Kotamballi N. Chidambara Murthy Central Research Laboratory, Ramaiah
Medical College and Hospital, MSRIT Post, Bengaluru, India
Wee Sim Choo School of Science, Monash University Malaysia, Jalan Lagoon
Selatan, Bandar Sunway, Selangor, Malaysia
xx Contributors
Marta Corzo-Martínez Department of Production and Characterization of Novel

Dayanand Dalawai Department of Botany, Karnatak University, Dharwad, India
Vijayalaxmi S. Dandin Department of Botany, Karnatak University, Dharwad,
India
Mamali Das Department of Biotechnology, Alagappa University [Science Cam-
pus], Karaikudi, Tamil Nadu, India
Theophilus Clavell Davies Department of Geology, University of Nigeria,
Nsukka, Nigeria
Ludmilla de Carvalho Oliveira Department of Food Technology, School of Food
Engineering, University of Campinas, Campinas, São Paulo, Brazil
Victor de Freitas REQUIMTE/LAQV, Department of Chemistry and Biochemis-
try, Faculty of Sciences, University of Porto, Porto, Portugal
Mario Fernández de Frutos Madrid Institute for Advanced Studies in Food,
Madrid, Spain
J. Abraham Domínguez Avila Centro de Investigación en Alimentación y
Desarrollo A.C. Carretera a La Victoria km 0.6, Hermosillo, Sonora, Mexico
Natalia Drabińska Department of Chemistry and Biodynamics of Food, Institute
of Animal Reproduction and Food Research of Polish Academy of Sciences,
Olsztyn, Poland
Som Dutt Division of Crop Physiology, Biochemistry and Post Harvest Technol-
ogy, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India
Carlos Eduardo Barão Federal Institute of Paraná (IFPR), Paranavaí, Parana,
Brazil
Ana Évora REQUIMTE/LAQV, Department of Chemistry and Biochemistry, Fac-
ulty of Sciences, University of Porto, Porto, Portugal
Ana Faria REQUIMTE/LAQV, Department of Chemistry and Biochemistry,
Faculty of Sciences, University of Porto, Porto, Portugal
Nutrição e Metabolismo, NOVA Medical School, Faculdade de Ciências Médicas,
Universidade Nova de Lisboa, Lisboa, Portugal
Azeez Ayomide Fatai Department of Biochemistry, Faculty of Science, Lagos
State University, Ojo, Lagos, Nigeria
Contributors xxi
Milad Fathi Department of Food Science and Technology, College of Agriculture,

Isfahan University of Technology, Isfahan, Iran
Iva Fernandes REQUIMTE/LAQV, Department of Chemistry and Biochemistry,
Isabel C. F. R. Ferreira Mountain Research Centre (CIMO), Polytechnique Insti-

tute of Bragança, Bragança, Portugal
Instituto Politécnico de Bragança, Bragança, Portugal
Montserrat Fitó Cardiovascular Risk and Nutrition Research Group (CARIN),
Richard J. FitzGerald Department of Biological Sciences, University of Limerick,
Limerick, Ireland
José David Flores-Félix Microbiology and Genetics Department, University of

Salamanca, Salamanca, Spain
Spanish-Portuguese Institute for Agricultural Research (CIALE), Salamanca, Spain
Bartosz Fotschki Department of Biological function of food, Institute of Animal

Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn,
Poland
Thumballi R. Ganapathi Plant Cell Culture Technology Section, Nuclear Agri-

culture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai,
India
Paula García-Fraile Institute of Microbiology ASCR,v.v.i, Prague, Czech

Republic
Lab 210-213, Edificio Departamental de Biología, Microbiology and Genetics
Department, University of Salamanca, Salamanca, Spain
Flavia Gasperi Department of Food Quality and Nutrition, Research and Innova-
tion Centre, Fondazione Edmund Mach (FEM), San Michele all’Adige, Trento, Italy
Maria Inés Genovese Department of Food Science and Experimental Nutrition,
University of São Paulo, São Paulo, Brazil
Siddhesh B. Ghag UM-DAE Centre for Excellence in Basic Sciences, Mumbai,
Maharashtra, India
Giovanna Giovinazzo Institute of Sciences of Food Production (ISPA), National

Research Council, Lecce, Italy
xxii Contributors
Hector Alonzo Gomez-Gomez Department of Chemistry and Biochemistry, Insti-

tute of Biosciences, Sao Paulo State University – UNESP, Botucatu, São Paulo,
Brazil
Gustavo A. González Aguilar Centro de Investigación en Alimentación y
Manuel Gonzalez Ronquillo Departamento de Nutricion Animal, Facultad de
Francesco Grieco Institute of Sciences of Food Production (ISPA), National
Research Council, Lecce, Italy
Ricardo J. R. Guilherme QOPNA, Department of Chemistry, University of
Aveiro, Aveiro, Portugal
Moni Gupta Division of Biochemistry, Faculty of Basic Sciences, Sher-e-Kashmir
University of Agricultural Sciences and Technology, Chatha, Jammu, Jammu and
Kashmir, India
Sachin Gupta Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir
University of Agricultural Sciences and Technology of Jammu, Jammu, India
Janet A. Gutiérrez-Uribe Tecnologico de Monterrey, School of Engineering and
Science, Department of Bioengineering and Science, Campus Puebla, Puebla,
Mexico
Pádraigín A. Harnedy Department of Biological Sciences, University of Limer-
ick, Limerick, Ireland
Sumio Hayakawa Department of Cellular and Molecular Medicine, Medical
Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
Álvaro Hernáez Cardiovascular Risk and Nutrition Research Group (CARIN),
Muhammad Imran National Institute of Food Science and Technology, University
of Agriculture, Faisalabad, Pakistan
Department of Diet and Nutritional Sciences, Faculty of Health and Allied Sciences,
Imperial College of Business Studies, Lahore, Pakistan
Muhammad Jawad Iqbal National Institute of Food Science and Technology,
Faculty of Food, Nutrition and Home Sciences, University of Agriculture, Faisala-
bad, Pakistan
Contributors xxiii
Mahdi Irani Gum Group Knowledge Base Company, Technology Development

Center, Ferdowsi University of Mashhad, Mashhad, Iran
Mamoru Isemura Tea Science Research Center, University of Shizuoka, Suruga-
ku, Shizuoka, Japan
Tariq Ismail Institute of Food Science and Nutrition, Faculty of Agricultural
Seid Mahdi Jafari Department of Food Materials and Process Design Engineering,
Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Anna Jakubczyk Department of Biochemistry and Food Chemistry, University of
Life Sciences in Lublin, Lublin, Poland
Basil Jarvis Daubies Farm, Ross-on-Wye, UK
Department of Food and Nutritional Sciences, School of Chemistry, Food and
Pharmacy, The University, Reading, UK
Suellen Jensen Klososki Federal Institute of Paraná (IFPR), Paranavaí, Parana, Brazil
Alejandro Jiménez-Gómez Microbiology and Genetics Department, University of
Salamanca, Salamanca, Spain
Sandeep Baban Kalse Department of Post Harvest Engineering, Post Graduate
Institute of Post Harvest Management, Killa Roha, Maharashtra, India
Kristina Kappel Department of Safety and Quality of Milk and Fish Products,
Max Rubner-Institut, Kiel, Germany
Monika Karaś Department of Biochemistry and Food Chemistry, University of
Mehri Karim Department of Food Science and Technology, College of Agricul-
ture, Isfahan University of Technology, Isfahan, Iran
Mutiu Idowu Kazeem Department of Biochemistry, Faculty of Science, Lagos
State University, Ojo, Lagos, Nigeria
Hasim Kelebek Department of Food Engineering, Faculty of Engineering and
Natural Sciences, Adana Science and Technology University, Adana, Turkey
Soroush Rahimi Khoigani Department of Food Science and Technology, College
of Agriculture, Isfahan University of Technology, Isfahan, Iran
Nevzat Konar Faculty of Architecture and Engineering, Department of Food
Engineering, Siirt University, Siirt, Turkey
xxiv Contributors
Aleksandra Kozłowska First Faculty of Medicine, Department of Social Medicine

and Public Health, Medical University of Warsaw, Warsaw, Poland
Urszula Krupa-Kozak Department of Chemistry and Biodynamics of Food, Insti-
tute of Animal Reproduction and Food Research of Polish Academy of Sciences,
Olsztyn, Poland
Omer Kucuk Winship Cancer Institute of Emory University, Atlanta, GA, USA
Dinesh Kumar Department of Livestock Products Technology, College of Veter-
inary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhi-
ana, Punjab, India
Pavan Kumar Department of Livestock Products Technology, College of Veteri-
nary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhi-
ana, Punjab, India
Yogesh Kumar ICAR – Central Institute of Post-Harvest Engineering and Tech-
nology (CIPHET), Ludhiana, Punjab, India
Anam Layla Institute of Food Science and Nutrition, Faculty of Agricultural
Aurélien V. Le Gouic Department of Biological Sciences, University of Limerick,
Limerick, Ireland
Xunde Li Western Institute for Food Safety and Security, School of Veterinary
Medicine, University of California Davis, Davis, CA, USA
Giuseppina Pace Pereira Lima Department of Chemistry and Biochemistry, Insti-
tute of Biosciences, Sao Paulo State University – UNESP, Botucatu, São Paulo,
Brazil
José Moisés Laparra Llopis Madrid Institute for Advanced Studies in Food,
Madrid, Spain
Monica Rosa Loizzo Department of Pharmacy, Health and Nutritional Sciences,
University of Calabria, Rende, Cosenza, Italy
Ronan Lordan Department of Biological Sciences, University of Limerick, Lim-
erick, Ireland
Aline Carbonera Luvizon Faculdade Sudoeste Paulista – FSP, Avaré, São Paulo,
Brazil
Terrence Madhujith Faculty of Agriculture, Department of Food Science and
Technology, University of Peradeniya, Peradeniya, Sri Lanka
Om Prakash Malav Department of Livestock Products Technology, College of
Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University,
Ludhiana, Punjab, India
Contributors xxv
Sivakumar Manickam Department of Chemical and Environmental Engineering,

Faculty of Engineering, University of Nottingham, Semenyih, Malaysia
Cláudia Marques Nutrição e Metabolismo, NOVA Medical School, Faculdade de
Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal
Faculty of Medicine, University of Porto, Porto, Portugal
Edwin E. Martínez Leo Facultad de Ingeniería Química, Universidad Autónoma
de Yucatán, Mérida, Yucatán, Mexico
Natália Martins Mountain Research Centre (CIMO), Polytechnique Institute of
Bragança, Bragança, Portugal
REQUIMTE/LAQV, Faculty of Pharmacy, University of Porto, Porto, Portugal
Susan Mason Department of Wine Food and Molecular Biosciences, Faculty of
Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand
Nuno Mateus REQUIMTE/LAQV, Department of Chemistry and Biochemistry,
Meeta Mathur Department of Botany, Mithibai College, University of Mumbai,
Mumbai, India
Samira Mebdoua Laboratoire d’amélioration Intégrative des Productions
Végétales, Ecole Nationale Supérieure Agronomique, Alger, Algeria
Nitin Mehta Department of Livestock Products Technology, College of Veterinary
Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana,
Punjab, India
Rogério Mendes Portuguese Institute for the Sea and Atmosphere, Lisbon,
Portugal
Ana María Mendoza-Wilson Centro de Investigación en Alimentación y
Desarrollo, A.C., Coordinación de Tecnología de Alimentos de Origen Vegetal,
Hermosillo, Son., México
Igor Otavio Minatel Department of Chemistry and Biochemistry, Institute of
Biosciences, Sao Paulo State University – UNESP, Botucatu, São Paulo, Brazil
Faculdade Sudoeste Paulista – FSP, Avaré, São Paulo, Brazil
Shabir Ahmad Mir Department of Food Technology, Islamic University of Sci-
ence and Technology, Awantipora, Jammu and Kashmir, India
Remya Mohanraj Houston Community College, Houston, TX, USA
P. Monika Department of Biotechnology, Ramaiah Institute of Technology,
MSRIT Post, Bengaluru, India
xxvi Contributors
Efigenia Montalvo-González Laboratorio Integral de Investigación en Alimentos,

Instituto Tecnológico de Tepic, Tepic, Nayarit, Mexico
James D. Morton Department of Wine Food and Molecular Biosciences, Faculty
of Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand
Vahid Mosayebi Department of Food Science and Technology, College of Agri-
culture, Isfahan University of Technology, Isfahan, Iran
Hosakatte Niranjana Murthy Department of Botany, Karnatak University,
Dharwad, India
Research Center for the Development of Advanced Horticultural Technology,
Chungbuk National University, Cheongju, Republic of Korea
Hassan Hussein Musa Department of Medical Microbiology, Faculty of Medical
Laboratory Sciences, University of Khartoum, Khartoum, Sudan
Taha Hussein Musa Key Laboratory of Environmental Medicine, Ministry of
Education, School of Public Health, Southeast University, Nanjing, Jiangsu, China
Ricardo Fernández Musoles Valencian International University, Valencia, Spain
Emine Nakilcioglu-Tas Department of Food Engineering, Faculty of Engineering,
Ege University, Bornova, Izmir, Turkey
Androniki Naska Department of Hygiene, Epidemiology and Medical Statistics,
School of Medicine, National and Kapodistrian University of Athens, Athens,
Greece
Constantina Nasopoulou Department of Food Science and Nutrition, School of
the Environment, University of the Aegean, Myrina, Lemnos, Greece
Yumiko Oishi Department of Cellular and Molecular Medicine, Medical Research
Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
Maria Beatriz P. P. Oliveira REQUIMTE/LAQV, Faculty of Pharmacy, University
of Porto, Porto, Portugal
Cemal Orhan Department of Animal Nutrition, Faculty of Veterinary Science,
Firat University, Elazig, Turkey
Jorge Osorio Avalos Departamento de Nutricion Animal, Facultad de Medicina
Veterinaria y Zootecnia, Universidad Autonoma del Estado de Mexico, Toluca,
Estado de Mexico, Mexico
Semih Otles Department of Food Engineering, Faculty of Engineering, Ege Uni-
versity, Bornova, Izmir, Turkey
Kee-Yoeup Paek Research Center for the Development of Advanced Horticultural
Technology, Chungbuk National University, Cheongju, Republic of Korea
Contributors xxvii
Ibrahim Palabiyik Faculty of Agriculture, Department of Food Engineering,

Namik Kemal University, Tekirdağ, Turkey
Kasi Pandima Devi Department of Biotechnology, Alagappa University [Science
Campus], Karaikudi, Tamil Nadu, India
Arti Parihar Bellingham Technical College, Bellingham, WA, USA
Mordhwaj S. Parihar School of Studies in Zoology and Biotechnology, Vikram
University, Ujjain, Madhya Pradesh, India
So-Young Park Research Center for the Development of Advanced Horticultural
Technology, Chungbuk National University, Cheongju, Republic of Korea
Maike Passon Institute of Nutritional and Food Sciences, Molecular Food Tech-
nology, University of Bonn, Bonn, Germany
Niti Pathak Department of Botany, Deshbandhu College, University of Delhi,
Delhi, India
Kakoli Pegu Food Engineering and Technology Department, Institute of Chemical
Technology, Mumbai, India
Alejandra Pérez-Vega Cardiovascular Risk and Nutrition Research Group
(CARIN), Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
Chia-Wei Phan Mushroom Research Centre, University of Malaya, Kuala
Lumpur, Malaysia
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala
Lumpur, Malaysia
Tatiana Colombo Pimentel Federal Institute of Paraná (IFPR), Paranavaí, Parana,
Brazil
Haniyeh Rasouli Pirouzian Department of Food Science and Technology, Tabriz
University of Medical Sciences, Tabriz, Iran
Paolo Polidori School of Pharmacy, University of Camerino, Camerino (MC), Italy
Valeria Polzonetti School of Biosciences and Veterinary Medicine, University of
Camerino, Camerino (MC), Italy
Alejandra M. Preciado Saldaña Centro de Investigación en Alimentación y
Stefania Pucciarelli School of Biosciences and Veterinary Medicine, University of
Zahra Rafiee Department of Food Materials and Process Design Engineering,
Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
xxviii Contributors
Ashwani K. Rai Department of Botany, Banaras Hindu University, Varanasi, India

Pinky Raigond Division of Crop Physiology, Biochemistry and Post Harvest
Technology, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh,
India
Kishan Gopal Ramawat Department of Botany, University College of Science, M.
L. Sukhadia University, Udaipur, India
Seyed Mohammad Ali Razavi Food Hydrocolloids Research Centre, Department
of Food Science and Technology, Ferdowsi University of Mashhad (FUM),
Mashhad, Iran
Guillermo Reglero Department of Production and Characterization of Novel
Department of Production and Development of Foods for Health, IMDEA-Food
Institute, CEI (UAM-CSIC), Madrid, Spain
Natália Crialeison Balbo Vall Ribeiro Department of Food Science, Faculty of
Food Engineering, University of Campinas, Campinas, Brazil
Raúl Rivas Microbiology and Genetics Department, University of Salamanca,
Salamanca, Spain
Associated R&D Unit, USAL-CSIC (IRNASA), Salamanca, Spain
Lab 210-213, Edificio Departamental de Biología, Microbiology and Genetics
Department, University of Salamanca, Salamanca, Spain
Lizbeth E. Robles Jimenez Departamento de Nutricion Animal, Facultad de
Sílvia M. Rocha QOPNA, Department of Chemistry, University of Aveiro, Aveiro,
Portugal
Delia B. Rodriguez-Amaya Faculty of Food Engineering, University of Campi-
nas, Campinas, Sâo Paulo, Brazil
Universidade Federal da Fonteira Sul, Laranjeiras do Sul, Parana, Brazil
Rafael A. Rojas Herrera Facultad de Ingeniería Química, Universidad Autónoma
de Yucatán, Mérida, Yucatán, Mexico
Cristina M. Rosell Food Science Department, Institute of Agrochemistry and Food
Technology (IATA-CSIC), Paterna, Valencia, Spain
Michele Rosset Federal Institute of Paraná (IFPR), Colombo, Parana, Brazil
Jacqueline Ruiz Canizales Centro de Investigación en Alimentación y Desarrollo
A.C. Carretera a La Victoria km 0.6, Hermosillo, Sonora, Mexico
Contributors xxix
Vikineswary Sabaratnam Mushroom Research Centre, University of Malaya,

Kuala Lumpur, Malaysia
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala
Lumpur, Malaysia
Prajakta D. Sadawarte Food Engineering and Technology Department, Institute
of Chemical Technology, Mumbai, India
Kazim Sahin Department of Animal Nutrition, Faculty of Veterinary Science, Firat
University, Elazig, Turkey
Nurhan Sahin Department of Animal Nutrition, Faculty of Veterinary Science,
Firat University, Elazig, Turkey
Ângelo C. Salvador QOPNA, Department of Chemistry, University of Aveiro,
Aveiro, Portugal
CICECO- Aveiro Institute of Materials, Department of Chemistry, University of
Aveiro, Aveiro, Portugal
Liliana Santos-Zea Tecnologico de Monterrey, School of Engineering and Sci-
ence, Centro de Biotecnología-FEMSA, Monterrey, Mexico
Vicki Schlegel Department of Food Science and Technology, University of
Nebraska Lincoln, Lincoln, NE, USA
Ute Schröder Department of Safety and Quality of Milk and Fish Products, Max
Rubner-Institut, Kiel, Germany
Maira R. Segura Campos Facultad de Ingeniería Química, Universidad Autó-
noma de Yucatán, Mérida, Yucatán, Mexico
Serkan Selli Department of Food Engineering, Faculty of Agriculture, Cukurova
University, Adana, Turkey
Manzoor Ahmad Shah Department of Food Science and Technology, Pondicherry
University, Puducherry, India
Ramesh Kumar Sharma Food and Environment Issues, Bikaner, Rajasthan, India
M. Shivapriya College of Horticulture, University of Horticultural Sciences,
GKVK, Bengaluru, India
Vincenzo Sicari Department of Agricultural Science, Mediterranean University of
Reggio Calabria, Reggio, Calabria, Italy
Helena Silva Portuguese Institute for the Sea and Atmosphere, Lisbon, Portugal
Armando J. D. Silvestre CICECO- Aveiro Institute of Materials, Department of
Chemistry, University of Aveiro, Aveiro, Portugal
Brajesh Singh Division of Crop Physiology, Biochemistry and Post Harvest Tech-
nology, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India
xxx Contributors
Brij Pal Singh Dairy Microbiology Division, ICAR-National Dairy Research

Institute, Karnal, Haryana, India
Charalampos Siotos Department of Hygiene, Epidemiology and Medical Statis-
tics, School of Medicine, National and Kapodistrian University of Athens, Athens,
Greece
Subajiny Sivakanthan Faculty of Agriculture, Department of Agricultural Chem-
istry, University of Jaffna, Jaffna, Sri Lanka
Carmen G. Sotelo Instituto de Investigaciones Marinas (CSIC), Vigo, Spain
Soma Srivastava Division of Agricultural Engineering and Renewable Energy,
Central Arid Zone Research Institute, Jodhpur, India
Anna Stachniuk Laboratory of Separation and Spectroscopic Method Applica-
tions, Center for Interdisciplinary Research, The John Paul II Catholic University
of Lublin, Lublin, Poland
Surya Sudheer Department of Chemical and Environmental Engineering, Faculty
of Engineering, University of Nottingham, Semenyih, Malaysia
Hafiz Ansar Rasul Suleria UQ School of Medicine, Translational Research Insti-
tute, The University of Queensland, Brisbane, QLD, Australia
Department of Food, Nutrition, Dietetics and Health, Kansas State University,
Manhattan, KS, USA
Baby Summuna Division of Plant Pathology, Faculty of Agriculture, SKUAST-
Kashmir, Jammu, Jammu and Kashmir, India
Yasuo Suzuki Department of Nutrition Management, Faculty of Health Science,
Hyogo University, Kakogawa, Hyogo, Japan
Shrikant Baslingappa Swami Department of Post Harvest Engineering, Post
Graduate Institute of Post Harvest Management, Killa Roha, Maharashtra, India
Dorota Szostak-Węgierek Faculty of Health Science, Department of Clinical
Dietetics, Medical University of Warsaw, Warsaw, Poland
Elson Yi-Yong Tan Mushroom Research Centre, University of Malaya, Kuala
Lumpur, Malaysia
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala
Lumpur, Malaysia
Hiroki Tanabe Department of Nutritional Sciences, Faculty of Health and Welfare
Science, Nayoro City University, Nayoro-City, Hokkaido, Japan
B. Tejashree Department of Biotechnology, Ramaiah Institute of Technology,
MSRIT Post, Bengaluru, India
Omer Said Toker Faculty of Chemistry and Metallurgical, Department of Food
Engineering, Yildiz Technical University, İstanbul, Turkey
Contributors xxxi
Carlos Torres Department of Production and Characterization of Novel Foods,

Alexandros Tsoupras Department of Biological Sciences, University of Limerick,

Limerick, Ireland
Rosa Tundis Department of Pharmacy, Health and Nutritional Sciences, University

of Calabria, Rende, Cosenza, Italy
Julieta Valussi Cardiovascular Risk and Nutrition Research Group (CARIN),

Luis Vázquez Department of Production and Characterization of Novel Foods,
Gustavo R. Velderrain Rodríguez Centro de Investigación en Alimentación y

V. Venugopal Former Scientific Officer Food Technology Division, Bhabha
Atomic Research Center, Mumbai, India
Visiting faculty, Department of Food Science and Technology, Kerala University of
Fisheries and Ocean Sciences (KUFOS), Kochi, Kerala, India
Akhilesh Kumar Verma Department of Livestock Products Technology, College
of Veterinary Science, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan
Vishwavidyalaya evam Go Anusandhan Sansthan (DUVASU), Mathura, Uttar
Pradesh, India
Véronique Verrez-Bagnis Ifremer, Nantes, France
Shilpa Vij Dairy Microbiology Division, ICAR-National Dairy Research Institute,
Karnal, Haryana, India
Mónica A. Villegas Ochoa Centro de Investigación en Alimentación y Desarrollo
A.C. Carretera a La Victoria km 0.6, Hermosillo, Sonora, Mexico
Javier Villela-Castrejón Tecnologico de Monterrey, School of Engineering and
Science, Centro de Biotecnología-FEMSA, Monterrey, Mexico
Silvia Vincenzetti School of Biosciences and Veterinary Medicine, University of
Marco Vinceti Research Center for Environmental, Genetic and Nutritional Epi-
demiology (CREAGEN), University of Modena and Reggio Emilia, Modena, Italy
Department of Epidemiology, Boston University School of Public Health, Boston,
MA, USA
xxxii Contributors
Anshu Wali Division of Biochemistry, Faculty of Basic Sciences, Sher-e-Kashmir

University of Agricultural Sciences and Technology, Chatha, Jammu, Jammu and
Kashmir, India
Jianbo Xiao Institute of Chinese Medical Sciences, State Key Laboratory of
Quality Research in Chinese Medicine, Macau University, Taipa, Macau
Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, Würzburg,
Germany
Deepika Yadav Dairy Microbiology Division, ICAR-National Dairy Research
Institute, Karnal, Haryana, India
Ioannis Zabetakis Department of Biological Sciences, University of Limerick,
Limerick, Ireland
Ewelina Zielińska Department of Biochemistry and Food Chemistry, University of
Damian Zieliński Department of Ethology and Animal Welfare, University of Life
Sciences in Lublin, Lublin, Poland
Part I
Favorable Regimen for Good Health and
Epidemiological Studies
Health Benefits of Dietary Phenolic
Compounds and Biogenic Amines 1
Hector Alonzo Gomez-Gomez, Cristine Vanz Borges,
Igor Otavio Minatel, Aline Carbonera Luvizon, and
Giuseppina Pace Pereira Lima
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Polyamines/Biogenic Amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4 Processing Methods – Effects in the Content of Phenolic Compounds
and Polyamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5 Organic Versus Conventional Fruits and Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6 Beneficial Effects of Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7 Interaction of Phenolic Compounds and Polyamines on Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
8 Interaction of Phenolic Compounds and Polyamines on Human Health . . . . . . . . . . . . . . . . . . 13
9 Concentrations of Phenolic Compounds and Biogenic Amines in Some Foods . . . . . . . . . . 19
10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Abstract
The bioavailability of dietary phytochemicals may be influenced by several
factors as food matrix, cultivation conditions, host microbiota, and physiological
state. Phenolic compounds and polyamines have been associated with various
H. A. Gomez-Gomez · C. V. Borges · G. P. P. Lima (*)

Department of Chemistry and Biochemistry, Institute of Biosciences, Sao Paulo State University –
UNESP, Botucatu, São Paulo, Brazil
e-mail: [email protected]
I. O. Minatel
Department of Chemistry and Biochemistry, Institute of Biosciences, Sao Paulo State University –
UNESP, Botucatu, São Paulo, Brazil
A. C. Luvizon
# Springer Nature Switzerland AG 2019 3

J.-M. Mérillon, K. G. Ramawat (eds.), Bioactive Molecules in Food, Reference Series in
Phytochemistry, https://doi.org/10.1007/978-3-319-78030-6_27
4 H. A. Gomez-Gomez et al.
health benefits. However, their role in human health is dependent on interactions

with the gut microbiota and conversion to further bioactive compounds. Dietary
compounds have a wide range of effects that are play after the binding and/or
interaction between these compounds and other molecules.
Keywords
Phenolic compounds · Polyphenols · Polyamines · Biogenic amines · Fruit ·
Vegetable · Beverage · Gut microbiota
Abbreviations
ADC Arginine decarboxylase
AIH Agmatine iminohydrolase
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
CANSpdDC Carboxynorspermidine decarboxylase
CANSpdDH Carboxynorspermidine dehydrogenase
CPA N-carbamoylputrescine amidohydrolase
DAO Diamine oxidase
DDC 3,4-dihydroxyphenylalanine decarboxylase
DOPA 3,4 Dihydroxyphenylalanine
E4-P Erythrose 4-phosphate
GABA γ-aminobutyric acid
HDC Histidine decarboxylase
ODC Ornithine decarboxylase
PAL Phenylalanine ammonia liase
PAO Polyamine oxidase
PEP Phosphoenolpyruvate
POD Peroxidase
PPO Polyphenol oxidases
ROS Reactive oxygen species
SAM S- adenosylmethionine
SAMDC S-adenosylmethionine decarboxylase
SAT1 Spermidine/spermine-N1-acetyltransferase
SPDS Spermidine synthase
SPMS Spermine synthase
TAL Tyrosine ammonia-lyase
TGFβ Transforming growth factor-β
1 Introduction
Phenolic compounds are natural compounds occurring in vegetables, fruits, and

derived beverages that humans usually obtain through the diet. Structurally, phenolic
compounds are characterized by the presence of one or several phenolic groups,
conferring their complexity and allowing to classify them into flavonoids
1 Health Benefits of Dietary Phenolic Compounds and Biogenic Amines 5
(polyphenol) or nonflavonoids. This large class of compounds has a well-established

antioxidant activity and other beneficial effects on human health. However, its
interactions in human body after dietary ingestion should be better addressed. Poly-
amines are a group of aliphatic amines, ubiquitously produced by humans and
vegetables. These compounds are able to bind to many cellular macromolecules as
DNA, RNA, and proteins, conferring them the ability to promote cell proliferation,
signal transduction, and membrane stabilization. Phenolics and polyamines are
produced by vegetables, whereas in human organism, only polyamines can be
synthesized. Considering the possible interaction of these dietary components, this
chapter summarizes some phytochemicals in vegetables and the mechanisms by
which these compounds are absorbed, metabolized, and affect physiological func-
tions in humans.
2 Phenolic Compounds
Phenolic compounds constitute an important class of secondary metabolites, notably

in function of its recognized antioxidant activity, which confers quality to the food
and beneficial potential to the human health. These antioxidants neutralize the free
radicals, by inhibiting or interrupting the chain of initiation/propagation of oxidative
reactions, converting the free radicals in less harmful molecules, and repairing
the oxidative damages in human cells [1]. The functionality and stability of these
phytochemicals in the human body depends not only on the quantity, but also on
their location in the food matrix, on the presence of other bioactive compounds
and, mainly, on the binding and/or interaction between these compounds and other
molecules. Interestingly, the interaction between polyphenols and biogenic amines
metabolic pathways should be pointed out to emphasize the role of both molecules
on the healthy or harmful characteristics of a certain food.
Derived from the secondary plant metabolism, phenolic compounds constitute a
group of molecules with a diversity of functions in the plant and different beneficial
properties for health [2, 3]. The antioxidant activity and the free radical scavenging
capacity depend on their structure, number of hydroxyl groups (OH), and position on
the structure [4]. These heterogeneous characteristics are due to the complex meta-
bolic pathways, for which the phenolic compounds are synthesized and the shikimic
acid pathway is the most important biosynthesis route [5, 6].
By the shikimate pathway (present in plants and absent in animals), the binding of
primary metabolism occurs (carbohydrates production) with the secondary metabo-
lism, using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4-P, pentose
phosphate pathway), synthesizing chorismate, from which aromatic amino acids can
be synthesized, such as phenylalanine, tyrosine, and tryptophan. These amino acids
are used in the protein synthesis and also as secondary metabolites, including
phenolic compounds, meaning that this biosynthetic route may contribute as a
regulator of primary and secondary plant metabolism [7–9].
Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) catalyzes the synthesis of
trans-cinnamic acid from phenylalanine. In addition to PAL, tyrosine ammonia-lyase
Chorismate L-Arginine ADC Agmatine

↑UV-B ARG AIH
L - Phenylalanine L - Tyrosine L - Tryptophan Ornithine CP
PAL TH ODC CPA

GABA
↑UV-B
Cinnamic acid TAL L-DOPA 5-Hydroxytryptophan Putrescine
DAO
DDC
Δ1 Pyrroline SPDS
4 – Coumaric acid Dopamine Serotonine Spermidine

PAO
H2O2 + NH3 SPMS SPMOX

Stilbenes Phenolic acids
Flavonoids Aminopropylpyrroline Spermine
PAO
Fig. 1 Interaction of polyamines (primary metabolism) and phenolics (secondary metabolism) in

plants. ARG arginase, AIH agmatine iminohydrolase, ADC arginine decarboxylase, ODC ornithine
decarboxylase, CP N-carbamoyl putrescine, CPA N- carbamoylputrescine amidohydrolase, SPDS
spermidine synthase, SPMS spermine synthase, SPMOX spermine oxidase, DAO diamine oxidase,
PAO polyamine oxidase, PAL phenylalanine ammonia-lyase, TAL tyrosine ammonia-lyase, TH
tyrosine hydroxylase, DDC dopa decarboxylase
(TAL, EC 4.3.1.23) has great importance in this metabolic route. TAL catalyzes the
conversion of tyrosine to p-coumaric acid [9]. On the other hand, tyrosine can be
converted in DOPA (3,4 dihydroxyphenylalanine) by tyrosine dehydrogenase and in
dopamine (biogenic amine) by 3,4-dihydroxyphenylalanine decarboxylase (DDC;
EC 4.1.1.27) (Fig. 1). Both enzymes are relevant for phenolic compounds synthesis.
On the other hand, phenolic compounds may be oxidized by polyphenol oxidases
(PPO, EC 1.10.3.2 or EC 1.14.18.1), such as tyrosinase, cresolase, catecholase,
diphenolase, and phenolase or peroxidases (POD, EC 1.11.1.7). High POD and
PPO activities are related to the production of browning compounds, with decrease
in the commercial value and loss of quality, including nutritional benefits [10, 11]. To
reduce oxidative enzymes activities (PPO and POD), it may be necessary for
physical and chemical treatments to guarantee the nutritional quality, maintaining
some phytochemicals contents, such as anthocyanins, other phenolic compounds,
and antioxidant activity [12–15].
3 Polyamines/Biogenic Amines
Polyamines and/or biogenic amines have been investigated in food matrixes due to
the interest regarding the nutritional paradox and its possible action as antioxidants.
Previous studies indicate a possible role of polyamines, present abundantly in food
of the Mediterranean diet, with the prolongation of human life [16]. Diamines, as
putrescine, and polyamines (spermidine and spermine) can be formed through

the decarboxylation of specific amino acids [17]. The biogenic amines are the
aliphatic, cationic organic compounds of low molecular weight, formed after
amino acids decarboxylation or by amination and transamination of aldehydes and
ketones [18, 19], which include histamine, serotonin, tyramine, 2-phenylethylamine,
tryptamine, putrescine, cadaverine, and agmatine. In raw vegetables, the amines are
formed from the activity of some enzymes, while in processed foods they are formed
by the action of microorganisms, which can often be, depending on the amine found
and on the concentration, a signal of contamination [20].
In mammals, the polyamines can be synthesized, obtained from the diet, or
even, synthesized for intestinal microorganisms. In mammals, the putrescine bio-
synthesis comes from the ornithine decarboxylation by the action of ornithine
decarboxylase (ODC) (EC 4.1.1.17), forming the diamine putrescine. In plants,
there is an alternative pathway by the action of arginine decarboxylase (ADC, EC
4.1.1.19) forming putrescine. Arginine is decarboxylated by ADC, forming
agmatine, which forms N- N- carbamoylputrescine by the action of agmatine
iminohydrolase (AIH, EC 3.5.2.12). The enzyme N-carbamoylputrescine
amidohydrolase (CPA, EC 3.5.1.53) converts the product in putrescine (Fig. 1).
The suppression of the putrescine synthesis from arginine could increase the amount
of available arginine available for the synthesis of nitric oxide, which is responsible
for maintaining normal vascular functions [21], retarding the progression of vascular
diseases associated to the aging process. The diamine cadaverine is produced by
microorganisms and originated from lysine decarboxylation, by lysine decarboxyl-
ase [22].
Both in mammals and plant cells, spermidine is formed from the addition of
aminopropyl group in putrescine by the action of spermidine synthase (SPDS, EC
2.5.1.16) and spermine synthase (SPMS, EC 2.5.1.16) and adds another
aminopropyl group to the spermidine, forming spermine. In parallel, S-adenosyl-
methionine (SAM) is decarboxylated by SAM decarboxylase (SAMDC, EC
4.1.1.50) and provides aminopropyl groups to the formation of spermidine and
spermine. All three, ODC, ADC, and SAMDC, are regulatory enzymes (rate-limit-
ing) of the polyamines synthesis [23, 24].
The polyamines oxidation occurs via amines oxidases. Diamine oxidase
(DAO, EC 1.4.3.6) oxides putrescine or cadaverine in primary amino groups,
forming 4- aminobutanal, which cyclics, forming delta-1-pirroline and hydrogen
peroxide, and polyamine oxidase (PAO, EC 1.5.3.3) oxides spermidine and
spermine. Tri- and tetramines oxidations produce hydrogen peroxide (Fig. 1).
During the polyamines oxidation, there is also a formation of γ-aminobutyric
acid (GABA), a nonprotein amino acid that acts as a endogenous molecular
marker, with inhibitory action of neurotransmitters in animal cells [25]. The food
enriched with GABA became popular due to their functional effects and regarding
the health in the arterial pressure regulation, cardiac frequency, relieve of pain, and
anxiety [26].
Polyamines present a series of functions in cells from prokaryotes and
eukaryotes organisms. In plants, they have an important role in the growth,
differentiation, synthesis of nucleic acids and proteins, membrane stability,

resistance, senescence, programmed cells death, adaptive responses to the envi-
ronment, among others [27–30]. In addition, these molecules in mammals
are involved in some diseases as Alzheimer and cancer [31]. Studies demonstrate
increase in polyamines levels in the urine and serum [32], as well as
induce the activity of enzymes involved in the polyamines synthesis, as ODC in
tumor cells [33].
Biotic and abiotic stresses lead to oxidative stress, which is, many times,
quickly manifested with accumulation of reactive oxygen species (ROS). ROS
are important signaling molecules, but are toxic in high concentrations. ROS affect
many cell functions by damaging the nucleic acids, oxidizing proteins, and
causing lipid peroxidation [34]. The polyamines have a double role against the
ROS accumulation in stressful conditions and are considered osmoprotectors and
ROS scavengers. The polyamines putrescine, spermidine, and spermine are pos-
itively charged at physiological pH and can bond to membrane cell groups
negatively charged and prevent damages caused by stress factors in plants [35].
In addition, polyamines can interact with negatively charged molecules as DNA
and RNA through electrostatic bonding [36]. This bonding stabilizes the DNA
structure, mainly by bonding with spermidine and spermine [37, 38]. Spermidine
and spermine (not putrescine), when in high concentrations (above 10 mM),
provide protection against damages from DNA radiation [39]. However, in sub-
mM levels, it is ineffective and can slightly promote damages caused by gamma
radiation. Other studies affirm that the polyamines have the ability to induce the
DNA compaction and aggregation, besides acting as a scavenger of free radicals,
mainly OH generated by gamma irradiation [39, 40], thus becoming a DNA
protector cell.
The antioxidant action of some biogenic amines and polyamines correlates to the
quantity of amino groups in their chemical structures. Studies have shown that
amines as dopamine present a higher in vitro antioxidant capacity (through DPPH
test), comparing to other natural antioxidants, as ascorbic acid [41]. In addition, it
has been claimed that the increased intake of polyamines present in foods led to
augmented intracelular amounts of these metabolites, promoting vascular health in
humans. The increase in intracelular polyamines suppresses the enzymatic activity
required for their synthesis.
Some biogenic amines (histamine, tryptamine, tyramine, putrescine, and cadav-
erine) are undesirable in high concentrations in the food, because they can promote
respiratory problems, headaches, cardiac palpitations, hypertension, or hypotension,
among other allergic diseases. Simultaneously, some polyamines (e.g., putrescine,
spermidine, and spermine) in high concentrations can cause the proliferation of
cancer cells [42]. The association of polyamines to cancer growth/progression may
be related to polyamines catabolism. The excessive ingestion of food rich in poly-
amines, with a consequent increase of its content in the cells, can induce apoptosis,
possibly due to the increasing peroxide content which is the product of the degra-
dation of di- and polyamines by the oxidative enzymes DAO and PAO [43], leading
to the cell death.
The intake of polyamines by feed and to control of some diseases has been
studied by many researches groups, with the intention of decreasing or preventing
damages provoked by these amines. Cipolla et al. [44] observed, in patients with
prostate cancer, that reducing polyamine diet and partial intestinal decontamination
was beneficial to improving the life quality and pain control. The authors also
describe that 3 months after the end of this treatment, the pain came back.
4 Processing Methods – Effects in the Content of Phenolic

Compounds and Polyamines
Changes in the phenolic compounds and polyamines compositions are of great

complexity because they vary according to the structure and the type of analyzed
food, besides the cooking method used to its preparation. The most of the vegetables
and some fruits are preferably consumed after some type of thermal processing, which
causes favorable changes in the flavor and texture, increasing the food palatability.
Previous studies indicate that the retention of phytochemicals and the antioxidant
properties after the cooking vary considerably among the different vegetables,
among the methods used in their preparation, and mainly, among the specific analyzed
compounds [45, 46]. The phenolic compounds are generally considered stable to the
heat, and the occasional loses in the different thermal processes are probably attributed
to lixiviation. Studies indicate that rutine and other polyphenols present in vegetables
(e.g., asparagus) do not degrade when heated. The rutine’s stability to heat was
confirmed by recovering the compounds from the water used in the boiling process
[47]. Generally, methods that do not use water, e.g., steaming, lead to an increase in the
total phenolic compounds, flavonoids and phenolic acids in comparison to the in
natura vegetables [48]. This can be explained by the rupture of complexes among
the phenolic compounds and other compounds (e.g., proteins) generated by the
extraction by steam, resulting in a better availability of these compounds [49].
The polyamines are original from amino acids, such as arginine and ornithine,
which act as precursors and can suffer decarboxylation processes by the action
of putrefactive bacteria. This explains why a higher concentration of polyamines
is found after the fermentation of products as sauerkraut, some sausages, cheeses,
and wines [50–52]. In addition, it is known that the processing of vegetables, such
as the heating or freezing, can rupture the cell wall, leading to the liberation of these
compounds that were free or binding to the membrane, inducing an increase
of bioavailability of some compounds [49, 53]. However, for polyamines there are
no differences on the content, in organic or conventional green beans, after ther-
mal processing [46]. Besides this, organic green beans presented higher spermidine
and spermine contents. These data can be important when the relation of poly-
amines with diets for cancer patients is studies, as described by [44], or even when
it is wanted to obtain food with antioxidants, since these amines are considered
free radicals scavengers [54]. Thus, studies with polyamines acting as antioxidants
and their relation to diets should be deepened, due to the duality of these substances
function.
5 Organic Versus Conventional Fruits and Vegetables
Several studies highlight the benefits of ingesting of organic foods, due to the higher
content of some bioactive compounds and higher antioxidant activity, besides being
free of chemical residues [46, 55–57]. The plant exposition to a stressful environ-
ment, as attack by insects or fungi, can induce the production of natural defense
substances, as well as the phenolic compounds [58, 59]. The organic plant-based
food contains higher polyamines levels (that can be protection mechanisms against
many types of stress) in comparison to the conventional farming [57]. Studies with
fruits indicate an increased polyamine content and a protective effect against dam-
ages caused by biotic and abiotic factors in the membranes [60]. However, this
profile was not verified in collard green, where the highest putrescine levels were
found in the conventionally cultivated vegetables [57].
Currently, the pharmaceutical industry has searched for organic plants with higher
contents of bioactives. It is known that the interaction between different food
metabolites (as biogenic amines and polyphenols) and their biosynthetic pathways
can contribute to the healthy or harmful characteristics of the own food. In a research
developed in our laboratory using two jambu cultivars (Acmella oleracea cv.
Jambuarana and Nazaré), a medicinal plant used in the cosmetic and pharmaceutical
industries and also consumed by the population from North region of Brazil, the
polyamines levels was affected by the cultivation practice. Organic jambu (both
varieties) showed higher spermine content in leaves and spermine and spermidine
in inflorescences compared with the conventional fertilization. Besides the poly-
amines levels, the phenolic compounds content was increased by the organic
cultivation [61]. The results show that the organic cultivation can induce positive
alterations regarding the antioxidants levels.
Biotic or abiotic stress can induce high phenolic compound contents by increas-
ing the activity of the enzymes. Phenolic compounds act as cell protectors against
free radicals generated by stress [62, 63]. Some plant-based foods produced under
moderate stress, such as organic farming, tend to show higher antioxidant activity
due to phytochemical levels, such as phenolics, compared with conventional ones.
Organic food may contain more phenolic compounds due to the absence of agro-
chemicals, which collaborates in the defense against some types of stress [30].
An increased 139% in the total phenolic content of organic tomatoes in relation to
conventional farming, which may be a consequence of increased PAL activity, was
observed during development [64]. Even after the processing, this quality seems to
be maintained. Organic tomato juice presents higher phenolic compounds levels and
higher antioxidant activity when compared to those conventionally grown [65].
6 Beneficial Effects of Phenolic Compounds
Phenolic compounds play an important role in sensory characteristics and are respon-
sible for some organoleptic properties such as aroma, color, taste, bitterness, and
astringency in fruits and vegetables, as well as their derivatives [66, 67].In addition,
they are directly or indirectly related to quality and health benefits. Flavonoids, antho-
cyanins, tannins, and resveratrol (stilbene) have proven action against different diseases
in humans [68–71]. Quercetin shows beneficial properties and is found in onion, apple,
brassicas, among other vegetables. This flavonoid has antidiabetic, anti- inflammatory,
and anticancer activity [72]. Other flavonoids, such as kaempferol and catechin, act as
antioxidants. Studies demonstrated the inhibition of tumor cell growth, reducing the
progression of breast cancer in a rat model after treatment with both flavonoids [73, 74].
Some species of the genus Citrus, Brassica, Allium, and Mallus contain
kaempferol, a flavonoid with potential to be used in individuals with cardiovascular
diseases. In vitro studies showed its action on the decrease of cellular apoptosis
induced by LDL cholesterol in human cells and antithrombotic action [75–77].
Beneficial effects are enhanced with resveratrol, a molecule considered effect-
ive in preventing different pathological conditions, for example, in the reduction of
blood pressure and repression of lipid peroxidation, oxidative stress, and lipid levels
[78, 79]. In vitro studies demonstrate that this stilbene has a beneficial effect as
a coadjutant in the control of norovirus, a pathogenic agent involved in food diseases
(at great risk in the elderly and in the immunocompromised). In addition, the
resveratrol action has been demonstrated in the expression of genes that decrease
insulin resistance, meaning possible beneficial effects for diabetics [80, 81].
Anthocyanins are natural pigments found in fruits and vegetables. These com-
pounds provide blue, purple, and red colors and with proven activity as
nutraceuticals. Grapes and their derivatives are sources of antioxidant substances
and are rapidly absorbed, with various physiological activities in animal cells [82].
Cyanidin-3-O-β-glucoside is present in a large number of fruits and vegetables, and
studies have been demonstrated its protective effect against lipopolysaccharide-
induced lung injury by lowering cholesterol levels and suppressing the inflammatory
response [83]. This anthocyanin has also shown to decrease the negative effects of
free fatty acids, decreasing insulin resistance through the activation of cellular and
cytoprotective antioxidant genes [84], as well as having preventive or complemen-
tary activity against inflammatory chronic diseases [85, 86]. It was demonstrated by
in vitro study using Morus alba L. that the presence of cyanidin-3-O-β-glucoside,
together with cyanidin 3-rutinoside, has an inhibitory effect on the migration and
invasion of human lung carcinoma (A549) cells.
Malvidin-3-O-β-glycoside, the main anthocyanins in grapes, is a potent anti-inflam-
matory; it aids in the balance and inhibition of pro-inflammatory pathways, promoting
cardiovascular health benefits. When malvidin-3-O-β-glycoside is in combination with
peonidine, there is an increased beneficial effects activity. Studies in vitro and in vivo
showed decreases of transcription of genes encoding proinflammatory cytokines,
causing a decrease in anti-inflammatory activity in arthritic rats [87, 88].
Pelargonidine, another anthocyanin found in large quantities in grapes and deriva-
tives, has a neuroprotective effect, improving memory deficit by mitigating oxidative
stress, helping to reduce the symptoms of individuals with Alzheimer’s disease. In
addition, pelargonidine provides potential benefits against atherosclerosis in albino
mice. This compound acts against stress generated by environmental toxicants (urethane
and diepoxybutane), compounds classified as possible human carcinogens [89–91].
Delphinidin also has a beneficial activity and its anticancer action has been
demonstrated. In vitro study has demonstrated inhibition of proliferation and migra-
tion of ovarian carcinoma cells. This effect may be an important health response,
meaning a potential natural chemotherapeutic to prevent the development and
progression of cancer cells [92, 93]. In addition, it has regenerative effect on cells
exposed to UVB radiation (100 mJ/cm2), demonstrating great potential in protecting
skin damaged by the sun.
Several phenolic acids have been studied in reducing symptoms or diseases. It has
been demonstrated that p-coumaric acid has the ability of eliminating free radicals
and lowering LDL cholesterol [94]. Ferulic acid has been related to the protection of
oxidative stress in rat renal tissue caused by cisplatin (anticancer agent). The use of
this phenolic acid avoided renal damage and helped to recover the histological
parameters of the kidney under normal conditions [95]. Chlorogenic acid found in
sweet potatoes and coffee has antibacterial and anti-inflammatory activity and can
inhibit tumors. Besides that, this phenolic acid has protective effects on the liver and
antihypertensive function. In rats with the cerebral ischemia reperfusion injury,
chlorogenic acid improves pathological lesions in brain tissue, reducing mortality.
Studies demonstrated an effect against prostatic hyperplasia in mice, besides reduc-
ing the negative effects of high consumption of glucose and lipids, avoiding the
occurrence of diabetes, obesity, cardiovascular diseases, and cancer in humans
[96–98].
7 Interaction of Phenolic Compounds and Polyamines on

Plants
Besides the effect of some polyphenols on the polyamines, the plants can form
phenolamides, which are polyamines combined by amide bonding with some
phenolic acids such as coumaric, caffeic, and ferulic acids, through the substitution
of 1, 2, or 3 amino groups. This combination of the phenolic acids that occurs with
the aliphatic polyamines (putrescine, cadaverine, spermidine, and spermine) forms
the basic amides.
When occurs the bonding with the aromatics (tyramine, tryptamine, dopamine,
and octopamine) or with aliphatics, forms the neutral amides [99, 100]. The
phenolamides has been described as specific secondary metabolites and related to
the development of plants defense mechanism, and the structure of these molecules
are similar to the ones found in the insects toxins [101].
The phenolamides content can be affected in response to the pathogens attack,
as during the hypersensitive reaction towards fungi or virus diseases. Some plant
organs, as the leaves, can synthesize phenolamides as defense mechanism, decreas-
ing the pathogens action [99]. Reference [102] describes an increase of
phenolamides and decrease of the powdery mildew infection. Reference [103]
suggests that the low incidence of virus diseases in flowers and seed can be related
to the phenolamides content. However, in some cases of host-pathogen interactions,
especially during the hypersensitive reaction towards fungi or viruses, leaves of
vegetative plants synthesize some phenolic amides at the same time as defense
mechanisms, limit pathogen expansion.
Many foods can produce harmful substances from the oxidation of fatty acids
[104] with mutagenic and carcinogenic activities during the processing, the storage,
or by contaminants [105]. In order to fight the oxidation, synthetic or nat-
ural antioxidants can be used. Some species contain high phenolamides levels that
can be used as antioxidants. Reference [106] described phenolamides in Piper
and demonstrated a higher antioxidant capacity compared to alfa-tocopherol, and
feruperine showed an antioxidant activity almost as high as BHA (butylated
hydroxyanisole) and BHT (butylated hydroxytoluene).
8 Interaction of Phenolic Compounds and Polyamines on

Human Health
Studies that related the beneficial effects of the interaction of the phenolic
compounds with the polyamines indicate that phenolic compounds, as the
chatequins, interact with the biosynthetic pathways of polyamines. This reaction
occurs specifically reducing the production of histamine and putrescine due to
the inhibition of the histidine decarboxylase (HDC; EC 4.1.1.22) and ODC activities
and increase spermidine/spermine N1-acetyltransferase activity, which pro-
motes the polyamines catabolism [107, 108]. High activity of ODC is characteristic
of the tumor cells [109], and the inhibition of ODC expression may be one of several
targets involved in the antiproliferative effects of resveratrol [42]. Resveratrol and
piceatannol (Piceat) (derived from resveratrol) also were able to reduce the ODC
levels in Caco-2 colorectal cancer cells [110]. This enzyme is directly related to
the cellular proliferation and carcinogenesis. Similar results were observed by [42],
where resveratrol induced a decrease in the ODC activity in Caco-2 cells and a
significant decrease of intracellular putrescine content. However, it did not affect
the SAMDC activity, not affecting the content of spermidine and spermine.
Polyphenols has the ability to targeting polyamines or biogenic amines. Besides
the possible synergic activity of both compounds, some classes, as flavonoids,
have shown the capacity of blockage key enzymes from biogenic amines biosyn-
thetic pathways [108]. Dopamine and serotonin play critical role in neuropsychiatric
disorders, such as depression, schizophrenia, and Parkinson’s disease. Both biogenic
amines are essential for serotonergic and dopaminergic systems to regulate the
neurotransmission. Most of the psychotropic drugs used in the treatment of neuro-
psychiatric disorders, including antidepressants, antipsychotics, anxiolytics, and
psychostimulants, exerted their pharmacological actions by interfering with seroto-
nergic and dopaminergic transmission. Thus, deregulations in their levels (amines)
are a key step in the control of neurological diseases. It is interesting to note that
epigallocatechin gallate and epigallocatechin showed the ability to interact with and
bind to the enzyme Dopa decarboxylase (converts L-Dopa and L-5-
hydroxytryptophan into dopamine and serotonin, respectively), leading to its irre-
versible inhibition [111]. Histamine is other biogenic amine that can be regulated by
epigallocatechin gallate. To produce histamine neurons, mast cells, basophils, and

monocytes/macrophages, among others, produce the enzyme HDC that catalyzes the
conversion of histidine in histamine. A dual effect of epigallocatechin gallato was
observed against histamine. The first was demonstrated in rat basophilic leukemia
cells that epigallocatechin gallato prevented histamine release from mast cells [112].
The second effect was observed in mammalian cells, where epigallocatechin gallato
demonstrated a potent inhibitory effect of HDC activity [113].
Interestingly it has also been reported that high concentrations of some phenolic
compounds affect biogenic amine production by inhibiting lactic acid bacterial
growth [114]. Low levels of biogenic amines in some types of wines can be related
to the presence of large phenolic compounds quantities (e.g., anthocianins and
stilbenes) that, by inhibiting the activity of naturally present bacteria, can reduce
the formation of histamine, tyramine, and putrescine.
Usually the polyphenols are not absorbed by small intestine, and it has been
estimated that only 10% is absorbed and the remaining 90% is metabolized in the
large intestine by gut microbiota or excreted [115]. The polyphenols conversion in
the human gut is a complex process and should be considered when assessing the
bioavailability of plant compounds. For example, compounds as procyanidins are
poorly absorbed in its original molecular structure, but the human gut microbiota
might play an important role in the absorption process. This interaction was observed
in the conversion of catechin and epicatechin [116]. Bacterial strains Eggerthella
lenta and Flavonifractor plautii, isolated from human feces, showed the capacity
to convert both compounds in active metabolites. The strain Eg. lenta was able
to cleave the heterocyclic C-ring of both (+)-catechin and ()-epicatechin to produce a
common precursor, 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol,
that was subsequently converted in 5-(30 ,40 -dihydroxyphenyl)-γ-valerolactone and
4-hydroxy-5-(30 ,40 -dihydroxyphenyl) valeric acid by F. plautii strain (Scheme 1). To
produce catechin metabolites, bacterial strains promote cleavage and/or
dehydroxylation of C and A rings of catechins molecules and the B ring is preserved.
Despite the well-known antioxidant capacity induced by polyphenols, several of
these compounds act as redox-dependent poisons against type II enzymes [117].
This poisoning effect is the capacity of enhance DNA cleavage by topoisomerase II
and depends on the number of -OH groups on the B and C-rings. Considering that
many anticancer drugs target the type II enzyme, this feature can represent an
interesting mechanism by which dietary polyphenols or its metabolites act pre-
venting cancer [118].
Two of the most consumed beverages worldwide are coffee and tea, and both
beverages are source or phenolic compounds. These beverages have shown several
beneficial effects on health, in part due to the high content of phenolic substances.
Tea is a rich source of catechins, epicatechin, epigallocatechin, epicatechin gallate,
and epigallocatechin gallate, compounds which can promote diverse effects in
humans. Catechins and its metabolites may be involved in the insulin signaling
pathway, regulation of various transcription factors, and inhibition of pro-oxidant
enzymes such as nitric oxide synthase, lipooxygenase, cyclooxygenase, and xan-
thine oxidase [119]. Similarly to tea, the coffee consumption may induce different
OH OH
OH OH
A C A C
HO O HO O
B B
(+)-Catechin OH OH
OH
(-)-Epicatechin
OH
Eggerthella lenta
Common precursor
Flavonifractor plautii
OH
OH
HO
HO
H
O HO
O
O
OH
5-(3',4'-Dihydroxyphenyl)-γ-valerolactone 4-Hydroxy-5-(3',4'-Dihydroxyphenyl) valeric acid
Scheme 1 Conversion of (+)-catechin and ()-epicatechin by human intestinal bacteria. Both

compounds are converted by Eggerthella lenta in a common precursor, which is further converted
by Flavonifractor plautii to active metabolites that retain the polyphenol B-ring (gray background)
(Adapted from: Kutschera et al. [116])
health benefits, according to the processing method used to obtain the beverage.
Green coffee beans contain large amounts of chlorogenic acids, a family of water-
soluble phenols formed by the esterification of ()-quinic acid with one or more
cinnamic acids. However, with the roasting process of coffee grains, the
chlorogenic acid progressively decreases in relation to light to dark roasting
degree. In addition to effects in phenolic compounds, the roasting process induces
the formation of Maillard reaction products (high molecular weight products).
Some of these high molecular weight compounds (such as melanoidins, which also
have antioxidant capacity) increase in dark roasting compared to light roasting
[120]. It is well known that polyphenols and melanoidins present in foods and
beverages are involved in their antioxidant activity [121]. Therefore, the antiox-
idant capacity of coffee beverages is not only contributed by the components
originally present in green grains, but also by the components generated during
the roasting process.
Coffee is probably the richest dietary source of chlorogenic acid, with a single
dose of espresso coffee supplying 24–423 mg [122]. Nonespresso beverages are also
rich sources, and many regular coffee drinkers can easily consume 1–2 g per day of
this phenolic compound, surpassing even the intake coming from fruit and vegeta-
bles. In addition to chlorogenic acid, other polyphenols, phenolic acids, and
melanoidins have been linked to several health benefits, such as suppression of
postprandial hyperglycemia and hyperinsulinemia (by inhibiting digestive enzymes)
[123] and protection of DNA oxidative damage (by decreasing deoxyribose degra-
dation, DNA scission, and inhibition of 8-hydroxydeoxyguanosine formation) [121].
Probably these effects are associated to direct interactions with membrane structures,
enzymes, and receptors, besides the antioxidant capacity. The oxidative stress and
the resulting oxidative damage are closely related to the development of inflamma-
tion and chronic diseases. In addition to the effects described above, the oxidative
scavenger activity of coffee beverage has been shown in humans. The plasma
antioxidant activity measured on subjects (1 and 2 h after the ingestion of 200 ml
of coffee) exhibited a significantly higher antioxidant capacity than the controls that
did not ingest coffee [124].Further, healthy subjects consuming instant coffee
(800 mL/day, for 5 days), co-extracted from green and roasted grains, also showed
a significant protection against oxidative damage [125]. In these subjects, urinary
marker of lipid peroxidation (8-isoprostaglandin F2α) and plasma marker of func-
tional protein modifications induced by nitric oxide (3-nitrotyrosine) were decreased
by 15.3% and 16.1%, respectively [125].
The main drawback to using polyphenols as agents to prevent or treat diseases
is their poor bioavailability and the variable bioaccessibility in the human body.
Thus, to improve the desired effects in health promotion, consumption of several
polyphenols or the association of polyphenols and drugs has been recommended
[126–128]. In addition, it is necessary to consider that dietary fruits and vegetables
provide other bioactive compounds, beside polyphenols, that help the human body
to prevent chronic diseases as cancer, diabetes, hypertension, obesity, and cardio-
vascular disease [129–132]. An interesting mechanism of anticancer activity induced
by polyphenols was showed in compounds coming from cocoa samples. The
flavanols and procyanidins from cocoa were able to induce nonapoptotic cell death
in cancer cell line Caco-2 [133]. This effect was mainly induced by decreasing ODC
and SAMDC activities, two key enzymes of polyamine biosynthesis. High expres-
sion of ODC is an important characteristic of tumor cells and tumor development;
therefore, the interaction of polyphenols with polyamines biosynthesis are interest-
ing targets for cancer prevention.
The human intestinal tract contains high levels of polyamines, which are derived
from the diet and/or from de novo production by host and its microbiota. Besides
that, humans cells have the ability to ubiquitously produce polyamines as putrescine,
spermidine, and spermine, from amino acid arginine, which is converted in ornithine
by ODC. These endogenous molecules play an essential role in the process of cell
development, growth, and differentiation. However, deregulated levels of poly-
amines at low concentrations are linked to cell growth defects and at high concen-
trations to toxic effects and carcinogenesis. Diets based on foods and beverages rich
in arginine may induce increased synthesis of polyamines. Further, knowing that

arginine is the amino acid precursor of polyamines and nitric oxide, it is expected
that the enzymes arginase 1 and nitric oxide synthase compete for arginine and
promote a bypass in metabolic pathways. The immune response, mainly induced by
M1 macrophages, is dependent of nitric oxide to induce the production of pro-
inflammatory cytokines. Spermine can inhibit M1 macrophage activation by
suppressing the expression of ODC. However, even blocking nitric oxide pathway
and M1 macrophage activation and the synthesis of anti-inflammatory transforming
growth factor-β (TGFβ) and IL-10 were not altered [134].
In addition, polyamines have been associated to multiple effects in the central
nervous system, acting as neurotransmitters or neuromodulators. Injuries in the central
nervous system, such as ischemia and trauma, are associated to increased activities of
ODC and spermidine/spermine-N(1)-acetyltransferase. The increased activity of these
enzymes results in accumulation of putrescine, a candidate marker of brains injury.
Increased amount of putrescine seems to be more associated to brain injury than
spermine and spermidine, once levels of the former two were unaltered in three
different models of forebrain ischemia and traumatic brain injury [135]. On the other
hand, the administration of polyphenols and their supposed interaction with poly-
amines biosynthesis were demonstrated in animal model of ischemia-induced neuronal
injury [136]. Neuronal damages and the polyamines content (in the ischemic tissue)
were decreased after epigallocatechin gallato administration. In this study, authors
speculated that although administration of epigallocatechin gallato did not show
complete neuroprotection, it seems to be a promising strategy for attenuation of global
ischemia-induced neuronal injury [136]. In this same study, the levels of spermine and
spermidine were unaltered in the injured brain tissue, whereas the levels of putrescine
were significantly decreased. It is well known that the spermidine may be synthesized
from the putrescine, so is not unexpected the maintenance in its levels.
The spermidine biosynthesis is mediated SAMDC and aminopropyltransferase
spermidine synthase (SPDS) through the addition of aminopropyl group (donated
by decarboxylated S-adenosylmethionine (dcSAM)) to putrescine molecules
(Scheme 2). The following steps are capable to convert spermidine to spermine
through the spermine synthase (SPMS). When necessary, spermine and spermi-
dine can be recycled to spermidine and putrescine, respectively, by spermidine/
spermine-N1-acetyltransferase (SAT1) followed by oxidation by polyamine oxidase
(PAO) [137]. In addition to dietary polyamines consumed by each individual,
the gut microbiota of these individuals may be responsible for spermidine biosyn-
thesis [138–140]. Several bacterial strains have the capacity to produce spermine via
SAMDC and SPMS enzymes, or the orthologues enzymes carboxynorspermidine
dehydrogenase (CANSpdDH) and carboxynorspermidine decarboxylase
(CANSpdDC) that convert sym-norspermidine in spermidine [138].
The relevance of human microbiota in polyphenols and/or polyamines absorption is
remarkable. In a human study, volunteers that consumed orange juice containing
hesperetin-7-O-rutinoside showed increased concentrations of hesperetin-7- O-glucu-
ronide in the plasma and urine [141]. Probably the disaccharide glycoside moiety of
hesperetin-7-O-rutinoside was cleaved by colonic bacteria releasing hesperetin, which
O
H2N
OH
Arginine
N
H2N NH2
Arginase
H H H O
H H H
H N
O H N H
+
O
H O H N
+
N+ N+ H
H S
O O Ornithine H H H
O sym-norspermidine
S-adenosylmethionine H N
N N (SAM)
N N H (CANSpdDH)
ODC (CANSpdDC)
N AMD1
H H NH2
H2N PAO
Putrescine
NH NH
H H NH2
O + SPDS
S
+
N H SAT1 O N1-Acetylspermidine
H
O H
O NH2
Decarboxylated SAM H2N
N NH
N (dcSAM) PAO
Spermidine
N N NH NH
NH NH2
N SPMS SPMOX O N1-Acetylspermine
SAT1
H H
NH NH2
H2 N NH
Spermine
Scheme 2 Polyamines biosynthetic pathway in human body. Arginine is the precursor amino acid
converted to ornithine by arginase. Putrescine is synthesized through ornithine decarboxylase (ODC)
or S-adenosylmethionine decarboxylase (SAMDC). S- adenosylmethionine (SAM) is decarboxylated
(dcSAM) and the molecule provides aminopropyl groups to produce putrescine. After that, putrescine
is converted in spermidine by spermidine synthase (SPDS) and/or spermine by spermine synthase
(SPMS). Spermine can be recycled to spermidine directly by spermine oxidase (SPMOX). Spermine
and spermidine can be recycled to spermidine and putrescine, respectively, by spermidine/spermine-
N1-acetyltransferase (SAT1) followed by oxidation by polyamine oxidase (PAO). Alternatively, gut
microbiota can synthesize spermidine (blue route) from sym-norspermidine by expressing orthologues
enzymes carboxynorspermidine dehydrogenase (CANSpdDH) and carboxynorspermidine decarbox-
ylase (CANSpdDC) (Adapted from: Brooks [137])
was acted upon by UDP-glucuronosyltransferases in the colonocytes, producing

hesperetin-7-O-glucuronide. In addition, an unidentified hesperetin-O- glucuronide
was found in plasma, showing that these metabolites passed into the portal vein to
reach the blood stream [141]. Hesperetin and its conjugated metabolites are the major
forms of hesperidin in human plasma. It occurs after hesperidin hydrolysis in the
gastrointestinal tract and conjugation during absorption. In several situations, the
metabolites have similar, or even, higher physiological effect than their precursors.
An interesting example of this effect was observed in spontaneously hypertensive rats.
The hesperetin-7-O-β-D-glucuronide exerted hypotensive, vasodilatory, and anti-
inflammatory activities, similarly to hesperetin. On the other hand, the metabolite
hesperetin-30 -O-β-D-glucuronide had little effect on these parameters [142].
The interaction of polyphenols and polyamines, as observed for catechins metab-
olites, was also described for hesperetin and naringerin [143]. Both compounds
produced a remarkable reduction of B16-F10 melanoma cell, in vitro and in vivo,
and the proposed mechanism was by lowering of the intracellular levels of poly-
amine, spermidine and spermine.
9 Concentrations of Phenolic Compounds and Biogenic

Amines in Some Foods
The phenolic compounds and the polyamines are present in variable quantities
in many foods and the knowledge of its content can influence positively or nega-
tively in various diseases. Alterations in the content of these phytochemicals are
verified depending on the food matrix used, on the method of cultivation, and on the
treatment and/or on the processing the raw material is submitted [57, 61, 144].
In addition, following previous considerations, it should be useful to actively
promote the production of functional beverages and foods, having a modified
balance between amines and polyphenols, by using different agricultural manage-
ment practices and processing methods.
10 Conclusion
Regular consumption of phenolic compounds and polyamines rich food has been
shown to improve the human health. There is a need for detailed investigation of the
complexities of effects on the absorption of these compounds, as well as their
metabolism and de novo synthesis in the human organism. The gut microbiota
produces metabolites, different from those that can be generated by human enzymes,
and in most cases, reduces the activity of dietary compounds; however, sometimes
the metabolites formed exhibit enhanced properties.
The gut microbiota and the dietary components are involved in a complex
metabolic metabolism. Interactions between these components are essential for
human responses to food-based interventions and to achieve desired health
improvement.
Acknowledgment The authors are grateful to the National Council for Scientific and Technolog-
ical Development (CNPq, Brazil) (305177/2015-0), Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES) (02441/09-8) and the São Paulo Research Foundation (FAPESP)
(2016/22665-2)
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Intake of Mediterranean Foods
2
Charalampos Siotos, Marco Vinceti, and Androniki Naska
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2 Mediterranean Diet: Definition and Assessment of Adherence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3 Mediterranean Diet and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4 Spatial and Time Differences in Food Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.1 Sources of Dietary Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5 Monitoring the Intake of Mediterranean Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6 Monitoring Adherence to the Overall Mediterranean Dietary Pattern . . . . . . . . . . . . . . . . . . . . . . 43
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Abstract
The traditional Mediterranean diet is characterized by: (a) high consumption of
cereals, vegetables, fruit, nuts, legumes, fish, and seafood; (b) the use of olive oil
as the main, if not the only, added lipid; (c) moderate consumption of milk and dairy
products; (d) moderate intake of alcohol, in the form of wine and preferably during
meals; and (e) low consumption of meat and meat products. The prevalent consump-
tion of olive oil and the low consumption of animal products are reflected in the high
ratio of monounsaturated to saturated fat intake, typical of the dietary pattern in the
region. There is increasing evidence from observational and experimental
C. Siotos · A. Naska (*)

Department of Hygiene, Epidemiology and Medical Statistics, School of Medicine, National and
Kapodistrian University of Athens, Athens, Greece
e-mail: [email protected]; [email protected]
M. Vinceti
Research Center for Environmental, Genetic and Nutritional Epidemiology (CREAGEN),
University of Modena and Reggio Emilia, Modena, Italy
Department of Epidemiology, Boston University School of Public Health, Boston, MA, USA

30 C. Siotos et al.
epidemiological studies, further enriched by the conclusions of their systematic

reviews and meta-analyses, that adherence to the Mediterranean dietary pattern
promotes health and reduces the risk of premature death from chronic degenerative
diseases. Mediterranean countries and especially the European ones have experi-
enced a “westernization” process of their food habits, and have increased the per
capita supply of non-Mediterranean foods (animal fats, vegetable oils other than
olive oil, sugar, and meat) and decreased the supply of legumes and alcoholic
beverages, including wine. The evidence that Mediterraneans are gradually departing
from their traditional eating habits does not only refer to the adult population in the
region, but it has also been reproduced in large-scale nutritional surveys among
children, adolescents, and young adults – the trend-setters for future generations.
Next to the effect on people’s health, the gradual abandoning of the traditional
Mediterranean diet cannot support sustainable development in the way the Mediter-
ranean diet does. Being adjusted to the cultural, climatic, and other environmental
characteristics of the region, the Mediterranean diet is protective and helpful to
biodiversity, accessible and economically affordable, and contributes to food and
nutrition security.
Keywords
Mediterranean diet · Score · Food intake · Health
1 Introduction
The Mediterranean basin has historically been a crossroad of civilizations and this is
reflected in the culture, the scenery, the flora, and the food resources. Some plants, like
the olive tree, wheat, and the grapevine, have been in this area for millennia, whereas
citrus fruit, tomatoes, eggplants, corn, rice, beans, and potatoes were imported at
different time periods. Over the years, this large variety of plant foods has been
integrated in people’s culinary habits paving the way to what has become in the
twentieth century the worldwide famous Mediterranean diet [1].
The countries bordering the Mediterranean Sea have their own dietary traditions, and
olive oil occupies a central position in all. Without the presumption of a scientific
definition, the Mediterranean diet could be considered as the dietary pattern found in
the olive oil growing areas of the Mediterranean region in the late 1950s and early 1960s
[2]. Traditionally, in addition to olive oil, the daily intake included a high intake of
cereals, fruit, nuts, vegetables, and legumes combined with a low intake of dairy
products, meat, and meat products. In the traditional Mediterranean diet, fish and seafood
intake depended on the vicinity to the sea and ethanol intake was moderate and mainly in
the form of wine during meals [3]. This combination of intakes is rich in components
with well-established cardioprotective functions: the liberal use of olive oil guarantees an
increased intake of monounsaturated fatty acids and tocopherols; fish, vegetables, and
nuts are good sources of n-3 and n-6 polyunsaturated fatty acids; the high consumption
of vegetables, fresh fruit, and cereals provide fiber, a variety of vitamins (such as vitamin
C, vitamin E, and beta-carotene), important minerals (potassium, for instance), and other
beneficial substances (such as polyphenols and anthocyanins) [4, 5]. The limited
2 Intake of Mediterranean Foods 31
consumption of meat and dairies provided few saturated fatty acids and the consumption
of locally grown products, such as the wild green leafy vegetables, conveys additional
benefits due to their high flavonoid content [6].
Contrary to other also highly cited dietary regimes, the Mediterranean dietary
pattern has not been developed by health professionals on the basis of analytical
evidence for health-promoting dietary choices. It is rather a natural experiment, a
holistic lifestyle that existed in the region for years and was discovered in the second
half of the twentieth century after ecological observations that people in this region
experienced mortality rates which were far lower than those of more developed and
affluent countries [7]. The first systematic attempt to investigate dietary patterns in
the Mediterranean region dates back to 1948 on the island of Crete. At that time, the
Greek government was worried about the post-war conditions and invited the
Rockefeller Foundation to undertake an epidemiological study in order to provide
advice on how to raise the population’s standard of living. The study assembled data
collected from 128 Cretan households. The final report was published in 1953 and
provides a succinct but vivid description of people’s diet. In particular, the investi-
gators wrote: “olives, cereal grains, legumes, fruits, wild greens and herbs, together
with limited quantities of goat meat and milk, game and fish, consist the basic Cretan
food. . .no meal was complete without bread. Olives and olive oil contributed heavily
to the energy intake. Food seemed literally to be “swimming” in oil. . .” and
concluded that the food consumption levels “. . .were surprisingly good. On the
whole, their food patterns and food habits were extremely well adapted to their
natural and economic resources, as well as their needs” [8].
2 Mediterranean Diet: Definition and Assessment of

Adherence
Overall, the traditional Mediterranean diet has the following characteristics:
• High consumption of cereals, vegetables, fruit, nuts, and legumes

• High consumption of fish and seafood
• Use of olive oil as the main, if not the only, added lipid
• Moderate consumption of milk and dairy products
• Low consumption of meat and meat products
• Moderate intake of alcohol, in the form of wine
The traditional Mediterranean diet has predominantly been a plant-based diet

with olive oil as the principal added lipid, allowing a high intake of monounsaturated
and a low intake of saturated fatty acids – the latter contributed 7% to 8% of total
energy intake [2].
The operationalization of the Mediterranean dietary pattern has been achieved
through various computational scores, which are used to reflect the aggregated dietary
exposure particularly when diet-disease associations are evaluated. The first and more
often cited index to assess adherence to the Mediterranean diet among adults and elderly
is the Mediterranean diet score calculated on the basis of the aforementioned
32 C. Siotos et al.
characteristics of the Mediterranean diet [9]. According to this score, individuals receive
a value of þ1 if their daily intake of foods frequently included in the traditional
Mediterranean dietary pattern (i.e., vegetables, legumes, fruit and nuts, cereals, fish
and seafood, and monounsaturated to saturated fat ratio to reflect the high olive oil
intake) is above the gender-specific median value of the study sample. Correspondingly,
individuals are assigned a value of 0 if their daily intake of the aforementioned foods is
below the gender-specific median. Hence, an individual receives þ1 value if his daily
intake of vegetables is above the median vegetable consumption in this study sample.
Similarly, the individual will receive an additional þ1 value if his intake of fruit and nut
is also above the sample median. However, if the individual’s daily intake of fish and
seafood is below the sample median, he will receive the value of 0. In addition,
individuals receive values of þ1 if their daily intake of milk and dairies, meat and
meat products is equal to or lower than the gender-specific median and are assigned
values of 0 otherwise. Thus, an individual with daily meat intake below the sample
median will receive an additional þ1 value, but if his daily intake of milk and dairies is
above the sample median, he will receive a 0 value. Lastly, a value of þ1 is further given
to individuals consuming a moderate amount of alcohol (i.e., between 5 and 25 g per
day for women and between 10 and 50 g per day for men) and a value of 0 otherwise.
Finally, all values received are added and the total score ranges between 0 (minimal
adherence to the traditional Mediterranean diet) and 9 (maximal adherence to the
traditional Mediterranean diet) (Fig. 1).
Variations of this score, as well as other indices to assess conformity to Mediter-
ranean dietary traditions are also available in the literature [10–15]. Although these
employ various scoring techniques, they are all based on the dietary characteristics
described above, which capture the essence of the Mediterranean dietary profile.
Among children and adolescents, adherence to the Mediterranean diet is usually
assessed through the KIDMED index [16], which comprises of 16 questions combining
principles of a Mediterranean dietary pattern (i.e., eating fruit, vegetables, and legumes
regularly; using olive oil at home), together with general dietary guidelines for children
(for instance, always to have breakfast). Respondents receive positive scores if they
conform to principles of the Mediterranean diet and follow nutritional guidelines and
negative scores otherwise. The total score of the KIDMED index for children and
adolescents is usually classified into three levels: equal or higher than 8 indicates an
optimal adherence to Mediterranean diet; scores between 4 and 7 denote a necessity for
improvement in order to adjust intake to the Mediterranean diet; and scores below or
equal to 3 point to a diet of very low quality. Next to the KIDMED index, other scores
have also been proposed in the literature to assess adherence to the Mediterranean diet
among children and adolescents [17].
3 Mediterranean Diet and Health
The ecological observation that adults living in the Mediterranean area have displayed
favorable health statistics induced the classic Seven Countries Study which has
prospectively investigated long-term incidence and mortality from coronary heart
Med diet score = 0
For 1 to 6
1 Vegetables No Yes
2 Legumes Intake > median
3 Fruit and Nuts
4 Cereals and
products
5 Fish and score = score score = score+1
seafood
6 Mono -
unsaturated to
saturated fat
ratio
For 7 and 8
No Yes
Intake < median
7 Meat and
products
score = score score = score+1
8 Milk and dairies
No Yes
T1<Value<T
9 Alcohol (g/day)
Male: T1=10, T2=50
score = score score = score+1
Female: T1=5, T2=25
Med diet score
Fig. 1 Schematic presentation for the estimation of the Mediterranean diet score (Med diet
score) [9]
disease in cohorts of men aged 40–59 years in seven countries, including three
Mediterranean ones: Italy, Greece, and Croatia [18]. The baseline survey was carried
out between 1958 and 1964 and information on diet was collected through food diaries
in random subsamples of the 16 study cohorts. The study primarily provided evidence
34 C. Siotos et al.
for a strong positive association between the average saturated fat intake and overall
mortality or mortality from coronary heart disease [19]. Although the Seven Countries
Study mainly contributed to the understanding of the importance of the type of fat
consumed on coronary heart disease risk, it certainly assisted in putting the whole diet
of populations around the Mediterranean into the limelight. Indeed, the health indices
observed in the region could not be explained by differences in education levels,
financial status, or health care systems, simply because in this area in the 1960s,
socioeconomic indicators were much lower than those in more developed areas of the
world. Therefore, attention has focused on the overall diet of Mediterranean
populations through a holistic approach, as one of the key explanatory factors [19].
The interest of the scientific community in the traditional diet of populations in
the Mediterranean region and its health promoting effects thrived after mid 1990s
and the publication of a prospective study conducted among elderly Greeks residing
in rural areas [20]. The researchers recorded the usual dietary intakes of about 180
males and females aged 70 years and over and observed that higher adherence to
Mediterranean diet (as assessed by the score described above) was significantly
associated with a reduced risk of death from any cause by 17% per one unit increase
in the score (95%CI: 1–31%). Authors additionally evaluated the association of each
one of the components of the Mediterranean diet with overall mortality and noted
that the individual components of the diet score had weak and generally nonsignif-
icant associations with survival, whereas the overall score had a substantial and
statistically significant effect.
In several case-control and prospective cohort studies, as well as experimental
studies, mostly conducted among Mediterranean populations in Europe, the Medi-
terranean diet has been associated with a reduced incidence of coronary heart
disease, stroke, and cancer, and with beneficial effects on cardiovascular disease-
related markers [21–23]. Dietary intakes following the principles of Mediterranean
diet have also been associated with self-perceived mental and physical health, global
cognitive function, as well as an overall higher quality of life [24–26]. The consistent
evidence from large-scale observational studies for its protective effects lead a group
of investigators in Spain to the undertaking of the “Prevención con Dieta
Mediterránea” (PREDIMED) study, a large nutritional trial assessing the long-term
effects of Mediterranean diet on the primary prevention of cardiovascular disease.
Men (55–80 years of age) and women (60–80 years of age) with no cardiovascular
disease at enrolment were eligible for inclusion in the study if they had either type II
diabetes mellitus or at least three of the following risk factors: smoking, hyperten-
sion, elevated low-density lipoprotein cholesterol levels, low high-density lipopro-
tein cholesterol levels, overweight or obesity, or a family history of premature
coronary heart disease. About 7500 individuals at high cardiovascular risk were
randomized to follow: (a) a Mediterranean diet supplemented with freely provided
extra virgin olive oil, (b) a Mediterranean diet supplemented with mixed nuts again
provided to participants at no cost, or (c) the control diet (advice to reduce all dietary
fats). The main findings of the trial showed that a Mediterranean diet supplemented
with extra virgin olive oil reduced the incidence of major cardiovascular events
by approximately 30% [hazard ratio (HR) 0.70; 95% CI: 0.54, 0.92) and by 28%
[HR 0.72; 95% CI: 0.54, 0.96] when supplemented with nuts, in comparison to the
control diet [27]. Although PREDIMED was planned as a 6-year trial, it was stopped
after a median follow-up time of 4.8 years (interquartile range, 2.8–5.8 years) on the
basis of early statistically significant results on the beneficial effect of following a
Mediterranean diet on premature mortality. Subsequent analyses identified further
benefits on blood pressure, insulin sensitivity, lipid profiles, lipoprotein particles,
inflammation, oxidative stress, and carotid atherosclerosis [4, 28], providing strong
experimental support for the advantages of consuming a Mediterranean diet.
The bulk of publications around the health-promoting effects of the Mediterra-
nean diet have been systematically reviewed through several meta-analyses. In a
review of prospective cohort studies that investigated possible associations of
adherence to the Mediterranean diet and overall or disease-specific mortality, Sofi
et al. [29] observed that a two-point increase of adherence to the Mediterranean diet
was associated with a significant protection against premature death and the inci-
dence of cardiovascular and other chronic degenerative diseases. These findings
were further confirmed in 2010 in a meta-analysis which considered the studies of
the earlier analysis, together with seven prospective studies published in 2009 and
2010 (one study for overall mortality; three studies for cardiovascular incidence and
mortality; one study for cancer incidence and mortality; and two studies for neuro-
degenerative diseases) [21]. The meta-analysis included 50,000 incident cases or
deaths from any cause and/or cause-specific. Authors observed that a two-point
increase in adherence to the Mediterranean diet was associated with a significant
reduction of overall mortality (pooled relative risk (RR): 0.92; 95% CI: 0.90, 0.94,
cardiovascular incidence and mortality (pooled RR: 0.90; 95% CI: 0.87, 0.93),
cancer incidence and mortality (pooled RR: 0.94; 95% CI: 0.92, 0.96), and occur-
rence of neurodegenerative disease (pooled RR: 0.87; 95% CI: 0.81, 0.94). There
was no evidence for publication bias in the included results [21].
In their systematic review of randomized controlled trials assessing the effect of
diets with unrestricted fat intake and following the principles of the Mediterranean
diet to CVD risk compared to any control diet, Liyanage et al. [30] retrieved six
studies among adults with follow-up periods longer than 3 months. The studies
reported effects on major vascular events (n = 477), death from any cause
(n = 693), and vascular deaths (n = 315). When data from all studies were
combined, a Mediterranean-type diet protected against major vascular events
(pooled RR: 0.63, 95% CI: 0.53–0.75), coronary events (pooled RR: 0.65, 95%
CI: 0.50–0.85), stroke (pooled RR: 0.65, 95% CI: 0.48–0.88), and heart failure
(pooled RR: 0.30, 95% CI: 0.17–0.56), but not for all-cause mortality (pooled RR:
1.00, 95% CI: 0.86–1.15) or cardiovascular mortality (pooled RR: 0.90, 95% CI:
0.72–1.11). Schwingshackl and Hoffmann [31] reviewed observational (case-con-
trol and cohort) studies to assess the effect of adherence to Mediterranean Diet on
overall cancer mortality, incidence of different types of cancer, as well as fatality
among cancer survivors. An overall population of 1,784,404 individuals from 56
studies was included in this analysis. The highest score of adherence to Mediter-
ranean diet was associated with a lower risk of death from cancer at any site
(pooled RR: 0.87, 95% CI 0.81–0.93), colorectal cancer (pooled RR: 0.83, 95% CI
36 C. Siotos et al.
0.76–0.89), breast cancer (pooled RR: 0.93, 95% CI 0.87–0.99), gastric cancer
(pooled RR: 0.73, 95% CI 0.55–0.97), prostate cancer (pooled RR: 0.96, 95% CI
0.92–1.00), liver cancer (pooled RR: 0.58, 95% CI 0.46–0.73), head and neck
cancer (pooled RR: 0.40, 95% CI 0.24–0.66), pancreatic cancer (pooled RR: 0.48,
95% CI 0.35–0.66), and respiratory cancer (pooled RR: 0.10, 95% CI 0.01–0.70).
Based on a small number of studies among cancer survivors, there was no
association between the highest adherence to Mediterranean diet and risk of
death (three cohort studies), or cancer recurrence (one cohort study). In particular,
the pooled RR for death from cancer among patients was 1.01 when individuals
with highest adherence to Mediterranean diet were compared to those with lower
adherence (95% CI: 0.81–1.26).
In order to summarize the evidence and evaluate the validity of the association
between adherence to the Mediterranean diet and multiple health outcomes reported
by several systematic reviews, Dinu et al. [32] reviewed evidence across meta-analyses
of observational studies and randomized clinical trials. Thirteen meta-analyses of
observational studies and 16 meta-analyses of randomized trials investigating the
association between adherence to Mediterranean diet and 37 different health outcomes
were analyzed. The evidence for a beneficial effect of Mediterranean diet was robust and
supported by a strong statistically significant finding, large simple sizes, and not
considerable heterogeneity among studies for overall mortality, cardiovascular diseases,
overall cancer incidence, neurodegenerative diseases, and diabetes. For most of the site-
specific cancers, as well as for inflammatory and metabolic parameters, the evidence
was only suggestive or weak. No evidence was reported for bladder, endometrial and
ovarian cancers, as well as for low density lipoprotein-cholesterol levels.
Furthermore, investigations among individuals residing in non-Mediterranean
regions such as Denmark and the Netherlands in Europe, Melbourne in Australia,
China, and the US provided additional evidence that adherence to the principles of
the Mediterranean diet affects the survival of elderly people [33–37]. In these studies,
an increase in a diet score derived from the key features of the traditional Mediter-
ranean diet was associated with a significant reduction in overall and cause-specific
mortality. The evidence that the beneficial effect of adherence to Mediterranean diet
on the risk of premature death prevails also among individuals in areas distant to the
Mediterranean basin rejected the possibility of an association confounded by nondiet-
related factors, as well as the probability of effect modifiers shaping the health-
promoting effects of the Mediterranean diet. Thus, one could safely argue that it is
not the climatic, social, and cultural conditions that shaped this association, that the
evidence for an independent effect of diet on overall survival is reinforced.
The findings of epidemiological studies are additionally supported by accumulat-
ing evidence from laboratory studies, which point to the converging effects of
Mediterranean diet and olive oil (particularly the virgin or extra virgin olive oil
that has not undergone excessive process) on the homeostatic control of genes
implicated in cardiovascular risk, such as lipid metabolism, vessel protection, and
blood pressure control, immune-inflammatory pathways, metabolic regulation, and
detoxification of reactive species [5]. The impressive growth of the omic technologies
provides new insight in the molecular and metabolic effects of functional components
of olive oil and other Mediterranean foods. Transcriptomic fingerprints revealed

molecular targets in the area of primary prevention and clinical management of
cardiovascular disease, as well as other degenerative age-related disorders [38].
4 Spatial and Time Differences in Food Intake
4.1 Sources of Dietary Data
In spite of the catholic recognition of Mediterranean diet as a health-conducive pattern,

several populations in the region seem to be gradually abandoning it. Data to monitor
food consumption over time and among different regions can be derived from three
main sources: (a) the Food and Agriculture Organization assembled Food Balance
Sheets (FBS), (b) the Household Budget Surveys (HBS), and (c) Nutrition Surveys,
the latter collecting detailed, quantitative data on the usual food and beverage intake of
a large – and often representative – sample of the target population.
The FBS provide information on food quantities assumed to be available for
human consumption in the country, estimated on the basis of the annual food
production, imports and exports, changes in stocks, agricultural and industrial uses
within a country, as well as losses during storage and transportation (http://www.fao.
org/faostat/en/#data/FBS). The data refer to the early stages of the food chain and the
per capita supply is obtained by dividing the respective food quantity by the
population partaking of it. The total population estimates, however, refer as a rule
to resident population only, while nonresident population, such as tourists, illegal
immigrants, refugees, etc., are generally not included. This omission may therefore
result in an underestimation of the total partaker population and an overestimation of
the various per-person food supplies. The accuracy of FBS data are further depen-
dent on the reliability of the underlying basic statistics, which vary in terms of
coverage and accuracy. Although import and export data are generally accurate, in
some cases there may be some trade across national boundaries that goes
unrecorded. Data on own food production are collected in some countries, but this
information can be substantially under-recorded where there is a thriving economy
in home production. Waste and food given to pets may also be sources of error.
Hence, given the nature of the FBS and their inherent limitations, the data are
interpreted to provide information on annual per capita supply of foods to the
population.
Household budget surveys are systematically conducted by National Statistical
Offices and aim at collecting, among others, data on food availability at household
level. Household budget surveys provide information on products available for
consumption to a nationally representative sample. The members of the participating
households are asked to record information on all foods and beverages available in the
household during a reference period, including purchases, contributions from own
production, and food items offered to members as gifts. The survey is implemented
over a period of a year, with due attention to capture seasonal variation in food intake.
Information on the demographic and socioeconomic characteristics of the household
38 C. Siotos et al.
members is also recorded, allowing analyses on the effect of socioeconomic determi-

nants on food choices (http://www.hhf-greece.gr/DafnesoftWebV2/). The HBS are not
primarily designed to collect nutritional information and the food data bear limitations,
which need to be considered when they are used for the purpose of nutritional
surveillance. In most cases, no records are collected on the type and quantity of
food items and beverages consumed outside the home (at restaurants, canteens, and
similar establishments, for example); food losses and waste, foods given to pets, as
well as meals offered to guests are not consistently collected; and gender- and age-
specific estimations of food consumption require the application of statistical model-
ing [39]. The data collected though the HBS provide information on the daily
availability of foods and beverages to a nationally representative sample of each
country’s households.
The individual-based nutrition surveys constitute the optimal method for
assessing dietary patterns, quantifying determinants of food choices, and evaluating
diet-disease associations. Being expensive and labor intensive, however, represen-
tative individual-based surveys are undertaken regularly in only a limited number of
countries, usually those with robust economies and years of experience in this field.
Furthermore, the implementation of various protocols and data collection methods
limits the potential to undertake comparisons between countries.
Notwithstanding their caveats, countries with no routine information on the food
consumption of large samples of their population, as well as those interested in
comparing their national dietary patterns over time or with those of other
populations, have traditionally used the FBS or the HBS data [40, 41]. They both
provide a resource for conducting comparative nutritional analyses and could help
highlighting issues such as (a) the dietary patterns prevailing in different countries
and their sociodemographic determinants; (b) time trends in the food habits of
populations; and (c) the evaluation of nutrition action plans, interventions, and
related strategies implemented at national or international level [42].
5 Monitoring the Intake of Mediterranean Foods
Making use of FBS data referring to the periods between 1961 and 1963 and
between 1998 and 2000, Balanza et al. [43] calculated changes in total energy,
energy provided from macronutrients, and specific food groups for three European
regions: Southern or Mediterranean (including Spain, Portugal, Italy, Greece,
France, Cyprus, and Albania), Northern (combining the UK, Sweden, Norway,
Finland, Germany, Ireland, Denmark, and Iceland), and Eastern Europe (comprising
the Czech Republic, Slovakia, Poland, Bulgaria, Hungary, and Romania). Over the
study period, the supply of total energy and energy from lipids increased consider-
ably in all European areas, while the percentage of energy from carbohydrates
decreased. The greatest changes have occurred in Mediterranean Europe where,
contrary to traditional eating habits, a significant reduction in the energy supplied
by cereals (30%) and wine (55%) and an increase in the supply of milk (78%) and
dairy products (24%) was noted. Overall, such changes led to an increase of 20% in
the total supply of calories, comprising an increase of 48% in calories from lipids,
mostly of animal origin and a fall of 21% in calories from carbohydrates.
In another comparison of FBS data referring to the periods 1961–1965 and
2000–2004 and covering Mediterranean countries in Europe, Africa and the Mid-
dle-East, as well as countries in other regions (North and Central Europe, North and
South America, Asia, and Australia), Vareiro et al. [41] confirmed previous findings
that the greatest changes in food supplies took place in Mediterranean Europe, with
an increase in the per capita supply of non-Mediterranean foods (animal fats,
vegetable oils other than olive oil, sugar, and meat) and a decrease in the supply of
legumes and alcoholic beverages, including wine. However, although gradually
abandoning some of its typical characteristics, the Mediterranean countries of
Europe still recorded higher per capita supplies of olive oil, vegetables, fruit, and
fish than other areas. Interestingly, during the same periods, northern European
countries tended to increase the consumption of Mediterranean foods such as olive
oil and fruit.
According to an analysis of HBS data collected in the 1980s and early 1990s
including nationally representative samples of households from several European
countries [44], in Greece and Spain the mean daily population intake exceeded the
WHO recommendations of at least 400 g of combined fruit and vegetable intake per
day. However, the correlation between the proportion of low fruit and low vegetable
consumers was unexpectedly weak (Spearman’s correlation coefficient: þ0.18),
suggesting differential preferences toward the consumption of fruit and vegetables
among the surveyed populations. Moreover, more than 50% of the households in the
surveyed populations were likely to consume less than the recommended daily
vegetable intake of three portions, and this applied even to the two Mediterranean
populations included in this analysis. In particular, 56% of the households in Greece
and 76% of the households in Spain failed to report three portions of vegetables per
day.
Using FBS data collected from 1961 up to 2013 (http://www.fao.org/faostat/en/
#data/FBS), Tables 1 and 2 present the annual supplies as well as the average change
in the annual supply of food groups, components of the Mediterranean diet. Table 1
presents the supply of foods of livestock production and milk (animal foods);
vegetables, fruit, cereals and cereal products, beans, peas, and other legumes (plant
foods); and all types of fish, crustaceans, cephalopods, and mollusks (fish and
seafood) in 15 countries surrounding the Mediterranean basin. Table 2 presents
data for the supply of olive oil and wine. In general, annual supplies reflect local
production and food preferences shaped by cultural norms. For instance, the supply
of olive oil is highest in the three main olive oil producing countries (Greece, Italy,
and Spain), while the supply of wine is low in countries with a high percentage of
Muslims in the population (Algeria, Egypt, and Turkey). In the 50-year period
between 1961 and 2013, all Mediterranean countries recorded an increase in the
supply of animal foods, fish, and seafood. The supply of plant foods increased in the
majority of countries; a decrease between 1961 and 2013 was however recorded in
Cyprus (approximately 80 kg/capita) and in Spain (about 65 kg/capita) and to a
lesser extent in Israel (7 kg/capita). On average, all countries recorded a small – and
40
Table 1 Supply of animal and plant foods, fish and seafood (kg/capita/year) in Mediterranean countries in 1961 and 2003 and average changes per year
Animal foods Plant foods Fish and seafood
1961 2013 Average annual 1961 2013 Average annual 1961 2013 Average annual
changea change changea
Albania 112.7 365.65 4.86 325.76 541.75 4.15 1.92 4.54 0.05
Algeria 56.19 162.24 2.04 194.64 505.87 5.99 3.31 3.91 0.01
Bosnia and 120.52b 206.17 4.08 335.8b 491.74 7.43 0.16b 4.25 0.19
Herzegovina
Croatia 197.12b 296.45 4.73 213.76b 310.86 4.62 2.6b 18.22 0.74
Cyprus 91.51 186.98 1.84 340.16 256.94 1.60 5.73 20.78 0.29
Egypt 40.35 88.37 0.92 305.46 554.01 4.78 3.74 22.05 0.35
Greece 121.12 327.88 3.98 422.77 486.04 1.22 16.21 18.34 0.04
Israel 185.19 288.74 1.99 454.88 447.73 0.14 18.83 22.98 0.08
Italy 175.63 325.29 2.88 415.76 432.41 0.32 11.56 20.58 0.17
Lebanon 87.17 154.14 1.29 419.02 440.34 0.41 3.95 10.26 0.12
Malta 175.63 271.11 1.84 300.25 435.2 2.60 10.7 27.79 0.33
Montenegro 412.63c 433.7 3.01 476.9‡ 513.12 5.17 7.44c 11.2 0.54
Spain 103.59 256.15 2.93 367.51 302.61 1.25 24.87 35.28 0.20
Tunisia 54.6 143.84 1.72 300.09 555.45 4.91 4.09 13.54 0.18
Turkey 196.01 229.05 0.64 503.27 589.44 1.66 2.4 5.7 0.06
Source: Food Balance Sheet data available at http://www.fao.org/faostat/en/#data/FBS (accessed on September 2017)
Animal foods: All types of meat and milk
Plant foods: Vegetables, Fruit, Cereals and their products, Beas, Peas and other Pulses
Fish and seafood: All types of fish, Crustaceans, Cephalopods, and Mollusks
a
Estimated as: (Supply in 2013) – (Supply in 1961)/number of years in between
b
1992
c
2006
C. Siotos et al.
Table 2 Supply of olive oil and wine (g/capita/year) in Mediterranean countries in 1961 and 2003
and average changes per year
Olive oil Wine
1961 2013 Average 1961 2013 Average
annual changea annual changea
Albania 1,110 570 10.4 1,660 7,970 121.3
Algeria 870 1,730 16.5 20 10 0.2
Bosnia and 0b 220 10.5 5,100b 1,880 153.3
Herzegovina
Croatia 610b 710 4.8 40,410b 13,800 1,267.1
Cyprus 2390 2,320 1.3 12,150 15,700 68.3
Egypt 40 80 0.8 70 50 0.4
Greece 14,550 13,550 19.2 33,710 18,690 288.8
Israel 110 2,150 39.2 4,460 860 69.2
Italy 9,120 9,960 16.2 109,590 30,550 1,520.0
Lebanon 2,840 3,180 6.5 2,110 2,700 11.3
Malta 90 1170 20.8 930 15,370 277.7
Montenegro 600c 510 12.9 17,980c 20,770 398.6
Spain 8,200 10,930 52.5 59,210 20,980 735.2
Tunisia 6,720 3,150 68.7 2,490 2,680 3.7
Turkey 3,800 1,280 48.5 960 390 11.0
Source: Food Balance Sheet data available at http://www.fao.org/faostat/en/#data/FBS (accessed on
September 2017)
a
Estimated as: (Supply in 2013) – (Supply in 1961)/number of years in between
b
1992
c
2006
in many cases negligible – increase in the supply of fish and seafood. Regarding olive
oil, some countries recorded remarkable reductions in the per capita supply between
1961 and 2013. The per capita annual supply of olive oil reduced by 3.6 kg in Tunisia,
2.5 kg in Turkey, and 1 kg in Greece. The changes in the per capita annual supply of
wine were more variable – some countries recorded an increase (such as Malta and
Montenegro), while others recorded a substantial decrease (Italy and Croatia).
Yearly trends in food supply are graphically presented in Figs. 2–6 for six
countries (Algeria, Greece, Italy, Spain, Tunisia, and Turkey) selected on the basis
of geographical coverage and observations from Tables 1 and 2. In the years between
1961 (when the Mediterranean diet was registered through the Seven Countries
Study) and 2013 (the latest year available in the database), the daily supply of animal
foods (livestock products and milk) was steadily increasing (Fig. 2). A slight
decrease in the daily supply of animal foods was recorded in Italy in the period
between 1983 and 1993, but in 2013, per capita supplies were higher than the ones in
early 1960s. The pattern of changes over time is more variable in the case of foods of
plant origin (Fig. 3). Overall, the daily supply increased in Algeria and Tunisia,
remained rather stable in Italy and Greece and since late 1980s decreased in Spain.
The consistency in the daily supply of fish and seafood present in Table 1 is also
42 C. Siotos et al.
450
400
350
300
250
200
150
100
50
Algeria Greece Italy Spain Tunisia Turkey
Fig. 2 Trends over time in the supply of animal foods (kg/capita/year) in Mediterranean countries
750
650
550
450
350
250
150
1961
1963
1965
1967
1969
1971
1973
1975
1977
1979
1981
1983
1985
1987
1989
1991
1993
1995
1997
1999
2001
2003
2005
2007
2009
2011
2013
Fig. 3 Trends over time in the supply of plant foods (kg/capita/year) in Mediterranean countries
evident through Fig. 4, where changes per year are presented. Fish intake in Spain is
popular, since not only the daily supply constantly outweighs that of the other
Mediterranean countries, but it increased steadily leading to 10 kg/capita higher
daily supply in 2013 in comparison to 1961. Greece has been leading the daily
supply of olive oil since the early 1960s (Fig. 5), followed only by Italy and Spain
after the late 1970s. However, the daily supply of olive oil has been constantly
decreasing in Greece since mid-1970s, and although it remains the highest, the
difference with the other Mediterranean countries in Europe is narrowing. Over
time, changes in the daily supply of wine are presented in Fig. 6. Values are very low
among countries with large percentage of Muslims in their population and they
probably reflect the consumption of wine by tourists visiting these countries. Dif-
ferences over the years were not remarkable in Greece and Spain, while in Italy the
50
45
40
35
30
25
20
15
10
5
0
Fig. 4 Trends over time in the supply of fish and other seafood (kg/capita/year) in Mediterranean
countries
25
20
15
10
Fig. 5 Trends over time in the supply of olive oil (kg/capita/year) in Mediterranean countries
supply of wine continuously decreased since mid-1970s and is now closer to that
recorded in other European Mediterranean countries.
6 Monitoring Adherence to the Overall Mediterranean

Dietary Pattern
The ecological evidence that Mediterranean populations are gradually departing

from their traditional eating habits has been reproduced in large-scale nutritional
surveys among children, adolescents, and adults [45, 46]. In a report presenting
harmonized data for nutrition and health indices in Europe based on studies with
44 C. Siotos et al.
140
120
100
80
60
40
20
Fig. 6 Trends over time in the supply of wine (kg/capita/year) in Mediterranean countries
wide population coverage, individuals in the Mediterranean region reported higher

intakes of red meat and saturated fatty acids and lower intakes of plant products in
comparison to their European counterparts [47]. Publications presenting changes
over time in the dietary habits of populations participating in the Seven Countries
Study further support that the Mediterraneans are abandoning their traditional eating
choices. In early 1990s, Kafatos and colleagues conducted a follow-up study of 245
surviving men in Crete aged 70–89 years, who had participated in the Seven
Countries Study in the 1960s [48]. A representative subsample of 21 among them
further provided 3-day weighed food records, which indicated increases in the intake
of saturated and decreases in monounsaturated fatty acids [48]. De Lorenzo and
colleagues [49] studied differences in food habits in Nicotera, one of the south Italian
rural areas of the Seven Countries Study, between 1960 and 1996. In the second
assessment, 80 inhabitants of Nicotera (43 males, 37 females, aged 40–59 years)
with no indication of chronic disease and not under regular medication or on a
special diet were selected at random. Based on dietary data collected through a food
frequency questionnaire, authors estimated the participants’ Mediterranean Ade-
quacy Index and noted that the mean index decreased from 9.4 for males and 11.4
for females in 1960 to 2.8 and 2.5, respectively, in 1996. Regarding specific food
items, authors observed an increase in the consumption of animal foods, cakes, pies,
cookies, and sweet beverages among both men and women. Not disregarding
methodological characteristics of the 1996 study that may limit direct comparisons
with the 1960 data, results indicate a significant reduction to the extent through
which the population adhered to the Mediterranean diet. Similar results were also
observed in Crevalcore (northern Italy) and Montegiorgio (central Italy), the other
two rural Italian cohorts participating to the Seven Countries Study [49], as well as to
other studies among adult Mediterranean populations [45].
Lower adherence to the traditional Mediterranean diet has also been consistently
reported among children, adolescents, and young adults, the trend-setters of dietary
habits and lifestyle choices of future generations. In their review of studies assessing the
adherence to Mediterranean diet among children and adolescents, Iaccarino Idelson et al.
[46] analyzed the results of studies which applied the KIDMED index and were mainly
conducted in European Mediterranean countries (49 out of the 55 eligible studies).
Adherence varied widely both within and between Mediterranean populations. Based on
studies covering large samples, poor adherence to Mediterranean diet varied from 1.6%
in Spanish children to 62.8% in Greek adolescents; average adherence from 28.0% in
Greek adolescents to 73.8% in Italian adolescents residing in rural areas; and good
adherence from 4.3% in Greek 10–12 year olds to 53.9% in Spanish children.
According to most of the studies, adherence to Mediterranean diet was directly associ-
ated with physical activity (and possibly with diet adequacy) and inversely with
sedentary behavior. Gender, age, the family’s socioeconomic status, and the children’s
body weight were possible determinants of the level of adherence.
Low consumption of plant foods and a prevalent intake of commercial sweet and
savory cereal products have been the main components explaining poor to average
adherence. In their review of the diet of preschool children in the Mediterranean
countries of the European Union, Pereira-da-Silva et al. [50] reported that in the
majority of countries, young children consumed frequently fruit and vegetables, but
also sugared beverages and snacks. The majority of children reported high energy
and high protein intakes mainly from dairy products, together with excessive sodium
intake. The consumption of energy-dense foods and overweight were recorded as
early as in toddler and preschool ages. Furthermore, most children reported low
adherence to the Mediterranean diet, which was associated with being overweight or
obese, lower maternal educational level, and parental unemployment.
In a study among 1740 Italian children 8–9 years old who were recruited from
regions all over the country, a low consumption of fruit, vegetables, and legumes,
and a high intake of commercially baked goods for breakfast and sweets were
identified as contributors to the low adherence. About one-third of the sample
reported that hindrances to the consumption of fruit, vegetables, and legumes were
affecting their ability to follow a Mediterranean diet. In this study, only 5% of the
children were identified as high adherers based on the KIDMED index [51].
In the cross-sectional analysis of the IDEFICS study, Tognon and colleagues [17]
assessed adherence to the Mediterranean diet of 16,220 children aged 2–9 years old
from eight European countries (Sweden, Germany, Hungary, Italy, Cyprus, Spain,
Belgium, and Estonia), using a Mediterranean Diet Score based on the relative fre-
quency of food group consumption. One point was given for intakes higher than the
median relative frequency for (a) potatoes, vegetables, and legumes; (2) fruits and nuts;
(3) cereals; or (4) fish; and one point if intakes were below the median for: (5) dairy and
(6) meat products. The final score added up to a maximum of 6 points and a score equal
or higher to 3 was interpreted as high adherence to a Mediterranean-like dietary pattern.
In this study, the largest proportion of children with high intake frequencies of cereals,
vegetables, potatoes, fruits, and nuts was recorded in Sweden. More than half of the
Swedish children (56.7%) reported high adherence to components of the Mediterranean
diet, followed by the Italians (37.5%) and the Germans (35.1%). The lowest levels were
observed in Cyprus, where 75.8% of children had score values below 3 [17]. The
46 C. Siotos et al.
highest prevalence of children with a score higher than 3, which could be interpreted as
average to high adherence, was recorded among the Italian preschool boys (55.9%) and
the lowest among the Spanish school-aged girls (26.0%). Apparently, higher adherence
to a Mediterranean-like dietary pattern was generally not associated with living in a
Mediterranean country or in a highly educated or high-income family. Differences in
adherence between boys and girls or age groups varied between countries without any
general pattern. It should be noted, however, that the results on the level of adherence
also varied according to the index used: for example, the proportion of children with
high adherence ranged from 24.2% in Cyprus to 56.7% in Sweden using the score
described above, and from 29.4% in Germany to 49.3% in Italy using a different score,
formulated on the basis of an adaptation of the Mediterranean diet score [46].
In the multi-center IDEFICS study, different food groups contributed to high
adherence levels in each country. Hence, among Italian children, the low adherence
could be attributed to a low consumption of vegetables and legumes (prevalence of
high consumers: 36.2%), while in Sweden high adherence was achieved through a
low consumption of dairy and meat products (prevalence of high consumers of
dairies: 24.6%; and prevalence of high consumers of meat: 30.9%). The consump-
tion of olive oil and vegetable oils is still more prevalent among children in the
Mediterranean region than in other European countries – the prevalence of children
above the median for the unsaturated to saturated fatty acid ratio was equal to 72.6%
in Italy and to 58.7% in Cyprus. The children in Spain have consistently been
identified among the highest consumers of fish and other seafood.
Some alarming findings have been reported from studies conducted in Greece and
Cyprus. In a representative sample of 554 Greek children and 358 adolescents,
Kontogianni and colleagues assessed adherence to the Mediterranean diet using the
KIDMED index [52]. Only 11.3% of children and 8.3% of adolescents had an optimal
score (8). Among children, KIDMED scores did not differ between boys (mean SD:
5.4 1.9) and girls (5.4 1.7), but in adolescents, the index tended to be higher among
females (4.9 2.1) than among males (4.6 2.0) ( p-value: 0.07). In a study of 1140
Cypriot children aged 9–13 years old using the KIDMED score too, only 8.7% of the
boys and 5.3% of the girls were classified as high adherers (score 8) and 38.5% of
boys and 36.3% of girls had a KIDMED score ranging from 0 to 3 [53]. In the context of
the GRECO study, including a representative sample of Greek children aged
10–12 years old, Farajian et al. [54] reported that only 4.3% of the children had an
optimal KIDMED score (8) and about half of the children (46.8%) were classified as
poor adherers (3). The score values did not differ between boys and girls, normal
weight, overweight, or obese children. Children with higher KIDMED scores resided in
semiurban or rural regions and reported higher physical activity levels.
7 Conclusion
The Mediterranean diet is characterized by a high intake of plant foods (fruit,

vegetables, cereals, nuts, legumes) and fish, together with a low intake of products
of animal origin (meat, milk and diaries). Olive oil is the most frequent – if not the
only – lipid added to food, and alcohol intake is moderate and mostly in the form of
wine, preferably during meals. There is increasing evidence from observational and
experimental epidemiological studies that adherence to the Mediterranean dietary
pattern promotes health and reduces the risk of premature death from chronic
degenerative diseases. These conclusions are further supported by large systematic
reviews and meta-analyses. Nonetheless, Mediterranean countries and especially the
European ones have experienced a “westernization” process of their food habits, and
have increasingly been exhibiting patterns of food choices similar to non-Mediter-
ranean ones. In a period when other western countries experience a nutrition
transition that favors the Mediterranean dietary pattern [47], traditional food choices
in Italy, Greece, and other Mediterranean regions are abandoned. This could be
attributed to the globalization of the food supply, the overall improvement in
socioeconomic conditions in Europe that made food (especially of animal origin)
more affordable, and the urbanization of life, which primarily affects the younger
generations.
The westernized diets – particularly followed by the youth in the region – have
been associated with higher disease and mortality rates in North Europe and USA
[55, 56]. Unfavorable changes in disease rates may eventually appear and may
lead to an increase of the demand and the cost of health services, an issue of
concern in era of limited resources both at the individual and governmental levels.
Moreover, these food choices cannot support sustainable development, in the way
the Mediterranean diet does. Being adjusted to the cultural, climatic, and other
environmental characteristics of the region, the Mediterranean diet is protective
and helpful to biodiversity; accessible and economically affordable; can lead to a
cut down of country’s expenses for food imports; and contributes to food and
nutrition security, as well as to the health of present and future generations [57, 58].
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Flavonoids – Food Sources, Health Benefits,
and Mechanisms Involved 3
Aleksandra Kozłowska and Dorota Szostak-Węgierek
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2 Flavonoids: Classification, Sources, and Dietary Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3 Flavonoids Action and Current Research and Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1 Antioxidant and Anti-Inflammatory Action of Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.2 Cardioprotecive Action of Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.3 Antidiabetic Action of Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.4 Anticancer Action of Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.5 Flavonoids and the Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.6 Flavonoids: Promising Natural Compounds Against Erectile Dysfunction . . . . . . . . . . . 68
3.7 Flavonoids: Promising Natural Compounds Against Cataract . . . . . . . . . . . . . . . . . . . . . . . . 69
3.8 Flavonoids: Promising Natural Compounds Against Viral Infections . . . . . . . . . . . . . . . . 69
3.9 Flavonoids and Their Therapeutic Benefits in Inflammatory Bowel Disease . . . . . . . . . 71
4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Abstract
In recent years, growing attention has been focused on the use of natural sources
of antioxidants in the prevention of chronic diseases. Flavonoids are the examples
of such substances. It is a group of bioactive compounds that are widely distrib-
uted in many plant-based foods and beverages. Flavonoid-rich products include,
A. Kozłowska (*)
First Faculty of Medicine, Department of Social Medicine and Public Health, Medical University of
Warsaw, Warsaw, Poland
D. Szostak-Węgierek
Faculty of Health Science, Department of Clinical Dietetics, Medical University of Warsaw,
Warsaw, Poland

54 A. Kozłowska and D. Szostak-Węgierek
among others, berries, citrus fruits, grapes, cherries, dock, arugula, onions,
artichokes, soybeans, cowpeas, black beans, parsley, oregano, and tea. Flavo-
noids exhibit a wide range of positive effects, such as strong antioxidant, anti-
inflammatory, and antiplatelet activities. They may contribute to the prevention of
chronic diseases, including metabolic disorders, diabetes, and cardiovascular
disease, because of their beneficial effect on blood lipids, blood pressure, plasma
glucose levels, and also stabilization of athetosclerotic plaque. Furthermore,
evidence from epidemiological, animal, and in vitro studies support protective
effects of foods and dietary supplements rich in flavonoids against some types of
cancer, Alzheimer’s disease, Parkinson’s disease, some viral infections, cataract,
erectile dysfunction, and inflammatory bowel disease. Consumption of flavo-
noids with diet appears to be safe. There is a growing body of evidence that a diet
rich in these substances is beneficial for health and its promotion is thus
justifiable.
Keywords
Flavonoids · Antioxidants · Bioactive compounds · Chronic diseases ·
Prevention · Neurodegenerative diseases · Cataract · Erectile dysfunction ·
Inflammatory bowel disease · Type 2 diabetes
1 Introduction
This chapter provides the current state of knowledge about the role of flavonoids in
prevention and treatment of chronic diseases (CD). It is a comprehensive review of
literature on both flavonoids as the whole group and also flavonoid subclasses. We
believe that the presented data will be useful for both researchers and practitioners in
medicine and dietetics.
The history of research on flavonoids goes back several decades. In 1930, a new
substance was isolated from lemon juice by Hungarian Nobel laureate Albert Szent-
Gyorgyi, who also discovered vitamin C. At that time, it was believed to be a
member of a new class of vitamins and was referred to as vitamin P. Later, it became
clear that this substance was a flavonoid (rutin) and till now at least 6000 different
types of flavonoids have been identified [1, 2].
Flavonoids are secondary metabolites of higher plants and represent a large
class of phenolic compounds found in fruits, vegetables, herbs, cocoa, and some
beverage products [3]. They have several important functions in plants, such as
providing protection against harmful UV radiation or plant pigmentation. In
addition, they regulate gene expression and modulate enzymatic action. They
also have antiviral and antibacterial properties, as well as high antioxidant capac-
ity. These compounds are primarily synthesized for the plants’ own defense
against oxidative stress. However, they maintain antioxidant properties also ex
planta and hence contribute to the pharmaceutical and dietary attributes of plant
foods. Therefore, antioxidant properties of flavonoids are all-important for both
plant and human biology [4, 5].
3 Flavonoids – Food Sources, Health Benefits, and Mechanisms Involved 55
2 Flavonoids: Classification, Sources, and Dietary Intake
Flavonoids constitute a class of polyphenols. All naturally occurring flavonoids

possess three hydroxyl groups, two of which are on the ring A at positions five
and seven and one is located on the ring B, position three. They can be subdivided
into different subgroups depending on the number of the carbon atom of the C ring to
which the B ring is attached and also the degree of unsaturation and oxidation of the
C ring. Flavonoids in which the B ring is linked in position 3 of the C ring are called
isoflavones. Those in which the B ring is linked in position 2 can be further
subdivided into several subgroups on the basis of the structural features of the C
ring. These following subgroups are: flavones, flavonols, flavanones, flavanols or
catechins, and anthocyanins. Some authors have additionally distinguished
flavanonols, chalcone, and neoflavonoids subclasses [6]. Table 1 presents the basic
structure of flavonoid and examples of specific substances in each flavonoid
subclass.
Important dietary sources of flavonoids are vegetables, fruits, seeds, and some
cereals, together with wine, tea, and certain spices (Table 2). The most abundant
sources are berries, cowpeas, dock, kale, dark chocolate, parsley, oregano, capres,
green and black tea. It is important to note that the presence of flavonoids in
vegetables and fruits may greatly vary depending on the crop variety, processing,
climate, seasonality, plant species, manufacture, and storage. Differences in flavo-
noid contents between plant species are usually moderate, although in a few cases
very high amounts have been observed, e.g., in certain berries and tea prepared from
leaves of the Quingmao tree [7].
Table 1 Basic structure of flavonoid and subclasses of flavonoids (Authors’ selection based on
Refs. [5–8])
Basic structure of flavonoid
Subclass Examples of compounds

Anthocyanins Pelargonidin, cyanidin, delphinidin, peonidin, petunidin, malvidin
Flavanols Epicatechin, catechin, epicatechin gallate (ECG), gallocatechin,
epigallocatechin (EGC), epigallocatechin gallate (EGCG)
Flavanones Naringenin, naringin, hesperetin, eriodicytol
Flavones Sinensetin, isosinensetin, nobiletin, tangeretin, luteolin, apigenin, chrysin,
galangin
Flavonols Kaempferol, quercetin, fisetin, isorhamnetin, myricetin
Isoflavones Daidzein, genistein, daidzin
Table 2 Content of flavonoids in chosen foodstuffs (mg/100 g foodstuff) (Authors’ selection based
on Ref. [7])
Range of flavonoids’
content Products (flavonoids mg/100 g)
>1500 mg/100 g Parsley, dried (4854.49), oregano, Mexican, dried (1545.79)
300 mg–1500 mg/ Elderberries (518.13), Capres, raw (493.03), chokeberry (368.66)
100 g
100 mg–300 mg/ Cowpeas (277.41), parsley, fresh (233.16), currants, black (167.47),
100 g blueberries (158.51), blackberries (137.66), green tea, brewed (121.27),
black tea, brewed (119.32), cranberries (113.58), dark chocolate
(108.60), cocoa, dry powder (106.68), dock (102.20)
70 mg–100 mg/100 g Kale (92.98), fennel (84.50), currants, red (79.49), kumquats (79.26),
black currant juice (78.04) white tea, brewed (74.60)
40 mg–70 mg/100 g Arugula (69.27), cabbage, red (64.34), peppermint, fresh (60.48),
grapefruit (55.40), lemons (53.38), oolong tea, brewed (52.37), limes
(48.60), grapes, red (48.35), thyme, fresh (47.75), raspberries (47.58),
onions, red (44.87), oranges (43.49), wine, table red (40.84), cherries
(40.00)
10 mg–40 mg/100 g Strawberries (34.31), pecans (34.01) beans, black (28.00), radishes
(26.52), wheat, purple (25.85), Orange juice (24.13), celery hearts, green
(22.60), artichokes (22.20), chives (21.67), pears (21.53), peppers, hot
chili (21.17), broadbeans, cooked (20.63), lettuce, red (19.40), pink
grapefruit juice (17.97), buckwheat (15.38), asparagus, cooked (15.16),
milk chocolate (15.04), pistachio (14.37), plums (14.23), cress, fresh
(14.00), apple (13.73), bananas (13.69), hazelnuts (11.96), chicory,
green (11.79), spinach (11.44), almonds (11.00), beans, kidney, red
(10.87), apricots (10.67), chard (10.43), endive (10.10)
<10 mg/100 g Sorghum, red (8.43), Brussels sprouts, cooked (7.68), peppers, green
(6.98), beer (3.34)
Extent of biological effects of flavonoids depend not only on their content in

food, but also on bioavailability that is influenced by the source type (food or
pharmaceutical formulation), consumers’ characteristics, such as gender and age,
their interindividual variations, including physiological and molecular factors,
composition and activity of gastrointestinal microflora, and also differences in
mechanisms of absorption and biotransformation of different compounds [2]. The
main food sources of substances from particular subgroups are shown in Table 3.
There are several databases of flavonoids contents in foods which can be used to
assess their consumption with human diet. Most studies estimate flavonoids intake using
food composition tables such as the USDA databases and the online Phenol-Explorer
database (www.phenol-explorer.eu). However, these tables might be of limited use
because only a restricted number of foods have been analysed for their polyphenol
content using different analytical techniques. For this reason, the results of nutritional
assessments may vary depending on the sort of database used. It should be emphasized
that many surveys performed in 1990s could underestimate the actual intake of flavo-
noids as the databases of flavonoid content of foodstuffs were incomplete at that time. For
Table 3 Content of flavonoid subclasess in chosen foodstuffs (mg/100 g foodstuff) (Authors’

selection based on Ref. [7])
Anthocyanins (mg/100 g)
Elderberry juice 411.40 Blackberries 90.64 Pecan nuts 25.02
concentrate
Chokeberry 349.79 Red currants 75.02 Red table wine 23.18
Bilberries 285.21 Red cabbage 63.50 Black grapes 21.63
Chickpeas 262.49 Raspberries 40.63 Pears 12.18
Black currants 154.77 Red bilberries 40.15 Morello cherries 7.45
American bilberries 141.03 Strawberries 27.76 Hazel nuts 6.71
Flavanols (mg/100 g)
Green tea, brewed 116.15 Blackberries 42.50 Apples 9.17
Black tea, brewed 115.57 Cooked broad 20.63 Peaches 8.60
beans
Dark chocolate 108.60 Pecan nuts 15.99 Apricots 8.41
Cocoa, dry powder 52.73 Red table wine 11.05 Apple juice 5.96
Flavanones (mg/100 g)
Dried Mexican oregano 412.13 Limes 46.40 Grapefruit juice 18.98
Grapefruit 54.50 Oranges 42.57 Artichokes 12.51
Lemons 49.81 Orange juice 18.99
Flavones (mg/100 g)
Dried parsley 4523.25 Artichokes 9.69 Chicory 2.85
Dried oregano 1046.46 Green pepper 4.71 Lemons 1.90
Fresh parsley 216.15 Celeriac 3.90 Red grapes 1.30
Flavonols (mg/100 g)
Fresh capers 493.03 Goji berries 31.20 Chicory 8.94
Dried parsley 331.24 Fresh 21.59 Buckwheat 7.09
cranberries
Elderberry juice 108.16 Cooked 15.16 Dried and 6.91
concentrate asparagus sweetened
cranberries
Sorrel 102.20 Blackcurrants 11.53 Fresh figs 5.47
Rocket lettuce 69.27 American 10.59 Cooked brussel 5.24
bilberries sprouts
Red onions 38.34 Morello 9.41 Apples 3.40
cherries
Isoflavones (mg/100 g)
Soy flour 166.66 Soybeans, raw 48.95 Clover, red 21.0
Soybeans mature seeds 103.56 Miso 41.45 Soybeans, cooked 17.92
(Europe)
Natto 82.29 Soy yoghurt 33.17 Sufu 13.75
Tempeh 60.61 Tofu, different 28.91 Pistachio nuts 3.63
types
proper interpretation of the results of assessment of flavonoids intake with diet, a kind and
year of release of used database should be considered.
Recent evidence suggests that the total daily intake of flavonoids varies among
nations and cultures. Habitual dietary intake of flavonoids is variable and in USA,
estimated daily intake was on average 200.1 8.9 mg/d, while in Europe, mean
intakes among European adults ranged from 506 mg/d (the Central region) to
348 mg/d (the Northern region) and the 301 mg/d (the Southern region) [9, 10].
The mean total flavonoid intake in the Polish population was equal to 403.5 mg/day,
when calculated with the use of the Phenol-Explorer database, and 525 mg/day,
when the USDA databases were used [11]. These results contrast with our own
results that demonstrated that the average daily flavonoid consumption among Polish
students was equal to 801 mg [12].
3 Flavonoids Action and Current Research and Trends
It seems that a diet rich in flavonoids may be beneficial for human health [3].
Emerging evidence from epidemiological and randomized controlled trials support
protective effects of foods and dietary supplements rich in flavonoids against chronic
diseases, including coronary heart disease, type 2 diabetes mellitus, cancer,
Alzheimer’s disease, Parkinson’s disease (PD), cataract, erectile dysfunction, and
inflammatory bowel disease. In this subsection, we summarized results of epidemi-
ological and animal studies, human clinical trials, and also in vitro studies on both
flavonoids and flavonoids subclasses, that showed the main effects and mechanisms
of flavonoid action.
3.1 Antioxidant and Anti-Inflammatory Action of Flavonoids
Flavonoids possess many biochemical properties, but the best investigated effect of
almost all subclasses of flavonoids is their antioxidants activity. Almost every group
of flavonoids has a capability to act as antioxidants. It was reported that flavones and
catechins seem to be the most powerful flavonoids for protecting the body against
reactive oxygen species [13, 14].
Antioxidant activity of flavonoids depends on their functional hydroxyl groups
that can mediate antioxidant effects by scavenging free radicals and/or by chelating
metal ions. Mechanisms of antioxidant action include suppression of reactive oxy-
gen species (ROS) formation, either by inhibition of enzymes, or by chelating trace
elements involved in free radical generation, scavenging ROS, and upregulation or
protection of antioxidant defenses [2]. The chelation of metals seems to be crucial in
the prevention of radical generation which damage target biomolecules. Some
flavonoids are capable of inhibiting the enzymes involved in ROS generation, for
example, microsomal monooxygenase, glutathione S-transferase, mitochondrial
succinoxidase, NADH oxidase [15]. Antioxidant action of flavonoids may also
result from activation of antioxidant enzymes, such as catalase, glutathione perox-

idase, and heme oxygenase-1 (HO-1), which have radical scavenging ability.
Flavonoids also may exhibit also anti-inflammatory action what can be mediated
by inhibition of both the activity and production of various proinflammatory sub-
stances and enzymes, such as tumor necrosis factor α (TNF-α), cyclooxygenase
(COX), and lipoxygenase (LOX) [16]. Moreover, flavonoids may play a role in
resolution of inflammation also by inhibition of activation of nuclear factor kappa-
light-chain-enhancer of activated B cells (NF-κB), which is responsible for control
of transcription of DNA, cytokine production, and cell survival, and also by sup-
pression of prostaglandin E2 (PGE2), which impairs T cell receptor signaling [17].
3.2 Cardioprotecive Action of Flavonoids
The well-recognised antioxidant properties of flavonoids resulted in the interest in

their potential role in prevention of cardiovascular disease. Interestingly, the direct
mechanisms are not well understood. It seems that the action of flavonoids is
multifaceted and depends on parallel processes. Among the main mechanisms,
there are: antioxidant and anti-inflammatory activities, regulation of blood pressure,
decreases in cholesterol levels, protection of LDL against oxidative modification,
and antiplatelet effects.
Lipid peroxidation is a common consequence of oxidative stress. High blood
concentration of oxidatively modified low-density lipoproteins (ox-LDL) is one of
the most important factors wich accelerates development of atherosclerosis. Flavo-
noids protect lipids against oxidative damage by various mechanisms. Because of
their antioxidant and chelating properties, flavonoids inactivate reactive oxygen
species (ROS) and this way counteract plasma LDL oxidation and ameliorate
inflammation of the blood vessel endothelium [18].
Antiarteriosclerotic action of flavonoids is related also to the reduction of inflam-
mation in the blood vessel wall through inhibition of the influx of leukocytes. It has
been reported that some flavonoids exert also antiplatelet aggregation effects through
various mechanisms, among which the inhibition of the arachidonic acid-based
pathway seems to be the most representative [19]. They decrease activity of enzymes
that participate in the formation of prostaglandins leukotrienes and thromboxane A2,
substances that mediate inflammation and aggregation, from arachidonic acid. This
results in decrease in inflammation and platelet aggregation. Inhibition of these
enzymes results also in protection of LDL against oxidation and regulates capillary
pressure back to normal [20].
Beyond of protection of blood vessels against ox-LDL, antiatheromatous action
of flavonoids results also from suppression of 3-hydroxy-3-methylglutaryl-coen-
zyme A reductase (hHMGR) activity. This enzyme plays a key role in the synthesis
of cholesterol in the human body and thereby influences its plasma levels. Inhibition
of its activity lowers intracellular cholesterol concentrations and results in the
following increase in expression of LDL receptors. This in turn raises the cellular
lipoprotein uptake and removal of cholesterol from the circulation. Based on the
criteria of inhibition of hHMGR activity, many cholesterol-lowering drugs, such as

statins, have been developed, and their efficacies in controlling blood cholesterol
levels have been well recognized [20].
Anthocyanins, found in berries, blackcurrants, red grapes, plums, and cherries, are
a good example of a flavonoids which reduce blood cholesterol level in patietns with
high CVD risk. In a 6-month study, low-density lipoprotein cholesterol (LDL-C)
concentrations were reduced following anthocyanins supplementation (320 mg/d)
in 122 hypercholesterolemic patients, while the similar result was observed in another
study performed among 58 diabetic patients who received purified anthocyanin
supplementation (320 mg/d). This intervention significantly decreased serum LDL
cholesterol (by 7.9%; P < 0.05), triglycerides (by 23.0%; P < 0.01), and increased
HDL cholesterol (by 19.4%; P < 0.05) compared with placebo after the 24 weeks
[21, 22]. Another randomized, double-blind, placebo-controlled clinical trial
(80 hyperlipidemic patients) showed that 2 months supplementation with extract
from fruit rich in anthocyanins, whortleberry fruit (350 mg capsule every 8 h) reduced
serum levels of total cholesterol, triglyceride, and LDL-C by 27.6%, 19.2%,
and 26.3%, respectively and increased that of HDL-C by 37.5% [23]. In turn, a 8-
weeks dose-response study (50 g freeze-dried blueberries, which is egual to approx-
imately 350 g fresh blueberries), performed in 48 participants with metabolic syn-
drome, demonstrated beneficial effects on plasma oxidized LDL (ox-LDL)
and combined serum malondialdehyde (MDA) and hydroxynonenal (HNE)
concentrations, which are biomarkers of lipid and lipoprotein oxidation and have
also been associated with coronary artery disease. The decreases in ox-LDL and
combined MDA and HNE were greater in the blueberry supplemented group (28
and 27%, respectively) than in controls (9 and 9%) (P = 0.009 and P = 0.005)
(Basu et al. 2010).
It is also important to emphasize the remarkable role of anthocyanins in reduction
of biomarkers of CVD risk. Results of four out of five analyzed recent studies
suggested that increased habitual anthocyanins intake is significantly associated
with a reduction in risk of coronary heart disease (CHD) by 12–32% in multivariate
analyses [24–28].
Catechins, next to anthocyanins, play an important role in amelioration of
cardiovascular disease risk factors. Catechins are the generic terms of flavanols, as
well as the most important tea flavonoids. It was found that the intake of catechins at
a rate of 576 mg per day for 24 weeks can significantly decrease body fat mass
among 40 obese or near-obese children. Catechins could regulate the RNA and
protein expression of fatty acid metabolism enzymes in the liver. They may affect the
activity of metabolic enzymes, which can improve the oxidation of fatty acids and
the inhibit fatty acid synthesis. This specific action of catechins may result in
reduction of lipid levels in the blood and liver, as well as may decrease body fat
deposition, thus reducing early onset of cardiovascular disease in children [29].
Quercetin, a flavonoid from the flavonol subclass, may be also effective as a
cardioprotective agent. It was shown that its anti-inflammatory, antioxidant, and
antiapoptotic effects can effectively protect against myocardial damage in rats.
Compared with the control group, mRNA and protein levels of TNF-alpha and
IL-1beta and also malondialdehyde (MDA) content in myocardial tissue of rats in

both the low-dose (100 mg quercetin/kg/daily) and high-dose (400 mg quercetin/kg/
daily) groups decreased significantly. Antioxidant enzymes, such as superoxide
dismutase (SOD) and catalase (CAT) activities increased significantly. The cell
apoptosis index was significantly reduced [30]. In turn, human intervention studies
demonstrated positive effects of 6 week treatment with quercetin-rich onion skin
extract (162 mg/d quercetin) on blood pressure and endothelial function in 70
overweight-to-obese patients with (pre-) hypertension [31]. The mechanisms respon-
sible for the BP-lowering effect remain unclear. The putative pathways include
improvement of vascular function in an endothelium-dependent or endothelium-
independent manner, decrease in oxidative stress, and/or interference with the
renin–angiotensin–aldosterone system. Importantly, flavonoids can improve endo-
thelial function by stimulating the nitric oxide (NO) bioavailability, a key regulator
of vascular function, leading to improvements in vascular tone and blood pressure
regulation [31, 32].
However, while many studies demonstrated significant inverse associations
between flavonoid intake and CVD incidence, the Framingham Offspring Cohort
failed to support such observation [28]. The results showed that there was no
significant association between flavonoid intake and CVD incidence after multivar-
iable adjustment. This finding, based on a long-term follow-up of middle-aged and
elderly Americans, suggests that the relationship between flavonoid intake and CVD
risk is not clear and requires further investigation.
Data from intervention studies suggest that there is a wide interindividual vari-
ability in flavonoid metabolism as 15–99% of their ingested dose was recovered as a
wide range of flavonoid metabolites. It implies that flavonoids’ metabolism may play
a critical role in explaining the differential responses in CVD risk biomarkers
observed in clinical trials (responders vs nonresponders) [33–35].
This diversity in responsiveness to flavonoids intake may relate to a number of
factors, where the microbiome is likely to be critical and it plays a key role in
flavonoids metabolism. Furthermore, it is likely that intake alters the composition
and function of the gut microbiome and conversely, that the microbiota may enhance
the metabolism of flavonoids, but this bidirectional relationship has not been studied
enough yet [34]. To understand the importance of metabolism, particularly micro-
biome-mediated biotransformation, in explaining the CV health effects of flavo-
noids, a combination of epidemiological studies and dietary intervention trials are
needed.
3.3 Antidiabetic Action of Flavonoids
Flavonoids may exert beneficial effects in diabetes by various, parallel intracellular

signaling pathways in pancreatic β-cells, liver, adipose tissue, and skeletal muscles.
These compounds are able to influence β-cell mass and function, as well as energy
metabolism and insulin sensitivity in peripheral tissues. Flavonoids can increase
glucose uptake through translocation of GLUT4 vesicles to the cell membrane. They
may also enhance glucose uptake in response to insulin through the stimulation of
adenosine monophosphate-activated protein kinase (AMPK) and other kinases such
as ERK1/2 and p38 mitogen-activated protein kinase (p38MAPK) [36].
Epidemiological studies and meta-analyses suggested an inverse relationship
between the consumption of flavonoid-rich diets and development of diabetes.
A recent study evaluated the relationship between dietary intake of total flavonoid
intake and type 2 diabetes (T2DM). This analysis, consisting of six prospective
cohort studies including 18,146 cases and 284,806 participants, reported that the
highest intake of total flavonoids could reduce risk of diabetes by 9%, when
compared with the lowest. Moreover, an increase in the total flavonoids intake of
500 mg/d was associated with a significant risk reduction of 5% [37]. Furthermore,
in another epidemiological study, it was shown that high consumption of flavonoids
from flavanones subclass was also associated with a reduced risk of diabetes. After
multivariable adjustment, the authors observed a 31% reduction in new-onset dia-
betes in the highest compared with the lowest tertile of flavanones intake in near
6 years of follow-up (18,900 person-years) [38]. Population-based studies have also
suggested that flavonoids-rich foods, such as soy (isoflavones, genistein), tea
(flavanols), citrus fruit (flavanones), and blueberry (anthocyans), exerted beneficial
effects on blood pressure, lipid metabolism, insulin resistance, and glucose uptake,
which are related to the development of T2DM [39–42].
Numerous in vitro and animal studies also support a beneficial effect of dietary
flavonoids on glucose homeostasis. The supplementation of a dietary apple/kale
extract (AKE), which is rich in flavonoids, significantly improved both blood
glucose levels and oral glucose tolerance test (OGTT) in mice. Furthermore, in
situ uptake of glucose was significantly inhibited by AKE. Authors concluded that
AKE exhibits antidiabetic properties by a dual mechanism, including the inhibition
of α-glucosidase and sodium-dependent glucose transporter 1 (SGLT1) [43]. Some
studies support the concept that naringenin is a potent insulin sensitizer in vivo.
Fibroblast growth factor 21 (FGF21) regulates energy homeostasis and the metabolic
adaptation to fasting. After naringenin supplementation of diet of both genotypes of
wild-type mice, FGF21-positive and FGF21-negative, improvement of glucose
tolerance was observed [44]. Moreover, in streptozotocin-induced diabetic rats fed
high-fat diet, naringenin improved postprandial hyperglycemia. Oral intake of
naringenin (25 mg/kg body mass) exerted significant inhibition of intestinal α-
glucosidase activity in vivo and thereby delayed the absorption of carbohydrates
in T2DM rats, thus resulting in significant lowering of postprandial blood glucose
levels [45].
In a study performed in women (n = 1997), it was shown that the highest intakes
of both flavones and anthocyanins, when compared with the lowest, were linked to
reduced risk of diabetes. These flavonoids consumption was also inversely associ-
ated with biomarkers of inflammation, insulin concentration, and also value of
HOMA-IR (homeostasis model assessment of insulin resistance) index. Participants
with high intakes of flavones had lower HOMA-IR and insulin concentrations
and higher adiponectin concentrations, with respective differences of 0.1
(P-trend = 0.04), 0.5 μU/mL (P-trend = 0.02), and 0.6 μg/L (P-trend = 0.01)
comparing extreme quintiles of intake [46].Thus, the described results suggest that
total flavonoids, their particular subclasses, and specific substances have the poten-
tial to serve as natural plant bioactive compounds for dietary prevention strategies
against T2DM.
3.4 Anticancer Action of Flavonoids
Flavonoid compounds display a remarkable spectrum of biological activities, includ-

ing those that may affect such stages of carcinogenesis as initiation, promotion, and
progression. In the initiation and promotion stages, they include: inactivation of
carcinogens, inhibition of cell proliferation, enhancement of DNA repair processes,
and reduction of oxidative stress. In the progression phase, flavonoids may induce
apoptosis, inhibit angiogenesis, exhibit antioxidant activity, and also induce cyto-
toxic or cytostatic action against cancer cells [47].
Preventing carcinogen metabolic activation is one of the most important mech-
anism by which flavonoids can exert their chemoprevention effects. Flavonoids may
interact with phase I metabolizing enzymes (e.g., cytochrome P450), which meta-
bolically activate a large number of procarcinogens. Flavonoids inhibit certain
cytochrome P450 isozymes, such as CYP1A1 and CYP1A2, and thus protect against
cellular damage by carcinogens. Another important mechanism of action is possi-
bility induction of phase II metabolizing antioxidant enzymes such as glutathione
S-transferase (GST) and UDP-glucuronyltransferase (UDP-GT).
Stimulation of apoptosis is the next important mechanism by which flavonoids
may be recognized as anticancer substances. Dysregulation of apoptosis plays a
critical role in oncogenesis. Flavonoids were shown to induce apoptosis in some
cancer cell lines, while sparing normal cells. It was recently reported that flavonoids
possess antiproliferative and apoptosis-inducing activities in several cancer human
cell lines, i.e., gastric, colon, breast, lung liver, head, and neck cancers [48–55]. The
molecular mechanisms by which flavonoids induce apoptosis have not been
clarified.
The anticancer effects of flavonoids may be also explained by the cell cycle
inhibition. Cyclin-dependent kinases (CDKs) are recognized as key regulators of cell
cycle progression. Various types of cancers exhibit hyperactivation of CDKs due to
mutation of CDK genes or CDK inhibitor genes. These disturbances may be
constrained by flavonoids [47]. Moreover, such flavonoid as quercetin stimulates
the inhibition of HMGB1-induced TNF-α and IL-1β mRNA expression, which
suggests that quercetin modulates cell signaling that in turn regulates the action of
proinflammatory cytokines. The HMGB1 protein, which is present outside the cell,
can increase the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β),
and other inflammatory mediators from monocytes (Khan et al. 2016). All of the
described mechanisms help to explain the chemopreventive effects of flavonoids
against carcinogenesis.
Increasing evidence from both epidemiological and laboratory studies suggests
that the high dietary intake of flavonoids reduces the risk of developing certain types
of cancers. It concerns, among others, the breast cancer. In general, the prevalence of
the breast cancer is lower in Asian than in North American and European countries
[56]. It may be explained by higher intake of soy in Asian populations, that is an
abundant source of isoflavones. A meta-analysis of 22 observational studies showed
that high isoflavone intake was linked to reduced breast cancer risk rather in Asian
than in Western populations. The protective effect of isoflavones in Asian subjects
concerned both pre- and postmenopausal women [57]. On the contrary, a population-
based prospective cohort study performed in Japan (15,607 women) showed a
beneficial effect only in postmenopausal females [58]. These results suggest that
soy and isoflavone intakes may have a protective effect mainly against postmeno-
pausal breast cancer in Asian populations, but their beneficial action is possible also
in premenopausal women.
High consumption of total flavonoids was also reported to be associated with a
significant reduction of ovarian cancer risk by 18% [59], as well as of the risk of
smoking-related cancers, such as aerodigestive tract cancer (33% risk reduction) and
lung cancer (16% risk reduction) [60]. Guo et al. demonstrated that higher green tea
consumption (above 7 cups/day) was linearly linked to reduced prostate cancer risk.
It was concluded that green tea catechins were effective for preventing prostate
cancer [61].
The epidemiological data presented above concern only prevention and the
results of the studies are not always unambiguous and apply only to some types of
tumors. The present review of literature suggests that results may be promising but
are inconclusive. Further, prospective cohorts assessing dietary flavonoid intake and
studies using other methods to evaluate exposure (i.e., markers of consumption,
metabolism, excretion, bioavailability, drug–drug interactions) are needed to over-
come the limitations emphasized in observational studies testing dietary intakes and
determine consumption levels required to achieve health benefits [62, 63].
Results of laboratory studies which consider flavonoids as anticancer agents are
very promising. Recently, epigallocatechin-3-gallate (EGCG) – a major flavonoid
constituent in green tea – has received much attention as a potential cancer chemo-
preventive agent with DNA topoisomerase I (Topo I) inhibitory activity [64]. Topo I
is a crucial enzyme that works to relax supercoiled DNA during replication, tran-
scription, and mitosis. In a number of human solid tumors, the intracellular level of
Topo I is higher than that in normal tissues. It seems that controlling the Topo I level
is essential in treating cancers and a large number of Topo-directed agents (e.g.,
camptothecin, topotecan, and irinotecan) are currently in clinical use. Topo I inhib-
itors exert their antitumor activities by stabilizing the cleavable Topo I–DNA ternary
complex, blocking rejoining of the DNA breaks, and inhibiting enzyme binding to
DNA. It should be emphasised that EGCG possessed low cytotoxicity with much
higher half maximal inhibitory concentration (IC50) to human tumor cell lines than
the traditional Topo-directed agents [64].
Another interesting substance is kaempferol, flavonoid from flavonols subclass. It
was recently reported that this flavonoid possesses antiproliferative and apoptosis-
inducing activities in several cancer cell lines, i.e., human cervical cancer cells (SiHa
and Hela), human osteosarcoma cells, cholangiocarcinoma cells, human breast
carcinoma (MCF-7) cells, human bladder cancer cells (5637, T24), human stomach
carcinomacells (SGC-7901), and human lung carcinoma cells (A549) [48–54]. It
was also found that kaempferol may play an important role as a potential therapeutic
agent for cancer metastasis [54]. The antimetastatic action of kaempferol is related to
inhibition of matrix metalloproteinases (MMPs) that are regarded as major critical
molecules assisting tumor cells during metastasis, because of excessive degradation
of the extracellular matrix, and thus facilitation of cancer cells invasion [65].
These findings demonstrate that kaempferol and epigallocatechin-3-gallate may
be novel anticancer agents but more detailed studies should be performed to confirm
their anticancer effects for clinical use.
The biological properties of citrus flavonoids and their effectiveness in cancer
prevention have been widely studied. Recenty, Wang et al. have reported on anti-
cancer ability of flavonoids composition of citrus fruits varieties [13]. In vitro tumor
cytotoxicity of the citrus extracts were measured on three gastric cancer cell lines, i.
e., SGC-7901, BGC-823, and AGS. For the cytotoxicity of individual compounds,
the highest correlation coefficient with cytotoxicity of extracts on all three gastric
cancer cell lines was shown for nobiletin, a flavonoid from flavones subclass [13].
Not only citrus exctract but also apple exctrat was shown to have a positive effect
in vivo. George et al. demonstrated that preexposure with apple flavonoids protected
from various carcinogen-induced toxicity in normal human bronchial epithelial cells
significantly reduced toxic effects of various carcinogens, especially nicotine-
derived nitrosamine ketones, by inhibiting DNA damage response signaling and
initiated DNA repair mechanisms [66].
Other promising phytochemicals are Grape Seed Proanthocyanidins (GSPs).
GSPs have been shown to have cytotoxic activity against various cancer cell lines
with no significant toxic effects on normal cells. Treatment with GSPs significantly
reduced the viability and induced apoptosis of human head and neck squamous cell
carcinoma (HNSCC) cells, including HNSCC cell lines derived from the oral cavity,
larynx, pharynx, and tongue [55].
A number of human, animal, and cell-culture studies reported the potential effects
of genistein on different types of cancer, including breast, prostate, and ovarian
cancers. Genistein belongs to isoflavone subclasses. The mechanism of its influence
on cancer is complex and requires detailed investigations. Depending on the cancer
type, the same molecule may exert adverse or beneficial effects. Among breast cancers,
there are three subtypes: Triple Negative BC (TNBC, which do not express estrogen,
progesterone, or HER2 receptors), HER2 (that possess HER2 receptors), and ER (that
possess estrogen receptors). It was demonstrated that genistein plays different roles in
this triple scenario [67]. Some studies showed favorable effects of both high and low
doses of genistein as they decreased proliferation of ERα-β and HER2 breast cancer
cells [68–70]. However, it was reported by other authors that high doses of genistein
may enhance markers of proliferation, such as FGFR2, E2F5, BUB1, CCNB2,
MYBL2, CDK1, and CDC20 [71]. On the other hand, genistein and daidzein and
their metabolites may interact with ATP-binding cassette (ABC) G2 efflux transporter,
which is also called the breast cancer resistance protein (BCRP) and mediates the
disposition and excretion of numerous endogenous chemicals and xenobiotics. These
isoflavones may serve the BCRP as substrates, inhibitors, and/or modulators of gene
expression. When acting as BCRP inhibitors, these substances may induce reversal of
multidrug resistance and may enhance effectiveness of chemotherapy. On the other
hand, they may bring about also negative outcomes, such as increase in the toxicity or a
decrease in the efficacy of specific medications [72].
3.5 Flavonoids and the Nervous System
Enhancing cognitive abilities has become an important scientific challenge, recently

driven by the interest in preventing age-related cognitive decline and sustaining
normal cognitive performance in response to cognitively demanding environments.
Effectiveness of flavonoids in prevention of age-related neurodegenerative diseases
has been much investigated in the recent years. It concerns particularly dementia,
Parkinson’s (PD), and Alzheimer’s diseases (AD). It seems that flavonoids can
modulate neuronal function. Diets rich in these substances were shown to benefi-
cially affect maintenance of human cognitive functions, probably through protection
of neurons, enhancement of their function and regeneration [73].
The main pathological mechanisms of neurodegenerative diseases, including AD,
are both increased oxidative stress and inflammation [74–76]. It seems that flavo-
noids may protect effectively against oxidative neuronal damage and may also
decrease inflammation and thus have positive effects on the nervous system.
The multiplicity of beneficial effects of flavonoids exerted on the nervous system
seems to result from variety of mechanisms. One of them is regulation of the
neuronal signal cascade what brings about inhibition of cell apoptosis caused by
neurotoxic substances, such as amyloid β (Aβ) or 6-hydroxydopamine, (6-OHDA).
This promotes neuronal survival and differentiation. Another mechanism is
improvement of the cerebral blood flow, what can induce angiogenesis and growth
of new neurons in the hippocampus. Furthermore, flavonoids may increase the
amount of the brain-derived neurotrophic factor (BDNF) which is crucial to adult
neurogenesis, synaptic growth, and neuronal survival. All of these processes are
crucial for maintenance of neuronal and cognitive brain functions [77, 78].
The neurodegenerative process in Alzheimer’s diseases is characterized by the
presence of cerebral extracellular deposition of amyloid-β plaques, intraneuronal
neurofibrillary tangles, and cerebral atrophy [79]. The enzyme β-Secretase 1
(BACE1) controls the rate-limiting step of Aβ generation. The newly synthetized
Aβ rapidly aggregates to form neurotoxic amyloid fibrils. Shimmyo et al. demon-
strated that four flavonols (myricetin, quercetin, kaempherol, and andmorin) and one
flavone (apigenin) could inhibit BACE-1 activity in both the cell-free system and
neuronal cells. Each flavonoid significantly decreased concentrations of amyloid β
peptide-40 (Aβ1-40) and amyloid β peptide-42 (Aβ1-42) in neurons. These results
showed that flavonoids may reduce neuronal Aβ level and that this phenomenon is
related to BACE-1 inhibition [80].
Some studies also show that catechins, which are found in tea, may prevent the
formation of amyloid-β plaques, enhance cognitive functions, and may slow down
the onset and progression of AD. These substances (or their metabolites) can pass the
blood-brain barrier (BBB) and exert neuroprotective effects. Several studies dem-
onstrated that EGCG and other flavonoids, such as luteolin, could reduce toxic levels
of Aβ in the brain, and also mitochondrial dysfunction in AD brains [81]. EGCG also
prevented Aβ peptides-induced neurotoxicity, elongation of the fibrils, and stabili-
zation of the formed fibrils in cell cultures, and also enhanced clearance of phos-
phorylated tau protein in primary neurons in rats [82–84].
Parkinson’s disease (PD) is a progressive neurodegenerative disease, character-
ized by selective loss of dopaminergic neurons in the human midbrain region known
as the substantia nigra pars compacta (SNpc). The factors that trigger the onset of
this disease still remain unknown. Some evidence supports the role of oxidative
stress and imbalance in the natural antioxidant defense system. It is presumed that
these factors may contribute to the formation and/or accumulation of abnormal or
“toxic” proteins in the neuronal cells, which can lead to neuronal death [76].
Flavonoids such as quercetin glycosides, rutin, and isoquercitrin have distinct
features in upregulating the production of intracellular antioxidant enzymes such as
superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) and
glutathione in a 6-hydroxydopamine (6-OHDA-) induced in PC-12, rat pheochromo-
cytoma cells [85, 86]. It can protect neuronal cells from oxidative stress through
elevation of intracellular antioxidant enzymes. Apart from that, the neuroprotective
effects of flavonoids were further demonstrated in animal studies using tangeretin [87].
Tangeretin, which is found mainly in citrus fruit, given to mice passes the BBB and
protects the nigrostriatal pathway against adverse effects of 6-hydroxydopamine.
Not only research performed on animals but also on humans confirm an important
role of flavonoids (or their metabolites) in PD’s. In a prospective study, it was
observed in men that in the highest quintile of total intake of flavonoids there was
a 40% lower PD risk than in the lowest quintile. However, the authors admitted that a
protective effect of other constituents of plant foods should be taken into account,
and that it seems that the whole dietary pattern is of a great importance [88].
Effectiveness of flavonoids in prevention of age-related cognitive impairment,
particularly among patients at risk, has been much investigated in the recent years.
Research investigating the relations between cocoa flavanols and cognition shows
dose-dependent improvements in general cognition, attention, processing speed, and
working memory. Moreover, cocoa and cocoa-derived food could also improve
normal cognitive functioning and exert a protective role on delaying or preventing
age-related deterioration of cognitive decline [89].
In another study, compared to the consuming a low cocoa flavanol (48 mg) drink,
administration of intermediate (520 mg) and high (993 mg) cocoa flavanols drinks
over an 8-week period was associated with reduction of some measures of age-
related cognitive dysfunction in healthy aged participants. Interestingly, such cog-
nitive beneficial effects were paralleled by improvements in insulin resistance,
suggesting a role of glucose sensitivity in modulating cognitive function in these
patients. These data suggest that the habitual intake of flavanols can support healthy
cognitive function with age [90]. These results were consistent with the findings of
the study performed by Samieri et al. who showed that high flavonoid intake in
midlife helped to maintain good health and well-being during aging among women.
In this large prospective study (13,818 women from the Nurses’ Health Study),
higher intake of several flavonoid subclasses (i.e., flavonols, flavones, flavanones,
flavan-3-ol polymers, and anthocyanins), as well as total flavonoids in the age-
adjusted analyses were associated with better odds of healthy aging. Healthy aging
was defined as survival to more than 70 years with maintenance of four health
domains (no major chronic diseases or major impairments in cognitive or physical
function or mental health). In analyses of each component of healthy aging, such
flavonoid subclasses as flavones and flavanones were significantly associated with
two of four domains in authors’ definition of healthy aging, although associations
were generally weaker than for overall healthy aging. For example, compared with
women in the lowest quintile, women in the highest quintiles of flavone and
flavanone intakes had 32% and 24%, better odds of no mental health limitations,
respectively, and 23% and 15%, better odds of no physical function limitations,
respectively [91].
Collectively, it seems that regular consumption of foods rich in flavonoids
reduces the risk of neurodegenerative diseases and counteracts or delays the onset
of age-related cognitive disorders. Flavonoid compounds have been found to acti-
vate the endogenous antioxidant status in neuronal cells and hence protect them
against neurodegeneration. Studies also show that catechins may prevent the forma-
tion of amyloid-β plaques and enhance cognitive functions, and thus may be useful
in treating patients with Alzheimer’s disease or dementia.
3.6 Flavonoids: Promising Natural Compounds Against Erectile

Dysfunction
It should be emphasized that such disorders as Parkinson’s and Alzheimer’s diseases,

stroke, and cerebral trauma often cause erectile dysfunction (ED) by decreasing
libido or causing inability to initiate the erectile process [92]. Besides of neurological
diseases also, endothelial dysfunction may result in erectile disorder. Pathophysiol-
ogy of ED is multifactorial and and is usually triggered by impaired release of NO.
Although NO-mediated vessel relaxation plays a central role in erection, other
mediators such as cyclic-guanine monophosphate (cGMP), acetylcholine (ACh),
prostaglandins, and bradykinins are also important. Angiotensin-I-converting
enzyme (ACE) deactivates bradykinin, a vasodilator which is involved in erectile
function via the release of NO and relaxation of corpus cavernosum. The conversion
of angiotensin I to angiotensin II and deactivation of bradykinin can also induce high
blood pressure which in turn additionally impairs erectile function [92, 93].
Oboh et al. have shown a relationship between ED and oxidative stress due to
excessive generation of free radicals [94]. In an animal study, flavonoid and other
polyphenols-rich extract inhibited angiotensin-I-converting enzyme (ACE) and argi-
nase activities – key enzymes linked to erectile dysfunction – and oxidative stress
markers in rats’ penile tissues [94].
Furthermore, in a prospective study performed among 25,096 men from the

Health Professionals Follow-Up Study, it was shown that higher intakes of flavo-
noids from such subclasses as flavanones, anthocyanins, and flavones were signif-
icantly associated with a reduction in the risk of erectile dysfunction in men below
70 years of age but not in older [95].
3.7 Flavonoids: Promising Natural Compounds Against Cataract
Cataract is the major ophthalmic dysfunction of lens in which the clear lens gradually
develops into a cloud opaque and impairs vision. Cataract is one of the secondary
complications of diabetes affecting more than 20 million people and accounts 51% of
all blindness globally [96, 97]. It appears that flavones apigenin,and chrysin play an
important role in attenuation of sugar-induced cataractogenesis. Aldose reductase (AR)
is a key enzyme of polyol pathway that is responsible for the reduction of glucose and
galactose. Under normoglycemic condition, less than 3% of glucose is metabolized
through polyol pathway. However, in the case of hyperglycemia more than 30% of
glucose enters polyol pathway that leads to excessive accumulation of sorbitol in the
lens, what in turn results in several adverse pathological changes which ultimately
accelerate the process of cataractogenesis and other diabetic complications. The
mechanism of anticataract action of apigenin and chrysin includes inhibition of AR,
what was shown in sugar-induced lens cataract animal model studies [98, 99].
A panel of ten dietary flavonoids from different subclasses was evaluated as
possible inhibitors of sugar-induced cataractogenesis using bovine lens organ culture
studies. The results of lens organ studies clearly indicate that addition of rutin and
silibinin in the cultured lenses containing glucose significantly prevented the progres-
sion of lens opacity and maintained the lens transparency as compared with other tested
flavonoids. It clearly demonstrated the efficacy of rutin and silibinin as promising
agents that lead to inhibition of glycation reaction and amelioration of sugar-induced
cataractogenesis. The findings of this study may be useful for designing and develop-
ment of the novel safe molecules for the management of diabetic cataract [96].
Anthocyanins also seem to protect against cataract. It is commonly regarded that
in the pathogenesis of this disease apoptosis of the lens epithelial cells plays an
important role, similarly as oxidative stress seems to be the major cause of lens
opacity. It was demonstrated that anthocyanin from black soybean may protect
human lens epithelial cell line (HLE-B3) from oxidative stress. This substance was
shown to reduce HLE-B3 cell death in oxidative stress circumstances [100].
3.8 Flavonoids: Promising Natural Compounds Against Viral

Infections
Research focused on antiviral agents isolated from plants started in 1950s, when the
activity of 142 plants against influenza A virus was evaluated in embryonated eggs.
Twelve of them suppressed virus multiplication [101]. Nowadays, the main
directions of the research include the efficacy of flavonoids in the treatment of

hepatitis B virus (HBV), herpes simplex virus (HSV), and human immunodeficiency
virus (HIV) infections.
One of the examples is amelioration of chronic hepatitis by flavones. Luteolin-6-
C-β-D-boivinopyranosyl-30 -O-β-D-glucopyranoside and chrysoeriol-6-C-β-D-
boivinopyranosyl-40 -O-β-D-glucopyranoside, flavones isolated from Alternanthera
philoxeroides, an aquatic plant originated from South America, were shown to have
significant anti-HBV activities. These substances inhibited the secretion of HBsAg
from in the HepG2.2.15 cells [102]. Similarly, in HepG2.117 cells, EGCG inhibited
HBV replication through impairing HBV replicative intermediates of DNA synthe-
sis, thereby reducing the production of HBV [103]. Furthermore, in the culture of
hepatoma HepG2 cells, it was shown that EGCG treatment raised lysosomal acid-
ification, and thus enhanced autophagy, which appeared to inhibit HBV replication
[104]. Thus, botanical agents may be attractive sources of a new anti-HBV drugs.
Behbahani et al. found that kaempferol and kaempferol-7-O-glucoside have
strong HIV-1 reverse transcriptase inhibitory activity. These compounds exerted
their potent inhibitory effects, at a concentration of 100 μg/ml, on the early stage
of HIV replication in target cells [105]. Furthermore, quercetin exerted a significant
effect on the pharmacokinetics of ritonavir in rats. Liang et al. have suggested a new
strategy of treatment that might result in higher brain distributions, reduced toxicity,
and improved efficacy of HIV-1 protease inhibitors (HPIs) which are key compo-
nents of the highly effective antiretroviral therapy (HAART) currently used to treat
HIV-1 infection. Among the eight investigated flavonoids, quercetin was shown to
exert the strongest effect on the accumulation of ritonavir in human brain-microvas-
cular endothelial cells (HBMECs), by improving the intracellular accumulation of
ritonavir by 76.9% [106].
Numerous studies reported anti HSV-1 and HSV-2 effects of flavonoids, espe-
cially of EGCG and quercetin, in animal and cell-culture studies [8, 104, 107–110].
Oral infection with herpes simplex virus type 1 (HSV-1) or genital infection with
HSV type 2 (HSV-2) represent the most common infectious diseases in humans.
Isaacs et al. found that EGCG can inactivate HSV virions by binding to the envelope
glycoproteins gB and gD, which are essential for HSV infectivity. Moreover, the
EGCG digallate dimers theasinensin A, P2, and theaflavin-3,30 -digallate inactivated
HSV-1 and HSV-2 more effectively than did monomeric forms. These dimers are
stable at vaginal pH, indicating their potential to be antiviral agents against HSV
infections [111]. In addition, modification of green tea flavanol EGCG with palmi-
tate increased the effectiveness of EGCG as a potent inhibitor of herpes simplex
virus 1 [108]. Hung et al. have suggested possible mechanisms whereby quercetin
may exert its anti-HSV activity. They revealed that quercetin inhibits the infection of
HSV-1, HSV-2 and acyclovir-resistant HSV-1 mainly by blocking viral binding and
penetration in the beginning of infection. They also reported that quercetin
suppressed NF-κB activation, which is essential for HSV gene expression [112].
Flavonoids may also influence autophagy that plays a crucial role in the regulation of
viral replication. Enhancement of autophagy is therefore regarded to be a potential
therapeutic strategy for viral infection [113].
3.9 Flavonoids and Their Therapeutic Benefits in Inflammatory

Bowel Disease
Ulcerative colitis (UC) and Crohn’s disease are two major manifestations of inflam-
matory bowel disease (IBD). An increasing number of studies have associated the
intake of flavonoids-rich foods, especially catechin-rich food, with the prevention
and treatment of IBD. Mechanisms by which flavonoids benefit IBD have not yet
been entirely recognized, while human experiments demonstrating that catechins can
improve IBD are still scarce so far. However, there is abundant evidence showing
that flavonoids may work through a combination of parallel processes such as
oxidation inhibition, alteration of cellular signaling, and regulation of intestinal
flora [114, 115]. Such flavonoids as catechins epicatechin (EC), epicatechin gallate
(ECG), epigallocatechin (EGC) and its stereoisomer gallocatechin (GC), EGCG and
its stereoisomer gallocatechin gallate (GCG) could significantly reduce the oxidative
damages of the colon by promoting the activation of the antioxidative agents, such as
glutathione peroxidases (GPO) and glutathione (GSH). In addition, reduction of the
inflammation by regulation of the infiltration and proliferation of immune related-
cells, such as neutrophils, colonic epithelial cells, macrophages, and T lymphocytes
by catechins can provide benefits to IBD. Such anti-inflammatory properties of
catechins as regulating the activation or deactivation of inflammation-related oxida-
tive stress-related cell signaling pathways, such as NF-κB, MAPKs, transcription
factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), signal transducer and the
activator of transcription 1/3 (STAT1/3) pathways can be also important for IBD
treatment. Finally, catechins can also stabilize the structure of the gastrointestinal
microecological environment via promoting the proliferation of beneficial intestinal
bacteria and regulating the balance of intestinal flora, which seems be beneficial for
IBD [115].
Catechins may be effective agents to relieve IBD without obvious side effects in
animal and in vitro studies [114]. It was demonstrated that EGCG applied orally at
the dose of 6.9 mg/kg body weight, in combination with piperine which was used to
enhance the bioavailability of EGCG, inhibited colon inflammation in colitis in
murine model of colitis. Additionally, it significantly reduced the loss of body
weight, improved the clinical course, and increased overall survival in comparison
with untreated groups [116]. Reduction of tissue concentrations of malondialdehyde
and increase in activity of SOD as well as GPO have been shown. In another study
by Vasconcelos et al., EC (10 mg/kg) was also shown to benefit colitis induced by
2,4,6-trinitrobenzenesulfonic acid (TNBS) in acute and chronic rat models. In the
chronic colitis model, EC treatment was linked to lower macroscopic and micro-
scopic lesion scores and increased glutathione levels, decreased COX-2 expression
and increased cell proliferation [117].
Extensive laboratory and epidemiological studies have generally reaffirmed that
catechins, depending on their structures and dose, exert preventive effects against
chronic inflammatory diseases [118, 119]. However, it should be emphasized that
relevant evidence indicated that in the case of application of high concentrations of
catechins their pro-inflammatory capacity may exceed the benefitial effect on gut
inflammation. A high dose of EGCG was also demonstrated to act as a prooxidant,

instead of antioxidant, by generating ROS, causing even damage to cellular DNA in
various kinds of cells [119, 120]. The growing body of evidence indicates that
dietary catechins and their condensed sources may play a beneficial role in IBD;
however, further clinical and epidemiological trials are greatly needed.
4 Summary
Epidemiological, cell cultures, animal, and human studies support positive effects of
flavonoids on health. Over the past decades, flavonoids have attracted interest due to
their presumed roles in various beneficial activities, such as antihypertensive, anti-
bacterial, antiinflammatory, antioxidative actions. They were also shown to be protec-
tive agents against atherosclerosis. These compounds have also strong
chemopreventive potential including anti-inflammatory, antimutagenic, and anti-
carcinogenic activities. Moreover, increasing numbers of studies have demonstrated
that flavonoids may protect against cancer of varios locations, including the stomach,
colon, breast, lung, and liver. Furthermore, their consumption may improve postpran-
dial hyperglycemia, protect LDL particles against oxidative modification, reduce
neuroinflammation, and improve lipid profile in hyperlipidemic patients. However,
there is a great need for better understanding of the underlying molecular mechanisms.
Research challenges and future studies required in flavonoid research include trials
to understand the bidirectional relation between flavonoid metabolism and the micro-
biome. Findings on potential associations between dietary flavonoids intake and chronic
diseases risk should ultimately be integrated with studies using appropriate methods to
evaluate exposure (i.e., markers of consumption, metabolism, excretion, bioavailability)
to overcome the limitations of observational studies. Moreover, detailed research on
dietary intakes would provide better insights into the role of specific classes of
flavonoids and would show which of them are the most important in disease prevention.
It would also indicate the most appropriate level of their consumption. Finally, future
reaserch should include prospective clinical trials that would consider evaluation of the
extent of absorption and metabolism to establish dose-response relations.
At the present state of knowledge, it should be recommended to consume
abundant dietary sources of flavonoids, such as fruits and vegetables, legumes,
nuts, spices, and herbs every day.
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Bioactive Molecules, Nutraceuticals,
and Functional Foods in Indian Vegetarian 4
Diet and During Postpartum Healthcare
Jaya Arora and Kishan Gopal Ramawat
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2 Food Habit Influenced by Caste System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
3 Indian Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4 Bioactive Molecules, Nutraceuticals, and Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
5 Vegetarian Diet in Postpartum Healthcare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Abstract
Traditionally the majority population of the Indian subcontinent is vegetarian since
ancient times due to social and religious guiding principle of the society. Consump-
tion of whole grains of cereals and pulses, fruits, vegetables, and milk products is
associated with vegetarian diet. Essentially all meals consist of flatbread, cooked
pulses, a vegetable, curd/butter milk, and/or rice. Pulses are consumed in very large
quantity, and several salted snacks and sweet preparations are available in the market.
Beneficial effects of such products are now being revealed and scientifically vali-
dated by modern tools and techniques. In this review, we presented a concise and
comprehensive scenario about vegetarian diet in Indian region enforced by caste
system associated with religion. This leads to progression of hale and hearty brain
and intellectual community of Brahmins consuming only vegetarian diet rich in
fruits. Brahmin is a varna (class, caste) in Hinduism specializing as priests, teachers
(acharya), and protectors of sacred learning across generations. Marriages within the
caste further helped in gradual evolution of Brahmins (a kind of hybridization
between superiors). Vegetarian diet is maintained from birth to until death. There is
a well-defined and programmed vegetarian herbal diet starting from postpartum care,
J. Arora (*) · K. G. Ramawat (*)

Department of Botany, University College of Science, M. L. Sukhadia University, Udaipur, India

80 J. Arora and K. G. Ramawat
and beneficial effects of such diet are discussed in light of scientific information
available. Effect of bioactive molecules present in these foods and safety aspect are
also discussed.
Keywords
Vegetarian diet · Postpartum · Functional foods · Bioactive molecules · Caste
system · Hindu · Brahmin diet
1 Introduction
Vegetarian diet refers to nonconsumption of meat or animal products including sea

animals, fish, and eggs. However, in India milk and milk products are consumed by
vegetarians. This tradition of vegetarian diet is part of Hindu philosophy, religion,
and society where cruelty to animals is forbidden. Indians in general and majority of
Hindus in particular are vegetarian since time immemorial. About 30–40% of the
population of the Indian subcontinent is vegetarian, varying between 10% and 62%
according to the region [1–4], whereas it is around 10% elsewhere in the world such
as Sweden (10%), Austria (9%), Australia (2–11%), and Brazil (7.6%) [4]. Vegetar-
ianism in Western countries is a luxury associated with higher socioeconomic
position, greater physical activity, and lower rates of smoking which may result in
the observed health benefits [2, 3], whereas in India, it is a tradition- and caste-based
lifestyle since centuries. In contrast, vegetarianism is of recent origin in the Western
world [5]. A well-planned vegetarian diet has all the essential components required
for a healthy living [6].
Traditionally Indian foods can be divided into three categories: cooked vegeta-
bles, milk, and fresh fruits. Ayurveda means the science of life. It is an ancient
system of healthcare and longevity. Ayurveda takes a holistic view of human
beings, their health and illness. It aims at positive health, which has been defined
as a well-balanced metabolism coupled with a healthy state of being. Ayurvedic
concepts are thus deep rooted in day-to-day Indian culture, and hence food is
considered as responsible for physical, mental, and temperamental state of an
individual [7, 8].
Several recent studies show that vegetarian diet is associated with protective
effect of diet on blood cholesterol [9–11], blood pressure [11–14], fasting blood
sugar [15, 16], and cross-sectional evidences suggesting beneficial effects on blood
insulin [17, 18], C-reactive protein [17–19], many amino acids [20], and insulin-like
growth factors [17, 21]. Consumption of vegetarian or nonvegetarian diet results
in different bowel microorganisms [22], which may have beneficial implications
for diabetes, cardiovascular disease, and some cancers. Vegetarian diet has also been
associated with low rate of mortality, new types of cardiovascular diseases [23–25],
and diabetics [15]. Specific works showed reduction in incidence of prostate
cancer [26] and colorectal cancers [27, 28], whereas increased incidences of cancer
as well as diabetes and cardiovascular diseases were correlated with nonvegetarian
diet [29–33]. These studies have shown an advantage for vegetarian diet over
4 Bioactive Molecules, Nutraceuticals, and Functional Foods in Indian. . . 81
nonvegetarian diet in disease prevention and long-term health. India is a vast country
in which food, dialect, and dress change after every 500 km. Shridhar and
co-workers [3] found significant cardiovascular health benefits associated with the
vegetarian diet in four geographic regions of India. Traditionally food preparations
and basic ingredients are more or less same but with different or variable spices and
condiments and cooking medium [ghee (cleared butter) or any of the vegetable oils:
mustard, ground nut, sesame, or coconut]. Being an old civilization, these cooking
practices have been long established about processing of food, its preservation
techniques, and their therapeutic effects. This diet has profound effect, as it contains
body-healing chemicals, antioxidants, dietary fibers, and probiotics, on the devel-
opment and social behavior of the population. The food is rich in functional
components and provides prophylactic effect to various diseases such as diabetes,
cardiovascular diseases related to high cholesterol, and obesity. These effects are
further enhanced by processing the food as sprouting, malting, and fermentation
[34]. In this chapter, we have reviewed the information about development of
socioreligious faith to consume vegetarian diet in majority of the Indian population,
and vegetarian diet is blended with herbal medicine during postpartum care for long-
term benefit of the mother and the newborn. Available scientific information about
these foods is also discussed.
2 Food Habit Influenced by Caste System
Composition of Indian food is influenced by Aryan belief. Aryans were heterogeneous

group of people who lived mainly in northern ancient India. They migrated from
Indo-Iranian borderlands about 3000–8000 years ago with their cultures and customs
[35] and laid down caste system and its governing Vedas for society. The four Vedas,
namely, Rigveda, Samaveda, Yajurveda, and Atharva veda, describe the use of different
cereal grains and their use in our daily life. Aryans believed that food was not simply
meant for body nourishment, but was the basic part of a cosmic moral cycle [36].
The present-day Indian population is descendents of two ancient groups known as
“Ancestral North Indians” and “Ancestral South Indians” [37, 38]. The earliest
archaeological evidence for agriculture in the northern region is 8000–9000 years
old (Mehrgarh in present-day Pakistan) and involved wheat and barley derived from
crops originally domesticated in West Asia [39, 40]. In contrast to this, agriculture in
southern India is about 4600 years old, mainly cultivating mung bean, horse gram,
and local millets (pearl millet, ragi, sorghum) which are not related to west Eurasian
agriculture [41]. This is also supported by linguistic correlation of north Indian
population speaking Hindi derived from Sanskrit (which is Indo European lan-
guage), whereas Dravidian languages are not related to any of the Eurasian language
[42]. There are 4635 anthropologically well-defined human populations in India;
532 are tribes, including 72 ancestral tribes with the hunter and gatherer lifestyle.
Each population differs in terms of language, culture, physical features, and, most
importantly, genetic architecture. The total population count of India has reached
1.21 billion [43, 44].
The exact date of origin of caste system is not clear but Rigveda contains written
description of the system. Caste system was a social arrangement based on
occupation. There were four broad categories of castes in a Hindu society, Brah-
mins, Kshatriya, Vaishya, and Sudra, having a specific job to perform: preaching
and teaching, ruler or warrior, agriculture or business, and laborers, respectively.
Brahmin is a varna (class, caste) in Hinduism specializing as priests, teachers, and
protectors of sacred learning across generations. It is believed that caste system
was associated with Indo-Aryan, who established themselves as superior caste and
pushed the original Dravidian population to southern part and Sri Lanka. These
conclusions are based on mitochondrial DNA and Y chromosome markers [45].
Brahmins and Vaishya were forbidden to eat nonvegetarian food and supposed to
eat cereals, fruits, and vegetables by this social system. Now we know that high
consumption of fruits and vegetables is associated with reduced risk of stroke and
good health. Eating vegetarian diet rich in fruits and vegetable has/had a beneficial
effect on brain functioning associated with arranged marriages within the caste
resulting in a kind of forced breeding or hybridization between elites. This has a
long-term consequential effect on the evolution of Brahmins as preachers and
teachers of the society, whereas Kshatriya and Sudra were allowed to eat non-
vegetarian diet including spiced food which results in high metabolism and muscle
building required for their work [7, 46, 47]. Under this social system, vegetarian
food is consumed during all rituals from child birth, in marriages to until death.
Hindu rituals are not discussed here.
3 Indian Foods
Since Hippocrates, whose well-known dictum stated “Let food be thy medicine and
medicine be thy food,” diet has been key for health. It is imperative that food is not
medicine for quick change, but long-term preventive measures are important for process
of aging and health. Therefore, it is truly said that “you are what you eat” [48]. We have
come to know day by day with new reports that bioactive molecules present in food
have direct impact on our long-term health besides their nutritional value. It is better to
consume what is produced in the region. Long-term consumption of a locally grown
food has direct impact on metabolism and well-being of the population.
Whole grains or whole flour is staple food in the Indian subcontinent. Several
food plants native to this subcontinent and/or cultivated since ancient times are
wheat, rice, barley, cowpea, sugarcane, citrus, mango, onion, eggplant, pigeon
pea, Aegle, and jujube [41]. There are several preparations of grains (wheat, rice,
pulses, pearl millet, maize, pseudocereals), fermented grains/coarse grains (idli,
dhokla, dosa, kanji), fruits and green vegetables, and milk products (milk, curd,
butter milk, lassi, milk cake, and cottage cheese) [7, 49]. A typical meal consists
of flatbread made of whole wheat grain flour, boiled and salted-spiced pulse, a
cooked vegetable, and curd or butter milk. Boiled rice may or may not be
included. Porridge prepared from a mixture of rice or wheat semolina and pulse
is also a common practice. Pulses are consumed in large quantities in daily life as
whole, split, or ground into flour making several types of preparations as boiled
and cooked and fried and salted and a variety of desserts. Some representative
preparations shown here (Fig. 1) are (A) dal, (B) khaman dhokla, (C) sev,
(D) kofta, (E) dahi vada, (F) sambar vada, (I) laddu, and (J) barfi. Besides this,
frequent fasting or consumption only of pseudocereals and fruits is common
practice in this region. There are convincing evidences that effect of whole
extract of medicinal plants is more effective than isolated molecules. Similarly
the disease protection offered by all grains in prospective epidemiological studies
far exceeds the protection from isolated nutrients and phytochemicals in all
grains [50, 51]. Therefore, the concept of functional foods includes foods or
food ingredients that exert a beneficial effect on human health and/or reduce the
risk of chronic disease beyond basic nutritional functions [52]. Generally, alcohol
is not associated with vegetarian diet and forbidden for some caste by social and
religious directives.
All grains and millets are the ideal base for a wholesome diet. This is because all
variants of grains and millets are low-cost, nutritious, and locally available foodstuff.
Moreover, all grains affect glucose and insulin responses, and on the other side, they
have slower rate of digestion due to the presence of more dietary fiber, resistant starch,
and oligosaccharides in grains [53]. All grains are rich in antioxidants; phenolic
compounds; phytate; phytoestrogens such as lignan, plant stanols, and sterols; and
vitamins and minerals. Epidemiological studies establish that all grains intake is protec-
tive against cancer, cardiovascular diseases, diabetes, and obesity [54]. Asian Indians
may be benefitted from ancient whole-grain diet by decreasing the overall amount of
dietary carbohydrate and increasing carbohydrate quality in food [55]. Nutrition plays a
significant role in the prevention of disease such as diabetes (low glycemic index),
cholesterol-lowering effects, ability to decrease the risk of heart diseases, and their
protective effects against some cancers. Whole-grain cereals not only contain a large
number of nutrients and bioactive compounds, but consumption of pulses provides
complementary nutrients and a synergistic health benefit. Epidemiological evidence
shows a consistent inverse association between whole-grain intake and the risk of
chronic disease [56]. Among these bioactive compounds, γ-aminobutyric acid
(GABA), a non-proteinogenic amino acid with several health benefits such as anti-
diabetic and hypotensive effects (a well-known blood pressure-lowering compound)
and depression and anxiety reduction, is of particular interest [57].
India is the largest producer (26%), consumer (nearly one third), and importer of
pulses in the world [58]. During 2014–2015, pulses were grown in an area of 25.23
million hectares with a total production of 17.38 million tons and consumption of
22.68 million tons. Pulses have high contents of protein (20–25%), carbohydrates
(55–60%), calcium, and iron. Being vegetarian, pulses constitute the major source of
protein in Indian diet. Before the Green Revolution of the 1960s in India, traditional
meals and recipes were derived from whole-grain carbohydrates and also included
amaranth, barley, millet, and other ancient grains that have been grown on the Indian
subcontinent for the past few millennia. With more so-called Westernization, food
habits are gradually changing particularly in urban populations with increased
consumption of refined flour [59].
A B
Boiled salted food
C D
E F
G H
Deep fried, salted snacks
Whole and split pulses
I Desserts
J
Fig. 1 Pulses are consumed in large quantities as whole, split, or ground into flour in daily life
as boiled and cooked and fried and salted and prepared as various desserts. Some representative
preparations are presented here: (a) dal, (b) khaman dhokla, (c) sev, (d) kofta, (e) dal,
(f) mixture, (g) Dahi Vada, (h) Sambar Vada, (i) laddu, (j) barfi
4 Bioactive Molecules, Nutraceuticals, and Health Benefits
Food provides not only essential nutrients for growth and development but also
bioactive molecules for prevention of diseases. Consumption of fruits, vegetables,
cereals, and pulses is associated with prevention of several diseases including serious
illnesses like cardiovascular disease and cancer. It was inferred that the additive and
synergistic effects of phytochemicals present in fruit and vegetables were responsi-
ble for their potent antioxidant and anticancer activities and that the benefit of a diet
rich in fruit and vegetables is attributed to the complex mixture of phytochemicals
present in whole foods. This effect cannot be attained by providing purified bioactive
molecules individually [50, 60, 61].
Nutraceutical, a word created by combining “nutrition” and “pharmaceutical,”
was coined in 1979 by Dr. Stephen L. DeFelice, founder and chairman of the
Foundation for Innovation in Medicine, Cranford, New Jersey, USA [62]. It is
a food or food product that provides health and medical benefits, including the
prevention and treatment of disease. They are the product isolated or purified from
foods and generally sold in medicinal forms not usually associated with food and
demonstrated to have a physiological benefit or provide protection against chronic
disease. Most of the Indian food preparation contains spices and condiments, and
these have various bioactive molecules and exert antioxidant effect. Some of the
most frequently used are turmeric, asafetida, saffron, cardamom, ginger, clove,
fenugreek leaves and seeds, coriander leaves and seeds, mustard leaves and seeds,
chillies, nutmeg, mace, cinnamon, nigella seeds, etc. These all are proven effective
medicinal herbs and impart beneficial effects on health.
Indian food habits are in practice for at least the last two millennia and mainly
comprised of cereals, pulses, fruits, vegetables, and milk [59]. More and more recent
researches, particularly epidemiological studies, suggest a beneficial effect of such
diet due to increased antioxidant potential of such foods resulting in reduced
incidence of cancer, cardiovascular diseases, and stroke. This property is mainly
attributed to fruits, and now we recommend eating five fruits a day or one apple a day
keeps the doctor away. Slowly and slowly molecular mechanism and mode of action
of bioactive molecules present in such foods are being unraveled. Health-promoting
effects of isoflavonoids of pulses, antioxidants, and dietary fiber and various carbo-
hydrates and bioactive lipids and consumption of fruits and vegetable are described
in detail in this book; hence, details are not presented here.
Cereals (principally corn, wheat, and rice and sorghum and millets to a lesser
extent) are staple food in many developing countries of the southern hemisphere
with consumption of more than 400 million tons in 2017 [63]. Predominant Indian
diet is rich in cereals, pulses, oils, and spices which are source of dietary fiber,
vitamin E, carotenoids, and phenolic compounds. This diet exerts strong antioxidant
activity (μg α-tocopherol equivalent/100 g fresh weight) and antioxidant activity of
some of the prominent Indian foods such as rice (80–102), whole wheat (220–500),
spinach (750–890), amaranth (620–810), coriander leaves (610–750), and fenugreek
leaves (520–690) [64]. Daily intake of about 40 g dietary fiber is recommended for
an adult which varies in India due to socioeconomic conditions from 60 to 70 g
depending upon the type of cereal consumed. Dietary fibers in major cereals and
pulses consumed in India are rice (8.3), wheat (17.2), pearl millet (20.5), and mung
(15.2), while the soluble dietary fiber content of foods is reported to vary widely:
cereals, 8–20%; legumes, 20–50%; vegetables, 25–67%; and fruits, 20–65% [64,
65]. Cereals are not only rich in soluble and non-soluble carbohydrates but also
contain vitamins, minerals, phenolics, carotenoids, tocopherols, tocotrienols, antho-
cyanins, and phytosterols and impart health beneficial effects like immunomodula-
tory effect; antioxidant activity; antiproliferative and hepatoprotective effects [66,
67]; protection against coronary heart diseases, type 2 diabetes, and cancer; and
antiaging, antiallergic, and cholesterol-lowering abilities [68, 69].
Pulses (beans, peas, and lentils) have been consumed for at least 10,000 years and
are among the most extensively used foods in the world. Pulses are main source of
protein in a vegetarian diet and chickpea is consumed all over the globe. In Indian
diet, chickpea (Bengal gram, Cicer arietinum) is consumed in large quantities not
only in vegetable but in sweet and salted fried preparations, followed by mung bean
(green gram, Vigna radiata), arhar (pigeon pea, Cajanus cajan), urad (black gram,
Vigna mungo), lentil (Lens culinaris), and cowpeas (Vigna unguiculata). Pulses have
several health-promoting components, including vegetable protein, complex carbo-
hydrate, dietary fibers, vitamins, minerals, oligosaccharides, isoflavones, phospho-
lipids, and antioxidants. Its regular consumption has been associated with protective
effects against various diseases. Major bioactive phytoconstituents such as phytic
acid, lectins, sterols, saponins, dietary fibers, resistant starch, oligosaccharides, and
carotenoids and isoflavones have shown protective effects to various human diseases
such as diabetes, obesity, cancer, osteoporosis, and cardiovascular diseases [70–73].
A balance between antioxidants and free radicals is necessary to maintain good
physiological functioning of the body, and therefore, great attention has been given
to understand the mechanism of free radical action and protective effect of anti-
oxidants present in food. If free radicals overpower the body’s ability to control
them, a condition known as oxidative stress ensues [74]. Thus free radicals alter the
metabolism of lipids, proteins, and DNA leading to deranged physiology or
diseased state. Therefore, search for natural and safe antioxidants has been inten-
sified in recent years. A significant role of oxidative stress has been correlated with
several diseased conditions like atherosclerosis, arthritis, certain cancers, and
process of aging. Oxidative stress is now thought to make a significant contribution
to all inflammatory diseases (arthritis, vasculitis, glomerulonephritis, lupus ery-
thematous, adult respiratory disease syndrome), ischemic diseases (heart diseases,
stroke, intestinal ischemia), hemochromatosis, acquired immunodeficiency syn-
drome, emphysema, organ transplantation, gastric ulcers, hypertension and pre-
eclampsia, neurological disorder (Alzheimer’s disease, Parkinson’s disease,
muscular dystrophy), alcoholism, smoking-related diseases, and several others
[75]. It is needless to mention that an excess of oxidative stress can result in the
oxidation of lipids and proteins, which always causes changes in their structure and
consequently functions.
Fruit consumption is directly related to society’s economic status and is variable
in different countries. Anticancerous phenolics from Phyllanthus, tea polyphenols
(epigallocatechin gallate) as antioxidant and anticancer, radioprotective litchi phe-
nolics/flavonoids, hypoglycemic Syzygium, quercetin and hydroxyl cinnamates of
sweet cherries, xanthones of mangosteen, ellagitannins of pomegranate, ursolic acid
of sea buckthorn, muscle relaxative watermelon, anti-cholesterolemic soluble fiber
and sterols, cardioprotective saponins, ACE-inhibitory potato hydrolysates, anti-
pancreatic cancerous ascorbic acid, and carotenoids including pro-vitamin A are
some of the well-known examples [76]. Selected tropical fruits and vegetables and
their bioactive molecules and biological effects are presented in Tables 1 and 2.
Some of them are grown since millennia, while some are improved recently from
their wild relatives. The commonly consumed fruits are mango, banana, papaya,
guava, custard apple, pomegranate, apple, sapota, orange, and watermelon. These
fruits and vegetables contain diverse bioactive molecules with health benefits rang-
ing from antioxidants, immunomodulators, antidiabetic, and hepatoprotective to
anticancer. The other prominent food-based bioactives include tea polyphenols
(epigallocatechin gallate) as antioxidant and anticancerous, β-carotene, lycopene
and isoflavones for prostate cancer prevention, omega-3 and -6 fatty acids, licorice,
quercetin, curcumin as anticancer (antiangiogenesis), curcumin (anti-inflammatory
and anticancer).
In an extensive literature survey, Bhattacharya [59] observed that Indian food
habit has gradually shifted from large quantities of indigestible fiber carbohydrate,
small amounts of digestible carbohydrate, moderate fat, and moderate protein to an
increasing intake of high-fiber and refined carbohydrates associated with a decreas-
ing intake of animal proteins. Major shift in intake of high-fiber carbohydrate was
observed from1775 to 1947. After 1947, slight change in food habit was observed
with increased frequency of small quantities of refined carbohydrates and improve-
ment in protein intake associated with socioeconomic conditions of the country.
Therefore, Indian vegetarian food is not a single ingredient but a cocktail of many
nutritional materials and herbs to impart nutraceutical effect on human health
because of its long-term continuous consumption from childhood.
5 Vegetarian Diet in Postpartum Healthcare
Pregnancy is a unique situation in the life of to-be mother, and diet during this not
only affects the health of the mother but also the newborn and has bearing on
health throughout life. Similarly, nutrition and functional foods given during the
postpartum care have long-term effect on growth, health, and metabolism of the
mother and newborn. The postpartum period is commonly defined as beginning
1 h after delivery of the placenta and lasting 6 weeks [122]. The postpartum
period is a time of increased physical, emotional, and social change for the
parents of newborn. In many Southeast Asian cultures including India,
Table 1 Selected subtropical fruits, their bioactive molecules and biological activities
Plant species,
name Part used Bioactive molecules Biological activity References
Aegle Coumarins such as Antihyperglycemic, [77, 78]
marmelos, marmelosin, chronic diarrhea,
bael umbelliferone, and dysentery, peptic
scopoletin ulcers, as a laxative
Artocarpus Artocarpin, Chemoprevention of [79, 80]
heterophyllus, moracin C colorectal cancer,
jackfruit antioxidant activity,
anti-inflammatory
activity
Carica Papain, carotene Antioxidant activity [81, 82]
papaya,
papaya
Citrus Ascorbic acid, citric Antioxidant, [83]
reticulata, acid, carotene antiallergic,
orange improved lipid
profile
Fragaria sp., Flavonoids, Antioxidant, anti- [84, 85]
strawberry anthocyanins, inflammatory,
hydrolyzable antiatherosclerotic,
tannins anticarcinogenic,
(gallotannins, antidepressant
ellagitannins), properties
phenolic acids
(hydroxycinnamic,
hydroxybenzoic
acids), condensed
tannins, vitamin C,
folate
Malus, apple Chlorogenic acid, Antioxidants, [86, 87]
epicatechin, antiproliferative,
procyanidin B2, breast cancer
phloretin and treatment
quercetin,
vitamin C, benzoic
acids (gallic acid),
hydroxycinnamic
acids (chlorogenic
acid), flavanols
(catechin),
flavonols (rutin),
chalcones
(phloridzin),
terpenes (ursolic
acid)
Mangifera sp., Phenolic Antioxidant activity, [88–91]
mango compounds, analgesic,
mangiferin, antidiabetic,
vitamins A and C, antisclerotic,
β-carotene and antimicrobial,
(continued)
Table 1 (continued)
Plant species,
xanthophylls, cis-9, antiviral; cardio-,
cis-15- hepato-, and
octadecadienoic neuroprotective;
acid anti-inflammatory;
antiallergic; memory
improving;
radioprotective
against X-ray,
gamma, and UV
radiation
Musa sp., Flavonoid fraction Immunomodulatory [92]
banana activity
Psidium Flavonoids, Antidiarrheal [93]

guajava, carotenoids, activity, antioxidant
guava polyphenolic activity, antidiabetic,
compounds, antimicrobial
pentacyclic activity
triterpenoids
Phyllanthus Phenols, gallate, Cardioprotective [94, 95]
emblica mucic acid 1-ethyl effect, antioxidant
Indian 6-methyl ester 2-O-
gooseberry gallate
Punica Polyphenols, citric Antiatherogenic, [96, 97]
granatum, acid, ascorbic acid, antioxidant,
pomegranate ellagitannins, antihypertensive,
ellagic acid anti-inflammatory,
cardioprotective,
anticancer
Rubus sp., Phenolics, tannins, Antioxidants, [98, 99]
blackberry, stilbenes, caspase-mediated
raspberry, flavonoids, apoptosis in human
dewberry anthocyanins; breast
vitamin C, alpha- adenocarcinoma
tocopherol; flavonol cells
glycoside, apigenin,
cyanidin-3-
glucoside
Syzygium Polyphenols: Antidiabetic, [100, 101]
cumini, jamun resveratrol, antioxidant,
anthocyanins, antihyperlipidemic,
ellagic acid/ antiulcer,
ellagitannins hepatoprotective,
antiallergic,
antiarthritic,
antimicrobial, anti-
inflammatory,
antifertility,
antiplaque,
(continued)
Table 1 (continued)
Plant species,
radioprotective,
nephroprotective,
antidiarrheal
activities
Ziziphus Ascorbic acid; Aphrodisiac, [102, 103]
jujuba, jujube thiamine; hypnotic-sedative,
riboflavin- anxiolytic,
bioflavonoids; anticancer
pectin; mauritine A; (melanoma cells),
amphibine H; antifungal,
jubanine A and B; antibacterial,
mucronine D; antiulcer, anti-
nummularine B; inflammatory,
sativanine E; antifertility/
ziziphines A to Q; contraception,
betulinic acid; hypotensive,
jujubosides A, B, cardiotonic,
A1, and B1; antioxidant,
saponins immunostimulant,
wound healing
properties
postpartum period is considered important from point of view of recovery, by

offering a period of confinement ranging from 10 to 45 days. Many activity and
dietary restrictions are applied during this period, and the main emphasis is given
on proper nutrition.
The following traditions are followed:
1. Covering of head: By north Indian traditions, the mother is supposed to cover the
head with scarf to prevent heat loss and catching cold.
2. Daily oil massage is given to the mother and newborn by experienced elderly lady
or nurse to impart strength to the body. Sesame oil for the body and coconut oil
for the head (or cleared butter for all purposes) are used which are absorbed
quickly.
3. Hot/lukewarm water is used for bath.
4. Diet is fortified with cleared butter whether it is bread, mixed porridge, wheat
porridge, or vegetables. Too much emphasis is given on consumption of milk,
cleared butter, and high-calorie diet (sweet preparations) prepared with herbs.
5. Decoction of cumin, carom, and/or curcuma in milk is given during initial days in
the morning.
6. Like headscarf, belly binding with a large cotton cloth is also practiced to
facilitate repositioning of the uterus, perfectly position the baby during feeding,
and put the stomach muscles back together.
Table 2 Some of the subtropical vegetables, their bioactive molecules and biological activities
Plant species, Principal nutrient Bioactive
part used source molecules Biological activity References
Brassica Lectins, essential Immunomodulatory, [104, 105]
oleracea, oils, hepatoprotective,
cauliflower, glucosinolates antioxidant,
inflorescence anticancer effects
Capsicum Capsaicin Stimulation of [106]

annum, chilli, gastrointestinal
fruit defense and
absorption,
stimulation of
salivary, intestinal,
hepatic, and
pancreatic secretions
Chenopodium Vitamin A, oxalic Anthelmintic, [107]
album, bathua, acid antimicrobial,
leaves antioxidant
Cyamopsis Flavonoids, Anticancer, [108, 109]

tetragonoloba, isoflavonoids antioxidant,
cluster bean, (daidzein, antidiabetic,
immature pods genistein), antiulcer,
luteolin, quercetin, cytoprotective,
kaempferol anticholinergic,
hypolipidemic
Daucus carota, Polyacetylenes, Potential treatment [110, 111]
carrot, root carotenoids (beta- for chronic
carotene and lymphocytic
lutein) leukemia
Lagenaria Sterols, Controlling diabetes, [112]
siceraria, terpenoids, hypertension, liver
bottle gourd, flavonoids, diseases, weight loss,
fruit saponins and other associated
benefits
Momordica Cucurbitane-type Antidiabetic, [113, 114]
charantia, triterpenoids, immunomodulatory
bitter gourd, triterpene effect, antioxidant,
fruits glycosides, anti-inflammatory,
phenolic acids, anticancer,
flavonoids, antibacterial, anti-
essential oils, obesity activity
saponins,
momordicosides
Q U, charantin
Raphanus Raphanusamide, Antiulcer, [115]
sativus, radish, cyanidin, vanillic antimicrobial,
root acid, kaempferol, antifungal, anti-
quercetin inflammatory,
diuretic, antioxidant,
anticarcinogenic,
(continued)
Table 2 (continued)
Plant species, Principal nutrient Bioactive
part used source molecules Biological activity References
antitumor promotor,
and anti-HIV
activities
Solanum Polyphenols, Prevention of cancer [116, 117]
lycopersicum, β-carotene, and
tomato, fruit lycopene, neurodegenerative
vitamin C, and cardiovascular
quercetin, rutin, diseases, improving
hydroxycinnamic immunity
acid derivatives
Solanum Flavonoids, Analgesic, [118]
melongena, tropane, antipyretic,
brinjal, fruit glycoalkaloids, antioxidant, anti-
arginine, inflammatory,
lanosterol, hypolipidemic,
gramisterol, hypotensive,
aspartic acid antiplatelet,
intraocular pressure
reducing
Spinacia Ascorbic acid, Protective effect [119–121]
oleracea, folate, against metabolic
spinach, leaves phylloquinone, syndrome, anti-
alpha-tocopherol, obesity, anti-
and the inflammatory,
carotenoids lutein, anticancer,
violaxanthin, hypoglycemic, and
zeaxanthin, and lipid-lowering effect
beta-carotene
In Asia, the dietary choices of women during pregnancy and the postpartum
period are principally influenced by traditional healthcare system and cultural beliefs
whispered for these periods. It is believed that a “hot-cold balance” should be
maintained in order to stay healthy and that this can be achieved by following special
prescriptive diets during pregnancy and the initial postpartum period, commonly
termed “the confinement period” [123, 124]. Several studies indicate that maternal
diet during pregnancy and the postpartum period has not only important effects on
the offspring’s health but also the diet of a lactating mother which influences the
composition of breast milk [125–127].
Vegetarian diet is considered safe for pregnant women and long-term health of the
newborn [128]. The major population in the Indian subcontinent is vegetarian, and
health of babies is not a problem in general except in cases of malnutrition.
Traditionally postpartum care includes hygienic condition with feeding mother
with several nutrients and functional foods as enumerated in Table 3. All these
herbal plants are used in 50–200 g quality in several preparations for postpartum
4
Table 3 Bioactive molecules and biological activity of plants used in diet therapy during postpartum healthcare (compiled from [130–134])
Biological activity
Total
Plant species polyphenols DPPH% SOD% FRAP
(family), part Bioactive molecules (mg GAE/g) inhibition inhibition (mM Fe+2/g) Others/remarks
Aegle marmelos Furocoumarins, flavonoids, 95.33 2.31 80.25 1.03iu 54.94 0.88 884 5.13 Treatment of chronic
(Rutaceae), fruit rutin, and marmesin; a diarrhea, dysentery, and
number of essential oils peptic ulcers, as a laxative
Anethum Essential oils, coumarins, 4.73 0.23 75.53 2.91 37.25 1.18 62 1.13 Carminative, stomachic, and
graveolens flavonoids, phenolic acids, diuretic
(Apiaceae), fruit and steroids
Anogeissus High molecular weight 300.0 4.55 86.94 2.57 84.95 1.62 975 6.86 Reduction in serum total
latifolia polysaccharic acid (ghattic cholesterol
(Combretaceae), acid)
gum
Areca catechu Alkaloids of the pyridine 393.33 3.77 72.71 0.91 43.23 0.74 1003.33 9.55 Diuretic, digestive,
(Arecaceae), nut group, ß-sitosterol, anthelmintic, astringent, and
leucocyanidins, catechu cardiotonic
(tannins, catechin, gallic
acid)
Asparagus Steroidal saponins, essential 4.13 0.30 26.26 0.71 32.44 0.58 80 1.26 Gastric ulcers and dyspepsia
racemosus oils, asparagine, arginine, and as a galactogogue
(Asparagaceae), tyrosine, flavonoids
root tuber (kaempferol, quercetin,
Bioactive Molecules, Nutraceuticals, and Functional Foods in Indian. . .
rutin)
Butea Butrin, isobutrin, butin, 426.66 8.31 87.17 1.39 80.53 1.42 79.0 1.26 Antimicrobial, wound
monosperma palasitrin, and butein healing, antifungal,
(Fabaceae), gum antidiarrheal,
hypoglycemic,
hepatoprotective,
(continued)
93
94
Table 3 (continued)
Biological activity
Total
antioxidant, antihelminthic,
anticonvulsive
Cinnamomum (E)-Cinnamyl acetate, trans- 58.6 0.29 74.31 1.53 79.00 2.53 222 1.98 Benefit in respiratory,
zeylanicum alpha-bergamotene, and digestive, and gynecological
(Lauraceae), bark caryophyllene oxide ailments
Cocos nucifera Phenols (catechins, 2.46 0.12 3.14 0.37 10.7 0.96 03 0.32 Antihelminthic, anti-
(Arecaceae), epicatechins, tannins, and inflammatory,
endosperm flavonoids), triterpenes, antinociceptive, antioxidant,
steroids antifungal, antimicrobial,
and antitumor activities
Curculigo Saponins, sapogenins, 11.06 0.92 30.18 0.71 46.32 1.22 90 2.64 Heating, aphrodisiac,
orchioides phenolic glycosides, a alternative, appetizer
(Amaryllidaceae), triterpene alcohol
tuberous roots
Curcuma amada Curcuminoids (curcumin, 14.93 1.06 84.88 2.04 12.50 0.54 74 1.12 Antibacterial, insecticidal,
(Zingiberaceae), demethoxycurcumin, antifungal, and antioxidant
rhizome bisdemethoxycurcumin)
Curcuma longa Curcuminoids (curcumin, 16.66 0.42 78.39 1.23 24.05 0.14 72 1.76 Anti-inflammatory,
(Zingiberaceae), demethoxycurcumin, anticancer, antibacterial,
rhizome bisdemethoxycurcumin) antiviral, antioxidant,
cardioprotective,
hepatoprotective
J. Arora and K. G. Ramawat
4
Desmodium Pterocarpans (gangetinin, 13.53 0.94 85.62 1.32 25.48 049 208.5 3.56 Febrifuge, digestive, anti-
gangeticum gangetin, desmodin), catarrhal, antiemetic,
(Fabaceae), roots isoflavones (desmocarpin, hepatoprotective
genistein)
Elettaria 1,8-Cineol 3.86 0.12 62.33 1.27 30.18 0.80 02 0.46 Antioxidant activity, anti-
cardamomum inflammatory, antimicrobial
(Zingiberaceae), potency
seeds
Embelia ribes Embelin 12.0 1.04 89.99 1.78 23.18 0.46 633 3.60 Anthelmintic, carminative,
(Myrsinaceae), and stimulant
fruits
Gmelina arborea Gmelo furan-a 10.26 0.30 89.68 1.53 44.43 1.29 777 2.52 Rejuvenative, aphrodisiac,
(Lamiaceae), roots furanosesquiterpenoid, memory enhancer
sesquiterpene, gmelinol,
ceryl alcohol,
hentriacontanol-1,
β-sitosterol, n-octacosanol
Litsea glutinosa Steroids and terpenoids, 70.0 0.12 79.82 0.84 82.15 1.83 638 7.63 Antioxidant activities,
(Lauraceae), bark butenolides, litsealactone, analgesic property, arrest
tannins, actinodaphnine, bleeding, aphrodisiac
boldine,
heteropolysaccharide
polyuronide
Mesua ferrea Mesuol, mammeisin, 66.66 0.32 87.47 0.87 46.77 1.02 900 3.45 Antioxidant, antineoplastic,
(Clusiaceae), mesuagin, mammeigin, antispasmodic,
flowers anthraquinones, xanthones, anticonvulsant,

mesuaferrins hepatoprotective,
immunomodulatory
(continued)
95
96
Table 3 (continued)
Biological activity
Total
Mucuna pruriens L-Dopa 38.66 1.15 89.85 1.76 17.99 0.42 868 4.04 Male infertility, nervous
(Fabaceae), seeds disorders, and also as an
aphrodisiac, antioxidant
activity
Myrica esculenta Myricanol, 45.33 2.04 81.27 3.26 75.39 0.98 800 3.02 Antimicrobial, wound
(Moringaceae), aproanthocyanidin, healing, antifungal,
bark β-sitosterol, taraxerol, hepatoprotective,
myricadiol antioxidant, antihelminthic
Myristica fragrans Trimyristin, myristic acid, 12.0 1.04 89.99 1.78 23.18 0.46 633 3.60 Aromatic, aphrodisiac,
(Myristicaceae), myristicin, safrole, and diarrhea, anticonvulsant,
seeds elimicin analgesic, anti-
inflammatory, antidiabetic,
antifungal antibacterial,
antioxidant
Myristica fragrans Lignin(macelignan), 40.66 1.15 88.96 1.32 14.2 0.38 588 3.51 Inhibits the enzyme
(Myristicaceae), flavonoids, phenolics, xanthine oxidase
aril of fruits tannins, alkaloids, and
saponins
Ocimum basilicum Terpenoids, alkaloids, 9.00 0.72 63.68 1.80 5.04 0.14 08 0.53 Hepatoprotective, antitoxic,
(Lamiaceae), flavonoids, tannins, saponin immunomodulatory,
leaves glycosides antihyperglycemic,
antifungal, hypolipidemic,
anti-inflammatory,
antibacterial
4
Piper cubeba Diarylcyclobutane 15.53 1.85 87.47 0.92 9.72 0.96 485 6.80 Cough, swelling,
(Piperaceae), fruits neolignans, indigestion, dysmenorrhea,
dibenzylbutyrolactone erectile dysfunction
lignan, O-ethyl cubebins,
monoacetate of
dihydrocubebin
Piper longum Alkaloids, tannins, 4.13 0.23 45.57 0.12 37.13 0.98 50 0.96 Antioxidant, antibacterial
(Piperaceae), roots flavonoids activity
Piper nigrum Piperine 5.13 0.61 57.22 1.05 31.88 0.73 19 0.40 Stimulate digestive enzymes
(Piperaceae), fruits in the pancreas, antioxidant
Quercus infectoria Gallic acid, tannic acid 645.33 10.2 90.20 2.4 90.34 0.35 1115 9.29 Astringent, diarrhea,
(Fagaceae), galls leucorrhea, uterine prolapse,
tightening the loose or
sagging tissues
Rubia cordifolia Anthraquinone (munjistin, 9.60 0.87 71.98 2.21 49.32 0.59 75 1.10 Blood detoxifying, anti-
(Rubiaceae), roots purpurin, and inflammatory, hemostatic,
pseudopurpurin), rubiadin antidysenteric, antipyretic,
analgesic, anthelmintic
Solanum Alkaloids, sterols, saponins, 4.86 0.65 84.41 1.21 24.9 062 520 2.29 Antiasthmatic,
surattense flavonoids hypoglycemic,
(Solanaceae), hepatoprotective
roots antibacterial
Smilax chinensis Kaempferol and its 9.26 0.81 87.36 1.54 55.05 0.83 55 1.62 General health tonic,
(Liliaceae), roots glycosides, rutin, engeletin syphilis, skin diseases and
women’s health concerns

Solanum indicum Flavonoids, steroid, tannin, 14.13 0.80 72.68 1.27 25.11 0.70 733 3.60 Antioxidant, anticancer,
(Solanaceae), saponins anti-inflammatory, diuretic,
roots diaphoretic
(continued)
97
98
Table 3 (continued)
Biological activity
Total
immunomodulatory,
neuroprotective effects
Symplocos Phenolic glycosides 86.0 0.05 75.29 0.10 63.33 1.54 884 4.50 Anticancer,
racemosa (symplocoside), hepatoprotective,
(Symplocaceae), triterpenoids (betulinic acid, antioxidant, antiandrogenic
bark acetyloleanolic acid, effect, anti-inflammatory,
oleanolic acid) flavonoids wound healing activity,
(quercetin) antidiabetic effects
Syzygium Clove oil (eugenol, 101.33 0.23 88.13 0.58 55.05 1.05 675.33 5.36 Antibacterial, antifungal,
aromaticum trans-β-caryophyllene) insecticidal, antioxidant,
(Myrtaceae), anticarcinogenic activities
flower bud
Terminalia Tannins (gallic acid, 166.0 0.35 78.88 0.85 84.66 2.24 1009 8.98 Antioxidant, antidiabetic,
chebula chebulagic acid, hepatoprotective, anti-
(Combretaceae), punicalagin, chebulanin, inflammatory,
fruits corilagin, neochebulinic antimutagenic,
acid, ellagic acid, chebulinic antiproliferative,
acid), chebulinic acid, radioprotective,
ellagic acid, anthraquinones cardioprotective,
antiarthritic neuroprotective
activities
4
Trachyspermum Thymol, carvone, limonene, 12.46 0.98 88.39 1.01 0.26 0.02 955 7.63 Stimulant, antispasmodic,
ammi (Apiaceae), dillapiole and carminative
seeds
Tribulus terrestris Flavonoids, flavonol 10.2 0.53 82.64 0.98 1.10 0.06 560 2.80 Diuretic, aphrodisiac,
(Zygophyllaceae), glycosides, steroidal antiurolithic,
fruits saponins, and alkaloids immunomodulatory,
hypolipidemic,
hepatoprotective, anti-
inflammatory
Vitex negundo Flavonoids, flavone 5.46 0.12 53.96 0.96 4.25 0.10 180 1.26 Antinociceptive, anti-
(Verbenaceae), glycosides, volatile oil, inflammatory, antitumor,
seeds triterpenes, tannins, and antioxidant, antimicrobial,
lignin antiandrogenic, anti-
osteoporotic,
hepatoprotective,
antihyperglycemic activities
Zingiber officinale Flavonoids, alkaloids, 11.4 0.40 87.11 0.61 33.78 0.53 47 0.65 Immunomodulatory,
(Zingiberaceae), saponins, steroids, antitumorigenic,
rhizome terpenoids, tannin antioxidant, anti-
inflammatory, antiapoptotic,
antihyperglycemic, anti-
lipidemic and antiemetic
actions
99
period besides food fortified with fat and sugar to suppress bitter taste of herbs.
Ginger is used in separate preparation as considered hot and lodh (Symplocos
racemosa) in separate preparation being considered as cold. Hot and cold prepara-
tions are given separately, hot preferably first. All these preparations are divided into
equal proportions to last 40–45 days. Increased milk drinking associated with
galactogogue like Asparagus racemosus roots facilitates milk production. Bioactive
molecules present in these herbal ingredients impart nutrition (high calories) and
functional molecules which are helpful in developing immunological, antioxidative,
and galactogogue effects. These plants contain a wide variety of free radical-scav-
enging molecules, such as phenolic compounds (e.g., phenolic acids, flavonoids,
quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids,
amines, betalains), vitamins, terpenoids (including carotenoids), and some related
endogenous compounds, which have high antioxidant activity [129]. Most of the
herbs and foods given during postpartum care in India showed strong antioxidant
activity [130]. Other prominent biological activities include stimulant, improves
digestion, blood purifier, progesterone production, balance of hormonal cycle,
emotional balance, tonic for nerves and stress and wound healing activities. Some
of them are well-established medicinal plants used in Ayurveda such as fruits of
Aegle marmelos and Terminalia chebula; rhizome of Curcuma longa and Zingiber
officinale; roots of Asparagus racemosus, Rubia cordifolia, Smilax chinensis, and
Desmodium gangeticum; and seeds of Mucuna pruriens and Vitex negundo. Details
of their active constituents are given in Table 3. Dried ginger is traditionally used in
several food preparations including tea as well as major ingredient in postpartum
food preparation. Ginger is known to impart antioxidant, antimicrobial, and anti-
inflammatory effects, and modern tools of molecular targets validated these effects
[131]. In all 38 plants are described in Table 3. These plants contain very diverse
chemicals and their effects as an individual is neither known completely nor is
possible to describe in this chapter. It is the combined effect of these herbs that is
more important, and we know that such formulation imparts synergistic effect on
human health.
Diet therapy includes preparations of medicinal herbs in the form of decoc-
tions, infusions, and cold extracts. The herbs given during this period are tradi-
tionally known to strengthen the body and mind and prevent disorders such as
postpartum depression (PPD), body aches, insomnia, indigestion, and oxidative
stress [135].
Several reports describe the effect of maternal diet particularly rich in high fat on
composition of human milk. Bioactive lipids in milk include triacylglycerides,
diacylglycerides, saturated and polyunsaturated fatty acids, and phospholipids. Ben-
eficial activities of milk lipids include anticancer, antimicrobial, anti-inflammatory,
and immunosuppression properties. Fats are good media for solubility of many
organic compounds and facilitate their absorption [136, 137]. Maternal diet has
been shown to influence fatty acid composition of breast milk, with changes
appearing within 8–10 h after a meal is consumed [138–140]. Fatty acids with a
chain length greater than C14:0 originate from the maternal diet or body stores, while
fatty acids up to C14:0 originate from de novo synthesis in the breast [138, 141].
The term conjugated linoleic acid (CLA) describes a mixture of positional and
geometric isomers of linoleic acid (C18:2n-6) which contain a conjugated double-
bond system instead of the more common isolated double bonds. Rumenic or cis-9,
trans-11-octadecadienoic acid (cis9,trans11-C18:2) is the most regular CLA isomer
and is frequently regarded as the biologically most pertinent one [142]. Humans
receive CLA through diet (dairy products and ruminant meat), and it is presumed
that the amount of CLA in the milk of breastfeeding women can be augmented
by increasing the amount of organic dairy nutrients within their diet [143].
CLA content and both rumenic acid and TVA in human breast milk were increased
in the milk of lactating mother consuming diet rich in organic products as compared
to control (mothers) eating the same products obtained through conventional
methods [144, 145].
Alternative to household preparations, several Ayurvedic formulations are also
available for postpartum care such as shatavari kalpa granules (A. racemosus) for
lactation and dashmool (ten roots, the combination of ten roots is used widely in
Ayurveda for many health conditions related to anti-inflammatory, antirheumatic or
antiarthritic, analgesic, antispasmodic, adaptogenic, antioxidant, neuroprotective,
anti-paralytic, uterine tonic, uterine detoxifier). Out of these ten roots, five (Aegle
marmelos, Desmodium gangeticum, Gmelina arborea, Solanum surrentense,
Tribulus terrestris) are used in the household preparations. Besides, there are several
herbal preparations that are also available for the newborn mainly for digestive
system correction, gas, and flatulence.
6 Conclusions
It is increasingly clear that the regular consumption of traditional food has a long-
lasting beneficial effect rather than changed diet regimen. Vegetarian diet has at least
no harmful effect. Pulses are traditionally consumed all over India, and now we have
new evidence that beneficial bioactive peptides are produced during processing and
cooking; hence, the effect is nutraceutical besides nutritional. Similarly, coconut oil
is consumed traditionally in almost all food preparations in Kerala state of India,
while north Indian consumed sesame or mustard oil. Slight variation in food habits
are due to what is produced in that area is preferably consumed in the area,
irrespective of the general belief. There is no record of higher disease incidence in
Kerala because of the consumption of saturated fat in high amount. Beneficial effects
of mustard and sesame oil are being established now, whereas these are used since
centuries and soybean or sunflower oil is of recent introduction.
In this review, we have tried to portrait the scenario prevailing in a country like India
in relation to vegetarianism and postpartum care influencing the whole life of a newborn.
In India, vegetarianism is centuries old and strictly followed by caste system and
practiced by religion in well-to-do families. In contrast to this, those who are below
poverty line families, they do not follow strict diet rules by caste and religion, and they
have their own tribal rules or no rules, hence are not strictly vegetarian. Several herbal
ingredients used during postpartum care need to be evaluated scientifically using

modern pharmacological techniques to ascertain their effects.
This will also provide information about necessary inputs required for better
health of the mother and child. Recent studies demonstrated health beneficial effects
of these centuries-old herbal preparations, and more insight into mode of action and
mechanism of action using modern pharmacological tools will shed light on bene-
ficial effects of these herbs and foods.
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Challenges in Optimal Utilization of
Bioactive Molecules Clinically 5
Kotamballi N. Chidambara Murthy, M. Shivapriya, P. Monika, and
B. Tejashree
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
2 Alternation in Content of BAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
2.1 Development and Agriculture: Growth of Plant, Season, Postharvest Handling . . . . 114
3 Hurdles in Release of Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
3.1 Food-Food Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
3.2 Food-Drug Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.3 Types of Drug-Nutrient Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4 Models Used to Study Utilization of BAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5 Challenges in Optimizing Mode of Delivery of BAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Abstract
It is an established fact that the plant-based food is known to provide several
health benefits beyond basic nutrition to human. However, with increased
demand for food, excessive use of pesticides, chemicals, and hormones to
increase the yield and quantity has led to serious issues with use of plant-based
diet. Additionally, dependency on few major crops, use of convenient, and ready
K. N. Chidambara Murthy (*)

Central Research Laboratory, Ramaiah Medical College and Hospital, MSRIT Post, Bengaluru,
India
M. Shivapriya
College of Horticulture, University of Horticultural Sciences, GKVK, Bengaluru, India
P. Monika · B. Tejashree
Department of Biotechnology, Ramaiah Institute of Technology, MSRIT Post, Bengaluru, India

110 K. N. Chidambara Murthy et al.
to eat type of food have also lead to excess of energy and high consumption of
carbohydrates which resulted to unexpected increase in incidence of obesity,
diabetics, and other metabolic syndromes, globally. In view of such issues with
plant-based diet, recently a lot of emphasis is laid to the use of health promoting,
low calorie and mixed variety of plant-based diet, and to have more benefits.
Traditional indigenous and exotic plants are enriched with beneficial nutrients
and are sold in today’s market as functional food or nutraceuticals. Market value
for this class of consumer market is close to 200 billion $ and is expected to grow
at a rate of 7.5% compound annual growth rate. However, in this journey from
food to nutrition and nutraceuticals, some key components have been neglected,
which is critical to get optimum benefits. In this chapter, we would like to discuss
some of the key challenges in achieving optimum concept of health promoting
molecules from farm to fork. Often, we measure the content of these in plant
either after harvesting or during storage and based on which we assume the surety
of health benefits. Either due to lack of facility or collaborative efforts, we are not
considering critical factors like the right time for harvest of commodity, right
storage and processing technique, and right cooking and formulation protocol
along with selection of right base and ingredients for food. Some of these aspects
are discussed in detail along with problems in achieving the same. Additionally,
the chapter also provides necessary information on how to test and ascertain the
quality of nutrition and its delivery for optimal benefits.
Keywords
Bioactive molecules · Bioavailability · Bio-efficacy · Food-food interactions ·
Nutraceuticals
1 Introduction
Bioactive molecules (BAM) in plants: Why and how they exist? Every creature in this
universe has been provided with primary means of survival and existence. It is the
immune system in human and animals, toxins, or other chemicals in microbes and in
plants through secondary metabolites [1]. These secondary metabolites in addition to
protecting plants against pest and diseases and abiotic stresses will also serve as
metal transporting agents, as agents of symbiosis between microbes and plants, as
sexual hormones, and as differentiation effectors. They have a wider variety of
diverse chemical structure and biological functions that are species specific. In
addition to structural reinforcement of tissue and tree, some of the secondary
metabolites have special functions such as chemoattractants for pollinators, indica-
tion of ripening, indication of spoilage, etc.
Recent studies have also shown that plants have chemical and physical signal
molecules to communicate to each other and sense the environmental alterations [2,
3 ]. Therefore, it is evident that secondary metabolites act as primary defense system
in plants and protect them from both biotic and abiotic stresses. Since secondary
metabolites are produced in very low concentration, for commercial application of
5 Challenges in Optimal Utilization of Bioactive Molecules Clinically 111
potent components, production of secondary metabolites will be induced by means

of external factors like stress. Secondary metabolite content in most plants can be
enhanced by biotic and abiotic elicitation technique, which is most commonly used
in biotechnological route to producing plant metabolites. Some of the examples
include production of vincristine and vinblastine by C. roseus cell cultures devel-
oped from shake flasks to scale up through bioreactor [4]. Other phytochemicals
produced using such biotechnological route include medically important molecules
like Taxol, berberine, podophyllotoxin, scopolamine, and nutraceuticals like antho-
cyanin, betacyanin, gingseng, geraniol, etc. [4, 5].
Formation of secondary metabolites: All the secondary metabolites are usually
formed from conjugation and other reaction of primary metabolites, specifically
amino acids, carbohydrates, and proteins. Among the biochemical pathways,
shikimic acid, malonic acid, mevalonic acid, and methylerythritol phosphate
(MEP) pathways are responsible of formation of secondary carbon-containing mol-
ecules. It is through photosynthesis and carbon source CO2 compounds and through
different pathways that compounds such as phenolics, nitrogen containing com-
pounds, and terpenes are formed. For example, most of the terpenoids are formed
from basic unit IPP (isopentenyl diphosphate) and a basic unit C5H8, either from
mevalonic acid or methylerythritol phosphate (MEP) pathway from sequential
combination of three acetyl CoA units to form 3-hydroxy-3-methyl-glutaryl-CoA
(HMG-CoA) followed by mevalonic acid and finally isopentenyl diphosphate (IPP).
This is formed by dephosphorization in the case of mevalonate pathway. In the case
of MEP pathway, it is formed from combination of glyceraldehyde 3-phosphate and
pyruvate, through 1-deoxy- D-xylulose 5-phosphate and methylerythritol phosphate
(MEP). Similarly, most of the secondary metabolites are formed through established
pathways (Table 1). Some of the high-value compounds are produced in plants by
induction of stress, alteration in microenvironment, use of elicitors (biotic and
abiotic), and genetic manipulation through biotechnological tools. Table 2 shown
is the list of secondary metabolites produced through biotechnological routes.
Table 1 Formation of secondary metabolites in plant through different pathways and precursors
for the same
Initial
Class of secondary metabolite precursor Pathway
Purine alkaloids Histidine and
purine
Imidazole alkaloids Histidine
Terpenoids, indole alkaloids, quinoline alkaloids, Shikimate/ Shikimate pathway
quinazolidine alkaloids, acridine alkaloids Shikimic aid
Flavanoids, stilbenes, coumarins, tannins (condensed), Cinnamate, Phenylpropanoid
lignans chalcone pathways (PPP)
Polyketides, saturated fatty acids Acetyl CoA, Mevalonic acid
mevalonate pathways
Vitamin B1, B6, isoprenoid Glucose Deoxy xylulose
pathway
Table 2 Secondary metabolites and bioactive compounds produced through biotechnological

route
Name of the
metabolite Route of production Application Reference
β-Carboline Suspension culture Administration of BCA is [6, 7]
alkaloids currently available
experimental model to study
essential tremor
Gallotannins Root culture Prevention of gushing in beer [8]
and carbonated beverages
Withaferin A Shoot culture To study intermediate filament [9]
proteins
Berberine Root culture Antibacterial alkaloid [10]
Vanillin Genetically modified Flavoring agent [11]
organism(Pycnoporus
cinnabarinus)
Catechin Hairy root culture Antibacterial agent [12]
Shikonin from Stirred tank bioreactor Inhibit cancer cell glycolysis [13]
Lithospermum
erythrorhizon
Vinblastine Air lift reactor Chemotherapeutic agent [14]
from
Catharanthus
roseus
Capsaicin from Immobilized cell reactor + Digestive stimulant and for [15]
Capsicum precursor isocapric acid treatment of rheumatic
disorders
Diosgenin from Suspension cell culture Precursor for medicinal [16]
Dioscorea steroids
doryophora
Terpenoids Metabolic engineering of Flavor, fragrances, and [17]
mevalonate pathway cosmetics
Lycopene Metabolic engineering of Antioxidant, antitumor agent [18]
non-mevalonate isopentenyl
synthesis pathway
Chemically, three major groups of secondary metabolites are produced in plants,

namely, terpenes, phenolics, and N- and S-containing compounds. Terpenes com-
posed of 5-C isopentanoid units are toxins and feeding deterrents to many herbi-
vores. Phenolics synthesized primarily from products of the shikimic acid pathway
have several important defensive roles in the plants as well as in humans. Members
of the third major group, i.e., N- and S-containing compounds, are synthesized
principally from common amino acids, and these have several biological properties
in both plant and human [19].
Secondary metabolites role in plant: Unlike primary metabolite’s presence,
distribution of secondary metabolites within the species and plant part is very
specific. They perform several functions in plant which includes protection of
plant against stress, pathogens, and interaction between plants and other organisms
[20]. Defense is the primary role of these metabolites in plants, which is executed in
multiple ways which includes being toxic to organisms (like pathogenic microbes,
insects, and animals), formation of barrier for pathogens, production of enzymes that
degrade pathogens, and acquiring immunity after first infection. Antimicrobial
compounds which are released whenever there is an interaction from microbes are
termed as phytoanticipins; these will be released from constitutively stored pre-
cursors during microbe attack [20]. Some of the compounds in this category are
saponins, glucosinolates, cyanogenic glucosides, and benzoxazinone glucosides.
2 Alternation in Content of BAM
Bioactive molecules are meant for protection of plant under various stress and
diseased conditions. Use of these for human health is one of the ancient systems
of medicine which originated in Indian and other South Asian countries centuries
back. The same has got great significance in the last few decades, due to established
benefit in several diseases including chronic diseases. Although benefits from plant-
based formulation is attributed to combined effect of multiple constituents, each of
the plant is known to have a specific molecule known to be key ingredient for
activity, and often they are termed as marker compound or active principles. As these
are naturally produced or accumulated in plants, their concentration and distribution
depend on several factors related to both plant and environment. These factors
include processing of plant materials as food or formulation along with genetic
factors of consumer and other factors including the bioavailability and bio-efficacy.
Factors influencing the efficacy of bioactive molecules are summarized in Fig. 1.
Optimal utilization of bioactive molecules (BAM)
Plant relate Handling & Processing Consumer or Human
Plant variety & species Form of Raw, Semi- Health condition

consumption Cooked & Cooked
Agriculture practice Disease or GI Tract, Liver
Water, Lipid &
Base of Food Mixed base
Soil type/ base Mutation in metabolic
genes
Part of the plant Ingredients
used in food Physiological
Season of cultivation & conditions of organs
Collection Food –Food
interaction
Stress factors Psychology of Patient
Food –Drug
interaction
Fig. 1 Various factors affecting optimal utilization of bioactive molecules (BAMs) from food
2.1 Development and Agriculture: Growth of Plant, Season,

Postharvest Handling
Accumulation of secondary metabolites at different stages of growth depends on

both intrinsic and extrinsic factors. Accumulation of phenolic compounds increases
with development, and the highest accumulation is observed during flowering stage.
In the case of Hypericum perforatum L., increased content of naphthodianthrones at
higher temperature and light intensity has been reported [21]. In the case of model
plant Arabidopsis thaliana, it was observed that both content and composition of
glucosinolate changed during different stages of development. Dormant and germi-
nating seeds had the highest concentration (2.5–3.3% by dry weight) of
glucosinolates, followed by inflorescences, siliques (fruits), leaves, and roots. Con-
tent of aliphatic glucosinolates predominated in most organs; indole glucosinolates
make up nearly half of the total composition in roots and late-stage rosette leaves.
Seeds possessed much higher concentrations of several types of aliphatic
glucosinolates than other organs. Content of methylthioalkyl and hydroxyalkyl
glucosinolates and compounds with benzoate esters was high in seed compared to
other organs. Based on the age, older leaves had lower glucosinolate concentrations
than younger leaves indicating maturity will enhance the secondary metabolites.
Content of phytochemicals declined during seed germination and leaf senescence
[22]. These results indicate that depending on role of secondary metabolites, accu-
mulation varies with growth and quantity of accumulation will not be same in
different parts. Therefore, selection of plant part and stage of development is critical
to get optimum content for preparation of herb-based products.
Another important aspect of phytochemical variation is seasonal changes, as
mentioned above; accumulation of secondary metabolites is greatly influenced by
light, temperature, and length of day and night. These aspects are well established
using model plants in laboratory studies; varied content of anthocyanin in grapes in
different season and climate has been reported [23]. Accumulation of antimicrobial
compounds in bulb and leaf extracts of Tulbaghia violacea, Hypoxis hemerocallidea,
Drimia robusta, and Merwilla plumbea in different season has been reported by [24].
Results of the study suggest that accumulation of antimicrobial compound was
highest in winter and autumn seasons. Total phenolics were found to be highest in
spring compared to other seasons. Content of condensed tannin, gallotannin, and
flavonoid levels, depending on the sample, was either higher in spring or winter
except for H. hemerocallidea (corm) which had higher gallotannin levels in autumn.
Total saponin content was highest in winter in all the plant studies [24]. Comparison
of phytochemicals composition in different parts of commercial broccoli cultivars in
spring and fall provided interesting findings in content of glucosinolates, vitamin C,
total phenol, and total flavonoid contents and antioxidant activity. The highest total
glucosinolate content was found in the florets of plants grown in both seasons.
Phenols and flavonoids were highest in leaves, while vitamin C was highest in
stems, suggesting that broccoli leaves and stems may be good sources of phyto-
chemicals. All phytochemical contents were generally higher in florets in the spring
than in the fall but were higher in leaves and stems during the fall than the spring
[25]. These results indicate that health benefits we get from the plant-based diet have
some relation with season. This could be the reason for practice of seasonal plant-
based diet in many cultures.
Similar to environment, nutrients supplemented to plants will also have great
influence on the accumulation of secondary metabolites. Content of asiaticoside,
asiatic acid, and quercetin 3-O-glucuronide was different in three cultivars of
Centella asiatica grown under different soil nutrition condition. Salt stress is
known to increase several secondary metabolites in plants; this includes sorbitol
(Lycopersicon esculentum), flavonoids (Hordeum vulgare), jasmonic acid
(Lycopersicon esculentum), tropane alkaloids (Datura innoxia), anthocyanins
(Grevillea spec.), trigonelline (Glycine max), and glycine betaine (Triticum
aestivum). Similarly draught stress which sometime occurs naturally is known to
increase the accumulation of morphine alkaloids, glycosides, epicatechins, rutine,
flavonoids, and anthocyanins, which are well known for the biological activity in
human [26]. Under saline stress two cultivars of broccoli, namely, “Naxos” and
“Parthenon” have shown accumulation of the highest phenolics [27]. Several plants
have demonstrated varied accumulation of secondary metabolites when cultivated
under different light and temperature, indicating direct effect from altered photosyn-
thesis and several biosynthetic pathways. Accumulations of lycopene in tomato [28],
carotenoids in Brassicaceae [29], and flavonoids and terpenoids in citrus [30] are
some of the examples. The same approach is used in the case of plant cell culture to
produce high-value secondary metabolites in reactors. These constitute the plant-
and environment-associated factors that influence the accumulation of secondary
metabolites.
Postharvest-associated factors also play major role in deciding the content of
secondary metabolites in food ingredients. This is very important as most of the food
ingredients will be stored (transported) under different conditions after harvest
before consumed. This includes storage by producers, transportation, storage in
market place, and household storage (refrigeration and room temperature). The
time between the harvest and consumption of fruits and vegetables will be several
weeks and the same is few weeks to months in the case of cereals and pulses. Hence,
it is important to evaluate and understand changes in phytochemical content during
postharvest handling and storage, which will have great impact on health beneficial
activity of ingredients. Modified atmospheric packaging plays an important role in
preserving the phytochemical content during postharvest and storage [31]. The level
of phytochemicals present in fruit and vegetables may vary within and across
cultivars. Available literature correlates the level of phytochemicals with many
factors including cultivar type, environmental and agronomic conditions, harvest
and food processing operations, and storage factors [32]. Postharvest storage of the
vegetables and fruits in cold rooms increases the stress and upregulates the antiox-
idant pathway and antioxidant production; phytochemical content and also the
weight of the vegetable commodities were observed to be significantly different
from room temperature-stored vegetables/fruits and cold room-stored vegetables/
plants [33]. Total phenolic content gets declined during curing and storage of fruits/
vegetables due to anthocyanin degradation; anti-inflammatory factors and
antioxidant activity are reduced during the course of storage [34]. Further, studies
also indicated that phytochemicals in fruits such as anthocyanins, phenolics levels
changed during ripening and senescence [35]. The optimization of food processing
and storage factors is an essential step to reduce the degradation of phytochemicals
for potential health benefits.
Processing: Each of the plant food ingredients is subjected to one or the other
processing before consumption. This may be cutting or size reduction, germination,
drying in the case of those consumed fresh, steaming, boiling, frying, or cooking.
These physical processes are known to greatly influence the fate of phytochemicals.
One of the studies has shown variation in content of phenolic acids, isoflavones,
flavones, and glucosinolates when analysis was done using fresh and fresh-cut samples
including tomato, carrot, grape, eggplant, and broccoli. Content of phytochemicals
reduced in cut tomato compared to fresh and similar results was observed in the case of
eggplant. In other vegetables there was no significant variation between fresh and cut
samples. Content of ascorbic acid, carotenoids, anthocyanins, and volatile principles
(monoterpenes) decreases when fruit/vegetable is cut in to pieces and stored for a long
time before use, irrespective of temperature as these are sensitive to light, temperature,
and air [36]. Impact of different processing techniques on biological activity and
phytochemicals composition is as shown in Table 3.
Three major medicinal plants, namely, Ocimum basilicum, Senna petersiana, and
Hypoxis hemerocallidea have shown higher antimicrobial activity in fresh form
compared to stored samples suggesting loss in either content or biological activity
[60]. Another study has shown that heating and storage can greatly influence the
phenolic content and biological (antioxidant) activity of blue berry extracts [61].
Cooking or formulation: This is another key factor in deciding quality and
quantity of phytochemical content in food we finally consume. There are case-
specific issues concerned with cooking, which essentially has three major processes,
namely, shallow frying, deep frying, semi-boiling and pressure or complete cooking.
In all these processes, using temperature-sensitive ingredients like ascorbic acid,
some vitamins and enzymes are deactivated. On the other hand, cooking will also
help in percolation of active compounds from peelings to edible parts as seen in the
case of potato, cashew, and other crops. Content of glucosinolate and sulforaphane is
greatly reduced in broccoli florets after cooking, and therefore it is recommended to
be consumed raw for optimum health benefits [62]. In addition, cooking will also
lead to alteration in chemical nature of phytochemicals like breakdown (pigments),
complexation (glucosides), and polymerization (polyphenols). It is also important to
consider the biological effects of converted compounds, which may be useful or
sometimes harmful (formation of acrylamide is most common example).
3 Hurdles in Release of Bioactive Compounds
In addition to plant-related factors, other factors, which are known to limit or control
the bioaccessibility of compounds, are complex nature of plant materials, food
matrix, and other factors related to cooking and formulation of food [63]. These
Table 3 Impact of processing on component of food ingredients and biological benefits

Impact of processing
Sl. Plant Bioactive Processing (+) or ( ) on
No source/s molecule/s method/s used bioactive molecule/s Reference
1 Oranges Phenolics High temperatures Affects the content of [37]
and grape or freezing some polyphenols,
fruits including flavonoids,
due to the
extractability
alteration by the
rupture of the cell
wall
2 Kale Phenolics Peeling, washing, Increase in levels of [38]
And red chopping, boiling, phenolics, as well as
cabbage frying, and baking antioxidant activity
(traditional oven or after cooking
microwave)
3 White Phenolics Boiling, steam - boiling results in a [39]
cauliflower techniques and stir decrease of the total
frying phenolic content
- steam techniques
and stir frying
promoted an increase
of these compounds
4 Tomatoes Phenolics Boiling, baking, Significant reduction [40]
and frying in total phenolic
content
5 Kale Phenolics Steam cooking Higher contents of [38]
And red bioactive compounds
cabbage and higher
antioxidant activity
6 Potato Anthocyanin Microwave Preserves or causes a [41]
“cultivar” cooking, steamed little increase in the
(Solanum pressure cooking, anthocyanin content
tuberosum boiling, and baking
L.) in a hot air oven
7 Catechin Heating Reduction in [42]
bioactive compound
by epimerization
8 Blueberry Anthocyanin Blanching Increase in the [43]
fruits retention of
Anthocyanin levels
(23% instead of
12%) and the total
anthocyanin content
of juice from
blanched blueberry is
twice the
nonblanched one
(continued)
Table 3 (continued)
9 Fruits and Catechin, Blanching Affects color, flavor, [44]
vegetables quercetin, texture, and loss of
and its the nutritional and
glycosides functional quality by
promoting the
oxidation of phenolic
compounds
10 Blackberry Phenolics Pasteurization Phenolics is lost [45]
11 Mushroom Phenolics Sanitizing using Disappearance of [46]
sodium phenolics and the
hypochlorite formation of their
oxidation products
12 Red onion Flavanols Sanitizing using Losses of flavonols [47]
slices and sodium (23%) and
anthocyanins hypochlorite anthocyanins (13%)
due to leaching.
13 Pineapple, Phenolic Sanitizing using Significant increase [48]
banana, content and ozone (O3) in total phenolics
And guava flavanoids Whereas in bananas
and pineapples, the
flavonoid content
increased in response
to ozone treatment
For guava fruits, the
flavonoid content
increased and total
phenolic decreased
inversely
14 Campbell Phenolics, Microwave - microwave heating [49]
grapes flavonoids, heating, and ultrasonication
and ultrasonication and increased bioactive
anthocyanin blanching contents in non-cold
stabilized grape juice
than in cold
stabilized juices
- blanching
decreased bioactive
compounds both in
non-cold stabilized
and cold stabilized
juice
15 Pumpkins Phenols and High pressure No differences in the [50]
carotenoids processing total bioactive
(lutein, molecule contents
a-carotene,
and b-
carotene)
(continued)
Table 3 (continued)
16 Red bell Vitamin C Pulsed electric field Minimal loss of [51]
Peppers treatment vitamin C due to
improved mass
transfer during
drying
17 Beetroot Betalain Pulsed electric field 90% recovery of [52]
tubers treatment metabolite
(B.
vulgaris)
18 Oil Rapeseed Pulsed electric field Increased content of [53]
tocopherol treatment polyphenols and
Polyphenol tocopherols
19 Carrot β-Carotene Boiling Increase in [54]
bioavailability
20 Kiwifruit Lutein, neo- Pasteurization by No effect on [55]
puree lutein, Conventional heat bioaccessibility
β-Carotene,
Neoxanthin,
Violaxanthin
21 Milk- and α- High pressure No effect (α- [56]
soy-based Tocopherol, processing tocopherol)
Fruit γ- Decrease (γ- and δ-
Beverages Tocopherol, tocopherol)
and δ-
Tocopherol
22 Orange Ascorbic Pasteurization Ascorbic acid, total [57]
juice acid, flavanones, total
phenolics, carotenoids, and
flavanones, provitamin A values
and of pasteurized orange
Carotenoids beverage were lower
than those of
fermented juice.
Total phenolic
remained unchanged
and was similar to
that of original juice
23 Fruits and Anthocyanin Thermal Anthocyanin [58]
vegetables Processing pigments readily
degrade during
thermal processing
which can have a
dramatic impact on
color quality and
may also affect
nutritional properties
(continued)
Table 3 (continued)
24 Orange Vitamin C, High Losses in total [59]
juice carotenoids, pasteurization vitamin C
and were < 9%,
flavanones treatments with the
higher temperatures
tended to show the
higher decrease in
the content of both
forms of vitamin C.
Whereas increase in
naringenin and
hesperetin
are intermediate to plant- and human-related factors, and major ones include release
of bioactive molecules from food matrix, nature of BAM from source, food-food
interaction, and food-drug interactions.
Release of bioactive molecules from food matrix: Bioactive molecules are found
in plants, fruits, and vegetables and include heterogeneous group of molecules such
as polyphenols, carotenoids, tocopherols, phytosterols, and organosulfur compounds
[63] with different chemical structures (hydrophilic, hydrophobic). These com-
pounds vary in their distribution in nature (specific to particular vegetable or
ubiquitous), concentration in food and human body, site and mode of action,
efficiency against oxidative species, and their specificity.
There are many clinical evidences indicating the link between the chronic diseases
such as cancer and cardiovascular diseases with diet, especially with plant-based food
[64, 65]. Although the composition of the nutrients and other bioactive compounds in
food will be mentioned on the labels printed on the package, the bioavailability of the
bioactive molecules in human body differs. It is dependent on various factors, which
include chemical nature of the nutrients, their release from food matrix, interaction
with other food components, presence of suppressors and cofactors, formation of stable
components from which nutrients are released, and so on. Recent evidence also
suggests that the natural matrix of food and also the microstructure of processed
food influence the bioavailability of bioactive molecules in vivo [66].
Food composition database (FCD) provides the information regarding the amount
of energy, proteins, fats, vitamins, minerals, and other nutrients present in food. FCD
provides the information which is the based on certain chemical assays performed. In
the case of complex food, it is based on the nutrient composition of ingredients.
FCDs have been methodically compiled over the years in many countries and
provide information about the nutrients contained in most consumed foods [67].
FCDs are generally used to assess the nutrient content of diets and to derive nutrition
guidelines. They are also often utilized in food policy recommendations and nutri-
tion monitoring.
The FDA has defined bioavailability as the rate and extent to which the active
substances or therapeutic moieties contained in a drug are absorbed and become
available at the site of action [54]. This definition has to be also applied for the
bioactive molecules present in food. Book on Indian nutrition that is known as
Indian Food composition Table published by the National Institute of Nutrition,
Hyderabad, on January 2017 provides extensive details on most of the food ingre-
dients used in different regions of India.
Another term that is commonly used is bioaccessibility, which is defined as the
amount of an ingested nutrient that is available for absorption in the gut after
digestion [68]. If the amount of recovered nutrient is of relevance, then
bioaccessibility term comes into use. However, bioavailability is measured in
blood/plasma in vivo, and it will be affected by several factors such as physiological
state, dosage, etc.
After consumption, the nutrients that are present in a food or drink will be
released, absorbed into the bloodstream, and circulated in the body, and also few
of the times, it is transported to their target tissues. Nutrients differ in their bioavail-
ability, which is mainly decided by their solubility and affinity for binding with
proteins and biological molecules either in blood or in different organs. Release of
the nutrient from the food matrix, effect of digestive enzymes, binding and uptake by
the intestinal mucosa, transfer across the gut wall to the blood or lymphatic circu-
lation, systemic distribution and deposition, metabolic and functional use, and
excretion can affect nutrient bioavailability. It is mediated by external (characteris-
tics of the food matrix, chemical form of the nutrient, etc.) and consumer internal
(gender, age, nutrient status, and life stage) factors. The bioavailability of macronu-
trients (carbohydrates, proteins, and fats) is usually very high, e.g., more than 90% of
the amount ingested will get absorbed and utilized.
Bioaccessibility is the first step to make a nutrient bioavailable, which mainly
depends on availability of nutrients for absorption. During this step, the nutrient is
released from the food matrix and converted into a chemical form that can bind to
and enter the gut cells or pass between them. Chewing, enzymatic digestion of the
food in the mouth, mixing with acid and enzymes in the gastric juice, and releasing
into the small intestine are the unit operations of the process by which the nutrients
are rendered bio-accessible. The small intestine is the major site of nutrient
absorption. Enzymes of the pancreatic juice continue breaking down the food
matrix. Certain procedures involved in food preparation like cooking, chopping,
or pureeing collaborate with mastication and enzymes to the digestibility of food
matrices.
The oral bioavailability is limited due to the restricted release of the components
from plant matrix, solubility of nutritional molecules in gastrointestinal fluid, the
permeability across intestinal epithelial cells, as well as the enzymatic and chemical
reactions occurring within the gastrointestinal tract. Following four essential steps
are necessary for the effective absorption of bioactive compounds:
1. Release from food matrix

2. Incorporation into bile-salt micelles
3. Absorption by epithelial cells

4. Incorporation into the chylomicrons with secretion into lymphatic system
As mentioned in previous section, processing of food also has an impact on the

chemical constituents, physical and sensory properties of the final product. In
addition to cooking, industrial food processing technologies may influence the
content of bioactive compounds leading to changes in their functional properties
(e.g., bioavailability, bioaccessibility, and bioactivity) and potential health benefits.
Loss of phytonutrients in foods becomes more significant as food is processed,
stored, packaged, and transported. Some of the processes like frying, puffing,
semi-cooking, and steaming are known to increase the bioavailability of compounds
which are less sensitive to heat. Similarly, processing such as grinding, fermentation,
and/or mild heating may improve bioavailability, most likely as a result of disruption
of the cell walls of plant tissues, dissociation the nutrient-matrix complexes, or
transformation into more active molecular structure. Therefore, attention should be
given to the degree of disintegration of the initial tissue structure because of its
impact on food quality, functionality, and deterioration. As the demand for func-
tional food increases, intense research efforts for the development of new processing
technologies is conducted with the goal of ensuring maximal nutritional and func-
tional properties, as well as the overall quality of a product.
The bioavailability of nutrients and bioactive compounds present in plant prod-
ucts (fruits and vegetables) is presently an extremely important area of food and
nutrition research. The objective of these studies is to determine the contribution of
actual foods as sources of specific nutrients (e.g., antioxidants) and to establish
processing conditions that maximize the health benefits. Although several photo-
chemicals are known to be important in promoting human health, the bioavailability
of these bioactive molecules will vary with respect to their microstructure. For
example, food microstructure seems to be quite relevant in the bioavailability of
several antioxidants. Some of the phytochemicals for which bioavailability has been
established and extensively researched includes carotenoids, polyphenols, and
folates.
Carotenoids are a family of fat-soluble plant pigments that provide red and
orange colors to fruits and vegetables. Their function is to absorb light in photosyn-
thesis, protecting plants against photosensitization. Dietary carotenoids are consid-
ered to be beneficial in the prevention of a variety of diseases, including certain
cancers and eye disorders. The 5 principal carotenoids found in human plasma as a
result of ingestion of plants are α-carotene, β-carotene, cryptoxanthin, lutein, and
lycopene, but over 600 carotenoids have been identified to date in plant and marine
organisms. Our research has demonstrated both radical scavenging and
hepatoprotective activity of carotenoid-rich marine microalgae [69, 70]. Carotenoids
present in a wide variety of plants are partially concentrated in chromoplasts or
chloroplasts in different ways. The extent of release from the food matrix is highly
variable depending on whether carotenoids are non-covalently bound to protein or
fiber or not. In general, release of carotenoids from plant foods occurs only when the
cells in the food matrix are disrupted, as is usually the case during food preparation,
processing, and/or mastication, but not during digestion, at least in the ileum of
humans [71]. One of the studies suggests that size reduction in the case of carrot
(pureed fresh carrot) has shown highest release of carotenoids and its isomers
compared to raw or boiled carrot [72].
Lycopene, a carotenoid responsible for the distinctive red color of ripe tomatoes,
is usually located within cell membranes, and its release is determinant on the
bioavailability. Epidemiological studies have suggested that consumption of lyco-
pene may protect against CVD and reduce the risk of several types of cancer, most
notably those of the prostate, breast, lung, and digestive tract [73]. Food processing
like cooking or heating may improve lycopene bioavailability by breaking down cell
walls, which weakens the bonding forces between lycopene and the tissue matrix,
thus making it more accessible. One of the studies indicate that bioavailability of
lycopene is higher in tomato paste compared to consumption of fresh one [74].
Another clinical study suggests that severely homogenized (blending for 2.5 min
using the same blender, followed by processing in a high-pressure homogenizer at
200 bar) tomato had shown the highest amount of lycopene and carotenoid content
in serum along with increased antioxidant activity. This was in comparison with
treatment of mild homogenization (blending for 2 min) and raw tomatoes to volun-
teers in food [75]. Clinical study has also shown that lycopene is more bioavailable
in its cis form from tangerine compared to all trans form as in the case of tomato
juice, which is more of chemical factor [76]. These results indicate that along with
physical, chemical factors also plays critical role in deciding bioavailability of
lycopene and other phytochemicals.
Xanthophylls are yellow pigments found in plants and are known for various
biological effects, which includes lutein, zeaxanthin, capsanthin, canthaxanthin,
astaxanthin, echionine, and β-cryptoxanthin. These are oxygenated carotenoids
that are synthesized within the plastids. The xanthophylls lutein and zeaxanthin
accumulate in the eye lens and macular region of the retina and have been implicated
in helping to protect the eye against oxidative damage and cataracts. For optimal
absorption of xanthophylls, they must be released from their food matrix and then
transferred to lipid micelles in the small intestine. This requires the presence of
dietary fat in the small intestine, which stimulates the gallbladder to release bile acids
(i.e., emulsifiers). Therefore, fat or fat base is important for all nonpolar phytochem-
icals including fat-soluble vitamins.
Polyphenols represent a wide variety of compounds belonging to several classes,
for example, hydroxybenzoic and hydroxycinnamic acids, anthocyanins, pro-
anthocyanidins, flavonols, flavones, flavanols, flavanones, isoflavones, stilbenes,
and lignans. These are ubiquitously found in all the plants used in diet and nutritional
supplements. As mentioned before, phenolic compounds (or polyphenols) are sec-
ondary metabolites of the pentosephosphate, shikimate, and phenylpropanoid path-
ways in plants. Reported bioavailability of polyphenols is highly variable depending
on their structure and conjugation. Among polyphenols, phenolic acids account for
about one third of the total intake, and flavonoids account for the remaining two
thirds. The most abundant flavonoids in the diet are flavanols (catechins plus
proanthocyanidins), anthocyanins, and their oxidation products. Chemical structure
is the primary determinant of bioavailability of polyphenols. Studies in human have

shown that excretion of polyphenols through urine varies from 0.5 to more than 50%
depending the source and structure. Over 27% of caffeic acid was detected in urine
after administration of 1000 mg, 24% in the case of hesperidin, 19.8% in the case of
genistein from soy milk, and 6–8% of naringin from grapefruit. Most of the poly-
phenols takes 1–2 h to reach peak plasma concentration, except those absorbed after
degradation. Based on the elimination rate and half-life of polyphenols, it is essential
for repeated administration or consumption of sources preferably once in 24 h [77].
Folates and folic acid are forms of vitamin B that are necessary for the production
and maintenance of new cells, especially important during periods of rapid cell
division and growth, such as throughout infancy and pregnancy. Inadequate folate
intake has been associated with development of birth defects [78]. Leafy green
vegetables such as spinach and turnip greens, dry beans and peas, and some other
fruits and vegetables are rich natural sources of folate in nature; folates are cova-
lently bound to macromolecules. Bioavailability of folate from food source mainly
depends on factors like nature of food matrix, extent of deconjugation of poly-
glutamyl folates in the intestine, and stability of certain folates during digestion and
in presence of other dietary constituents which may either increase or reduce the
stability. Apart from this, in the case of fortified supplements, stability of folate used
is also a major concern toward optimum utilization. McNulty and Pentieva from the
University of Ulster, UK, have discussed details of these factors along with exper-
imental details in estimating folate bioavailability and bio-efficacy in acute and
chronic model [79]. They have concluded that chronic studies on food supplemen-
tation can give better understanding on the bioavailability of folate, and this needs to
be conducted using robust and highly controlled experiments which can ensure
controlled source and delivery of folate equivalent to folic acid. It was also pointed
that stability of folate and its analogues in plant sources (green leafy vegetables) after
cooking is another major limiting factor.
3.1 Food-Food Interactions
Humans consume verity of food to meet daily needs of energy depending on the
ethnicity and region. Additionally different types of foods are consumed on different
occasions, which generally consist of several verities made out of multiple ingredi-
ents originating from either plant, animal, or microbial source and several chemicals;
processed or semi-processed ingredients are also used in the food preparation. As
each of the ingredients will have multiple phytochemicals, there will be a good
chance of interaction between two or more molecules resulting in useful or delete-
rious outcome. The classical example of useful interaction is enhancement in iron
bioavailability in the presence of ascorbic acid or vitamin C. Conversely, interfer-
ence of phytic acid in absorption of essential minerals and nutrition is the established
example for negative outcome.
Food supplements (e.g., nutrients, vitamins, hormones, amino acids, antioxi-
dants) and functional food (e.g., phytosterols or omega-3 fatty acids enriched
food) occupy a position between food and drugs. Botanical materials represent a
large segment of this class of products (e.g., tulsi, Withania, soy isoflavones, yam, or
hop extracts). In addition to food-food interactions, the interaction between phyto-
chemicals of food and toxic contaminants is another less studied concept. A team of
toxicologists made an attempt to study this concept in order to understand the
interactions which can lead to additive or subtractive or even synergistic effects,
under a project named FOODINTER in Belgium. The communication between
scientists and stakeholders (authorities, producers, and consumers) plays an impor-
tant role in this particular regard. This will help to ensure the safety from food-food
interaction and food-contaminants interactions, which accidentally get in to food
during storage of food ingredients and processing or preparation of food. Therefore,
it is necessary to conduct risk assessment of chemicals, natural compounds, and
environmental contaminants present in food supplements which could interact
between them or with micro- or macronutrients of normal human diet.
Interaction studies have been performed using existing in vitro models (based on
culture of various cell types, prokaryotes and eukaryotes) with mixture of active
substances at different concentrations. The project selected Ginkgo biloba, St John’s
wort, maca, black radish, garlic, and soy isoflavones, which are most commonly
used as health supplements to understand the interactions. They studied several key
phytochemicals from each of these commodities along with seventeen trace elements
As, Ba, Bi, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Sr, Ti, Tl, Zn, and Hg, over 23
mycotoxins, more than 15 PHAs, organochlorine pesticides (OCPs), polychlorinated
biphenyls (PCBs), and of polybrominated diphenyl ethers (PBDEs), and dioxins
were also analyzed in the selected samples. Evaluation parameters like cytotoxicity,
inhibition of metabolizing enzymes, and results provided very specific information
regarding various contaminants that needs to be considered for limitation in all the
products containing selected plants/extracts derived from them. There is a need to
create such database on all the major food ingredients used globally. Parameters for
analysis and study interactions should be based on the location of cultivation,
agriculture and postharvest practices, processing technique used, and method of
food preparation [80].
3.2 Food-Drug Interactions
Food-drug interaction is defined as the alteration in the pharmacodynamic, pharma-

cokinetic properties of the drug due to the interaction with the nutrients present in the
food or change in the nutritional composition in of food (in vivo) due to its
interaction with drug. Food-drug interactions can result in two main clinical effects:
either a decreased bioavailability of a drug, which predisposes to treatment failure, or
an increased bioavailability, which increases the risk of adverse events and may even
precipitate toxicities [81]. For example, grapefruit juice is a selective intestinal
CYP3A4 inhibitor, cytochrome P-450 3A4, in the small intestine, resulting in a
significant reduction of drug pre-systemic metabolism [82]. The overall exposure of
some drugs can be increased by more than fivefold when consumed with grapefruit
juice and increases the risk of adverse effects. The use of certain drugs may affect GI
tract function and may lead to a loss of bodily electrolytes and fluids. People in risk
due to the food-drug interactions are usually elderly people, cancer patients, and
people with malnutrition, who are more prone for mutation in related to metabolism.
However, elderly people are exposed to the highest risk as 30% of the prescribed
drugs are consumed by them due to the decrease in physiological function with age
[83]. Pharmacokinetic particularities associated with aging are absorption (changes
in gastric pH, decreased gastrointestinal blood flow), distribution (decreased lean
body mass, water, serum albumin concentration, and binding serum proteins), and
elimination (reduced renal function). Therefore, drug bioavailability, volume of
distribution, clearance, and half-life of drugs are modified with aging. Water-soluble
drugs become more concentrated, and fat-soluble drugs will have longer shelf life
due to slow release of the drugs from fat tissue. Food-drug interaction is a serious
issue in drugs with very narrow therapeutic window, like chemotherapeutic, immu-
nomodulators, and drugs acting on CNS.
3.3 Types of Drug-Nutrient Interactions
Based on nature and mechanisms, drug-nutrient interactions are classified into four
types:
Type I interactions are ex vivo bio-inactivation, which refer to interactions between

the drug and the nutritional element or formulation through biochemical or
physical reactions, such as hydrolysis, oxidation, neutralization, precipitation,
or complexation. They usually occur in the delivery device or outside the body.
Type II interactions affect absorption. They cause either an increase or decrease of
the oral bioavailability. The precipitant agents may modify the function of
enzymes (type A interactions) or transport mechanisms (type B interactions)
that are responsible for biotransformation. Complexation, binding, and/or other
deactivating processes occur in the gastrointestinal tract as a result of interaction
between drug/food ingredients (type C interaction) and reduce absorption.
Type III interactions affect the systemic or physiologic disposition and occur after
the drug or the nutritional element has been absorbed from the gastrointestinal
tract and entered the systemic circulation. Changes of the cellular or tissue
distribution, systemic transport, or penetration to specific organs or tissues can
occur.
Type IV interactions refer to the elimination or clearance of drugs or nutrients, which
may involve the antagonism, impairment, or modulation of renal and/or
enterohepatic elimination.
Predicting drug interactions during drug development is a challenge for pharma-

ceutical industries and regulatory agencies. Since the publication of the US Food and
Drug Administration’s (FDA’s) first in vitro and in vivo drug interaction guidance
documents in 1997 and 1999, researchers and clinicians have gained a better
understanding of drug interactions [84]. This knowledge has enabled the FDA and
the industry to progress and begin to overcome these challenges. The FDA has
continued its efforts to evaluate methodologies to study drug interactions and
communicate recommendations regarding the conduct of drug interaction studies,
particularly for CYP-based and transporter-based drug interactions, to the pharma-
ceutical industry. A drug interaction website was established to document the FDA’s
current understanding of drug interactions [http://www.fda.gov/cder/drug/
druginteractions/default.htm].
Host factors (includes genetic factors) include genetic and pathological factors
that are associated with adverse, toxic effect of selected food ingredients in individ-
uals. Pathological conditions associated with malabsorption mainly associated with
mucosal layer. This can be classified as premucosal, which includes impaired
digestion and bile acid/enzyme deficiencies; mucosal-associated factors like reduced
absorption, bowel resection, and diseases affecting absorption; postmucosal-associ-
ated factors including altered nutrient transport and vascular or lymphatic abnormal-
ities. Factors affecting the bioavaibality in human is also depicted in Fig. 2 for better
understanding. These conditions can be treated with altering the nutrition in terms of
the use of high protein diet, parenteral feeding and designer food with easily
digestible and absorbable nutrients [85]. Genetic factors include mutation in meta-
bolization genes such as P450 (CYP2D6), methylene tetrahydrofolate reductase
Fig. 2 Factors affecting/influencing bio-availability in human

gene, GST, N-Acetyl transferase, and so on [86]. Impact of metabolizing enzymes on

the activity and content of biologically active compounds are shown in Table 4.
4 Models Used to Study Utilization of BAM
There are several in vitro and in vivo models available to understand the utilization
of bioactive molecules from food ingredients. Prerequisite for bioavailability study is
that we need to identify one or two marker compounds and its metabolites for which
there should be very sensitive quantification or analytical method available. Flavo-
noids, polyphenols, terpenoids, and coumarins are some of the compounds exten-
sively subjected for bioavailability studies in various models.
Among in vitro models, digestion of samples using gastric and intestinal enzymes
at both gastric and intestinal pH is most widely used for both food and phytochem-
icals. It is commonly referred as “in vitro digestion model” [98]. Enzymes of
porcine, rabbit, or human origin are used for this study, and some models have
shown that the use of minor components such as bile salt, phospholipids, gastric
lipase, and emulsifier is also considered for better results [99]. Simulated salivary
fluid (SSF, pH 7.0), simulated gastric fluid (SGF, pH 3.0), and simulated intestinal
fluid (SIF, pH 7.0) are used in these experiments, which give good indication of
release of absorbable form of compound of interest. Use of artificial permeable
membrane (also referred as artificial immobilized membrane-AIM), which simulates
the permeability of intestinal lining is other model used commonly to estamale
bioavailability of natural molecules. This is prepared by covalently binding mem-
brane-forming lipids to an amorphous silica substrate which mimics both hydropho-
bic and hydrophilic nature of absorbing cells. Another model used is reconstituted or
isolated cell membrane, which is more useful to understand specific type of molec-
ular transport across the membrane. Compartment model is a commonly used model,
which has two or more compartments connected through semipermeable membrane
and movement of molecule from one chamber to another [100].
Common cell-based model used for bioavailability study is CaCo2 cell line,
which is a colon adenocarcinoma cell. This cell line is generated to represent
mucosal cells, which is primary site of absorption for GI tract. Primarily CaCo2
cells are used to understand the ability of nutrients to cross the intestinal barrier, as
well as to study their transport mechanisms. Advantage with this model is it is quick
and can provide good idea on permeation and absorption across the intestinal
membrane, and it does not require ethical clearance [101]. Other ex vivo models
include ligation of the intestine of animals like rat, rabbit, porcine, and pig. In this
model, the intestine is ligated on one end, and solution containing food is added
inside, and it is immersed in solution mimicking biological fluids [98, 100].
In situ model includes intestinal perfusion in animal, which is mainly done
through microsurgery. It this done by cannulation of desired region of the intestine
(proximal) after anesthetizing animals and collecting the sample flowing in
the intestine at distal end. In this model any region of the small and large intestine
can be studied [102]. In vivo animal and human models are the most realistic
Table 4 Major metabolizing enzymes known to interfere with bioactive molecules and their
effects (+) or ( ) on the bioactive molecules
Sl. Bioactive Effect (+) or ( ) on bioactive
No Metabolizing enzyme/s molecule/s molecule/s Reference
1 Transporter enzyme of Bioactive Reduces oral bioavailability [87]
intestinal or liver cells (e. molecules
g., P-glycoproteins
2 Clearance enzymes Bioactive Inhibitory potency against the [88]
molecules target
3 β-Glucuronidase, Resveratrol Significantly reduced activities [89]
β-glucosidase, β- of these fecal and host colonic
galactosidase, mucinase, mucosal enzymes compared to
and nitroreductase control animals (21%, 45%,
37%, 41%, and 26%,
respectively). The reduced
bacterial enzyme activity was
associated with a significant
reduction in colonic tumor
incidence in the resveratrol-fed
rats compared to control rats
4 COX-2, NF-κB, AP-1, Resveratrol Inhibition of enzymes [90]
TNF-α, IL6, and VEGF
(vascular endothelial
growth factor)
5 GSTT2 and COX-2 Quercetin Modulation of enzymes [91]
involved in detoxification and
inflammation in LT97 human
adenoma cells that could
contribute to the
chemopreventive potential of
polyphenols after degradation
in the gut
6 β -glucuronidase, β - Resveratrol Modulating activity of [92]
glucosidase, β - bacterial enzymes and
galactosidase, mucinase, inhibitory effect on DMH-
nitroreductase induced colon carcinogenesis
in rats
7 Cytochromes P450 Flavanoids Induce the expression of [93]
several CYPs and modulate
(inhibit or stimulate) their
metabolic activity. In addition,
some CYPs participate in
metabolism of flavonoids.
Flavonoids enhance activation
of carcinogens and/or
influence the metabolism of
drugs via induction of specific
CYPs
8 Lipoxygenase, Flavanoids Inhibition of key enzymes [94]
phospholipase, and involved in the prostaglandin
cyclooxygenase. biosynthesis
(continued)
Table 4 (continued)
Sl. Bioactive Effect (+) or ( ) on bioactive
No Metabolizing enzyme/s molecule/s molecule/s Reference
9 Kinases Flavanoids Inhibit these enzymes by [95]
competing with ATP for the
binding to the catalytic site.
These modes of inhibition
provide an explanation for a
molecular basis of flavonoid
anti-inflammatory effects
10 Cytochrome P450 Quercetin Inhibition of gene expression [96]
monooxygenases of CYP1 family enzymes
through blocking aryl
hydrocarbon receptor which
plays an important role in the
cancer chemopreventive
properties
11 Cytochrome P450 Kaempferol Prevents CYP1A1 gene [97]
monooxygenases transcription induced by
prototypical aryl hydrocarbon
receptor ligand
models, which involves repeated collection of blood and urine sample at regular
intervals to understand the concentration of free-circulating compounds. Based on
the loading concentration, both bound and organ absorbed concentrations can be
calculated [103].
5 Challenges in Optimizing Mode of Delivery of BAM
As explained in previous sections, phytochemicals or BAM is present in different

cellular compartments, which needs to be released from plant cell in to food matrix
from where it needs to be released to GI tract for absorption. In this journey, BAM
should be stable and withstand the conditions of postharvest, food processing and
finally resist gastric pH and microenvironment to successfully be available for
absorption. Some of the major challenges in this journey are as follows:
• Good agriculture practices to produce optimum BAM concentration.

• Best postharvest practices to protect or enhance the content of BAM.
• Best food processing methods to retain the quality and quantity of BAM.
• Minimizing the anti-nutritional or inhibitors of BAM activity through physical
(soaking, grinding, etc.) or biological (fermentation, germination, etc.) process.
• Avoiding the combination of ingredients or food, those are known to interfere
with either absorption or biological activity.
• Use of combination of ingredients or nutrients, which are known to synergize the
activity or absorption of each other.
• Finally, to understand the genetic variation (metabolizing, transporting, and cell

signaling genes) in individual who have problem in absorption and utilization of
specific BAM.
6 Summary
It is a well-established fact that plant-based food can provide more benefits beyond
primary nutrition to human. Often we give more importance to primary nutrition and
focus on providing food security through agriculture and food industry practices.
With increase in incidence of chronic disease, it is essential to provide more quality
food which can provide good content of phytochemicals to support key biological
functions like enhanced immunity, increase radicals scavenging ability, detoxify
xenobiotics, and avoid infections. To achieve this, it needs integral efforts of
agriculturist, food processing industry, food scientists, cook, and finally smart
consumer. Often, aspects related to safe and efficient delivery of BAM is neglected
due to lack of evidences, integration of experts and also complexity of processes
involved. Therefore, it is essential to integrate the interdisciplinary expertise to
achieve optimum outcome. This can greatly help in achieving good health through
nutrition and can greatly help to reduce the burden of chronic diseases globally.
Acknowledgment Authors would like to acknowledge the support of the management of Gokula
Education Foundation (medical) for their constant support and encouragement for research and also
would like to acknowledge the help of research collaborators Prof. Bhimanagouda S Patil and Dr. G.
K. Jayaprakasha, at VFIC, Texas A&M University for their support for research.
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Bioactive Food Components in the
Prevention of Cardiovascular Diseases 6
Arti Parihar and Mordhwaj S. Parihar
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2 Risk Factors of Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3 Diet and Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4 Types of Food Components Used for Prevention of Cardiovascular Disease . . . . . . . . . . . . . 142
5 Bioactive Food Components in the Prevention of Cardiovascular Disease . . . . . . . . . . . . . . . 142
5.1 Carotenoids (Lycopene) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
5.2 Phenolic Acids and Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5.3 Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Abstract
Cardiovascular diseases (CVDs) remain one of the leading causes of death
globally. The risk factors such as lipids, lipoproteins, and inflammation play a
critical role in CVD. Lifestyle factors directly influence the risk of CVD. Under-
standing of the risk factors and the disease-causing mechanisms will lead to novel
therapeutic treatments. Emerging data have explored the utility of natural food-
based strategies in the management of diseases. Increasing interest has been
grown up in recent years on the health-related products of food industry and to
understand how foods products can help and maintain the individual cardiovas-
cular health. Plant extracts rich in bioactive components could be used as the
A. Parihar (*)
Bellingham Technical College, Bellingham, WA, USA
M. S. Parihar
School of Studies in Zoology and Biotechnology, Vikram University, Ujjain, Madhya Pradesh,
India

138 A. Parihar and M. S. Parihar
functional ingredients for providing many health benefits. The recent advances in
health benefits of bioactive components provide novel therapeutic approaches,
which have played an important role in the reductions of CVD worldwide. This
chapter presents a critical review of the potential benefits of bioactive foods
consumed through diet to reduce the incidence of cardiovascular disease.
Keywords
Cardiovascular disease · Coronary heart disease · Atherogenesis · Dyslipidemia ·
Inflammation · Active ingredients · Bioactive components of foods
1 Introduction
Cardiovascular diseases (CVDs) cover a wide group of disorders that involve the
cardiac muscles and the vascular system. CVDs are regarded as the most common
human health problem throughout the world. The mortality rates differ considerably
from country to country with Japan and other Mediterranean countries having the
lowest rates [1]. CVD is the major cause of the death increasing day by day in
developed and developing countries [2, 3]. Heart failure is more common in countries
with lower socioeconomic status and with those who tend to adopt unhealthier
lifestyle, such as smoking and careless dietary habits [4–6]. CVD alone is responsible
for taking 17.7 million lives every year, and atherosclerosis has been recognized as the
prominent symptomatic anomaly responsible for CVD-related deaths [7]. The most
important CVDs includes coronary heart disease, cerebrovascular disease, peripheral
arterial disease, heart failure, rheumatic heart disease, congenital heart disease, deep
vein thrombosis, and pulmonary embolism. Heart attacks and strokes that usually
caused by a blockage of blood flow to the heart or brain are the most important acute
manifestations of CVD in human. The causes of this global epidemic have been
largely explained to originate from lifestyle factors which directly impact the novel
pathways of CVD risk [8]. The increased lipids in the blood and build-up of fatty
deposits on the inner walls have been strongly correlated with the increased incidence
of CVD. Usually, combinations of risk factors are involved in causing the heart attacks
and strokes. The most important factors associated with the incidence of CVD are lack
of physical activity, drinking alcohol, use of tobacco, unhealthy diet, obesity, hyper-
tension and hyperlipidemia. Diabetes mellitus is regarded as another epidemiological
factor associated with an increasing prevalence of CVD [9, 10].
Atherosclerosis, mainly located in the intima of middle sized and large arteries, is
the major cause of myocardial infarction, heart failure, and stroke. Dyslipidemias in
vascular endothelium [11] and cholesterol deposition are the major contributors of
atherosclerosis. Low-density lipoprotein (LDL) cholesterol when oxidized is pro-
inflammatory and immunogenic and acts as an independent risk factor for CVD. The
increase in oxidized LDL cholesterol results in endothelial dysfunction and directly
influences the development of atherosclerosis. The decline in high-density lipopro-
tein (HDL) cholesterol increases the susceptibility to atherosclerosis substantially
while the increase in the HDL cholesterol may reduce the incidence of coronary
6 Bioactive Food Components in the Prevention of Cardiovascular Diseases 139
heart disease (CHD) [12] and reduces the risk of CVD [13]. Appropriate levels of
HDL cholesterol may also be responsible for clearing oxidized LDL cholesterol
from the tissue by obstructing the monocytes attachment to the endothelium layer of
blood vessels. Maintaining the HDL cholesterol level may also initiate the nitric
oxide release that stops vascular bed atherogenesis [14]. Since the built up of
atherosclerosis lesions require a long period of time, therefore, initiation of early
lipid management may help prevention of atherosclerotic vascular diseases [15]. To
manage the lipid level in patients with dyslipidemia, alternatives therapies have been
designed in recent years [16]. Lifestyle modifications have been suggested as the
primary prevention strategy in managing cardiovascular risk whereas food supple-
ments were prescribed to patients to reduce the risk factors and symptomatic relief
from CVD. The natural foods obtained from medicinal plants have been tried that
might confer some benefit on some patient. However, more research is required that
may include clinical trials with long follow-up outcomes to conclude their effective-
ness against CVD illnesses.
The past few years have witnessed the extensive expansion of research on the
association between dietary food and CVD. Certain foods have been recognized
showing the effectiveness in reducing the risk of chronic diseases. These specific
foods and food components appear as therapeutic strategies in the reduction and
prevention of the risk of CVD. In recent years, a considerable importance has been
given to functional foods. Apart from their basic nutritional effects, the functional foods
exhibit an important role in disease prevention, or slowing the progression of many
chronic illnesses. These functional foods work on two basic principles either possess a
component with positive health benefits or remove a component with negative effects
on the body functions. The relationship between the nutritional value of food compo-
nents and the prevention of several chronic diseases led to increase in their demand in
the market. However, to sustain in the market these foods must be safe, healthy, and
delicious. They may be composed of a single compound that physiologically active or
may include the addition of other food components to make them functional including
omega fatty acids, prebiotics, phytochemicals, and bioactive peptides.
Bioactive food components are physiologically active constituents that are pre-
sent in minute quantities in plant products and lipid-rich foods [17]. They are being
extensively studied to evaluate their beneficial effects on health. These compounds
vary widely in chemical structure and function and are grouped accordingly. Scien-
tific evidence indicates that those certain bioactive food components participate in
the prevention of CVD [17, 18]. Oral supplements of bioactive food components
when taken along with the routine diet may increase the absorption of nutrients that
will have clinical benefit in some diseases. Usually, bioactive components of foods
are taken in addition to a healthy diet but they do not serve as the substitution to
conventional food [19]. Their consumption as part of basic nutrition exerts useful
physiologic effects in reducing the risk of diseases [20]. They may act at the different
metabolic pathways that control various metabolism including lipid disorders in
humans. By virtue of their targeted actions on various metabolic pathways, they are
believed to have the therapeutic advantages for reducing the risk of CVD by
combating the inflammation and dyslipidemias [21]. They have well-illustrative
beneficial biological effects such as antilipidemic, antihypertensive, antiglycemic,

antithrombotic, and antiatherogenic. With the perceptible health benefits of bioactive
components against various diseases, a wide array of the metabolomics and physi-
ological relevance of these compounds were established [22]. A recent study in the
United States, adults showed the associations of suboptimal intakes of dietary factors
with mortality due to CVDs [4]. These functional foods, apart from providing basic
nutritions, provide therapeutic benefits in the management of chronic diseases [23].
In this chapter, we thoroughly examined the beneficial effects of bioactive food
components in reducing the risk factors of CVD including hypertension,
dyslipidemias, oxidative stress, and inflammation.
2 Risk Factors of Cardiovascular Disease
A number of risk factors such as changes in lifestyle, age, physical inactivity, blood
pressure, smoking, alcohol, obesity, hyperlipidemia, and diabetes have been listed
for the pathogenesis of CVD [24–26]. Inflammation has been regarded as the
principal molecular mechanisms responsible for atherogenesis [27–29]. During
inflammation nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κ
B), a family of transcription factors that regulate varied processes in immune cells,
initiates encoding of a number of genes responsible for cytokines and chemokines
production [30]. The NF-κB signaling and cytokine secretion are found increased in
atherosclerosis [31]. The IkappaB kinase/NF-kappaB (IKK/NF-κB) signaling path-
way plays an important role in inflammation. In addition, this signaling pathway also
regulates many other biological functions, including growth and survival of cells.
These authors further elaborated that the activation of IKK/NF-κB pathway, which is
an important regulator of inflammation, in cardiovascular tissues produce an exces-
sive inflammatory response that causes cardiomyopathy leading to heart failure.
Oxidative stress is another risk factor affecting the cardiovascular tissue in many
ways. Oxidative stress can influence the functioning of endothelial layer of blood
vessels, thereby leading to cardiovascular disease [32]. Reactive oxygen species
(ROS) can have a direct effect on cardiac cells by oxidizing cellular constituents and
disrupting the functions of proteins and enzymes [33]. The increase in ROS during
the myocardial ischemia, hypoxia, and reoxygenation is the principal causes of
reperfusion injury in cardiac tissues. During reoxygenation, increase in ROS cause
direct oxidative damage to cellular components [34]. The obesity has been exten-
sively correlated with the incidence of CVD, particularly among women [35].
Studies have shown an important connection between the obesity and dyslipidemia
and the metabolic syndrome [36]. Dyslipidemia is mainly characterized by elevated
levels of LDL-cholesterol and decline in HDL-cholesterol [37]. Hypercholesterol-
emia with total cholesterol level above 190 mg/L is the major form of dislipidemia
and considered as one of the major risk factors for the CVD.
Hypertension is one of the major risk factors for cardiovascular-related illness. It
is believed that hypertension is the single greatest contributor to cardiovascular
disease [38]. With aging population throughout the world, the prevalence of
hypertension has a steep increase [39]. In addition to its detrimental effects on

cardiovascular tissues, the hypertension is also associated with other cardiovascular
risk factors, such as metabolic syndrome [40] and renal disease [41]. Changes in
lifestyle, lowering sodium intake, reducing alcohol consumption, and weight reduc-
tion or physical exercise may lower blood pressure and thus reduce CVD [42].
Cigarette smoking is another important risk factor for CVD. An estimated 34.7% of
all deaths resulting from cigarette smoking is related to CVD. Tobacco use cause
impairment in endothelium-dependent vasodilation in the coronary microcirculation.
The vasodilation response which was partly initiated by the release of nitric oxide
(NO) was reduced in individuals who smoked [43]. Sera from smokers contain a
reduced expression of endothelial nitric oxide synthase (eNOS) [44], whereas a brief
exposure to tobacco smoke has caused the production of peroxynitrite (ONOO )
[45]. Consumption of tobacco has been linked with the increase in the oxidation of
LDL cholesterol, platelet aggregation, and impairment of endothelial layer [46].
Smoking for long-term increases the prevalence of hypertension, stroke, and ath-
erosclerosis. Alcohol consumption is also associated with CVD. Although moderate
consumption of alcohol cause lower risk of coronary heart disease, excess consump-
tion of alcohol is detrimental to cardiovascular tissue [47]. These studies suggest that
lifestyle changes such as physical inactivity, tobacco smoking, and heavy alcohol
consumption are the risk factors contribute to CVD. The oxidizing chemicals,
nicotine, carbon monooxide, and other particulate matters are believed to be
accountable for cardiovascular disease.
3 Diet and Cardiovascular Disease
The life style and dietary patterns are often directly related with the development of
hypertension, diabetes, and CVD. The nutritional diets may have a direct impact on
the functioning of circulatory system physiology, thus affecting the occurrences of
these diseases [48]. The metabolic abnormalities and the risk factors for CVD
including dyslipidemia, central obesity, and hypertension are mostly depend upon
the intake of an excess of total energy such as consuming high calorie or fatty meal
[49, 50]. Individuals with dyslipidemia and high cholesterol levels were advised
to take a diet rich in bioactive substances, fiber, and antioxidants and minimize the
consumption of saturated and trans fatty acids. The dietary interventions that
interfere with the reduction of plasma cholesterol and triglyceride levels may be
effective in the prevention and management of CVD [51]. The diet consisting of
nutritionally poor foods with high calorie and highly processed with deficiency of
functional foods has been found to enhance systemic inflammation [52]. Food rich in
fruits and vegetables along with moderate amounts poultry, fish, and meat has been
associated with the reduction of inflammation thus prevent the occurrence of CVD.
The direct correlation between serum total cholesterol and the heart attack and stroke
was known since long time. Similarly, the importance of the Mediterranean diet in
reducing the risk of coronary heart disease (CHD) [53–58] was also known since
many years ago. Several studies were conducted describing the lifestyle and diets of
a population in the Mediterranean region and their relation to the rates of heart
diseases [59, 60]. The traditional Mediterranean diet consisting of cereals, olive oil,
fish, legumes, fruits, vegetables, dairy and meat products and moderate quantity of
wine is quite beneficial for the heart. Consuming such diet provides a low risk of
CHD and prevents the heart disease. Studies in individuals who opted for Mediter-
ranean food [61] have shown a reduction in the incidence of CHD. Similarly, the
choice of the Mediterranean diet with extra-virgin olive oil or nuts among individ-
uals who are at high cardiovascular risk showed a reduction in CVD [62]. The
studies performed on the composition of diet that should be useful for the prevention
of CVD is limited. Thus, there is an urgent need to undertake such meticulously
designed studies on the dietary food components that may be useful in reducing the
lifestyle generated cardiovascular risk.
4 Types of Food Components Used for Prevention of

Cardiovascular Disease
A critical role of lifestyle and diet in the prevention and treatment of CVD has
become widely accepted. Besides classical food components, i.e., carbohydrates,
proteins, fats, vitamins, and minerals, the diet prescribed to promote health and
prevent diseases also includes the foods derived from medicinal plants known by
various terms such as medicinal foods, bioactive foods, functional foods, or thera-
peutic foods [63], the consumption of which is believed to reduce the risk of many
diseases including CVD [64]. The functional food refers to products with certain
health benefit and reduced the risk of diseases. Foods such as fruits vegetables,
cereals, fish, and red wine can be considered as functional foods that can prevent or
cure several diseases. Medicinal food refers food product that can be considered to
have therapeutic value in treating or preventing certain diseases and plays beneficial
effects on physiological functions of a specific tissue. Medicinal foods are consid-
ered important in reducing the risk of certain diseases. Bioactive food components
refer to constituents in foods supply with the basic human nutritional needs and
taken with diet or as supplements. These food components exhibit the power to
regulate one or more processes of metabolism that exerts the health benefits in
reducing the risk of chronic human diseases [65]. These food components taken in
appropriate quantities must be a part of the standard diet and consumed on a regular
basis in order to beneficially affecting at various metabolic targets.
5 Bioactive Food Components in the Prevention of

Cardiovascular Disease
One of the most favorite topics in the modern world is how to keep healthy and
reduce the diseases caused by aging or changes in lifestyle. The most efficient ways
to decrease the risk of common diseases, including CVD and cancer, are by limiting
intake of carbohydrate/fat enriched food, limiting consumption of alcohol, limiting
salt intake, and adding the plant-derived food items, including vegetables, fruits,
whole grains, legumes, nuts, and oils in the routine diet. Generally, bioactive foods
are referred to those compounds that have potent antioxidative, anti-inflammatory,
antithrombotic and immunomodulatory properties. These compounds possess the
property to protect the body against the inflammation, cholesterol accumulation in
the cardiovascular tissues, and protect against the oxidative stress, thus prevent the
development of cardiovascular diseases and cancer as well as other pathological
conditions such as neurodegenerative diseases [18]. Some of the most important
bioactive components found in fruits and vegetables that possess the potential for
promoting a healthy metabolism and in the prevention of diseases are flavonoids,
carotenoids, organosulfur compounds, phytoestrogens, tocopherols, and L-ascorbic
acid (Table 1). Figure 1 shows the schematic presentation of source and effects of
some of the important bioactive compounds. The detailed account of occurrences
and biological effects of these bioactive compounds and their role in the prevention
of CVD are described individually in the following sections.
5.1 Carotenoids (Lycopene)
Carotenoids belong to a class of natural fat-soluble pigments found mainly in

plants. The dietary carotenoids including lycopene, beta-carotene, lutein, beta-
cryptoxanthin, zeaxanthin, and astaxanthin provide health benefits in decreasing
the risk of many chronic diseases. Lycopene, the major carotenoid obtained from
plants such as tomatoes, watermelon, and pink or red grapefruit, is very important in
attenuating the risk of CVD. Lycopene extracted from plant sources predominantly
exists in an all-trans isomer; however, cis-lycopene is the more bioavailable form
[66]. The isomerization of trans-cis isoforms occurs with exposure to light of heat or
in the gastric cavity under acid conditions [67]. Heat generated during cooking and
processing converts some of the trans-lycopene to cis-lycopene increasing its
bioavailability in the tissue [68, 69]. Thus the bioavailability of lycopene is greater
in tomato paste and tomato puree than from fresh tomatoes [70, 71]. Thus the
bioavailability of dietary lycopene is greater after cooking or when consumed with
oil and other dietary fats [72, 73]. In addition, other nutrition present in tomatoes
such as carotenes and lutein would also be absorbed by cells. Thus consuming whole
processed tomato products will have more advantages than consuming lycopene
alone. Although some studies concluded that consumption of a carotenoid-rich diet
have no positive effect on plasma antioxidant status or markers of oxidative stress
[74], but the majority of epidemiological and clinical studies have shown beneficial
effects of lycopene or tomato-based food preparation in cardiovascular health.
Lycopene supplementation has been shown to improve biomarkers associated with
CVD. Studies involving a large group of people reported the association of higher
intake of tomato with a reduced risk of CVD and myocardial infarction [75].
Lycopene level in the blood serum also improved the common carotid artery
intima-media thickness which is an indicator of early atherosclerosis [76]. The intake
of lycopene caused the reduction in cholesterol synthesis and enhanced the
Table 1 List of important bioactive food components that may influence cardiovascular diseases
Bioactive
Category components Source Cardiovascular effects References
Carotenoids Lycopene Tomatoes, Hypolipemic, inhibitor of [77–80]
(carotenoid) watermelon, proinflammatory and
and pink or prothrombotic factors
red grapefruit
Flavonoids Genistein Soybean, soy Antiatherosclerotic by [105]
(Isoflavone) products inhibiting the expression of
ICAM-1 and VCAM-1 and
NFk-B on human endothelial
cells
Sulforaphane Cruciferae Anti-inflammatory and [119]
(isothiocyanate) Family antioxidant by stimulating
Nrf2
Apigenin Celery, Myocardial ischemia/ [110, 112,
(flavone) parsley, and reperfusion injury reduction, 113]
chamomile inhibition of lymphocyte
proliferation, and
proinflammatory cytokine
expression
Quercetin Onions and Antiplatelet aggregation [121, 122]
(Flavanols) shallots activity, reduction of
mycocardial infarction
Resveratrol Grapes, red Reduction in LDL oxidation, 106–109
(polyphenolic wine vasorelaxation, reduction of
compound) platelet aggregation,
antiatherosclerotic,
Anthocyanins Fruits and Antiatherosclerotic, [131–141]
(glycosides of vegetables reduction in CVD mortality,
anthocyanidins) protect DNA damage
Hesperetin Citrus plants Block oxidized LDL- [94]
(flavanone) induced endothelial
apoptosis
Catechins Green tea Reduction in blood [91, 93]
(Flavanol) cholesterol;
antihypertensive;
triglyceride, total cholesterol
and low-density lipoprotein
cholesterol
Vitamins Ascorbic acid Fruits and Prevent HDL from lipid [144]
vegetables oxidation
Alpha- Oils, nuts, and Prevent HDL from lipid [147–150]
tocopherol leafy green oxidation
vegetables
Abbreviations: ICAM-1 Intercellular adhesion molecules-1, VCAM-1 Vascular cell adhesion mol-
ecule-1, Nrf2 nuclear factor-erythroid 2, LDL Low-density lipoprotein, HDL High-density lipopro-
tein, CVD Cardiovascular disease, DNA deoxyribonucleic acid
Fig. 1 Schematic diagram illustrating the role of bioactive components of food in the prevention of
cardiovascular diseases (CVDs). As shown, bioactive components act on cells or tissue by targeting
many physiological and metabolic processes. Together regulating multiple processes, these com-
ponents of food prevent or cure cardiovascular diseases
degradation of LDL [77]. Oxidative damage related parameter such as lipid perox-
idation and LDL oxidation were significantly declined in the subjects who were
prescribed lycopene supplementation [78]. Similarly, individuals who were treated
with lycopene (40 mg/day) for 6 weeks showed decline in triglyceride levels and
LDL cholesterol levels whereas the HDL cholesterol was significantly increased
[79]. The increase in HDL cholesterol and decline in total cholesterol/HDL choles-
terol ratio was also reported in a study comprised of healthy men and women
consuming the soy-tomato beverage that contain 21 mg lycopene/day on an average
daily [80]. These studies suggest that lycopene or tomato-based products with a
significant amount of lycopene improves the cardiovascular health by reducing the
triglycerides, scavenging LDL, and maintaining HDL level thus reducing the risk of
CVD.
5.2 Phenolic Acids and Flavonoids
Flavonoids are a group of polyphenolic compounds found in significant amount in

many fruits, vegetables, grains, and beverages. They are classified as flavonols,
flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. The flavonols
include compounds namely isorhamnetin, kaempferol, myricetin, and quercetin.

Apigenin and luteolin compounds are grouped under flavones. Flavanones include
compounds eriocitrin, hesperetin, and naringenin whereas flavan-3-ols include com-
pounds such as catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallo-
catechin gallate, and gallocatechin. Anthocyanidins include compounds such as
cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin, whereas
compounds daidzein and genistein are grouped under isoflavones. These compounds
have varied chemical structure and biological functions and are grouped accordingly.
The isoflavones genistein, daidzein, and glycitein occur predominantly in legumes.
Genistein and daidzein found in soy, and resveratrol occurs in grape skin and red
wine, are nonsteroidal polyphenolic compounds and considered as a phytoestrogen.
The flavonoids contain sulfur are grouped under organosulfur compounds, promi-
nent among are sulforaphane found in crucifer plants and γ-glutamyl-S-allyl-L-
cysteines and S-allyl-L-cysteine sulfoxides found in garlic (Allium sativum L.,
family Liliaceae). Anthocyanins are a group of most studied flavonoid occurs in a
wide variety of plant kingdom. Anthocyanins are providing the bright red-orange to
blue-violet colors in many fruits and vegetables. Their consumption has been
estimated to be up to ninefold higher than that of other dietary flavonoids [81].
Phenolic acid and flavonoids have three most important health benefit effects on
the prevention of cardiovascular tissue. The most important effect is attributed to
their antioxidant activity [82]. Secondly they have prominent effects on the preven-
tion of atherosclerosis [83], and lastly, they have a significant effect on the platelet
aggregation [84]. In vitro studies indicate that flavonoids inhibit the oxidation of
LDL and decrease the thrombotic tendencies [85]. They delay the development of
atherosclerotic plaque and prevent the development of atherosclerosis by reducing
the endothelial dysfunction. Another mechanism by which flavonoids help prevent
CVD is through their effect on platelet aggregation. Flavonoids interact with mem-
brane lipids and modifying the membrane fluidity [86], which is partly responsible
for the antiaggregatory and disaggregatory effects on human platelets.
Catechins are polyphenolic compounds found in food and in all kinds of tea with
cardioprotective properties [87]. Catechins have an important role in the prevention
of cardiovascular disease [88, 89]. Catechins have been shown to reduce the
accumulation of cholesterol and its oxidation products in the walls of arteries, thus
help in smooth blood circulation [90]. Supplementation of catechins may reduce the
serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol, thus
preventing atherosclerosis [91]. Catechins may improve the vascular endothelial
environment by eliminating ROS [92]. In addition, catechins exhibit very strong
antihypertensive activity [93]. Both ( )epigallocatechin gallate and hesperetin fla-
vonoids block oxidized LDL-induced endothelial apoptosis thus may function as
antiatherogenic agents [94]. However, there is no clear evidence to suggest a
beneficial effect of tea catechins on the prevention of CVD.
Phytoestrogens are polyphenolic compounds occur in many legumes, beans, nuts,
soybeans, seeds of sesame and so on, and mimic the properties of estrogens. These
compounds include certain isoflavonoids, flavonoids, stilbenes, and lignans [95].
The best-studied dietary phytoestrogens are the soy isoflavones and the flaxseed
lignans. Isoflavones, such as genistein and daidzein, occur in soybeans, legumes,

lentils, and chickpeas. Lignans are the most abundant nonflavonoids occur in most
cereals, linseed, fruits, and vegetables. The phytoestrogens are the most intensively
studied bioactive components of food with regard to their health benefits. Isoflavone
content of foods such as soybeans is sold as nutritional supplements. Good scientific
evidence supports the observation that phytoestrogens may play a beneficial role in
reducing the risk of cardiovascular disease [18]. Diet is the chief source of
phytoestrogens in human. The bioavailability of phytoestrogens in body organs
may be dependent on the metabolism of intestinal bacteria. After ingestion in their
beta-glycosidic forms, most of these phytoestrogens are hydrolyzed in the intestine
to their aglycones. Then in the intestinal wall and liver aglycones are absorbed and
glucuronidated [96, 97].
Isoflavones have beneficial biological effects in the cardiovascular system. They
exert antiestrogenic effects by competitive inhibition at the estrogen receptor and
help maintain cellular proliferation, vascular reactivity, lipid profiles, and thrombosis
[98]. Epidemiologic studies reported that consumption of dietary isoflavones from
soy products protects women not only from cardiovascular disease but also from
breast and uterine cancer [99–102]. The lipid-lowering functions of isoflavones will
have profound effects on cardiovascular protection. Although very little evidence
presented to show the protective roles of phytoestrogens for cardiovascular disease,
clinical studies involving Japanese women concluded that intake of high isoflavone
was correlated with the reduced risk of cerebral and myocardial infarctions [103].
Phytoestrogens intake could delay the progression of atherosclerosis in vascular
tissue by their pathophysiologic effects on lipid profile, reactive oxygen species
production, inflammation, and tissue damage [104]. Atherosclerosis is initiated
when monocytes bind to the endothelium layer of blood vessel, migrate into the
tunica intima, and later develop into the foam cells. Lipid-induced and oxidant-
sensitive transcription of adhesion molecules and chemokines promote the monocyte
binding to endothelium. Genistein has been reported to be capable of inhibiting
the expression of intercellular adhesion molecules-1 and vascular cell adhesion
molecule-1 (ICAM-1 and VCAM-1) on human endothelial cells cocultured with
monocytes [105], thus protecting against atherosclerosis. Resveratrol, a phytoestro-
gen found in high concentration in red wine, has been proposed to be the agent
responsible for cardiovascular protection [106]. Its protective role in the cardiovas-
cular system occurs by mechanisms of stimulation of reduction of low-density
lipoprotein oxidation, vasorelaxation, suppression of platelet aggregation, anti-
atherosclerotic properties, and also providing defense against ischemic-reperfusion
injury [107]. This compound has the ability to stimulate Ca++-activated K+ channels
so as to increase endothelium nitric oxide signaling, thus exerting vasorelaxant
activity [108, 109].
Apigenin, a flavonoid found in many vegetables such as celery (Apium
graveolens), parsley and chamomile have demonstrated to possess the cardio-
protective effects. Apigenin ameliorates myocardial ischemia/reperfusion injury
via the inactivation of p38 mitogen-activated protein kinase [110]. In a cardiac
hypertrophy model, the supplementation of apigenin reduces hypertension,
cardiomyocyte cross-sectional area, and serum angiotensin II [111]. Similarly, in an

autoimmune myocarditis mice model, dietary apigenin cause inhibition of lympho-
cyte proliferation thus mediating the protection of cardiac tissue [112]. Apigenin
caused reduction of LPS-induced mortality in mice by inhibiting proinflammatory
cytokine expression [113] and decrease heart injury by suppressing sphingosine
kinase 1/sphingosine 1-phosphate signaling pathway [114]. In a recent study, Li et al.
[115] demonstrated that apigenin protects cardiac tissue damage, cardiac injury,
cardiomyocyte cell death, and cardiac dysfunction against LPS- induced toxicity
by its anti-inflammatory and antioxidant effect.
Organosulfur compounds are chiefly found in cruciferous vegetables, as well as
in garlic and onions. Vegetables belong to Cruciferae family such as cabbage,
broccoli, and kale contain rich amount of sulfur-containing compounds known as
glucosinolates. Isothiocyanates are biologically active breakdown products of
glucosinolates. Different types of glucosinolates are found in different cruciferous
vegetables, each of which upon hydrolysis forms a different isothiocyanate [116].
For example, glucosinolate glucoraphanin, most abundantly present in 3-day-old
broccoli sprouts, is converted to the isothiocyanate sulforaphane by the endoge-
nous enzyme, myrosinase. Sulforaphane protects the chronic diseases by
upregulating the “phase 2 response” known as Kelch-like ECH-associated protein
1- nuclear factor erythroid 2 p45-related factor 2-antioxidant responsive element
(Keap1-Nrf2-ARE) pathway. Sulforaphane has been regarded as one of the most
potent known naturally occurring inducers of cytoprotective phase 2 enzymes
[117]. Glucosinolates are rapidly hydrolyzed by myrosinase, generating metabo-
lites when raw cruciferous vegetables are cut while processing the food. While
cooked vegetables inactivate myrosinase, so as to prevent the glucosinolates
breakdown. Light steam cooking for about 5 min will preserve some of the
myrosinase and thus allow the conversion of isothiocyanate. Most of the absorp-
tion of glucosinolates occurs in the small intestine, however, a large proportion of
it reaches the colon [118] where it generates a broad range of metabolites
depending on the pH and the presence of cofactors. The sulforaphane has been
shown to target pro-inflammatory pathways by stimulating Nrf2 induced antiox-
idant enzymes [119] and downregulating the expression of NF-κB target genes
which code for proinflammatory mediators, such as tumor necrosis factor-α
(TNF-α), interleukin-1 (IL-1β), and IL-6 [119].
Allium vegetables are recognized to be a good source of organosulfur compounds
[120] and its supplementation in human exhibits antiplatelet aggregation activity.
Similarly, the quercetin, the main polyphenol found in onions and shallots, also
inhibited platelet aggregation in vivo [121] and in vitro [122]. Higher intake of
cruciferous and allium vegetables (per serving 75 g/d) were associated with lower
risk of atherosclerotic vascular disease mortality [120]. This study was conducted
in older Australian women with 15 years of follow-up and concluded that higher
cruciferous and allium vegetables intakes render lower risk of atherosclerotic vas-
cular disease (ASVD) mortality in old individuals and protect vascular health.
Resveratrol has recently become a well-known potent antioxidant and is found in
red grapes, blueberries, cranberries, and other types of Vaccinium berries [123].
It is also known for its antithrombotic, anti-inflammatory, anticarcinogenic, and

lifespan elongation effects, as well as its positive role in protection against insulin
resistance [18, 124–126].
Anthocyanins are the polyphenolic compounds, possessing a characteristic
C3–C6–C3 carbon structure and present in fruits, vegetables, berries, and red wine
[127]. Anthocyanins are readily absorbed and metabolized in the tissue. These
phenolic compounds found in the circulation and urine as intact, methylated,
glucuronide derivatives, and/or sulfoconjugated forms [128–130]. Their anti-inflam-
matory and antioxidative properties show beneficial effects in the cardiovascular
system. Compounds containing anthocyanin have been shown to reduce atheroscle-
rosis in rodents [131]. Epidemiological studies have examined the relationship
between total anthocyanin intake and risk of developing CVD. In a 16-year fol-
low-up study period of consuming strawberry daily (intake of 0.2 mg/d of anthocy-
anins), the postmenopausal women participating in the Iowa Women’s Health Study
showed a significant reduction in CVD mortality [132]. Intake of blueberries also
showed a significant decrease in coronary heart disease mortality. Moderate con-
sumption of red wine will have a preventive effect in CVD-related mortality [133,
134]. The beneficial effects of anthocyanins in the prevention of CVD are strongly
linked to the protection against reactive oxygen species-induced oxidative stress.
Other mechanisms of anthocyanin beneficial role on cardiovascular tissues may be
via providing protection from DNA damage, inhibiting enzyme, regulating immune
responses through increased cytokine production, and exhibiting anti-inflammatory
activity [135–139]. There is a significant reduction in ischemia in patients with
vascular diseases who were consuming anthocyanin [140]. Inhibition of
platelet activity and experimental coronary thrombosis in vivo was significantly
achieved by commercial grape juice (10 mL/kg) [141]. These studies suggest that
anthocyanins have a wide range of protective effects against CVD. However,
epidemiological studies are insufficient to provide comprehensive information
about their usefulness in CVD.
5.3 Vitamins
A substantial body of evidence has presented describing the possible role of several
vitamins in the reduction of CVD risk. L-ascorbic acid or vitamin C found in a wide
variety of fruits and vegetables has received a considerable attention in the past two
decades as a powerful dietary antioxidant with a possible role in heart health. It is
well known that HDL participates in the removal of cholesterol from sites of
atherosclerotic lesion. In addition, the HDL also performs other functions that
potentially have cardioprotective properties. Some of the beneficial functions of
HDL include the inhibition of platelet activation, anticoagulant and profibrinolytic
activities, preservation of vascular endothelial function, and protection of LDL from
oxidation [142]. However, HDL is also vulnerable to lipid oxidation with ensuing
loss of some cardioprotective properties [143]. Vitamin C has been shown to prevent
the lipid oxidation in HDL and thus conserving the cardioprotective properties
of this lipoprotein [144]. Vitamin C also has been found to inhibit the oxidation of
LDL-protein, thereby contributing to reduce atherosclerosis [145]. Although the
cardiovascular other functions of vitamin C in cardiovascular diseases are not fully
understood, it has been linked to improve the lipid profiles, protect arteries from
arterial stiffness, and improve the endothelial function [145]. Vitamin E that includes
tocopherols and tocotrienols was found in many types of oils, nuts, and leafy green
vegetables and exhibit cardiovascular protective property [146]. Several studies have
demonstrated the protective role of vitamin E in preventing HDL from lipid oxida-
tion with subsequent cardioprotective benefits [147–150]. Initial studies found no
correlation between serum or plasma vitamin E concentrations and death from
cardiovascular diseases [151, 152]. Epidemiological studies in Finnish men found
no association between serum vitamin E level and a coronary end point [153]. An
extensive study on vitamin E supplementation was carried out in the USA involving
87,425 female nurses [154]. Their findings concluded that the benefit function of
vitamin E supplementation was only apparent with the continued consumption for
greater than 2 years. The benefit of vitamin E intake appeared to occur with both
supplemental and dietary vitamin E. Overall studies suggest that there exists a
relationship between consumption of vitamin E and the incidence of CVD [146].
6 Conclusion
Changes in lifestyle, dietary habits, and environmental and physiological factors

directly influence the risk factors of CVD. The health benefits of fruits and vegeta-
bles are known since ancient times. However, investigations on their bioactive
components and their physiological and metabolic targets attracted the significance
of these compounds in preventing or treating certain chronic human diseases.
Studies have shown that fruitful changes in lifestyle and regular consumption of
recommended bioactive food components will help prevent chronic cardiovascular-
related illness. Bioactive components of food are a challenging area in terms of its
ability to regulate the metabolic process and control chronic diseases. Exact mech-
anism of their effects and appropriate doses on various signaling pathways needs to
work out. The appropriate human dose and the risk of side effects of most of the
bioactive food components are not known. Awareness among people and confidence
among physicians on dietary recommendations to patients to prevent or to treat the
disease are warranted. The key is to encourage people to make habit of consuming
bioactive food components as part of their daily diet so as to prevent or eliminate
lifestyle or age-related chronic diseases.
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Capsicum annuum Bioactive Compounds:
Health Promotion Perspectives 7
Muhammad Imran, Masood Sadiq Butt, and
Hafiz Ansar Rasul Suleria
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2 Chemistry of Capsicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3 Health Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.1 Anticancer and Antimalignant Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.2 Cardiovascular Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.3 Antidiabetic Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.4 Antiobesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
3.5 Antiaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
3.6 Anti-inflammtory Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3.7 Capsaicin in Urological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
M. Imran
National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan
Department of Diet and Nutritional Sciences, Faculty of Health and Allied Sciences, Imperial
College of Business Studies, Lahore, Pakistan
M. S. Butt
National Institute of Food Science and Technology, Faculty of Food, Nutrition and Home Sciences,
University of Agriculture, Faisalabad, Pakistan
H. A. R. Suleria (*)
UQ School of Medicine, Translational Research Institute, The University of Queensland, Brisbane,
QLD, Australia
Department of Food, Nutrition, Dietetics and Health, Kansas State University, Manhattan, KS, USA
e-mail: hafi[email protected]

160 M. Imran et al.
Abstract
Capsicum annum L. commonly known as bell pepper exhibits proven health as
well as medicinal significance. It can be consumed either in fresh or processed
form and is rich source of vitamin C, provitamin A, and calcium. Array of
bioactive compounds especially antioxidants in its phytochemical profile make
it an ideal choice for preventing cell damage, cancer insurgence, diabetes prev-
alence, cardiovascular disorders, cataracts, Alzheimer’s, and Parkinson’s disease.
Major antioxidant compounds in capsicum are carotenoids, tocopherols, and
capsaicinoids (capsacicin). Their anticancer role is attributed to their ability to
act as scavengers of singlet molecular oxygen, reactive oxygen species (ROS),
peroxyl radicals, and reactive nitrogen species (RNS). Capsaicinoids intake
effectively reduced the triacyclglycerols, plasma total cholesterol (PTC), and
non-high-density lipoprotein cholesterol, and thereby helps in the prevention of
cardiovascular ailments. It also exhibit effective and proactive contribution
against age-related ailments. Capsaicin exposure expressively repressed the ini-
tial adipogenic differentiation, maturation, and lipogenesis of adipocytes. Capsa-
icin also has ability to target the TRPV1 receptors in the C-fibers lead to their
stimulation followed by desensitization that helps to improve the neurogenic
bladder. So, it may serve as a potential emerging treatment for patients who are
nonrespondent to conventional therapy especially those with neurogenic bladder.
Keywords
Bell pepper · Capsaicinoids · Antioxidant · Neurogenic bladder
List of Abbreviations
18α-GA 18 alpha-glycyrrhetinic acid
ABCA1 ATP-binding cassette transporter
ABCG1 ATP-binding cassette transporter-G1
ABCG5 ATP-binding cassette transporter-G-5
AdipoR2 Adiponectin gene/protein and its receptor
ADP Adenosine diphosphate
ALCAM Activated leukocyte cell adhesion molecule
AMPK Activation of activated protein kinase
Apo-A1 Apolipoprotein-A1
apoM Apolipoprotein M
ATP Adenosine triphosphate
BAT Brown adipose tissue
BCC Basal carcinoma cells
BUN Blood urea nitrogen
C/EBPα C-enhancer-binding proteins
Ca2+ Calcium
CaMK-II Calmodulin-dependent protein kinase II
Capz Capsazepine
CCMSs Capsaicin-chitosan microspheres
7 Capsicum annuum Bioactive Compounds: Health Promotion Perspectives 161
CD36 Cluster of differentiation-36

COX-2 Cyclooxygenase-2
CRP C-reactive protein levels
CRT Calreticulin
Cx43 Connexin 43
DCs Dendritic cells
DHC Dihydrocapsaicin
DNA Deoxyribonucleic acid
EC-LPS Lipopolysaccharide from Escherichia coli
EMT Epithelial mesenchymal transition
eNOS Endothelial nitric oxide synthase
ERK Extracellular signal-regulated kinases
FABP4 Fatty acid binding protein-4
FADD Fas-associated protein with death domain
FAK Focal adhesion kinase
GC Gastric cancer
GDM Gestational diabetes mellitus
Glu Glutamate
GSH Glutathione
GSSG Oxidized glutathione
HDL-C High density lipoprotein
HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase
HO-1 Heme oxygenase-1
HSL Hormone sensitive lipase
HUVECs Human umbilical vein endothelial cells
ICD Immunogenic cell death
IL-1β Interleukin-1 beta
IL-6 Interleukin-6
KA Kainic acid
Klf2 Kruppel-like factor 2
LDL-C Low-density lipoprotein-cholesterol
LDL-R Low-density lipoprotein receptor
LPS Lipopolysaccharide
MDA Malondialdehyde
MS–MS Mass spectrometry
NDO Eurogenic detrusor overactive
NET Neuroendocrine tumor cells
NF-κB Nuclear factor-kappa B
NOD/SCID Nonobese diabetic/severe combined immunodeficiency
NPC1 Niemann-Pick C1 protein
OH Hydroxyl
PC-3 Pancreatic cancer
p-CaM Adhesion molecule
PCR Polymerase chain reaction
PPARdelta Peroxisome proliferator-activated receptor delta
162 M. Imran et al.
PPARgamma Peroxisome proliferator-activated receptor gamma

PPARα Peroxisome proliferator-activated receptor-alpha
PPARγ Peroxisome proliferator-activated receptor gamma
PPARγ Peroxisome proliferator-activated receptor-gamma
PTPϵ Protein-tyrosine phosphatase ϵ
RNS Reactive nitrogen species
SOD Superoxide dismutase
SRA-1 Steroid receptor RNA activator 1
SRB1 Scavenger receptor class B member 1
TG Triglycerides
TIMP-1 Tissue inhibitors of metalloproteinases-1
TNF-α Tumor necrosis factor-alpha
TRP Transient receptor potential
TRPV1 Transient receptor potential vanilloid subtype 1
UCP2 Uncoupling protein 2
UV Ultraviolet
VEGFA Vascular endothelial growth factor-A
VLDL-C Very low-density lipoprotein- cholesterol
WT Wild-type
1 Introduction
The bell pepper (Capsicum annum L.), a member of Solanaceae family, is most
commonly consumed fruity vegetable and well known for its antioxidant signifi-
cance [1]. Solanaceae family include over 90 genera and 2000 species, among them
Capsicum annum, Capsicum chinense, Capsicum baccatum, Capsicum pubescens,
and Capsicum frutescens are most common. Bell pepper exhibits medicinal as well
as food value in all over the world, and both warm and dry climate are considered
suitable for its cultivation [2]. About 30 species are included in the genus Capsicum,
out of which only five are domesticated while rest of these are considered as wild.
Major cultivars include C. baccatum, C. chinense, C. frutescens, C. annuum, and C.
pubescens. Capsicum chinense is known as pungent fruit among the domesticated
species [3]. These are also a rich source of neutral phenolic compounds, especially
luteolin, quercetin, and capsaicinoids [4]. The utilization of bioactive compounds
plays key health-promoting functions such as provide protection against oxidative
damage to cells, cancer insurgence, diabetes prevalence, cardiovascular disorders,
cataracts, Alzheimer’s, and Parkinson’s disease [1]. These chemical compounds also
prevent oxidation of structural lipids of brain cells and are vital for their proper
functioning [4].
Different consumable forms of pepper are fresh, processed (sauces), or preserves
(dehydrated powder). Its industrial utilization is mainly due to its unique composi-
tion and aromatic properties. Capsicum annuum has worldwide existence [5] and has
different types of peppers such as sweet bell and hot peppers, cherry, serrano, and
cayenne [6, 7]. These serve as a domineering nutrient source in human diet [8, 9]. It
has a significant source of ascorbic acid (vitamin C) and carotene (provitamin A). As
per estimates, about 60% and 100% recommended daily amounts (RDA) for vitamin
A and C can be achieved by the intake of 50–100 g fresh pepper. Ripened pepper
fruit contain phytochemicals with antioxidant and anticancer functions [10]. Among
the different antioxidants found in pepper, chlorophyll, carotenoids, tocopherols, and
capsaicinoids are most important constituents [8].
The pungent values of chili fruit are due to the presence of alkaloids compound, i.
e., capsaicinoids, which is in capsicum placenta during its early maturity and can be
used in the human diet as food, medicine, and pharmaceutics. Capsaicin along with
dihydrocapsaicin collectively constitute about 90% of the capsicum capsaicinoids,
while the rest include nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin,
norcapsaicin, and nornorcapsaicin [11].
Carotenoids a colored pigment found in peppers impart yellow to red color. It also
exhibits antioxidant properties and prevents tissues damage by acting as singlet
molecular oxygen, reactive oxygen species (ROS), peroxyl radicals, and reactive
nitrogen species (RNS) scavenger [12] (Table 1).
2 Chemistry of Capsicum
Flavonoids along with some other polyphenols are pervasive phytochemicals and
considered as essential functional ingredients found in different types of peppers
(green, sweet, and hot). So far various attempts have been made to detect, quantify,
and characterize different types of flavonoid in peppers [14, 15]. They are also rich in
glycosides as well as aglycones of quercetin, myricetin, luteolin, kaempferol, and
apigenin. Structural studies of flavonoids showed that position and numbers of
hydroxyl groups along with a double bond at the C2-C3 position are mainly
responsible for their strong antioxidant and anticancer activities. Role of structure
in determining antioxidant potential confirms the myricetin as one of the most potent
flavonoids found in pepper [16, 17]. Mass spectrometry (MS–MS) fragmentation
and ultraviolet (UV) spectra-based structural study was carried out to identify C- and
O-glycosides in peppers. Results showed that the pericarp of pepper fruit contains
four quercetin (3-O-rhamnoside, 3-O-rhamnoside-7-O-glucoside, 3-O-glucoside-7-
O-rhamnoside, and quercetin glycosylated), two luteolin O-glycosides (apiosyl-
acetyl-glucoside and 7-O-2-apiosyl-glucoside), five luteolin C-glycosides (6-C-
hexoside, 8-C-hexoside, 6-C-pentoside-8-C-hexoside, 6-C-hexoside-8-C-pentoside,
and 6,8-di-C-hexoside) along with two apigenin C-glycosides (6-C-pentoside-8-C-
hexoside and 6, 8-di-C-hexoside) [18].
Flavonoid levels vary and mainly depend on genetics, developmental stage,
and ecological situations. Such as red fruits of most cultivars have shown highest
quantities of flavonoids. On the other hand, green fruits were found to be rich in
quercetin 3-O-R-L-rhamnopyranoside whose level decreased during ripening
[19]. Bioactive compounds include both essential and nonessential substances
of natural occurrence in food and are well known for their impact human health.
164 M. Imran et al.
Table 1 Nutritional Nutrient 1.80 g of capsicum

composition of Capsicum
Water 0.145 g.
annum [13]
Energy 5.724 kcal
Carbohydrate 1.019 g
Fiber, total dietary 0.490 g
Protein 0.216 g
Fat (Total lipid) 0.311 g
Ash 0.109 g
Calcium 2.664 mg
Iron (Fe) 0.140 mg
Sodium (Na) 0.540 mg
Copper (Cu) 0.007 mg
Phosphorus (P) 5.274 mg
Selenium (Se) 0.158 mcg
Manganese (Mn) 0.036 mg
Magnesium (Mg) 2.736 mg
Zinc (Zn) 0.045 mg
Potassium (K) 36.252 mg
Saturated fatty acids (SFA) 0.059 g
Monounsaturated fatty acids (MUFA) 0.050 g
Thiamin 0.006 mg
Vitamin B6 0.037 mg
Niacin 0.157 mg
Riboflavin 0.017 mg
These are usually present in insignificant amounts in various foods and influence
many cellular and physiological functions [20]. The bell pepper exhibits excel-
lent antioxidant activity due to the presence of phytochemicals, such as flavo-
noids, capsaicinoids, phenolic compounds, carotenes, and ascorbic acid [21, 22].
Presence of these valuable micronutrients in bell pepper increases its significance
against emerging diseases, especially dementia, diarrhea, and dysentery, along
with many other ailments [23, 24]. However, quantities of these bioactive com-
ponents in bell pepper depends on their farming conditions, state of maturity at
harvesting, and varietal purity, and postharvest management [21, 24]. In red
pepper, capsanthin, cryptocapsin, and capsorubin represent oxygenated caroten-
oids and exhibit strong antioxidant activity [25]. These phytochemicals pose
generous ability to counteract free radicals involved in lipids oxidation, proteins,
and cell deoxyribonucleic acid (DNA) damage and chronic human ailments [26].
In addition to capsaicinoids and carotenoids, flavonoids also exhibit antioxidants,
anti-inflammatory, antiallergic, and antibacterial significance [14, 27, 28]
(Table 2).
Table 2 Bioactive compounds and their potential metabolic roles

Name Chemical structure Metabolic role References
Capsaicin HO
H
Anticancer [29, 30]
N
O
O
Luteolin OH Antidiabetic [4]
OH
HO O
OH O
Dihydrocapsaicin HO Cardiopreventive [31]
H
N
O
O
Capsaicin HO Antirheumatoid [32]
H
N and
O antiosteoarthritis
O
Capsazepine HO
S
Cl Anticancer [33]
HO
N N
H
β-Carotene Antitumorigenesis [34]
3 Health Perspectives
3.1 Anticancer and Antimalignant Activities
Meticulous in vitro and in vivo probing in mice demonstrated that capsaicin sup-
presses basal carcinoma cells (BCC) (5637 and T24) and nonobese diabetic/severe
combined immunodeficiency (NOD/SCID) mouse by inhibiting their proliferation,
persuading cell cycle detention at G0/G1 phase, and diminishing ROS production. It
also enhanced the Forkhead box O3 also known as FOXO3a and enhanced the
catalase and superoxide dismutase concentration [29, 35]. Capsaicin stimulates
autophagic process in BC cells by altering redox homeostasis, changing adenosine
diphosphate/adenosine diphosphate (ADP/ATP) ratio, and inducing mitochondrial
depolarization along with activation of activated protein kinase (AMPK) pathway.
This enhanced capsaicin-prompted cell decrease in BC cells, proving that capsaicin
promoted autophagy serves as a prosubsistence progression. Furthermore, BC cells
showed distinctive mesenchymal properties of the epithelial-mesenchymal transition
(EMT), such as overextended appearance and enhanced expression of the integrin-
like kinase, anti-apoptotic Bcl-2 proteins, vimentin, and α5 and β1 integrin subunits
166 M. Imran et al.
on capsaicin treatment. It rises CD24 and vascular endothelial growth factor-A

(VEGFA), promotes the expression of Dhh/Ptch2/Zeb2 members of the Hedgehog
signaling pathway and tissue inhibitors of metalloproteinases-1 (TIMP-1), and
decreases CD44 and activated leukocyte cell adhesion molecule (ALCAM) mRNA
expression levels. Amantini et al. [36] reported the involvement of the Hedgehog
signaling pathway in the CPS-influenced autophagy and EMT phenotype. While
increased resistance to commonly used BC curing drugs such as mitomycin C,
gemcitabine, and doxorubicine was observed in capsaicin-resilient EMT-positive
BC cells.
Capsaicin administration at the rate of 200 μM significantly inhibited the colon
cancer cells proliferation; repressed TNF-α, IL-1β, IFN-γ, IL-10, and IL-1ra synthe-
sis and exudation in a dose-dependent manner; and stimulated the IL-6 in human
peripheral blood mononuclear cells (PBMCs) and HT-29 or RKO cells [37]. In
addition its intake effectively reduce the proliferation of renal carcinoma cells,
attenuation of transient receptor potential vanilloid type 1 (TRPV1) representative
opponent capsazepine (CPZ), induction of apoptosis, upregulation of proapoptotic
genes (Fas-associated protein with death domain (FADD), c-myc, Bax and cleaved-
caspase-3, -8, and -9), and downregulation of antiapoptotic gene Bcl2. It also
stimulated the p-38 and the Jun N-terminal kinase (JNK)-MAPK pathways [38].
Capsaicin in gastric cancer (GC) cell lines exerts its effect via significantly
suppressing cell progression, but in GC cell lines, it causes change in histone
acetylation. Subsequent investigations confirmed the vital role of hMOF, i.e., a
major histone acetyltransferase for H4K16 in capsaicin-tempted epigenetic vicissi-
tudes. Reduction in hMOF activity was noticed in GC tissues, which could be
reinstated by capsaicin both in vivo and in vitro [39].
Immunogenic cell death (ICD) is due to early calreticulin (CRT) surface expo-
sure. Cisplantin and capsaicin can persuade the cell apoptosis in human osteosar-
coma cells (MG-63). Capsaicin treatment causes CRT translocation from the
intracellular space to the cell surface which increased the MG-63 cells phagocytosis
and IFN-γ secretion stimulation [40], induces apoptosis, lowers the bladder cancer
lines growth, and downregulates the tNOX expression. Moreover, capsaicin effec-
tively decreases the expressions of various protein involved in cell cycle progres-
sion, thereby enhanced the phenomenon of cell cycle arrest, repressed the
extracellular signal-regulated kinases (ERK) activation, reduced paxillin and focal
adhesion kinase (FAK) phosphorylation, and cell migration [41].
Capsazepine (Capz) is a synthetic analog of capsaicin having anticancer effects.
The mode of action of the capsaicin against prostate cancer cells involves; inhibition
of signal transducer and activator of transcription-3 (both constitutive and induced)
activation, suppression of the upstream janus kinases (JAK1/2) and c-Src activation
and low concentration level of oncogenes protein products, which in turn induce
apoptosis is, minimize proliferation and invasion. The phosphatase inhibitor
pervanadate overturned the Capz-induced STAT3 inhibition, representing that the
consequence of Capz based on a protein tyrosine phosphatase. Moreover, siRNA-
mediated knockdown of protein-tyrosine phosphatase ϵ (PTPϵ) reversed the Capz-
induced initiation of PTPε and reticence of STAT3 activation, indicating that PTPε is
crucial for Capz-dependent STAT3 dephosphorylation. Finally, intraperitoneal Capz

administration decreased tumor growth in a xenograft mouse prostate cancer model
and reduced p-STAT3 and Ki-67 expression [42].
Capsaicin treatment of U251 cells decreased cell viability and altered the punctate
patterns of LC3. In U251 human glioma cells, dihydrocapsaicin (DHC) exerts its
effect by inducing apoptosis in a dose- and time-dependent manner and through
decreased cell viability. The apoptosis effect of capsaicin is mainly attributed to
mitochondrial depolarization, caspase-9 and -3, and enhanced ROS synthesis along
with increase in calcium (Ca2+) level [42].
3.2 Cardiovascular Role
Different body organs such as brain, gut, bladder, dorsal root ganglia, and sensory
nerves contain transient receptor potential vanilloid subtype 1 (TRPV1), which
serves as a receptor for capsaicin. TRPV1 activation results in increased intracellular
calcium signaling which in turn induce various physiological effects. It is also
involved in swelling, oxidation trauma, and associated discomfort [38, 43].
Capsaicinoids intake significantly reduced the plasma total cholesterol, non-high-
density lipoprotein cholesterol, and triacyclglycerols but not cause any change in
high-density lipoprotein cholesterol level. Fecal excretion of total acidic sterols is
increased by dietary capsaicinoids possibly by increasing cholesterol 7-alpha-
hydroxylase and decreasing liver X receptor α. Capsaicinoids reduced the choles-
terol absorption by decreasing plasma campesterol/cholesterol ratio as confirmed by
plasma sterol analysis. Capsaicinoids impede cyclooxygenase-2 (COX-2) expres-
sion, thereby positively influence the endothelium-dependent relaxations but nega-
tively affect the endothelium-dependent contractions.
In atherosclerotic plaque mice, dihydrocapsaicin (DHC) treatment expressively
reduced the cellular cholesterol content but increased the apoA1-mediated choles-
terol efflux. Results also showed decreased in plasma triglycerides (TG), low-density
lipoprotein (LDL-C), very low-density lipoprotein (VLDL-C), interleukin-beta (IL-
1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and C-reactive protein
(CRP) levels while apoA1 and high-density lipoprotein (HDL-C) levels in plasma
were significantly increased. Results of another study reported a significant reduc-
tion in atherosclerotic lesion in apoE / mice when treated with DHC. Moreover,
combined treatment with liver X receptor alpha (LXR-alpha) siRNA and peroxisome
proliferator-activated receptor gamma (PPARγ) siRNA induces the upregulation of
DHC on ATP-binding cassette transporter (ABCA1), ATP-binding cassette trans-
porter-G1, ATP-binding cassette transporter-G-5 (ABCG5), scavenger receptor class
B member 1 (SRB1), Niemann-Pick C1 protein (NPC1), cluster of differentiation-36
(CD36), low-density lipoprotein receptor (LDL-R), 3-hydroxy-3-methylglutaryl-
CoA reductase (HMGCR), apolipoprotein-A1 (Apo-A1), and apo-E expression
markedly stopped but decreased the DHC regulation on steroid receptor RNA
activator 1 (SRA-1) expression distinctly compensated. LXRα siRNA treatment
168 M. Imran et al.
demonstrated nonsignificantly affect the DHC-induced PPARγ expression but with

significantly decreased DHC-based LXRα expression [31].
3.3 Antidiabetic Role
Peripheral neuropathy is a chronic diabetes complication mellitus and is character-

ized by severe pain. Antidepressants and antiepileptic administration serves as
effective drug treatment options. Nociceptive sensory neurons, as well as other
tissues containing transient receptor potential vanilloid 1 (TRPV1), are triggered
via capsaicin-regulated modulation. TRPV1 activation induces calcium influx and
promotes Kruppel-like factor 2 (Klf2), uncoupling protein 2 (UCP2), LXRα, endo-
thelial nitric oxide synthase (eNOS), peroxisome proliferator-activated receptor delta
(PPAR delta), and gamma (PPAR gamma) expression. In addition, TRPV1 activa-
tion provokes Ca influx which minimizes shear stress and stimulates eNOS, COX-2,
thrombomodulin, and NRF2-responsive antioxidant enzymes expression along with
decreased expression of proinflammatory proteins. LXRα activation via TRPV1-
mediation validates effective cholesterol transfer thereby prevent plaque buildup. In
gastrointestinal (GI) tract, capsaicin-based stimulation of TRPV1 increases meta-
bolic rate via compassionately promoted activation of brown fat. TRPV1 activation
demonstrated defensive antioxidant property through the improved expression of
uncoupling protein-2 (UCP2) in the liver and vascular endothelium during non-
alcoholic fatty liver disease and hyperglycemia perspective, respectively [44, 45].
Capsaicin supplementation effectively decreased the fasting lipid metabolic syn-
dromes and postprandial hyperglycemia as well as hyperinsulinemia in women with
gestational diabetes mellitus (GDM) and also reduced the prevalence of large-for-
gestational-age newborns [46].
Capsaicin reduces viability and propagation, and induces apoptotic death in
pancreatic neuroendocrine tumor (NET) cells. In addition, it also causes loss of
mitochondrial membrane potential; minimizes ATP, reactive oxygen species (ROS),
and mitochondrial Bcl-2 protein synthesis; and increases cytochrome c levels [47].
In male KKAy mice, 0.015% capsaicin supplementation significantly reduced the
plasma and/or liver fasting glucose/insulin and triglyceride concentrations, along
with decreased macrophage intrusion and inflammatory adipocytokine genes expres-
sion (e.g., monocyte chemoattractant protein-1 and interleukin-6). In addition, it also
causes increase in adiponectin gene/protein and its receptor (AdipoR2) expression as
well as activation of hepatic AMP-motivated protein kinase, i.e., a marker of fatty
acid oxidation. It decreases metabolic dysregulation in diabetic and/or obese KKAy
mice through improved adiponectin and its receptor expression [48].
Apolipoprotein M (apoM), mainly found in kidney and liver tissues, is linked
with increased prevalence and development of diabetes and atherosclerosis compli-
cations. Dihydrocapsaicin (DHC) induction significantly decreased the atheroscle-
rotic plaque formation in apoE / mice. Treatment of HepG2 cells with 0, 25, 50,
and 100 μM DHC for 24 h effectively decreased apoM expression at both protein
and mRNA level in HepG2 cells in a dosage- and time-dependent way. DHC
treatment negatively influences the Foxa2 expression but positively affects the
LXRα expression in HepG2 cells. In addition, overexpression of Foxa2 markedly
recompensed the inhibition effect induced by DHC on apoM expression. Treatment
of C57BL/6 mice liver with DHC had significantly lower expression of apoM and
Foxa2 while had higher expression of LXRα [49].
3.4 Antiobesity
Brown fat-specific thermogenic uncoupling protein-1 and bone morphogenetic

protein-8b expression in white adipose tissue (WAT) are stimulated by capsaicin.
Browning of WAT is triggered by capsaicin as it promotes expression and activity of
sirtuin-1 through TRPV1 channel-based raise of intracellular Ca2+ and protein
kinase II and AMP-stimulated kinase phosphorylation. Meanwhile, it also improved
PPARγ 1 coactivator α expression along with increased metabolic and ambulatory
activity. Another possible mechanism involved in WAT browning may include
capsaicin-based stimulation of sirtuin-1-dependent deacetylation of PPARγ and the
transcription factor PRDM-16 and assisted PPARγ-PRDM-16 interaction [50].
Brown adipose tissue (BAT) is involved in energy balance and body fatness
regulation because this is the site of sympathetically activated adaptive nonshivering
thermogenesis. Acute cold exposure activates the BAT in humans which in turn
causes cold-induced increase in whole-body energy expenditure. BAT metabolic
activity was found to be lower in old and obese peoples. BAT serves as energy
dissipating activity in the body therefore negatively correlated with body fatness and
is considered as an effective proactive approach against body fat accumulation.
Repeated exposure to cold induces stimulatory effects through the activation of
transient receptor potential (TRP) channels, which are chemosensitive receptors
for various naturally occurring herbal and food ingredients. Capsaicin and its analog
capsinoids, demonstrative agonists of TRPV1, mimic the effects of cold to decrease
body fatness through the activation and recruitment of BAT [51].
Capsainin treatment of bone marrow mesenchymal stem cells (BMSCs) for 6
days followed by 2 days of adipogenic induction demonstrated a significant decrease
in the cell viability and proliferation in BMSCs. The capsaicin-treated cells were
collected at 2nd, 4th, and 6th days for analysis. After capsaicin exposure, dose and
time-dependent reduction in cell viability and proliferation was observed in BMSCs.
Interestingly, capsaicin increased the production of reactive oxygen species (ROS)
and reactive nitrogen species (RNS) in order to induce cell cycle arrest at G0-G1
along with increased apoptosis. Capsaicin exposure effectively reduced the initiation
of adipogenic differentiation, lipogenesis and maturation with associated repression
of PPARγ, C-enhancer-binding proteins (C/EBPα), fatty acid binding protein-4
(FABP4), and stearoyl-CoA desaturase-1 (SCD-1). Capsaicin-based stimulation for
ROS and RNS synthesis hinders the adipogenic mesenchymal stem cells discrep-
ancy through different possible mechanisms such as inhibit proliferation, induce
apoptosis, or cell cycle arrest [52].
170 M. Imran et al.
Previous studies reported the involvement of TRPV1 pathway in weight man-

agement via enhanced intracellular Ca2+ concentration, although the possible mech-
anism by which dietary capsaicin involved in controlling obesity is not known.
Connexin43 (Cx43) molecules take part in adipocyte differentiation by facilitating
Ca2+ transfer between coupled cells. Role of TRPV1-mediated changes in Cx43-
facilitated adipocyte-adipocyte interaction is not completely understood. In a study,
effort was made to explore either Cx43 take part in TRPV1-facilitated adipocyte
lipid break down in cultured 3 T3-L1 preadipocytes and visceral adipose tissues
from humans, wild-type (WT) and TRPV1-deficient (TRPV1 / ) mice or not.
Findings indicated that capsaicin treatment results in the TRPV1 and Cx43
coexpression along with increased Ca2+ influx in 3 T3-L1 preadipocytes and
endorsed lipid breakdown in cell as revealed by Oil-red O staining. No effect was
observed when capsazepine, a TRPV1 antagonist, and 18 alpha-glycyrrhetinic acid
(18α-GA), a gap-junction inhibitor, were administered. Regular intake of capsaicin
cause weight reduction of perirenal, mesenteric, and testicular adipose tissues in WT
mice fed a high-fat diet. Capsaicin improved the expression levels of cell adhesion
molecule (p-CaM), connexin 43 (Cx43), calmodulin-mediated protein kinase II
(CaMK-II), PPARδ, and hormone-sensitive lipase (HSL) in mesenteric adipose
tissues from WT mice fed a high-fat diet, db/db mice, as well as obese humans;
however, these effects were not observed in TRPV1 / mice. Continuing capsaicin
consumption reduced the serum lipids level and body weights of WT mice, but not
effect high-fat fed TRPV1 / mice. These findings inveterate that capsaicin-medi-
ated TRPV1 stimulation improved Ca2+ invasion in Cx43-assisted adipocyte-adipo-
cyte interaction which in turn endorses lipolysis both in vitro and in vivo. Cx43 is
upregulated by capsaicin intake through activation of TRPV1 to improve visceral fat
remodeling [53].
In mice, capsinoids inhibited fat buildup in vivo and in vitro. Liver, a chief site for
lipid breakdown, was exposed to capsinoids, and the result showed a significant
decrease in HMG-CoA reductase, CPT-1, FAT/CD36, and GLUT4 levels which in
turn increase lipid breakdown in both adipose tissues and liver. Moreover, Oil red O
staining also demonstrated that capsinoids inhibit fat buildup in the adipocytes [54].
Tan et al. [55] develop capsaicin-chitosan microspheres (CCMSs) through ion-
cross-linking and spray drying and exploit its antiobesity suitability in rats (obese).
CCMSs impact on body mass, Lee’s index, fat, and serum lipids levels were
examined. Furthermore, mRNA expression of PPARα, PPARγ, leptin, UCP2,
GPR120, FTO, and adiponectin in the liver was assessed by quantitative real-time
polymerase chain reaction (PCR) while protein expression of adiponectin, leptin,
PPARα, UCP2, and hepatic lipase in serum was assessed by enzyme-linked immu-
nosorbent assay. CCMSs were prepared with 85.17% entrapment efficacy and 8.87%
mean drug loading capability. CCMSs demonstrated to be more efficient in control-
ling body weight, body fat, body mass index (BMI), organ index, fat/body mass
ratio, and serum lipids when compared with Orlistat, capsaicin, and chitosan micro-
spheres. The CCMSs downregulated the leptin expression but improved the expres-
sion of UCP2, PPARγ, PPARα, and adiponectin. Results showed that total
cholesterol, fasting glucose serum levels, and triglyceride levels were also decreased
by capsaicin treatment. Immunoblot exploration and reverse transcription-polymer-

ase chain reaction (RT-PCR) indicated higher expression of adiponectin and other
adipokines containing PPAR-α, PPAR-γ, visfatin, and adipsin, but decreased tumor
necrosis factor-α and IL-6 expression [56].
Hydrogen peroxide (H2O2) at 300–1000 μM level favorably and effectively
stimulated capsaicin-mediated high threshold afferents but not low threshold
stretch-sensitive afferents, which were only stimulated by expressively greater
H2O2 levels. The TRPV1 apponents, capsaicin motivated 86% of high threshold
afferents. The transient receptor potential cation channel (TRPA1) antagonist, HC-
030031, but not the TRPV1 antagonist, capsazepine or the TRPM8 antagonist, M8-
B, significantly inhibited the H2O2-facilitated stimulation of high threshold afferents.
Dimethylthiourea and deferoxamine not altered the H2O2 effect on high threshold
afferents. The results illustrate that H2O2-induced enduring instigation of the most of
capsaicin-sensitive high threshold afferents, while not effect threshold stretch-sen-
sitive afferents, when used in the concentration range determined in swelling or
reperfusion after ischemia [57].
Study was planned with BALB/c mice and they were subjected to ethanol (5 g/kg
body weight) for 6 month to induce oxidative stress. Mice were distributed in four
groups and provided with curcumin (0.016%) or capsaicin (0.014%) containing diets
with or without ethanol. During this observation period, behavioral disorder was
observed in one mouse fed on an alcohol-treated normal diet. Capsaicin treatment
significantly decreased the amount of malondialdehyde and phosphatidylcholine
hydroperoxide levels in the brain tissue extract but catalase and superoxide
dismutase activity remain unaffected [58].
Different cellular processes especially inflammatory injury and antioxidant
homeostasis depend on Heme oxygenase-1 (HO-1), i.e., rate-limiting enzyme in
the heme metabolism. Use of capsaicin monitor serum creatinine and blood urea
nitrogen (BUN) concentrations along with tissue histology improves cisplatin-
induced renal dysfunction. Moreover, capsaicin treatment decreases the expression
of inflammatory mediators and oxidative stress markers for renal injury. It also
persuades HO-1 expression in HK-2 cells and kidney tissues. Shielding properties
of capsaicin were entirely revoked by either knockdown in HK-2 cells or treatment
with the HO inhibitor HO-1 or ZnPP IX [59].
Capsaicin hinders almost 2.5–9% and 5–20% of complex-I activity and 8–75% of
complex-III activity in Bx-panreatic cancer (PC-3) and AsPC-1 cells respectively,
which was mainly caused by superoxide dismutase, catalase, and EUK-134. While,
in normal HPDE-6 cells, capsaicin treatment not effect complex-I or complex-III
activities. Capsaicin treatment effectively reduced the adenosine triphosphate (ATP)
concentration in BxPC-3 and AsPC-1 cells and mitigated by catalase or EUK-134
(synthetic superoxide dismutase). Oxidation of mitochondria-specific cardio lipid
was considerably higher in capsaicin-treated cells. BxPC-3 derived ρ(0) cells lack
mitochondrial DNA and were found to be entirely resilient to capsaicin facilitated
ROS synthesis and apoptosis. Findings tell that cytochrome c release, as well as
caspase-9 and caspase-3 cleavage caused by mitochondrial membrane potential
disruption were effectively obstructed by EUK-134 and catalase in BxPC-3 cells.
172 M. Imran et al.
The capsaicin treatment reduces glutathione level and decreases the enzymatic
performance and expression. Transient transfection results in overexpression of
catalase which provides protection against capsaicin-induced ROS production and
apoptosis. Besides, reduced in SOD activity with an increase in GSSG/GSH levels
was detected in capsaicin (2.5 mg/kg) treated mice tumors [60].
Capsicum oleoresin (75 mg/kg bw/day) is found to be deleteriously linked with
serum cholesterol and triglycerides levels in hypercholesterolemic gerbils [61]. The
red pepper or its active component capsaicin showed a significant reduction in liver
cholesterol and is responsible for enhanced fecal excretion of both free cholesterol
and bile acids in female albino rats. This hypocholesterolemic effect of capsaicin is
likely to be responsible for the presence of common vanillyl moiety [62].
Reduced weight gain and ameliorated hypertrophy of the liver and adipose tissues
were found in male mice treated with high-fat diet, red paprika, and capsanthin. Red
paprika and capsanthin treatment also improved serum lipid profile and adipokine
secretion, and ameliorated hepatic steatosis by hindering fatty acid oxidation and
gluconeogenesis. In epidydimal fatty tissue, red paprika and capsanthin inhibited
adipogenesis and decreased lipid droplet size [63].
3.5 Antiaging
Redox status of the red blood cell is characterized by lower glutathione (GSH)/
oxidized glutathione (GSSG) ratio. In all age groups, GSH level is significantly
influenced by capsaicin treatment which in turn causes a major shift in GSH to
GSSG ratio and changes the cell redox status. The results assertively prove the
antioxidant efficacy of capsaicin and also highlight its ability to modulate the redox
status of red blood cells. This result proposes that food components that serve as
antioxidant increases GSH level and possibly exhibit effective and proactive contri-
bution against age-related ailments [64, 65].
The glutamate (Glu) induced neurotoxic effect negatively affects the cell viability
but capsaicin treatment improved the situation. Use of glutamate (Glu) with capsa-
icin effectively decreased the ROS synthesis and apoptotic neuronal death by a
combined treatment with both phytochemicals. The lower mRNA concentration of
cytoplasmic glutathione peroxidase, Cu/Zn and Mn superoxide dismutases, Bcl-x(L)
and elevated mRNA contents of interleukin-1β, and tumor necrosis factor-α were
successfully reinstated by after treatment with capsaicin and/or resveratrol [66].
Antioxidant activity in the brain and blood of kainic acid (KA) facilitated status
epilepticus model (30 mg/kg) was increased by using capsaicin (0.33 mg/kg or 1 mg/kg).
Moreover, capsaicin treatment effectively decreased the KA-mediated increase in
cytokines IL-1β and TNF-α level in the brain. Results confirmed that combined use
of KA and capsaicin (1 mg/kg) because more reduction in apoptotic cell death in the
cornu ammonis sections of the hippocampus when compared with their individual
use [67]. Acetone (80%) extracted free polyphenols in red Capsicum annuum var.
aviculare (Tepin) induced lipid peroxidation in brain and liver as well as exhibited
the higher Fe(II) chelating ability, hydroxyl (OH) radical scavenging ability than the
bound polyphenols. These free polyphenols caused a significantly higher inhibition

in the malondialdehyde (MDA) production in the brain and liver homogenates in a
dose-dependent manner [4].
3.6 Anti-inflammtory Role
Atherosclerosis refers to chronic vascular inflammation characterized by combina-

tion of endothelial malfunctioning, leukocyte stimulation, lipid accrual, excessive
generation of inflammatory mediators, and ROS. Capsaicin plays protective role for
human umbilical vein endothelial cells (HUVECs) against oxLDL-facilitated dys-
function. Apo B disintegration along with associated diene formation of the Cu-
regulated LDL oxidation is responsible for antioxidant potential of capsaicin. In
HUVECs, capsaicin causes collapse of mitochondrial membrane potential and
obstructs oxLDL induced ROS synthesis, and caspase-3 stimulation. It also avoids
foam cell synthesis in macrophage RAW 264.7 cells [32, 53, 68].
Escherichia coli–derived lipopolysaccharides (EC–LPS) were used at the level of
1 μg/ml to induce inflammation via stimulation of primary peripheral blood mono-
nuclear cells (PBMCs) and U-937 macrophages. Magnetic bead kit analysis showed
that nonivamide decreased the EC-LPS-induced release of IL-6 and TNF-α in
PBMCs and U-937 macrophages. This anti-inflammatory mechanism may involve
MAPK pathway but was independent of NF-κB pathway. In addition, use of
trigeminally active compound and an antagonist of TRPV1 or TRPA1 in combina-
tion for treating U-937 eliminated the anti-inflammatory activity. Results confirmed
that nonivamide exhibited similar anti-inflammatory activity as observed in capsa-
icin and t-pellitorine. In U-937 macrophages, the tested compounds demonstrated an
antiprovocative effect by preventing the EC–LPS-induced activation of the MAPK
pathway. In addition, the TRP channel activation performs a vital role in anti-
inflammatory capacity of capsaicin and nonivamide [69].
Capsaicin limits lipopolysaccharide (LPS)-facilitated IL-1β, IL-6, and TNF-α
formation in a time- and dose-dependent manner. Furthermore, it increases LXRα
expression via PPARγ route. Control of LXRα stimulation by siRNA reduced the
inhibitory effect of capsaicin on LPS-induced IL-1β, IL-6, and TNF-α synthesis. In
addition, LXRα siRNA repealed the inhibitory effect of capsaicin on p65, nuclear
factor (NF-κB) protein expression. So, anti-inflammatory effects of capsaicin are
LXRα-based, as LXRα link the capsaicin facilitated PPARγ stimulation and NF-κB
inactivation in LPS-motivated inflammatory reaction [70] (Table 3).
3.7 Capsaicin in Urological Disorders
Neurogenic bladder is a urological ailment that utterly disturbs the comfort of

patient’s life and is common in patients with severe sclerosis, spinal cord injury,
and other neurological pathologies. Capsaicin has also been evaluated as substitute
treatment of neurogenic bladder disorder [71]. Neurogenic bladder is characterized
174 M. Imran et al.
Table 3 Health perspectives of capsicum annum

Disorders Mechanisms References
Anticancer Induced cell cycle detention at G0/G1 phase [29, 35]
Enhanced FOXO3a expression
Altered histone acetylation [39]
Inhibited the colon cancer cells proliferation [37]
Decreased the formation and secretion of IL-1β, TNF-α, IL-10,
IFN-γ, and IL-1ra
Stimulated the secretion of IFN-γ [40]
Induced apoptosis, and downregulated the tNOX expression
Suppressed the activation of ERK [41]
Reduced the paxillin and FAK phosphorylation
Enhanced regulation of Dhh/Ptch2/Zeb2 members of the [36]
Hedgehog signaling pathway, improve CD24, VEGFA and
TIMP1 and lower CD44 and ALCAM mRNA expression
Reduced the proliferation of renal carcinoma cells [38]
Attenuated transient receptor potential vanilloid type 1
(TRPV1) representative antagonist capsazepine (CPZ)
Stimulated P-38 and JNK MAPK pathways
Cardiovascular Reduced the triacyclglycerols, total cholesterol, non-high- [53]
density lipoprotein cholesterol, and in plasma
Stimulated cholesterol 7α-hydroxylase expression but
negatively influenced hepatic X receptor alpha
Antidiabetic Mediated activation of TRPV1-expressing neurons [44, 45]
hyperglycemia perspective
Reduced reduces mitochondrial Bcl-2 protein production [47]
Increased cytochrome c levels
Reduced triglyceride and fasting glucose amounts [48]
Increased adiponectin gene/protein and its receptor (AdipoR2)
expression
Activated hepatic AMP-activated protein kinase
Antiobesity Improved bone morphogenetic protein-8b and brown fat- [50]
specific thermogenic uncoupling protein-1 expression
Triggered browning of WAT
Repressed initiation of adipocytic differentiation, lipogenesis, [52]
and maturation
Repressed PPARγ, C/EBPα, FABP4, and SCD-1
Inhibited fat accumulation and significant decrease in HMG- [54]
CoA reductase, CPT-1, FAT/CD36 and GLUT4 levels
Decreased the levels of malondialdehyde and [58]
phosphatidylcholine hydroperoxide levels
Enhanced the concentrations of catalase and superoxide
dismutase activity
Antiaging Decreased the KA-facilitated rise in cytokines IL-1β level and [67]
TNF-α declined apoptotic cell death
Anti- Suppressed ROS production [32, 53]
inflammatory
Inhibited the EC-LPS-induced activation of the MAPK [69]
pathway
by two severe conditions namely neurogenic detrusor overactive (NDO) and

detrusor hyperreflexia that results in urgency and rise in urinary frequency, and
sometime more severe complications [71, 72]. Overactive bladder is a clinical
disorder that looks like neurogenic bladder [73], but its causes are not linked with
neurological or urogenital sicknesses [74, 75].
Results of the previous findings reported that use of an alcoholic solvent may
cause annoyance and pelvic discomfort in more than half patients, thereby decreased
the capsaicin effectiveness [76]. The capsaicin or resiniferatoxin (RTX) effect in
urinary tract is related to the action on TRPV1 receptors in sensory fibers and
urothelial cells [77]. In vitro study was conducted by using bladder urothelial cells
from non-neurogenic overactive bladder patients as well as from healthy volunteers.
Results showed significant improvement in TRPV1 expression and activation along
with higher capsaicin sensitivity in nonneurogenic overactive bladder patients [29,
78]. Possible mechanism by which capsaicin exerts its beneficial effect on the
bladder activity is its ability to target and activate the TRPV1 receptors in the C-
fibers. The use of both capsaicin and RTX is still not a routine clinical practice
clinical use of RTX, and capsaicin is not very common but it may serve as a potential
substitute for neurogenic bladder patients who are nonrespondent to conventional
treatment with oral antimuscarinic drugs [71].
4 Conclusion
Capsicum annuum is well known all over the world owing to its taste along with
characteristic smell and taste. It is also a promising source of carotenoids,
capsaicinoids, flavonoids, and vitamin precursors. There is also an emerging trend
to use of capsicum annum polyphenols in food-based products to curtail the various
human degenerative disorders due to its antioxidant and free radical scavenging
properties. This fruit has been proven very effective as antioxidant, anticancer,
cardioprotective role, oxidative stress prevention, antiobesity, antidiabetic, and anti-
microbial activities. Capsaicin is another bioactive compound which is present in
higher concentrations in this fruit as compared to other bioactive moieties. The
composition of capsicum annum polyphenols is significantly varied with genotype,
soil type, temperature, maturity stages, and processing conditions. Capsaicin mark-
edly suppressed the production and secretion of TNF-α, IL-1β, IFN-γ, IL-10, and IL-
1ra in human cancer cell lines. Additionally, capsaicin lowered the proliferation of
renal carcinoma cells; induced apoptotic cell death; attenuated transient receptor
potential vanilloid type 1 (TRPV1); upregulated the proapoptotic genes including c-
myc, FADD, Bax, and cleaved-caspase-3, -8, and -9; and downregulated the Bcl-2
gene. Capsaicin has been proven effectual to reduce the higher glucose levels,
cholesterol and triglycerides concentrations as well as significantly decreased the
CPT-1, HMG-CoA reductase, FAT/CD36, and GLUT4 concentration. These
compounds from capsicum annum markedly caused reduction in level of the cyto-
kines IL-1β and TNF-α in the brain. Moreover, capsaicin also suppresses the
176 M. Imran et al.
lipopolysaccharide (LPS)-facilitated IL-1β, IL-6, and TNF-α formation. Further, it

also enhanced the expression through PPARγ pathway. The current book chapter
summarizes the therapeutic role of Capsicum annuum polyphenols against human
syndromes.
Acknowledgments The authors are thankful to Functional and Nutraceutical Food Research
Section, National Institute of Food Science and Technology, University of Agriculture, Faisalabad,
Pakistan. The project was partially supported by Higher Education Commission, Pakistan, under
Pak-US Science and Technology Cooperation Program Phase IV with a project entitled “Establish-
ment of Functional and Nutraceutical Food Research Section at the National Institute of Food
Science and Technology, University of Agriculture, Faisalabad, Pakistan.”
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Sesame: Bioactive Compounds and Health
Benefits 8
Niti Pathak, Asani Bhaduri, and Ashwani K. Rai
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2 Bioactive Compounds in Sesame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.1 Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2.2 Minor Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
2.3 Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
2.4 Phytates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
2.5 Polyunsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
2.6 Short-Chain Peptides, Protein Hydrolysates, and Their Functional Properties . . . . . . 191
3 Quantitative Insight in Lignan and Tocopherol Content: Interspecific and Intraspecific
Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4 Biotechnological Approaches for Sesame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
4.1 Genome Sequencing of Sesame Crop: Unraveling the Oil Biosynthetic Pathway . . . . 192
4.2 Functional Gene Expression: “Lignan Biosynthetic Genes” . . . . . . . . . . . . . . . . . . . . . . . . . 193
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Abstract
Sesame is a valuable oilseed crop that contains various nutritionally rich bioactive
compounds including lignans, tocopherol homologues, phytosterols, etc. Lignans
are the product of oxidative coupling of β-hydroxyphenylpropane. Sesame has a
N. Pathak
Department of Botany, Deshbandhu College, University of Delhi, Delhi, India
A. Bhaduri
Cluster Innovation Centre, University of Delhi, Delhi, India
A. K. Rai (*)
Department of Botany, Banaras Hindu University, Varanasi, India

182 N. Pathak et al.
combination of glycosylated lignans and oil-dispersed lignans. Based on their

medicinal and pharmacological properties, the most important lignans are
sesamin, sesamol, sesamolin, and sesaminol. Tocopherols (vitamin E compounds)
are the lipid-soluble free radicals and constitute a major part of human diet. In
sesame seeds, α-, γ-, and δ-tocopherols are found as tocopherol homologues. In
addition to lignans and tocopherols, sesame is an important source of phytosterols,
phytates, polyunsaturated fatty acids, and bioactive peptides. However, utilization
potential of many of these compounds has not yet been fully understood. This
chapter delves into the presence of multifarious bioactive components in sesame
seeds, their biosynthetic pathway, and functional importance.
Keywords
Bioactive compounds · Sesame · Sesamin · Sesamolin · Sesamol · Pinoresinol ·
Tocopherol · Phytosterols
Abbreviations
CYP81Q1 Sesamin synthase
DIR1 Dirigent protein
DMPQ 2,3-Dimethyl-5-phytyl-1,4-hydroquinol
VTE1 Tocopherol cyclase
γ-TMT γ-Tocopherol methyltransferase
1 Introduction
Sesame (Sesamum indicum, family Pedaliaceae) is considered as one of the earliest

domesticated crops and oilseed plants known to mankind with its multifarious uses. It is
found in the tropics and subtropics but is most common in the narrower belt closer to the
equator, mostly north of it [1]. Basically, sesame is a crop of the developing countries in
more southern latitudes. For a long time, it is being used in religious rituals in India,
Egypt, and Persian region [2, 3]. Sesame is regarded as “queen of oilseeds” because of
its oil quality [4, 5], sterols, and antioxidative agents, i.e., methylenedioxyphenyl
compounds, sesamin, sesamolin, and tocopherols that act as nutraceuticals and impart
resistance to oil against oxidative deterioration. Further, the composition of dry
decorticated sesame seed per 100 g includes edible portion, i.e., water (3.75 g), energy
(2640 kJ; 631 kcal), protein (20.45 g), fat (61.21 g), carbohydrate (11.73 g), dietary fiber
(11.6 g), high amounts of Ca (60 mg), Mg (345 mg), P (667 mg), K (370 mg), Fe
(6.36 mg), Zn (6.73 mg), vitamin A (66 IU), thiamin (0.70 mg), riboflavin (0.09 mg),
niacin (5.80 mg), folate (115 μg), alpha-tocopherol (1.68 mg), and no ascorbic acid [6].
The presence of oxalic acid in sesame seeds makes it little bitter in taste.
The sesame oil is comprised of 83–90% unsaturated fatty acids that contain
glycerides of oleic acid (36–54%) and linoleic acid (38–49%). Other components
are saturated fatty acids (myristic acid, 0.1% or less; palmitic acid, 8–12%; stearic
acid, 3.5–7%; arachidonic acid, 0.5–1%). The unsaponifiable matter (1.2%) includes
tocopherols and the lignans sesamin (0.1–0.6%), sesamolin (0.25–0.3%), sesamol,
8 Sesame: Bioactive Compounds and Health Benefits 183
Fig. 1 Seed color variation in two sesame species cultivated in India
and sesaminol, which give the oil its resistance to rancidity. Extracted sesame cake
varies in color from light yellow to grayish black, depending on the dominant seed
coat color. Its chemical composition also varies according to cultivar, method of oil
extraction, and the presence of testa. The sesame cake has ample amount of calcium
and phosphate; protein content ranges from 35% to 47% but is deficient in lysine.
Sesame seed color show variation in different species which could partly be
attributed to changes in composition of bioactive phenolics i.e., lignans and tocoph-
erols (Fig. 1). The present chapter provides detailed and updated information on the
bioactive components present in sesame seeds, their importance, and health benefits.
In particular, sesame lignans and tocopherols have been focused at length: the
biosynthetic pathway, the gene regulation, and the biotechnological approaches to
enhance the concentration of lignans and tocopherols in sesame crop.
2 Bioactive Compounds in Sesame
Bioactive compounds are beneficial components in food and are accountable for
disease-preventing properties. They include a range of chemical compounds including
phenolics, carotenoids, phytosterols, and polyunsaturated fatty acids. These com-
pounds are often utilized as antioxidants and other purposes such as inhibiting
cholesterol absorption, blocking the activity of bacterial toxins, etc. Our recent review
on value addition in sesame crop has elaborated the production of value-added
products such as sesame oil and meal, thereby enhancing its economic importance [7].
The target candidates for this study are bioactive compounds such as lignans, tocoph-
erols, phytates, phytosterols, and polyunsaturated fatty acids (Fig. 2).
2.1 Phenolics
Natural phenolic compounds are secondary metabolites, which are widely distrib-
uted in the plant kingdom. Plant phenols are noted for their role in prevention of
Fig. 2 Distribution of bioactive components in sesame seeds
various ailments associated with oxidative stress such as cardiovascular and neuro-
degenerative diseases and cancer [8]. Phenolics are characterized by the presence of
aromatic ring (at least one) coupled with a few hydroxyl groups. The antioxidative
potential of phenolics is very high as these compounds can produce stable radical
intermediates utilizing electrons. Due to their antioxidative potential, they play an
important role in the stabilization of edible oils and protection from off-flavor
formation [8]. Recent findings suggest that phenolics could play an important role
in conditions associated with oxidative stress [9, 10]. The antioxidant property of
sesame seed and its oil along with various health properties is attributed to the
presence of lignans such as sesamin, sesamolin, sesaminol, sesangolin, 2-episalatin,
and tocopherol isomers [11]. Sesame oil is extremely resistant to oxidative rancidity
due to the presence of chemically related compounds sesamol and sesamol dimer.
2.1.1 Lignans: Chemistry and Biosynthesis

The common feature of many natural products is recognized as a C6C3 unit, i.e., a
propylbenzene or phenylpropanoid skeleton [12]. In a review of natural resins,
Haworth was first to suggest (1936) [13] that the class of compounds derived from
two C6C3 units having β,β0 linkage (8–80 bond) should be called as lignans. Lignan is
actually a constituent of lignin, a generic name for the compound resulting from two
p-hydroxyphenylpropane molecules. Sesame seed contains two major groups of
lignans: (1) oil-soluble lignans (sesamin, sesamolin, sesaminol, sesamolinol, and

pinoresinol) and (2) glycosylated water-soluble lignans (sesaminol triglucoside,
pinoresinol triglucoside, sesaminol monoglucoside, pinoresinol monoglucoside,
and two isomers of pinoresinol diglucoside and sesaminol diglucoside) [14–16].
The antioxidative property of sesame seed is associated with lignan components
present in the oil and are unique to sesame. The lignans, sesamin, and sesamolin and
their derivatives prevent oxidation of the oil and give it a long shelf life and stability
[17]. Lignans and tocopherols are also reported to act in coordination resulting in
enhanced vitamin E activity [18]. Namiki [19] suggested that the bioactive com-
pounds present in sesame seed cannot individually describe the high oxidative
stability of roasted sesame oil, but the cumulative effect of all the components of
sesame oil protects the roasted sesame oil from oxidative deterioration.
The oil fraction of most oilseeds comprises mainly of triacylglycerols (95–99%)
acting as a dispersing media for a wide range of lipophilic and amphiphilic second-
ary metabolites, together known as the unsaponifiable fraction of the seed. These
unsaponifiable portions can be extracted with lipophilic solvents after saponification
of acyl lipids [20] and are of importance as the presence of the lignans may be taken
as marker for genuineness of the oil. Other constituents, tocopherols and
tocotrienols, are strong antioxidants and provide stability to the oil. Also, lignans,
especially sesamin, have currently been recognized for possessing interesting phys-
iological bioactivities against chronic diseases.
Sesaminol, a water-soluble glycoside along with fat-soluble sesamin and
sesamolin, constitutes the major lignans of sesame seeds. The isomer of sesamin
called episesamin is generated in the process of refining sesame oil. The functional
methylenedioxyphenyl group of most of these lignans especially sesamin,
sesamolin, and sesangolin initiates the activities [21, 22], and these molecules in
turn utilize their effect via inhibition of liver microsome oxidases [23].
Sesamin Biosynthesis
Phenylalanine and tyrosine are precursors for various plant substances such as
tannins, polymeric lignin, and lignans. Phenylalanine is converted to cinnamic
acid by the enzyme phenylalanine ammonia lyase (PAL). This is followed by a
series of hydroxylation, methylation, and reduction leading to production of
coumaric acid, caffeic acid, ferulic acid, and eventually E-coniferyl alcohol [24].
However, it has also been claimed that [14C]-labeled tyrosine is incorporated into
sesamin when administered to the cell suspension cultures of Sesamum indicum [25].
E-coniferyl alcohol has also been shown to undergo stereoselective coupling
to synthesize (þ)-pinoresinol in Sesamum indicum seeds (Fig. 3). (þ)-Pinoresinol
is then metabolized further, and methylenedioxyphenyl group is added in matur-
ing sesame seeds which result in production of (þ)-piperitol and (þ)-sesamin and
(þ)-sesamolin [26]. A 78 kDa dirigent protein is known to assist in a stereoselective
bimolecular coupling to produce (þ)-pinoresinol [27]. Lignan synthesis is develop-
mentally regulated and depends upon the stage of seed maturity. The most mature
seeds of 8 weeks efficiently convert (þ)-pinoresinol into (þ)-piperitol and (þ)-
sesamin, while younger seeds have higher conversion to (þ)-sesamolin [28].
Fig. 3 Diagrammatic representation of sesamin biosynthetic pathway depicting conversion of

coniferyl alcohol to pinoresinol with unstable intermediate piperitol followed by conversion to
sesamin. Sesamin is further converted to sesamolin. Boxes indicate action of enzymes
Recently, it is shown that the synthesis of sesamin from pinoresinol is catalyzed by

CYP81Q1 in two steps [29].
2.1.2 Tocopherol Chemistry and Biosynthesis

Tocochromanols have both the hydrophobic and hydrophilic elements – these bio-
molecules generally have a lipophilic isoprenoid side chain linked to the membrane
lipids and a polar chromanol ring toward the membrane surface. Tocochromanols
inhibit membrane lipid peroxidation and scavenge reactive oxygen species. It is a
well-known fact that antioxidants neutralize free radicals, thereby preventing DNA
damage. Being scavenger of reactive oxygen species, tocopherols minimize free
radical attack and interrupt lipid peroxidation. In this way, these molecules protect
cell membranes, enable lipid repair and replacement, and are useful in preventing
cancer and heart diseases [30]. Tocopherols are known to play a role in plant
metabolism, for instance, sugar transport from leaves to phloem [31].
Tocopherols are an important group of plant phenolics that have antioxidative
activity and nutritional values [32]. They belong to a family of molecules that have a
chromanol ring (chroman ring with an alcoholic hydroxyl group) and a 12-carbon
aliphatic side chain containing two methyl groups in the middle and two more
methyl groups at the end. Plants synthesize eight different vitamin E forms, includ-
ing α-, β-, γ-, and δ-tocopherols and α-, β-, γ-, and δ-tocotrienols [33]. All tocoph-
erols and tocotrienols consist of a chromanol ring and varying number of methyl
Fig. 4 Diagrammatic representation of tocopherol biosynthetic pathway depicting conversion of p-

hydroxyphenylpyruvate to homogentisate, which gets converted to methylated hydroquinol and
finally to tocopherol homologues
groups on the chromanol ring. Metabolic fate and biological activities of tocols
depend upon their structural features. The entire isoforms act as lipid antioxidants,
and α-tocopherol has the highest vitamin E activity [34, 35]. The difference between
tocopherols and tocotrienols is that tocopherols have a saturated tail, while
tocotrienols have an unsaturated tail.
Higher plants like dicots have tocopherols in almost all parts including roots,
stems, leaves, flowers, fruits, and seeds [36, 37]. However, the total tocopherol
content and different forms of tocopherols in these tissues differ considerably.
Among the tocopherols, α-tocopherol is the predominant form in photosynthetic
tissues such as stems and leaves. In most seed crops, α-tocopherol is present only in a
minor form, and γ- and δ-tocopherols tend to predominate [38].
Tocopherol Biosynthesis
The hydroquinone ring of tocopherol is derived from the shikimate pathway
of aromatic amino acid synthesis (Fig. 4). Biosynthesis of homogentisate, the
precursor for tocopherol, tocotrienol, and plastoquinone, is catalyzed by p-
hydroxyphenylpyruvate dioxygenase (HPPD) [39]. After attachment of the hydro-
phobic side chain by homogentisate phytyl transferase (HPT1/VTE2) [40, 41] and on
methylation (VTE3) [42, 43], 2,3-dimethyl-5-phytyl-1,4-hydroquinol (DMPQ) is
formed, which is converted to γ-tocopherol by tocopherol cyclase (VTE1) [44, 45].
Tocopherol cyclase is considered as a key enzyme in tocopherol biosynthesis. Final

methylation by γ-tocopherol methyltransferase (γ-TMT, VTE4) results in the pro-
duction of α-tocopherol.
2.1.3 Sesame Seed: Lignan and Tocopherol Bioactivities

Sesamin and sesamolin were initially considered as the major lignans in sesame
seeds [46]. Sesaminol, another major lignan, was identified later from sesame seeds
[47]. These seed components possess unique properties, and regular human con-
sumption helps in lowering blood lipids [48] and arachidonic acid levels [49]. The
sesame seed lignans are also known to reduce cholesterol level by inhibiting its
absorption and synthesis simultaneously [50]. These lignans are anticarcinogenic
[51] and anti-inflammatory [52] and are known to increase hepatic fatty acid
oxidation [53]. They have immunomodulatory activities [54] and possess antihyper-
tensive [55, 56] and neuroprotective effects against hypoxia or brain damage [57].
In addition to lignans, multiple tocopherol homologues [α-tocopherol (αT),
δ-tocopherol (δT), and γ-tocopherol (γT), tocotrienols] are also present in sesame
seeds, which possess antioxidative and health-promoting properties. Sesame lignans
are found to exhibit synergistic effect with tocopherols on vitamin E activity and act
as specific inhibitor of fatty acid metabolism in humans [58]. These nutritional
properties of the sesame seeds contributed by the principal factors lignans and
tocopherols have promoted their use in the daily diet worldwide.
Sesame lignans have antioxidant and tocopherol-sparing activities [59–62]. They are
reported to reduce cholesterol level [48, 50, 63] and exhibit antihypertensive [64] and
anti-inflammatory activities [65] as well as affect lipid metabolism by enhancing gene
expression and hepatic enzyme (acyl CoA oxidase, carnitine palmitoyltransferase,
bifunctional enzyme, and 3-ketoacyl-CoA-thiolase) activities involved in fatty acid
oxidation [66, 67]. On the other hand, lignans reduce the activities of enzymes involved
in lipogenesis (acetyl-CoA carboxylase, fatty acid synthase, ATP citrate lyase, glucose-
6-phosphate dehydrogenase, and pyruvate kinase) by altering the gene expression [66].
Hence, sesame plays a crucial role in minimizing the vulnerability and increasing the
safety against atherosclerosis, cancer, and cardiovascular diseases [48, 64, 68].
Thus, sesame has both preventive and therapeutic values in a variety of chronic
diseases, owing to its rich lignan content and its antioxidative, anticholesterolemic,
and antihypertensive properties. Sesame lignans, especially sesamin, are absorbed in
human body, undergo enterohepatic circulation, and are converted into strong
antioxidative metabolites. Intestinal bacteria metabolize them into other bioactive
compounds such as mammalian lignans enterolactone and enterodiol compounds,
which the role in breast, prostate, and colon cancers, bone health, and cardiovascular
diseases is understudy [69–71].
In traditional Chinese and Indian systems of medicine like Ayurveda, sesame
occupies an important position with diverse pharmaceutical application. In China,
sesame oil is used in treatment of toothaches and gum diseases [72]. In India, it is
being used as an antibacterial mouthwash to relieve anxiety and insomnia and in the
treatment of blurred vision, dizziness, and headache [72]. Sesame oil is also noted
for burn-healing effect [73]. Moist-exposed burn ointment (MEBO) a purely herbal
formulation from China contains sesame oil as an active ingredient along with β-
sitosterol, berberine, and other plant extracts in trace amount [74] and is usually used
in managing the burns of the face, neck, and hand [73].
The main antioxidant activity of α-tocopherol is to break the radical chain in
membranes and lipoproteins [75]. Its antioxidant potential along with various func-
tions at the molecular level mitigates the possibility of cardiovascular diseases and
cancers [76, 77]. Other tocols that are present in lesser amount can also perform the
antioxidative and biological activities. Gamma-tocopherol (γ-T), for instance, is
more potent in decreasing platelet aggregation and LDL oxidation and delaying
intra-arterial thrombus formation than that of α-tocopherol [78, 79]. Tocotrienols
inhibit cholesterol biosynthesis [80] and are found effective in reducing the tendency
of breast cancer [81]. Thus, tocopherols have high antioxidant, antitumor, and
hypocholesterolemic potential. γ-Tocopherol is the major tocopherol in sesame,
whereas α- and δ-tocopherols are present in very small amounts. It has been reported
that γ-tocopherol is a more effective antioxidant compared to other tocopherols [82]
but has poor vitamin E activity in biological systems [76].
Tocopherols terminate the recyclable chain reaction of polyunsaturated fatty acid
(PUFA) radicals produced by oxidation of lipid [83]. The lipid peroxy radicals that
are scavenged by tocopherols are converted into tocopheroxyl radicals, which with
the help of ascorbate and other antioxidants are recycled back as the corresponding
tocopherol [84]. In this manner, a tocopherol molecule can undergo in multiple lipid
peroxidation chain-breaking events before it is finally degraded.
2.2 Minor Phenolics
In addition to tocopherols and lignans, sesame contains other phenolics like

naphthoquinone and phenolic acids in trace amounts [85–88]. Sesamol, which is
known as a strong free radical scavenger [89], is also present in sesame oil. It is a free
phenol with methylenediphenoxy group.
2.3 Phytosterols
Phytosterols (sterols and stanols) are plant triterpenes with preventive functions
in many diseases especially cancer. They have been shown to possess antioxidant
[90], anti-inflammatory [91], and antibacterial properties [92]. Similar in structure
to cholesterol (phytosterols have extra methyl group at C-24 position), these plant
sterols, when digested, compete with cholesterol for small intestine absorption leading
to lowering of the cholesterol level in blood [93]. The recommended functional foods
usually contain phytosterols extracted from plant sources, or at times processed foods
have phytosterols as supplement and are sold as cholesterol-lowering foods. Although
corn and legumes are used to extract phytosterols, it is the sesame seeds that have the
highest (400–413 mg 100 g1) amount of phytosterols [94].
In a recent study, Gharby et al. [95] noted that β-sitosterol constitutes a major
portion of phytosterols in sesame seed and oil, and campesterol and stigmasterol are
the other important sterols present. Compared to other phytosterols, β-sitosterol has
been studied more extensively for its beneficial and physiological effect on human
being. β-Sitosterol lowers cholesterol level [96], enhances immunity, and has anti-
inflammatory properties [97]. The other major component is campesterol, which
accounts for about 17.8% of the total sterols. Stigmasterol and Δ5-avenasterol
measure about 6.4% and 10.2%, respectively, in sesame oil. Minor sterols present
are Δ7-stigmasterol and Δ7-avenasterol. The total sterol content in sesame seed oil is
approximately 540 mg/100 g oil.
2.4 Phytates
Phytic acid is a bioactive compound with wide distribution in plant foods. Due to its
molecular structure, phytic acid has affinity to polyvalent cations such as minerals
and trace elements. Phytic acid is one of the most important sources of phosphorus in
plant seeds, and sesame is no exception. In fact, sesame seeds are richer in phytate
than the commonly known legumes. In oil seeds such as sunflower, soybean,
sesame, linseed, and rapeseed, the phytic acid content ranges from 1% to 5.4%
compared to 0.2–2.9% in legumes. A defatted sesame meal has much higher phytate
concentration than that of soybean meal [98]. Graf and Dintzis [99] have measured
5.36% phytic acid content in sesame seeds. Often, phytates are termed as anti-
nutrient for preventing mineral absorption from meal, but it is also seen that phytates
have anticancerous and hypocholesterolemic activities [100, 101].
2.5 Polyunsaturated Fatty Acids
Fatty acids are carboxylic acids with long-chain hydrocarbon side groups derived
from or contained as esterified molecular form in lipids (fat, oil, or wax) of microbes,
animals, and plants usually ranging from 1 to 30 carbon atoms in length attached to a
terminal carboxyl group [102]. Polyunsaturated fatty acids (PUFAs) are long-chain
fatty acids containing two or more double bonds introduced by specific desaturase
enzymes. Over the years, vegetable oils rich in various PUFAs have emerged as
potential dietary elements for normal growth and development of human beings with
considerable biomedical significance. LC-x-3-PUFAs are important for human
health in maintaining the cellular membrane by regulating cholesterol synthesis,
transportation [103], and eicosanoid synthesis [104].
Sesame is a high-energy food containing approximately 50% oil. The fatty acid
composition of sesame oil is highly desirable with about 80–85% unsaturated acids
and only 15–20% of saturated acids. Sesame oil consists mainly of linoleic
(35–50%) and oleic (35–50%) acids, with small amount of palmitic (7–12%) and
stearic (3.5–6%) acids, but with only traces of linolenic acid [105, 106]. Studies
indicate that a high intake of n-6 fatty acids shifts the physiologic state to one that is
pro-thrombotic and pro-aggregatory, characterized by increase in blood viscosity,

vasospasm, and vasoconstriction and decrease in bleeding time. The n-3 fatty acids,
however, have anti-inflammatory, antithrombic, hypolipidemic, and vasodilatory
properties [103]. Sesame in combination with soybean oil increases the vitamin E
activity along with nutritive value of the lipid [19, 107].
Sesame varieties differ greatly in fatty acid contents of their seeds, and this under-
standing could lead to crop engineering with an aim to develop better quality edible oil in
future [108]. Indian sesame germplasms have lower level of saturated fatty acids
compared to other sesame varieties with palmitic acid being the prominent one. C18:1
and C18:2 are the major unsaturated fatty acids present in Indian varieties [109]. In
unsaturated fatty acids, while the content of C18:1 and C18:2 is quite high in sesame oil
(38–49% and 17–43%, respectively), the C18:3 content is below par (<1%) [110]. A
critical analysis of different cultivars is the need of hour, if we are assertive to improve the
nutritional quality of oil by engineering the fatty acid biosynthetic pathway.
2.6 Short-Chain Peptides, Protein Hydrolysates, and Their

Functional Properties
The bioactive polypeptides are amino acid chains joined by amide or peptide bonds,
with molecular weight not exceeding beyond 20 kDa. These are protein fragments
that have independent function in various biochemical, physiological, or cellular
processes. While some of these peptides are formed naturally, it is the artificial
protein hydrolysates that are much in use and are structurally and functionally a
diverse group of peptides. Proteins can be digested by proteases, or specific fragment
can be created in bio-fermenters utilizing microbes [111]. Sesame food preparation is
also an important source of protein [112]. Protein hydrolysates are widely in use as
nutritional supplement, functional ingredient, food flavor enhancer, pharmaceuticals,
and cosmetics [113, 114]. They display hormone- or drug-like activities and based
on the mode of action can be classified as antimicrobial, antithrombotic, antihyper-
tensive, opioid, immunomodulatory, mineral binding, and antioxidative [115]. Use
of papain for producing sesame protein hydrolysates having better functional prop-
erties, high storability, and emulsifying properties than that of the original sesame
protein lysate has been studied by Bandyopadhyay and Ghosh [116].
3 Quantitative Insight in Lignan and Tocopherol Content:

Interspecific and Intraspecific Variation
Sesame crops are of high nutritional value owing to the presence of antioxidants
lignans (sesamin, sesamolin, and sesamol) and tocopherols (α-, γ-, and δ-forms).
Separation of tocopherol homologues in sesame by RPLC on triacontyl (C30) station-
ary phase has been successfully optimized. The reproducibility of the procedure is in
the range 1.7–3.9%, and recovery ranged from 91% to 99% [117]. Lignans and
tocopherol homologue contents in a wide collection of 143 sesame lines (wild species,
landraces, introgressed lines, and cultivars) collected from diverse agroecological

zones of India have been determined by reverse-phase HPLC (RP-HPLC) [118].
Screening of sesame germplasm revealed an exploitable level of variation in major
bioactive compounds. High levels of sesamin and γ-tocopherol suggested for the
efficient introduction of these lines in the trait enhancement of other oilseed crops.
4 Biotechnological Approaches for Sesame
Sesame crop is relatively superior in oil quantity and quality compared to many
major oil crops. The oil content varies from 35% to 60%, but mostly it is about 50%
of seed weight [119]. Therefore, genetic engineering of sesame directed toward
creation of sesame oil having diverse composition of lignans and tocopherols with
high nutritional value is an important biotechnological aspect. With high genetic
diversity, sesame becomes a promising oilseed crop for performing genetic manip-
ulations to obtain high yield and quality oil [120].
4.1 Genome Sequencing of Sesame Crop: Unraveling the Oil

Biosynthetic Pathway
Higher oil content compared to other oilseed crops, combined with small genome
related to oil quality enhancement, makes sesame an invaluable model plant for
studying oil biosynthesis. Wang et al. [121] have done de novo assembling of the
genome, where a set of 12 transcriptomes and 29 resequenced accessions provided
a large resource for exploring the mechanisms underlying different oil content
between sesame and soybean, as well as among the sesame accessions. The results
reveal the presence of whole genome duplication and absence of the Toll/interleukin-
1 receptor domain in resistance genes. Genes and oil biosynthetic pathways respon-
sible for high oil content were determined employing comparative genomic and
transcriptomic analyses. That has indicated for the expansion of type 1 lipid transfer
genes by tandem duplication, the contraction of lipid degradation genes, and the
differential expression of essential genes in the triacylglycerol biosynthesis pathway
in the early stage of seed development [121].
Resequencing data study in 29 sesame accessions from 12 countries suggests that
the high genetic diversity of lipid-related genes is linked with the wide variation in
oil content. Additionally, the results shed light on the pivotal stage of seed develop-
ment, oil accumulation, and potential key genes for sesamin production, an impor-
tant lignan with numerous health benefits. Recently, two more sesame landraces and
the chloroplast genome have been sequenced [121, 122] enriching the sesame
genome dataset. A recent genome-wide association study has identified SiNST1as
the candidate gene for lignin and cellulose biosynthesis, and this gene could indi-
rectly be associated with sesamin and sesamolin content [123]. Thankfully, a few
open access platform like Sinbase is also being maintained where all these datasets
are managed for further analysis and utilization by sesame researchers [121].
4.2 Functional Gene Expression: “Lignan Biosynthetic Genes”
High-throughput sequencing technology has provided ample data on DNA sequences for
the genomes of many plant species. Expressed sequence tags (EST) of many crop species
have been generated, and thousands of sequences have been annotated as putative
functional genes using powerful bioinformatics tools. Impact on breeding programs
could be reached with this approach, because quality of crop plants is a direct function
of their metabolite content [124] and quality of plant tissues determines their commercial
value with respect to flavor, fragrance, shelf life, physical attributes, etc. [125].
Comparative analysis of expressed sequence tags from Sesamum indicum and
Arabidopsis thaliana developing seeds has been done by Suh et al. [126]. The group
could identify the genes involved in accumulation of seed storage products and in the
biosynthesis of lignans, sesamin, and sesamolin. Their study could also identify the
identical and different gene expression profiles during sesame and Arabidopsis seed
development and the genes specific to sesame seeds [126]. Sirato-Yasumoto et al.
[67] reported that sesamin content in sesame seeds was controlled by polygenes,
because F2 populations originating from reciprocal crosses between high sesamin
and normal sesamin varieties showed a continuous distribution in sesamin content,
and correlation coefficients between F2 and F3 generations were positive and highly
significant for sesamin content.
Ono et al. [29] reported that CYP81Q1 is a single gene encoding (þ)-piperitol/
(þ)-sesamin synthase in the Sesamum indicum genome, suggesting that CYP81Q1
is a single enzyme, which catalyzes the formation of (þ)-sesamin from
(þ)-pinoresinol. The CYP81Q1 homologue (CYP81Q3) showed no activity of
(þ)-sesamin biosynthesis, due to (þ)-sesamin deficiency in this species, whereas
S. radiatum showed dual activity of the enzyme. Further, expression profile
of CYP81Q1 gene was temporally consistent with the accumulation pattern of
(þ)-sesamin during seed development. Hata et al. [127] detected sesamin
(using ultra-performance liquid chromatography-fluorescence detection) in ses-
ame leaves of two Japanese sesame varieties “Gomazou” and “Kin-goma,” which
differed in sesamin content of the seed, and probed genotypic differences. The
higher sesamin content of “Gomazou” leaves correlated well with that of seeds and
the expression of the sesamin biosynthetic gene CYP81Q1, indicating that geno-
typic difference of CYP81Q1 gene expression affected leaf sesamin contents.
To comprehend sesame domestication, we examined the expression of sesamin
synthase (CYP81Q1) during capsule maturation in three wild Sesamum spp. and four
sesame cultivars [128]. Among the cultivars, only S. indicum (CO-1) exhibited
transcript abundance of sesamin synthase and high sesamin content similar to
S. malabaricum, whereas other cultivars had low expression, indicating that sesamin
synthase was not favored during domestication.
5 Conclusions
Sesame is no more an “orphan” crop – the widespread collection of varieties and

landraces coupled with extensive research in every aspect has made the crop as the
perfect model system amidst other oilseeds. The superior quality of sesame seed oil
containing a variety of lignans and tocochromanols merits a higher place among the
oilseed crops being consumed worldwide. The diversity of sesame cultivars and their
characterization has given sesame researchers enough impetus to create genetically
engineered sesame varieties with high yield of secondary metabolites. Sesame seeds
are microcapsules for health promotion and disease prevention in humans and an
sustained effort in this area of oilseed research would be of immense value to the
plant breeders as well as consumers.
Acknowledgments Ashwani K Rai gratefully acknowledges the National Academy of Sciences,

India, for awarding NASI-Senior Scientist Platinum Jubilee Fellowship. Niti Pathak wishes to thank
Dr. K V Bhat, NBPGR for the work carried out in his lab.
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Nutritional Quality of Mangifera Species
9
Luis M. Anaya-Esparza and Efigenia Montalvo-González
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
2 Description of M. species Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
3 Traditional Uses and Health Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
4 Postharvest Quality Parameters of Mangifera Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
5 Nutrimental Quality in Mangifera Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
6 Phytochemicals Present in Mangifera Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
6.1 Phytochemicals Present in Mango Pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
6.2 Phytochemicals Present in Mango Peel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
7 Other Mangifera Species with Potential for Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Abstract
Mango is known as the king of the fruits; its nutritional importance, unique flavor,
and delicious taste impart this status as super fruit. Furthermore, it is commer-
cially cultivated in different tropical and subtropical areas in the world. Mangifera
indica is the most important fruit of this genus; over 60 different species of edible
mangoes are grown worldwide; however, the most of them are not marketable
L. M. Anaya-Esparza
Laboratorio de Microbiología de Alimentos, Departamento de Ciencias Pecuarias y Agrícolas,
Universidad de Guadalajara, Centro Universitario de los Altos,
Tepatitlán de Morelos, Jalisco, Mexico
E. Montalvo-González (*)
e-mail: efi[email protected]; [email protected]

202 L. M. Anaya-Esparza and E. Montalvo-González
and underutilized and commonly are denominated as wild mango species, but
they exhibit higher nutritional values. Mango species present morphological,
physiological, sensorial, and nutritional differences among them. However,
most of Mangifera species are characterized by their strong aroma, intense peel
coloration, attractive fragrance, and delicious taste. Also, mango fruit is consid-
ered very healthy, good source of energy, and easily digestible.
Mango pulp and peel are a good source of carbohydrates, dietary fiber, vitamins
(B, C, and E), minerals (Ca, P, Fe, Na, and K), and bioactive compounds (poly-
phenolic compounds, flavonoids, mangiferin, lupeol, and carotenoids). Most of
Mangifera species have good nutrimental quality; their consumption may contrib-
ute nutrition. In general, the commercially (M. indica) and wild mango species can
be considered for many purposes including for processing and for consumption
because mango may be considered an excellent source for improving nutrition.
However, enhanced knowledge of the status of such species and information on
their health benefits is critical in efforts to promote these valuables mango species.
Keywords
Mangifera species · Wild mango · Nutritional quality · Bioactive compounds ·
Health benefits
Abbreviations
AA Ascorbic acid
DF Dietary fiber
DRI Dietary reference intake
DW Dry weight
IDF Insoluble dietary fiber
SDF Soluble dietary fiber
TA Titratable acidity
TDF Total dietary fiber
TSP Total soluble polyphenols
TSS Total soluble solids
1 Introduction
Mango fruit has been recognized as the “king of the fruits,” having socioeconomic
importance in many countries and/or localities. Mango fruit is cultivated commercially
in more than 87 countries around of the world, but also, mango ranks among major
fruits worldwide [1, 2]. Furthermore, mango is a rich source of bioactive compounds
and essential macro- and micronutrients; it is a powerful nutritive fruit, containing most
of the essential substances needed by the human health. Its nutritional importance,
unique flavor, and delicious taste impart this status as super fruit [3].
Mangifera is a genus that belongs to the Anacardiaceae family in the order of
Sapindales, which is native to the Southeast Asia, in particular originated in India,
Indo-Myanmar border, and Bangladesh [4]. Kostermans and Bompard [5] listed 58
9 Nutritional Quality of Mangifera Species 203
species in the genus Mangifera, but today, this number is considerably increased [4];
majority of which were distributed in Asia. Currently, mango is cultivated in
different tropical and subtropical areas worldwide [6, 7], and it has several species,
varieties, and cultivars [8]. Around the world there are more than 150 mango
varieties, and the main producers are India, China, Thailand, Indonesia, and Mexico
[9]. Previous data are about the best-known species, Mangifera indica, a commer-
cially important member of this genus, where the most representative varieties
worldwide are “Tommy-Kent,” “Tommy Atkins,” “Haden,” “Keitt,” and “Ataulfo”
varieties [10]. However, there are another consumable, but not marketable wild
mango species in the genus Mangifera such as M. casturi, M. zeylanica, M. odorata,
M. lalijiwa, M. caesia, M. foetida, M. laurina, and M. pajang, among others [11];
they represent a great potential for food, industrial, and pharmaceutical use. Unfor-
tunately, the wild mangoes are vulnerable and in danger of extension [12–16].
2 Description of M. species Fruits
Mango is a tropical and exotic fruit with many species, varieties, and cultivars [1].
According to Hidayat et al. [6], the classification of Mangifera species is still labile; it
is because of the complexity of the Mangifera genus. Furthermore, several mango
fruit characterizations including pulp color (light, yellow, gold, and orange), pulp
texture (soft, intermediate, or strong), fiber quantity in pulp, juiciness of pulp (slightly
juicy, juicy, or very juicy), pulp aroma (absent, mild, intermediate, or strong), and
eating quality (poor, good, very good, or excellent) have been developed [17]. For
this reason, mangoes are grouped under two broad categories as wild and cultivated
[18]. The fruit description of some edible Mangifera species is presented below.
Mangifera indica fruit is more or less compressed, oblong or sub-uniform, fibrous,
highly variable in size, shape, weight, and peel coloration and has fleshy drupe with
sweet juice and large and compressed seed. In the same way, fruits of M. andamanica
are oval shaped and small in size and weight (11–15 g); their flesh is yellow, fibrous,
and juicy with sweet taste (22 Brix) [19]. Likewise, M. caesia fruit is an ovoid-
oblong drupe, necked at base, and medium sized (12–20 cm), with whitish, yellow-
ish, greenish, or pale brownish, smooth, thin skin (1 mm); the pulp is white, soft,
juicy, and fibrous, with a peculiar sourish taste; it can be sweet, acidic, or both and has
strong smell at maturity [20, 21]. Additionally, the fruits from M. odorata are
obliquely ellipsoid-oblong or oblong drupe, are medium sized (10–13 cm), and
have an average weight of about 200–320 g with a thin skin (3–4 mm) but also are
characterized by their strong odor; the pulp is yellow to orange yellow, firm, fibrous,
and juicy and has sweet to acidic, sweet taste (21 Brix). The fruit peel has greenish
purple color to canary yellow at maturity [19, 20, 22, 23].
With respect to M. sylvatica, the fruit is obliquely ovate, small (8–10 cm long;
27 g), and compressed; the pulp is yellow in color, has a sweet and sour taste, is very
aromatic, and is almost fiberless. Their skin is very thin but has a big kernel (40% of
its weight). The fruits look very much like that of the common mango (M. indica)
[24–26]. M. foetida fruit is obliquely ovoid-oblong or almost globose drupe, small to
medium in size, dirty dark olive green or yellowish green in color with brown
lenticels, fibrous, juicy, savory, and with strong smell and has skin of 5 mm thick
and pale yellowish-white flesh when immature that turns into yellow or golden
yellow when ripe [20, 21]. M. laurina fruit is a drupe-like small mango, obliquely
oblong, small in size (5–7 cm), and medium green turning greenish yellow to yellow
in color at maturity; flesh is yellow, watery, sweet, with a strong resinous taste, juicy,
very acidic, and fibrous [20, 22, 23]. On the other hand, M. pajang fruit is a big drupe
(2–3 kg), brownish, globose to broad-ovoid, 15–20 cm across, and roughish. Flesh is
yellow, fibrous, acidic to acid-sweet, and middy fragrant [20, 21].
Furthermore, the fruits of M. zeylanica have an average weight of 150 g; they
have a thin skin and are watery, sweet, and pleasant in flavor [22]. Also, the fruit of
M. casturi is produced in large racemes of ten or more; fruits are small (50–84 g)
compared to other mango species; immature fruits are green, and when ripe the color
changes to brown or purple black, and it has a shiny surface. The flesh is orange with
fiber; its taste is unique (slightly sweet) similar to lychee fruit [21, 22]. The fruits
from M. lalijiwa exhibited a medium size of fruits (250 g) with green skin; the flesh
is white pale yellow with particular brown honey pockets in the flesh. Fruits are very
sweet and aromatic with a distinguish honey flavor [21].
In the last years, some research has demonstrated the relationship of some wild
mango species, for example, M. odorata and M. foetida also have close relationship
based on internal transcribed spacer nuclear ribosomal DNA [4]. Some authors have
mentioned that M. odorata is a hybrid result from M. indica and M. foetida [27].
Also, a close relationship between M. laurina and M. sylvatica to M. indica was
previously reported [8].
Mango species exhibited morphological, physiological, and sensorial differences
among them. In general, the mango fruit is a simple, large, more or less compressed,
fleshy, and resinous drupe. It varies in size, shape, color, taste, and nutritional value.
However, most of the Mangifera species are characterized by their strong aroma,
intense peel coloration, attractive fragrance, and delicious taste.
3 Traditional Uses and Health Importance
Mango fruits can be eaten immature green, mature green, or ripe [20] and usually are
consumed fresh with or without peel; however, due to the excellent organoleptic
attributes that it exhibits, the pulp is used as an ingredient to elaborate food and
beverage products such as juices, nectars, puree, jam, jelly, yogurt, wine, and others
[2]. On the other hand, in traditional folk medicine, mango pulp is consumed as an
antiparasitic, laxative, and stomachic, among others [1, 28]. According to the World
Health Organization, traditional medicine system based on the use of plants is an
important source of health care [29]. In recent years, secondary metabolites with
known pharmaceutical activities have been extensively investigated as a source of
medical agents [30, 31]. In particular, Mangifera species exhibited several biological
compounds as mangiferin, mangiferic acid, mangiferol, ellagic acid, steroids, alka-
loids, terpenoids, saponins, tannins, and others [31, 32]. In this context, the intake of
Table 1 Physicochemical parameters of different flesh Mangifera species at green mature stage
(GSM) and consumed mature stage (CMS)
Parameter Maturity stage pH AT (% citric acid) TSS ( Brix)
M. indica cv. Tommy-Kent GSM 3.48 4.26 10.7
CMS 4.07 2.39 15.1
M. indica cv. Tommy Atkins GSM 3.62 4.12 10.9
CMS 3.91 2.08 16.7
M. zeylanica GSM 3.53 2.19 11.5
CMS 4.06 1.26 17.7
M. casturi GSM 3.11 5.02 7.2
CMS 4.12 2.40 20.4
M. lalijiwa GSM 3.23 5.49 9.4
CMS 4.37 1.67 17.2
M. odorata GSM 3.90 2.83 6.7
CMS 4.12 2.49 10.6
Source: Barbosa-Gámez et al. [3]
mango pulp promotes strengthening of the body’s defenses, mainly due to the
presence of bioactive compounds in fruit pulp and peel as discussed below.
4 Postharvest Quality Parameters of Mangifera Species
It has been demonstrated that the majority changes related to the quality in mango
fruit occur during its ripening [33, 34]. Barbosa-Gámez et al. [3] evaluated the
changes in physiochemical parameters (pH, titratable acidity, and total soluble
solids) of the pulp of M. casturi, M. lalijiwa, M. odorata, M. zeylanica, and M.
indica cv. Tommy-Kent and Tommy Atkins, which are harvested in two maturity
stages (green mature stage and consumed mature stage). In general, changes in pH,
titratable acidity (TA), and total soluble solids (TSS) were observed in both maturity
stages as shown in Table 1. In all M. species studied, an increase in pH and TSS
content was reported when green to consumed mature stage, while TA decreases.
Furthermore, numerous physical and physiological changes of mango ripening
involving surface color, shape, size, shoulder growth, specific gravity, and firmness
have been correlated with the fruit maturity stage [1].
5 Nutrimental Quality in Mangifera Species
Fruits play an important role in human nutrition by providing additional sources of

energy and bioactive compounds, and their consumption is recommended by many
health organizations [35]. Mango is a popular and economically important fruit
worldwide mainly for its excellent organoleptic properties and nutritional value [32].
According to Masibo and He [36], mango fruit is unique because each of its parts
(pulp, peel, and kernel) is utilizable. In the literature, mango is described as a fruit with
high amounts of water (72–86%) and carbohydrate (9–25%) and low protein
(0.9–5.1%) and lipid (0.2–2.7%) content as presented in Table 2; these values are
depending on each Mangifera species and can vary with the location of cultivation,
variety, and stage of maturity.
In particular, differences in nutrimental contents were observed between
Mangifera species. M. zeylanica exhibited the highest water (86%) and lipid content
(2.7%). On the other hand, M. foetida exhibited the lowest water (72%) content
but the highest carbohydrate (25%) content. With respect to the protein content,
M. lalijiwa (4.7%), M. odorata (4.7%), and M. casturi (5.1%) showed similar values.
Furthermore, some Mangifera species as M. lalijiwa, M. odorata, M. casturi,
M. zeylanica, and M. sylvatica exhibited better nutrimental values compared to
M. indica cv. “Tommy-Kent” and “Tommy Atkins.” The nutrimental quality of the
fruit is influenced by species, variety, nutritional status, and environmental conditions
during growth of the parent plant [1].
The mango pulp is considered very healthy, good source of energy (63–92 kcal),
and easily digestible (Table 2). Furthermore, mango fruit has been considered as
a functional food because it is a source of dietary fiber [37]. The importance of
dietary fiber (DF) content in fruit and its implications in human nutrition and health
as prebiotic and regulating the glucose and lipid levels (cholesterol and tri-
acylglycerols) in blood have been reported previously [38]. The total (TDF), soluble
(SDF), and insoluble (IDF) dietary fiber values in some Mangifera species pulp at
two maturity stages are shown in Table 3. In general, Barbosa-Gámez et al. [3]
reported a decrease in total dietary fiber content when unripe to ripe mango fruit in
all Mangifera species is evaluated. However, all mango species exhibited high
amounts of dietary fiber, but M. lalijiwa showed the highest content in immature
stage (41 g/100 g DW) and M. zeylanica in mature stage (20 g/100 g DW).
Vitamins and mineral contribute a major part of nutrimental content of fruits;
these compounds are essential nutrients that are required for various biochemical
and physiological processes in the body. In the case of mango, significant values of
vitamin C, vitamin E, B vitamins (Table 4), calcium, phosphorous, iron, sodium,
and potassium (Table 5) were found. Ascorbic acid (AA) or vitamin C is the most
important water-soluble antioxidant, usually present and highly bioavailable in
tropical fruits [39]. According to the Institute of Medicine’s Food and Nutrition
Board [40], the dietary reference intake (DRI) for vitamin C is 90 mg/day for males
and 75 mg/day for females. In the case of mango, vitamin C content was dependent
of each species and varies from 47 to 400 mg per 100 g of edible portion; the
highest vitamin C content was found in wild mango species (M. zeylanica,
M. pentandra, and M. pajang). Daily consumption of 100 g/day of mango
(e.g., M. indica by popular) pulp could ensure an intake of 100% of the DRI.
Furthermore, an interesting values of vitamin E (3.4–7.8 mg/100 g) and B vitamins
as niacin (0.6–329 mg/100 g), niacinamide (8–149 mg/100 g), pyridoxine
(4.7–86.2 mg/100 g), riboflavin (0.04–97.6 mg/100 g), and thiamine
(0.05–8 mg/100 g) were found in different Mangifera species, which are another
important vitamin groups for health care [41].
9
Table 2 Nutrimental composition and energy value (per 100 g of edible portion on fresh weight basis) of different Mangifera species
Mango species Energy (kcal) Moisture (g) Ash (g) Total protein (g) Total fat (g) Carbohydrates (g) Total fiber (g) References
M. indica cv. Tommy- 66.3 83.5 3.4 4.04 1.5 9.2 – [3]
Kent
M. indica cv. Tommy 75 85 2.3 3.7 1.8 11.0 – [3]
Atkins
M. caesia 47.8–64 81.2–86.5 0.6 1.0 0.2 14.6–11.9 2.4 [20]

M. foetida 76 72.5–78.5 0.8 0.8–1.4 0.2 17.9–25.4 1.8 [20]
M. odorata 69.3–83 79–80 0.7 0.9–1.0 0.1–1.8 15.6–19 1.4 [20]
M. casturi 89.9 85.1 5.3 5.1 1.5 14 – [3]
M. lalijiwa 89.2 81.2 2.3 4.7 1.6 11.1 – [3]
M. odorata 63.9 80.1 2.7 4.7 2.3 6.1 – [3]
M. zeylanica 92.3 86.3 3.6 4.2 2.7 12.8 – [3]
M. sylvatica 59 85 1.93 5 3.2 1.95 2 [26]
207
Table 3 Soluble dietary fiber (SDF), insoluble dietary fiber (IDF), and total dietary fiber (TDF)
contents (% dry weight) in pulp of different Mangifera species
Mango species Maturity stage SDF IDF TDF
M. indica cv. Tommy-Kent GSM 5.6 8.9 14.5
CMS 5.3 8.1 13.3
M. indica cv. Tommy Atkins GSM 4.7 11.5 16.2
CMS 4.2 5.2 9.4
M. zeylanica GSM 12.5 18.0 30.5
CMS 11.0 8.6 19.6
M. casturi GSM 10.3 15.0 25.3
CMS 6.1 12.5 18.6
M. lalijiwa GSM 6.9 11 17.9
CMS 5.1 8.3 13.4
M. odorata GSM 11.8 29 40.8
CMS 7.3 8.3 15.6
Source: Barbosa-Gámez et al. [3]
Mango is a major source of carbohydrates, dietary fiber, vitamins, and minerals

with high energy value but low in calorie. Fresh as well as processed form of mango
fruit is an important part of people’s diet. Furthermore, most of Mangifera species
have good nutrimental quality; their consumption may contribute nutrition in many
countries around the world. Also, some of these wild mango species have been
domesticated for its use and commercialization.
6 Phytochemicals Present in Mangifera Species
In addition to their delicious taste, refreshing flavor, aroma, and nutritional value,
mango fruit provides “bioactive compounds” to improve human health [34]. Bioactive
compounds are defined as “inherent non-nutrient constituents of food plants with
anticipated health promoting/beneficial and/or toxic effects when ingested” [42].
According to Raman et al. [7], the concentration of these compounds depends on
many factors (e.g., varieties, maturity stage). In general, mango fruit has bioactive
compounds in roots, leaves, bark, seeds, peel, and pulp. The types of compounds
include β-carotene, isoflavones, vitamin C, mangiferin, gallic acid, lupeol, kaempferol,
quercetin, and α-tocopherol, among others [43–45]. All these compounds have great
potential for use in food and pharmaceutical industry (Raman et al. [7]). As mentioned
above mango fruit is consumed with or without peel; for this reason we will focus on
the bioactive compounds present in edible part (pulp and peel) of mango fruit.
6.1 Phytochemicals Present in Mango Pulp
Mango, like most fruits, is an important source of natural compounds considered

as bioactives [34]. In the edible part of mango fruit, compounds as polyphenols,
9
Table 4 Vitamin content in different Mangifera species pulp (mg/100 g DW)

Mango specie Ascorbic acida Vitamin Ea Niacina Niacinamidea Pyridoxinea Riboflavina Thiaminea References
M. indica cv. Tommy-Kent 140 5.19 104.2 28.2 15.1 28.2 5.1 [3, 28]
M. indica cv. Tommy Atkins 64.5 7.8 81.1 46.5 14.6 21.1 4.8 [3]
M. caesia 125 7.4 1.2 0.16 0.05 [20, 46]
M. foetida 122 0.6 0.04 0.09 [20, 45, 46, 48]
M. laurina 135 [46]

M. odorata 47 329.2 149 86.2 97.6 7.2 [3, 46, 48]
M. pajang 403 [48]
M. casturi 100 4.8 87 66 46 25 8 [3]
M. lalijiwa 97.6 3.8 50 8 4.7 7.2 3.1 [3]
M. pentandra 400 [46]
M. longipetiolata 322 [46]
M. zeylanica 400 160 36 30 30 6.8 [3, 46]
a
Results are expressed as mg per 100 g DW
209
Table 5 Mineral content in different Mangifera species pulp (mg per 100 g DW)
Minerals
Mango specie Ca P Fe Na K
M. indica 10 0.13
M. caesia 7 17 0.3 1 120
M. foetida 16 19 0.2 2 361
M. odorata 9 13 0.4 2 187
Source: Lim [20]
phytosterols, isoflavones, and β-carotene, among others, have been identified as

shown in Table 6.
Polyphenols are among the most extensive groups of phytochemicals present in
fruits. Mango is an excellent source of dietary antioxidants as phenolic compounds.
The concentration of phenolic compounds in mango pulp varies with species and/or
variety, ranging from 200 to 3000 mg of gallic acid equivalents per 100 g of edible
portion (Table 6), which exhibited a great antioxidant capacity [46] (Table 7).
According to Palafox-Carlos et al. [34], the major phenolic compounds found
in “Ataulfo” mango pulp are chlorogenic, gallic, protocatechuic, and vanillic acid. In
addition, the authors mentioned that the gallic acid (39%) showed the highest contri-
bution on antioxidant capacity followed by chlorogenic acid (21%), while major
phenolic compounds in M. indica cv. Keitt pulp are gallic acid; mono-, tetra-, and
penta-galloyl glucoside; hydroxybenzoic acid; and gallotannins [47]. The identified
phenolic compounds coincide with those reported by Masibo and He [36] for mango
pulp. Furthermore, Masibo and He [36] mentioned that the flavonoids are the most
abundant polyphenols in our diet; unfortunately, information about flavonoid content
in Mangifera species is scarce, and the values reported for some mango species ranged
from 100 to 500 mg per 100 g of edible portion. Khoo and Ismail [48] have reported
the presence of isoflavones as daidzein and genistein in M. foetida, M. pajang,
and M. odorata pulps. These compounds can act as phytoestrogens, which may
serve as health-promoting compounds in consumer’s diet. M. odorata possessed the
highest daidzein and genistein content (11.6 mg/100 g), followed by M. pajang (9.02 mg/
100 g) and M. foetida (6.81 mg/100 g). In all cases daidzein content was higher than
genistein content. According to the authors, the variability of the isoflavone content in
mango fruits may be influenced by several internal and external factors [7]. Recently,
López-Cobo et al. [49] in three mango cultivars (Keitt, Osteen, and Sensation) from
Mangifera indica reported the presence of Alk(en)ylresorcinols and p-coumaric acid.
Mangiferin is a xanthone that exhibits great potential as antioxidant; it is a
pharmacological active phytochemical and a natural polyphenolic antioxidant [50].
Mangiferin content of mango pulp ranged from 0.078 to 4 mg per 100 g. Further-
more, another phytochemical, lupeol, a well-known triterpene, is also found in
several medicinal plants and fruits, including mango. The lupeol content in mango
pulp from different mango species ranged from 0.006 to 0.181 mg per 100 g of
edible portion. Some authors have mentioned that mangiferin and lupeol can act as
an anti-inflammatory, antidiabetic, and cholesterol-lowering agent [50, 51].
9
Table 6 Bioactive compounds present in different ripe Mangifera species pulp

Total phenolic Total flavonoid Total isoflavones Mangiferin Lupeol (mg/ Total carotene
Mango specie (mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g) 100 g) (mg/100 g) References
M. indica cv. 524a 4.4b 10.67 [3, 28, 50]
Tommy-Kent
M. indica cv. 420a 0.006 [3, 61]
Tommy Atkins
M. indica Ataulfo 80–174 0.010 0.030–0.060 [34, 44]
M. indica B Green 0.253 0.006 [43]
M. indica 0.217 0.181 [43]
Dashehari
M. indica Chousa 0.078 0.023 [43]

M. caesia 2637 550 [46]
M. foetida 2918 550 6.81 4.81 [45, 46,
48]
M. laurina 144 176 [46]
M. odorata 257 202 11.6 3.95 [3, 46, 48]
M. pajang 7055 256 9.02 [48]
M. casturi 1538a [3]
M. lalijiwa 595a [3]
M. pentandra 676 118 [46]
M. longipetiolata 263 129 [46]
M. zeylanica 676a 118 [3, 46]
a
Total soluble polyphenols
b
Mango puree
211
Table 7 Antioxidant capacity by different methods in Mangifera species pulp

IC50
Mangifera species ABTS DPPH (%) (mg/mL) FRAPa References
M. indica 73 10.21 0.64 [46]
M. indica cv. Tommy-Kent 39.20a 5.10a 0.37a [3]
M. indica cv. Tommy Atkins 209.4a 1.46a 12.1a [3]
M. caesia 92 8.14 0.66 [46]
M. foetida 17 43.22 0.62 [46]
M. laurina 56 13.32 0.64 [46]
M. odorata 88.30a 37 20.16 0.52 [3, 46]
M. pajang 19 38 0.49 [46]
M. zeylanica 150a 2.91a 13.15a [3]
M. lalijiwa 130.8a 15.27a 6.94a [3]
M. pentandra 56 13.27 0.65 [46]
M. longipetiolata 90 8.33 0.61 [46]
M. casturi 156a 13.54a 11a [3]
a 1
mmol kg dry weight
The attractive color of mango pulp is mainly due to the presence of abundant β-
carotene, but also, carotenoids are a potent antioxidant with several human health
benefits [52]. Carotene content in Mangifera species ranged from 0.006 to 10.7 mg per
100 g of edible portion. M. foetida and M. odorata are Mangifera fruits which are
considered as underutilized tropical fruits. But, the flesh of these mango species
exhibited an important total carotene content as reported by Khoo et al. [45]; further-
more, carotene content of M. foetida (4.81 mg/100 g) and M. odorata (3.95 mg/100 g)
is comparable to other commercial mangoes. Ajila et al. [53] mentioned that the
yellow-orange flesh or ripened mango is attributable to the presence of carotenes. In
this context, many of the wild mango species exhibited yellow-orange color pulp,
indicating the possible presence of carotenes in their pulps [48].
Furthermore, Vilela et al. [54] mentioned that ripe mango pulp from M. indica cv.
“Tommy Atkins” and other cultivars is a rich source of phytosterols and other lipophilic
phytochemical (Table 8). The major groups of lipophilic compounds in mango pulp are
sterols (947 mg/100 g DW) and fatty acids (949 mg/100 g DW), followed by the steryl
glycosides (201 mg/100 g DW) [54]; some of these lipids have been termed as
“bioactive lipids” because of their potential benefits for human health [55].
6.2 Phytochemicals Present in Mango Peel
Mango peel has been recognized as source for obtaining valuable components [56, 57 ]
as phytochemicals (polyphenols, carotenoids) and vitamins (E and C) with different
health-promoting properties [53], mainly by its antioxidant activity [34]. Moreover,
mango peels are great source of proteins, carbohydrates, and dietary fiber [58–60],
which make it suitable to be processed for value-added applications in functional foods
Table 8 Compounds identified in the lipophilic extracts of ripe mango pulp

Compound Mangifera indica cv. Tommy Atkins (mg per 100 g db)
Fatty acids 940
Saturated 324
Dodecanoic acid 4
Tetradecanoic acid 26
Pentadecanoic acid 2
Hexadecanoic acid 228
Heptadecanoic acid 8
Octadecanoic acid 28
Eicosanoic acid 2
Docosanoic acid 8
Tetracosanoic acid 10
Pentacosadiynoic acid 8
Unsaturated 612
Hexadec-9-enoic acid 91
Heptadec-9-enoic acid 16
Octadeca-9,12-dienoic acid 48
Octadeca-9,12,15-trienoic acid 131
cis-Octadeca-9-enoic acid 205
trans-Octadec-9-enoic acid 122
Diacids 1
Long-chain aliphatic alcohols 104
Sterols 947
β-Sitosterol 571
Campesterol 149
Stigmasterol 68
Steryl glucosides 201
Others 195
α-tocopherol 64
Source: Vilela et al. [54]
and nutraceuticals [61] as discussed below. Currently, mango peel flour is used as
functional ingredient in food products, mainly in bakery products [62].
Ediriweera et al. [63] reported that major lipophilic compounds identified in M.
zeylanica peel (chloroform extract) were 1H-cycloprop[e]azulen-7-ol-decahydro-
1,1,7-trimethyl-4-methylene, β-sitosterol, 9,12-octadecadienoic acid, caryophyllene
oxide, phenol-3-pentadecyl, and α-tocopherol, among others. Also, Kim et al. [47]
informed about the presence of unsaturated fatty acids as oleic acid, linoleic acid,
and ethyl linoleate in peels from Mangifera indica L. cv. “Irwin.”
Hassan et al. [60] reported in Mangifera pajang K. a content of TDF of 72%
(SDF: 33.4% and IDF: 38.8%). Ajila et al. [64] stated mango peel to be a rich source
of fiber, and 30–50% SDF and 50–70% IDF can be considered as a well-balanced
range for maximum health benefits, due to each fraction that has different
physiological effects [65]. Similar results were reported in mango peels by Ajila and
Prasada Rao [66] in “Badami” and “Raspuri” varieties (TDF ranged from 40% to
72% in both cases). Furthermore, DF has been associated with polyphenol com-
pounds and called “antioxidant dietary fiber” [66].
With respect to ascorbic acid, green and ripe mango (Mangifera indica
var. “Chokanan”) peel contained an ascorbic acid of 109 and 52 mg per 100 g
(DW), respectively [62]. Previously, Ajila et al. [53] reported an AA content of
34 and 39 mg per 100 g (DW) in raw and ripe peel of mango “Raspuri” and
“Badami” cultivars, respectively. Also, Sogi et al. [61] informed an AA value of
75 mg/100 g (DW) in mango (Mangifera indica cv. “Tommy Atkins”) peel.
According to Ayala-Zavala et al. [37], mango peel exhibits great agro-industrial
potential for use as functional ingredient or as anti-browning additive in food
processing due to high content level of AA in the samples that act as
natural antioxidant.
Tocopherol species are present in foods; α-tocopherol is most important to
human health [67]. Ajila et al. [56] reported the α-tocopherol content for raw
(10.4 mg ATE per 100 g DW) and ripe (23 mg ATE) mango peels from M. indica
var. Badami. Nonetheless, Abbasi et al. [67] reported a high amount of vitamin E
in peels (ranged from 7 to 43 mg per 100 g DW) than in pulp (ranged from 0.87 to
4.12 mg) of nine “Chinese” mango (M. indica) varieties, and they mentioned that
the variations in results indicate that phytochemical composition in fruits may
greatly be affected by genetic diversity within/among the cultivars and other
factors as maturity stage and harvesting time. These high values show that the
consumption of mango peel can contribute at the dietary needs for intake of
vitamin E.
Concerning with total soluble polyphenol (TSP) compounds, García-Magaña
et al. [58] reported TSP of 6.8 and 4.2 g GAE (per 100 g db), respectively,
in “Ataulfo” and “Tommy Atkins” mangoes. Hassan et al. [60] informed a
concentration of 9.8 g GAE (per 100 g DW) in Bambangan (M. pajang K.)
mango. Furthermore, Sáyago-Ayerdi et al. [68] characterized the hydrolyzable
polyphenol profile in the peels of the “Ataulfo” mango; they reported that mango
peel may contain series of gallotannins between 5 and 13 units that possess
high antioxidant activity. This means that the peel of the mango species can be
considered an excellent source of antioxidants. Shieber et al. [50] reported
the presence of mangiferin, quercetin, and kaempferol and their related
conjugates in mango peel from M. indica cv. “Tommy Atkins.” Prasad et al. [69]
identified six phenolic compounds (pyrogallic acid, gallic acid, catechin,
epicatechin, mangiferin, and rutin) from M. pajang peels. Blancas-Benitez et al.
[70] informed about the presence of chlorogenic acid (82%) and vanillin acid
(17%) in “Ataulfo” mango peel (M. indica). On the other hand, Barreto et al. [71]
studied 16 varieties of mango (M. indica) peel, and they reported minimal differ-
ences in the profiles between cultivars; however, there was considerable variation
in the amounts of the major phenolic compounds. These findings demonstrated
that every mango species is genetically different and unique, as has been pointed in
this study.
7 Other Mangifera Species with Potential for Consumption
Other commercially valued species that produced edible fruits are Mangifera similis,
M. quadrifida, M. griffithii, M. altissima, M. gebede, M. macrocarpa, M. rufocostata,
M. flava, M. applanata, M. macrocarpa, M. duperreana, M. oblongifolia,
M. kashiana, M gracilipes, M. sclerophylla, M. merilli, M. rumphii, M. rigida,
M. quemanga, and M. superba, among others [72]. The ripe fruits are acid-sweet
and have a pleasant flavor [20]. Additionally, there are many cultivars or varieties
from M. indica that exhibit a great potential for their commercialization. According to
many authors, Mangifera family includes many wild Mangifera species [1, 2].
Unfortunately, nutrimental information about these mango species are scarce. Naik
et al. [73] opine that it is the large variability that has hindered the production of the
commercial varieties on a large scale. However, it is true that this large biodiversity
has not been exploited to the full potential [19].
8 Conclusion
Evidence showed that some of the wild Mangifera species such as M. caesia,
M. foetida, M. odorata, M. casturi, M. lalijiwa, M. zeylanica, M. sylvatica, and
others are healthy fruits to consume specially from the nutrimental viewpoint and are
similar or better nutrimental source than the popular M. indica. In general, the
commercially (M. indica) and wild mango species can be considered for many
purposes including for processing and for consumption because mango may be
considered an excellent source for improving nutrition. However, enhanced knowl-
edge of the status of such species is necessary for the conservation of these valuable
species. Also, this chapter offers a better understanding of the nutrimental and
functional potential of these fruit species.
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Part II
Proteins and Their Biological Activity
Plant Proteins from Legumes
10
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2 History of Legumes Cultivation and Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.1 Near East and Western Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2.2 America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.3 Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
2.4 Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
3 Origin of Protein in Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
3.1 An Evolution Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
3.2 Adaptation for N2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
3.3 The Nitrogen Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
3.4 How to Explain the Efficiency of a Tripartite Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
4 Protein Levels Compared to Other Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
4.1 Specific Proteins of Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
4.2 Absolute Nutritional Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
4.3 Digestibility (AntiNutritional Factors) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
5 Food Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
5.1 Traditional Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
5.2 Modern Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
6 Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
6.1 The Proteins of Food Allergies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
6.2 Main Allergens from Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
6.3 The Identified Allergens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
6.4 Effect of Food Processes on Allergen Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
C. Bennetau-Pelissero (*)
Department of Life Science and Health, University of Bordeaux, Bordeaux, France

224 C. Bennetau-Pelissero
Abstract
Legumes are part of the human edible panel since prehistory times but the
remains that reached our last centuries were all from a period posterior to fire
domestication. In all parts of the world where human civilizations developed,
pulses were associated with cereals and the combination of their proteins man-
aged to cover the essential amino-acid requirements of Humans and animals.
Legumes gathering more than 19,000 different species, all present high protein
content due to specific symbiosis with rhizobia and arbuscular mycorrhizae
present in the soils. These associations are thought to originate from first symbi-
otic events dating from more than 60 million years before present. They allow the
plants to fix nitrogen that is used for protein biosynthesis. The nutritional value of
actual pulses is generally higher than that of other crops especially since domes-
tication and the genetic selection processes operated by humans. Beside proteins
with suitable amino-acid profiles, legumes also contain digestible carbohydrates
and some of them also contain fat. In some cases, these fat include polyunsatu-
rated fatty acids that increase further the nutritional value of the corresponding
legumes. However, if such valuable plants managed to survive along geological
periods, it is because their evolution with their environmental pressure lead them
to develop anti-nutritional substances to protect themselves from their predators.
Here will be discussed some of these anti-nutritional substances, the so-called
tannins, phytic acid, saponins, phytoestrogens, lipoxygenase, hemagglutinin,
trypsin inhibitor, as well as allergens. Because all these substances are basically
useful for the crops, it is only during processing that they should be removed.
Therefore, a special focus is made on traditional versus modern recipes and
industrial food processing. Their respective impacts on basic nutritional compo-
nents (amino-acids, fats, carbohydrates, vitamins, and minerals) as well as on the
anti-nutritional factors listed above are examined. Basically, wet processing
which was most frequently developed in the past, associated orf not with fermen-
tation or germination, is also the most efficient in removing all anti-nutritional
factors.
Keywords
Legumes · Prehistoric domestication · Proteins · Amino-acid profiles ·
Anti-nutritional factors · Tannins · Saponins · Phytic Acids · Phytoestrogens ·
Oligosaccharids · Hemagglutinins · Lipoxygenases · Tripsin inhibitors ·
Allergens
1 Introduction
Legumes have been used for thousands of years in human nutrition and constitute
one of the pillar bases of human civilization. Among other early domesticated pulses
are lentils (Lens culinaris, L. Medic.), pea (Pisum sativum, L.), chickpea (Cicer
arietinum), broad bean (Vicia Faba, L.), common bean (Phaseolus vulgaris, L.),
10 Plant Proteins from Legumes 225
lupine (Lupineus albus, L.), peanut (Arachis hypogaea, L.), cowpea (Vigna
unguiculata, L. Walp.), bambara groundnut (Vigna subterranean, L. Verdc), or soy
(Glycine max, L. Merr). In all continents, pulses were domesticated although the
archeological traces of these events are not always easy to show. The origin of high
protein content and quality in legumes is the symbiotic nitrogen fixation by rhizobia
and mycorrhizae in plant root nodules. This leads to increased production of protein
and injects nitrogen into agricultural systems. Nitrogen from rhizobia symbiosis also
provides residual soil nitrogen to subsequent non legume crops. Therefore, legumes
have been used in crop rotations for thousands of years. The proteins of legumes are
not only concentrated but also exhibit interesting amino-acid profiles that make them
attractive for animals and humans. Therefore, and to allow plant survival to pest and
predators, legumes progressively developed an arsenal of biochemical “weapons” to
limit their palatability and digestibility. Thus beside excellent theoretical nutritional
characteristics, legumes developed anti-nutritional factors such as tannins, phytic
acid, saponins, oligosaccharides, hemagglutinins, lipoxygenases, protease inhibi-
tors, or certain polyphenols. These substances help preventing large consumption
of plants in raw and forced humans to invent cooking processes and specific recipes
including fermentation or germination. These traditional processing practices have
recently been renewed thanks to modernization of processing tools and industriali-
zation of the food preparation. These new processes can act only partially on anti-
nutritional agents. This may be a cause of increasing allergy prevalence among
human consumers. Nowadays, modern recording tools allow to show that in all
continents and with all legumes these allergic manifestation due to pulses consump-
tion exist and possibly rise. This review tends to go through all these aspects giving
perspective to the issue of plant proteins in human future.
2 History of Legumes Cultivation and Consumption
Domestication of a crop leads to the appearance of novel traits which can be

favorable to harvest and to human uses. According to Caracuta et al. [1] experiments
conducted on wild modern peas, chickpeas and lentils prove that neither harvesting
of wild stands or cultivation of wild legumes results in profitable yields [2–4].
Therefore the added value got from the actual crops, directly comes from domesti-
cation. Among other traits, the seed-coat should be thinner and smoother in domes-
ticated stocks to facilitate water penetration and germination [5]. However, using this
mutation as a trait of domestication, while exploring antic farming sites, remains
controversial, because several cultured legumes including lentils or grass pea do not
exhibit great differences with their wild relatives [6, 7]. Oppositely, increase in seed
size is considered to be one of the major domestication-traits even though this
increase in seed-size does not occur at the early stage of domestication but rather
later as a result of crop improvements [1]. Legumes have been associated to cereals
as basic foodstuff in the human history for as long as agriculture and settlement
started. The remains from excavations of the Neolithic period already, shows
associations of wheat varieties and barley to lentils and peas in the Near East region
Fig. 1 Diagrammatic representation of the appearance of the main crops during human history as
revealed by remains found in ancient human settlements
where cereal cultivation was shown to be first performed [8], then came chickpea
(Fig. 1). The present chapter shows that all over the world and in all continents,
legumes were domesticated to be part of human protein intake. They were always
associated to cereals which allow the best amino-acid combination to fulfill human
nutritional requirements.
2.1 Near East and Western Europe
2.1.1 Lentils
According to Zohary and Hopf [8], lentils (Lens culinaris L.) are found in remains
which were determined to be as old as agriculture itself. Lentils seem to be closely
associated with the start of wheat and barley cultivation in the Near East. The
presence of small lentil-seeds was shown among remains retrieved from pre-
farming zones of North Syria dated from 10,000 to 9,500 Cal BP (Calibrated
Before Present). A few traces of the use of lentils are then found in Iraq, Iran,
Anatolia, and Jordan. These traces are from the Neolithic period and more pre-
cisely from the ninth millennium Cal BP. and consist of small lentil-seeds
(2.5–3.0 mm in diameter). This size does not allow to conclude on the wild or
cultivated origin of the seeds. Larger amounts of lentil-seeds were also discovered
in later phases of the Neolithic settlement in the Near East. According to Zohary
and Hopf [8], they were found: in Syria (8,250 to 7,950 Cal BP) [9], in Turkey
(7,800 to 7,000 Cal BP) [10], and in Iran (7,500 to 7,000 Cal BP) [11]. At that time,
lentil-seeds had already attained 4.2 mm in diameter which is characteristic of a
development under domestication. Lentils spread to East and then to Western
Europe during the Bonze and the Iron ages, respectively. Because the remains of
lentil-seeds in France seems to be less evident in early and late Bronze age than
they are in Germany, Hungary, and Switzerland, it is assumed that the use of lentil-
seeds spread from East to West during this particular period. However, only the
seed-size of lentils can help to diagnose for domestication. Unfortunately this trait
evolves very slowly and is affected by environmental factors, and therefore, it is

difficult to ascertain that we would be able to determine the one set of Lens
domestication in the future.
2.1.2 Peas
Peas of the Pisum gender were found to be common in the Neolithic agriculture
settlements in Europe. They were found to be closely associated with the wheat and
barley products. According to Zohary and Hopf [8], remains of peas were discovered
in early Neolithic farming villages of the Near East (9,000 to 8,000 Cal BP).
Preserved, since they were carbonized, pea seeds were already present in aceramic
jars of north Iraq, southeast Turkey, and in the prepottery B level in Jericho.
According to the remains, the use of these legumes most probably spread over the
Near East since remains dated from the eighth millennium Cal BP were found in this
area. Remains dated from 7,400 to 7,000 Cal BP [12] show the smooth seed coat
characteristic of domesticated peas. Genetic approaches show that the actual species
Pisum sativum (L.) most probably derives from two main wild species P. elatius and
P. humile which is smaller.
2.1.3 Cicer
Chickpea (Cicer arietinum L.) as other early European cultivated crop seems to
originate from the Near East and deriving from a progenitor C. reticulatum.
According to Garrard [13], remains of C. arietinum were found in Neolithic settle-
ments of the Euphrate area and close to the natural distribution area of its putative
progenitor. Later (10,000 Cal BP), it is recorded in Jericho far from its original
region, and this is an indication of domestication. Establishment of the domesticated
forms and replacement of the wild ancestral populations of European legumes is
thought to have occurred in the Near East. This was performed within a relatively
short time. Chickpea, however, appears as an exception among all other “founder
crops.” When all other crops including legumes are germinating in autumn,
flowering in late winter/early spring and maturating in early summer, chickpea is a
spring sown crop. Although the wild precursor was probably following the general
cycle pattern, chickpea was deliberately sown in late winter (February) and this is
considered to be a strong trait of domestication. As mentioned in [14], the winter
cycle is found in all wild progenitors of the “founder crops” of Near Eastern
agriculture without exception because their harvest yield is far better following
winter germination. Plants are then able to use the winter rainfall for growing. It
appears that chickpea sowing was delayed to avoid the Ascochyta disease whose
pathogen agent is Didymella rabiei. This fungus has the potential to cause total yield
loss on chickpea, and its occurrence is high since 9 fields over 10 can be attacked in a
given area. In recent tests, only one out of ten actual varieties was able to resist to the
fungal attacks when sowed in winter. The disease does not affect the legume sowed
in late winter because of early dryness, and this allows maintaining lower (0.95 tons/
ha rather than 3.0 tons/ha) but consistent yields.
2.1.4 Faba
Broad bean belongs to the section Faba of the genus Vicia. They appear today as big
beans even though it is though that the progenitor of the actual broad bean was
smaller and most probably derives from the Vicia faba var. minor [15]. Quite
recently [1], reported the discovery of large amount of Faba seeds in sites located
in the Galilee region of Israel. According to carbon duration, the seeds were
collected 11,000–10,000 Cal BP and are the oldest Faba seeds ever found so far.
The most ancient datation corresponds with a period when water supply was good
enough for production and harvesting. The authors argue saying that these Faba
seeds were most probably cultured under human input as early as the following
millennium (10,000 Cal BP.) since seeds remains are still of good size indicating
good water supply in a country where dryness was gaining. The earliest remains of
broad beans found in Western Europe were from the Iberic peninsula and were dated
from the late Neolithic times. In contrast, few carbonized remains of broad beans
appear in several Bronze Age sites of the East Mediterranean and Aegean. All
Bronze Age broad beans have relatively small seed and thus most probably belong
to Vicia faba var. minor.
2.2 America
2.2.1 Phaseolus
The common bean Phaseolus vulgaris (L.) presents multiple variations of fresh and
dried varieties. Those include string beans, green beans, French beans, kidney beans,
or pinto beans. These varieties and others provide a third of daily dietary protein in
some cultures of Africa and of Americas. P. vulgaris is originating from Mesoamer-
ica and South America. Its domestication is much more recent than the European
legumes since it is thought to start about 8,000 years before present time [16]. Plant
domestication is often associated with a suite of morphological changes. In the case
of common bean, domestication has led to increases in seed and leaf sizes, as well as
to changes in growth habit and other features. Moreover, these morphological shifts
occurred not once but twice, as common bean was domesticated independently in
Mesoamerica (probably in what is now Mexico) and the Andes [17]. Currently, the
domesticated gene pool of the species seems to be organized into four Mesoamerican
and three Andean races [18, 19]. These two pools were domesticated independently.
However, although the four Mesoamerican races Durango, Jalisco, Mesoamerica,
and Guatemala differ in ecological adaptations, geographic ranges, morpho-agro-
nomic traits, allozyme alleles, and random amplification of polymorphic DNA
(RAPD) markers [18, 19], they probably all derive from a single domestication
event. Indeed, they all present the same predominant phaseolin electrophoretic type
S, and similar amplification fragment length polymorphism (AFLP) patterns [20,
21]. In parallel there are three Andean races: Nueva Granada, Peru, and Chile, which
also differ in morpho-agronomic characters, allozymes, and phaseolin types [19].
This would support multiple domestications, but their geographic ranges overlap,
and they seem to be similar in AFLP patterns [18]. This supports a single origin.
2.2.2 Lupineus
The discovery of the origin of lupine in South America is quite recent. According to
Atchison et al. [22], Lupineus mutabilis, also called tarwi, was domesticated once
and not in the putative south-central Andean core area of early agriculture, but rather
in northern Peru, most likely in the Cajamarca region. This area is included, or close
to, the distribution area of L. piurensis. Therefore, it can be assumed that L. piurensis
is the most likely wild progenitor of the modern lupine L. mutabilis. Demographic
analyses suggest that tarwi split from L. piurensis around 2,600 Cal BP. and suffered
a classical domestication bottleneck. The earliest unequivocal archaeological evi-
dence of domesticated tarwi seeds is from the Mantaro Valley, central Peru 1,800
Cal BP. According to the actual theory, lupine then spread North and South from the
initial area of origin in Peru. Therefore, the pulse went south to Bolivia and north to
Ecuador and Colombia. Lupine arrived then in Europe with Spanish conquistadores,
i.e., in the early fifteenth century.
2.2.3 Arachis
According to Bonavia [23], the geographic area of origin and domestication of the
peanut would be the Huarmey valley, near the Peruvian coast. Most of the
archeological records of fruits of Arachis hypogaea date from approximately
3,500–4,500 years BP. However, based on the distribution and biology of wild
species and landraces of Arachis, the region of origin of the peanut may have been
in the south of Bolivia and the northwest of Argentina [24]. According to Grabiele
[25], radiocarbon-dated macro-botanical remains were dated from approximately
Cal 7,840 year BP. They appeared as wild Arachis species or peanut fruits in early
domestication stages. They were recovered in buried preceramic sites in the lower
western slopes of the Andes in Northern Peru. However, since this region is not
considered as a plant domestication center, the first Arachis species may have
been first cultivated elsewhere in South America earlier than Cal 8,000 year BP.
According to Grabiele et al. [25], A. monticola may be an intermediate tetraploid
ancestor from which A. hypogaea has arisen upon domestication. In addition,
A monticola was most probably obtained from the two diploid species: maternal
(A. duranensis) and paternal (A. ipaensis) wild diploid species of Arachis.
2.3 Africa
2.3.1 Cowpea
The African Vigna studied here are cowpea [V. unguiculata (L.) Walp.] and bambara
groundnut [V. subterranea (L.) Verdc.] [26]. Vigna unguiculata is most probably
originating from sub-Saharan Africa. It was introduced in America during the
seventeenth century by Spanish conquistadores and is now largely cultured and
consumed as black eyed pea in USA and Brazil. According to Kongjaimun et al.
[27], cowpea was domesticated from wild cowpea in Africa. In 2007, D’Andrea
et al. [28] reported the discovery of cowpea remains in Kintampo settlements in
Ghana. Kintampo is a Later Stone Age (LSA) tradition of West Africa dating to
3,600–3,200 Cal BP [29]. One seed collected in the B6B site in the Horizon 4 was
submitted for radiocarbon dating by AMS. The resulting date was of 3,410 60 Cal
BP, at 95.5% c.i. (TO11883). This date confirms a Kintampo association and is
consistent with other determinations obtained from the site. Although to date, the
earliest remains are originating from Western Africa, a Northern or North-Eastern
origin has been argued based on the absence of true ecologically wild cowpea in
West Africa [30]. However, the paucity of available data from eastern African
Countries has precluded a final assessment. Based on the botanical evidences, it is
thought that Vigna unguiculata then spread in Asia. There, the cultivated cowpea or
its weedy relative was subsequently selected for the vegetable crop, yardlong bean
[Vigna unguiculata subsp. sesquipedalis]. The subspecies’ main characteristic is the
length of its pods which can range between 35 and 60 cm long. Nowadays, cowpea
constitutes a major dietary source of protein for many sub-Saharan populations.
When mixed with cereals, protein quality is significantly improved [31].
2.3.2 Bambara Groundnut

The main groundnut originating from Africa is Bambara Vigna subterranean. It is an
important food legume grown widely in semi-arid Africa and closely related to
cowpea (V. unguiculata). Bambara shares much of its area of cultivation and origins
of genetic diversity with cowpea [32]. According to Philippson and Serge Bahuchet
[33], the Bambara African name * /j℧g℧ appeared in the Proto Bantu language
probably incorporated thank to the immigration of people from Proto Benue
Congo origin. This immigration is dated from a climate deterioration that scourged
Sahara and Sahel and which is dated 7,100–6,900 Cal BP. Naming a plant does not
mean that it was domesticated and cultured but at least recognized as a define plant
probably because of human usage. Bambara groundnut appears as two botanical
forms; var. spontanea which is most likely the wild forms and var. subterranea
comprising the cultivated forms. Vigna subterranean spontanea is restricted to an
area from Nigeria to Sudan, with a center of diversity around Cameroon. Vigna
subterranean subterranea is found in many parts of the tropics and particularly in
sub-Saharan Africa. Both the wild and cultivated forms bear 11 pairs of chromo-
somes [34]. As mentioned here, the use of this legume by human populations of
West Africa probably dates from the early proto Bantu period which seems to
coincide with Ceramic Late Stone Age but this still remains to be substantiated by
archeobotany.
2.4 Asia
2.4.1 Glycine
The main legume in Asia is identified as soybean Glycine max. China is the country
of origin of soybean. Deng in [35] reports that soybean was found to be present in the
Neolithic site of Baligang in the Shijiahe period (Cal 4,500 BP). According to
Hymowitz [36], it was domesticated and therefore cultivated in the Eastern half of
North China 3,100 Cal BP. More precisely, soybean domestication probably started
in Liaoning province because the wild soybean grows everywhere and the stages of
evolution are apparent [37]. Glycine max belongs to the subgenus Soja, which also
contains G. soja and G. gracilis. G. soja, is a wild species of soybean, found in many
different environments in many Asian countries [38]. According to cytological,
morphological, and molecular traits, G. soja is most probably the ancestor of
G. max. On the contrary, G. gracilis is most probably semi-wild form of G. max,
which phenotypic traits place it as an intermediate in the speciation between G. max
and G. soja. According to Willis [39], G. max ancestors produce tiny, hard seeds that
are useless for food unless properly prepared. Therefore, the initial use of soya bean
ancestors was essentially as green manure in crop culture rotations. Since then, and
until 1915, Manchuria in North-Eastern China has been the heart of soybean
production in China [40]. The ancient character for soybean (shu) seems to appear
on Zhou dynasty bronze vessels dated around 3,020 Cal BP [36]. Confucius
documents dated from 2,500 Cal BP mentioned soybean as one of the five staple
grains of China. These were foxtail millet (Setaria italica), broomcorn millet
(Panicum miliaceum), rice (Oryza sativa), wheat (Triticum aestivum), and legumes
essentially soybeans (Glycine max).
3 Origin of Protein in Legumes
3.1 An Evolution Process
Legumes are particularly rich in proteins since they produce amino acid from
ammonia. This NH3 is supplied by rhizobia symbiotic organisms which produce it
from aerial Nitrogen (N2). Archeobiology established that the symbiosis between
legumes (Fabaceae) and nitrogen-fixing bacteria, the so-called rhizobia, appeared
Cal. 60 million years BP [41, 42]. This long association probably explains why
adaptative processes gave rise to about 19,500 legume species [43]. Molecular and
genetic studies suggest that rhizobia bacteria progressively associated to the more
widespread and much older endo-mycorrhizal symbiosis [44]. The association
between arbuscular mycoorhizal fungi and plants involve recognition factors gen-
erated by the fungus and named Myc factors [45]. As for mycorrhizal association,
the plant-rhizobia interaction is most generally initiated by a mutual recognition of
molecular signals released by both symbiotic partners. It is characterized by varying
degrees of specificity pre-determined by the nature and profile of seed/root exudates
from the legume, as well as nodulation factors from the rhizobia [46, 47]. The plant
molecules involved in the recognition step are flavonoids [48, 49].
3.2 Adaptation for N2 Fixation
Nowadays, the association between rhizobia bacteria and mycorrhizal fungi give birth to
root nodules able to capture the aerial nitrogen and to inject it into the plant metabolism.
Therefore, legumes receive the bulk of nitrogen fixed by rhizobia in the form of
ammonia, which is incorporated into organic form before being exported from nodules.
The formation of effective root nodules with compatible soil rhizobia allow to reduce
atmospheric N2 into NH3 for bacterial and plant use. The symbiosis requires the close
association of the bacteria and the plant and the nitrogenase enzyme complex that
reduces N2 to NH3 is oxygen labile. Rhizobia as other soil bacteria are obligate aerobes
and require oxygen for respiration and metabolism. Therefore, they should combine two
opposite situations under nitrogen-fixing conditions: oxygen for their own metabolic
requirements and anaerobic conditions for nitrogen fixing. To achieve this paradox, the
plant and the bacteria produce a micro-aerobic environment around nitrogen-fixing
rhizobia in nodules. The outer cells of the nodules play as a barrier to gaseous diffusion
limit the rate of oxygen into the central infected tissue. The outer cells, their bacteroids,
and plant mitochondria consume oxygen as fast as it can enter the nodules. The oxygen
is directly targeted to mitochondria and bacteroids via plant hemoglobins the so-called
leghemoglobins. This insures a low oxygen ratio in the vicinity of the nitrogenase
enzyme complex [50]. If leghemoglobin transcription is prevented (RNAi), this leads to
a higher steady-state level of free oxygen locally a lower ATP/ADP ratio and a complete
absence of nitrogenase activity [51].
3.3 The Nitrogen Uptake
The symbiosis between legumes and rhizobia, at its most basic level, results in the
exchange of reduced carbon from the plant for reduced nitrogen from the bacteria.
Thanks to its photosynthesis the plant produces sucrose which is the primary source
of reduced carbon for nodule metabolism [52]. In nodules, phosphorenolpyruvate
carboxylase and malate dehydrogenase activities divert carbon flux away from
glycolysis to form malate. Malate is considered to be the primary source of carbon
for rhizobia bacteroids. During nodule development, bacteroids are programmed to
fix N2 and not to assimilate NH3 into an organic form. The assimilation of NH3 is left
to the plant, and during nodule development, genes such as those encoding gluta-
mine synthase, glutamate synthase, and aspartate amino transferase are induced.
These enzymes can incorporate NH3 into amino acids for export from nodules [53].
Noteworthy, in legumes of tropical origin, like Glycine max and Vigna unguiculata,
nitrogen is exported from nodules into the plant metabolism as ureides [54]. The
nitrogen exportation from the bacteroids to the plant mainly occurs via ammonia
rather than via any other more elaborated substance, i.e., amino acid. This transfer
most probably occurs in a passive way. However, Glutamate, Asparate, or Glutathi-
one may play a role in the active transport of nitrogenic molecules through the
symbiotic membrane separating the bacteroids and the plant.
3.4 How to Explain the Efficiency of a Tripartite Symbiosis
A co-evolution process is generally put forward to explain the specificity of the

association between legumes and their symbionts [55]. However, according to
Martínez-Romero [56], in some cases there may rather be a constant selection of

micro-symbionts by the plant. The symbiont is selected for its large and fast
capabilities for genetic change or of acquisition of symbiotic genes [57]. Rapid
changes in symbiotic nucleic acid material could have enabled bacteria to adjust and
adapt to the diversification burst of legumes that occurred during the earth evolution.
Many studies have been conducted in order to decipher the mechanism of the
tripartite association between the plant, the bacteria (rhizobium), and the fungus
(mycorrhizae). According to Maillet et al. [58], mycorrhizal fungi of the
genus Glomus and rhizobia secrete very similar lipo-chitooligosaccharide signal
molecules, the so-called Nod factors in rhizobia and Myc factors lipo-chitooligo-
saccharides in arbuscular endo-mycorrhizal fungi. All Nod factors are lipo-chitooli-
gosaccharides with a β-1,4-linked N-acetyl-D-glucosamine backbone of which the
nonreducing sugar moiety is substituted with an acyl chain [59]. In legumes,
rhizobial Nod factors are detected thanks to two different Lys-M-domain receptors
to kinase. These proteins have an extracellular domain containing three Lys-M
domains, a transmembrane domain, and an intracellular kinase domain. Mutations
in either of the genes encoding for these receptors can block Nod factor induced
responses, suggesting that these proteins operate in conjunction [60].
4 Protein Levels Compared to Other Plants
As mentioned previously, legumes are rich in proteins because they can use nitrogen
fixed by their symbiotic nodules to produce endogenous proteins. Therefore when
compared to other plant families, they tend to contain a higher rates of protein
(Fig. 2). Legumes proteins are classified according to their solubility properties in
water, salted water, or ethanol/water solutions. Albumins are soluble in water while
globulins are soluble in salt water solutions, and prolamins are soluble in ethanol/
water solutions.
4.1 Specific Proteins of Legumes
The most abundant proteins in grain legumes are globulins. These are classified as
2S, 7S, and 11S globulins according to their sedimentation coefficients (S) [61].
The two main storage proteins in soybean (Glycin max) are glycinin (11S) and
β-conglycinin (7S) [62]. They can reach 41% of the total of grain weight. The
glycinins are hexamers with molecular masses of 320,000 and 375,000 Da. The
subunits are composed of specific acidic polypeptide chain which molecular mass
is 40,000 Da linked by disulfide bonds. In lupine (Lupinus alba), the storage
proteins are conglutin [63]. They have been classified into four groups: α, β, γ,
and δ conglutins. The α–conglutin is a hexameric protein containing monomeric
unit either acidic: (α) or basic: (β) subunits with molecular weight between 42,000
and 52,000 Da and between 20,000 and 22,000 Da, respectively. The α-conglutin
(11S) may account for about 35–37% of the total seed storage proteins in white
Fig. 2 Protein content of several plants used as human food
lupine seeds [64]. The most abundant lupine globulin is β-conglutin, also named
vicilin, which represents about 44–45% of white lupine seed storage proteins [64].
The β-conglutin (7S) is a trimeric protein. Each of its monomer is formed by three
polypeptides of low (17000–20,000 Da), medium (25,000–46,000 Da), and high
(53,000–64,000 Da) molecular weight. The γ-conglutin accounts for about 5% of
the amount of proteins in mature white lupine seeds [64]. The γ-conglutin (7S) is a
tetrameric protein of about 50,000 Da composed of two subunits with molecular
weight between 17,000 and 29,000 Da linked by disulfide bonds. Finally, δ-
conglutin is the least abundant lupine conglutin, representing 3–4% only of total
conglutin in white lupine [63]. δ-Conglutin (2S) is a monomeric protein consisting
of two subunits of around 4,000 and 9,000 Da, respectively. These subunits are
linked by two disulfide bonds. In pea (Pisum sativum), the globulin are named
vicilin and convicilin (7S) and legumin (11S) [61]. Each group represents 4% and
3%, respectively, of the total weight of pea seeds. According to Barać et al. [65],
vicilin appears as a globulin constituted from subunits with molecular weight of
47,300; 35,000; and 28,700 Da. Three minor subunits also appear in SDS-PAGE
profiles of Tris-extracts with molecular weights of 37,000; 33,300; and 31,800 Da.
Beside, two subunits of convicilin are identified with molecular weight of 77,900
and 72,400 Da. Legumin was identified as a trimer. Each monomer has a molecular
weight of about 63,500 Da. These units associate acidic subunits of 40,890 Da and
basic subunits of 22,300 and 23,100 Da. In broad bean (Vicia faba), the protein
content accounts for 25–30% of the total weight, 75% of which are storage
globulins legumin (11S) and vicilin (7S). The former is two- to threefold more
abundant than the latter. Legumin is then the main storage proteins [66]. As in pea,
legumin in V. faba is a polypeptide with a molecular weight of 60,000–61,000 Da,
which is cleaved in vivo into two components: the α-subunit of 36,200 Da (acidic)
and a β-subunit of 22,000 Da (basic). The native legumin molecule is composed of
six α-β units connected by disulfide bonds [66]. V. faba legumin in vivo is a
hexamer of the basic polypeptide with a molecular weight of 328,000 Da [67].
Vicilin in Vicia faba has a molecular weight of 150,000 Da according to Derbyshire
et al. [67], which is cleaved into subunits of molecular weight comprises between
55,500 and 33,100 Da (46,000, 43,100 and 33,300). In the common bean
(Phaseolus vulgaris), the main globulin is called phaseolin (7S) and is a trimer
formed from three subunits of 46,000; 49,000; and 50,500 Da [68, 69]. P. vulgaris
also contains a legumin like globulin with a molecular weight of 340,000 Da.
Immuno-determinations of the legume proteins show some cross-reactions
between species like between peas and broad beans.
4.2 Absolute Nutritional Value
The nutritional value of different legumes can vary significantly as a result of

their peculiar composition [70]. As examples, the quantity and variety of dietary
fibers and starch, the protein composition, the rate of several anti-nutritional
substances, and the phytochemical content of legumes can influence their dietary
value. Legumes usually contain bioactive compounds, including enzyme inhib-
itors, hemagglutinins (lectins), phytoestrogens, oligosaccharides, saponins, and
other phenolic compounds [70]. These substances can play metabolic roles in
animals and humans who consume these foods frequently. The consumption of
phytochemicals can be either beneficial or adverse and globally require additional
investigations. They can also act synergistically or antagonistically with other
components of the diet, and their mechanisms of action still have to be deciphered
for health and diseases understanding. Some components are also known to
improve their digestibility with food processing and this will be discussed later
on. The absolute nutritional value of a legume takes into account its nutrient
profiles as well as their digestibility.
4.2.1 Amino Acid Profiles

Leguminous proteins are globally low in the essential sulfur containing amino acids,
methionine, cystine and cysteine, methionine, as well as in tryptophan (see Table 1)
and are therefore considered to be an incomplete source of protein [80]. This is true
either for humans or for domestic animal nutrition. Therefore, traditional associa-
tions are observed all around the world in human civilizations like dhal with rice in
India, beans with corn tortillas in Mexico, tofu with rice in Asia, sorghum and
cowpeas in Africa, Bambara groundnut and maize kernels in Zimbabwe, or rice and
beans in Southern Africa and Latin America.
For domestic animals, the association is frequently corn with soybean or wheat
and soybean [81]. For nutritional balance, legumes and cereals are to be consumed in
the ratio 35:65 [82]. Although, this chapter is essentially focused on proteins from
legumes, the traditional edible species also contain fibers, carbohydrates, and fat in
different proportions (Table 2).
Table 1 Amino acid composition of several traditional edible legumes
236
Lentils Pea Chickpea Fava Bean Lupine Peanut Cowpea Bambara Soybean MungBean
L. P. C. V. P. vulgaris L. A. V. V. G. max V. mungo
culinaris sativum arietinum faba mutabilis hipogea unguiculata subterranea
% protein 25.13 26.0 24.4–25.4 32.34 20.99 38.2 22.31** 23 18.8 38.1* 27.5
AA in % Seed Flour Seed Seed Extruded Flour Nut Hydrolysate Nut Flour Seed
protein flour
Asp 10.76 10.4 10.9–11.5 9.45 11.53 8.41 12.03 11.1 14.61 11.89 13.5
Tre 3.12 3.66 2.7–3.0 3.18 4.43 4.32 3.03 3.45 4.43 5.07 3.15
Ser 4.84 4.37 3.3–3.7 4.5 6.24 5.95 5.96 4.60 6.85 5.42 4.95
Glu 14.20 16.6 17.3–17.8 14.79 14.77 26.12 19.32 18.30 20.95 19.65 21.7
Pro 2.40 5.56 3.8–4.1 3.72 5.53 4.27 4.85 3.98 5.36 4.81 4.23
Gly 3.2 4.43 3.4–3.6 3.81 3.95 3.71 6.92 3.78 4.65 4.37 4.26
Ala 3.92 4.53 4.7–5.2 3.63 3.76 2.81 5.20 4.25 5.14 4.27 4.35
Cys 0.88 0.68 0.4–0.6 0.78 0.95 1.26 1.26 0.75 2.4 1.6 0.75
Val 3.98 5.2 4.1–4.6 3.81 4.81 3.52 4.21 5.98 6.24 3.4 5.20
Met 0.82 0.86 4.1–4.6 0.66 1.24 0.32 1.05 1.58 0.64 0.77 1.92
Ile 3.08 3.8 4.5–4.8 3.45 3.81 3.16 3.1 4.58 5.45 3.96 4.74
Leu 6.68 6.36 8.1–8.5 6.54 7.62 7.41 6.22 7.64 10.21 6.76 8.36
Tyr 2.52 3.05 2.6–2.8 2.91 3.57 4.28 4.27 3.16 3.13 3.53 3.27
Phe 4.36 4.54 5.0–5.3 3.75 5.72 3.25 4.09 6.60 7.69 4.33 5.66
Lys 5.72 8,58 6.7–7.0 5.55 7.10 7.59 3.58 7.28 8.02 9.08 4.19
His 2.40 3.4 2.9–3.2 2.31 3.67 3.06 2.17 3.20 3.86 3.81 2.49
Trp 0.80 0.5 0.8–0.9 0.81 1.19 0.31 0.79 ND 0.6 0.5 0.97
Arg 7.52 13.76 8.0–8.5 9.18 6.00 10.86 9.84 7.30 7.48 7.57 6.33
References [71] [72] [73] [74] [75] [72] [76] [77] [78] [72] [79]
* Protein given for seed and AA for flour
** Calculated as the mean of 7 different cultivars
C. Bennetau-Pelissero
Table 2 Average percentages of different constituents in several crops adapted from [82]
Crop Carbohydrates Proteins Fat Fibers
Barley 83% 11% 2% 4%
Oats 74% 12% 5% 10%
Rye 82% 13% 2% 3%
Millet 75% 15% 5% 10%
Wheat 78% 14% 2% 3%
Soybean 30% 42% 21% 10%
Chickpeas 58% 25% 5% 12%
Lupines 50% 40% 7% 43%
Lentils 60% 33% – 11%
4.3 Digestibility (AntiNutritional Factors)
All legumes presenting high protein contents together with other valuable nutrients
(polyunsaturated fatty acids, minerals, fibers) have a theoretical high nutritional
value. However, if such plants get through the geological times until domestication,
it is because despite their nutritional value they manage to limit their destruction by
potential predators, by limiting their crude digestibility and appetence. Looking
closely to their composition, it can be found that all legumes contain anti-nutritional
factors that limit their digestibility and therefore their nutritional interest as crude
matter for animal predators in the wild. As we will see further, the consumption of
these legumes raise significantly when humans were able to apply basic cooking
practices. The main anti-nutritional factors being inventoried here are tannins, phytic
acids, oligosaccharides, lipoxygenases, hemaglutinins, anti-protease factors.
4.3.1 Tannins
Tannins (Fig. 3) mainly contained in the seed coats [83] are defined as water-soluble
polymeric phenolic compounds exhibiting molecular weights from 500 to 3000 that
can combine with proteins, cellulose, gelatin, and pectin to form insoluble com-
plexes [84].
They currently protect the grains against insects, birds, and fungal attacks. The
tannins content of legumes can be rather variable between species but also inside the
same variety (Table 3).
In addition, it was shown that tannins affect the availability of amino acids, the
utilization of protein, and they inhibit the activities of digestive enzymes [87].
Therefore, they tend to reduce the nutritional qualities of plants for their predators
[88]. In domestic animals fed sorghum rich in tannins, it was shown that there were
significant inverse relationships between tannin content and the mean digestibility of
all AA [89]. When fava bean hulls tannins were added to casein diet, the apparent
fecal digestibility of total and individual amino acids was decreased in rats [90]. The
digestibility of proline, glycine, and glutamic acid were the most affected. It was
thought to be due to the interactions of tannins with the proline-rich proteins secreted
by the parotid gland since increasing amount of tannin-rich fava bean hulls caused a
Fig. 3 Chemical structures of some anti-nutritional substances from legumes
Table 3 Tannin content in some legumes

Product Tannin content (g/kg) References
Chickpea (Cicer ariterium) 0.6–2.7 [85]
Cowpea (Vigna sinensis) 1.4–10.2 [85]
Pea (Pisum sativum) 0.6–10.5 [85]
Pigeonpea (Cajanus cajan) 3.8–17.1 [85]
Dry beans (Phaseolus vulgaris) 0.3–12.6 [85]
Kidney beans (Phaseolus vulgaris) 5.3–17.55 [86]
Faba bean (Vicia faba) 0.5–24.1 [85]
Urd bean (Vicia mungo) 8–12 [87]
linear increase in both the relative size of the parotid glands and in the quantity of
proline-rich proteins in the rat’s gland [90]. In addition, proline-rich proteins are
secreted in the saliva and bind dietary tannins in the oral cavity. It was suggested that
this phenomenon protects dietary and endogenous protein (digestive enzymes and
proteins of the luminal side of the intestinal tract). However, if the salivary secretion
is not sufficient, the tannins interactions with digestive enzymes can reduce protein
and amino acid digestibility from tannin-containing diets [90, 91].
4.3.2 Phytic Acid

Cereals and legumes contain phytic acid (myoinositol 1,2,3,4,5,6-hexa-
kisdihydrogen phosphate) at levels ranging from 0.4% to 6.4%, w/w. Phytic acid
Table 4 Phytic acid content in several legumes

Phytic acid (g/kg
Legumes Phytic acid (g/kg) protein) References
Soyabeans (Glycine max) 26 68–76 [93]
Soyabean meal (Glycine max) 32–41 62–78 [94]
Common bean (Phaseolus 8–11 459–578 [95]
vulgaris)
Chick pea (Cicer arieticum) 5–12 29–47 [94]
Pigeon pea (Cajanus cajan) 7–17 29–72 [94]
Mung bean (Vigna radiata) 10–15 45–57 [94]
Urd bean (Vigna mungo) 13–15 46–54 [94]
Lentils (Lens culinaris) 7 27 [94]
(see Fig. 3) is the most preeminent form of phosphate storage form in most seeds. It
accounts for 60–90% of total seed phosphorus [92]. In dicotyledonous seeds, such as
legumes, nuts, and oilseeds, phytic acid is found associated with proteins. The
amount of phytic acid in legumes can vary greatly (see Table 4).
Phytic acid has been reported to interfere with the action of pepsin on several
vegetable and animal proteins in vitro. Its anti-proteolytic action probably goes
through phytate:protein interaction that forms complexes at low pH [96]. Phytic
acid was shown to inhibit trypsin activity in some but not all studies. Phytate was
also shown to significantly (up to 25%) inhibit in vitro digestion of casein [97]. In
addition, fermentation processes of millet, reducing phytate content by 23–26%,
improved in vitro digestibility by 14–26%. Using microbial phytase, which metab-
olize phytate, in poultry or swine, improves phosphorous bioavailability and reduces
the environmental impact of animal manure. Phytase also increased threonine and
valine digestibility [98] and phytate concentration was negatively correlated with the
inherent protein and amino acid digestibility of animal feed. From the animal studies,
it seems that it is the ileal digestibility that is improved. The effects of phytase
supplementation on protein and amino acid digestibility of proteins in several animal
species may be explained by the release of protein from the protein-phytate com-
plexes which are natural in feedstuffs. A second effect can be the prevention of
formation of protein-phytate complexes directly in the gut. A third effect can be the
reduction of the negative effect of phytic acid on pepsin and trypsin activities as well
as to the reduction in endogenous amino acid losses [99].
4.3.3 Oligosaccharides
Legumes are classically rich in oligosaccharides of the raffinose family, namely,
raffinose, stachyose, and verbascose (see Fig. 3 and Table 5).
According to Devindra et al. [106], raffinose family oligosaccharides are galacto-
oligosaccharides that can account for more than 50% of the total soluble sugars in
some cases. These carbohydrates cannot be hydrolyzed and absorbed, since the
human small intestine does not exhibit the appropriate α-galactosidase activity.
Therefore, these sugars are digested by the microorganisms present in the large
240
Table 5 Oligosaccharide content of several legumes

Genus Species (sample nb.) Sucrose Raffinose Stachyose Verbascose References
Lens Lentils 12.15 5.63 39.54 11.51 [100]
Pisum Pea (n = 3) 30.7 1.9 cd 12.3 2.3c 31.2 3.8a 23.9 6.7c [101]
Cicer Chickpea (n = 17) 18.71 0.22 7.35 0.05 15.35 0.07 0.80 0.12 [102]
Vicia Broad bean (n = 3) 45.1 1.4bd 6.6 1.8bd 14.2 2.6d 31.8 5.0b [101]
Phaseolus Kidney bean (n = 5) 38.8 5.2db 7.2 3.0ad 48.9 4.5e 2.4 0.5a [101]
Lupineus White lupine (n = 4) 24.10 0.56 11.97 0.41 51.87 2.52 8.77 1.07 [103]
Arachis Peanuts (n = 2) 14.22 0.32 0.90 0.015 1.58 0.02 0.42 0.005 [104]
Vigna Cowpea (n = 3) 29.3 1.8c 5.0 0.1ad 57.8 1.7c 5.6 1.0a [101]
Vigna Bambara (n = 3) 1.71 0.22 1.19 0.18 [105]
Glycine Soybean (n = 6) 59.6 3.1a 7.7 1.6a 30.9 3.9a 1.6 0.4a [101]
Vigna Mung bean (n = 4) 27.8 0.7c 6.1 0.7abd 23.3 1.9da 36.9 1.8b [101]
Means followed by the same letter within a column are not significantly different at P <0.05.
intestine. This leads to flatus formation [107] because the indigestible raffinose
family sugars are fermented anaerobically by the gut flora. This causes intestinal
discomfort, nausea, abdominal rumbling, and diarrhea [108]. Therefore, these oli-
gosaccharides which are important for the plant yields can be considered as anti-
nutritional agents.
4.3.4 Hemagglutinins
Hemagglutinins are proteins belonging to the lectin family. These molecules are
involved in the defense mechanisms of plants via their antifungal activities [109].
They can represent a large fraction of pulse seed proteins especially in beans. These
proteins are known to bind carbohydrate moieties which can be present on cell
membranes. As such, they can induce cell agglutination. Phytohemagglutinins
(PHA-P) are tetrameric structures associating two types of polypeptide chains called
PHA-E and PHA-L. These peptides bind preferentially either to erythrocytes or to
leukocytes, respectively. Thus, five possible tetrameric isolectins of approximately
120 kDa can be formed, i.e., E4, E3L1, E2L2, E1L3, and L4 randomly [110]. A large
channel is present in the middle of the tetramer that may protect the protein from
proteolytic degradation [111, 112]. The carbohydrate-binding sites of lectins can
recognize not only monosaccharides but also oligo- and polysaccharides. Both PHA
subunits (“E” and “L”) contain an N-glycosylation binding sites. PHA-E is specific
for bisected complex-type N-glycans with an outer Gal and bisecting GlcNAc, while
PHA-L specifically binds tetra- and triantennary complex-type N-glycans with β6-
branching [113, 114]. Close to the carbohydrate binding sites are located the divalent
cations Ca2+ and Mn2+, which maintain an active stable conformation with a great
affinity for sugars [115]. Lectins are resistant to heat and to digestive enzymes, and
they also can bind to the surface of enterocytes. This phenomenon can result in toxic
reactions because of changes in intestinal permeability [116]. It was shown that PHA
can strongly bind to the brush border membrane of the small intestine, and
undigested PHA has a dose-dependent effect on hyperblastosis, and tissue growth
[117]. High doses of dietary lectins also induce the abnormal development of
intestinal microvilli in rats [118] as well as the disruption of enterocytes’ plasma
membrane repair. This effect usually results in necrotic death of the wounded cells
[119]. Blood bioavailability of lectins is poor in normal situation, but it can increase
when the intestine barrier is altered.
In their study [120], Putszai and co-workers shown that lectin from Paseolus
vulgaris were able to decrease casein digestibility in rat. The net protein utilization of
crude bean was 11 and that of casein could be reduced in a dose-dependent manner
when adding increasing amount of lectin extracted from Phaseolus vulgaris. In
humans, the oral acute toxicity of Phaseolus vulgaris lectins is characterized by
nausea, vomiting, bloating, and diarrhea [121]. The legumes’ lectins are not always
as toxic, and several species express moderate agglutinating effects. Finally, oral
administration of certain lectins such as those from Phaseolus vulgaris were found to
induce immunoglobulin (Ig)E-mediated reactions, and sometimes simultaneously
with IgG-mediated reactions [112, 122]. These phenomena take part into the allergic
reactions observed in human with several edible pulses. This will be detailed later.
4.3.5 Lipoxygenase
Lipoxygenases are non-heme, iron-containing enzymes widely distributed in plants,
fungi, and animals [123]. These enzymes catalyze the dioxygenation of polyunsat-
urated fatty acids PUFAs into cell signaling agents used as autocrine, paracrine, or
endocrine signal molecules. Multiple lipoxygenase genes were identified in plants
(at least eight in soybean, Glycine max), in animals (at least seven in the mouse), and
in humans (five pseudogene characterized) [123]. Their importance is not correlated
to their concentration, and cellular effects are now known to be crucial. In plants,
lipoxygenases can be found in all organs. In plants, these substances have been
shown to have a role in the vegetative growth, wounding, herbivore, and pathogen
attack responses and also in mobilization of storage lipids during germination [124].
The role of lipoxygenases in plant defense mechanism was shown to be due to their
implications in the synthetic metabolism of signal molecules such as Jasmonic Acid
and their methylated parent compounds in sorghum [125], in arabidopsis attacked by
aphids [126] or in the pulse Pisum sativum [127]. Lipoxygenase (Lox) enzymes play
a role in the development of unpleasant flavors in foods containing legumes by
oxidation of polyunsaturated fatty acids. This is particularly true for soybean which
contains significant levels of these substances [128]. The action of lipoxygenase is
thus deleterious to the palatability of legumes rich in fatty acid such as soy or peanut
and these enzymes can therefore be considered as anti-nutritional factors.
4.3.6 Saponins
Saponins constitute a class of glycosides found essentially, but not exclusively, in
plants. These substances include a steroidal or triterpene aglycone linked to one, two,
or three saccharide chains. The carbohydrate chains can vary in size and complexity
via ester and/or ether linkages (Fig. 4).
The most common sugars linked to the sapogenin moiety are galactose,
arabinose, xylose, and glucose. Saponins express amphiphilic properties thanks
to the lipophilic and lipophobic characteristics of the aglycone and carbohydrate
moieties, respectively. Saponins occur in numerous edible plants, including
legumes (soya, peas, and beans), root crops (potato, yams, asparagus, and
alliums), or medicinal herbs (ginger). In grain legumes, saponin contents vary
between 0.5% and 5% dry weight, with soybean exhibiting the highest rate
(5.6%) [129]. In vitro, saponins were shown to inhibit, carrier-mediated galactose
transport but not that of L-glucose [130]. Polyethylene glycol 4000, which
transfer through the endothelial barrier is known to proceed via an extracellular
mechanism, is also increased in vitro [130]. This indicates that saponins inhibit
active transport and simultaneously increase the general permeability of the
enterocyte barrier. This phenomenon is observed in vitro at saponins concentra-
tions ranging from 0.3 to 8 mM. Not all saponins exhibit the same potency.
Therefore, it appears that some saponins are able to increase the permeability of
the small intestinal mucosal cells, facilitating the uptake of substances to which
the gut would normally be impermeable. This effect, occurring at rather high
concentration, is likely to have in vivo consequences when the saponins ingestion
is recurrent. These consequences may be deleterious and justify the anti-
Fig. 4 An example of saponin. Here is Bacopa saponins C with its carbohydrate moiety and its
steroidal moiety
nutritional status of saponins. However, saponins also exhibit anti-inflammatory,

immunomodulatory and antifungal and antimicrobial activities and are protecting
agents for the plants.
4.3.7 Anti-Protease Factors

Proteinase inhibitors are ubiquitously produced since their role is to regulate the
proteolytic activity of their target proteinases. They are key factors in living organ-
ism interactions, and they appear essential looking at microorganisms and plants
interactions, or examining microorganisms and animals interactions. They are also
important in the plants and animals interactions. Therefore, many vegetables includ-
ing legumes, cereals, potatoes, and tomatoes contain protease inhibitors that act on
trypsin, chymotrypsin, or carboxypeptidases [92]. Maybe because of its high protein
content, soybean is the richest source of dietary trypsin inhibitors and contains the
Kunitz inhibitors (see Table 6) and the Bowman-Birk inhibitors [108].
The Kunitz inhibitors have a molecular weight of about 21.5 kDa with two disulfide
bridges and act mainly against trypsin. The Bowman-Birk inhibitors have a molecular
weight of about 8 kDa formed by 60–90 AA residues and numerous disulfide bonds.
They mainly inhibit chymotrypsin and trypsin at independent binding sites. The
Bowman-Birk inhibitor (BBI) family is the most widespread group in common bean
(Phaseolus vulgaris) as well as in other legumes such as soybean (Glycine max) and
pea (Pisum sativum). Variants of these two main types of inhibitors have been
characterized with different amino acid sequences, electrophoretic mobility, specific-
ity, and sensitivity to thermal inactivation. The actions of soybean inhibitors were
reported to be similar in rat and humans on trypsin and chymotrypsin [129]. According
to Lin et al. [136], Bowman-Birk inhibitors inhibit trypsin via binding with lysine or
arginine at the P1 residue. Soybean, as one of the most concentrated legumes in anti-
protease, has been largely studied and therefore the anti-nutritional effects of anti-
Table 6 Values of trypsin inhibitor activities in some legumes

Trypsin inhibitor Trypsin inhibitor activity
Legumes activity (mg/g) (mg/g protein) References
Soybean (Glycine max) 16.7–27.2 34.7–122.6 [131]
Pea (Pisum sativum) 2.7 11.9 [132]
Kidney beans 4.6 13.5–62.3 [86, 133]
(Phaseolus vulgaris)
Chick pea (Cicer 1.7–8.5 8–39.5 [134]
ariterium)
Cowpea (Vigna 1.7 6.4 [135]
sinensis)
protease will be largely illustrated from data obtained on this legume. Many studies
reported the deleterious effects of raw soy-protein compared to processed matters on
protein digestibility and animal growth [137, 138]. Raw soybean protein preparations
can cause pancreas hypertrophy and hyperplasia in susceptible animals since the
exposure to soybean trypsin inhibitors results in the increased synthesis and secretion
of proteases (such as trypsin, chymotrypsin, and elastase) [139]. Trypsin and chymo-
trypsin being rich in sulfur-containing amino acids such as methionine and cysteine,
the result of this hyper-secretion is a diversion of these essential AA from the synthesis
of body tissue proteins. This can result in an alteration of growth in domestic animals
since soy protein is also deficient in these amino acids [140]. Direct infusion of the
Bowman-Birk inhibitor purified from soybean into the duodenum of men significantly
increased the pancreatic secretion of trypsin, chymotrypsin, and elastase. This effect
was similar to that observed in rats [108]. Still according to Liener [108], the increased
secretion of sulfur-AA-rich proteases would be due to the suppression of the negative
feedback regulation of pancreatic secretion via an increased release of cholecystokinin
from intestinal mucosa. This release is not only controlled by the protease secretion
[141]. Because of their depressive effects on growth and protein digestibility, protease
inhibitors from legumes can be considered as anti-nutritional factors. The studies
earlier showed that heating and food processing can have beneficial effects on these
anti-nutritional substances.
5 Food Processing
As seen above, legumes are at the same time rich in highly nutritive compounds
and also in anti-nutritional factors. Their human consumption has been signifi-
cantly detected during the Pre-neolithic and Neolithic periods when fire was
already domesticated. Therefore, these pulses were likely to be cooked before
consumption. Indeed, it can be easily demonstrated that the anti-nutritional factors
detailed earlier can be partially or totally inactivated by food processing. Tradi-
tional heating, boiling, cooking, soaking, simmering, germinating, or fermenting
all have been studied and were shown to improve the digestibility of the proteins in
legumes. Modern practices including industrial defatting, roasting, extrusion, or
microwave cooking will be analyzed regarding their effect on anti-nutritional

factors and bioactive polyphenols.
5.1 Traditional Processing
Legume seeds can be eaten raw as for chickpeas or broad beans when they are still
green and at a tender stage (unripe stage) [132]. To improve conservation, legume
seeds are traditionally dried to be consumed at distance of the harvest. Most farmers
in developing countries naturally dry their mature beans under the sun [142].
Because seed coats contain tannins and other anti-nutritional factors, seeds are
often dehulled [142]. Traditional dehulling was performed using a stone grinder
and shaking the resulting grains over large flat baskets. More recently threshing,
shelling, and grading of legume seeds were mechanically improved using machines
[142]. After drying, seeds can be stored for several months before processing. Then
two kinds of processes can be separated: the non-fermentation processes and the
fermentation processes.
5.1.1 Traditional Non-fermentation Processes

These include sprouting of the fresh seeds and traditional soaking and cooking
practices in boiling water. In [143], Khokhar and Chauhan studied domestic methods
of processing and cooking the moth bean. Their studies included soaking in plain
water, soaking in mineral salt solution, ordinary cooking of soaked seeds, sprouting,
and ordinary cooking of sprouts. They described the soaking procedure as follow.
After seed cleaning from broken seeds, dust, and other foreign materials, soaking
was performed in tap water or mixed salt solution for 12 h at tropical temperature
(24 C). A seed to water ratio of 1:5 (w/v) was used. The unimbibed water was
discarded. The soaked seeds were rinsed twice with water and then dried in air. The
ordinary cooking procedure was described as follow. Presoaked seeds, after rinsing
in water, were put in pans and tap water was added in a 3:1 v:w ratio. Cooking was
performed for 60–90 min in water until seeds became soft when felt between fingers.
Water was discarded before seeds were dried in air. Khokhar and Chauhan [143] also
described the domestic sprouting procedure as follow. The soaked seeds were
germinated in clean dishes lined with wet vegetal mesh for 60 h at 25 C, with
frequent watering. Sprouts were then rinsed in water and dried under the sun and
cooked till soft like soaked samples mentioned above. For soybean [142], non-
fermentation processes include the preparation of soy “milk,” soy-cheese (Tofu), and
soy-sheet (Yuba). In all cases, soybeans are cleaned and soaked in water for at least
6 h at room temperature. They can be dehulled before soaking or cooking. For soy-
juice, the traditional practice usually includes a precooking step in water lasting
10–30 min. This step usually allows eliminating dust residues with the first water
discard. Seeds are then crushed in the water before the final cooking step, lasting
additional 10–30 min. Then the water is filtered to collect the juice. The filtered
residue (Okara) can be kept for animal feeding. Tofu was traditionally prepared from
the juice after cooling. Curdling agents are added to the juice. These can be calcium
chloride, calcium sulfate, magnesium chloride, magnesium sulfate, calcium acetate,

or calcium lactate according to Prabhakaran et al. [144]. In China, calcium salts
mined from mountain quarries have been used for over 2000 years [145]. In Japan,
the traditional curdling agent was sea salt that contains small quantity of magnesium
chloride (MgCl2). The curd is then drained on a wooden frame with holes in the
bottom and coated with fabric. The soybean curd is then pressed using a specific lead
to squeeze out the water. This water constitutes the tofu whey. According to
Maneepun [142], Yuba production is a domestic practice only performed at small
scale. During thick soy “milk” prolonged cook, a film is formed at the surface.
Boiling is prolonged until the sheet of film is thick enough, and then it is removed
from the surface of soy “milk” by using a bamboo stick. The wet sheet is placed over
a sheet of thick cloth, and air-dried by hanging. Yuba is used for wrapping meat and
vegetable fillings in Chinese cuisine.
5.1.2 Nutritional Characteristics After Traditional Non-fermentation

Processing
According to the study by El Adawi on chickpea [132], boiled chickpea seeds were
not significantly different from raw peas in total protein, total amino acid, and total
carbohydrate contents. Boiling significantly decreased the non-protein nitrogen, ash,
and fat contents due to their diffusion into the cooking water. Crude fiber was
significantly increased by boiling and or soaking associated to cooking. Water-
soluble vitamins and minerals tend to leak into the water and significantly decrease
in chickpeas on boiling [132]. Equally, Mubarak et al. [79] tested domestic processes
on the anti-nutritional content of mung bean. Among them, they tested traditional
cooking steps such as dehulling, soaking, boiling, and germination. They showed
that the protein content was only slightly affected by soaking and boiling. All
treatments significantly reduced the stachyose and raffinose content of mung bean
and germination reduced it to nothing. Meanwhile, starch content was only reduced
by germination. When considering the anti-nutritional factors, it appears that trypsin
inhibitors and hemagglutinins were destroyed by boiling but only reduced by
dehulling, soaking, or germination. Tannins and phytic acids were significantly
reduced by all treatments, the most effective being germination. Polyphenols
under their glycosylated form also leak into the soaking and cooking water [146]
Germination of chickpea seeds resulted in a significant increase in crude protein,
non-protein nitrogen, and crude fiber compared to raw seeds, while ash was not
significantly affected. Germination also significantly decreased the fat and total
carbohydrate contents. To be more precise, raffinose, stachyose, and verbascose
were completely eliminated by germination and cooking in water for the latter. The
same results were obtained on soybean sprouts [147]. According to El-Adaway
[132], trypsin inhibitor activity was significantly decreased ( 82%) by boiling.
Sprouting was less efficient since it decreased trypsin inhibitor activity by only
34%. Hemagglutinin activity was completely destroyed by cooking and was drasti-
cally reduced (77%) by germination. This was also observed by Khalil & Mansour
[148] on fava bean seeds. According to El-Adaway [132], tannins ( 52%), phytic
acid ( 71%), and saponins ( 48%) in chickpeas were significantly reduced by

simple boiling. Germination was less effective than boiling in reducing tannins
and saponins but more effective in reducing phytic acid. This could be due to the
phytase activity during germination. Polyphenols in legumes are generally present
under their glycosylated forms and as such are soluble in water. Therefore, in all the
preparation associating soaking, boiling, or simmering in water, these polyphenol
concentrations can be reduced. For isoflavones, the longer the water contact the
lower the remaining concentrations [149]. If the soy “milk,” Tofu, and Yuba are
considered, the successive soaking, cooking, and simmering in water classically
included in their traditional recipes were most likely to reduce their isoflavone
concentrations. This is sustained by the study by Liu et al. [150] showing that the
vast majority of rural Chinese women were exposed to isoflavone levels lower than
15 mg/day when the actual exposure in modern Asian countries is usually over
20 mg/day and may go up to 120 mg/day. The estrogenic activities of isoflavones
were observed at already 45 mg/day in American women [151].
5.1.3 Traditional Fermentation Processes

Legumes were fermented either traditionally or recently using several microbial
strains from Lactobacillus, Bacillus, Aspergillus, Rhyzopus, Actinomucor, and
Saccharomyces genders [152]. Traditionally fermented soy-food was quite numer-
ous and still include Natto, Miso, Tempeh, fermented Tofu (Sufu), and soy-sauce
(Si-iu). Natto is produced from soybeans cooked for 2–4 h in renewed water and
dried after water discard. Beans are then wrapped in straw and seeded with Bacillus
subtillis natto. Fermentation is prolonged for 24–48 h. Traditionally in Japan, Miso
is made from soybean cooked in renewed water for 2 to 4 hours before grinding.
Ground soy paste is then mixed with Koji (Aspergilus oryzae) developed on rice
and/or wheat and other microbial strains together with salt and fermented in
anaerobic conditions for 6–8 month. Miso tends to resemble the Chinese soy
paste (Tao-cheow) [142]. Tao-cheow is made by incubating soybean paste with
Aspergillus oryzae or other microbial strain like Lactobacillus delbrulckii,
Pediococcus halophilus, Saccharomyces sp. for 3–4 months. After this first stage
of Koji fermentation, a liquid is formed and separated for production of soy sauce.
The resulting paste is packed and pasteurized and can be kept for several years with
the best flavor after a year. Tempeh is an Indonesian soy-food prepared from
soybeans rinsed and precooked 2–3 times in renewed water before being light
dried and seeded with Rhyzopus oligosporus. Traditionally, precooked seed were
packed in banana leaves and the fermentation was performed for 24–48 h at
tropical temperature 24–26 C, with high hygrometry [149]. Si-iu production as
described in [142] includes two fermentation steps. The first stage is koji produc-
tion using Aspergillus orizae in an aerobic solid-state fermentation for 3–4 months.
The second stage is aromi fermentation by mixed cultures of halophilic yeast and
lactic acid bacteria. This is an aerobic fermentation in 20–22 percent (w/v) brine
solution leading to a bright reddish-brown colored sauce with pleasant aroma and
salty taste [142].
5.1.4 Nutritional Characteristics After Traditional Fermentation

Processing
Fermentation usually induces phytate hydrolysis because microorganisms possess
phytase enzymes, which hydrolyze phytic acid into inositol phosphates [153]. This
phenomenon is valuable because myoinositol phosphates with less than five phos-
phate groups (i.e., IP-1–IP-4) do not inhibit zinc absorption [154], and those with
less than three phosphate groups do not affect nonheme iron absorption [155, 156].
Microbial phytases originate either from the microflora present on the surface of
legumes or from inoculates used for processing. The phytate reduction can vary and
can reach 90% in soya beans, cowpeas, and lima beans. When tannins content are
high, the phytase activity can be inhibited, and fermentation is less-effective.
Fermentation also improves protein quality and digestibility as well as vitamin B
content. Fermentation reduces adverse microbial development and improves food
safety. Fermentation also produces small organic acids that improve iron and zinc
absorption. This leads to lower pH and increases the activity of endogenous phytase
from legume flours [157]. Ibrahim et al. [158] also showed in cowpea that fermen-
tation with Rhizopus oligosporus dramatically reduced trypsin inhibitor activities. It
was the same when fermentation was performed using a lactic acid bacteria Lacto-
bacillus plantarum DSM 20205. Note that again in all recipes soya beans were
traditionally cooked for long durations (2–4 h) in water and that the water was
discarded. Therefore, glycosylated isoflavones were most likely eliminated with the
water revealing that the isoflavone exposure probably raised dramatically in recent
times in soy consuming countries where industrial processing were developed.
Indeed, in industrial Asian countries, the modernization of human soy-food pro-
cessing occurred 50 years ago, i.e., about two human generations ago.
5.2 Modern Processing
Soy being widely used in industrial countries, either for animal or human feeding, is
probably the legume which has been the most intensively processed to adapt to
different people tasting and uses. New processes are still extensively developed to fit
new demands [145]. As an example, food companies developed soy cakes, soy
flakes, soy protein concentrates or isolates, meat substitutes proteins and extenders,
and improved the quality of final products. Modern processes include high pressure
cooking, autoclave cooking, microwave cooking, and extrusion. Solvent extractions
are also modern processing.
5.2.1 Non-Fermented Modern Legume Products

According to Noguchi [145], modern Tofu is made from grade soybeans soaked
overnight in water. After water discard, boiling water is poured on the beans which
are pulverized into a mash. The paste is then ladled into boiling water and allowed to
boil gently for about 10 min. Then the mixture is filtered to obtain “soy milk” and the
residual material is called Okara. In Western countries, the process can be even
simpler since after boiling for 1–2 min, softened soya beans are ground in cooking
water before being filtered to collect “soy milk” and Okara. In Japan, modern
cooking of the beans were developed consisting in quick steaming at high temper-
ature (45 seconds at 180 C) before crushing and water addition. Subsequent
filtration leads to the separation of “soy milk” and Okara. A small amount of either
calcium sulfate (CaSO4) or magnesium chloride (MgCl2) from gypsum is added to
coagulate “soy milk.” The curds are then gently removed from the top of the whey
and poured into molds lined with cloth. The containers have many draining holes in
their bottom to evacuate whey. Other derivatives from “soy milk” or Tofu are Yuba
(soymilk sheets) and Shimi-tofu. Yuba is made heating thick “soy milk” in pans to
evaporate the water. The thin film formed on the surface of “soy milk” is then gently
removed and dried. Shimi-tofu is prepared from soy protein curd cooled to below
0 C. During this step, small ice crystals grow, before being thawed to expel excess
water. A dried matter is then formed having a sponge-like texture named Shimi-tofu.
Beside these modern making of traditional products, new technologies allow
producing new types of products based on legumes. The Fig. 5 summarizes the
different ways followed to obtain different modern products from soy.
Soy cakes are essentially used for animal feeding. They are obtained from soy oil
processing, after the oil extraction by pressing and/or using solvents like hexane. For
human feeding, soy meal and oil are the most frequent soy-product, from which most
other processed soy-based products derive [142]. New technologies have been
introduced in the soybean industry that have and will have significant impact on
farming methods, storage, and distribution of legume-based products. The industrial
Fig. 5 Different soy-based products and their pathway of production following modern processing
processes always start with cleaning the seeds to remove foreign material. After
drying and cracking into pieces, seeds are dehulled. They are heated and rolled into
“flakes” before oil extraction. Oil is extracted from soybean flakes using hexane and
then degummed and refined for edible and non-edible uses. The protein-rich flakes
are toasted, dried, and ground in meal. Soybean meal can be mixed with corn for use
in animal feeding. It is also processed into soy flour, soy concentrates, or soy protein
isolates. The degumming process of soy oil gives lecithin, which is used in the candy
and baking industries. Full fat soy flour from soybean is also used for baking, for
soy-based beverages, snack foods, as well as for traditional foods (soy sauce, Tofu,
and Miso). Texturized vegetable protein (TVP) obtained by extrusion of defatted soy
flour can be found in different forms (granules, flakes, chunks, or slices). TVP has a
long shelf life and can be kept at room temperature for several months. TVP should
be re-hydrated with equal quantity of water and can be used as a meat substitute in
processed foods (baked goods, meat products, protein drinks, soup bases, and
gravies). Microwave and autoclave cooking also are modern processes which are
used to prepare canned legumes.
5.2.2 Fermented Modern Legume Products

According to Nogushi [145], modern Miso is no more prepared from soya bean
boiled for several hours in renewed water but rather from steamed beans. The
process still implies long-term fermentation with Koji from soya, rice, or wheat
and with other microorganisms. Although soy sauce still requires time to be brewed
as it is for fine wines and cheeses, modern soy sauce is made from roasted wheat
grains crushed and soybeans softened by steaming. A special seed starter is then
added to the wheat and soybean mixture and incubated for 3 days. The resulting Koji
is then combined with brine to form Moromi. Moromi is then fermented in large
tanks until it reaches its full flavor and then pressed into fabrics to extract the raw soy
sauce. The latter is then refined and pasteurized before its packaging in bottles.
5.2.3 Nutritional Quality of Legume Under Modern Processing

According to Siulapwa and Mwambungu [159], after oil solvent extraction, soybean
seeds moisture is higher than that of row beans and that of extruded full-fat soy
seeds. These have lower ash content when compared to raw seeds or to seed
extracted with solvent. The extruded product exhibit lower fat and higher protein
content than raw seeds and seeds extracted with solvent. Extrusion and solvent
extraction reduce crude fibers, Ca++, and phosphorous content. Both processes
reduce significantly trypsin inhibitors activities. The processes increase arginine
proportion while they decrease methionine. Raw seeds had lower amount of all the
nonessential amino acids apart from tyrosine when compared to solvent extracted
seeds and extruded seeds. In cowpea (Vigna unguiculata), Ibrahim and coworkers
[158] showed that long-time soaking (16 h) in bicarbonate solution caused remark-
able reduction in the anti-nutritional factors. Pressure cooking was more effective
than ordinary cooking to remove phytates. This was confirmed by Khokhar et al.
[143] on moth bean (Vigna aconitifolia) cooked under pressure after soaking in
mineral salt solution. Finally, El Adawi [132] showed on chickpea (Cicer arietinum)
that microwave cooking reduced non-protein nitrogen, ashes, fibers, and oligosac-
charides but had no significant effect on total protein and carbohydrate content
including starch. In their study, they also show that this process also significantly
reduced trypsin inhibitors and hemagglutinin activities as well as tannins, saponins,
and phytic acid. Chickpea autoclaving led to the same reduction of anti-nutritional
compounds except for trypsin inhibitors for which it was shown to be more efficient.
However, both cooking processes significantly depressed the B vitamins content
(Riboflavin, Thiamin, Niacin, and Pyridoxine) sometimes at rates over 50%. Min-
erals including Na, K, Ca, Mg, P, Mn, Zn, Cu, and Fe tend to leak into the water
while boiling. However both microwave and autoclave cooking tend to better
preserve their concentrations compared to other practices. These results were
remarkably confirmed by Mubarak [79] while working on mung bean seeds
(Phaseolus aureus). The effect of fermentation was studied in beans (Phaseolus
vulgaris) [160]. The parameters followed were anti-nutritional factors (α-amylase
inhibitor, chymotrypsin inhibitor, cyanogenetic glycosides and lectins) and also
fibers (total dietary fiber -TDF-, insoluble -IDF- and soluble -SDF-). Beans were
treated by natural and lactic acid fermentation. Autoclaving was added or not after
the fermentation process. All treatments decreased the SDF content, while the IDF
content were not modified in processed beans. Cellulose content was reduced by
treatments and resistant starch increased in processed beans, except after lactic acid
fermentation. Fermentation with Lactobacillus plantarum increased pectic polysac-
charides and Klason lignin. Microorganisms reduced the solubility of dietary fibers.
According to Noguchi [145], cooking soybean mash for Tofu and “soy milk” in
water for at least 10 min before filtering “soy milk” is crucial since anti-nutritional
enzymes of the beans are inactivated during boiling. This is confirmed by Chen and
co-workers [161] who showed a progressive decrease of trypsin inhibitor activities
(Kunitz and Bowman Birk factors) in “soy milk” with or without added salt. In their
experiments, 10 min cooking approximately decreased TI activities between 40%
and 30% of their initial value. One hour cooking reduces it between 20% and 15% of
the initial value. However, not all modern processing of “soy milk” and Tofu
especially those developed in the West include such step. Consequently, it has
been shown in 1997 in Japan [162] that soybean products retain 2.5–12.5% of the
initial trypsin inhibitor activity of the whole soybean and that “soy milk” is the food
which is the most concentrated. Thus humans are consuming some active trypsin
inhibitor in their daily lives. In [163], the phenolic composition of mung beans
(Vigna radiata) and yellow soybeans (Glycine max) were followed under soaking
and fermentation using Lactobacillus plantarum CECT 748 T. It was shown that
soaking induced leaking of conjugated isoflavone for soybeans and increased
apigenin derivatives in mung beans. On the other hand, fermentation converted
glycosylated isoflavones of soybean into bioactive aglycones and increased the
bioactive vitexin in green beans. These data are, in accordance with those from
Fernandez-Lopez et al. [149], showing that glycosylated isoflavones tend to be
progressively removed from soybean matrices during prolonged cooking and sim-
mering in water. These steps were included in soybean traditional recipes. However,
nowadays, modern processes tend to replace boiling in water by steaming and
cooking times and contacts of seeds with water were dramatically reduced in modern
practices. In addition, extruded soy still contain high amount of isoflavones although
they tend to be deglycosylated during the extrusion process [164]. This means that
isoflavone exposure of soy-consumers was considerably lower in ancient times than
it is nowadays when people consume modern processed soy-products. Because of
their estrogenic effects, isoflavones tend to reduce the fertility of the soy consumers
(animals and most probably humans) and can therefore be considered as anti-
nutritional factors. In addition, the modern environment contains many other endo-
crine disruptors of anthropoid origin which can act synergistically with isoflavones
on reproduction or estrogen-dependent tumor growth [165]. Isoflavone can also
exert beneficial effects on several health end-points (bone preservation, prostate
cancer protection, breast cancer occurrence, hot flashes relief. . .) and should be
kept for specific health application for specific consumers [149, 166].
6 Allergy
Food allergies are adverse reactions to a harmless food that occur when the immune
system reacts to proteins normally present in food without any incidence and that are
recognized as foreign substances in some individuals. Then, the immune system
triggers a response to neutralize them. Allergic responses vary from person to person
and a protein may be allergenic in one individual but not in others. Nowadays,
according to Verma et al. [167], eight foods or food groups account for over 90% of
food allergies (peanuts, soybeans, cow’s milk, hen’s egg, fish, crustacean, wheat, and
tree nuts). According to Maria John et al. [168], soybean is listed among the eight
first allergenic diets for humans and animals due to the presence of many kinds of
allergens whose action can be amplified by the presence of other anti-nutritional
factors. Overall, allergenic events recorded after consumption of legumes in decreas-
ing order of frequency may be peanut (Arachis hypogaea), soybean (Glycine max),
lentil (Lens culinaris), chickpea (Cicer arietinum), pea (Pisum sativum), mung bean
(Vigna radiata), pigeon pea (Cajanus cajan), and lupine (Lupineus alba). This takes
into account both the allergenic power and the overall consumption at world scale. In
Spain in 2015, [169] reported that among 455 adults, the prevalence of legumes
allergy was 6.9% (77% women). Legumes involved were: lentil 27% of episodes;
bean, 19%; peanut, 16%; soybean, 14%; chickpea, 13%; pea, 8%; and mungo bean,
3%. Anaphylaxis due to food allergies has a prevalence of about 6–8% in children
and 4% in adults [167].
6.1 The Proteins of Food Allergies
Food reactions can be classified into three groups: the non-immunologic reactions
and the immunologic reactions that can be subdivided again according to the type of
reaction involved. The non-immunologic reactions include food intolerance or
gluten sensitivity whereas immunologic reactions can either induce immunoglobulin
E (IgE)-mediated symptoms or non-IgE-mediated gastrointestinal symptoms. These

phenomena mostly occur early in life, and food allergy frequency usually decreases
with age. Legumes usually provoke IgE-mediated reactions. These reactions occur
because foreign antigens have been able to stimulate the antigen-presenting cells at
the gut level. This means that proteins were able to resist to food processing and then
to gastric degradation by local acid and protease. The antigen presenting cells then
process the antigens and present them to the Major Histocompatibility Complex
(MHC). This activates naive CD4+ lymphocytes of the Th2-type.
These immune cells secrete IL-4 and IL-13, which promote specific IgE secretion
by plasma cells. This leads to the IgE antibodies to bind to mast cells. Re-exposure to
the same food antigens causes the antigen-IgE antibodies complex to bind to mast
cells. This directly provokes mast cells degranulation and release of histamine.
Histamine and other mediators (prostaglandin D2, and cysteinyl leucotriene) can
provoke vasodilatation, smooth muscle contraction, and mucus secretion that all lead
to development of allergenic symptoms [170]. As mentioned earlier, allergenic
proteins share common physicochemical and immunological properties. Class 1
food allergens such as legume allergens are stable in gastric fluid; their molecular
mass range between 10 and 70 kDa; they are water soluble glycoproteins and can
lead to sensitization via gastrointestinal tract [171]. Class 2 food allergens are
sensitive to heat and to gastric acid and enzymes, thus sensitization does not occur
via oral route. By means of IgE cross reactivity, they can cause allergenic response in
people sensitized via inhalant allergens like pollen [172]. The dose of exposure does
not seem to be related to either the sensitization process or to the allergenic
susceptibility. Less than one microgram levels can induce an allergenic reaction
[167] even if some authors report that the minimal level of exposure is over 500 μg
of protein but not below this [173]. Glycosylation contributes in the allergenicity of a
protein and it seems that the glycan moiety can also interact with IgE antibodies. The
structural features of the proteins which are responsible for their allergenic properties
are still difficult to predict. In the case of legumes, during digestion, allergens lose
conformational form and exist as linear epitopes and sensitized individuals via
gastrointestinal tract [67]. To summarize, the legume allergens resist to gastric
fluid and proteases, they are heat stable and are glycosylated proteins. They present
epitopes that bind to IgE. These can be linear epitopes remaining after food pro-
cessing and gastric attack, they induce biologically active reactions.
6.2 Main Allergens from Legumes
Several classes of proteins have been involved into legume allergy. These are storage
proteins which are considered to be stable to heat and to gut enzymes. Associated to
anti-nutritional compounds such as saponins or hemagglutinins, they can cross the
gut barrier and can induce immune response. Storage proteins presented earlier in
this review are primarily localized in the seed, nut, or kernel. They are classified in
Cupins, Prolamins, Pathogenesis Related proteins (PR-proteins), and Profilins.
6.2.1 Cupins Superfamily

The cupins superfamily share two conserved consensus sequence and a β-barrel core
domain. Cupins include allergenic seed storage proteins of the vicilin and legumin
family. These storage proteins are present in several legumes known for their
allergenic properties, i.e., soybeans (Gly m5, Gly m6), peanuts (Ara h1, Ara h3),
lupine (Lup a), pea (Pis s1), and broad bean (Vign r2, Vign r3).
6.2.2 Prolamin Superfamily

The prolamin superfamily is widely distributed among plants and not only in
legumes. Prolamins include cereal seed storage proteins, several important types of
allergens from legumes, tree nuts, cereals, fruits, and vegetables. This family
includes chickpea allergens (Cic a2S Albumin), which is a 2S protein. It also
includes the nonspecific lipid transfer proteins, the cereal alpha-amylase, and some
protease inhibitors.
6.2.3 Pathogenesis Related Proteins (PR-Proteins)

PR-proteins stand for more than 10 different types of protein which increase in plants
in response to environmental stresses or pathogens. PR-proteins are generally small
in size, stable in acidic conditions, and resistant to proteolytic degradation. In
legumes, some allergenic proteins were shown to belong to this category such as
Vig r1 from mung bean, Ara h8 from peanut, and Gly m4 from soybean.
6.2.4 Profilins
Profilins are small proteins from 12 to 15 KDa. They are found in the cytoplasm of
eukaryote cells and therefore they exhibit highly conserved sequences. Plant pro-
filins are involved in a large proportion of cross-reactions between allergenic sources
and especially in cross-reactions with pollens. As Class 2 allergens, they are
considered to affect 10%–35% people in Europe. Some identified profilin-related
legume allergens are Ara h5 from peanut and Gly m3 from soybean.
6.3 The Identified Allergens
In [167], Verma et al. gave a comprehensive table with the allergens from legumes
which were known in 2012. In addition, main legumes are classified according to
their decreasing importance of allergenic effects (frequency and severity) together
with the number of known allergens in Fig. 6. In 2016, Bouakkadia et al. [174]
completed the list that was increased to 15 allergens in peanuts, to 14 substances in
soybean, and to 4 different compounds in lentils.
6.4 Effect of Food Processes on Allergen Proteins
Verma et al. in [175] published a comprehensive review and table showing the
impact of different food processes on the inhibition of legume proteins playing as
Fig. 6 Classification of the main legumes according to their increasing allergenic properties. The
known allergens are mentioned for each pulse
food allergens. They classified the processes into four major categories: roasting,
boiling, autoclaving, microwave heating. They reviewed the main allergens from
peanuts (Arachis hypogea), soybean (Glycine max), lentils (Lens culinaris), lupine
(Lupinus alba), pea (Pisum sativum), French bean (Phaseolus vulgaris), chickpea
(Cicer arietinum), and mung bean (Vigna mungo). The effect of cooking has to be
tested since it is not easy to predict. In some cases, cooking practices lead to
elimination of the epitopes and to a reduction of the IgE reactivity. In that case,
food processing affects the structural and allergenic properties of allergens by
altering their stability or other physicochemical properties. On the opposite, in
other cases, there is an increase of immunoreactivity of the antigens which leads to
a higher IgE reactivity, a greater release of histamine and other mediators to finally
increase the allergenic response. This can be explained since three-dimensional
structures of proteins are generally correlated with their activity. Different temper-
ature treatment can induce variable changes in protein structure. As an example,
when heated at 70–80 C, proteins lose their secondary structure whereas at
80–90 C, new bonds and rearrangements of disulfide bonds can occur. At higher
temperature (90–100 C), protein aggregates can be formed [176]. Over 100 C, new
bonds between lysine residues and other substances may be created leading to the
formation of adducts [177]. Protein digestibility and absorption usually increases
with heat treatments. However, in some cases, thermal processing may also lead to
formation of neoantigens that were not originally present and that can enhance the
allergenic response. These neoantigens can result from the Maillard reaction, of
protein with sugar residues upon heating. From the review of Verma et al. [175], it
appears that boiling and autoclaving are overall good ways to reduce or to eliminate
allergens although these procedures were not always efficient especially during short
treatments on lentils, lupine, or French beans allergens. It should be noted that the
traditional recipes which included prolonged cooking or simmering in water were
empirically designed to get rid of anti-nutritional factors but can also be considered
as efficient on allergens. Finally it can be summarized the following elements:
Microwave heating is efficient in reducing soybean allergens but not lupine aller-
gens. Autoclaving was reported to decrease the allergenicity of lentils, pea, chickpea,
lupine, and peanuts. Boiling is efficient in reducing the immune reactions of peanuts,
mung bean, chickpea, and soybean antigens but the duration of the treatment is
crucial and generally as to be prolonged for better efficiency. Roasting decreases
mung bean allergenicity but increases that of peanut. As a consequence, food
processing should be taken into account while comparing the prevalence of allergic
reactions to various legumes over different world regions.
7 Conclusion
Proteins from legumes are undoubtedly of remarkable nutritional interest although

they are better combined with cereal proteins to cover the animal food requirements.
These combinations are known to exist since the domestication of crops anywhere
human civilizations developed. Therefore, considering the actual expansion of the
human population, pulses can be considered as a solution for future sustainable
nutrition. Their ability to fix and enrich the soils in nitrogen as well as their capacity
to produce protective and signal molecules to prevent or face pest attacks give them
advantages when phytochemical treatments are due to be reduced. However, if these
invaluable plants reached our modern times, it is because they also manage to protect
themselves from their natural predators. Therefore, co-evolution of plants with
microorganisms, insects, and herbivores along the geological times conducted to
the intrinsic production of many different defensive substances some of which can
be considered as anti-nutritional factors. These factors being essential for the plant
growth and survival, genetic improvement should respect them for better field
production rates. Therefore, the removal of these factors, which were developed
by the plants to be detrimental for their consumers, can only be achieved at the
transformation level. Future food processing methods will have to be imagined to
reduce these anti-nutritional factors as economically as possible for both environ-
ment and market. Lessons should be taken from the ancient times and from the
traditional practices and recipes used as early as the domestication of legumes
started. Crossing different data obtained from modern practices or from those
developed in the past, it appears that the traditional food processes, being essentially
wet, managed empirically or not to eliminate most of the deleterious compounds
including phytoestrogens, anti-nutritional substances, and allergens.
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Soybean Bioactive Molecules: Current
Trend and Future Prospective 11
Brij Pal Singh, Deepika Yadav, and Shilpa Vij
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
2 Soy Isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
2.1 Soy Isoflavones for Women Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
2.2 Soy Isoflavones as Anticancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
2.3 Soy Isoflavones for Cardiovascular Disease (CVD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
2.4 Soy Isoflavones for Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
2.5 Soy Isoflavones as Antimicrobial Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
3 Soy Bioactive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
3.1 Effect of Soy Bioactive Peptides on Blood Pressure Regulation . . . . . . . . . . . . . . . . . . . . 276
3.2 Effect of Soy Bioactive Peptides on Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
3.3 Antimicrobial Soy Bioactive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
3.4 Soy Bioactive Peptides as Immune System Modulator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
3.5 Effect of Soy Bioactive Peptides on High Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
3.6 Soy Bioactive Peptides for Weight Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
3.7 Anticancer Soy Bioactive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
4 Soy Saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
4.1 Health Effects of Soy Saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
5 Soy Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
5.1 Health Effects of Soy Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
6 Future Prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
Abstract
Bioactive food components or functional foods have recently received significant
attention because of their widely advertised positive effects on health beyond
basic nutrition. Soybean, a leguminous crop native to East Asia, is renowned for
B. P. Singh (*) · D. Yadav · S. Vij

Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India
e-mail: [email protected]; [email protected]; [email protected]

268 B. P. Singh et al.
high protein and often used to replace the animal proteins in an individual’s diet,
due to fact that is only vegetable food contains all the essential amino acids.
Therefore, FDA authorized a health claim for soy protein that 25 g of soy protein
per day may reduce the risk of heart disease. Besides, soybean comprises
isoflavones, phytosterols, saponins, and other basic nutritive constituents, such
as lipids, vitamins, minerals, oligosaccharides, and biological active peptides, that
are of strong therapeutic values. The potential health benefits of soybean/soy
bioactive components include protect heart health, anticancer, reduce the effects
of menopause, promotes bone health, improve metabolism, and decrease the risk
of diabetes. Fermentation is considered as one of the best means to eliminate
unpleasant beany flavors, which limit the wide consumption of soybean. Soy
isoflavones appear to have estrogen-like activity because they structurally resem-
ble to estrogen and bind to estrogen receptors. Daidzein, genistein, and glycitein
are three major glycosidic forms of isoflavones found in soybeans responsible for
most of health benefits. Soy bioactive peptides are specific fragments of major
soy proteins β-conglycinin and glycinin, and they can be released by enzymatic
hydrolysis, food processing, and/or fermentation. Apart from that, saponins
derived from soybean appear to have strong cancer inhibitory properties. More-
over, several experimental trials revealed the ability of soy phytosterols to lower
cholesterol. In spite of remarkable biological functions of soy bioactive com-
pounds, their large-scale production and commercialization are limited. There-
fore, there is much required need to emphasize large-scale production,
mechanism of action, and bioavailability of these components. Henceforth, this
chapter comprises the current scenario and future prospective of major soy
bioactive compounds and their associated health benefits.
Keywords
Soybean · Phytoestrogens · Isoflavones · Bioactive Peptides · Lunasin ·
Saponins · Phytosterols · Angiotensin-converting enzyme
1 Introduction
Soybean (Glycine max) is considered one of the widely consumed legume crops in
the world. Usually, soybean seeds consist of about 36.5% protein, 19.9% lipids, 30%
carbohydrates, and 9.3% dietary fiber. Soybean has the highest Protein Digestibility
Corrected Amino Acid Score (PDCAAS) among legumes, indicating that soy
protein provides most of the amino acids required for human nutrition [1]. Soybean
has been regarded as an important protein source for Eastern people. The recent
knowledge of soy nutritional and functional properties has considerably increased
the interest and consumption of soy products in the Western world. The consumer is
looking for more nutritious and healthy products, concerning with the consequences
of his/her food choices and life styles. Several studies have demonstrated the
associated advantages of the use of soy products in preventing heart disease, obesity,
blood cholesterol, cancer, diabetes, kidney disease, osteoporosis, blood pressure
11 Soybean Bioactive Molecules: Current Trend and Future Prospective 269
Fig. 1 Soybean bioactive

components and associated
health benefits
regulation, and menopause symptoms. Soybean and soy-based products contain

phytochemicals such as isoflavones, saponins, phytosterols, and bioactive peptides
that promote health (Fig. 1) [2]. Food and Drug Administration (FDA) authorized
“Soy Protein Health Claim” on October 26, 1999, that 25 g of soy protein a day may
reduce the risk of heart disease. Since then market is very much responsive to this
health claim and soy foods had penetrated rapidly into Western cultures and diets [3].
Nutritional supplements based on soy (Glycine max L.) extract are a rapidly growing
segment in the food and health care market [4]. Soy also benefits bone health, which
is a concern for aging people [5]. The types of soy products available in market
include soy-based cheeses, tofu, soy yoghurt, soy ice cream, soy sauce, and soy
vegetable burgers. Also soybean flour, soybean concentrates, and isolated soy pro-
teins are some of ingredients used in the food industry.
Although soy-based foods provide a range of health benefits to consumers, the
consumption of soy milk or other soy-based products is hindered due to the presence
of unpleasant off-flavors in soybeans. These characteristic flavors are caused by
n-hexanal and pentanal, which occur in beans as a product of breakdown of
unsaturated fatty acids [6]. In addition to these aldehydes, various oligosaccharides
including raffinose and stachyose present in soybean may cause a gastrointestinal
discomfort to consumers [6, 7]. Raffinose and stachyose are α-galactosides
comprising three and four monomeric units respectively, and are nondigestible in the
gut due to the absence of α-galactosidase in the human intestinal mucosa. Conse-
quently, intact oligosaccharides pass directly into the lower intestine where they are
metabolized by bacteria that possess this enzyme, resulting in the production of
gases [8]. Fermentation is one of the best ways to eliminate such problem and also to
improve the acceptability. Fermentation improves the bioavailability of isoflavones,
assists in digestion of protein, provides more soluble calcium, enhances intestinal
health, and supports immune system. Moreover, nowadays, soy milk has been
emerging as an interesting alternative to dairy products for lactose-intolerant popu-
lation because it is free from lactose. Also, soybeans are less expensive and more
abundant than bovine milk, and do not contain cholesterol [9].
Soybeans comprise the highest concentration of isoflavones among all plant
foods. The associated health benefits of isoflavones include cancer prevention,
reduced risk of osteoporosis, valuable role in chronic renal disease, and protection
against cardiovascular disorders. Besides isoflavones, phytocompounds such as
phytosterols and saponins have anticancer, hypocholesterolemic, and immunosti-
mulatory activities in human beings. Soy bioactive peptides are specific fractions of
soy proteins which have beneficial effect on living organisms. Usually, biological
activities of soy peptides are encrypted in proteins, once they released from their
parental proteins through hydrolysis, they may influence the major body systems
such as cardiovascular, digestive, nervous, and immune. Henceforth, this chapter
comprises the current scenario of major bioactive compounds of soybean and their
potential health benefits. Future prospective in the field of soy bioactive molecules as
technological and functional properties has also discussed.
2 Soy Isoflavones
Isoflavones are a group of naturally occurring heterocyclic polyphenols mostly

found in soybean. They are present in soy and soy-derived products such as soy
milk, meal, sauce, and tofu, both as free aglycones and as conjugates. Isoflavones
are also known as phytoestrogens because they are found in plants and appear to
have estrogen-like activity. They are structurally similar to estrogen and bind to
estrogen receptors. Interest in soy isoflavones has increased in recent decades, due
to their potential preventive activity against human chronic diseases, such as
cardiovascular disease, cancer, osteoporosis, menopausal symptoms, and cognitive
function [10–12]. Twelve isoflavones have been reported to be present in soybean,
consisting of four chemical forms with each form having three compounds,
such as malonyl-β-glucoside, acetyl-β-glucoside, and aglycones [13]. Three
major groups of isoflavones found in soybeans are genistein, daidzein, and
glycitein [14]. Isoflavones generally exist in soybeans and soy foods
as aglycones (daidzein, genistein, and glycitein), β-glycosides (daidzin,
genistin, and glycitin), acetylglycosides (600 -O-acetyldaidzin, 600 -O-acetylgenistin,
and 600 -O-acetylglycitin), and malonylglycosides (600 -O-malonyldaidzin,
600 -O-malonylgenistin, and 600 -O-malonylglycitin) (Fig. 2) [15].
Glycoside forms Aglycone forms

Biologically inactive Biologically active
OH OH OH
OH O O
CH2OH
O CH3 OH
OH
O O HO O
OH
OH Genistin Genistein
HO
OH O
OH b-estradiol
O
CH2OH
O
OH OH
HO O
O O
OH
OH Daidzin Daidzein
HO O
CH3 OH Equol
CH3 OH O
O
CH2OH O
O
O
OH
HO O
O O
OH
OH Glycitin Glycitein
Fig. 2 Molecular structure of isoflavones. Isoflavones presents in a biologically inactive glycoside

form (genistin, daidzin and glycitin) in soybean. β-Glucosidases cleave the glucosyl residue and
generate biologically active aglycones form (genistein, daidzein, and glycitein) of isoflavones
during digestion. Daidzein can be further metabolized into equol
The bioavailability of isoflavones can be influenced by their chemical form in foods

and the susceptibility to degradation during heating [16]. Genistein and daidzein, in
soybean, are mainly present in the form of their glycosides. Thereafter the consumption
of soybeans or soy-based food products, intestinal microorganism’s hydrolyzed iso-
flavones to its unconjugated form i.e., daidzein, genistein and glycitein which are more
estrogenic and have better bioavailability [17]. Moreover, equol is a byproduct of
bacterial metabolism of daidzein. Equol is reported to have more potent estrogenic
activity, greater affinity for estrogen receptors, unique antiandrogenic, and antioxidant
activity [18]. Earlier studies have documented that malonyl genistin is the most
abundant form of isoflavones in soybean, followed by malonyl glycitin, daidzin,
daidzein, genistein, and glycitin. It is generally believed that isoflavones hydrolyzed
glycosides to their corresponding aglycones prior to gastrointestinal absorption. Iso-
flavones when ingested are metabolized extensively in the intestinal tract, absorbed,
transported to the liver, and undergo enterohepatic recycling. Intestinal bacterial glu-
cosidases cleave sugar moieties and release the biologically active isoflavones, daid-
zein, and genistein. These active isoflavones can be further biotransformed by bacteria
to the specific metabolites, equol [19], desmethylangolensin, and p-ethylphenol [20].
All these phytoestrogens are then eliminated, mainly by the kidney, and therefore share
the physiological features and behavior of endogenous estrogens [21].
2.1 Soy Isoflavones for Women Health
Ingestion of isoflavones containing soybean foods has reported as an alternative for

hormone replacement therapy for post-menopausal women. Several epidemiological
evidence suggest that isoflavones are beneficial for breast cancer and osteoporosis.
Osteoporosis is an age-related disorder that affects most of the aged population in the
world [22]. Osteoporosis most often occurs after menopause in women, when the
ovaries stop producing estrogen. Soy isoflavones have the potential to overcome
bone-specific effects, and evidence from epidemiologic studies supports that dietary
isoflavones reduced osteoporotic bone loss induced by menopause through decreas-
ing the bone resorption and stimulating the bone formation. Isoflavones can act as an
antiresorptive and bone-sparing agent in preventing osteoporosis. The phenolic rings
in their structures are critical structural elements to bind estrogen receptors (ERs) and
exert estrogen-like effects [23]. Animal studies, as well as double-blind placebo-
controlled trials in humans, suggest that genistein can help restore bone protection
[24]. In contrary, the ingestion of high concentrations of isoflavones has reported to
adversely affect the reproduction in several animal species. Daily ingestion of soy
products has been reported to lengthen the menstrual cycle and suppress the usual
midcycle surge in pituitary gonadotropins in premenopausal women [25].
2.2 Soy Isoflavones as Anticancer
The bioactive forms of isoflavones, especially the aglycones, have benefits against
cardiovascular diseases and cancers and function by acting as antiestrogens [26].
Breast cancer is the one of most frequently diagnosed cancer in female worldwide
and occurs as an interaction of genes and diet. It has been reported that the
consumption of phytoestrogens enriched soy food/soy products is traditionally
high in Japan, where the annual breast cancer incidence rate is only 0.033%, whereas
this figure reached to 0.08% in Western European countries and in the USA, where
people consumed less soybean [27]. Genistein and daidzein, two principal compo-
nents in soy products, have been focused nowadays for their role in cancer preven-
tion. Genistein is a well-known tyrosine kinase inhibitor [28] and inhibits
topoisomerase and angiogenesis. Additionally, isoflavones have been thought to
act as anticancer agents in part by their ability to scavenge oxidants involved in
carcinogenesis. Genistein, which possesses weak estrogenic activity, has been
shown to act in animal models as an antiestrogen and, therefore, may play a
protective role in hormonally influenced cancers, such as breast cancer. Studies
have shown that injection of the isoflavone daidzein reduced N-methyl-N
nitrosourea-induced mammary carcinogenesis by approximately 20% [29]. These
findings highlight the importance of daidzein as an anticancer agent and may offer
therapeutic potential against colon cancer. Moreover, there are also many reports
about the protective effect of soy against oxidative stress, which can also lead to
numerous disorders, e.g., neurodegenerative conditions, chronic inflammation, and
cancer [30]. An in vitro study on human intestinal Caco-2 cells revealed that
genistein, daidzein, and equol are able to reduce LPS-induced inflammatory

responses from intestinal cells, interfering with NF-kB dependent molecular mech-
anisms [31].
2.3 Soy Isoflavones for Cardiovascular Disease (CVD)
In vitro investigations have demonstrated the hydrogen-donating abilities of

isoflavones and metabolites, inhibition of lipid peroxidation, and the ability to
interact with the oxidants such as hypochlorous acid and peroxynitrite [32]. Cell
culture studies suggest that isoflavones may enhance the cellular antioxidant
network by increasing metallothione in mRNA levels, inhibiting peroxynitrite-
mediated LDL oxidation by delaying tyrosine nitration [33], and activating gluta-
thione peroxidase [34] (thereby increasing levels of cellular reduced glutathione)
or inhibiting superoxide production, and thus cell-mediated LDL modification
[35]. A number of studies have shown that the consumption of soy is anti-
atherogenic, and the bioactive components in this regard are the isoflavones
[36–38]. These polyphenols have the potential to scavenge lipid-based peroxyl
radicals, and it is possible that the prevention of lipid peroxidation is an important
mechanism underlying the protective effects of soy consumption. This contention
is supported by the inhibition of copper-dependent LDL oxidation by addition of
purified forms of the isoflavones, genistein, daidzein, and biochanin [39]. It has
also been reported that genistein can inhibit LDL oxidation, which plays a
potential role in atherosclerosis [40], by scavenging reactive oxygen species
(ROS) or blocking generation of ROS involved in numerous pathological events.
The shift of LDL particle size to a larger, less atherogenic pattern as a result of soy
protein consumption and improvement in endothelial function independent of
lipoprotein changes represent two other potential soy-mediated protective mech-
anisms [41, 42].
2.4 Soy Isoflavones for Diabetes Mellitus
Isoflavones content in soy foods possess antioxidant activity and α-glucosidase

inhibitory activity, which have proved effective in the treatment of type 2 diabetes
mellitus through lowering of the blood glucose level [43]. Similarly, Lee [44]
reported that genistein and soy protein isolate contribute to alleviating the adverse
effect of diabetes mellitus by enhancing the lipid metabolism as well as the hepatic
antioxidant defense system. Genistein and soy protein isolate supplements may be
beneficial for correcting the hyperglycemia and preventing diabetic complications.
However, their mechanism of action is yet to be elucidated. A well-controlled study
on 32 postmenopausal women with type 2 diabetes documented beneficial effect
with high isoflavones soy protein supplement. The high isoflavones soy protein
supplement has significantly reduced fasting insulin and insulin-resistance values in
this population [45].
2.5 Soy Isoflavones as Antimicrobial Agent
The antibacterial activity of flavonoids against S. aureus, including antibiotic-resis-

tant strains, and S. epidermidis has been reported by Harborne and Williams (2000),
and there is an increasing interest in the use of plant flavonoids for treating human
diseases [46]. The prokaryotic type II topoisomerases (DNA gyrase and topoisom-
erase IV) are targets for broad-spectrum antibiotics [47]. Genistein is a topoisomer-
ase II inhibitor and has been shown to stimulate topoisomerase IV-mediated DNA
cleavage in E. coli [48]. In addition, genistein inhibits the release of inflammatogenic
substances by activated macrophages [49] and neutrophils [50], thereby contributing
to the downregulation of inflammatory responses. According to Verdrengh and
coworkers, genistein may represent a new type of antistaphylococcal agent, whose
activity appears to involve the stabilization of the covalent topoisomerase II-DNA
cleavage complex [51].
3 Soy Bioactive Peptides
Bioactive peptides can be defined as a specific fragments of proteins that have

positive impact on body functions or conditions and may ultimately influence health
[52, 53]. Mellander [54] was the first to suggest that casein-derived phosphorylated
peptides enhanced vitamin D–independent bone calcification in rachitic infants [55].
Numerous bioactive peptides from different sources having various biological func-
tions have been identified since the first observation of bioactive peptides. Soybean
is economically the most important source of vegetable protein for millions of people
and a potential source of bioactive peptides. Besides, bioactive peptides have been
isolated and characterized from other food protein sources, including milk, egg, fish,
oyster, cereal, and radish seeds [56–58]. Soybeans, an excellent source of dietary
peptides, have antihypertensive, anticholesterolemic, and antioxidant activities, and
appear to prevent cancer [59, 60]. Soybean contains around 40% of protein, and the
major soy proteins are known as β-conglycinin and glycinin, which account for
65–80% of total proteins [61]. β-Conglycinin and glycinin are the precursor of most
of the peptides isolated from soy bean [62]. Several peptides have been isolated,
purified, and characterized from soybean having ACE-inhibitory, antioxidant, anti-
cancer, immunomodulatory, hypocholesteromic, antibacterial, and insulin-modulat-
ing activities (Table 1) [67, 78–81].
It has well established now that bioactive peptides can be produced from different
food proteins by enzymatic hydrolysis during gastrointestinal digestion and by
fermentation of food [52]. Enzymatic hydrolysis has been recognized as one of the
most common method for bioactive peptide production. Single and/or multiple
specific or nonspecific proteases, i.e., pepsin, bromelain, trypsin, chymotrypsin,
and papain, can be used efficiently for bioactive peptide production [82, 83].
Many bioactive peptides have been experimentally generated using various com-
mercial proteases [84]. These enzymes are also used in combination to release more
effective and stable bioactive peptides [85]. In addition, several enzymes of plant
Table 1 List of some potential bioactive peptides derived from soybean/ soy based products
Peptides sequences Biological effects
Val-Pro-Pro ACE inhibitory [63, 64]
Ile-Pro-Pro ACE inhibitory [63, 64]
Thr-Pro ACE inhibitory [63]
Ala-Phe-His ACE inhibitory [55]
Ile-Phe-Leu ACE inhibitory [65]
Trp-Leu ACE inhibitory [65]
Ile-Ile ACE inhibitory [66]
Ile-Phe-Tyr ACE inhibitory [66]
Leu-Phe-Tyr ACE inhibitory [66]
Phe-Phe-Tyr-Tyr ACE inhibitory [66]
Tyr-Val-Val-Phe-Lys ACE inhibitory [67]
lle-Pro-Pro-Gly-Val-Pro-Try-Trp-Thr ACE inhibitory [67]
His-His-Leu Antihypertensive [7]
Pro-Gly-Thr-Ala-Val-Phe-Lys Antihypertensive [68]
Ala-Asp-Phe-Val-Leu-Asp-Asn-Glu-Gly-Asn-Phe-Leu-Glu-Asn- Antioxidant and ACE
Gly-Gly-Thr-Tyr-Tyr-Ile inhibitory [53]
Pro-Gly-Thr-Ala-Val-Phe-Lys Antimicrobial [69]
Ile-Lys-Ala-Phe-Lys-Glu-Ala-Thr-Lys-Val-Asp-Lys-Val-Val-Val- Antimicrobial [69]
Leu-Trp-Thr-Ala
His-Thr-Ser-Lys-Ala-Leu-Leu-Asp-Met-Leu-Lys-Arg-Leu-Gly-Lys Antimicrobial [70]
His-Cys-Gln-Arg-Pro-Arg and Gln-Arg-Pro-Arg Immunomodulatory [71]
Gln-Arg-Pro-Arg Immunomodulatory [71]
His-Cys-Gln-Arg-Pro-Arg Immunomodulatory [72]
Gln-Arg-Pro-Arg Immunomodulatory [72]
Leu-Pro-Tyr-Pro Hypocholesterolemic [73,
74]
Leu-Pro-Tyr-Pro-Arg Hypocholesterolemic
[71]
Ile-Ala-Val-Pro-Gly-Glu-Val-Ala Hypocholesterolemic
[74]
Leu-Pro-Tyr-Pro-Arg Antiobesity [75]
Pro-Gly-Pro Antiobesity [75]
X-Met-Leu-Pro-Ser-Try-Ser-Pro-Try Anticancer [76]
Ser-Lys-Trp-Gln-His-Gln-Gln-Asp-Ser-Cys-Arg-Lys-Gln-Lys-Gln- Anticancer (Lunasin) [77]
Gly-Val-Asn-Leu-The-Pro-Cys-Glu-Lys-His-Ile-Met-Glu-Lys-Ile-
Glm-Gly-Arg-Gly-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp
origin, papain and pronase, can be used for protein hydrolysis of soy flour and wheat
flour [86]; soy protein hydrolysates have been enzymatically prepared by several
commercially available proteases, alcalase, flavourzyme, trypsin, papain, protease,
and peptidase [87]. Enzymatic digestion of β-conglycinin and glycinin hydrolyzed
more active amino acid R groups that lead to increased antioxidant activity. It has
been reported that additional advantage of hydrolysis can be the development of
hydrophobicity since proteolysis unfolds the protein chains. Moreover, hydrolysis

leads to production of small bioactive peptides, and the bitterness of peptides of
below 1000 Da is much less than fractions with a higher molecular mass [88].
Further, structural and chemical changes that occur during processing of food
proteins may also lead to release of bioactive peptides [89]. Apart from that,
fermentation is also an efficient way to produce bioactive peptides and food grade
hydrolyzed proteins. Lactic acid bacteria (LAB), a large group of beneficial bacteria
widespread in nature and also found in our digestive systems, are generally used for
bioactive peptide production. They are best known for their role in the preparation of
fermented products, not only because of their physiological significance but also
because of their technological importance in texture and flavor development [90].
The proteolytic system of lactic acid bacteria, e.g., Lactococcus (L.) lactis, Lacto-
bacillus (Lb.) helveticus, and Lb. delbrueckii ssp. bulgaricus, has proteinases that are
capable of releasing a large number of different oligopeptides (4–8 amino acid). The
oligopeptide transport system, main route for nitrogen entry into the cell and
peptidases, located intracellularly is required for complete degradation of accumu-
lated peptides [91, 92]. Lactic acid bacteria, possessing α-galactosidase enzyme, can
hydrolyze the soy oligosaccharides (sucrose, raffinose, and stachyose) during fer-
mentation and reduce its beany flavor and flatulence [6, 7, 93]. During fermentation
of soy milk, proteins are degraded into simpler forms like oligopeptides, dipeptides,
and tripeptides and serve as a good source of bioactive peptides. Comparatively
microbial fermentation is the cheapest process instead of enzymatic hydrolysis for
bioactive peptide production because microorganisms are a cheap source of pro-
teases and are recognized as safe. Upon administration bioactive peptides may affect
the major body systems, i.e., cardiovascular, digestive, immune, and nervous. The
beneficial effects of bioactive peptides include antimicrobial, antioxidative, anti-
thrombotic, antihypertensive, antidiabetic, anticancer, antiobesity, and immunomod-
ulatory [94–97].
3.1 Effect of Soy Bioactive Peptides on Blood Pressure Regulation
Antihypertensive peptides are the greatly studied bioactive peptides in soy foods.
Antihypertensive or angiotensin-converting enzyme (ACE) inhibitory peptides
block the first step in the rennin-angiotensin system and interrupt the negative
feedback effects of angiotensin II. The rennin-angiotensin system regulates blood
pressure and fluid balance in body. ACE is a nonspecific dipeptidyl carboxy peptidase
and converts the inactive decapeptide angiotensin I by cleaving dipeptide from the C-
terminus into the potent vasoconstricting octapeptide angiotensin II in the rennin-
angiotensin system (RAS), which has a tendency to increase blood pressure [98].
Besides, ACE also catalyzes the degradation of bradykinin, a blood pressure–lowering
nonapeptide in the kallikrein-kinin system (Fig. 3) [99–101]. Inhibition of ACE is
considered to be a useful therapeutic approach in the treatment of hypertension.
Hypertension is also considered as a main cause of several risk factors, such as heart
failure, stroke, coronary heart disease, and myocardial infarction.
Fig. 3 Role of angiotensin-

converting enzyme (ACE) in
blood pressure regulation
Soy bioactive peptides, i.e., VPP (valyl-prolyl-proline), IPP (isoleucyl-prolyl-

proline), and TP (Tyrosin-Proline), are known to have ACE-inhibitory activity and
blood pressure lowering effect [63]. Similarly, bioactive peptides from fermented
soy product (miso and doenjang) have been shown ACE-inhibitory activity in
spontaneously hypertensive rats, mainly associated with tripeptides (VPP and IPP)
[64]. Moreover, an ACE-inhibitory tripeptide Ala-Phe-His have been isolated from
soybeans fermented by Bacillus natto and Bacillus subtilis [55]. It has also observed
that peptides prepared from alcalase hydrolysis of soy protein significant decrease
systolic blood pressure (SBP) in spontaneously hypertensive rats [102]. Fractionated
whey from fermented soy milk with probiotics, L. casei, L. acidophilus,
L. bulgaricus, S. thermophilus, and B. longum, has reported to exhibit antihyperten-
sive activity [103]. Two ACE-inhibitory peptides have also been isolated having Ile-
Phe-Leu and Trp-Leu amino acid sequence [65]. Fermented soybean extract were
also used for the production of ACE-inhibitory peptide [104]. Shimakage and
coworkers identified novel ACE-inhibitory peptides, such as Ile-Ile, Ile-Asp, Ile-
Phe-Tyr, Leu-Phe-Tyr, Leu-Tyr-Tyr, Phe-Phe-Tyr-Tyr, and Trp-His-Pro, from prote-
ase-treated natto and soy milk [66]. Alauddin et al. [105] has recently identified the
ACE-inhibitory peptide and studied its hypotensive effect from processed soy milk
(PSM); the ingestion of PSM may lower high blood pressure and ameliorate
cardiovascular diseases related to hypertension without causing adverse side effects
in spontaneously hypertensive rat. Nakahara et al. [106] identified two antihyper-
tensive peptide Seryl–tyrosine (Ser-Tyr) and glycyl–tyrosine (Gly-Tyr) from
fermented soy sauce. Angiotensin-I converting enzyme (ACE-I) inhibitory activity
of prepared soy whey proteins were fractionated into different fractions using
ultrafiltration and found that unfractionated whey protein had the highest ACE-I
inhibition activity. The study also indicated that soy–whey protein fraction
(>50 kDa) had good solubility, emulsion activity, and stability, while the
unfractionated whey protein exhibited the strongest ACE-I inhibition percentage

(19%) [107]. Recently, around 33 peptides were identified from soy milk fermented
by a Lactobacillus plantarum strain, having ACE-inhibitory activity [53].
3.2 Effect of Soy Bioactive Peptides on Oxidative Stress
Antioxidants are recognized as a substances that can significantly decrease the

unfavorable effects of reactive species. Reactive species can be formed during
cellular metabolism of human system and plays important roles in cell signaling,
apoptosis, gene expression, and ion transportation. However, when the production of
these molecules is uncontrolled, poor cellular defenses lead to damaging of protein,
lipid, and nucleic acids by the process called oxidative stress. However, human
beings possess defense and repair systems for oxidative stress, but these innate
antioxidative systems are usually not enough to prevent them from the oxidative
stress [108]. Apart from several natural antioxidative compounds and vitamins,
food-derived bioactive peptides have now been explored as an effective antioxidant
agent. Antioxidant food supplements or bioactive peptides may be used to help the
human body and animals to reduce the oxidative damage [109]. It has been noticed
that several amino acid residues, i.e., histidine, tyrosine, tryptophan, phenylalanine,
proline, and leucine, contribute for antioxidant capacity of peptides [110].
Antioxidative peptides can inhibit oxidation through multiple pathways, includ-
ing inactivation of reactive oxygen species, scavenging free radicals, and chelation
of pro-oxidative transition metals [111]. Several amino acids, such as Tyr, Met, His,
Lys, and Trp, are generally accepted as antioxidants. On the same line, antioxidative
peptide from β-conglycinin fraction of soy protein has been isolated by Chen et al.
[112] using protease of microbial origin. Similarly, Coscueta et al. [113] reported
that hydrolysis of soybean meal protein isolate has potentially enhanced ABTS and
ORAC antioxidant capacity by treatment with corolase PP. Antioxidative
peptides have been produced from soy protein isolate through hydrolysis with a
food grade pancreatic trypsin/chymotrypsin with an antioxidative capacity of
113 mg TEAC/g [114].
In a recent study, Lactobacillus plantarum strain has been used as a starter to
ferment soy whey in order to study antioxidative potential. The results demonstrated
that fermented soy whey possessed more radical scavenging capacity as compared to
unfermented, which is directly linked to generation of bioactive peptides during
fermentation [115]. Similarly, Sanjukta et al. [116] reported 3.1–24 folds increment
in total antioxidant activity of two soybean varieties of Sikkim Himalayan region of
India by fermentation with Bacillus subtilis. Recently, we have also identified 35
antioxidative peptides from soy milk fermented by a Lactobacillus plantarum strain
[53]. Bioactive peptides has also identified in soybean-based infant formulas having
around 90% soy protein isolate. Around 120 antioxidant peptides were characterized
by using ultrafiltration followed by HPLC-ESI-Q-ToF-MS/MS [117]. Antioxidant
activity of lunasin (a soy peptide) has also been reported as a potent scavenger of
peroxyl and superoxide radicals. The protective role of lunasin on cell viability and
antioxidant defenses of human Caco-2 cells has also been studied and found that
direct antioxidant action of lunasin on enterocytes exposed to oxidizing species
makes this peptide a promising agent to preserve the integrity of intestinal mucosa
against oxidative damage–related diseases [118].
3.3 Antimicrobial Soy Bioactive Peptides
The growing problem of resistance to conventional antibiotics and the necessity for
new antibiotics has stimulated an interest in the development of antimicrobial
peptides (AMPs) as human therapeutics [119]. AMPs are peptides that can kill
microorganisms, and they often exhibit a broad spectrum of activity against Gram
Positive and Gram Negative bacteria [120]. Antimicrobial peptides are widely
distributed in nature and are essential to the immune system. They are the organism’s
first line of defense against colonization by exogenous microorganisms, and they
play a fundamental role in regulating bacterial populations on the mucosa and other
epithelial surfaces. Therefore, using natural sources of antimicrobial compounds has
enormous potential because they have characteristics such as low toxicity and high
specificity. The mechanisms of these natural antimicrobial compounds can be better
understood if we compare their modes of action against bacterial (unicellular) and
animal (multicellular) cells. Bacterial cells have a layer rich in negatively charged
phospholipids pointing toward the external environment, facilitating their interac-
tions with peptides, most of which are positively charged. In contrast, animal cells
are mainly composed of uncharged lipids in the outermost layer, and the negatively
charged regions are pointed toward the cell interior (cytoplasm) [121, 122]. Soy
proteins are excellent source of bioactive compounds. The biological activity of the
glycinin and β-conglycinin hydrolysates was confirmed, and glycinin peptides were
found to produce stronger antimicrobial effects than β-conglycinin peptides [123].
Antimicrobial peptides ranging from 15 to 40 amino acids in length, most of which
are hydrophobic and cationic, are generally involved in innate immunity. These
peptides include two or more positively charged residues provided by arginine,
lysine, or, in acidic environments, histidine, and a large proportion of hydrophobic
residues. They can exhibit an efficient role in the host defense against the most
frequent pathogenic bacteria that interact directly with them and therefore scaveng-
ing them. Such peptides provide protection against bacteria, fungi, and viruses by
acting on the cell membranes of the pathogens [124]. Recently, we have studied
antimicrobial activity of bioactive peptide fractions derived from fermented soy
milk. Five kilo dalton fraction has showed highest activity against most of the tested
pathogens, among them highest activity has reported against E.coli (12 0.57)
followed by S. dysenteriae (11 0.57), L. monocytogenous (10 0.57), and
B. cereus (10 0.57 mm) [125]. Two soy peptides, i.e., PGTAVFK and
IKAFKEATKVDKVVVLWTA, have tested against Pseudomonas aeruginosa and
Listeria monocytogenes biofilms; both the peptides showed strong inhibitory activity
against Listeria monocytogenes [69]. In a recent study, a soy meal fermented by a
Bacillus subtilis strain has found to produce antimicrobial peptides with great
antimicrobial activity against Vibrio alginolyticus and V. parahaemolyticus [70].
3.4 Soy Bioactive Peptides as Immune System Modulator
Soy bioactive peptides have also been reported to enhance immune function. Two
peptides from soy protein hydrolysate, i.e., His-Cys-Gln-Arg-Pro-Arg and Gln-Arg-
Pro-Arg, have been found to exhibit phagocytosis stimulatory activity [72]. Usually,
immunomodulatory peptides can enhance immune cell functions, such as lympho-
cyte proliferation, natural killer (NK) cell activity, antibody synthesis, and cytokine
regulation. A soy protein hydrolysates prepared from insoluble soy protein has
displayed highest immunomodulatory activity on proliferation of murine splenic
lymphocytes and phagocytic effect of peritoneal macrophages [87]. Similarly, a
phagocytic enhancing peptide has been isolated from trypsin digests of α-subunit
of β-conglycinin soy proteins [61]. Lunasin, a soy peptide, exerts synergistic effects
with cytokine Il-12 or Il-2 on modulating the expression of a number of genes in NK
cells, resulting in strong NK activation with enhanced cytotoxicity, which is associ-
ated with higher levels of IFNγ and granzyme B expressed by both CD56 bright and
dim populations [126]. Lunasin and other lunasin-like peptides, purified from
defatted soybean flour, inhibited inflammation in LPS-induced macrophage by
suppressing NFkB pathway. Out of three purified peptides (5, 8, and 14 kDa), the
5 kDa peptide inhibited most potently the proinflammatory markers including
interleukin-6 production, nuclear factor-kappa B, cyclooxygenase-2 expression,
nitric oxide production, inducible nitric oxide synthase, nuclear translocation, and
p50 nuclear translocation [127]. Recently, Tung et al. [128] studied stimulatory
effects of lunasin on innate immune cells by regulating expression of a number of
genes that are important for immune responses. Lunasin-treated conventional DCs
(cDCs) not only expressed elevated levels of costimulatory molecules (CD86,
CD40) but also exhibited upregulation of cytokines (IL1B, IL6) and chemokines
(CCL3, CCL4). Lunasin-treated cDCs induced higher proliferation of allogeneic
CD4+ T cells when comparing with medium control treatment in the mixed
leukocyte reaction (MLR).
3.5 Effect of Soy Bioactive Peptides on High Cholesterol
Hypercholesterolemia, also called high cholesterol, is the presence of high levels of

cholesterol in the blood. It is a significant risk factor in the development of heart
diseases and one of the major causes of deaths worldwide. Hypercholesterolemia is a
state of high blood lipids and elevated levels of lipoproteins in the blood. Dietary
proteins can modulate the effect of high serum cholesterol concentrations. The
hypocholesterolemic activity of food protein and peptides involves stimulation of
bile acids secretion, changes in cholesterol metabolism in the liver, hormonal effects,
and regulation of cholesterol receptors [129, 130]. The effects of soy-based product
on cardiovascular diseases (CHD) have considered through its impact on blood

cholesterol. It has been reported that the released bioactive peptides lower the
cholesterol levels when soy protein was subjected to protease digestion in the
digestive tract [131]. It has now been well established that the dietary intakes of
20 g of soy protein per day for 5 weeks would be effective in reducing the CHD risk
among high-risk, middle-aged men [132]. Similarly, the effect of fermented soy milk
on rats fed a high cholesterol diet was investigated to clarify the cholesterol-lowering
function [133]. Wang et al. (2008) reported that soy protein reduced the level of
circulating triglycerides and cholesterol in hypocholesterolemic subjects [134].
Similarly Sugano et al. [135] documented that soy protein hydrolysate of peptic
digest has a stronger serum cholesterol lowering effect in comparison to intact soy
protein in rats. Soy peptides may also bind to phospholipids and exert serum
cholesterol lowering activity in humans [136]. Keeping the cholesterol lowering
effect of soy protein in mind, Food and Drug Administration (FDA) approved a
health claim linking foods that are naturally rich in soy protein reduce coronary heart
disease [137].
LPYP, a tetrapeptide, has been isolated from soy protein glycinin with hypo-
cholesterolemic effect [73]. Zhong and coworkers studied the in vivo hypo-
cholesterolemic effect of soy peptides [138], and they found that cholesterol
micellar solubility inhibitory rate of soy peptides was more than 45%. Similarly,
Ferreira and coworkers (2010) reported cholesterol-lowering effect of β-conglycinin
and glycinin soy proteins in rats fed a high-cholesterol diet [139]. A recent study
showed effect of a symbiotic fermented soy product supplemented with okara (a by-
product from soybean) on cardiovascular disease risk markers in healthy men. In a
randomized two groups study, subjects consumed daily 100 g of soy-based product
fermented with Lactobacillus acidophilus La-5, Bifidobacterium animalis subsp.
lactis Bb-12, and Streptococcus thermophilus (starter culture), and LDL-C mean
decreased significantly resulting in a significant improvement of the LDL-C/HDL-C
ratio was observed [140]. Three peptides from soy glycinin, IAVPGEVA,
IAVPTGVA, and LPYP, have been studied by Lammi et al. [74] for their hypo-
cholesterolemic activity. HepG2 cells treated with these peptides interfere with the
catalytic activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR) and
modulate the cholesterol metabolism through the activation of the LDLR-SREBP2
pathway, increasing the ability of HepG2 cells to uptake the LDL.
3.6 Soy Bioactive Peptides for Weight Management
A condition when excess fat has accumulated in body to the extent that may have an
adverse effect on health called obesity. Obesity has now been considered as a major
health problem in most of the countries, which is associated with higher incidence of
CVD, type 2 diabetes, obstructive sleep apnea, cancer, osteoarthritis, and depression
[141]. Consumption of dietary proteins, i.e., soy, casein, and whey protein, has
reported antiobesity effect. An in vitro study revealed that soy protein has greater
antiobesity effect in comparison to casein and whey protein in obese rats/or mice
[142]. Soy protein may also lead to lower hepatic deposits of triglycerides [143].
Two peptides Leu-Pro-Tyr-Pro-Arg and Pro-Gly-Pro from soybean glycinin protein
have strong antiobesity potential [75]. Several animal studies showed that soy
protein ingestion lead to lipid-lowering effect by reducing hepatic cholesterol con-
tent and enhancing removal of LDL [144]. Soy proteins/peptides have directly
affected the hepatic cholesterol metabolism and LDL receptor activity. Evidence
from animal models suggested that soy protein has strongly influenced lipogenesis in
the liver. Similarly, dietary soybean proteins have reported to reduce the concentra-
tions of plasma triglycerides in rat liver. These effects have mainly associated with
the reduction of hepatic lipogenic enzymes, such as glucose-6-phosphate dehydro-
genase, malic enzyme, fatty acid synthetase, and acetyl-CoA carboxylase (ACC),
which concluded that soy protein reduces liver triglycerides by inhibition of hepatic
fatty acid synthesis in liver [145]. Soy protein can improve insulin resistance and
lipid levels by activation of PPAR (peroxisome-proliferator activated receptors),
which is the main transcription factor responsible for the regulation of expression
of genes involved in glucose homeostasis, lipid metabolism, and fatty acid oxidation.
On the same line, Jang and coworkers (2008) studied in vitro antiobesity effect of
soy peptides derived from black soybean. They observed that the diet-induced obese
mice fed a high-fat diet along with soy peptides gained less body weight in
comparison to control [146].
3.7 Anticancer Soy Bioactive Peptides
Lunasin, a peptide isolated from soybean, is well known for their anticancer effect
[77]. According to reports, lunasin can prevent cellular transformation induced by
chemical carcinogens and the viral oncogenes RAS and E1A [147, 148]. Hsieh and
coworkers (2010) reported that lunasin has been strongly inhibited cell proliferation
and cancerous foci formation in cells treated with 7,12-dimethylbenz (a) anthracene
(DMBA) and 3-methylcholanthrene-treated (MCA) [149]. Similarly, an in vivo
study also documented anticancer effect of lunasin induced by DMBA [150]. As
safety point of view reports claimed that lunasin does not affect the growth rate of
normal and established cancer cell lines in spite of cancer-preventive properties. Soy
proteins has several mechanisms for cancer prevention including increased mam-
mary gland differentiation, decreased activation of procarcinogens to carcinogens,
and regulation of genes in signal transduction pathways underlying tumor initiation,
promotion, and/or progression [151]. Rayaprolu and coworkers [152] reported
anticancer properties (more than 65%) of soy protein isolates against human colon
(HCT-116, Caco-2), liver (HepG-2), and lung (NCL-H1299) cancer cell lines.
Similarly, oral administration of lunasin has reported to reduce liver metastasis by
56–94% [153]. Downregulation of inflammation related genes was reported by
Hwang et al. [154] when breast cancer MCF7 cells were treated with fermented
soybean extracts. Similarly, Yang et al. [155] reported that lunasin can effectively
suppress allergic airway inflammation.
4 Soy Saponins
Saponins are compounds containing a steroid or triterpenoid aglycone linked to one

or more oligosaccharide moieties [156]. These high-molecular weight glycosides are
found in many plant-derived drugs. Saponins, which derive their name from their
ability to form stable, soap-like foams in aqueous solutions, constitute a complex and
chemically diverse group of compounds [157]. Soy saponins are amphiphilic com-
pounds and categorized as triterpenoic saponins [158]. Soybeans and soy-based
products contain approximately 1–5% saponins. It has been estimated that there
are at least 40 different saponins in soybeans. Most of the soy saponins identified till
date have a soyasapogenol conjugated at the 3-position with a 20 -glycosylated
glucuronic acid residue [159].
4.1 Health Effects of Soy Saponins
Soy saponins are well known for the inhibition of cancer cells [160]. Clinical studies
have suggested that saponins affect the immune system in ways that help protect the
human body against cancers [157]. At high concentrations, saponins cause cell
damage by disrupting the cell membrane or inducing apoptosis [161]. The
membranolytic activity of soybean saponins as indicated by their interaction with
human colon carcinoma cells explains their role as anticarcinogens. Previous reports
indicated that soybean saponins suppress the growth of colon tumor cells in vitro,
and a 2% crude soybean saponins diet inhibited a carcinogen-induced colonic
aberrant formation in rats. Soybean saponins have been shown to contain 2,3-dihydro-
2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), and DDMP (1 mg/mL) possesse-
s a strong radical scavenging activity equivalent to 17.1 units of superoxide
dismutase [162]. Soybean saponins significantly inhibit the release of prostaglandin
E2 (PGE2), nitric oxide (NO), tumor necrosis factor α (TNFα), and monocyte
chemotactic protein- 1 (MCP-1) in a dose-dependent manner [163]. They also
downregulate the expression of cyclooxygenase-2 (COX-2) or inducible nitric
oxide synthase (iNOS) at mRNA/protein levels. The anti-inflammatory properties
of soybean saponins are mediated by the necrosis factor-κB (NF-κB) signaling
pathway, blocking the degradation of inhibitory proteins known as IκB-α. Kang et
al. [163] concluded that the anti-inflammatory action of soybean saponins may be
useful for developing preventive agents against inflammatory diseases as well as
suppressing tumor progression. On the same line, Tsai and coworkers (2010)
reported that soy saponins decreased the number of viable human colon cancer
cells in a dose-dependent manner and suppressed 12-otetradecanol-phorbol-13-
acetate-stimulated PKC activity. Cells treated with saponins developed cytoplasmic
vesicles and the cell membrane became rougher and more irregular and eventually
disassembled [158].
Apart from those other beneficial properties of soy saponins includes hypo-
cholesterolemic, hemolytic, and immunostimulatory activities [11]. Moreover, soy
saponins were reported to have antiviral and antioxidative activities. The blood
cholesterol-lowering properties of soybean saponins are also of particular interest in

human nutrition. Saponins cause a depletion of body cholesterol by preventing its
reabsorption, thus increasing its excretion. Studies on rats have shown soybean
saponins to have an anabolic effect on bone components, suggesting its role as a
nutritional factor in the prevention of osteoporosis [135]. Nishida and coworkers
(1993) reported that the inhibition of radical-initiated lipid peroxidation by soy
saponins was responsible for its anticarcinogenic properties [164].
5 Soy Phytosterols
Phytosterols are naturally occurring compounds which are found in all foods of plant
origin. Phytosterols have the similar basic function in plants as cholesterol in animals,
and they play a key role in cell membrane function. Therefore, they are best known for
reducing blood cholesterol levels. Phytosterols comprise a wide variety of molecules
that are structurally similar to cholesterol. They are naturally found in pulses, vegetable
products, oils, and dried fruits [165]. Soybean phytosterols consist mainly of β-sitos-
terol, campesterol, and stigmasterol; β-sitosterol content being highest among them.
5.1 Health Effects of Soy Phytosterols
Several experimental trials revealed the ability of phytosterols to reduce LDL-

cholesterol, without significantly altering HDL-cholesterol or triglycerides in gen-
eral [165]. It was reported that phytosterols such as β-sitosterol offer anticancer
effects [166], prostatic hyperplasia-lowering effects [167], and stimulation of a
plasminogen activating factor [168]. In particular, there are many reviews on
cholesterol-lowering activity of phytosterols. It has been demonstrated that soybean
phytosterols are able to reduce serum cholesterol by inhibiting the absorption of
cholesterol. Therefore, phytosterols are often supplemented into functional foods to
improve human health. The FDA has also authorized a food claim for phytosterols:
“Food containing at least 0.65 g per serving of plant sterol esters, eaten twice a day
with meals for a daily total intake of at least 1.3 g, as part of a diet low in saturated fat
and cholesterol, may reduce the risk of heart disease” [169–171]. Lerman and
coworkers (2010) concluded that individuals at high CVD risk benefit from a soy
phytosterol containing medical food; thereafter, conduction of a 12-week random-
ized trial for subjects received a phytochemical-enhanced diet [172]. Similarly, a diet
program containing 30 g of soy protein and 4 g of phytosterols was subjected to 27
postmenopausal women per day for 12 week, and observed that total cholesterol,
low-density lipoprotein cholesterol, and triacylglycerol have decreased significantly
[173]. Moreover, mild-to-moderate hyperlipidemic patients were selected in a study
and treated with placebo soy milk powder enriched with phytosterols. After 3 months
of trail, the serum total cholesterol, low-density lipoprotein cholesterol, and non-
high-density lipoprotein cholesterol levels decreased by 9.3%, 11.4%, and 12.6%,
respectively [174].
6 Future Prospective
Over the last two decades, scientific research on soybean-derived bioactive com-
pounds, such as isoflavones, peptides, and protein hydrolysates, has been detonated,
which displays a broad scope of functions. However, these bioactive molecules are
less potent in comparison to pharmaceutical drugs; conversely, they have minimum
or no side effects because nature has provided the mechanism for their metabolism
and utilization or excretion. Usually, bioactivity of compounds in vitro cannot be
directly related to its in vivo effect because they may likely encounter degradation
and modification in the intestine, vascular system, and liver. Therefore, they need to
remain active during digestion and be transported through the intestinal wall into the
blood. However, several differences are observed between in vitro models and in
vivo studies [175, 176]. Therefore, bioavailability of bioactive compounds is one of
the major areas of research which requires focus. It was evident that only a small
portion of bioactive molecules are sufficient to exert the specific function at tissue
level once they pass through the intestine barrier [177]. For instance, a numbers of
mechanisms for absorption of peptides are available, such as paracellular route,
passive diffusion, transport via carrier, and endocytosis. The lymphatic system is
also a possible route of peptides absorption; here absorption of peptides is mainly
affected by their permeability via the capillary of the portal circulation and lipid
solubility [88]. The small peptides absorbed more readily instead of large peptides.
They are able to cross the intestinal barrier and exert their biofunctionality at the
specific target organs [178–180].
In spite of remarkable array of biological functions by soy bioactive molecules, it
may be surprising that only a few have commercialized and reached to the market.
Therefore, new strategies are needed to establish economical and efficient industrial
scale production of bioactive compounds with different action. However, the high
cost and multistep process limited the large-scale production of bioactive com-
pounds. Therefore, advance and precise technologies are needed for hydrolysis,
separation, and purification of bioactive molecules with high potency and yield.
Also, downstream processing such as drying, cellular disruption, extraction, and
production cost–related studies of bioactive molecules need specific attention.
Moreover, bioinformatics approach should be explored for prediction and analysis
of specific bioactivity of molecules. Molecular studies are also needed to examine
the mechanisms by which these compounds exert their actions.
7 Conclusions
Soybean has a great potential of supplying bioactive compounds that are function-
ally significant. However, the raw beans itself are not accepted throughout because it
contains some antinutrients that can disrupt digestion activities in the stomach,
leading to cramping and associated discomfort. Therefore, these bioactive compo-
nents can be incorporated in functional and fresh foods, dietary supplements, and
even pharmaceuticals with the aim of delivering specific health benefits. Microbial
fermentation is also an effective way to increased bioactivity and acceptability of

soybean. The effect of soybean/soy-derived bioactive components on emerging
lifestyle complications, such as metabolic syndrome and cardiovascular disorders,
has increased demands worldwide. Apart from that, soybean products can be good
substitutes for animal products because, unlike some other beans, soybean offers a
complete protein profile. Soybeans contain all the essential amino acids except
methionine, which must be supplied in the diet because they cannot be synthesized
by the human body. Besides the potential health effect of soybean-derived bioactive
molecules, the bioavailability and safety of these products should be ensured as most
priority. More definite clinical studies are needed to ensure any side effect. Advanced
molecular and bioinformatics approaches should also be explored for target-specific
studies of soy bioactive molecules.
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Wheat Bran Proteins
12
René R. Balandrán-Quintana and Ana María Mendoza-Wilson
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
2 Overview on Wheat Grain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
2.1 Protein Synthesis at Different Stages of Grain Development . . . . . . . . . . . . . . . . . . . . . . . . 299
3 Wheat Bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4 Wheat Bran Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
4.1 Distribution of Proteins Among the Various Layers of the Wheat Bran . . . . . . . . . . . . 303
4.2 Proteins from Aleurone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
5 Bioactive Properties of Protein and Peptides Isolated from Wheat Bran . . . . . . . . . . . . . . . . . . 307
6 Potential of Wheat Bran Proteins for Other Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Abstract
Wheat pericarp, known as bran after milling, is the protective envelope of the
grain. It is constituted by seven layers, grouped into three major sections: inner
(aleurone), intermediate, and outer bran, respectively. Thousands of proteins are
expressed during the wheat grain development, many of them in pericarp,
so wheat bran protein content is 15%. Aleurone proteins are mostly storage and
metabolic enzymes, while those of intermediate and outer bran layers are mainly
involved in stress/defense. Bioactivity of wheat bran proteins has been scarcely
explored. Some evidences suggest a role in regulating fat metabolism,
which could be preventive of obesity and nonalcoholic fatty liver disease.
R. R. Balandrán-Quintana (*) · A. M. Mendoza-Wilson

Centro de Investigación en Alimentación y Desarrollo, A.C., Coordinación de Tecnología de
Alimentos de Origen Vegetal, Hermosillo, Son., México

296 R. R. Balandrán-Quintana and A. M. Mendoza-Wilson
Antihypertensive peptides have been isolated from wheat bran, as well as soluble
proteins of low molecular weight with antibacterial properties. Other uses for
wheat bran protein extracts, such as enzyme inhibitors in food processing or as
reservoirs of proteins for nanoparticle self-assembly, are discussed.
Keywords
Cereals · Bioactive peptides · Wheat bran proteomics · Wheat pericarp · Pericarp
proteins · Wheat aleurone · Agro industrial by-products
Abbreviations
2-DE Two-dimensional electrophoresis
ACE Angiotensin I converting enzyme
AMP Adenosine monophosphate
AMPK AMP-activated protein kinase
Arg Arginine
ATP Adenosine triphosphate
CD Degrees day after anthesis
DAA Days after anthesis
DiFA (AT) Dehydroferulic acid (aryltetralin form)
DiFA (BF) Dehydrodiferulic acid (benzofuran form)
Gln Glutamine
Ile Isoleucine
Leu Leucine
NASH Nonalcoholic steatohepatitis
Phe Phenylalanine
PPO Polyphenol oxidase
Pro Proline
Thr Threonine
Tyr Tyrosine
Val Valine
1 Introduction
In this chapter, proteins from wheat bran are reviewed. Wheat is the third most
important cereal world-wide, aside from rice and maize, in terms of production [1].
However, it represents the first supply of proteins and energy for human beings,
either directly or indirectly, since it is processed to fabricate a variety of foods and
also is used for livestock feed [2, 3].
The most common processing method for the wheat grain is milling, from which
flour is obtained as the main product while bran and germ are major by-products [4].
Traditionally, most of the produced bran has been intended to animal consumption,
whereas the remaining fraction has found applications in a few food groups as a
source of dietary fiber [5]. In recent decades, an increasing interest for wheat bran
12 Wheat Bran Proteins 297
has arisen since many potential benefits to human health, derived from its consump-
tion, have been discovered [5–7]. In this context, the wheat bran proteins, despite
being of better quality than those of the flour, are underutilized because most are
enclosed in cells, surrounded by a cell wall made up of cellulose and hemicelluloses,
besides being complexed with polysaccharides, so their bioaccessibility is poor. This
entrapment and/or complexation also make the extraction and isolation of proteins a
difficult task [8].
Proteins from wheat endosperm have been well studied because of their techno-
logical importance [9], but not those of bran. The state of the art in this respect
is that wheat bran proteins are distributed among the different layers, aleurone
having the most part of them. The classes of proteins range from storage proteins
to enzymes involved in cellular metabolism and stress/defense. Most of the proteins
in aleurone are globulins, whereas those in middle and outer layers are mainly
albumins with metabolic and defense activity [10]. Until now, with the assistance
of mass spectrometry and bioinformatics, a great diversity of proteins have been
identified from the thousands of spots revealed in two-dimensional electrophoresis
(2-DE) gels from bran of mature and developing grains [11–16]. Distribution of
proteins among the different layers that compose the bran has been possible to
elucidate through proteomic studies assisted by electronic, fluorescence, and optical
microscopies [10]. It is also known that pericarp proteins are under strict genetic
control because no significant differences in their expression have been found
between species or between growing conditions.
Although at the present time not all the proteins which are contained in the 2-DE
Coomassie-stained spots have been identified, the available information has permit-
ted to explain in some detail how the wheat bran proteome is interrelated to provide
the grain with an arsenal of chemical compounds and enzymes. New evidences
showing the advantages that could be taken from the properties of wheat bran
proteins, highlighting those of importance to human health, are also discussed.
2 Overview on Wheat Grain
Common wheat (Triticum aestivum) is classified within the family of grasses

Poaceae, subfamily Pooideae, and represents only one of the nine species
of Triticeae tribe that conform to this subfamily, among which also are bar-
ley (Hordeum vulgaris), rye (Secale cereale), and triticale (Triticosecale sp.),
as well as other wheat species known by their common name as Emmer or farro
(T. dicoccum); hard Macaroni (T. durum); Einkorn the small spelt (T. monococcum);
Spelt (T. spelta), and Rivet or cone (T. turgidum). The name T. aestivum comprises
several forms of hexaploid wheat, although not necessarily all are common or have
the necessary characteristics for the bread making. Within each species, there are
also subspecies or varieties. The terms variety and cultivar are sometimes used
interchangeably; however in a botanical sense, a variety (or subspecies) is the result
of natural selection, whereas a cultivar is a plant that has been domesticated, i.e., is
the product of a selection made by man, so it does not represent an evolutionary
Fig. 1 Schematic representation of the major morphological components of the wheat caryopsis
and their distribution by grain weight
status [17]. In this chapter, when reference is made to a cultivar, the taxonomic name
will be written, followed by the name of the cultivar in quotation marks.
Wheat grain is a one-seeded fruit or caryopsis, which is constituted by three major
morphological components [18] (Fig. 1):
1. Embryo
2. Starchy endosperm
3. Pericarp
The embryo is the reproductive organ from which a new plant is born after
fertilization. Embryo represents the smallest percentage of the grain weight, only
3%, and its chemical composition is based on lipids and proteins. The starchy
endosperm is the most voluminous part, representing approximately 83% of the
grain weight, and is composed basically of starch and proteins. The proteins of the
endosperm possess chemical characteristics that grant them the functionality
required for the elaboration of bread. This quality makes wheat a unique cereal
and is one of the main sources of energy and protein in human food [19]. Pericarp
constitutes approximately 14% by weight of the caryopsis. It is an important source
of dietary fiber and bioactive compounds, among which phenolic compounds
stand out because of their antioxidant capacity [20, 21]. It also contains important
amounts of vitamins, minerals, and proteins (Table 1).
Table 1 Chemical Amount (per 100 g, unless

composition of wheat Component otherwise indicated) References
pericarp or bran
Water 9.9 g [22]
Protein 15.6 g [22]
Total lipid (fat) 4.25 g [22]
n-Fatty acids 0.66 g [23]
Monoglycerides 0.26 g [23]
Diglycerides 0.22 g [23]
Triglycerides 0.20 g [23]
n- 0.23 g [23]
Alkylresorcinols 0.05 g [23]
Sterols
Fiber, total dietary 42.8 g [22]
Sugars 0.41 g [22]
Xylose 47 mol% [21]
Arabinose 24 mol% [21]
Glucose 15 mol% [21]
Minerals
Potassium, K 1,182 mg [22]
Phosphorus, P 1,013 mg [22]
Magnesium, Mg 611 mg [22]
Carbohydrates, by 64.51 g [22]
difference
Vitamins
Thiamin 0.52 mg [22]
Riboflavin 0.58 mg [22]
Niacin 13.6 mg [22]
Vitamin B-6 1.3 mg [22]
Folate, DFE 79 μg [22]
Vitamin A 9 IU [22]
Vitamin E (alpha- 1.49 mg [22]
tocopherol) 1.9 μg [22]
Vitamin K
Phenolics (μg/g cell wall)
Trans-ferulic acid 3,600 [21]
5-80 DiFA (BF) 1,000 [21]
Cis-ferulic acid 1,000 [21]
8-0-40 DiFA 1,000 [21]
8-80 DiFA (AT) 1,000 [21]
2.1 Protein Synthesis at Different Stages of Grain Development
As in any other biological system, cereal proteins are synthesized in response to an

intricate molecular signaling system. In general terms, the ultimate aim of such a system
is the formation of a new seed, which is equipped with the necessary to generate another
plant and to defense itself against a hostile environment. Embryo, aleurone, and
endosperm act in coordination and in a differentiated way after fertilization. From the
embryo, the new plant is born, while the endosperm and aleurone provide all the reserve
substances and enzymes necessary for its development [12].
There are variations in literature regarding the number and duration of the stages
through which the wheat grain develops. In order to give an overview of the proteins
that are expressed along the wheat grain formation, here will be adopted the three-
stage development [3, 16]. Duration of each stage is given in days after anthesis
(DAA), i.e., days after the flowering period, and sometimes is also expressed as
degrees day after anthesis ( CD), i.e., the accumulated daily average temperature, in
Celsius degrees. These three stages are:
1. Early development (lag phase) [0–16 DAA; 0–250 CD]

2. Accumulation of storage compounds (filling phase) [14–28 DAA]
3. Maturation-desiccation [28 DAA to maturity]
The events occurring in the early development stage and lasting of each one are:
A. Fertilization and development of coenocytic endosperm (0–1 DAA).

B. Cellularization (3–6 DAA; 50–100 CD).
C. Differentiation (7–8 DAA; 115–135 CD). In this event, the formation of main
cell types occurs, such as transfer cells, aleurone, starchy endosperm, and cells
surrounding the embryo.
D. Cell wall initiation and intense mitotic activity (8–10 DAA; 135–165 CD).
E. End of cell division (at 16 DAA; 250 CD).
Protein expression starts from the beginning of wheat grain formation, as evidenced
by the 492 Coomassie-stained spots that have been observed in 2-DE gels from wheat
grains in the early development stage (first 2 weeks, from ovary fertilization to
280 CD) [16]. From these spots, 249 proteins were identified, 52.6% corresponding
to enzymes involved in cellular and carbohydrate metabolism; 17.4% to environmen-
tal and information processing; 14.6% to genetic information processing; 13.4% to
stress and defense responses, and 0.4% to cellular processes, whereas 1.6% were
unknown. The most important changes in the proteome, which is defined as “the
assortment of proteins produced at a specific time in a particular cell or tissue type”
(https://www.nature.com/scitable/definition/proteome-297) at the early stage were
observed at 125–195 CD, when proteins with molecular mass 10–110 kDa were
expressed and identified as storage proteins (glutenins and gliadins) [16].
Gliadins (prolamins) are not only the major storage proteins in the wheat grain but
also determine the viscoelasticity of dough, so their study has been fundamental for
targeting the quality of wheat [24]. Thus, proteome differences between wheat
cultivars having different gluten quality properties have been studied. The analysis
and identification of proteins in developing grains of T. aestivum “Jimai 20” and
T. aestivum “Zhoumai 16” by 2-DE, assisted by MALDI-TOF/TOF-MS, resulted in
variable patterns of protein expression. From 174 Coomassie-stained spots, 84
unique proteins were identified. Fourteen protein spots accumulated in higher
abundance in “Jimai 20” than in “Zhoumai 16,” the former having superior gluten
quality. Among proteins, the NAD-dependent isocitrate dehydrogenase, triticin

precursor, LMW-s glutenin subunit, and replication factor C-like protein were
identified, which are likely to be associated with superior gluten quality [24].
In general, proteins identified in the early stage are involved in the cellular
metabolism, such as the enzymes of amino acid synthesis, energy production, cell
wall initiation, etc., or in DNA repair and replication, cytoskeleton, and structure.
At the beginning of the grain formation, also starch granules-associated proteins
are expressed. Stable expression of many proteins involved in signal transduction,
sugar metabolism, and stress-defense is carried out between 150–280 CD. By the
195 CD period, proteins responsible for folding and degradation are expressed,
whereas the starch granule-associated proteins continue increasing constantly their
expression up to 280 CD. Heat shock proteins (HSP) are expressed in the early events
[16]. In other study, 138 unique proteins were identified by 2-DE and tandem MALDI-
TOF/TOF-MS in the 6–20 DAA development period of “Chinese spring” and “NIL-
31” wheat cultivars. Proteins involved in carbohydrate metabolism were the most
abundant (32.02%), followed by those of synthesis/assembly/degradation of proteins
(13.16%), stress/defense (10.53%), and energy production and transport (9.65%) [25].
Nevertheless the resolution attained by 2-DE, it has limitations, as for example
the difficulty for separating hydrophobic proteins. In this regard, other study
has permitted the identification of up to 1762 proteins in the early stage, comprising
from 4 to 12 DAA, by using isobaric tag for relative and absolute quantitation
(iTRAQ) and LC–MS/MS methods [26]. Most of the proteins were involved in
metabolism, including cell division, cytoskeleton, carbohydrate metabolism, lipid
metabolism, nitrogen metabolism, protein synthesis, signal transduction, translation,
and transport [26].
Thus, irrespective of the number of identified proteins, results obtained between
distinct methodologies are consistent in that the early stage is characterized by an
intense activity of enzymes involved in cellular metabolism, and this pattern is
similar among wheat species [25–28].
In the middle and late stages of grain development (21–42 DAA), by means of 2-DE
assisted by tandem MALDI-TOF/TOF-MS, 130 proteins have been identified in the
Coomassie-stained spots. According to differential functions, proteins were classified
and distributed as stress/defense (35.4%), carbohydrate metabolism (21.5%), protein
synthesis/assembly/degradation (3.1%), storage proteins (6.9%), energy production and
transportation (7.7%), photosynthesis (6.2%), transcription/translation (3.9%), signal
transduction (4.6%), and unknown function groups (10.8%) [29].
Proteome characterization of the elite wheat T. aestivum L. “Shaan 253” in the filling
stage (15–30 DAA) by using an iTRAQ approach, resulted in the identification of 859
differentially expressed proteins [30]. Results showed a down regulation over time of
proteins involved in energy and starch metabolism. On the other hand, storage proteins
(high and low molecular weight glutenins) were upregulated, especially at 25–30 DAA.
Most of the stress/defense proteins showed upregulation during grain filling, with high
expression levels at 30 DAA, i.e., alpha-amylase inhibitors, enzymes involved in the
response to drought and oxidative stress, serpins, and xylanase inhibitors, whereas
others, like some peroxidases and some HSP, were downregulated with age [30].
3 Wheat Bran
Bran is a slang word used for referring to cereal pericarp, the botanical tissue
enveloping the grain and that gives mechanical support and protection to the endo-
sperm [13] (Fig. 1). Wheat pericarp consists of seven layers, which together account
for approximately 14% in weight of the grain. For simplicity, authors frequently
group these layers to divide the pericarp into three great sections [31] (Fig. 1):
1. Inner pericarp. Consists of the aleurone, a monolayer of vegetative cells that

surrounds the endosperm and the germ. Although aleurone botanically belongs to
the endosperm, it is considered the innermost part of pericarp because it remains
adhered to it after grain milling [32].
2. Intermediate pericarp. Comprises three layers, composed by the cross cells, tube
cells, and testa, all of them made up from dead cells in the mature grain.
3. Outer pericarp. Also is composed of dead cells and comprise the hyaline layer,
beeswing and the outer layer.
Pericarp layers are deposited at different developmental stages of the wheat

grain, originally they were made up of living cells, each layer having a different
function [33]. In the last stage of development, when grain is desiccated to reach
the maturity, aleurone remains as a layer of vegetative cells. Aleurone constitutes an
essential source of minerals, chemical compounds, and proteins, necessary for the
growing of a new plant after fertilization [34]. On the other hand, the cells of
the intermediate and outer pericarp layers undergo cell death, serving as mechanical
support and protection for the mature grain [31]. However, they not only act
as a physical shield but also are dynamic structures since contain a variety of
enzymes intended to defend the grain against environmental stresses and pathogenic
attacks [10]. Thus, through the development of the wheat grain, a great variety of
proteins are expressed, many of them in the pericarp layers [13], resulting in that
wheat bran has a total protein content of approximately 15% in weight [22].
After milling, the wheat endosperm is recovered as the main product (the flour)
with an approximate yield of 72%, whereas pericarp and germ are obtained as by-
products, with average yields of 15% and 2%, respectively [32]. As said before, once
separated from endosperm and germ, in the miller’s argot, the pericarp is called bran.
Bran is obtained in the mill as flakes of 67 μm thick [35]. Mainly two classes of bran
are obtained, which are graded as human or animal consumption.
Animal consumption grade bran is mixed with impurities, such as straw and other
classes of offal. Human consumption grade bran is the purest class of bran and is
almost completely composed of bran flakes to which remainders of endosperm and
some germ particles are adhered [32].
Traditionally, approximately 90% of the wheat bran is used for animal consump-
tion, whereas the remaining 10% has found applications as ingredient in the elabo-
ration of processed foods, mainly bread and cereal snacks [36, 37], or is used for its
inclusion in healthy diets [5]. Wheat bran has been largely appreciated by its high
content of dietary fiber and more recently by the benefits to human health which are
attributed to a diversity of bioactive compounds (Table 1). Among the chemical

composition of wheat bran, proteins are highlighted, as these nutriments are present
in a relatively high proportion (more than 14% DW) [22, 37]. Also is known that
their essential amino acid composition is better than that of the flour [38]. However,
wheat bran proteins have been largely ignored in terms of a rational use, probably
because of the drawbacks arising during their extraction [8].
4 Wheat Bran Proteins
There is a copious literature on proteins from wheat endosperm. In fact, wheat endo-
sperm is by far one of the most studied protein systems because of their technological
and economical importance [9]. Wheat bran proteins have not been the subject of such
an extensive study but, fortunately, plant biochemistry has been in charge of providing
most of the information about it. Accumulated knowledge about classes, structures,
properties, and distribution of proteins in the wheat bran is important as allows under-
standing their role in the grain. Finding explanations to phenomena related to their
application, as well as predicting new uses, are also scope of interest in this subject.
It is worth mentioning the great impetus given by 2-DE to the large-scale study of
the functions of proteins. In 2-DE, the protein separation is performed in two steps,
by isoelectric focusing in the first one and then as a function of the molecular mass.
After staining, usually with the Coomasie blue reagent, a number of spots are
visualized on the gels, each one corresponding to a unique protein [39].
Proteins from the wheat pericarp or bran will be analyzed in this review in the
context of grain development, as well as in terms of potential benefits and applica-
tions, at the light of the newest available information. Due to the space constraint, the
analysis is not intended to be exhaustive but is expected to serve as a guide to
awaken the interest to innovate in relation to the uses of these proteins.
4.1 Distribution of Proteins Among the Various Layers of the

Wheat Bran
The methods used to prepare wheat bran for chemical characterization depends on
the purpose of such characterization and this issue is very important when it comes to
characterize proteins. Since bran has endosperm residues, if the purpose is to
characterize the bran proteins in bulk, or fractioning them according to solubility,
endosperm residues must be removed from samples by brushing [40] or by rapid
washing while immersed in water [41]. Further defatting with solvent of the washed
bran is also desirable. On the other hand, if bran will be used for technological
purposes, where the presence of endosperm is irrelevant, preparation (if any) will
probably consist in only homogenizing the particle size through sieving. In some
other cases, stabilization to inhibit enzymatic activities, mainly lipases and proteases,
is mandatory [42].
In proteomic studies, whole pericarp or its layers are invariably dissected by hand
after imbibing the wheat grains in water. The latter has the objective of facilitating the
manual dissection of individual layers. Manual separation of the bran into layers has
permitted to analyze the histology and chemical composition of each one. This
technique is advantageous because it permits to extract proteins from individual layers,
so cross-contamination between layers is avoided; however, this is a laborious task.
The study of proteins from wheat bran dates from the 1920s and the details known
until now have been gradually revealed. At that time, wheat bran was subjected to
successive extraction steps with different solvents in order to obtain the Osborne
fractions [43]. Wheat bran proteins were first classified as albumins, globulins,
and prolamins, according to their solubility in water, dilute salt solution, and 70%
alcohol, respectively [44]. In further studies, their amino acid content was reported
[45], as well as their biological value, assayed in murine model [46]. Since proteins
were obtained as a bulk, little or nothing was known about properties or functions of
individual proteins. Gel electrophoresis and gel chromatography have helped to
separate the proteins and elucidate their molecular masses [47]. In turn, separation
has allowed testing some more specific properties, for example, those related to
enzymatic activity [48].
Dissecting the bran in its layers and extracting proteins from each layer has been
a good approach to study their distribution in the grain pericarp. Microscopic
techniques have advanced to such an extent that it is now possible to observe the
precise sites in which the different chemical components of cells and tissues
are located. The high resolution of the current microscopes allows capturing in detail
the cellular compartments. It is also taken advantage of the specific interaction
of some cellular components with monoclonal antibodies and fluorescent molecules
or with molecules which develop well-defined colors [49]. This makes possible the
labeling of components of interest and, therefore, to identify their histological
location by fluorescence microscopy or conventional optical microscopy, respec-
tively [50].Intrinsic fluorescence of cellular components is exploited too, as is the
case of phenolic compounds and proteins. It is also common to couple one or
more microscopic techniques with the chemical labeling or the intrinsic fluores-
cence, to observe even more details. For example, in the particular case of wheat
bran, the resolution of the scanning electron microscope coupled to the sensitivity of
the confocal fluorescence microscopy, have allowed the fluorescence immunoloca-
lization of defense proteins in bran cross-sections [10].
Identification and elucidation of protein functions has greatly advanced in recent
decades thanks to proteomics, which relies on 2-DE, mass spectrometry, and nucle-
otide sequencing databases [51–53]. Proteomics is the study of all the proteins of a cell
or tissue, rather than individual proteins, in order to integrate the information and thus
respond to biological questions [53]. It is a tool which has permitted deepening on the
knowledge on distribution and functions of the wheat bran proteins [11].
Proteins in wheat bran are distributed among their different layers [10]. Those of
the outer layers are the most easily extracted because they are water soluble; these
proteins are basically defense-stress enzymes. Those in the middle layer are a little
more difficult to extract, most are oxidative-stress and defense-related proteins. On
the other hand, proteins from the aleurone layer are enclosed in the cells, so their
extraction is invariably assisted by cell wall-disrupting methods, such as shear force
and ultrasound, among others, or the use of hydrolytic enzymes [8]. Most of the
aleurone proteins act as storage proteins, although some also have hydrolytic
functions in protein and starch metabolism during germination and grain filling.
4.1.1 Proteins from the Outer Layers

The pericarp of the wheat grain represents the first line of defense against biotic and
abiotic stresses, providing a series of protective barriers:
1. Gives mechanical resistance against invasive attempts by insects and pathogenic

microorganisms;
2. Has a probable role in regulating the movement of water into dry grains, which is
important to germination, preharvest sprouting, expression of dormancy, and
conditioning for optimum milling performance [54];
3. Is sheltered with an arsenal of chemical compounds and enzymes, aimed at
repelling biotic attacks if mechanical damage cannot be avoided.
The strength of the wheat grain pericarp is provided by the combination of

individual mechanical properties of its layers [31]. In the caryopsis development,
pericarp plays a key role because it performs photosynthesis and supplies oxygen to
the endosperm, besides dissipating excessive energy during the grain-filling [55].
On the other hand, some of the chemical compounds acting against biotic stresses
are structural constituents of the mature grain coat, such as the phenolics and some
proteins [21]. Others, for example, many enzymes, are produced de novo or acti-
vated during one or more stages of the grain development. The components of
this protective machinery act not necessarily in isolation, but sometimes they do it
together or synchronously. Details on the complicated defense mechanisms of cereal
grains are out of the scope of this chapter. Here, the functions of those proteins
which are constitutive of layers of wheat bran will be reviewed.
Production of proteins in the course of caryopsis development obeys to gene
expression. Such expression is space differentiated and responds to programmed
events, which are necessary for the grain to reach a characteristic phenotype and to
be equipped for repelling biotic and abiotic stresses [56]. Aleurone and peripheral
layers are formed in the early stages of development of the wheat caryopsis [57].
Aleurone provides protection to the endosperm and acts as a reserve of plant
hormones, minerals, vitamins, and other nutrients, including proteins. Eventually,
these nutriments will be available in the mature grain to participate in the beginning
of a new life cycle. Peripheral layers are those comprising the outer and middle
pericarp, which, as said before, are the protective tissues of the grain. Some authors
include aleurone within the peripheral layers. In this review, this detail if is consid-
ered appropriate will be noted.
Proteomic studies on the peripheral layers (including the internal pericarp, hya-
line layer, testa, and aleurone) of hexaploid wheat grains, from anthesis to physio-
logical maturity (0–700 CD), have resulted in the identification of 207 proteins.
Classified in functional categories, the dominating proteins in peripheral layers are

those involved in metabolism (32%), storage (25%), stress/defense (17%), and
genetic information and processing (12%), whereas the remaining are unknown or
hypothetical (6%), involved in environmental information and processing (3%), ATP
interconversion proteins (3%), and proteins involved in biosynthesis of secondary
metabolites (2%) [15].
The enzyme profiles are in good correlation to the cellular events associated to
each development stage in cereal grains, with metabolic proteins having their
maximum expression at the early stages (up to 397 CD). Dominating proteins are
those involved in energy metabolism, including photosynthesis and ROS (reactive
oxygen species). At 204 CD, a significant decrease of protein folding, PS, and
signal transduction occurs, which is associated to the end of cell differentiation. The
maximum functional diversity has been found at 295 CD, in whose profile the largest
category corresponds to the carbohydrate metabolism (enzymes of the citric acid cycle,
glycolysis, and sucrose synthesis) as well as ribonucleoproteins, the latter involved in
posttranscriptional changes. At the 397–455 CD stage, when the grain color changes
from green to pale yellow, a drastic decrease in the profile of proteins belonging to
energy metabolism is observed, indicating that maturity is in progress [15].
In the late stages, proteins which indicate response to hypoxia, such as alcohol
dehydrogenase and pyruvate decarboxylase, are identified, as well as enzymes
involved in detoxification and oxidative stress. The two latter groups, which include
ascorbate-peroxidase, glyoxalase, and thioredoxin peroxidase, are also identified
in all stages of development. In the early stages, their presence is due to the
production of ROS by photosynthesis in the green pericarp, whereas in the late
stages probably help in maintaining the aleurone alive as this is the only tissue that
remains alive in the grain after maturity. Finally, in the latter stages (700 CD),
the protein profile is dominated by oxidative stress proteins and by storage and
defense-related proteins. Globulins 2, 3, 3B, and 3C have been identified, which are
probably specific to aleurone. Xylanase inhibiting proteins, as well as chitinases and
endo-chitinases are also dominating in the latter stages [15].
4.2 Proteins from Aleurone
The cereal grain aleurone is the main stock of phytic acid and minerals, besides to be
a reservoir of hydrolytic enzymes that act on starch and endosperm proteins during
the germination process [58]. Many of the nutrients and bioactive compounds
present in the wheat grain are concentrated in the bran [59], especially in the
aleurone layer. This layer is rich in B complex vitamins since 82% of all niacin,
61% of pyridoxine, and 37% of riboflavin are contained in it. Between 43% and 61%
of the total minerals are also contained in wheat aleurone, being particularly abun-
dant phosphorus, potassium and magnesium. Likewise, aleurone has 43.8% of
hemicelluloses (pentosans). Its protein content of 30% represents 15.3% of the
total protein content of the wheat grain, at the time that provides 30% of the total
Lys, the limiting essential amino acid in wheat [60].
Proteome from wheat aleurone is strongly genetically controlled as significant

differences between species have not been found in proteomic studies. Aleurone is
the layer of wheat bran with the most abundant variety of proteins. The number
of Coomassie-stained spots observed in 2-DE gels of wheat aleurone ranges from
approximately 700 to 1300. Proteins from aleurone and peripheral layers of
T. aestivum “Chinese Spring” and “Recital” did not show quantitative differences.
2-DE gels of the protein extracts revealed 518 to 754 Coomassie-stained spots in the
range of 12.6–76 kDa, irrespective to the cultivar [9, 10].
The proteomic study of aleurone cells, isolated from T. aestivum “Babber,”
resulted in 672 Coomassie-stained spots in the 2-D electrophoresis gels. Among
these spots, 387 (58% of the total) were identified as globulin-like storage proteins,
whereas the remainder 285 corresponded to proteins involved in carbohydrate
metabolism, protein synthesis, stress, and defense. The major proteins were 7S
globulin storage protein; xylanase inhibitors (XIP-I, XIP-III, TAXI-I); chitinases
(26 kDa endochitinase 1 precursor, chitinase class II); pathogenesis related-4
protein; secretory protein; alpha-amylase/subtisilin inhibitor; enolase; dehydroge-
nases (glucose-3-phosphate-, glucose ribulose- and formate-); cytosolic NADP
malic enzyme; aldose reductase, and heat-shock protein 70 (HSP70) [10].
Variation in the number of expressed proteins in aleurone between wheat
varieties is higher than that found between species. There have been observed
1258 Coomassie-stained spots in T. aestivum aleurone versus 1109 in T. durum,
with a total of 339 spots differing between genotypes. Among these 339 spots,
30.8% differed within T. aestivum varieties and 56.5% within those of T.durum,
whereas only 12.7% differed between T.aestivum and T. durum genotypes [14].
On the other hand, 1320 Coomassie-stained spots in 2-DE gels of aleurone from
T. monococcus have been observed. Eighty eight (91.5% of total) spots were
common between T.aestivum and T. monococcus, which indicates a highly
conservated genome A of the hexaploid wheat. Among the 88 spots that differed
significantly between species, 53 proteins were identified, 83% of which were
storage proteins. Other proteins were lipoprotein, glyceraldehyde-3-phosphate dehy-
drogenase, and 1-cis-peroxiredoxin [61]. Puroindolines are other class of proteins
found in aleurone and endosperm, which are products of the major locus that controls
the wheat grain hardness. It has been suggested that the action mechanism of
puroindolines in affecting the grain texture in soft wheat is by stabilizing the amylo-
plast membrane during desiccation and maturation. On the other hand, such stabiliza-
tion does not occur in hard wheat or only to a smaller extent, resulting in a more direct
contact between starch and the gluten protein matrix and so in a harder texture [62].
5 Bioactive Properties of Protein and Peptides Isolated from

Wheat Bran
Wheat bran proteins are underutilized when wheat bran is consumed by either
human or animals. This is because most of proteins are enclosed in the aleurone
layer, whose polysaccharide-constituted cell walls (which are resistant to digestion
in the human small intestine) reduce their bioaccessibility [63]. In the commercial
pig production, where the main diet consists in plant carbohydrates, a similar case
occurs since the nondigestible fiber reduces nutrient and energy digestibility [64].
Since extraction methods for wheat bran proteins are not efficient and non-profitable
[8], as far as is known at the present time there are no rationale use of them to take
advantage of their functional and nutritive properties. However, scientific evidences
regarding potential benefits of proteins or peptides from wheat bran to human health,
as well as proposals for their use in fields other than food, could revert this scenario.
Interesting discoveries on wheat bran proteins could impact or enhance the percep-
tion and interest of people in the consumption of wheat bran, or developing more
efficient methods for protein extraction.
It is known that the regular consumption of wheat bran results in reduction of
body weight, so it has been suggested as preventive of obesity. However, at the
present time, the compounds of bran which are responsible for diminishing body
weight have not been yet identified [65]. Fiber is a candidate, but other components
could also have a role. It is known the relation of obesity with the lipid metabolism.
Fat in excess, which is accumulated in adipose tissues, is further released as glycerol
and fatty acids by action of the pancreatic lipase, so the latter is the target of
inhibition for developing anti-obesity drugs [66]. In this regard, attention has been
directed to search for possible lipase inhibitors as responsible for weight reduction
associated to the uptake of wheat bran. The presence of a lipase-inhibitor component
solubilized in the aqueous phase of wheat bran was deduced when a loss of 77–94%
in the activity of porcine pancreatic lipase on triglyceride hydrolysis was observed
when assaying wheat bran at 1% concentration [67]. Such inhibition was reported to
occur only with the wheat bran and not when other fibers were assayed, like cellulose
or hemicelluloses. Also it was demonstrated that inhibition was independent of
adsorption effects on the fiber [67]. Later, it was reported that the lipolysis-inhibitor
compound could be a soluble protein [68], which was further corroborated [69].
In fact, two proteins with molecular weights of 24.4 and 27.5 kDa, which
inhibited the pancreatic lipase in vitro, were isolated from wheat bran and
germ, [70]. The inhibition mechanism was attributed to the interaction between
the proteins and the triglyceride substrate, hindering the adsorption of the enzyme
on the interface.
In other study, the ability of wheat bran to inhibit the hydrolysis of tributyrin,
a model lipid, by the calf pre-gastric lipase, has also been demonstrated in vitro [71].
Reductions in lipase activity up to 30.5% were observed with suspensions of
wheat bran, which was soaked by 24 h. Components responsible for the inhibitory
effects were not identified, but it was suggested that some low molecular weight
compounds, such as polysaccharides or proteins, could be involved [71].
The physiological relevance of lipase inhibition by wheat bran in humans remains
to be elucidated, but data suggest that wheat bran proteins likely play a role during
fat digestion.
Wheat bran proteins also could be effective protectors against bacterial infections.
Proteins with molecular weight <90 kDa, isolated from a soluble extract of wheat
bran by size exclusion chromatography, may interfere in the attachment of
enterotoxigenic E. coli to intestinal porcine epithelial cells [72], the latter used as
model of study. The identification of proteins by mass spectrometry resulted in
several low molecular weight protease inhibitors, such as Serpin-Z2B, Class II
chitinase, endogenous alpha-amylase/subtilisin inhibitor, and alpha-amylase/trypsin
inhibitor CM3. Also, evidence suggesting that Globulin 3 (one of the 7S storage
globulins of wheat) of 66 kDa could be one of the most firmly attached wheat bran
proteins to E. coli cells was demonstrated. Although no details on the molecular
events leading to the interfering process were studied, the information was placed in
the context of developing innovative anti-adhesion therapeutic agents to prevent
bacterial pathogenesis [72].
Other interesting fact about the wheat bran proteins is what has to do with
bioactive peptides. Interest for bioactive peptides is recent because many sources
of them have been identified, including cereals [73], in such a way that there are now
in the marketplace several products with a variety of declared bioactivities, such as
antihypertensive, antistress, immunomodulatory, among others [74]. By taking
advantage of the presence of endogenous proteases, bioactive peptides have been
generated by autolysis of wheat bran extracts (i.e., by stimulation of endogenous
proteolytic activity through promoting suitable conditions). Studies performed both
in vitro and with murine models have demonstrated several effects of these peptides,
which could be beneficial to human health.
The autolysis of wheat bran and shorts, both by-products of milling, the shorts
being constituted by a mixture of fine particles of bran, aleurone, and germ [32],
has resulted in the production of peptides with potential antihypertensive effect
[75].These peptides strongly inhibit the Angiotensin I converting enzyme (ACE),
which is a key enzyme in the regulation of blood pressure so that is the target of
inhibition for the development of antihypertensive drugs [76]. Shorts and bran
hydrolysates have shown ACE inhibitory activity (IC50) of 0.08 and 0.14 mg
protein/mL, respectively. The highest effect obtained is by autolysis of a mixture
of bran and shorts for 12 h, pH 3.2, 40 C, from which six peptides with ACE
inhibitory activity have been isolated: Leu-Arg-Pro, Leu-Gln-Pro, Ile-Gln-Pro,
Val-Tyr, Ile-Tyr, and Thr-Phe. The first two peptides are of additional interest as
they contain the branched amino acid Leu, which activates the pathway of AMP-
activated protein kinase (AMPK) [77, 78]. AMPK is thought to be important for
regulating fatty acid metabolism [79]. In this context, there is a pathological
condition known as the metabolic syndrome, whose liver manifestation is the
fatty liver disease of nonalcoholic origin [80, 81]. A progressive form of fatty
liver disease is the nonalcoholic steatohepatitis (NASH), which has as secondary
pathogenic factors the oxidative stress and the downregulation of AMPK [82,
83]. For these reasons, the effects of the Leu-Arg-Pro and Leu-Gln-Pro tri-
peptides obtained by autolysis of wheat bran have been investigated in murine
model on oxidative stress and the AMPK pathway [84].In general terms, the
administration of both peptides for 10 days to mice in which NASH was induced
by a high-fat diet resulted in a modulation of oxidative stress and overregulation
of AMPK. In addition, a significant decrease in the severity of NASH disease was
observed [84].
6 Potential of Wheat Bran Proteins for Other Uses
If using in bulk, wheat bran proteins could have some applications in food technol-
ogy [85]. Since long time, wheat bran proteins have been proposed as enrichers for
bread-making flour, because of their high digestibility, a well-balanced amino acid
profile, and a protein efficiency ratio comparable to that casein [86–88]. Results have
shown the feasibility of adding wheat bran protein-rich flour at levels up to 10%
with no negative effects on bread volume. On the other hand, sensory evaluation of
bread which was added with the protein-rich flour at levels between 15% and 25%,
resulted in lower scores for texture, color crust, and flavor, but these were not
objectionable [87].Wheat bran protein concentrates have also shown good water-
and oil-holding capacities as well as high dispersibility, the latter related with good
emulsification and foaming properties [40].
Inhibition of polyphenol oxidases (PPO) by proteins and peptides from several
sources has been reported [89–93]. PPO enzymes are responsible for the oxidation
of phenolic compounds, which eventually results in browning of fruits and vegeta-
bles [94], so are also target of inhibition for preventing enzymatic browning [95].
Under this scenario, hydrolysates obtained after proteolysis of aqueous extracts of
wheat bran proteins (i.e., the albumin fraction) have been assayed on buffered
extracts of apple. Kinetic data showed PPO inhibition up to 40% at hydrolysate
concentration of 1% (w/v). On the other hand, Lineweaver-Burk plots showed a
mixed-type inhibition, indicating that inhibitor or inhibitors interacted with both, the
enzyme and the enzyme-substrate complex [41]. Inhibitor molecules were not
identified, but authors reasoned about a possible role of proteins or peptides [41].
The globulin fraction of wheat bran proteins has also been tested as inhibitor of PPO,
using the amino acid l-Tyr as substrate [96]. Tyrosinase is a PPO broadly distributed
in organisms, including mushrooms [97], and is usually used as a model in PPO
inhibition studies [98]. Competitive inhibition of the activity of mushroom tyrosi-
nase by globulins from wheat bran, with an inhibition degree of 24% at 2 mM of l-
Tyr, has been reported [96].
The great variety of proteins found in wheat bran has motivated some researches
for finding alternative uses, beyond nutrition or the traditional food science appli-
cations. This is an interesting avenue in the context of an integral scheme for using
wheat bran, even more when emerging technologies are involved. Such is the case of
bionanotechnology, where proteins have shown to have a great potential for the
development of nanostructures with a diversity of applications [99–101]. At this
respect, the nanoencapsulation in order to protect or making drugs and bioactive
compounds more bioavailable is a promising approach in both the pharmaceutics
and food industries [102, 103]. Proteins from animal origin, like albumins, gelatin,
elastin, and those from milk and whey, have been the most studied as raw material to
fabricate protein nanoparticles by different methods [99]. Plant proteins have also
been studied, mainly soy proteins, zein, and wheat gliadins [101].
Recently, there have been some proposals for utilizing aqueous extracts of wheat
bran as source of water soluble proteins for nanoparticle formation. Nanotubes with
100 nm of internal diameter, outer diameter 200 nm, and more than 30 μm in length
have been obtained through the apparent self-assembly of peptides released after
proteolysis of wheat bran albumins in presence of calcium ions [104]. Since aqueous
extracts, once dialyzed and lyophilized, had a complex chemical composition
(protein accounted 45% DW), the structure of nanotubes was difficult to be attrib-
uted only to proteins. Instead, with the assistance of infrared spectroscopy analysis,
it was proposed a model in which polysaccharide-protein complexes, upon protease
action, behaved as sheets which were then gradually curved themselves with
the subsequent formation of a tubular nanostructure stabilized by calcium brid-
ges [104]. On the other hand, it was reported that undialyzed wheat bran aqueous
extracts subjected to cold gelation/desolvation results in formation of spherical
structures [105]. Cold-set gelation/desolvation is a method for fabricating nano-
particles [106] which is based in the alkali cold gelation of proteins. Cold gelation
consists in a first heating step, further cooling to room temperature and, finally,
adding divalent ions [107, 108]. In another more detailed study, it was shown the
protein nature of the nanoparticles and that these were formed during the heat
treatment, long before the addition of calcium, which is advantageous because one
step would be omitted during the process [109].
Protein-rich aqueous extracts of wheat bran are also investigated because of their
potential feasibility for acting as scaffolds for biomineralization in vitro [105].
Preliminary results show that a variety of minerals can be formed by just adding
calcium ions to aqueous extracts of wheat bran. Among the minerals, some which are
biologically induced, like calcium phosphates, are highlighted. The latter indicates
that phosphorus present in the extracts plays a role as precursor ion and that other
components of the extracts could act as the scaffold matrix. Experiments are in
progress to investigate the nature of the matrix and mechanisms of biomineralization.
In summary, in wheat bran there is a great diversity of proteins, which is derived
from a differential genetic expression of them during the development of the grain.
Most of these proteins are concentrated in the aleurone layer, where storage proteins
are predominating. In the layers of the intermediate and external bran, there are
proteins mainly involved in stress and defense. Although bioaccessibility of the
proteins of the aleurone layer is poor, those of the outer and intermediate bran layers
could be more accessible and present bioactivity related to the metabolism of lipids and
against enteropathogenic bacteria. Wheat bran proteins are also a source of antihyper-
tensive peptides, as well as peptides that activate the signaling pathway regulated by
AMPK. The latter is involved in the prevention of steatohepatitis of nonalcoholic origin
because of its involvement in the regulation of fatty acid metabolism. No less important
are the applications that wheat bran proteins could have in food technology, as inhibitors
of enzymatic darkening or for their functional and nutritional properties. Study of wheat
bran proteins could also be the scope of disciplines which are related to emerging
technologies, such as the bionanotechnology and biomimetics. In Fig. 2 a summary of
all this potential is represented.
As final reflection, the great amount and diversity of proteins that have been
identified in wheat bran suggest a variety of properties which are not exploited yet.
The newest evidences are pointing in direction to innovation possibilities which are
worth to be studied.
Fig. 2 Summary of the proposals for the use of wheat bran proteins
7 Conclusions
Underutilization of wheat bran proteins and the lack of proposals for their use make
the search for innovative alternatives a good opportunity area. Studies showing the
potential of wheat bran proteins or peptides in the prevention of hypertension and fat
metabolism-related diseases could give some guideline. Also, technological uses of
these proteins other than food, for example, in the fabrication of nanoparticles or
biomineralization, could be explored in depth in order to add value to wheat bran.
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Antihypertensive Peptides from Animal
Proteins 13
Z. F. Bhat, Susan Mason, James D. Morton, Alaa El-Din A. Bekhit, and
Hina F. Bhat
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
2 Management of Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
2.1 Pharmacological Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
2.2 Changes in Lifestyle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
2.3 Functional Foods and Food-Derived Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
3 Antihypertensive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
4 Mechanisms of Antihypertensive and ACE-Inhibitory Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
5 Production of Antihypertensive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
5.1 Chemical Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
5.2 Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
5.3 Microbial Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
5.4 Recombinant DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
5.5 Synthetic Antihypertensive Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
6 Antihypertensive Peptides as Functional Food Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
7 Antihypertensive Peptides from Meat Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Z. F. Bhat (*)
Division of Livestock Products Technology, Faculty of Veterinary Sciences and Animal Husbandry,
Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Jammu, Jammu
and Kashmir, India
S. Mason · J. D. Morton
Department of Wine Food and Molecular Biosciences, Faculty of Agriculture and Life Sciences,
Lincoln University, Christchurch, New Zealand
A. E.-D. A. Bekhit
Department of Food Sciences, University of Otago, Dunedin, New Zealand
H. F. Bhat
Division of Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of
Kashmir, Shuhama, Alusteng, Srinagar, Jammu and Kashmir, India

320 Z. F. Bhat et al.
7.1 Myofibrillar Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

7.2 Sarcoplasmic Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
7.3 Connective Tissue and Other Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
7.4 Effects of Aging and Cooking of Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
7.5 Meat Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Abstract
Hypertension is considered a major health problem throughout the world
among adults, adolescents, as well as children and several preventive and
therapeutic interventions are available. In addition to the pharmaceutical
drugs and lifestyle changes, significant milestones have been achieved in the
past decades in the identification of bioactive peptides from animal proteins
with useful antihypertensive activities. The antihypertensive properties of these
peptides are attributed to several mechanisms ranging from mineral-binding,
opioid-like and antithrombotic properties to inhibition of ACE (angiotensin-
converting enzyme). ACE-inhibitory peptides are the most widely studied
bioactive peptides with promising potential in hypertension management. In
addition to milk and dairy products, which are the major sources of antihyper-
tensive peptides, a remarkable increase has been observed in the documentation
of peptides from other animal proteins, such as meat, with demonstrated in vitro
and in vivo antihypertensive properties. Numerous opportunities exist in the
global market for the development of novel food products and additives based
on these antihypertensive peptides for the dietary management of hypertension.
This chapter reviews the antihypertensive peptides derived from meat proteins
and examines their possible role as a functional ingredient in foods for the
management of hypertension.
Keywords
Antihypertensive peptides · ACE-inhibitory peptides · Muscle proteins ·
Connective tissue proteins · Aging · Meat products
1 Introduction
Being the largest cause of death and leading cause of disability worldwide,
cardiovascular diseases are responsible for 17.3 million deaths per year globally
[1] and by 2030 this toll is expected to increase to more than 23.6 million deaths
worldwide [2]. Global deaths caused by cardiovascular diseases grew by 41%
from 1990 to 2013 [3] and are considered to be the cause of one third of female
deaths worldwide [4].
Despite of all prevention efforts and recent therapeutic advances, health
issues associated with atherosclerotic cardiovascular diseases are increasing [5].
13 Antihypertensive Peptides from Animal Proteins 321
For all racial and ethnic groups, hypertension is a leading independent risk
factor for cardiovascular diseases [6] and is thought to be the major preventable
cause of premature death throughout the world [7]. Being the leading cause of
mortality and morbidity among human adults globally [8, 9], epidemiologic
data indicates that approximately 40% of the human population aged above
25 years are affected by hypertension [10] and has been held responsible for
10.4 million deaths per year worldwide [11]. It is the major cause of death
among women compared with other metabolic and lifestyle risk factors [12].
Out of the annual 17 million deaths associated with cardiovascular diseases,
more than half are caused by hypertension [13]. In the last few decades, childhood
hypertension is constantly increasing and has become a major health problem in
children [14].
With at least a diastolic blood pressure of 90 mm Hg and a systolic blood
pressure of 140 mm Hg [15], the prevalence of hypertension is massive as
more than 1 billion people have hypertension worldwide and 80 million
people are affected in the USA alone [2, 16]. The number of the adult patients
with hypertension has increased from 594 million in 1975 to 1.13 billion in 2015
[17]. Based on the definition used, the childhood hypertension has been
recorded to exceed 30% in some reports [14]. Almost 90–95% of all the hyper-
tensive patients belong to the most common class of high blood pressure
known as “essential hypertension” which is recognized by an increase in a
patient’s blood pressure due to an unknown cause as no clear etiology is identified.
It seems to be caused by a complex interaction between environmental
factors and genetic predisposition. Essential hypertension is regarded as a
controllable risk factor of cardiovascular disease as it can be improved with
lifestyle choices like eating healthy foods, regular physical activity, decreasing
sodium intake, quit smoking, reducing alcohol consumption, and reducing the
stress level [18].
2 Management of Hypertension
2.1 Pharmacological Management
A range of pharmacologically active drugs are available on the market for the
treatment of hypertension which include calcium channel blockers, ACE (angio-
tensin converting enzyme) inhibitors, adrenergic inhibitors (such as α- and
β-blockers), diuretics, Ang II (angiotensin II) receptor blockers, direct vasodila-
tors, renin inhibitors, and mineralocorticoid receptor antagonists. Drugs that
inhibit renin-angiotensin system, which is a significant blood pressure regulator,
are extensively used for pharmacological management of hypertension. Hyperten-
sion can be safely treated in most patients by using pharmacological drugs;
however, these drugs have frequent side effects which may notably affect the
quality of life and is a major burden on global healthcare costs.
2.2 Changes in Lifestyle
Lifestyle choices and dietary recommendations are considered utmost important in

the management of hypertension. Unhealthy lifestyle and food eating habits
are considered to contribute strongly to the persistence and onset of arterial hyper-
tension [19]. The fast pace of modern times has increased preferences for
processed and fast foods due to convenience that has contributed significantly to
the spread of noncommunicable diseases like hypertension [20]. A substantial body
of evidence has demonstrated that certain individual dietary elements and dietary
patterns play a significant role in the development of hypertension [21]. Also, there is
convincing scientific evidence that the consumption of food rich in both energy and
dietary sodium is strongly associated with the development of hypertension [20, 22].
This has compelled many multinational food companies to reformulate their
products towards healthier and better nutritional versions [23].
Several reports on hypertension recommend early intervention for pre-hyperten-
sion. In addition to the pharmacological drugs, changes in lifestyle including weight
loss, reducing sodium intake, limiting alcohol consumption, increasing potassium
intake, avoiding smoking, and regular physical activity are advised for both
preventing the hypertension over the life span and for management of blood pressure
in hypertensive subjects [21, 24, 25]. Dietary strategies to control the blood
pressure in hypertensive patients and to lower the risk of hypertension in healthy
persons include diets low in sodium and rich in vegetables, fruits, and low-fat milk
products [21, 25].
2.3 Functional Foods and Food-Derived Peptides
Over the last few decades, efforts have been made consistently to produce designer and
functional foods that contain certain components with blood pressure lowering prop-
erties. These foods are intended to supplement or provide an alternative to
the pharmaceutical management of hypertension. Any food ingredient or additive
with antihypertensive properties, including food-derived peptides, could contribute to
the treatment or prevention of hypertension. Peptides with well-established in vitro
ACE-inhibitory activities may exert in vivo hypotensive effects if they reach the target
site in an active state [26, 27]. Fu et al. [28] suggested that daily consumption of aged
beef with naturally developed antihypertensive peptides may play a vital role in
maintaining normal blood pressure and indicated the possible use of these
peptides for the development of functional beef products, like patties, with antihyper-
tensive properties. Unlike synthetic ACE inhibitor drugs (such as captopril, benazepril,
perindopril, enalapril, trandolapril, and quinapril) which have frequent side effects such
as cough, hypotension, headaches, skin rashes, fatigue, fetal disorder and so on, food-
derived ACE inhibitors are believed to be stable and have minimal side effects [29].
Very few studies have been conducted on the side effects of food-derived ACE-
inhibitory peptides to date; however, it is believed if these natural peptides would
cause any side effects, those would be milder [30, 31].
3 Antihypertensive Peptides
Latent within the parent protein, bioactive peptides are specific stretches or frag-
ments of food proteins which can be released during food processing like enzymatic
hydrolysis or fermentation or during digestion inside the gut and have some phys-
iological benefits to human health beyond their nutritional capabilities. These short
sequence fragments are characterized with a low molecular weight and approxi-
mately 2–30 amino acid residues in length [32]. Released in vivo or in vitro from
food proteins, these peptides have the ability to affect various systems of body like
cardiovascular, digestive, immune, endocrine, and nervous system and attribute
various health effects including hypotensive, antimicrobial, cholesterol-lowering,
opioid, antioxidant, antithrombotic, immunomodulatory, cytomodulatory, and anti-
genotoxic activity.
Among the various bioactivities, antihypertensive property is widely studied and
several peptides from numerous sources have been reported. Proteins of animal
origin like meat, milk, whey, egg, blood, and fish proteins have been reported to
contain potential antihypertensive peptide sequences in their primary structure which
could become active when released during enzymatic or microbial hydrolysis [33].
Table 1 presents some of the identified peptide sequences with antihypertensive
properties from different animal proteins. Potentially bioactive ACE-inhibitory
peptides are normally short di- or tri-peptides as sequence length plays an important
role in the antihypertensive property of a peptide which could easily enter and bind
the deep-seated active site of ACE [58, 59]. However, peptide sequences of up to 14
amino acids have been reported to possess antihypertensive properties [60]. These
peptides usually contain aromatic or hydrophobic residues at their ultimate and
penultimate positions, with proline being particularly favorable residue in both
these positions [49, 59, 61, 62]. Besides the sequence length, the potency of the
ACE-inhibitory peptides is also determined by the three C-terminal residues, with
aromatic residues being most favored [62]. Several studies have suggested that
ACE-inhibitory peptides may also possess antioxidant properties due to the shared
requirements for both properties in terms of structure and length of peptide [63, 64].
The antihypertensive peptides have attracted global interest for the possible use of
their therapeutic potential for the development of antihypertensive foods for dietary
management of hypertension. Several new products have already struck the market
with antihypertensive claims (such as Ameal S, Danaten, and Evolus) and several are
under process of development and validation. Results of extensive in vivo clinical
trials are used to support the health claims of these products.
4 Mechanisms of Antihypertensive and ACE-Inhibitory

Peptides
Regulation of blood pressure in human body is a complex process involving several

metabolic pathways that makes the pathophysiology of hypertension highly com-
plex. It involves the interaction of genetic, environmental, and several other dietary
Table 1 Peptide sequences with antihypertensive properties from different animal proteins
IC50
Peptide sequence (μM) Protein source (protease used) Reference
MEVFVP 79.0 Flounder fish (Paralichthys Ko et al. [34]
VSQLTR 105 olivaceus) muscle (Kojizyme,
papain, pepsin, trypsin)
LHP 1.6 Camel milk proteins (α-amylase, Tagliazucchi
IY 2.1 pepsin, pancreatic and bile fluids) et al. [35]
AI 3.4
IPP 5.0
VY 7.1
LY 18.0
TF 18.0
FPGPIPK, IPPK, IVPN, – Buffalo skim milk (papain, pepsin, Mahmoud et
QPPQ trypsin) al. [36]
RVCL 175 Lizard fish (Saurida elongata) Wu et al. [37]
muscle (neutral protease)
LAFNPTQLEGQCHV – Sheep cheese whey (β- Corrêa et al.
lactoglobulin) (protease from [38]
Bacillus sp. P7.)
SWVE 33.88 Egg white proteins (protease from Pokora et al.
DILN 73.44 C. ficifolia fruit) [39]
GASSGMPG 6.90 Pacific cod skin (pepsin, papain, Ngo et al.
LAYA 14.5 trypsin, neutrase, alcalase, [40]
chymotrypsin)
GLPLNLP 18.7 Chum salmon (Oncorhynchus keta) Lee et al.
skin (α-chymotrypsin, trypsin, [41]
papain)
PLYV 0.05
DPHI 0.02
AER 0.11 Leatherjacket (Meuchenia sp.) Salampessy
muscle (papain, bromelain, et al. [42]
flavourzyme)
EQIDNLQ 0.24
WDDME 0.01
(g/L)
VPP, IPP – Emmental and gouda cheese
VPP, IPP, RYLG, RYLGY, – Cheddar cheese Stuknytè et
HLPLP, AYFYPEL, LHLPLP al. [43]
IQW 1.40 Egg white protein (ovotransferrin) Majumder et
LKP 2.80 (pepsin, thermolysin) al. [44, 45]
GL, GI, AI, VP, AY, GLN, – Skimmed goat milk (simulated Tagliazucchi
ALN, AEK gastric, pancreatic and bile fluids) et al. [46]
YQEPVLGPVRGPFPIIV – Bovine caseins (proteolytic extract Corrons et al.
from M. pomifera latex) [47]
PEQSLACQCL – Goat milk (β-lactoglobulin, β- Ibrahim et al.
QSLVYPFTGPI casein, κ-casein) [48]
ARHPHPHLSFM (pepsin)
(continued)
Table 1 (continued)
IC50
Peptide sequence (μM) Protein source (protease used) Reference
KHP >800 Sodium caseinate and purified α- Norris et al.
NP >800 casein (Aspergillus niger-derived [49]
ITP 10.0 prolyl endoproteinase)
WIQP 14.2
VLSRYP 36.7 Milk fermented with Li et al. [50]
LRFF 116.9 Kluyveromyces marxianus Z17
PFPEVFGK 108 Bovine αS1-casein (C12 Paul et al.
antihypertensive peptide) [51]
ESLSRLLG 46.7 Ostrich (Struthio camelus) egg Asoodeh et
white proteins (pepsin, pancreatin) al. [52]
YQDPRLGPTGELDPATQPI- 14.53 Koumiss (fermented mare’s milk) Chen et al.
[53]
VAVHNPVIV
PKDLREN 9.82
LLLAHLL 5.19
NHRNRMMDHVH 13.42
VLGPVRGPFP 137 β-casein (milk fermented with E. Quiro et al.
VVVPPF >1500 faecalis strains CECT 5727, 5728, [54]
LHLPLP 5.5 5826, and/or 5827)
LTQTPVVVPPF >1500
VRGPFPIIV 599
LHLPLPL 425
LVYPFPGPIPNSLPQNIPP 5.2
VLGPVRGPFPIIV >700
TGPIPN 316 Goat milk – β-casein (Subtilisin Geerlings et
SLPQ 330 alcalase) al. [55]
SQPK 354
RYLGY 0.71 Skim milk – casein-derived Contreras et
AYFYPEL 6.58 peptides (porcine pepsin A) al. [56]
YQKFPQY 20.08
FRADHPFL 3.2 Egg white protein hydrolysates Garcés-
RADHPFL 6.2 (BC pepsin 1:3000) Rimón et al.
YAEERYPIL 4.7 [57]
YRGGLEPINF >1000
ESIINF >1000
RDILNQ 435.7
IVF 33.9
YQIGL 173.8
SALAM 229.1
FSL 172.9
and physiological factors such as long-term high sodium intake levels, increased
sympathetic nervous system activity, increased RAS activity, low dietary calcium
and potassium intake, endothelial dysfunction, altered cellular ion channel, vessel
resistance variations due to vascular inflammation, and elevated activity of vascular
growth factors [65, 66]. The most widely studied pathways, with regard to food-
derived antihypertensive peptides, involve those reported to inhibit angiotensin
converting enzyme (ACE) in vitro. ACE is a key enzyme that regulates the blood
pressure of the body through the renin-angiotensin system. In addition to ACE-
inhibitory mechanism, food-derived peptides may help in lowering the blood pres-
sure through mechanisms that target rennin activity, calcium channels, arginine-
nitric oxide pathway, endothelin system function, Angiotensin (Ang) receptors,
vascular inflammation and oxidative stress, vascular remodeling, and sympathetic
nervous system [65, 67, 68]. As the peptides can trigger signaling processes by
binding the receptors in the gut, they may not need to be absorbed; however, many
peptides exert their antihypertensive effects only when present in relevant amounts
in the vascular system.
ACE inhibition is a better physiological target for treatment of clinical hypertension
due to its association with two types of blood pressure systems, the rennin-angiotensin
system (RAS) and kinin-nitric oxide system (KNOS). The angiotensin converting
enzyme (ACE), a heavily glycosylated membrane-bound zinc metalloprotease, is a
principle enzyme in RAS that controls the amount of fluids in the body and thus
regulates the blood pressure. Angiotensinogen is a prohormone synthesized in liver
which is cleaved by rennin produced by kidney to produce a decapeptide Ang I
(angiotensin I). Ang I is further cleaved by ACE to produce octapeptide Ang II
(angiotensin II) which is a potent vasoconstrictor and acts on vascular smooth muscles.
Further, ACE also causes inactivation of bradykinin and kallidin, the vasodilatory
peptides of KNOS. Therefore, by blocking the formation of Ang II and by reducing
the degradation of bradykinin and kallidin, ACE inhibitors can exert antihypertensive
effects. Although, ACE inhibitor pharmaceutical drugs are very much successful in
reducing the blood pressure, food-derived antihypertensive peptides are free from any
associated side effects and are believed to be safer than pharmaceutical drugs [65].
5 Production of Antihypertensive Peptides
Protein hydrolysates could be produced from protein sources by employing several

methods like enzymatic hydrolysis by using proteolytic enzymes, chemical hydro-
lysis process, and microbial fermentation [69]. Hydrolysates with ACE-inhibitory
properties have been generated from several kinds of proteins including proteins of
animal origin, like fish protein [70], meat protein [71, 72], milk protein [73], egg
protein [44], microbial proteins, like Saccharomyces cerevisiae protein [74], or plant
proteins, like sorghum grain protein [75]. These peptide hydrolysates could be
prepared economically from animal wastes and by-products that are rich in protein
and are generally processed into low-cost end products like meat meal, blood meal,
and so on [76].
5.1 Chemical Hydrolysis
Solutions of alkalis and acids could be used to cleave the peptide bonds of protein
substrates to produce different peptides. There is less control over the hydrolysis
which results into hydrolysates of different peptide profiles with reduced bioactiv-
ities and nutritional qualities. Higher variations in the end product and use of organic
solvents limit the high-end use of these hydrolysates.
5.2 Enzymatic Hydrolysis
Exogenous proteolytic enzymes are widely used to produce peptides of several

bioactivities by hydrolyzing various food protein substrates. Novel peptides
with higher antihypertensive properties have been discovered by using proteases
from various sources like alcalase, pepsin, flavourzyme, trypsin, actinidin, and
papain which are commercially available or by using some noncommercially
available proteases, such as Virgibacillus halodenitrificans SK1–3-7 protease
[77]. Due to the milder conditions and better control over the hydrolysis, enzy-
matic hydrolysis is considered a better method for the production of bioactive
peptides. The amino acid composition of the hydrolysates obtained is similar to
that of the protein substrates with few modifications depending on the type of
enzymes employed for the hydrolysis. This method is suitable for the production
of hydrolysates for food and pharmaceutical industries due to noninvolvement of
any toxic chemicals or organic solvents [78]. Protein hydrolysates of specific
bioactivities could be produced by controlling the conditions using specific
enzymes in comparison to chemical hydrolysis method. Protein hydrolysates of
different peptide profiles could be obtained by using different enzymes and
enzyme:substrate ratio.
5.3 Microbial Fermentation
Several microorganisms that are involved in the fermentation processes of foods

have the proteolytic capabilities and could produce protein hydrolysates with
bioactive peptides. The most common examples of these microorganisms are the
ones involved in the fermentation of dairy products [79]. The best known hypo-
tensive peptides IPP (Ile-Pro-Pr) and VPP (Val-Pro-Pro) were first isolated and
identified from fermented milk [76]. Several studies have been conducted for
the production of protein hydrolysates by utilization of microbial fermentation
[80, 81]. Antioxidative and antimicrobial peptides were produced from tannery
fleshings by fermentation using lactic acid bacteria [82]. Bacillus pumilus A1, a
keratolytic bacterium, has been reported to produce wool waste protein hydroly-
sates with antioxidative properties [80]. Bacillus subtilis A26, another proteolytic
bacterium, has been reported to produce protein hydrolysates through fermenta-
tion of several fish proteins [81].
5.4 Recombinant DNA Technology
Over the years, many novel genetic engineering-based techniques have been devel-
oped for the production of peptides at industrial scale. Recombinant DNA technol-
ogy is a preferred choice for the production of relatively large peptides consisting of
up to several hundred amino acids. This technology is well suited for the production
of large quantities of peptides from very inexpensive starting materials; however, it
typically requires a long and expensive research and development phase. There are
certain challenges in the production of peptides by these methods like degradation of
short hypotensive peptides by peptidases or proteases and the threat that expression
products may be harmful to the host. These shortcomings have been taken care of
by producing the peptides as a part of fusion proteins and afterwards separating
the target peptides by using enzymes and purifying them, though that adds to the
cost of production. Peptides with sequences FFVAPFPEVFGK, GHIATFQER,
HVLPVP, and HHL with antihypertensive properties have been successfully
expressed in E. coli [83]. A multimer, a precursor of 11 different hypotensive
peptides, was expressed and shown to release peptides during simulated gastroin-
testinal digestion [84].
5.5 Synthetic Antihypertensive Peptides
Peptides with antihypertensive properties could be conveniently synthesized for

therapeutic management of hypertension and is the most popular method of produc-
tion of peptides in a laboratory [85, 86]. The choice of the method depends on the
length and quantity of the desired product. While liquid-phase synthesis is a classical
approach for large-scale production of peptides for industrial purposes, the solid-
phase approach is now the standard method of production of peptides at the
laboratory scale and is most powerful method for synthesis of peptides composed
of about 10 to over 100 residues [85]. Peptides with several bioactivities have been
synthesized for their therapeutic applications and are currently being used for their
therapeutic contribution to the treatment of diabetes, obesity, immunosuppression
and gastrointestinal, cardiovascular, osteoporotic, antibacterial, or oncologic dis-
eases [87]. Synthesizing peptides or modifying peptides to improve their therapeutic
value is a cost-effective and convenient method of production; however, use of
synthetic peptides for the therapeutic purposes cause several and severe side effects
[88] which reduces their superiority over therapeutic drugs.
6 Antihypertensive Peptides as Functional Food Ingredients
The search for functional foods with antihypertensive properties has increased as
hypertension continues to increase worldwide. Several milk products like Calpis
(Calpis Co., Tokyo, Japan), Evolus (Valio, Helsinki, Finland), Ameal (Calpis Co.,
Tokyo, Japan), and Danaten (Danone, Paris, France) with scientifically proven health
claims are available in the market and exclusive rights of these manufacturers to use
these functional ingredients has proven to be a crucial factor in the ultimate success of
these products in the market [89]. Table 2 presents some of the commercially available
food products with antihypertensive claims. Using antihypertensive peptides as func-
tional ingredients for the development of food products needs certain considerations
including characterization of their physicochemical and sensorial properties. Figure 1
shows some of the essential steps in the development of meat peptide-based functional
foods with antihypertensive properties. One of the challenges of using peptides as
ingredients in food products is the bitter taste of these peptides composed mainly of
hydrophobic amino acids which are responsible for both bitterness and antihyperten-
sive properties [83]. However, several masking methods could preferably be used to
improve the acceptability. An optimal method for the production of peptides by
hydrolysis is required which could generate the output sufficient to meet the industrial
scale. The peptides need to retain their antihypertensive properties during the entire
stages of industrial level processing and remain stable during long-term storage. Little
information is available in the literature about the effects of various processing
methods on the activities of antihypertensive peptides. Research is required in some
areas like analyzing the interaction of food components and peptides during pro-
cessing, storage, and digestion.
Dehydration of peptides by using spray drying method has been reported to
induce changes in peptide composition and causing nonenzymatic browning and a
reduction in amino acid content [83]. Contreras et al. [90] studied the possibility of
production of two antihypertensive peptides viz. Ala-Tyr-Phe-Tyr-Pro-Glu-Leu and
Arg-Tyr-Leu-Gly-Tyr in a casein hydrolysate at an industrial scale. The peptides
were also incorporated into yoghurt and evaluated for stability during various
processing conditions and during storage under refrigerated conditions. The peptides
retained their ACE-inhibitory properties in vivo in spontaneously hypertensive rats
as well as under in vitro conditions. Four synthetic peptides (DFHINQ, GFHI, FHG,
and GLSDGEWQ) with ACE-inhibitory properties have been reported to retain
stability in water during heat treatment [91].
Another important challenge associated with antihypertensive food products is
the bioavailability of peptides which is responsible for differences between in vitro
and in vivo activities of peptides. The capacity of peptides to reach the target organ in
active form determines the physiological effect of the peptides. The final desired
activity of peptides depends on a series of processes involved in the oral adminis-
tration of peptides. These include the effect of GIT enzymes, brush border pepti-
dases, active intracellular peptidases during absorption through intestinal barrier, and
enzymes in the blood once they make it to the circulation [92, 93]. These processes
are more likely to affect the sequences which are responsible for the antihypertensive
properties of the peptides before they will elicit their response in the target organ.
Thus more scientists are interested in evaluating the different factors affecting the
bioavailability of different sequences responsible for hypotensive properties. Differ-
ent studies have been conducted on the effect of digestion on the hypotensive
peptides by using in vitro gastrointestinal simulation models. Several studies have
also analyzed the intestinal absorption by using in vitro intestinal epithelial
Table 2 Commercially available products with antihypertensive claims

Product Company Peptides Product type
Ameal S 120, Calpis Co. VPP, IPP Cultured milk, tablets,
Ameal S, Ameal (Tokyo, Japan) capsules, component
BP
Calpis Calpis Co. VPP, IPP Uncarbonated sweetened
(Tokyo, Japan) fermented milk beverage
Evolus Valio (Helsinki, VPP, IPP Calcium-enriched fermented
Finland) milk
Vasotensin Metagenics, USA LKPNM, LKP Tablets (bonito protein
hydrolyzate)
Peptide C12, C12 DMV FFVAPFPEVFGK Ingredient (casein protein
Peption International, The hydrolyzate)
Netherlands
Tensiocontrol Bioactor, The RADHPFL, IVF, Ingredient (egg protein
Netherlands YAEERYPIL hydrolyzate)
Biozate Davisco Foods Whey peptides Purified hydrolyzed whey
International, protein (powder form)
USA
Lowpept Innaves Biotech, RYLGY, Tablets, ingredient (powdered
Spain AYFYPEL casein hydrolyzate)
Casein DP Peptio Kanebo, Japan FFVAPFPEVFGK Soft drink (casein protein
hydrolyzate)
Danaten Danone (Paris, Tripeptides Fermented milk drink
France) (Lactobacillus helveticus
DN-119 90)
simulations involving the use of monolayer of intestinal cell lines. While evaluating
the effectiveness of hypotensive peptides, improvement in the bioavailability is
considered as an important goal.
7 Antihypertensive Peptides from Meat Proteins
With increasing awareness and concern about the health and safety, the quest to
replace synthetic drugs with natural compounds is of great interest. Antihypertensive
peptides from food proteins could provide an excellent alternative to synthetic drugs
[94] which is a major target for the treatment and prevention of clinical hypertension
[48]. Several human and animal studies have demonstrated the blood pressure
lowering capabilities of the food-derived peptides through ACE inhibition [95,
96]. Proteins from meat and meat by-products are considered as an excellent source
of peptides (Fig. 2) with both in vitro and in vivo ACE-inhibitory properties [97]. In
the last few decades, peptides derived from meat proteins from several sources, such
as chicken muscle [98], porcine muscle [99], and beef muscle [100], have been
found to reduce blood pressure through ACE inhibition [101]. Peptides with ACE-
Meat Protein Substrate
Ageing/Curing Extraction of Myofibrillar/ Microbial

(Endogenous enzymes) Sarcoplasmic/Stroma Fermentation
Proteins
Enzymatic
Hydrolysis
Protein Hydrolysates
Ultrafiltration/Fractionation
In vitro/ In vivo
Antihypertensive Testing
Peptide Purification
Peptide
Sequencing/Identification
Synthesis of Characterized
Peptides
Antihypertensive Activity
Assays
Bioavailability Trials
Incorporation into Food

Matrices
Fig. 1 Schematic diagram for the production of functional foods with antihypertensive properties
Myofibrillar
Proteins
Sarcoplasmic Antihypertensive Meat

Proteins Peptides Products
Stroma
Proteins
Fig. 2 Different meat protein substrates for the production of antihypertensive peptides
inhibitory properties have been reported from several skeletal muscle proteins
namely myosin, actin, tropomyosin, troponin, and collagen [85]. ACE-inhibitory
potential of these peptides is measured in vitro by IC50 value and in vivo by using
spontaneously hypertensive rats as an accepted animal model for human essential
hypertension [102–104]. Results from several studies have demonstrated that meat
protein hydrolysates may represent the source of natural peptides capable to reduce
ACE-I activity.
7.1 Myofibrillar Proteins
7.1.1 Contractile Proteins

Among all the meat protein-derived bioactive peptides, ACE-inhibitory peptides are
most extensively studied [102, 105]. ACE-inhibitory peptides are commonly pro-
duced from hydrolysis of skeletal muscle and connective tissue proteins of farm
animals like pig and poultry [106–108]. A large number of peptides with ACE-
inhibitory properties have already been reported from the enzymatic hydrolysates of
various meat proteins [27, 102, 105, 109]. Two ACE-inhibitory pentapeptides
sequenced as MNPPK and ITTNP were identified from the hydrolysates of pig
skeletal muscle proteins and were named as myopentapeptide A and B, respectively
[108]. These pentapeptides, corresponding to position 79–83 and 306–310 on the
myosin heavy chain, respectively, were shown to have ACE-inhibitory properties in
spontaneously hypertensive rats [110]. Six other peptides viz. PPK, MNP, NPP, ITT,
TNP, and TTN sharing the sequence parts of these two myopentapeptides also
showed the antihypertensive properties. An octapeptide VKKVLGNP
corresponding to position 47–54 on myosin light chain with temporary ACE-inhib-
itory properties was identified from the myosin protein hydrolysates [99]. One more
peptide sequenced as KRVITY isolated from pig skeletal myosin B treated with
pepsin and VKAGF isolated from porcine actin were reported to have significant
blood pressure lowering properties in vivo in rats [111, 112]. With IC50 value of
6.1 μM, KRVIQY was reported to retain its ACE-inhibitory activity after heating the
myosin B at 98 C for 10 min prior to hydrolysis by pepsin, suggesting the
bioavailability of the peptide even after cooking.
Seven ACE-inhibitory peptides with sequence LKP, LKA, LAP, IKW,
FKGRYYP, FQKPKR, and IVGRPRHQG were isolated from chicken muscle pro-
teins by thermolysin treatment [98]. Although, IC50 value of these peptides ranged
from 0.21 μM to 14 μM, however, the heptapeptide FKGRYYP with IC50 value
of 0.55 μM failed to produce antihypertensive activity in vivo in spontaneously
hypertensive rats. This indicated that in vitro ACE-inhibitory activity of the peptides
may not necessarily correlate with their in vivo ACE-inhibitory activity. Intracellular
peptidases or enzymes in the GIT or blood vascular system may have degraded
the peptide affecting its antihypertensive properties in vivo. Further, modification
of the peptides in the liver may also affect their in vivo antihypertensive
potential [102]. ACE-inhibitory properties of chicken breast muscle extract were
reported in vitro as well as in vivo in spontaneously hypertensive rats [104]. There
was a significant increase in the ACE-inhibitory activity of the extracts treated with
an Aspergillus protease and gastric proteases (trypsin, chymotrypsin, and intestinal
juice). All the three peptides identified with ACE-inhibitory properties possessed a
common sequence Gly-X-X-Gly-X-X-Gly-X-X, and the peptide with sequence
Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe showed the strongest ACE-
inhibitory activity with IC50 value of 42.4 μM. This peptide had a high affinity for
ACE and inhibited the enzyme in a noncompetitive manner. The Phe residue at the
C-terminus position and an aromatic residue at the antepenultimate position were
reported to play an important role in the inhibitory activity [113] supporting the
theory of presence of three C-terminal hydrophobic residues for the ACE-inhibitory
activity of a peptide. Binding of a peptide to ACE is strongly influenced by this
C-terminal tripeptide sequence and is required for interaction with three
hydrophobic subunits located on the active site of ACE [114]. Main inhibitors of
the enzyme ACE show hydrophobic residues or proline, lysine, or arginine as
C-terminal amino acids [115–117].
Two novel ACE-inhibitory peptides MNVKHWPWMK and VTVNPYKWLP
corresponding to positions 825–834 and 125–135 of myosin heavy chain, respec-
tively, were identified from chicken leg meat hydrolysate digested with pepsin. With
IC50 values of 228 μM and 5.5 μM, respectively, the peptides were suggested to be a
good starting substance for designing food supplements for hypertensive patients
[118]. Gu et al. [119] studied the production of ACE-inhibitory peptides from
muscles of black-bone silky fowl (Gallus gallus domesticus Brisson) hydrolyzed
with Alcalase 2.4 L (2.4 AU/g) and papain (320,000 U/g). A total of 29 peptide
sequences were identified and based on the ACE-inhibitory properties of different
fractions and prior literature, 11 peptide sequences with probable ACE-inhibitory
properties were selected, synthesized, and further analyzed for ACE-inhibitory
properties. Finally, two novel peptides Leu-Glu-Arg (IC50 = 45.62 2.40 μM)
and Gly-Ala-Gly-Pro (IC50 = 253.07 6.66 μM) with strong ACE-inhibitory
properties were found.
Beef myofibrillar protein extract was used to produce ACE-inhibitory peptides by
using five commercially available food-grade microbial protease preparations viz. HT
proteolytic (HT; 2 mg/mL) protease, acidic fungal protease (AFP; 20 mg/mL), fungal
protease 31,000 (F31 K; 10 mg/mL), fungal protease 60,000 (F60 K 4 mg/mL), and
fungal protease II (FPII; 4 mg/mL) [32]. The small peptide pool obtained from
hydrolysate by using gel permeation chromatography (nominally <15 residue pep-
tides) was reported to exhibit 89.0 2.9% ACE-inhibitory activity. Castellano et al.
[79] studied the production of ACE-inhibitory peptides from porcine myofibrillar
proteins hydrolyzed by Lactobacillus sakei CRL1862 and Lactobacillus curvatus
CRL705 isolated from traditional Argentinean sausages at 30 C for 36 h. Meat-
borne Lactobacilli were able to produce peptides with ACE-inhibitory properties and a
total of 50 and 18 peptides were characterized from L. curvatus CRL705 and L. sakei
CRL1862 protein hydrolysates, respectively.
7.1.2 Regulatory and Cytoskeletal Proteins
ACE-inhibitory peptides have been reported not only from muscle contractile pro-
teins but also from regulatory and cytoskeletal proteins like troponin, tropomyosin,
nebulin, and titin. An ACE-inhibitory peptide with sequence RMLGQTPTK was
isolated from porcine troponin-C hydrolyzed with pepsin and showed high resis-
tance to digestive enzymes, suggesting its possible in vivo ACE-inhibitory activity
[120]. Two novel ACE-inhibitory peptides with sequence Lys-Arg-Gln-Lys-Tyr-
Asp-Ile (KRQKYDI) and Glu-Lys-Glu-Arg-Glu-Arg-Gln (EKERERQ) were iso-
lated from porcine skeletal muscle troponin treated with pepsin with proven in
vivo antihypertensive properties in spontaneously hypertensive rats [103]. The
IC50 value of KRQKYDI and EKERERQ was reported to be 26.2 μM and
552.5 μM, respectively, and the peptide KRQKYDI showed the strongest ACE-
inhibitory activity among all previously reported troponin peptides.
Nearly 22 peptides, including the novel peptides MYPGIA and VIPEL, were
reported from porcine Longissimus dorsi muscle hydrolyzed by sequential action of
pepsin and pancreatin [121]. Among these, two pentapeptides with sequence
KAPVA (IC50 46.56 μM) and PTPVP (IC50 256.41 μM) corresponding to position
4784–4788 and 4216–4220 on titin, respectively, and tripeptide RPR (IC50 382 μM)
corresponding to position 1263–1265 on nebulin were also reported. Strong ACE-
inhibitory activity of these peptides (KAPVA, PTPVP, and RPR) was demonstrated
in vivo in spontaneously hypertensive rats after oral administration [122]. Although,
RPR showed far less ACE-inhibitory activity than KAPVA in vitro, it exhibited
comparable ACE-inhibitory activity to that of KAPVA in vivo. Higher in vivo
activity of peptides which show weaker in vitro activity might be explained by their
higher affinity to the target tissues or the possibility of involvement of a mechanism
other than inhibition of ACE [102].
7.2 Sarcoplasmic Proteins
An ACE-inhibitory hexapeptide (VLAQYK) with an IC50 value of 32.06 μM was

identified from enzymatic hydrolysates of sarcoplasmic protein extracts from beef
Biceps femoris [123]. The peptide exhibited strong ACE-inhibitory properties in
vivo in spontaneously hypertensive rats and also lowered blood total- and LDL-
cholesterol concentrations [124]. Based on its strong ACE-inhibitory properties in
vitro as well as in vivo, the authors proposed the use of this hexapeptide in clinical
applications and for development of functional foods. Four ACE-inhibitory peptides
identified as FHG, GFHI, DFHING, and GLSDGEWQ were produced from beef
sarcoplasmic proteins hydrolyzed by incubating with commercial enzymes thermo-
lysin þ proteinase A (pH 7.5/37 C), proteinase K (pH 7.5/37 C), trypsin (pH 7.6/
25 C), tyrosinase (pH 6.5/25 C), papain (pH 6.2/25 C), pepsin (pH 3.0/37 C),
and protease (pH 7.5/37 C) for 8 h [100]. The measured IC50 values of FHG, GFHI,
DFHING, and GLSDGEWQ against ACE were 52.9, 117, 64.3, and 50.5 μg/ml,
respectively.
Bernardini et al. [125] studied the possibility of production of ACE-inhibitory
peptides from sarcoplasmic proteins hydrolyzed from beef low-value brisket
cuts (Pectoralis profundus) with papain for 24 h. The 3 kDa peptidic fractions
showed higher ACE-inhibitory activity than 10 kDa fractions which was in agree-
ment with previous studies suggesting that peptide sequences with ACE-inhibitory
properties are generally composed of small number of residues and are less than
3 kDa [50]. The authors suggested the possible use of these ACE-inhibitory brisket
hydrolysates for the development of meat products, like patties, with antihyperten-
sive properties. Hydrolysates from different sources like milk and soy have already
been incorporated into the meat products like beef and pork patties [126–128]. A
unique heptapeptide with sequence FISNHAY was generated from porcine sarco-
plasmic proteins by L. sakei CRL1862 in fraction 27 which showed highest ACE-
inhibitory activity. This peptide had, rather than a proline residue, aromatic (tyro-
sine) and aliphatic (alanine) residues at the ultimate and penultimate C-terminal
positions, respectively [79].
7.3 Connective Tissue and Other Proteins
In comparison to other meat proteins, bovine elastin and collagen possess highest
frequency of peptide sequences with different bioactivities [129]. Peptides derived
from hydrolysis of bovine connective tissues have been reported to have strong
ACE-inhibitory properties [130–132]. Peptides with strong ACE and renin-inhibi-
tory activities were identified from collagen extracted from nuchal ligament of
bovine carcasses (GPRGF) and from cooked semitendinosus muscle (SPLPPPE,

EGPQGPPGPVG, and PGLIGARGPPGP) [28, 132]. Two ACE-inhibitory peptides
were produced and identified from collagen obtained from bovine Achilles tendon
hydrolyzed with bacterial collagenase. Both the peptides sequenced as
AKGANGAPGIAGAPGFPGARGPSGPQGPSGPP and PAGNPGADGQP-
GAKGANGAP retained 80% of the ACE-inhibitory properties after treatment
with common GIT enzymes [133]. Ryder et al. [32] studied the production of
ACE-inhibitory peptides from beef connective tissue protein extract by using five
microbial protease preparations. The small peptide fraction obtained from the hydro-
lysates showed 90.9 1.0% ACE-inhibitory activity.
O’Keeffe et al. [134] studied the ACE-inhibitory activity of porcine skin gelatin
hydrolyzed by Aspergillus niger prolyl endoproteinase for 4 h. Peptides were identified
by using UPLC-ESI-MS/MS and 166 sequences were identified, Met-Gly-Pro being
the most potent ACE-inhibitory peptide. Some of the peptides identified in the
hydrolysate like Gln-Phe, Tyr-Pro, Leu-Ala, Val-Pro, Ile-Pro, Ile-Ala, and Gly-Pro
were previously identified as strong ACE-inhibitors. Among these peptides Val-Pro,
Ile-Pro, Ile-Ala, and Gly-Pro were suggested to be resistant to GIT enzymes following
an in silico GI digest using PeptideCutter. The hydrolysate showed strong blood
pressure lowering properties in spontaneously hypertensive rats which was attributed
to the abundance of peptides with C-terminal Pro residue. The in vivo antihypertensive
properties of hydrolysates obtained from gelatin from different sources have been
reported by several workers [135–141]. Short peptides, such as Pro-Hyp, released from
gelatin have been reported to cross the intestinal barrier and enter the blood vascular
system after oral ingestion of hydrolysates by mice [142] and humans [143].
Inoue et al. [144] produced hydrolysates with ACE-inhibitory properties from
porcine liver with in vivo blood pressure lowering properties in spontaneously
hypertensive rats. Lafargaa et al. [72] studied the release of ACE-inhibitory peptides
from porcine and bovine meat proteins including collagen, hemoglobin, and serum
albumin, commonly found in meat by-products such as blood, bone, and low-value
meat cuts, using enzymes papain, pepsin, bromelain, ficain, and thermolysin.
PeptideCutter and BIOPEP were used for in silico cleavage of the proteins to predict
the release of over 30,000 peptides. Four novel ACE-inhibitory peptides viz. Asp-
Phe-Tyr, Ala-Pro-Pro-His, Ile-Ile-Tyr, and Ile-Phe-Tyr which were not present in
existing databases were identified. Sequence Glu-Tyr, an ACE-inhibitory dipeptide
corresponding to position 23–24 of bovine hemoglobin subunit alpha, was identified
which was previously reported from shark meat [145] and several natural products
including milk and fish [145, 146]. Fu et al. [131] identified two collagen peptides
with sequence GPRGF and VGPV from bovine connective tissue and were demon-
strated to be transported across Caco-2 cells, a monolayer of human intestinal cell
lines, suggesting the possible bioavailability of the peptides and likelihood to exhibit
the ACE-inhibition activity in vivo. Two peptides identified from the porcine
hemoglobin with sequence GFPTTKTYFPHF, corresponding to 34–46 fragment
of the α-chain, and VVYPWT, corresponding to 34–39 fragment of the β-chain, were
reported to have ACE-inhibitory properties, showing IC50 values of 4.92 μM and
6.02 μM, respectively [147].
Saiga et al. [141] studied the ACE-inhibitory properties of hydrolysates obtained

from collagen extracted from chicken legs. The hydrolysate produced with an Asper-
gillus species-derived enzyme showed the highest activity (IC50 = 260 μg/mL) with a
strong antihypertensive effect observed in spontaneously hypertensive rats adminis-
tered with low fraction. Peptides with a sequence Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro
exhibited highest ACE-inhibitory activity (IC50 = 29 μM). Two ACE-inhibitory
peptides with sequence GPL and GPV and IC50 values of 2.55 μM and 4.67 μM,
respectively, were purified from bovine skin gelatin hydrolysate [148].
7.4 Effects of Aging and Cooking of Meat
Encrypted in the meat proteins are several ACE-I inhibitory sequences which are
released during natural processes, like aging, dry curing, and fermentation, which
naturally induce proteolysis of both sarcoplasmic and myofibrillar proteins in the
muscles [149]. Fu et al. [28] studied the endogenous generation of peptides in beef
Longissimus thoracis (LT) and semitendinosus (ST) muscles during 20 days post-
mortem aging and reported the presence of a number of peptides with ACE-inhib-
itory properties. The activity was highest on day 10 in cooked ST (63 C) which
decreased at day 20, suggesting the presence of potent peptides on day 10 which
became accessible by cooking. Highest ACE-inhibitory activity was exhibited by
collagen-derived peptide sequences SPLPPPE, EGPQGPPGPVG, and
PGLIGARGPPGP. After 20 days postmortem, more peptides were identified due
to extensive degradation of proteins. Degradation of potent peptides by the endog-
enous enzymes may be the reason for the decrease in the ACE-inhibitory activity at
day 20 [150]. The authors also reported for the first time the release of low molecular
weight peptides (< 3 kDa) from aged beef with renin-inhibitory activity. The highest
concentration of low molecular weight peptides was observed in cooked beef aged
for 20 days and also exhibited highest renin-inhibitory properties. These results
suggest that generation of peptides and their related bioactivities depends on aging
time and cooking [151, 152] and may be ascribed to the release of certain active
peptides during different phases of aged or cooked beef [28]. Although, calpains,
which are mainly responsible for proteolysis, get inactivated at 55 C [151], other
endogenous proteases like cathepsins, metalloproteinase, and collagenase may still
cause proteolysis in beef during cooking, increasing the concentration of small
peptides [28, 153].
Liu et al. [154] studied the endogenous release of ACE-inhibitory peptides in
duck meat during postmortem aging and reported that the small peptides (<5 kDa)
produced due to the degradation of muscle proteins exhibited no ACE-inhibitory
activity. Endogenous enzymes, unlike exogenous enzymes, work in uncontrollable
manner and might only release a limited number of peptides [72] which might be
produced in too little quantities to induce ACE-inhibitory activity [154].
Sangsawad et al. [155] studied the effect of cooking methods on the production of
ACE-inhibitory peptides derived from simulated in vitro gastrointestinal digestion of
Korat crossbred and commercial broiler chicken breast. The highest ACE-inhibitory
activity was exhibited by Korat crossbred chicken digestas heated at 70 C for 0.5 h
while the lowest inhibitory activity was shown by the digestas of Korat crossbred
breast meat heated at 121 C for 1 h. In general, ACE-inhibitory activity of the
digestas also varied with the protein digestibility. The highest digestibility and ACE-
inhibitory activity was reported in samples cooked by mild thermal treatment. The
digestas heated at 70 C for 0.5 h had highest proportion of small peptides (<1 kDa)
whereas the digestas heated at 121 C for 1 h had highest proportion of large peptides
(>5 kDa). Peptide size has been reported to play an important role in ACE-inhibitory
activity with most of the peptides possessing 2–20 amino acids [30, 156]. Larger
peptides have difficulty in binding to the active site of ACE, resulting in decreased
inhibitory activity [157]. This may partly explain the lower ACE-inhibitory activity
of the digestas treated by high thermal treatment. High thermal treatment (>100 C)
has also been reported to induce several changes in proteins, like aggregation, cross-
linking, and oxidative modifications [158, 159] which reduce their susceptibility to
proteases, resulting in larger size peptides. Novel peptide sequences KPLLCS,
ELFTT, and KPLL with potent ACE-inhibitory properties were identified with
sequences homologous to that of myosin. The IC50 values of KPLLCS, ELFTT,
and KPLL were 0.37, 6.35, and 11.98 μM, respectively. Peptides ELFTT and KPLL
were reported to be resistant to in vitro plasmin hydrolysis for up to 60 min while
KPLLCS was hydrolyzed by plasmin after 15 min, suggesting the resistance of these
peptides to the blood proteases. Peptides need to resist the blood protease digestion
after absorption to exert the ACE-inhibitory activity in vivo (Table 3).
7.5 Meat Products
Meat is considered as a good source of peptides with different bioactivities [120,

122, 162]; however, dry-cured ham was the only product among the processed meat
products that was reported to be a natural source of peptides with antihypertensive
properties [163, 164]. Table 4 presents the identified peptide sequences with antihy-
pertensive properties from different meat products. Seven dipeptides sequenced as
RP, KA, AA, GP, AR, GR, and RR with ACE-inhibitory properties were produced
by the action of dipeptidyl peptidases purified from porcine skeletal muscle [169].
Since dipeptidyl peptidases remain active during the whole processing period of dry-
cured meat products, or at least a great part of it, the authors hypothesized that their
proteolytic action could contribute to the generation of peptides with ACE-inhibitory
activity in cured meat products. Vaštag et al. [170] also suggested the possibility of
production of meat protein hydrolysates with ACE-inhibitory properties during
ripening process of Petrovská Kolbása, a traditional dry fermented sausage. Later
three novel peptides possessing in vivo ACE-inhibitory properties were identified
from Spanish dry-cured ham [153, 163] and were reported to be highly stable during
in vitro gastrointestinal digestion and heat treatment [165]. AAATP was the identi-
fied peptide sequence with most potent ACE-inhibitory activity and effectively
decreased systolic blood pressure in spontaneously hypertensive rats [153].
ASGPINFT and DVITGA were other sequences that yielded a moderate ACE
Table 3 Peptide sequences with antihypertensive properties from different meat proteins
Peptide sequence Proteins Authors
LKP, LKA, LAP, IKW, FKGRYYP, FQKPKR, Chicken muscle Fujita et al. [98]
IVGRPRHQG proteins
GPL, GPV Bovine skin Kim et al. [148]
proteins
MNPPK, ITTNP, MNP, NPP, PPK, ITT, TTN, TNP Porcine skeletal Arihara et al. [108]
muscle proteins
VKKVLGNP, TNP, NPP, MNP, ITTNP, MNPPK, Porcine skeletal Nakashima et al.
PPK, TTN, KRQKYDI muscle proteins [110]
RMLGQTPTK, RMLGQTP Porcine troponin C Katayama et al.
[120]
GFXGTXGLXGF Chicken breast Saiga et al. [104]
muscle proteins
RMLGQTPTK, RMLGQTP, RMLGQ, RML, GQ, Porcine troponin Katayama et al.
TP, TK [160]
VLAQYK Beef muscle Jang and Lee
proteins [123]
GFXGTXGLXGF, GAXGLXGP Chicken collagen Saiga et al. [113]
Saiga et al. [141]
VKKVLGNP Porcine skeletal Katayama et al.
muscle myosin [99]
KRQKYDI, EKERERQ Porcine skeletal Katayama et al.
muscle troponin [103]
FHG, GFHI, DFHING, GLSDGEWQ Beef sarcoplasmic Jang et al. [100]
proteins
KRVIQY, VKAGF Porcine skeletal Muguruma et al.
myosin and actin [111]
KAPVA, PTPVP, ER, KLP, RPR, MYPGIA, Porcine muscle Escudero et al.
VIPEL, VLPE RVAPEEHP, VAPEEHPT, proteins [121]
LFDKPVSP, FDKPVSPL, ITTNPY, MLGQTP,
DQVFPMNPPK, MMVPI, NIIPA
VTVNPYKWLP, MNVKHWPWMK Chicken muscle Terashima et al.
proteins [118]
FISNHAY, AMQKIF, LKTAIQAAGYPDKV, Porcine Castellanoa et al.
SDHHIYL, IKNYPVVSIED, sarcoplasmic [79]
AAVYKALSDHHIY, MPQQIGVP, proteins
LSGGQSEEEASINL, TFSYGRALQA,
VGGASLKPEF, SPLPVIPH, EVGGEALGRL,
IKWGDAGATY, LEGKVLPGVDALS,
LVGGASLKPEF
LPP, APPH, NFY, IFY, IIY, LPP, IPP, IF, FY Bovine and porcine Lafargaa et al. [72]
muscle proteins
FQPS Kacang goat Mirdhayati et al.
muscle proteins [161]
KPLLCS, ELFTT, KPLL Chicken breast Sangsawad et al.
proteins [155]
SPLPPPE, EGPQGPPGPVG, PGLIGARGPPGP Beef collagen Fu et al. [28]
MGP, QF, YP, LA,VP, IP, IA, GP Porcine skin O’Keeffe et al.
proteins [134]
Table 4 Peptide sequences with antihypertensive properties from different meat products
Peptide sequence Product Authors
GNGGA, DVITGA, KDQGSYEDF, GVDNPGHPF, LNSLT, Spanish dry- Escudero
KAEEEYPDL, EEYPDL, ASGPINFT, AAATP cured ham et al. [153]
AAPLAP, AAATP, KAAAAP, KPVAAP, IAGRP, KAAAATP, Spanish dry- Escudero
PTPVP, PAPPK, AMNPP, IKLPP, VPPAK, KPGRP, PSNPP, cured ham et al. [165]
EAPPK, PAAPPK, KVLPG, TGLKP
PPK, PAP, AAP Iberian dry- Mora et al.
cured ham [166]
LGL, ALM, SFVTT, GVVPL, NSIM Parma dry- Dellafiora
cured ham et al. [167]
RVAPEEHPT, VAPEEHPT, IGGSI, LFDKPVSPL, LAPST, Parma dry- Paolella et al.
PGIAD, IVAPG, PSIV, DLTDY, SYELPDGQ, IDDL, cured ham [168]
LKGADPEDVITGA, TVKDLQHRL
(R)AVFPSIVGRPR(H), (R)LDLAGRDLTDYLMK(I), (R) Bresaola Ferranti et al.
AVFPSIVGRPR(H), (F)PSIVGRPR(H), (V)FPSIVGRPR(H), [149]
(R)FGVTITVHPEPR(V), (R)MDVSITGEPRPV(A), (R)
AVFPSIVGRPR(H), (V)TVKEDQVFPMNPPKFDKIED(M),
(R)AVFPSIVGRPR(H), (F)PSIVGRPR(H),
(V)FPSIVGRPR(H), (K)AGDNIKVEIPVLGRPKPT(V),
(L)NVLGRPGPPVGPI(K),
(R)LNVLGRPGPPVGPI(K),
(R)MDVSITGEPRPV(A), (R)AVFPSIVGRPR(H),
(V)FPSIVGRPR(H), (W)AAFPPDVGGNVDYK(N),
(W)AAFPPDVGGNVDYK(N), (V)
TVKEDQVFPMNPPKFDKIED(M), etc.
inhibition. Escudero et al. [165] studied the stability of ACE-inhibitory peptides

from Spanish dry-cured ham and reported that almost the same activity was retained
after applying diverse heating (50–117 C), times of processing (3–60 min), and
simulated in vitro digestion with gastrointestinal proteases. Peptides AAPLAP,
KAAAAP, KPVAAP, KAAAATP, and IAGRP were reported to be the most potent
ACE-inhibitory peptides. Gallego et al. [171] studied the stability of three ACE-
inhibitory peptides (KPVAAP, AAATP, and AAPLAP) naturally generated in dry-
cured ham during transepithelial transport in a Caco-2 cell monolayer. The results
showed that the peptides degraded during the assay, although, some of the KPVAAP
peptides remained intact and was also absorbed through the intestinal barrier,
suggesting the possible absorption and bioavailability of dry-cured ham peptides
in the blood stream to exert an antihypertensive action.
Production of peptides in dry-cured meat products depends on certain factors
such as activity of muscle endogenous enzymes, which is very much influenced by
genetics and muscle-type, and processing conditions including added ingredients
and ripening time [162]. Paolella et al. [168] studied the effect of maturation time of
dry-cured Parma hams on the peptide profile obtained upon simulated gastrointes-
tinal digestion. Parma ham is a well-known dry-cured meat product obtained from
heavy pig thighs, mildly salted and aged for a long period. A total of 81 peptide
sequences were identified in digested samples and most of the sequences mainly
originated from myofibrillar proteins (myosin and actin) and sarcoplasmic proteins
(such as pyruvate kinase, creatine kinase, fructose bisphosphate aldolase, etc.). A
higher amount of short sequences were found in the digesta with a total of 21
dipeptides and 11 tripeptides among the identified sequences. Several of the identi-
fied sequences were already known in the literature with demonstrated ACE-inhib-
itory properties. Dellafiora et al. [167] used hybrid in silico/in vitro approach for the
identification of ACE-inhibitory peptides from dry-cured Parma hams. Strong ACE-
inhibitory sequences, like LGL and SFVTT, were identified for the first time in
Parma dry-cured ham. On the basis of in silico results, a total of 25 peptides were
predicted as active, and five ACE-inhibitory peptides with sequence LGL, SFVTT,
GVVPL, ALM, and NSIM with IC50 values of 145, 395, 956, >1100, and
>1100 μM, respectively, were identified.
Mora et al. [166] evaluated the antihypertensive peptides generated naturally in
Iberian dry-cured ham and also studied the differences in the generated peptides
between traditional Spanish dry-cured (14 months of ripening) and Iberian dry-
cured ham (24 months of ripening). The extract obtained from Iberian dry-cured
ham was analyzed in vitro showing up to 97.7% of ACE-inhibition in some of the
fractions. A significant decrease of 12 mm Hg in systolic blood pressure of sponta-
neously hypertensive rats was also observed after 8 h of ingestion. Pro-Pro-Lys, Pro-
Ala-Pro, and Ala-Ala-Pro with strong ACE-inhibitory activities were the most
repeated sequences among 2632 peptides identified in Iberian dry-cured ham.
These sequences appeared a total of 322, 302, and 119 times, respectively, and
were mainly derived from the proteins troponin-T, myosin-heavy and myosin-light
chains. Mora et al. [172] characterized the peptide profile of three European hams
viz. Spanish Teruel, Italian Parma, and Belgian dry-cured hams. The peptides
generated in these types of hams were identified and quantified using a label-free
methodology to assess main differences in proteolysis between them. Peptide frac-
tions from Spanish Teruel exhibited highest ACE-inhibitory activity of 93% while
those from Belgian and Parma hams had ACE-inhibitory activity of 76% and 70%,
respectively.
The Bresaola della Valtellina, produced by salting and naturally curing specific
cuts of lean bovine hind quarters, is a Protected Geographical Indication from
Valtellina, Italy. There are other similar products which are traditionally produced
in other places such as Viande des Grison or Bunder fleish in Switzerland and Cecina
in Spain. Both myofibrillar and sarcoplasmic protein fractions of Bresaola get
extensively hydrolyzed by endogenous proteases during ripening, releasing a large
variety of peptides which could exert several biological activities if they survive the
digestive process. With this aim, Ferranti et al. [149] attempted to identify the bovine
muscle-derived proteins and peptides from two different Bresaola samples (with and
without the Protected Geographical Indication label) which could survive a static in
vitro model of digestion that included the sequential oral, gastric, and duodenal
phases. More than 170 peptides were identified which were released during in vitro
digestion from the major structural and sarcoplasmic muscle proteins. Most of the
identified peptides had reported antihypertensive properties or were precursors of
potentially antihypertensive sequences.
Pastırma, a Turkish dry-cured meat product produced from whole beef mus-
cles, was analyzed for antihypertensive activity and was reported to possess strong
ACE-inhibitory properties [71]. ACE-inhibitory activity of more than 86% was
shown from fractions corresponding to molecular masses between 900 Da and
1500 Da. ACE-inhibitory peptides were suggested to be generated due to the
proteolysis caused by endogenous enzymes and the çemen paste used in
production.
Recently, Fernández et al. [116] studied the possibility of generation of ACE-I
inhibitory properties in the dry-fermented sausage salchichón by using purified
proteolytic enzyme EPg222 and starter culture (P200S34) composed of
Pediococcus acidilactici (MS200) and Staphylococcus vitulus (RS34), separately
and together, followed by ripening for 90 days. EPg222 protease purified from
Penicillium chrysogenum Pg222, isolated from dry-cured meat products, has
been reported to have high proteolytic activity against myofibrillar proteins
under the conditions normally present in dry-cured meat products [173].
Batches inoculated with EPg222 were reported to have highest ACE-inhibitory
activities at 63 days of ripening. The ACE-inhibitory activities remained stable in
most cases even after in vitro simulated gastrointestinal digestion, suggesting the
significance of use of the enzyme EPg222 in association with the starter culture
P200S34 for the production of functional meat products with antihypertensive
properties. Mejri et al. [174] studied the effect of different starter cultures and
ripening times on the size, concentration, and antihypertensive capacities of
peptides in dry-fermented camel sausages. Sausages were inoculated with isolated
strains of S. xylosus and L. plantarum (batch A), S. xylosus and L. pentosus (batch
B), and S. carnosus and L. sakei (batch C) and were ripened up to 28 days. Both
peptide size and concentration were reported to be affected by the ripening time
and the inoculated bacteria. The ripening process resulted in an increased antihy-
pertensive capacity with highest bioactivities in the fractions with <3 kDa pep-
tides. Low molecular weight peptides (< 3 kDa) have been reported to contribute
to the development of flavor as well as antihypertensive activities in fermented
meat products [166].
8 Conclusions
A large number of peptides with demonstrated in vivo and in vitro antihypertensive

activities have been reported from different meat proteins. Endogenous generation of
peptides with antihypertensive properties has also been reported during natural aging
process increasing the value of meat as a functional food. Cured meat products like
dry-cured ham and pastırma might be considered as natural sources of peptides with
strong antihypertensive properties. Several meat-derived antihypertensive peptides
have been shown to retain their activity and bioavailability even after digestion and
cooking. Thus peptides derived from meat proteins provide an exciting option for the
development of functional foods with antihypertensive properties for preventive and

curative management of hypertension.
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Bioactive Peptides from Fish Protein
By-Products 14
Aurélien V. Le Gouic, Pádraigín A. Harnedy, and
Richard J. FitzGerald
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
2 Fish Protein Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
3 Processing of Fish Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
3.1 Protein Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
3.2 Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
4 Characterization of Fish Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
5 Bioactive Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
5.1 Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
5.2 Allergenicity and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
5.3 Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
5.4 Type 2 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
5.5 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
5.6 Other Bioactivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
6 Bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Abstract
The interest in fish processing by-products and underutilized catch for the
production of biofunctional food ingredients has increased in the last number of
decades. These marine-derived components contain a significant quantity of
protein, which is normally processed into low-value products such as animal
feed, fishmeal, and fertilizer. However, due to the global demand for high-quality
protein and the need for sustainable production and processing of landed material,
the valorization of proteins and other nutrients from fish processing by-products
has significantly increased. Fish processing by-products contain significant
A. V. Le Gouic · P. A. Harnedy · R. J. FitzGerald (*)

Department of Biological Sciences, University of Limerick, Limerick, Ireland
e-mail: [email protected]; [email protected]; dick.fi[email protected]

356 A. V. Le Gouic et al.
quantities of high-quality protein, which can be exploited as sources of essential

nitrogenous nutrients and biologically active peptides. Bioactive peptides, includ-
ing those from fish processing by-products, have been reported to possess the
ability to beneficially modulate physiological processes associated with non-
communicable diseases. These short peptides, which are encrypted within the
primary sequence of the parent protein and are released during food processing or
gastrointestinal digestion, could have a role in the prevention and management of
these diseases. This chapter reviews the recent literature on the processing and
utilization of proteins and protein hydrolysates from fish processing by-products
and underutilized fish species with a particular focus on their bioactive properties
and peptide sequences.
Keywords
By-products · Proteins · Hydrolysates · Biofunctional properties · Fish ·
Peptides · Noncommunicable diseases
1 Introduction
The ocean covers about 70% of the total earth’s surface and contains approximately
60% of all fish species. The other 40% are found in fresh water which comprises 1%
of the earth’s surface. The fishing industry represents an important economic activity
for many countries around the world. In 2003, marine fisheries supported 260
million jobs directly and indirectly [1]. The demand for fish and shellfish has
increased throughout the world. The amount of farmed fish doubled in the last
decade while that of captured fish has tended to stabilize. According to the Food
and Agriculture Organization (FAO), global fish production in 2016 was estimated
to be 174.1 million tonnes (mt) by both capture and aquaculture with 152.8 mt being
used for human food consumption and 21.3 mt for nonfood uses [2]. It has been
estimated that greater than 60% (by weight) of the fish which are processed are
represented by by-products, i.e., head, skin, bones, fins, trimmings, viscera, blood,
and roe [3]. The large quantity of fish by-products generated represents a potentially
significant source of pollution in developing and developed countries. Since 2014,
the European Commission, under Directive No 1392/2014 [4], regulates the discard
of all fish caught regardless of whether they are regulated by quota, smaller than the
minimum size and either dead or alive, and now imposes an obligation to land all
catch.
Fish processing by-products and underutilized catch contain a significant quantity
of protein which is normally processed into low-value products such as animal feed,
fish meal, and fertilizer [2]. However, given the increased global demand for high-
quality protein and the requirement for sustainable production and processing, there
is an increasing interest in the extraction and valorization of proteins and other
nutrients from fish processing by-products. The proteins within fish processing by-
products represent a source of high-quality protein which can be exploited for the
provision of essential nitrogenous nutrients. Therefore, various biotechnological
14 Bioactive Peptides from Fish Protein By-Products 357
approaches have, and are, being employed to extract valuable nutrients and bioactive
compounds targeted at enhancing human health. Bioactive compounds have a role in
the management and in the protection against a range of chronic noncommunicable
diseases (NCDs). Furthermore, by-product proteins may be used to generate biolog-
ically active peptides (BAPs). BAPs have the ability to modulate physiological
processes and thereby have a role in the prevention and management of disease.
Protein hydrolysates are obtained following the enzymatic conversion of
intact proteins into peptides. These protein fragments usually contain no more
than 20 amino acids. A large range of fish protein hydrolysates, generated using
food-grade proteolytic preparations, such as trypsin, Alcalase® 2.4L,
Flavourzyme ® 500L/1000L, Corolase ® PP, and Promod ® 144MG, have been
reported in the literature [5–7]. These hydrolysates, due to their physicochemical
properties, are a source of amino acids [8] which have applications in human and
animal nutrition, as well as in the pharmaceutical and cosmetic industries. Due to
their good nutritional composition, amino acid profile, and bioactive properties, fish
protein hydrolysates have many commercial applications [9].
This chapter reviews the recent literature on the processing and utilization of
proteins and protein hydrolysates from fish processing by-products and underutilized
fish species with a particular reference on their bioactive properties and peptide
sequences.
2 Fish Protein Composition
Fish consumption is linked with many health benefits due to their high protein levels
and also due to their content of unsaturated fatty acids, vitamins, and minerals [10].
Fish proteins are a particularly rich source of the essential amino acids valine and
lysine [11].
The chemical composition of foods has an important role in the supply of
essential nutrients for the maintenance of human health. The chemical composition
of fish by-products, i.e., levels of protein, ash, and lipid, differs significantly between
species.
Fish processing by-products are designated as parts of the fish which are
not generally used for human consumption, e.g., head, skin, and viscera. However,
the nutrients therein are recoverable and can be utilized after further processing.
These components may represent between 30% and 60% by weight of the starting
material. Consequently, these fish processing by-products represent a rich source of
biofunctional materials such as vitamins, minerals, polysaccharides, polyunsaturated
fatty acids (PUFA), enzymes, collagen, gelatin, and bioactive peptides with valuable
nutraceutical, pharmaceutical, and cosmeceutical applications [12].
Proteins are characterized by their amino acid sequence and secondary structure
and also by their tertiary structure, which may be highly ordered. Each type of
protein has a unique structure that determines its function in the organism in addition
to its technofunctional properties when utilized as a food source/ingredient [13]. The
nutritional value of food proteins is determined on the basis of their essential amino
Table 1 Essential amino acid content of reference food proteins, fish proteins/protein hydroly-
sates, and human daily requirements
Amino acid
Sodium Egg Tilapia requirements (mg/kg/
caseinatea whitea filletsb Tuna viscera d)a age category
Amino (mg/g (mg/g (mg/g hydrolysatesc (years)
acid protein) protein) protein) (mg/g protein) 1–2 3–18 >18
Thr 40.5 45.3 27.6 59.0 24 18–17 15
Met 32.0 68.4 42.0 – 22 17–16 15
Val 56.4 73.0 66.2 89.3 36 29–28 26
Ile 45.9 55.9 62.4 69.3 27 22–21 20
Leu 88.9 93.6 103.2 77.0 54 44–42 39
Phe 101.4 110.4 49.8 51.6 40 30–28 25
His 25.4 26.3 20.2 84.5 15 12–11 10
Lys 77.5 76.0 97.5 18.7 44 35–33 30
Trp 10.4 17.6 5.2 – 6 4.8–4.4 4.0
a
FAO/WHO/UNU (2007)
b
Vidotti et al. [14]
c
Villamil et al. [15]
acid content. Table 1 summarized the essential amino acid content of foodstuffs
compared to the daily requirement. In comparison to plant proteins, proteins derived
from animal sources (because of their high content of essential amino acids) are
considered nutritionally superior. Of these, egg white and milk proteins (casein) are
usually used as reference proteins for determination of protein quality. Proteins
derived from meat and poultry muscle are also considered as a source of high-quality
protein. It has been shown that the nutritional value of most fish proteins is equal to
or better than casein and the quality of fish proteins may exceed that of terrestrial
animal meat [16].
Myofibrillar proteins are structural proteins responsible for movement by their
capacity for contraction. They represent 65–75% of the total fish muscle, while
sarcoplasmic proteins (soluble proteins) represent 20–35% [17]. Myofibrillar pro-
teins are mainly composed of actin and myosin. The motility of fish is also stabilized
and regulated by other structural proteins including titin, nebulin, α-actinin, tropo-
myosin, and troponin (T, I, and C). The proportion and presence of these structural
proteins depend on the fish species. Myosin is the major protein in fish muscle
(40%); however, it is a protein easily destabilized when heated, especially the
myosin from cold-water fish species. It is a large protein, with a molecular mass of
470 kDa [18], and has an unusual structure as it has both fibrous and globular
properties whereas most food protein ingredients, such as proteins from egg, soy,
and milk, have globular structures and have a lower molecular mass. Myosin is
composed of two heavy chains of 220 kDa and two pairs of different light chains
(LCs), ranging from 17 to 22 kDa [19]. The myosin molecule is approximately
160 nm in length. The heavy chains interact via two domains: a globular domain
called “head” and a fibrous domain called “tail.” Actin accounts for 15–30% of the
myofibrillar proteins. The monomer form of actin is a globular protein (G-actin) of
Fig. 1 Diagrammatic representation of the actin-myosin complex. (Modified from Gordon

et al. [20])
43 kDa; however, globular actin monomers polymerize to form filaments of fibrous

actin (F-actin). Other small proteins associated with either actin or myosin include
titin, nebulin, tropomyosin, troponin, C-proteins, and M-proteins [18]. These pro-
teins play an important role in the stabilization of the muscle myofibril basic
structure termed the sarcomere (Fig. 1).
The denaturation of myofibrillar proteins by heat, chemical, and enzymatic
treatment leads to the generation of a gel [21]. The gelation properties of fish
myofibrillar proteins are exploited in the food industry to produce surimi or
surimi-like products. The connective tissue proteins, or stromal proteins, provide
the structural elements for connective tissue. The main protein in this group is
collagen which in general represents approximatively 3% of the total protein in
fish muscle and is present in the skin, bone, myocommata, and swim bladder [22].
However, some species, especially Chondrichthyes species, can contain a signifi-
cantly higher amount of connective tissue (10%) compared with a value of 17%
found in mammalian species [23].
3 Processing of Fish Proteins
3.1 Protein Extraction
To date, several methods have been used for the extraction of proteins from
various fish species. The specifics of these methods vary depending on the
parameters employed, e.g., pH, temperature, agitation time, homogenization,
weight-to-volume ratio, and the number of sequential extractions. The main

approach used to recover proteins from fish is the so-called pH shift process.
This technique employs homogenization of the fish sample in an acid (pH < 5.0)
or alkaline (pH > 9.0) solution. The protein extract is then separated from the
solid components by centrifugation where solids such as bones, scales, flesh, and
skin are removed. Depending on the sample, a mechanical disruption process may
also be employed prior to the homogenization step to disrupt the cells [24, 25].
The acid- or alkaline-solubilized protein is then precipitated by adjusting the pH
to its isoelectric point. The precipitate is separated by centrifugation and a protein
isolate is then obtained.
A second approach used for protein extraction is known as the surimi process.
The production of surimi consists of protein recovery from fish mince by a series of
sequential steps. Washing with cold water is essential to remove water-soluble,
sarcoplasmic proteins and impurities such as the skin, bones, scales, and connec-
tive tissue which can reduce the gelation ability and ultimately reduce product
quality. The number of successive washes and the volume of water required vary
between fish species, the freshness of the starting fish mince, and the quality of the
surimi required. It is common to use 0.1–0.3% (w/v) NaCl in the washing solution
to facilitate removal of water during the subsequent processing steps. Before the
dewatering step, the washed minced fish still contains a large quantity of impuri-
ties, which often consist of tissues from the skin and/or internal belly flap that need
to be removed. A purification step is required to obtain a product with good
organoleptic quality, appearance, and product safety. The water in the surimi
base obtained can be removed by a screw press, and the water content is reduced
from 91% to 80–84% (w/w) [26]. This method of extraction of proteins from fish is
less successful than the pH shift method as sarcoplasmic proteins are lost during
the process, leading to a reduced yield. A general flow diagram for these two
processes is provided in Fig. 2.
Enzyme-assisted extraction of food proteins using food-grade proteolytic
preparations relies on the intrinsic properties of enzymes, i.e., high specificity
and selectivity. The disadvantages of enzyme-assisted extraction are the high
cost which may be an issue in the case of production at commercial scale and the
inability of some enzymes to disrupt the cell membrane which may lead to low
extraction yield. A new approach to overcome these issues involves the use of
microwave irradiation during enzyme-assisted extraction. The procedure known as
microwave-assisted enzymatic extraction (MAEE) is recognized as having the
potential to increase the yield of protein extracted and, in some cases, the bioactive
properties of the resulting product. Nguyen et al. (2017) recently studied the
generation of fish protein hydrolysates by MAEE and the pH shift method from
rainbow trout (Oncorhynchus mykiss) using the proteolytic enzyme Alcalase
2.4L ® [27]. The MAEE approach was reported to improve the technofunctional
and bioactive properties of the hydrolysates when compared to those generated
using the pH shift method.
Fig. 2 Flow diagram representing the sequential steps involved in the pH shift (a) and surimi
process (b) for the extraction of proteins from fish. (Modified from Kristinsson et al. [24])
3.2 Enzymatic Hydrolysis
Enzymatic modification is a method which has been used to treat food proteins to
improve their technofunctional, physicochemical, bioactive, and organoleptic prop-
erties without alteration of their nutritive value [28]. As the degree of hydrolysis of
the protein affects the bioavailability and the bioactivity of the peptides generated,
the appropriate choice of the hydrolysis conditions (e.g., temperature, pH, enzyme
preparation, and enzyme to substrate ratio) is critical [29, 30]. Proteolytic enzymes
can be classified according to different characteristics, e.g., proteolytic enzymes can
be endo- or exo-acting proteinases/peptidases. For example, endoproteinases cleave
peptide bonds within the protein and release peptides or shorter fragments, while
exopeptidases remove single amino acid residues or small peptides from either the
C-terminus (carboxypeptidases) or N-terminus (aminopeptidases) by cleaving the
terminal peptide bonds [31].
It has been shown that enzymatically modified food proteins have improved
technofunctional as well as bioactive properties compared with intact proteins
[6, 32]. The generation of lower molecular mass peptides with reduced secondary
structures can improve the solubility, turbidity, gelling, emulsifying, and the foaming
properties as well as heat and pH stability of proteins [33]. The use of fish protein
hydrolysates for their technofunctional properties has been extensively reviewed
[34–36]. The modification observed in the technofunctional properties of food
proteins following hydrolysis is highly dependent on the hydrolysis conditions, such

as pH, incubation temperature, and duration of hydrolysis. Furthermore, the speci-
ficity of the enzyme used for the process and the operating parameters are respon-
sible for the extent of hydrolysis and the peptide profile obtained. Control of the
hydrolysis process is even more important when working with animal products since
certain animal parts may contain endogenous proteinases [37] or even enzyme
inhibitors [38]. These endogenous enzymes are mainly located in the gastrointestinal
tract and are associated with the fish viscera; however, some hydrolytic enzymes,
such as cathepsins, collagenases, alkaline proteases, and calcium-dependent pro-
teinases, are located in the muscle tissue itself [39]. This endogenous proteolytic
activity can increase the extent of hydrolysis independently from the exogenously
added proteolytic preparations. Fortunately, the endogenous proteolytic activity in
fish is generally not as high as that found in the muscle of terrestrial animals (bovine,
porcine, and caprine).
A number of food-grade proteolytic preparations exist on the market. These arise
from microbial, plant, and animal sources. Among the enzyme preparations avail-
able for the generation of protein hydrolysates from fish processing by-products, the
most commonly used are Alcalase (Bacillus licheniformis), Neutrase (Bacillus
amyloliquefaciens), Flavourzyme (Aspergillus oryzae), collagenase (Clostridium
histolyticum), Pronase E (Streptomyces griseus), papain (Carica papaya), and bro-
melain (Ananas comosus) and digestive enzymes from bovine and porcine gastro-
intestinal tracts (e.g., pepsin, trypsin, and chymotrypsin). Moreover, fermentation
and autolysis are processes which may be employed for the production of peptides
by the action of the proteolytic enzymes from the product itself or from the action of
the intrinsic microorganisms present. Furthermore, proteolytic enzymes have been
purified from different fish species, especially from viscera for use during fish
protein hydrolysate manufacture [15].
In some regions of the world, the endogenous enzymes from marine sources are
used to improve the shelf life as well as the bioactive and technofunctional properties
of proteins from fish and shellfish. Several examples of fermented fish, shellfish,
and seafood exist for application as flavoring agents and food supplements with
bioactive properties, such as Kapi, a fermented shrimp paste from Thailand, and
Bakasang, a fermented fish sauce from Indonesia [40–44]. However, the high salt
content and low pH and the presence of undesirable contaminants such as halophilic
microorganisms and biogenic amines (histamine) represent some important issues
for consumer safety linked to the consumption of these products. Therefore, the use
of enzymatic hydrolysis with exogenously added food-grade proteolytic prepara-
tions when carried out in a controlled environment with the use of thermal treatment
for enzyme inactivation and bacterial reduction represents a feasible approach for
the generation of bioactive peptides from fish by-products for human consumption.
Furthermore, it has been reported that the utilization of proteolytic enzymes releases
a higher quantity of bioactive peptides with lower molecular masses than when
fermentation processes are employed [45]. Additionally, enzymatic hydrolysis
requires significantly less time to reach the desired degree of hydrolysis than
fermentation processes. For example, the production of fish protein hydrolysates
takes between 4 and 6 h to reach the desired degree of hydrolysis [46], while it may
take weeks or months for the fermentation process to reach the equivalent extent of
hydrolysis [47].
4 Characterization of Fish Peptides
Fish proteins contain peptide sequences encrypted within their primary structure.
Some of these peptides have the potential to beneficially modulate some metabolic
pathways and consequently may play a role in disease prevention and health
enhancement. Bioactive peptides are released from proteins during normal gastro-
intestinal digestion which occurs in the digestive tract or during food processing with
the use of proteolytic enzymes (hydrolysis) or microorganisms (fermentation) [48].
The biological activity of food-derived peptides mainly depends on their structural
properties such as molecular mass and the physicochemical characteristics of the
amino acids within the sequence [49]. The biological activity of peptides present in
protein hydrolysates is highly dependent on the hydrolysis conditions (pH, time,
temperature), the enzymes used, and the enzyme-to-substrate ratio applied [50, 51].
Therefore, careful control of these conditions during the generation of bioactive
peptides is essential to optimize bioactive properties and, therefore, their ability to
enhance human health.
Type 2 diabetes mellitus (T2DM) and cardiovascular diseases are two of the main
NCDs responsible for more than 0.6 and 3.9 million deaths, respectively, in Europe
per annum [52]. Marine by-product-derived proteins represent a source of peptides
that may have the ability to modulate specific biomarkers associated with these
diseases, and therefore they have the potential for incorporation into functional
food or nutraceutical products for the prevention and management of these condi-
tions. Many studies have been conducted using several different food sources, such as
bovine and camel milk, cereals, insects, and marine sources, to generate bioactive
peptides with in vitro antioxidant, cardioprotective, antidiabetic, and appetite sup-
pressant properties [53–58]. These include peptides with the ability to modulate
specific pathways linked with blood pressure control and T2DM. This includes
modulation of the renin-angiotensin system (inhibition of renin and angiotensin-
converting enzyme (ACE)), stimulation of the incretin system (inhibition of
dipeptidyl peptidase-IV (DPP-IV), and stimulation of the secretion of glucagon-like
peptide (GLP-1)), as well as stimulation of the secretion of intestinal cholecystokinin,
which is linked to appetite suppression in vivo [59–62]. Food protein-derived pep-
tides have been shown to reduce oxidative stress associated with inflammation and
tissue damage in vivo, which are complications generally linked to cardiovascular
disease (ischemia), diabetes (diabetic food ulcer), and many other diseases such as
neurodegenerative diseases and cancer [63]. The main bioactivities currently inves-
tigated for peptides from fish by-products are based on the regulation of oxidative
stress and cardiovascular disease (Table 2). These include antioxidant, ACE inhibi-
tory, renin inhibitory, and anticoagulant activities. Several reports suggest that protein
hydrolysates generated from fish can modulate the immune response and inhibit
Table 2 Biological activities of protein hydrolysates and specific peptide sequences arising from fish processing by-products and underutilized fish species
364
Species Source Enzyme Biological activity Peptide(s) sequence Potency Ref

1
Atlantic salmon Trimmings Corolase PP ACE inhibitory GGPAGPAV1 ACE IC50 = 673.16 μM, [64]
(Salmo salar) DPP-IV inhibitory GPVA2 DPP-IV IC50 = 8139.11 μM,
Antioxidant PP3 ORAC = 5.47 μmol TE/μmol
GF4 peptide
2
ACE IC50 = 445.61 μM,
DPP-IV IC50 = 264.74 μM,
ORAC = 9.48 μmol TE/μmol
peptide
3
ACE IC50 = 1912.46 μM,
DPP-IV IC50 = 4343.48 μM,
peptide
4
ACE IC50 = 178.14 μM,
DPP-IV IC50 = 1547.15 μM,
peptide
1
Skin Flavourzyme DPP-IV inhibitory GPAE1 IC50 = 49.6 μM [65]
2
GPGA2 IC50 = 41.9 μM
1
Skin Alcalase 2.4L, papain ACE inhibitory AP1 IC50 = 0.005 mg/mL [66]
2
collagen VR2 IC50 = 0.014 mg/mL
Pectoral Pepsin Anti-inflammatory PAY NO inhibition [67]
fin (0.75 mM) = 63.80%
iNOS Inhibition
(0.75 mM) = 75.00%
PGE2 inhibition
(0.75 mM) = 45.33%
COX-2 inhibition
(0.75 mM) = 48.00%
TNF-α inhibition
(0.75 mM) = 58.28%
A. V. Le Gouic et al.
14
IL-6 inhibition
(0.75 mM) = 43.48%
IL-1β inhibition
(0.75 mM) = 64.89%
Alaska Pollack Backbone Pepsin Calcium binding VLSGGTTMAMYTLV [68]
(Theragra
chalcogramma)
1
Frame Trypsin Immunomodulatory WT1 Lymphocyte proliferation [69]
NGLAP2 (20 μg/mL) = 31.35%
2
NGMTY3 Lymphocyte proliferation
(20 μg/mL) = 32.96%
3
Lymphocyte proliferation
(20 μg/mL) = 35.92%
Skin Trypsin Metal chelating GPAGPHGPPG 11.52 nmol Ca/μmol peptide [70]
collagen (Ca, Fe, Cu) 1.71 nmol Fe/μmol peptide
0.43 nmol Cu/μmol peptide
Amur sturgeon Skin Alcalase 2.4L Antioxidative PAGT EC50 DPPH [71]
(Acipenser gelatin Cryoprotective
Bioactive Peptides from Fish Protein By-Products
radical = 5.38 mg/mL

schrenckii) EC50 ABTS
EC50 hydroxyl
Bluefin tuna Frame Pepsin ACE inhibitory GDLGKTTTSNWSPP ACE IC50 = 11.28 μM [72]
(Thunnus
thynnus)
Backbone Pepsin Antioxidant APTBP EC50 DPPH radical = 0.72 mg/ [73]
mL
EC50 hydroxyl radical = 0.21 mg/
mL
EC50 superoxide
365
(continued)
366
Table 2 (continued)
Bluefin Skin Papain Antioxidant FIGP EC50 DPPH radical = 0.118 mg/ [74]
leatherjacket mL
(Navodon EC50 hydroxyl
septentrionalis) radical = 0.073 mg/mL
EC50 oxygen radical = 0.311 mg/
mL
Blue whiting Whole fish Protamex, Antioxidant – [75]
(Micromesistius Flavourzyme 500L
poutassou)
Mince Endopeptidase from DPP-IV inhibitory – CKK release (1.0% [76]
Bacillus CKK stimulation hydrolysate) = 122.03 pM
amyloliquefaciens and
B. licheniformis
Mince Alcalase 2.4L/ DPP-IV inhibitory – DPP-IV IC50 = 1.49 mg/mL [77]
Flavourzyme 500L Insulin secretion
GLP-1 secretion
Boarfish (Capros Mince Protease AP ACE inhibitory – ACE inhibition [25]
aper) (1 mg/mL) = 85.8%
Half-fin anchovy Whole fish Pepsin Antiproliferative – IC50 DU-145 cell = 41.67 mg/mL [78]
(Setipinna taty) Antioxidant EC50 DPPH radical = 4.46 μg/mL
1
Leatherjacket Mince Papain ACE inhibitory EPLYV1 IC50 = 118 μM [29]
2
(Meuschenia sp.) Bromelain DPHI2 IC50 = 48.7 μM
3
Flavourzyme 500L AER3 IC50 = 420 μM
4
EQIDNLQ4 IC50 = 270 μM
5
WDDME5 IC50 = 31.6 μM
Loach Whole fish Papain Antioxidant – IC50 MCF-7 = 16 mg/mL [79]
(Misgurnus Antiproliferative IC50 Caco-2 = 10 mg/mL
anguillicaudatus) IC50 Hep-G2 = 13 mg/mL
1
14
Longtail tuna Dark Papain Antiproliferative LPHVLTPEAGAT1 IC50 MCF-7 = 8.1 μM [80]
2
(Thunnus muscle by- Protease XXIII PTAEGGVYMVT2 IC50 MCF-7 = 8.8 μM
tonggol) product
1
Whole fish Protease XXIII DPP-IV inhibitory PGVGGPLGPIGPCYE1 IC50 = 116.1 μM [81]
2
CAYQWQRPVDRIR2 IC50 = 78.0 μM
3
PACGGFWISGRPG3 IC50 = 96.4 μM
1
Olive flounder Mince Pepsin ACE inhibitory MEVFVP1 IC50 = 79 μM [82]
(Paralichthys VSQLTR2 SBP6h (40 mg/kg) = 44.25 mmHg
2
olivaceus) IC50 = 105 μM
SBP6h (40 mg/kg) = 34.25 mmHg
1
Pangasius catfish Skin1 and Alcalase 2.4L ACE inhibitory – IC50 = 3.2 μg/mL [83]
2
(Pangasius bone2 IC50 = 1.3 μg/mL
sutchi) gelatin
Rockfish Skin Flavourzyme Antioxidant – ACE IC50 = 0.82 mg/mL [84]
(Sebastes hubbsi) gelatin ACE inhibitory DPPH scavenging
(4 mg/mL) = 45.8%
Superoxide scavenging
(4 mg/mL) = 67.8%
Hydroxyl scavenging
(4 mg/mL) = 94.7%
Alkyl scavenging
(4 mg/mL) = 64.8%
Rohu (Labeo Roe Pepsin Immunomodulatory – Increasing of peritoneal [85]
rohita) macrophage, NK cell activity, and
T-cell subpopulation, enhancing
mucosal immunity (S-IgA) and
antibody production (IgA)
(continued)
367
368
Table 2 (continued)
Roe Protease N Antiproliferative – IC50 Ca9-22 = 0.85 mg/mL [86]
1
Sandfish Meat1 and Alcalase 2.4L Anti-inflammatory – NO scavenging (0.1 mg/ [87]
(Arctoscopus roe2 Collupulin MG mL) = 18.43%
2
japonicus) NO scavenging (0.1 mg/
mL) = 52.35%
1
Seabass (Lates Skin Alcalase 2.4L1 Antioxidant – DPPH = 6.77 μmol TE/g dw [88]
calcarifer) Protease from Metal chelating ABTS = 65.48 μmol TE/g dw
hepatopancreas of (Fe) FRAP = 2.57 μmol TE/g dw
Pacific white shrimp2 Fe2+ chelating = 1.98 μmol
EDTA/g dw
2
DPPH = 6.30 μmol TE/g dw
ABTS = 59.35 μmol TE/g dw
FRAP = 2.65 μmol TE/g dw
Fe2+ chelating = 3.43 μmol
EDTA/g dw
1
Skate (Okamejei Skin Alcalase 2.4L ACE inhibitory MVGSAPGVL1 IC50 = 3.09 μM [89]
2
kenojei) gelatin LGPLGHQ2 IC50 = 4.22 μM
Small red Viscera Crude enzyme from Antimicrobial FPIGMGHGSRPA Bacillus cereus = 0.6 mg/mL [90]
scorpionfish Trichoderma (MIC) Bacillus subtilis = 0.42 mg/mL
(Scorpaena harzianum Staphylococcus aureus = 0.5 mg/
notata) mL
Salmonella sp. = 0.72 mg/mL
Listeria innocua = 0.49 mg/mL
Escherichia coli = 0.8 mg/mL
1
14
Unicorn Skin Glycyl endopeptidase Antioxidant EPGPVG1 ABTS scavenging = 1.25 μmol [91]
leatherjacket from papaya LPGPAG2 TE/g peptide
2
(Aluterus LDGPVG3 ABTS scavenging = 1.22 μmol
monoceros) EGPLG4 TE/g peptide
3
ABTS scavenging = 1.36 μmol
TE/g peptide
4
ABTS scavenging = 4.95 μmol
TE/g peptide
Winter flounder – Synthetic peptide Antiviral – EC50 HSV-1 = 83 μg [92]
(Pleuronectes
americanus)
Yellowfin sole Frame α-Chymotrypsin Anticoagulant TDGSEDYGILEIDSR IC50 FX11a (1.0 μM) = 62.4% [93]
(Limanda
aspera)
A one-letter notation was used for amino acid sequences
IC50, inhibitor concentration that inhibits enzyme activity/activated coagulation factor 11 (FX11a) by 50%
EC50, effective concentration causing 50% of antioxidant/antiviral activity
Antioxidant value expressed in μmol Trolox equivalent (TE)/μmol of dry weight (dw)
MIC, minimum inhibitory concentration

SBP6h, systolic blood pressure after 6 h of oral peptide administration
369
cancer development, as well as having ACE inhibitory, antihypertensive, anticoagu-

lant, ion chelating, antioxidant, antimicrobial, antiviral, and appetite suppressant
activities [87, 94, 95]. These characteristics of peptides generated within marine-
derived protein hydrolysates are linked to their potential as ingredients or nutraceu-
tical products for the management of symptoms related to NCDs such as diabetes,
cardiovascular diseases, cancer, and chronic allergic diseases. It has been previously
reported that peptides with low molecular mass, mainly di- and tripeptides, have
potent bioactive properties [96, 97], while longer peptides, with more than 20 amino
acid residues, have been associated with technofunctional property improvements
[98]. The most common method used to separate peptides within protein hydrolysates
is reversed-phase high-performance liquid chromatography (RP-HPLC) [99]. The
techniques mainly used to separate peptides on the basis of molecular weight include
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel
permeation high-performance liquid chromatography (GP-HPLC) [100]. Further-
more, characterization of the amino acid sequence of the peptides is generally carried
out using liquid chromatography systems coupled with mass spectrometry (MS) or
tandem mass spectrometry (MS/MS) [54, 97, 101].
5 Bioactive Properties
Globally, the total mortality linked to NCDs was 38 million in 2012 and is estimated
to reach 52 million by 2030 [102]. As mentioned in previous sections, peptides
derived from proteinaceous marine processing by-products have been shown to
possess a range of bioactive properties. The potential health benefits of consuming
fish protein hydrolysates/peptides for the control of NCDs and oxidative stress,
allergenicity and inflammation, hypertension, and cancer will therefore be outlined.
5.1 Oxidative Stress
Oxidative stress is an important factor contributing to the development and

the progression of NCDs. It is characterized by the generation of reactive oxygen
species (ROS), including hydroxyl radicals (•OH), superoxide radicals (O2•), and
non-radical hydrogen peroxides (H2O2). High ROS levels have been associated with
the deleterious modification of nucleic acids (DNA and RNA), proteins, and lipids
and have been implicated in accelerating cellular aging and several human condi-
tions, such as atherosclerosis and neurodegenerative diseases [63]. In contrast, low
ROS levels have beneficial physiological effects linked to the regulation of cell
signaling, through the redox regulation of transcription factors, protein phosphory-
lation (kinase), and ion transfer [103]. The consumption of dietary components
containing natural antioxidants (such as vitamin C, polyphenols, and carotenoids)
has been shown to reduce oxidative stress by enhancing natural defenses [104]. On
the other hand, oxidative stress can contribute to a reduction in the proliferation of
cancer cells by inducing cell death. However, while cell death can be achieved by
radical-induced oxidative stress, some cancer cells have developed resistance which
indicates that cells develop sophisticated adaptation responses to oxidative stress
[105]. The peptides from fish processing by-product proteins having antioxidant
activity have recently been reviewed [95]. For example, Glu-Leu-Phe-Glu-Pro-Arg,
a hexapeptide generated by Alcalase hydrolysis of seabass (Lates calcarifer) skin
gelatin, was shown to scavenge hydrogen peroxide [106]. Table 2 provides a list of
fish protein by-product hydrolysates/peptides exhibiting antioxidant activity as
reported in the literature.
The in vitro determination of antioxidant activity of peptides can be performed
using various chemical reactions. The methods used to assess antioxidant capacity
can be classified according to whether they assess the transfer of either hydrogen
atoms (HAT) or electron (ET) [107]. The assays used to measure proton-donating
ability are represented by the oxygen radical absorbance capacity (ORAC), hydroxyl
radical, alkyl radical, and peroxide radical scavenging activity assays. The reported
ORAC activity of peptides derived from fish processing by-products ranges from
5.47 to 19.74 μmol TE/μmol peptide (Table 2). Compared to milk protein-derived
antioxidant peptides, the radical scavenging activity was approximately 2.1- to 7.5-
fold higher than the hendecapeptide, Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile
derived from a β-lactoglobulin A hydrolysate [108]. The ET-based assays used
to determine the reducing capacity of an antioxidant are the Trolox equivalent
antioxidant capacity (TEAC), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate)
radical cation (ABTS•+), FRAP (ferric reducing antioxidant power), and DPPH•
(2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical) assays. The DPPH radical
scavenging activity (EC50, concentration causing 50% of antioxidant activity) of
Phe-Ile-Gly-Pro, a peptide derived from a bluefin leatherjacket skin hydrolysate, was
reported to be 0.118 mg/mL. Interestingly, Song et al. (2011) reported that a potent
pepsin hydrolysate from half-fin anchovy (Setipinna taty) possessed a DPPH EC50
value of 4.46 μg/mL, while the synthetic antioxidant butylated hydroxytoluene
(BHT) is reported to exhibit an EC50 value of 22.78 μg/mL [78]. The antioxidant
potency of bioactive peptides has been attributed to the presence of specific amino
acids therein especially histidine residues. This has been attributed to the chelating
properties and the radical-trapping properties of the imidazole ring. Furthermore, the
presence of hydrophobic amino acid residues in peptides has been associated with an
increase in their accessibility to hydrophobic targets [109, 110].
5.2 Allergenicity and Inflammation
Allergy is a hypersensitivity response associated with an immune system reaction

toward an allergen. The major types of allergies include life-threatening anaphylaxis;
food, drug, and insect allergies; asthma; rhinitis; angioedema; eczema; urticaria; and
eosinophilic disorders [111]. Allergic reactions/diseases can be caused by environ-
mental factors, such as pollution, as well as genetic factors. The global prevalence of
allergic diseases is increasing with 200–250 million people suffering from food
allergy and about 300 million people suffering from asthma causing 250,000 deaths
annually [112]. The diagnosis of allergy is commonly performed using blood

or cutaneous tests. The cutaneous (skin prick) test is carried out by introducing
a series of punctures on a subject’s skin which are loaded with different suspected
allergens. The signs of inflammation, indicative of an allergenic response, are then
observed. Inflammation is one of the first responses of the immune system to
a challenge/infection [113]. The increase in blood flow into the tissue causes
redness, swelling, heat, and pain. Inflammation occurs when damaged tissues or
infected cells release eicosanoids and cytokines. Eicosanoids, which include pros-
taglandins, dilate blood vessels and increase the temperature locally, while leukotri-
enes recruit white blood cells (leukocytes). Cytokines, including interleukins and
chemokines, are responsible for the recruitment and communication between leuko-
cytes at the site of infection, while interferons shut down protein synthesis in infected
cells [114]. Cytotoxic and growth factors can also be released by the damaged
tissues. These molecules have the ability to recruit immune cells to the site of
challenge/infection and contribute to the healing of damaged tissue following
removal of the antigen [115].
The human immune system reacts against various chemical and biological threats
by two separate but interconnected systems. The first defense system, called the
innate immune system, consists of cells and proteins present in the circulation
system, and it is activated in the presence of an exogenous threat. This system
includes mucosal epithelial barriers, dendritic cells, and leukocytes [116]. The
second system, called the adaptive immune system, is a specific system which
involves B- and T-cells and the production of specific antibodies against the detected
threat. The adaptive immune system is activated when the innate immune system is
insufficiently effective or when it has been overcome [117]. Current treatments for
allergies consist of the use of medication such as steroids, adrenalin, and antihista-
mines [118]. Immunotherapy is also available to treat some forms of allergy by
injecting an inactive form of the allergen to stimulate the production of specific
antibodies by the adaptive immune system [119]. Even though immunotherapy
provides a longer-lasting effect, it also represents an expensive alternative to phar-
macological solutions.
Immunomodulatory peptides can modulate immune functions by enhancing
lymphocyte proliferation, antibody synthesis, and cytokine regulation [120]. More-
over, immunomodulatory peptides may have the ability to reduce allergic reactions
and enhance the mucosal immune system in the gastrointestinal tract. Immunomod-
ulatory peptides isolated from human milk, rice, and soybean tryptic hydrolysates act
to stimulate the innate immune system [121–123]. A review of literature reveals that
the mechanism of action of immunomodulatory peptides is relatively non-specific,
and this may be the reason why the exact mechanism and the in vivo destination of
these peptides are still unknown. Figure 3 depicts the main mechanisms of action of
anti-inflammatory peptides. The details of the anti-inflammatory peptides isolated
from fish by-products are reported in Table 2.
The main anti-inflammatory activity of bioactive peptides, as currently described
in the literature, involves the up- and downregulation of signaling proteins (cyto-
kines) such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) and
Fig. 3 Mechanisms of inflammation and potential sites of action of bioactive peptides. (Modified
from Cicero et al. [124])
adhesion molecules. Modification of the expression and translation of these compo-

nents has the ability to reduce inflammation. Chalamaiah et al. (2018) have recently
reviewed the area of immunomodulatory peptides from food protein hydrolysates
[125]. Subhan et al. (2017) demonstrated that peptides from fish scale collagen could
downregulate the expression of pro-inflammatory cytokines in vitro [126]. This
suggests that peptides isolated from fish scale collagen had a beneficial effect in
the control of inflammatory diseases.
As shown in Table 2, a number of peptides/hydrolysates derived from
fish processing by-products are reported to possess anti-inflammatory capacity. For
example, the tripeptide Pro-Ala-Tyr derived from an Atlantic salmon (Salmo salar)
pectoral fin peptic hydrolysate exhibits anti-inflammatory activity via the down-
regulation of NO/iNOS and PGE2/COX-2 pathways by 64–75% and 45–48%,
respectively, compared to the control group. The inhibition of the production of pro-
inflammatory cytokines, TNF-α, IL-6, and IL-1β, has been also reported (Table 2)
by Anh et al. [67].
5.3 Hypertension
The literature reports that dietary protein intake can contribute to reducing high
blood pressure, coronary heart disease, and other infarctions [127]. Blood pressure is
determined by measuring two values: systolic blood pressure (SBP), which measures
the pressure in blood vessels when the heart beats, and diastolic blood pressure,
which measures the pressure in blood vessels when the heart is at rest. The normal
values for systolic and diastolic blood pressures are 120 and 80 mmHg, respectively.
A range of mechanisms are involved in the control of blood pressure, including the
secretion of specific hormones, modulation of blood volume, and secretion of nitric
oxide by endothelial cells and the renin-angiotensin-aldosterone system (Fig. 4).
ACE, for example, catalyzes the conversion of angiotensin I to angiotensin II, a
hormone which leads to vasoconstriction and an increase in blood pressure [128].
Furthermore, ACE degrades the vasodilator molecule bradykinin. Consequently,
Fig. 4 Mechanisms of blood pressure control and the potential action of bioactive peptides.
(Modified from Cicero et al. [124]) : direct inhibition, : direct stimulation
ACE inhibitory agents can lower hypertension. Many studies have focused on the
ability of fish protein hydrolysates/peptides to inhibit ACE [66, 89]. ACE inhibitory
peptides are generally short sequences. Moreover, structural studies of the ACE
active site using X-ray crystallography show a lid-like extension formed by the
amino-terminal helix (α 1-3) that partially covers the active channel and leaves an
opening of almost 3 Å for substrate and inhibitor access [129]. Wu et al. (2006)
performed an in silico analysis of the ACE inhibitory activity of long (4–10 amino
acids) and short (2–3 amino acids) peptides. The importance of the type of amino
acid residue in the peptide sequence for ACE inhibitory activity was predicted [130].
It was concluded that the optimum amino acid residues for potent ACE inhibition
starting from the C-terminus were Tyr and Cys in the first position; Trp, Met, and His
in the second position; Leu, Ile, Val, and Met in the third position; and Trp in the
fourth position. Blood pressure is highly regulated in vivo and involves mechanisms
other than modulation of ACE activity. It is likely that bioactive peptides derived
from fish protein hydrolysates also beneficially modulate these systems.
Recently, research on nitric oxide synthase 3 (iNOS) suggested that stimulation of
the production of nitric oxide (NO) in endothelial cells has a beneficial effect on
blood pressure (Fig. 4) [131]. Ahn et al. (2015) isolated the tripeptide Pro-Ala-Tyr
from an Atlantic salmon (Salmo salar) pectoral fin peptic hydrolysate and demon-
strated that it possessed the ability to modulate the secretion of intracellular NO
in vitro [67].
Several reports from in vivo studies using spontaneously hypertensive rats
(SHRs) show hydrolysates/peptides derived from fish proteins having the ability to
significantly reduce hypertension. Ko et al. (2016) identified ACE inhibitory pep-
tides (Table 2) from hydrolysates of flounder fish (Paralichthys olivaceus) protein
which were shown to have hypotensive effects in vivo [82]. The in vitro ACE IC50’s
for two identified hexapeptides, Met-Glu-Val-Phe-Val-Pro and Val-Ser-Gln-Leu-
Thr-Arg, was 79 and 105 μM, respectively. These peptides were found to reduce
SBP in SHRs after 6 h oral administration (Table 2). Interestingly, the reduction in
SBP value obtained with the Val-Ser-Gln-Leu-Thr-Arg-treated group was similar to
the group treated with Captopril ®, a synthetic drug inhibitor of ACE. As shown in
Table 2, numerous peptides with ACE inhibitory activity have been derived from
marine by-products. These include hydrolysates/peptides from Atlantic salmon,
bluefin tuna, boarfish, leatherjacket, Pangasius catfish, rockfish, and skate.
5.4 Type 2 Diabetes Mellitus
Type 2 diabetes mellitus (T2DM) is characterized by insulin deficiency caused by

pancreatic β-cell dysfunction and insulin resistance [132], which arises from the fact
that the pancreatic cells produce and release insulin, but the quantity released
is insufficient. All these complications lead to hyperglycemia and many side effects
such as atherosclerosis leading to heart attack, stroke, and organ failure. Type 1
diabetes, also named “insulin-dependent diabetes,” is a malfunction of the pancreatic
cells that fail to produce and release insulin resulting from a cell-mediated autoim-
mune attack of pancreatic β-cells [133]. However, T2DM is the most common type
of diabetes accounting for 90–95% of all diabetes cases [132]. The occurrence of
T2DM has increased in developed countries and is associated with an unhealthy
lifestyle and obesity which contributes to higher rates of morbidity as diabetic
individuals generally have a higher risk of heart disease, kidney failure, blindness,
and nerve and circulatory damage [132]. The global prevalence of diabetes was 415
million adults aged over 20 years in 2015 (8.8% of the adult population), and this is
expected to increase to 642 million by 2040 (10.4% of the adult population) [134].
The countries with the highest incidences of diabetes in 2015 were China (109.6
million), India (69.2 million), the United States (29.3 million), Brazil (14.3 million),
the Russian Federation (12.1 million), Mexico (11.5 million), Indonesia (10.0
million), Egypt (7.8 million), Japan (7.2 million), and Bangladesh (7.1 million).
Several types of medication are currently employed for the management and control
of T2DM. The most common treatments involve the use of Metformin ® and
Gliclazide ® which decrease the release of hepatic glucose and increase insulin
secretion, respectively. Both medications are on the World Health Organization list
of essential medicines for the treatment of T2DM. Other treatments used in the
management of the disease include the injection of glucagon-like peptide-1 ana-
logues and the use of enzyme inhibitors which inhibit the activity of dipeptidyl
peptidase-IV (DPP-IV), α-amylase, and α-glucosidase. These treatment approaches
enhance the body’s response to reduce postprandial serum glucose levels. The
enzymes targeted for inhibition are linked to the reduction of glucagon release and
the stimulation of insulin synthesis, glucose absorption, and metabolism, as well as
appetite reduction [135–137]. DPP-IV degrades two incretin hormones, glucose-
dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). In
the presence of glucose, the incretin system stimulates pancreatic β-cells to secrete
insulin. Therefore, by inhibiting the action of DPP-IV, the incretin hormones are
maintained at a stable level in the circulation and continue to stimulate insulin
production [138]. Human in vivo studies have shown that the inhibition of DPP-IV
leads to a reduction in glycated hemoglobin (hemoglobin A1c) [139] as well as an
increase in circulating GLP-1 [140]. Consequently, this represents clear evidence of

the blood glucose-lowering effect of DPP-IV inhibitors. Although DPP-IV inhibitors
and GLP-1 analogues are interesting targets for blood glucose regulation, they
represent distinct drug classes with different mechanisms of action, route of admin-
istration, and clinical efficiency. However, some synthetic drugs with DPP-IV
inhibitory activity are now being used for the treatment of T2DM, such as sitagliptin
(Merck & Co. as Januvia ®, FDA approved in 2006), vildagliptin (Novartis as
Galvus ®, EU approved in 2007), and alogliptin (Takeda Pharmaceutical Company
as Nesina ®, FDA approved in 2013). While these drugs have proven to be efficient
in the management of T2DM, side effects including postprandial hypoglycemia,
nasopharyngitis, headache, nausea, heart failure, hypersensitivity, skin reaction, joint
pain, and adverse cardiovascular effects have been associated with their use [141].
Therefore, the use of natural sources to produce DPP-IV inhibitors is being explored
in order to reduce the side effects of antidiabetic drugs. Protein hydrolysates derived
from fish processing by-products have been reported to have in vitro DPP-IV
inhibitory activity, and numerous DPP-IV inhibitory peptides have been identified.
These include Gly-Gly-Pro-Ala-Gly-Pro-Ala-Val (624.7 Da) and Gly-Pro-Val-Ala
(342.4 Da) which can inhibit DPP-IV by 50% at a concentration (IC50 value) of 0.26
and 8.14 mg.mL1, respectively [64]. Recent in vivo studies on protein hydrolysates
derived from the underutilized fish blue whiting (Micromesistius poutassou) have
shown the ability to lower glucose and increase insulin level in mice [77]. Another
approach to regulate blood glucose is to stimulate the production of cholecystokinin
(CKK) by enteroendocrine cells in the duodenum. The secretion of CCK is linked to
gastric emptying, the stimulation of pancreatic secretion, and satiety [142]. Several
in vitro and in vivo studies testing intact proteins and their hydrolysates or
corresponding amino acid mixtures demonstrate this phenomenon. Sharara et al.
(1993) have shown that protein intake stimulates CKK secretion postprandially in
rats, whereas free amino acid intake had no effect [143]. Furthermore, Cudennec et
al. (2008) have shown that protein hydrolysates derived from blue whiting can
enhance the secretion of CKK and GLP-1 in vitro [144]. In vivo studies with the
same hydrolysates showed an increase in CKK and GLP-1 levels in the plasma of rat
[145] and in human [62]. Greco et al. (2017) in reviewing the effect of protein intake
on appetite have shown that the effect observed depends on the protein source [146].
Madani et al. (2015) showed that feeding obese rats with sardine proteins resulted in
reduced plasma glucose and reduced insulin resistance as well as higher plasma
GLP-1 levels compared to the group fed with casein [147]. A list of DPP-IV
inhibitory peptides and CKK-stimulating hydrolysates obtained from fish processing
by-products and underutilized fish species is provided in Table 2. The in vitro DPP-
IV inhibitory activity of two peptides, Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala, iden-
tified from Atlantic salmon skin hydrolysates had IC50 values of 49.6 and 41.9 μM,
respectively. Furthermore, Gly-Pro-Val-Ala identified from an Atlantic salmon trim-
ming protein hydrolysate possessed a DPP-IV IC50 value of 264.74 μM. It was noted
that the differences in amino acid residues in the peptide sequence had a major
impact on DPP-IV inhibitory activity. The peptide Ile-Pro-Ile which is a known
DPP-IV inhibitor and found in many dietary proteins was reported to have an IC50
value of 3.2 μM [148]. It is possible that fish processing by-products could be a

potential source of peptides with similar or higher levels of DPP-IV inhibitory
activity.
5.5 Cancer
Cell division is a normal physiological event that occurs in tissues. Cell proliferation
and cell death are highly regulated processes. Certain mutations in cellular DNA
destabilize this process and can ultimately lead to cancer. The process that transforms
normal cells into cancer cells is called carcinogenesis. It is characterized by a series
of changes at cellular and genetic level that reprogram the cell into an uncontrolled
division process leading to the formation of a tumor. This malignant mass can remain
at a particular site or spread throughout the body via an angiogenesis process and
metastasis diffusion.
Apoptosis is a form of programmed cell death and is one of the main mechanisms
used in cancer treatment. As apoptosis does not enhance immune response or
produce inflammation, it is a better method of treatment compared to classic
chemical chemotherapies. Therefore, selective induction and modulation of apopto-
tic pathways in cancer cells represent a promising approach for cancer therapy [149].
In mammals, two major apoptosis signaling pathways are involved in the activation
of cysteine proteases (caspases), the extrinsic death receptor, and the intrinsic
mitochondrial pathways [150]. These interlinked pathways involve pro- and anti-
apoptotic molecules that can trigger or regulate apoptosis. Therefore, the develop-
ment of antiproliferative peptides that specifically target these pathways has become
an interesting strategy for the development of anticancer therapies.
A large diversity of peptides with anticancer activity have been extracted from
various marine organisms, mainly sessile animals, such as sponges, molluscs, and
tunicates, which synthesize potent cytotoxic compounds to protect themselves
against predators. These compounds are currently being exploited for cancer therapy.
However, reports on the antiproliferative activity of peptides derived from fish protein
hydrolysates are limited. Chalamaiah et al. (2018) reviewed the area of anticancer
peptides from food protein hydrolysates [125]. Several studies have reported that free
amino acids have diverse effects on different cancer cells [151, 152]. Cys promoted
the proliferation of gastric and breast cancer cells. Asp and Arg stimulated the growth
of breast cancer cells, while Glu induced apoptosis in gastric cancer cells. Ala showed
an in vitro antiproliferative activity against gastric and breast cancer cells, while Pro
and Lys showed an antiproliferative activity against prostate cancer cells. These
reports suggest that the presence of specific amino acids in peptide sequences could
modulate their activity against different cancer cell metabolic pathways. Furthermore,
two peptides, Leu-Pro-His-Val-Leu-Thr-Pro-Glu-Ala-Gly-Ala-Thr and Pro-Thr-Ala-
Glu-Gly-Gly-Val-Tyr-Met-Val-Thr, isolated from tuna protein hydrolysates pos-
sessed antiproliferative EC50 values of 8.1 and 8.8 μM on the human breast cancer
cell line MCF-7 in vitro, respectively [80]. A list of the antiproliferative peptides
obtained from half-fin anchovy, loach, and rohu by-products is provided in Table 2.
To date, Alemán et al. (2011) have reported the highest antiproliferative EC50 value
(0.13 mg/mL) for a squid gelatin hydrolysate using Esperase ® on MCF-7 cell
line [153].
5.6 Other Bioactivities
Other biological activities involving peptides which possess antiviral and antimicro-
bial activity have been reported in the Antarctic fish (Pleuronectes americanus) and
small red scorpionfish (Scorpaena notate), respectively (Table 2). Mineral-binding
peptides have been identified from Alaska pollock and seabass. Mineral-binding
capacities are important in many metabolic processes, including nutrient absorption,
cellular proliferation, energy production, and oxygen transport [68, 70, 88]. Antico-
agulant activity had been reported in peptides extracted from yellowfin sole frame
[93] (Table 2).
6 Bioavailability
As already outlined, bioactive peptides can be released from food proteins during
food processing by fermentation or enzymatic hydrolysis as well as during normal
gastrointestinal digestion. Research has proven that fish processing by-products are a
potential source of bioactive peptides for the production of functional food ingredi-
ents [154]. However, more studies on the stability of the bioactive properties need to
be carried out. In order to be active at the target site in the human body, the peptides
must maintain their biological activity after passing through the gastrointestinal tract,
be resistant to extreme pH values and the action of gastrointestinal enzymes. An
in vitro simulated gastrointestinal digestion (SGID) approach is often carried out to
determine the stability of bioactive peptides following in vitro incubation with
gastrointestinal enzymes. The enzymes used are salivary amylase at pH 7.0 for the
oral phase, pepsin at pH 3.0 to simulate the gastric system, and a pancreatic enzyme
preparation composed of trypsin, chymotrypsin, elastase, lipase, and amylase at pH
7.0 to mimic the intestinal phase [155]. Hydrolysis of proteins by these enzymes can
release bioactive peptides. This has been shown when using the SGID approach for
the generation of bioactive peptides from different food sources such as cereals,
dairy products, and fish [156–159]. Moreover, in vitro SGID has shown that the
hydrolytic action of gastrointestinal enzymes has the potential to modulate the
bioactive properties of peptides generated following hydrolysis using non-
mammalian food-grade enzyme preparations [160]. For this reason, bioactive pep-
tides may be tested using in vitro gastrointestinal digestion to assess their potential
stability and bioavailability after ingestion. However, some bioactive peptides have
shown resistance to further digestion by gastrointestinal enzymes, such as the ACE
inhibitory peptide Leu-Leu-Pro from tilapia, which maintained its activity following
incubation with pepsin, pancreatin, and α-chymotrypsin [161]. The permeability of
biological membranes, which allow bioactive peptides to reach the circulation,
14
Fig. 5 Peptide transport mechanisms across intestinal cells. (Modified from Harnedy and FitzGerald [154])
379
depends on many factors such as peptide molecular mass and chemical stability and
hydrophobicity. The transport of peptides through the gastrointestinal tract and the
intestinal cell barrier is mediated via three main transport mechanisms. These
mechanisms are schematically represented in Fig. 5. These consist of (A) active
transport via the PepT1 carrier, which transports di- and tripeptides coupled with a
proton pump, (B) endocytosis-exocytosis which transports basic and hydrophobic
peptides via endocytosis vesicles, and (C) tight junction paracellular diffusion,
which transports intact oligopeptides through tight junction pores [154]. However,
active transport via PepT1 and passive paracellular diffusion are more efficient
routes than endocytosis-exocytosis-mediated transport as peptides may be hydro-
lyzed after endocytosis by intracellular enzymes into amino acids before reaching
the bloodstream. Furthermore, larger peptides and single amino acids have been
shown to be less easily absorbed by gastrointestinal cells than short peptides (i.e.,
containing two to six amino acids) [162]. Transfer across the gastrointestinal mem-
brane also depends on the amino acid sequence of the peptide [163]. Bioactive
peptides isolated from fish have been reported to be resistant to the gastrointestinal
digestion process and to be able to pass through intestinal membranes to reach the
bloodstream. For example, in vivo studies in hypertensive rats have shown that the
antihypertensive effects of bioactive peptides derived from fish, such as salmon,
sardine, sole, tuna, and Alaska pollock, remain stable after passage through the
digestion and assimilation processes. For example, Hou et al. (2016) showed that
Pro-Thr-Gly-Ala-Asp-Tyr derived from tryptic hydrolysates of Alaska pollock frame
could significantly enhance the humoral, cellular, and non-specific immune system
in immunosuppressed mice [164]. This indicates that this peptide was resistant to
digestion and was able to pass into the bloodstream. However, the stability of
bioactive peptides can be enhanced via several strategies which have been developed
by the pharmaceutical industry. Among these, encapsulation and structural modifi-
cation of peptides at C- and/or N- terminal residues, including glycosylation and
alkylation, have been shown to improve the bioavailability of peptides. Furthermore,
peptides containing Thr, Glu, Phe, and His amino acids seem to be absorbed
significantly faster compared to their free amino acid mixture equivalent [163].
The presence of a high percentage of Hyp and Pro amino acids also seems to
improve resistance to hydrolysis by gastrointestinal enzymes [165]. Some of these
approaches have been used to improve the bioavailability of fish-derived bioactive
peptides. For example, the encapsulation of rainbow trout peptides in biopolymer-
coated nanoliposomes was an efficient technique to maintain their antioxidant
capacity [166].
7 Conclusion
The study of fish protein for the generation of bioactive peptides has increased in the
last few years, and fish processing by-products as well as underutilized fish species
have been identified as potential sources for bioactive peptides. However, even
though several studies with regard to the extraction and hydrolysis of proteins
from fish and fish by-products, as well as the purification, characterization, and
identification of bioactive peptides, have been carried out, more research is required
to fully exploit and deliver their potential to consumers. While interesting studies on
the use of fish processing by-products as functional food ingredients have been
carried out, more research is needed in addressing the large-scale production of these
products, their bioavailability, compatibility with different food matrices, long-term
stability, and in vivo efficiency. Furthermore, it is necessary to determine the
mechanisms by which peptides and hydrolysates can mediate their physiological
effects. More nutrikinetic and metabolomic studies are required in order to under-
stand the relationship between the dose administered and physiological effect.
Marketing and economic studies are also required to establish consumer needs and
preferences. Finally, in vivo validation studies are required to generate health
promoting claims acceptable for international food safety agency approval.
Acknowledgments Aurélien V. Le Gouic and Pádraigín A. Harnedy were funded by the Depart-
ment of Agriculture, Food and the Marine, Ireland, under grant issue 14/F/873 and 13/F/467,
respectively.
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Edible Insects as Source of Proteins
15
Ewelina Zielińska, Monika Karaś, Anna Jakubczyk, Damian Zieliński,
and Barbara Baraniak
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
2 History and Cultural Acceptance of Entomophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
2.1 History and Tradition of Eating Insects by Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
2.2 Do Europeans Already Eat Insects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
2.3 Preferences of Consumers in Western Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
3 Examples of Popular Insect Species for Human Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
3.1 Diptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
3.2 Coleoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
3.3 Lepidoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
3.4 Orthoptera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
4 Nutritional Value of Edible Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
4.1 Energy Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
4.2 Protein and Essential Amino Acid Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 406
4.3 Lipids and Essential Fatty Acid Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
4.4 Fiber Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
4.5 Microelements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
4.6 Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
4.7 Bioactive Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
5 Processing Edible Insects for Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
5.1 Insect Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
5.2 Extracted Insect Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
5.3 Extracted Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
5.4 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
5.5 Functional Properties of Insect Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
E. Zielińska (*) · M. Karaś · A. Jakubczyk · B. Baraniak

Department of Biochemistry and Food Chemistry, University of Life Sciences in Lublin, Lublin,
Poland
e-mail: [email protected]; [email protected]; [email protected];
[email protected]
D. Zieliński
Department of Ethology and Animal Welfare, University of Life Sciences in Lublin, Lublin, Poland

390 E. Zielińska et al.
6 Why Should We Look for New Sources of Nutrients? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418

7 Environmental Impact of Entomophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
8 Safety Aspect of Edible Insects as Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
9 European Union Policy on Insects as Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
10 Insect Business . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
10.1 Insect Farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
10.2 Insect-Based Companies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
11 Insects as a Source of Nutrients for Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
12 Promising Opportunities of the Use of Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
13 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Abstract
The potential of insects as a source of protein for future food and feed is the object
of numerous studies. The nutritional value of edible insects is well established,
and other aspects of consumption thereof are investigated. In this chapter, we aim
to summarize the main features of insects as food. We briefly describe the history
of the usage of insects as food for humans and refer to the current acceptance of
insects by Europeans based on conducted surveys. We characterize the most
common insect species with the biggest potential to be used as food and feed in
the EU according to EFSA. We describe the nutritional value of insects and the
possibility of application thereof in the food and feed industry, keeping in mind
the safety of consumption. In addition, the ecological aspect of insect breeding is
discussed. A review of the growing edible insect market in Europe and the USA is
also provided. Moreover, we analyze the current legal status of insect intake in
Europe. We aim to make this chapter a current conclusion about the consumption
of insects.
Keywords
Entomophagy · Edible insects · Protein · Environment · Nutritive value ·
Functional properties · Insect products · Bioactive peptides · Preferences of
consumers
1 Introduction
The practice of eating insects is known as entomophagy. The term “entomophagy”

itself is relatively new in English and some other European languages. The Google
Ngram currently dates the first published mention of “entomophagy” to 1871, in a
volume entitled “Sixth annual report on the noxious, beneficial and other insects of
the state of Missouri” by Charles V. Riley. In French, the term “entomophagie” was
found in data from 1810 [1]. Today, we can find “entomophagy” in Oxford Dictio-
naries online, which defines the term as “the practice of eating insects, especially by
people.” The greater interest in entomophagy is reflected in the increase in the
number of publications. As reported by Evans et al. [1], the number of publications
15 Edible Insects as Source of Proteins 391
in the Web of Science containing “entomophagy” amounted to 16 in 2001–2010 and

as many as 49 in 2011–2015. In turn, 50 articles were published between 2016 and
September 2017.
The rising interest in edible insects is reflected in the emergence of companies
dealing with the production and processing of insects as well as the expanding
scientific literature. The resulting publications present the use of insects in feeding
farm animals, accompanying animals, and humans [2], whereas, research grants are
focused on the use of insects in the food industry in various forms [3]. The
consumption of insects as a whole can be difficult for Western consumers; therefore,
the easiest way for processing insects is grinding them to flour [4]. It is also possible
to isolate individual components of insects such as protein [5], fat [6], and chitin [7].
Eating insects by humans is not a new concept; it occurs globally [8] but is still
rare in Europe [9]. Why not eat insects? Is it worth it? The answer is simple –
definitely yes. Entomophagy has several advantages. First of all insects are a good
source of protein, essential fats, and antioxidant peptides [10–13]. Many insects are
rich in microelements such as iron, calcium, and zinc and in vitamins [14, 15].
Secondly, insect breeding is environmentally friendly. Insects emit significantly
fewer greenhouse gases (GHGs) and ammonia than most livestock [16]. Moreover,
insects require less space, feed, and water for breeding than livestock [17]. Economic
factors are also important. Insect rearing can be low-tech or very sophisticated,
depending on the level of investment [18]. For these three main reasons, insects
have been highlighted as an important food source in response to the growing
concerns about the future of world food security.
In this chapter, we describe the state of the knowledge of various aspects of insect
consumption and their potential to be used in the food industry. We will also discuss
the historical and cultural background of insect consumption, and we will focus on
the biology of popular species.
2 History and Cultural Acceptance of Entomophagy
Despite the lack of tradition and the reluctance of some consumers, edible insects are
increasingly becoming part of the diet of the people of not only Africa, Asia, and
Latin America but also the USA, Australia, and Europe [18]. In many countries, they
are considered not only as food providing nutrients but also as a delicacy. Although
for many Europeans, insects are still associated with something unpleasant, it should
be noted that in countries such as Thailand, Madagascar, and Mexico, they could
only be consumed by the royal elites and the rich [19].
2.1 History and Tradition of Eating Insects by Humans
Habits, geographic regions, cultures, and religious beliefs have the main influence on
food and diet practices. It is known that insects were consumed in ancient times, not
only by animals but also by our primate ancestors [20]. Already in the fourth century
BCE, cicadas (nymphs, in particular) were described by Aristotle as a delicacy in

Ancient Greece [21]. The first mention of insects as food ingredients can be found in
the Old Testament (Leviticus 11.20–23), where we can read: “All winged insects
moving on four feet you will regard as detestable for eating. Of all these winged
insects you may eat only the following: those with the sort of legs above their feet
which enable them to leap over the ground. These are the ones you may eat: the
various kinds of migratory locust, the various kinds of solham locust, hargol locust
and hagab locust. But all other winged insects on four feet you will regard as
detestable for eating” [22]. Noteworthy is the fact that only some insect species
were allowed to be eaten. Moreover, the New Testament (Matthew, 3, 4) mentions
locusts as the food of John the Baptist [22]. Entomophagy was described in the early
centuries AD in Greek literature by many philosophers; however, the approach to
insect consumption was not unequivocal because many Greeks in the first century
AD believed that cicadas should not be eaten because these insects were a swallow
food and themselves were scary and musical [23]. In turn, in the first century AD,
Roman historian Pliny the Elder (author of the encyclopedia – Historia Naturalis)
mentioned that cicadas were eaten in the East [23] and the Cossus, i.e., an insect
living on oak trees, was a meal valued by the Romans [23, 24]. In the early third
century AD, in one of his works (The Deipnosophistae of Athenaeus), which is
named the first cookbook, Athenaeus of Naucratis described grasshoppers and
cicadas that were eaten as an incentive to appetite [25]. As shown by the
abovementioned sources, the tradition of eating insects dates back several thousand
years, and the mention of insect food can be found not only in the literature of the
Christian religion but also in the Jewish and Islamic culture. As far as Christianity is
concerned, we find already mentioned data on insect eating in the Bible, whereas
species of kosher locusts were largely accepted in the Jewish culture in ancient times.
In the following years, the tradition of consuming insects among the Jewish diaspora
due to the lack of knowledge of the various types of insects known in the Torah as
“winged swarming things” significantly decreased. Probably, also the influence of
the Western culture was the reason for the disappearance of the tradition of eating
insects by Jews [26]. In the Islamic tradition, we can also find several references to
eating insects. El-Mallakh and El-Mallakh [27] find insects in the text of the Koran
(e.g., gnat, locust, lice, bee, ant, and spider) that have played an important role in
historical events or religious instructions. Only locusts, despite the association with
plagues, could be designed to eat (Sahih Muslim, 21.4801). Moreover, insects were
seen as low life forms (with the exception of bees) that had a negative influence on
the daily life of humans. In the early literature of the Far East, especially in the
Chinese literature, we can find information about insects as food. The Compendium
of Materia Medica, i.e., a Chinese herbology and medicine book written by Li
Shizhen during the Ming dynasty [28], provides information about the consumption
of insect tea prepared from feces of moths, e.g., Aglossa dimidiatus, Hydrillodes
morosa, and Nodaria niphona, which exhibited potential activity in treatment of
otitis media [29]. The same source of interesting data about insects provides infor-
mation that Megachile spp. China were used as medicine for cancer, Endoclita
sinensis (grape tree borer) was used to enhance health, and larvae and adults of
Apriona germari japonica (mulberry longhorn, mulberry borer) were consumed [30]
or used for treatment of cardiovascular diseases and brain fever [28]. As can be seen,
insects are an important part of human nutrition history. They were very important
sources of nutritional compounds not only for Africans, Asians, Americans, Latin
Americans, and Indians in the west of North America. Some species were more
important such as grasshoppers, caterpillars, beetle grubs and (sometimes) adults,
winged termites (some of which are very large in the tropics), bee, wasp, and ant
brood (larvae and pupae) as well as winged ants, cicadas, and a variety of aquatic
insects. It should be noted that these insects were eaten not only during the periods of
famine as the ultimate food, but they were also elements of the normal diet. Due to
the tradition of nutrition, taste, and nutritional compounds among some people such
as the Yukpa people of Colombia and Venezuela or the Pedi of South Africa,
traditional insect dishes are more preferred than meat dishes [31]. It should be
emphasized that insect consumption in many countries is not a new trend in nutrition
and is part of the culture and tradition of the East and tropical countries. Moreover,
the history of entomophagy is very old – from ants and larvae of beetles eaten in the
tribes of Africa and Australia to maintain a balanced diet to fried locusts and beetles,
which are popular also today in Thailand. It is estimated that insects are part of a diet
of about two billion people worldwide, and 1900 species have been documented in
the scientific literature as edible species. Similar to meat eaters who do not eat all
kinds of meat, insect consumers do not eat all insect species. The most populous
groups of insects include locusts, crickets, cicadas, beetles, wasps, caterpillars, leaf
and plant hoppers, termites, scale insects, true bugs, dragonflies, flies, ants, grass-
hoppers, and bees [18]. In cultures where insects are regarded as food components,
there are special local standards for the preparation thereof to make them suitable for
consumption [32].
2.2 Do Europeans Already Eat Insects?
Although it may seem that Europeans do not eat insects, this is not exactly true. Even
though many people claim that they would never eat insects, they actually consume
them every day. Insects are everywhere. In spite of farmers’ efforts, insects are found
in cereals, spices, vegetables, and fruits, and despite the enforced limits on the
specific insect content, they reach finished products such as flour, frozen food,
chocolate, ketchup, coffee, fruit juice, and many other foods. Calculations indicate
that each of us unknowingly consumes about 1 lb (500 g) of insects per year [33].
Furthermore, sometimes we eat insects consciously. The cochineal scale insect
(Dactylopius coccus) produces a red dye called carmine (E120) when it is crushed.
This dye is widely used in the food industry, e.g., in beverages, sauces, yogurt, and
baked goods. In turn, cheese from sheep milk casu marzu (literally “rotten cheese”)
is a Sardinian delicacy. It is left to ripen for so long that it starts to rot. The rotting
process attracts cheese flies that then lay their eggs on the cheese. The fly larvae eat
their way into the cheese, digesting fats along the way and making the hard cheese
soft and runny on the inside. The cheese is eaten with larvae, which are about 8 mm
long. However, the most popular and totally accepted product, although processed
through an insect, is honey. People may not realize anymore that it comes from an
insect and it is the vomit of some kind of larva [33]. Nevertheless, since honey is
delicious, perhaps a chance should be given to other insects.
2.3 Preferences of Consumers in Western Countries
Although there are historical data on the use of insects for nutrition and insects are
considered delicacies in many parts of the world, in most Western countries, eating
insects is regarded as a disgusting practice arousing distaste and associated with
primitive behavior [34]. In these cultures, insects are completely rejected as food
ingredients, because they are unclean, unhealthy, and thus involved in the risk
associated with food contamination and consuming insects [35]. The current grow-
ing scientists and consumers’ interest in insects as food should be noted. It seems that
there are three main barriers for the sector of edible insects: consumer acceptance,
technology, and regulation. Hoverer, a study conducted in Switzerland has indicated
nine main factors that have an influence on the willingness to consume insects. These
are convenience orientation, discernibility of insects in food, food neophobia,
expected food healthiness, perceived health benefits of meat, food technology
neophobia, need for familiarity, and binary variables: gender and prior consumption.
It is interesting that food neophobia does not play the most important role in the
willingness to consume insects [36].
The current European Union policy on the food sector is cautious about new food
sources. Classification of products as a “new food” and new food technologies as
well as legislation of edible insects in human diet is still under consideration [37].
Nevertheless, according to Lensvelt and Steenbekkers [38] and Caparros Megido et
al. [39], crickets and mealworms are most widely accepted as edible insects by
people from Western countries. Factors that have an influence on this trend include
increased interest in non-European cultures, developing ecology, environmental
concern, and increased consumer awareness of technological issues in food produc-
tion. In some countries, ten species of reared insects are mainly used as animal feed;
however, some of these species have recently been sold as food in China, Thailand,
the Netherlands, South Africa, Belgium, France, and the USA [40]. Commercial
insect consumption in Western countries is currently limited to experimental restau-
rants where insects are served as a delicacy and something different or original [41]
or to products that contain insecticides such as protein isolated as a nutrient ingre-
dient for athletes. There are many studies on consumer preferences, relationships
between human nutritional habits and entomophagy, or factors that have the stron-
gest influence on consumer acceptance of insects as food. The results are not clear
and depend on the countries and the awareness of participants in the experiments.
In recent years, the consumption of animal-based proteins, especially meat, aimed to
maintain a well-balanced diet has been identified as one of the most important topics
both for consumers and for food producers. It is known that the intensity of animal
production generates climate change, has a strong influence on the environment, and is
expensive [42]. For this reason, studies mainly focus on determination of consumers’
readiness to replace meat with other products in the diet [43]. Alternative nutritional
approaches are vegetarian, macrobiotic, or anthroposophical diets, in which meat is
either not eaten at all or very rarely and the protein is replaced by a vegetable healthy
protein. Last but not least, insects are considered a potential source of protein [44]. The
insects are not associated as food but as something nasty and food contamination.
However, results of a study conducted in Belgium have shown that more than 65% of
respondents disagree with the idea of including insects as food compounds [45].
Another study provided data from a representative survey of Dutch consumers on
their habits in preparation of meat meals, meat substitution, and reduction of this
ingredient in the diet, showing that the meal form, cooking skills, product familiarity,
and preferences for plant-based foods had an influence on the consumer preferences.
The most important factor that has an influence on acceptance of edible insects by
consumers is their invisibility and the way of preparation of the meal [39, 46]. The
results of the study with Dutch consumers, in which pictures of food with whole insects
and with protein isolated from insects but used as an ingredient of the product, e.g.,
pizza, indicated that the photos with visible insects were rated much more negatively
than those showing protein derived from insects and processed in the pizza. It seems that
the pizza flavor has a positive effect on accepting insects as food, as chocolate-coated
locusts were rated less negatively than fully visible insects [42]. The aim of another
experiment was to determine how the product preparation, individual traits (e.g., food
neophobia), and familiarity influence the acceptance of insects as food by consumers.
The Dutch respondents rated eight pictures with four dishes (familiar foods). There was
beef stew, curry, and brownie and spice cake in two variants: the mealworms were
ground and not visible in the picture, and the insects were visible in the dishes. The
results indicated that adding mealworms to familiar meals did not guarantee high
product acceptability. Moreover, the products with edible insects were ranked lower,
even if they were visually identical with products used as a control [47]. It would be
worth exploring whether insects would be more readily accepted as a snack food than a
part of the main meal. Since consumers have little experience with insects as food, it is
difficult to conclude that the taste of dishes is based only on the presentation of the meal
[48]. Hence, insects presented in various forms in a dish can cause different sensations
and perceptions of taste, which affect the readiness of consumers to eat insects. In turn, a
study conducted in Italy indicated that students engaged in a so-called bug banquet with
cookies made with “insect flour” willingly tried the product and claimed that they would
try other insects in the future. The main role in the decision to try a cookie made with
cricket flour was played by curiosity, but negative opinions of family members and
friends and the disgust factor prevented the consumers from eating the insects. In
general, respondents consider insects as a rich source of protein and other nutrients,
which may influence the desire to introduce them into the diet. This largely depends on
the market availability and regulatory framework, food category (e.g., bakery product
with insect flour), marketing strategies, methods of preparation, and culinary trends [49].
Another study provided some data about the sector of edible insects in the
Netherlands, the progress of the sector, and limitations in the legitimacy process.
Interviews were held with experts and stakeholders, including, e.g., industry experts,
researchers, government officials, and farmers in the emerging sector, about the
strengths, weaknesses, opportunities, and threats of the edible insect industry. It
should be noted that almost all respondents rated high the use of edible insects for
economic and social purposes. Edible insects can also constitute a new business
model in the Netherlands, be an alternative to soy and fishmeal, and provide a
solution to global protein deficiency. However, the development of this sector should
not be rushed and not limited to the feed market [37].
A study assessing the perception of entomophagy among the Belgian population
was carried out by Caparros Megido et al. [39]. The results indicated slight neo-
phobia, but consumers agreed to test insect preparations. Two species of edible
insects were tested (house crickets and mealworms); they were prepared as a snack
with known flavors and crispy texture. The results have suggested that people will be
eating, preparing, and cooking insects in the near future. It has been shown that
edible insects that are prepared as snacks with common flavors such as chocolate,
chili, caramel, or curry arise curiosity and are more likely to be eaten than raw
insects. It should be noted that, even in 2012, insects were not accepted as an
alternative or replacement of meat in Belgium [43].
Determination of the acceptance of edible insects as food and an alternative
source of protein was the aim of a study conducted by Gere et al. [50] in Hungary.
The results showed that less than 11% of respondents did not regard insects, algae,
soy, and whey as an alternative source of protein, but half of them knew algae and
whey as this kind of protein source, and soy had the highest familiarity scores.
Moreover, almost 60% of the respondents have heard about edible insects but do not
know them. Interestingly, those who intended to reduce their meat intake in the
coming year suggested insects as a replacement for fresh meat, but the general scores
of food neophobia were significantly higher than the scores of food technology
neophobia. The results confirm findings reported by other authors, i.e., in the case of
European consumers, food neophobia is a barrier for the consumption of insects.
Table 1 summarizes the acceptance of insects as alternative proteins among Western
consumers.
We can conclude that edible insects may be introduced to the diet of the Western
consumers in the future; yet, although the product design is important, it is insuffi-
cient to popularize insect consumption. Moreover, people’s knowledge about the
environment and positive aspects of edible insects as bioactive components of food
should be increased. In the future, edible insects may become such a delicacy for
European consumers as seafood, which in some European countries is a source of
disgust.
3 Examples of Popular Insect Species for Human

Consumption
The word insect derives from the Latin word “insectum,” meaning “with a notched
or divided body,” literally “cut into sections.” In the Online Etymological Dictionary
[55], we can also find: “The Latin word is Pliny’s loan-translation of Greek entomon
Table 1 Acceptance and methods of preparation of insects as alternative proteins among Western
consumers
Country, year of Method of Main research
study, and sample preparation question/intervention Provided information
Hungary, 2016, 400 No info Have you ever heard 89% of the
adult meat consumers about this alternative respondents know
[50] source of protein, and about the alternative
would you be protein sources. 60%
prepared to eat of the respondents
insects as a meat have heard about
substitute? insect consumption
and know what this
means. Insects can be
an alternative source
of protein for people
who want to reduce
meat consumption,
but neophobia is a
barrier
Italy, 2015, 109 Cookie with cricket Would you like to Almost all
people (53% female flour taste an edible insect? respondents tasted the
and 47% male), aged The reasons for the cookie and are willing
between 18 and decision to try other insects in
25 years old [49] the future. The
barriers include
negative opinions of
family members and
friends and the disgust
factor. Market
availability
(regulatory
framework), food
category (e.g., bakery
product with insect
flour) marketing
strategies, gastronomy
(preparation), culinary
trends, and education
have an influence on
insect consumption in
the future
German- and French- No info Predictors that are There are nine
speaking parts of currently used to significant variables
Switzerland, 2015, explain the reliably predicting
379 cases [36] willingness to the willingness to
consume insects consume insects:
convenience
orientation,
discernibility of
insects in food,
expected food
healthiness, need for
(continued)
Table 1 (continued)
familiarity, perceived
health benefits of
meat, food
technology
neophobia, food
neophobia, and
binary variables:
gender and prior
consumption. Food
neophobia was not
found to be the key
predictor of the
willingness to
consume insects but
shares its rank with
various predictors.
Further, the analysis
revealed one
significant two-way
interaction effect
Belgium, 2014, Burger with a Overall liking of the Females liked the
students interested mixture of beef, hybrid burger in beef burger better
in insect food, 79, lentils, and comparison with a than the burger
44% males, 56% mealworm beef burger containing
females [39] mealworms. Males
perceived no
significant difference
between the beef
burger and the
mealworm burger.
The major factors
influencing the
results were
knowledge of
entomophagy,
previous experience
with insect tasting,
and gender
Denmark and Italy, Insects Impact of individual Communication has
no info, 136 students and social benefits of an influence on the
from Denmark (61 eating insects on intention and
females), and 128 acceptance of insect- behavior and
from Italy (71 based food products. depends on the
females) [51] Effect of nation, gender, and
communication, also knowledge of
comparing messages entomophagy
based on individual Negative
versus societal associations with
benefits of insect insects have a strong
(continued)
Table 1 (continued)
consumption on the influence on
possibility to foster avoidance of
people’s willingness consumption of
to eat insect-based insect-based food
food
Netherlands, no info; Burger with “100% Consumer Unwillingness to eat
100 participants were ground beef,” “75% acceptance of new new food related
all occasional or ground beef and 25% food more with food
regular consumers of ground lamb brain,” appropriateness than
beef, whereas a “75% ground beef the actual taste of
minority claimed to and 25% ground frog meals. Consumers
have tasted the novel meat,” and “75% accept new
ingredients before ground beef and 25% ingredients in known
[52] ground mealworms.” foods with known
For brevity, these flavors
labels will be
mentioned as “beef
burger” and “novel
burgers”
Germany, 2014, 502 Insects as meat A study of The results indicated
adults [46] substitute, silkworm consumers’ that the willingness
(deep-fried), crickets willingness to eat to eat was
(deep-fried), different insect-based significantly higher
silkworm drink, processed and for processed food
cricket cookies, unprocessed food items such as drinks
cricket cookies choco and cookies than for
chip unprocessed food
items. The age of the
respondents has no
influence on the
willingness to eat
insects
Netherlands, no info, Eight mealworm How the product The respondents
976 adults from 18 to product images with preparation, accepted edible
94 years [47] differences: familiarity, and insects when these
mealworm visibility individual beliefs were added to
(visible/invisible), influence the familiar foods, and it
flavor (savory/sweet) consumer acceptance depended on the
carrier, and origin of insect-based food perceived
carrier (Western/ appropriateness of
Asian) mealworms as food
and the perceived
appropriateness of
the product
combination
Canada, no info, 134 Spring rolls filled A study of the first The disgust was
students from junior with carrots, celery, reaction to insects as reported, and “bug
high, high school, bean sprouts, sauce, a food idea banquets” were
and university [53] and whole cooked found to be the most
(continued)
Table 1 (continued)
crickets and effective way to
mealworms, roasted change attitudes
crickets and roasted toward insects as
seasoned food
mealworms, and
roasted mealworms
garnishing rice crispy
squares
Switzerland, 2015, Tortilla chips: made Could processed The results have
104, 55% men [54] with a traditional foods like cookies or shown that the
corn flour recipe and chips made with a positive experience
cricket flour as an small amount of with a processed
additional ingredient cricket flour be used insect product can
to increase the increase people’s
general acceptance of willingness to eat
insects as a food unprocessed insects.
source? The products with
edible insects as an
ingredient can taste
good, and processed
insect food may be a
first step to accept
insects as food by
western consumers
‘insect,’ which was Aristotle’s term for this class of life, in reference to their
‘notched’ bodies. First in English in 1601 in Holland’s translation of Pliny. In
zoology, in reference to a class of animals, 1753.”
Insects have a three-part body (head, thorax, and abdomen) with a chitinous
exoskeleton, three pairs of jointed legs, compound eyes, and two antennae. They are
among the most diverse groups of animals on the planet: there are more than one
million described species, which is more than half of all known living organisms.
Until recently, the total number of species is estimated at 6–10 million [56], but the
current estimates are up to 30 million [57]. Among the millions of insect species
living on earth, 2111 are considered as edible (Fig. 1) [58]. These facts show that insects
are an important source of protein in our diet. Members of Lepidoptera, Hemiptera,
Coleoptera, Diptera, Orthoptera, and Hymenoptera are the most commonly consumed
insects. The popularity of edible species varies among regions or cultures [59]. In
Europe, the consumption of insects is marginal, but the interest in insects is still
growing. Therefore, the Scientific Committee of the European Food Safety Authority
issued a scientific opinion “Risk profile related to production and consumption of
insects as food and feed” [60]. The report contains a list of insect species that were
found to have the biggest potential to be used as food and feed in the EU:
Fig. 1 Number of recorded

edible insect species per group
in the world [58]
Musca domestica: Common housefly

Hermetia illucens: Black soldier fly
Tenebrio molitor: Mealworm
Zophobas atratus: Giant mealworm
Alphitobius diaperinus: Lesser mealworm
Galleria mellonella: Greater wax moth
Achroia grisella: Lesser wax moth
Bombyx mori: Silkworm
Acheta domesticus: House cricket
Gryllodes sigillatus: Banded cricket
Locusta migratoria migratorioides: African migratory locust
Schistocerca americana: American grasshopper
At the same time, EFSA notes that this list does not need to be considered
definitive or exhaustive. This selection is undoubtedly supported by the large
number of scientific studies of these species.
3.1 Diptera
Flies (order Diptera) are a very popular insect order consumed by humans [58].
Musca domestica and Hermetia illucens are representatives of this order. They are
probably characterized by the largest reproductive capacity, shortest life cycles, and
rapid growth rates, which makes them one of the most attractive species of insects
for human consumption [61]. The black soldier fly (Hermetia illucens) can be used
commercially to solve a number of environmental problems associated with manure
and other organic waste, such as reducing manure mass, moisture content, and
offensive odors. At the same time, they provide high-value feedstuff for cattle, pig,
poultry, and fish [62]. H. illucens has become an object of interest to researchers who
work on the possibility of using this insect in animal feed [63–65]. It can also be used
in solving another problem: the increasing amount of food waste, which can cause
environmental pollution if not managed properly [66]. The black soldier fly exhibits
rapid food intake ranging from 25 to 500 mg of fresh matter/larva/day feeding on a
wide range of decaying organic materials, such as rotting fruits and vegetables, fish
offal, and animal manure and human excreta [67].
3.2 Coleoptera
Insects of this order are most commonly consumed in the world with 659
documented species being consumed (Fig. 1). Of the several edible groups within
this order, the most interesting is probably the family Tenebrionidae. The Tenebrio
molitor, Zophobas atratus, and Alphitobius diaperinus species listed by EFSA
belong to this family. Tenebrionids have a bad reputation as pests of meal, flour,
and other stored and packaged cereal foods, but, despite this, the yellow mealworm,
Tenebrio molitor, has been reared by zoos, aquaria, and commercial dealers as food
for animals since at least the eighteenth century [59]. Moreover, the mealworm
(T. molitor) is the subject of many studies. Its properties and nutritional value are
well known [4, 5, 14, 68–70]. The insect can be easily reared on many types of
vegetables and grains in a small space. There are even special devices for breeding
insects at home. A Livin Farms hive is a device that facilitates breeding of meal-
worms for the needs of your household. The system is divided into multiple
vertically stacked chambers beginning with pupae, placed into the topmost pupation
component, where they hatch into meal beetles (T. molitor). After the adult beetles
mate, they lay eggs that fall through into the egg layer where the mealworms are
hatched. Every week, the mealworms will be lowered into the next compartment
until they reach the sixth drawer for the weekly harvest. The mealworms can survive
on oats and vegetable scraps, while in-built smart systems help maintain ideal
microclimates for growth and remove foul odors. The weekly full harvest process
should take approximately 8–9 weeks for setup [71].
3.3 Lepidoptera
Caterpillars are the second most consumed insect order by humans, with 362
documented species being consumed (Fig. 1). Galleria mellonella, Achroia
grisella, and Bombyx mori are representatives of the EFSA list. They are eaten
in the larval form, and the largest size (up to about 60 mm) is reached by the
silkworm Bombyx mori. They are very high in fat but also in microelements such
as iron and zinc [68]. They are less popular for consumption in Europe, but they
are massively reared for the animal feed industry. Moreover, these larvae are the
subject of research on bioactive substances derived from their organism. Studies
on the antihypertensive properties of peptides from B. mori are being conducted
[72, 73]. B. mori and G. mellonella have been found to produce antimicrobial
peptides [74–77].
3.4 Orthoptera
Insects of this order such as grasshoppers, locusts, and crickets are known to all
insect-eating cultures. Two hundred seventy-eight species of crickets, grasshoppers,
and katydids are recorded as being consumed by humans (Fig. 1). Acrididae,
Gryllidae, and Tettigoniidae are the most commonly consumed insect families [61].
The house cricket Acheta domesticus is most readily available to Western con-
sumers. Studies have shown that, when kept at temperatures of 30 C or higher and
fed diets equal in quality to those used in bringing conventional livestock to market
condition, this cricket exhibits food conversion efficiency about twice as high as that
of broiler chickens and pigs. In addition, female crickets have much higher fecundity
than other animals. Each cricket lays 1200–1500 eggs over a period of 3–4 weeks
[59]. Another cricket Gryllodes sigillatus is also well known in Europe, for example,
as food for exotic pets. The tropical house cricket is probably native to Southwestern
Asia but has been spread by commerce to tropical regions worldwide [78]. Locusta
migratoria migratorioides and Schistocerca americana are members of the
Acrididae family. Despite the extensive practice of farming insects, these species
are more difficult in commercial breeding than the crickets mentioned above [18].
There is a prototype device for breeding crickets or grasshoppers at home. The
Lepsis is a vessel that can be used to grow insects for food. The product consists of
four individual units that are each designed to breed, grow, harvest, and kill
grasshoppers, and they combine to form a decorative kitchen product [79].
4 Nutritional Value of Edible Insects
The nutrient compositions of many edible insects were compiled and published in
numerous studies [80–85]. The nutritional values of insects are expressed as the
content of proteins (amino acid profile), fat, fibers, dietary energy, minerals, and
vitamins. The nutritional values of edible insects depend on many factors, e.g., the
origin of the insect, stage of metamorphosis, diet, and environmental factors (tem-
perature, humidity) [86]. Therefore, it is very difficult to generalize about the
nutritional value of the 2000 edible insect species.
4.1 Energy Content
The energy content in most edible insects is considerable due to the high levels of
protein and fat. The energy value depends on the stage of insects, i.e., larvae or pupae
are usually richer in energy than adults. Ramos-Elorduy et al. [87] analyzed 78 species
of insects and estimated their energy value in the range from 293 to 762 kcal/100 g.
On the basis of 113 literature reports, Rumpold and Schlüter [70] have found that
79.65% of insects are characterized by energy content above 400 kcal/100 g and
40.94% above 500 kcal/100 g. The mean energy content in edible insects is in the
range of 409.78–508.89 kcal/100 g (based on dry matter), with maximum energy
Table 2 Nutritional content of selected insect species compared with common protein
commodities
Protein
content (% in Energy content
Name (Latin name) dry mass) Fat (%) Fiber (%) (KCAL/100G)
Banded cricket (Gryllodes 70.00 [14] 18.23 [14] 3.65 [14] 452.00 [14]
sigillatus)
Black soldier fly 17.50–36.00 14.00 [80] 6.70 [89] 199.00 [80]
(Hermetia illucens) [81]
17.50 [80] 32.60 [90]
47.00 [89]
Common housefly (Musca 9.30 [89] 11.90 [89] – 552.40 [70]
domestica) 63.10–63.99 15.50–24.31
[70] [70]
Giant mealworm 41.50 [90] 36.20 [90] – –
(Zophobas atratus)
(Zophobas morio) 19.85–51.60 40.80–42.04 1.54–2.50 575.53 [70]
[81] [70] [81]
43.13–46.79 9.26–13.0
[70] [70]
46.80–55.16
[91]
Greater wax moth 15.39 [81] 51.40–60.00 8.92–19.52 650.13 [70]
(Galleria mellonella) 33.98–41.25 [70] [70]
[70]
House cricket (Acheta 7.50–23.70 2.41–6.71 1.18–4.03 135.10–170.90
domesticus) [80] [80] [81] [80]
15.60–73.60 9.80–24.00 6.20–22.08 414.41–455.19
[81] [70] [70] [70]
55.00–70.75 14.40–22.10 9.60–10.20
[70] [89] [89]
66.60–67.20
[89]
Lesser mealworm 48.60–60.00 8.50 [3] 4.40–9.10 –
(Alphitobius diaperinus) [81] [81]
61.70 [90] 24.30 [90]
29.00 [92]
Locust (Schistocerca 61.10 [70] 17.00 [70] 10.00 [70] 427.00 [70]
americana) (Schistocerca 76.00 [14] 12.97 [14] 2.53 [14] 432.00 [14]
gregaria)
Migratory locust (Locusta 62.20 [91] 12.61 23.61 364.70
migratoria) 13.00–28.00
f.m [18]
(continued)
Table 2 (continued)
Protein
content (% in Energy content
Name (Latin name) dry mass) Fat (%) Fiber (%) (KCAL/100G)
Mealworm (Tenebrio 11.42–30.38 5.40–19.94 1.39–21.00 130.00–482.00
molitor) [80] [83] [81] [80]
13.68–24.59 6.42–22.98 2.10 [83] 160.00–283.00
[83] [80] [83]
14.00–25.00 14.88–43.08 5.00–20.22 379.61–577.44
f.m. [18] [70] [70] [70]
20.00–68.60 25.00 [90]
[81]
47.00–65.29
[70]
Silkworm (Bombyx mori) 10.00–17.00 5.62–14.78 2–6.39 [70] 120.52–135.48
f.m. [18] [80] [80]
10.14–25.66
[80]
17.90–22.89
[81]
48.70–69.84
[70]
53.80–64.70 8.09–37.10 6.40 [89] 389.60–555.00
[89] [89] [70]
Commonly consumed protein source
Egg, whole, dried 12.56 [93] 9.51 [93] – 592.00 [93]
Beef, grass-fed, ground, 19.42 [93] 12.73 [93] – 198.00 [93]
raw
Chicken ground, raw 17.44 [93] 8.10 [93] – 143.00 [93]
Soybeans, mature seeds, 28.70–50.10 19.94 [93] 9.30 [104] 403.00–446.00
raw (Glycine max) [94] – 11.90 [94] [93, 94]
Broad beans, mature 22.00–38.20 1.53 [93] 25.00 [93] 320.00–341.00
seeds, raw (Vicia faba) [94] [93, 94]
Lentils/raw (Lens 24.63 [93] 1.06 [93] 10.70 [93] 352.00 [93]
culinaris)
Wheat, durum (Triticum 13.68 [93] 2.47 [93] 10.70 [93] 339.00 [93]
durum)
contents as high as 762.00–776.85 kcal/100 g in moth Phassus triangularis [70, 88].

Among the species of edible insects listed in Table 2, the maximum energy contents
was found in the greater wax moth (650.13 kcal/100 g) [10], and the minimum value
was estimated in the silkworm (B. mori) 128 7.48 kcal/100 g [80]. Evidently, the
energy content in most edible insects is high, even in comparison to meat.
4.2 Protein and Essential Amino Acid Profile
In recent years the protein content is a leading trend in the development of food
products. Protein is a very important basic macronutrient for humans, as it plays
many functions in the body and, above all, is a source of essential amino acids and
energy. The growing desire for unconventional non-animal-based protein sources
has led to an interest in pulses and insect-based protein. As a rich protein source,
edible insects are alternatives to meats within international dietary guidelines. The
total protein content of insect species is presented in Table 2.
The protein content of most edible insects has been shown in many studies to be
within the range of 13–77% [81, 82] or 9.3–80% [89, 95]. The protein level in
insects depends on many factors, e.g., their metamorphosis stages, feeding mixtures
used, varying water content, methods of preparation and processing applied before
consumption of insects (e.g., drying, boiling, or frying), and primarily methods used
from protein determination (Dumas technique or Kjeldahl method) [89, 90, 95].
Many studies have reported analysis of the nutritional value of edible insects;
however, these data are not always comparable due to the variations between insects
and because of the varying methodologies employed to analyze the compounds. The
Kjeldahl method is widely used for determination of the crude protein content in
food [18]. Therefore, many scientists use this method in their studies devoted to
insects. This procedure evaluates the total concentration of nitrogen (N), which is
converted to protein by multiplying it by the nitrogen-to-protein conversion factor of
6.25. Because insects contain many N-rich compounds that are not digested by
humans (chitin and proteins tightly embedded in its matrix), the Kjeldahl method can
overestimate the content of digestible protein. In view of the above, Jonas-Levi and
Martinez [81] proposed evaluation of digestible nitrogen by quantifying N in the
cuticle and subtracting it from the total nitrogen content and calculation of a new
N-conversion factor, which should be similar for all insect species and their devel-
opment stages.
The highest percentage of protein (80%) was found in the adult stage of the gypsy
moth (Porthetria dispar) and (78.8%) in the German cockroach (Blattella
germanica) [88]. Among insects recommended by EFSA, the cricket (Acheta
domesticus) was characterized by the highest protein content (73.60%) [81].
Comparison of the conventional protein source in human nutrition with insect
protein demonstrates that the content of proteins in many insects is comparable to
that in beef or chicken meat. As shown in Table 2, some insects are comparable to
eggs, meat, and plants. Moreover, insects are richer in protein than cereals, e.g.,
wheat 13.68%, pseudocereals: buckwheat 13.1% and amaranth 13.5% [96] and
legume seeds including dried peas (Pisum sativum) 14.2–36.1%, chickpeas (Cicer
arietinum)19.1–31.2%, beans (Phaseolus vulgaris) 15.2–36.0%, and soybeans (Gly-
cine max) 28.7–50.1% [94]. Finke et al. [97] investigated insects as a source of
protein used for feeding rats. They observed that the amino acid composition in
cricket protein (Acheta domesticus and Anabrus simplex) was equal or better than
that in soy protein. By contrast, silkworm protein showed a significant lower quality
than casein.
A major consideration with respect to the inclusion of insects in food products

relates to the quality of their dietary protein. Protein quality depends on the kind of
amino acids present in their sequence (essential or nonessential) and protein digest-
ibility. However, the quality of insect proteins in comparison to other animal and
plant proteins has to be assessed through the amino acid requirements in humans.
The most important aspect and characteristic of protein from a nutritional standpoint
is its amino acid composition. Phenylalanine, valine, threonine, tryptophan, isoleu-
cine, methionine, leucine, and lysine are classified as essential amino acids, and
histidine is a semi-essential amino acid.
Cereal proteins that are major components in human diets and animal feed
worldwide are rich in sulfur amino acids, but they are poor in lysine, tryptophan,
and threonine (e.g., maize) [98–100]. Similar to cereals, legumes (soybean, pea,
chickpea, lentil, beans) contain high amounts of essential amino acids such as
arginine, leucine, and lysine but are poor in sulfur-containing amino acids (particu-
larly methionine) and tryptophan [31].
The amino acid spectra in edible insect have been shown in many studies [70, 80,
89, 101–103]. Analysis of many edible insects has revealed that their essential amino
acid content is 10–30%, covering 35–50% of all types of amino acids, which is close
to the amino acid model proposed by the World Health Organization and FAO [89].
In some insect species, essential amino acids are very well represented [104], while
in others they are limited. It was reported in literature that insect proteins were low
in the Met þ Cys [103] and high in Lys, Trp and Thr [31], or Phe [105]. As
demonstrated by Williams et al. [89], Trp is a limited amino acid in ants, bee (Apis
mellifera), and silkworm (B. mori), Ile in caterpillar, and Lys in termites. The
essential amino acids (EAA) of selected insects compared to plant and animal
protein sources are shown in Table 3. The giant mealworm (Z. atratus) protein
is low in Met, silkworm (B. mori) in Met and Phe, and common housefly
(M. domestica) in Ile.
Generally, proteins derived from animal foods (meats, milk, eggs) are complete,
but protein malnutrition continues to be a problem in many countries around the
world. Insects are a promising alternative source for nutritional and functional
proteins. Insects are characterized by high protein quality with beneficial potential
for human nutrition. The great number of investigations on the health benefits of
edible insects associated with consumption thereof increase interest in developing
innovative technologies to expand the use of insects in food or feed products. Apart
from their nutritional properties, proteins from edible insects possess functional
properties that play an important role in food formulation and processing [18].
4.3 Lipids and Essential Fatty Acid Profile
Fats are one of the three main macronutrients, along with carbohydrates and protein.
Dietary consumption of fatty acids has an impact on human health. Some insects are
rich in fat, in a wide range from 4.56% to 60% (Table 2). The fat content is higher in
408
Table 3 Essential amino acid (EAA) content of selected insect species compared with common protein commodities [70, 93]
Summary of
the adult
EAA
Common requirements
Egg, Soybeans House cricket Giant housefly FAO/WHO/
whole, mature (Acheta Mealworm mealworm (Musca UNU (2017)
raw, Wheat, seeds. Silkworm domesticus) (Tenebrio (Zophobas domestica) [mg/g
EAA [mg/g protein] fresh durum Raw (Bombyx mori) (adults) molitor) morio) (larvae) protein]
Essential His 24.60 23.54 30.06 25.8–29.5 22.7–23.4 28.7–37.9 30.5 30.9 15
amino Ile 53.42 38.96 54.01 32.3–33 36.4–45.9 43.5–50.3 47.2 22.8 30
acids Leu 86.46 68.27 90.68 48.9–52.7 66.7–100 82.2–106.4 97 45.3 59
Lys 72.61 22.15 74.16 47.3–50 51.1–53.7 44.3–64.9 52.3 81.6 45
Met 30.25 16.15 14.99 12.5–14 14.6–19.6 12.7–19.5 10.7 36.6 16
Cys 21.66 20.91 17.95 8.6–9.1 8.33–9.8 6.8–10.9 7.6 6.6 6
Phe 54.14 49.78 58.15 28.4–29 31.7–30.2 26.2–43.7 34.5 55.8 30
Thr 44.27 26.75 48.40 28.4–31.2 6.3–7.6 34.2–41.8 39.6 35.5 23
Trp 13.30 12.87 16.20 6.8–7.5 31.1–36.1 8.0–11.0 9.1 49.5 6
Val 68.31 43.42 55.60 39.8–40.9 48.4–52.2 58.8–69.0 52.3 45.6 39
Total 469.03 322.807 460.21 278.80–296.90 317.33–378.50 345.4–455.4 380.8 410.2 269
EAA
E. Zielińska et al.
the larval stage than in the adult stage [82]. Larvae of the greater wax moth (~60%)
and mealworm (~43%) exhibit the highest amounts of fat [10, 90, 98, 100].
Lipids are a source of not only energy but also essential fatty acids (FA). Many
studies have found that replacing saturated fatty acids (SFA) with monounsaturated
(MUFA) and polyunsaturated fatty acids (PUFA) in the diet reduces the risk of
cardiovascular diseases. The evaluation of fat quality is a complex issue, since SFAs
elevate the LDL cholesterol concentration in serum, while MUFAs and PUFAs (in
particular, those from the n-6 family) have been shown to decrease LDL cholesterol
concentrations. Therefore, it is recommended that SFA should be replaced with PUFA
(n-3 and n-6) in the diet, and the total intake of SFA should not exceed 10% of energy
[106]. The fatty acids of many insects contain more PUFA and are comparable to those
of poultry and fish in their degree of unsaturation [70, 104]. In contrast, beef and pork
contain very low levels of PUFA but high content of MUFA [107]. The highest MUFA
proportion was found in beetle larvae, while PUFA levels were determined in adult
crickets [102]. The ratio of fatty acids recommended for human nutrition is SFA/MUFA/
PUFA 1.25:1.5:1. The ratio found in Zophobas morio by Kourimska et al. [105] was
1.9:1.4:1. The determined MUFA/PUFA ratio meets the requirements for human
consumption (1.4:1), but the amount of SFA is significantly higher. Similar results
were obtained by Zielińska et al. [14] (1.34:1.4:1.1) for Schistocerca gregaria. In turn,
Tzompa-Sosa et al. [11] obtained a lower ratio (1.4:1.6:1) in the case of this species. The
amount of SFA in the mealworm (T. molitor) was significantly lower. This is confirmed
by the results obtained by Zielińska et al. (2015) (1:1.7:1.2). Tzompa-Sosa et al. [11]
obtained a significant content of MUFA (1.1:2.3:1), whereas Bednářová et al. [91]
reported a greater amount of PUFA (0.7:0.8:1). A greater amount of PUFA was
determined by Zielińska et al. [14] in the case of Gryllodes sigillatus (1:1:1), but the
amount of SFA and MUFA was considerably lower than recommended. The quality and
quantity of fatty acids in foods are very important from the nutritional point of view. The
fatty acid composition in edible insects has been summarized in many studies [14, 68,
70]. The differences between reported values of fat contents in many studies may have
been associated with the variety of insects and with changing the insects’ feed compo-
sition [70, 95, 108]. The main MUFA detected in edible insects are palmitoleic acid and
oleic acid. The silkworm larva is a potential source of such fatty acids as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are important
to support a healthy cardiovascular system [109]. The highest content of essential
α-linolenic acid and oleic acid was determined in T. molitor larvae and A. domesticus
[11, 102, 110]. Therefore, the mealworm could be the most suitable insect of all
analyzed species for human consumption. PUFA that can be found in the fatty acid
spectra of edible insects are represented by linoleic, linolenic, arachidonic, and
eicosapentaenoic acids [70, 92, 103].
4.4 Fiber Content
The fiber content of edible insects was investigated in many studies and referred to as
crude fiber (CF), acid detergent fiber (ADF), and neutral detergent fiber (NDF)
[111–113]. According to Rumpold and Schlüter [70], the average fiber content in
edible insects ranges from 5.06% (termites) to 13.56% (true bugs). The average fiber
content in the species of edible insects listed in Table 2 ranges from 2% (silkworm)
to 22.08% (house cricket). The differences in the fiber content likely result from the
presence of different compounds that are bound to chitin. The exoskeleton of edible
insects contains a significant amount of different compounds, including chitin and
substances that are bound to chitin (e.g., protein, lipids, and other compounds) [111,
114]. Moreover, Finke [111] suggests that insects with harder cuticles do not
necessarily contain more chitin than softer-bodied insects, but rather they contain
higher levels of cross-linking proteins that are essential for sclerotization. Chitin is
considered as an indigestible fiber by humans, because chitinase found in human
gastric juices is inactive [115]. However, it was found that this enzyme is active in
the organisms of inhabitants of tropical countries where the consumption of insects
has a long-term tradition [18, 89, 105]. Removal of chitin improves the digestibility
of insect protein [111].
Chitin has been associated with defense against parasitic infections and some
allergic conditions; it acts as an anticoagulant and protects against certain pathogens
in the blood and skin. The positive activity of chitin involves significant reduction of
serum cholesterol, functions as a hemostatic agent for tissue repair, enhancement of
burn and wound healing, enhancement of pollutant removal from waste-water
effluent, improvement of the washability and antistatic nature of textiles, inhibition
of growth of pathogenic soil fungi and nematodes, and an increase in wheat, barley,
oat, and pea yields by as much as 20% [116–118].
4.5 Microelements
Many edible insects are a good source of macro- and micronutrients that are
necessary for a healthy organism [40, 119]. As shown in many studies, edible insects
are a rich source of phosphorus (P), magnesium (Mg), potassium (K), sodium (Na),
calcium (Ca), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), and selenium (Se).
Therefore, edible insects can supply necessary nutritive elements for human body
functions. Very important is the supply of Fe and Zn in the diet, given the worldwide
deficiencies in these minerals among humans. There are nutritional studies on the use
of insects (caterpillar cereal) in prevention of diseases associated with iron defi-
ciency diseases such as anemia and stunting [120, 121]. Most edible insects have
equal or higher iron contents than beef [104]. An excellent source of iron are mopane
caterpillar (Gonimbrasia belina) (31–77 mg/100 g) and locusts (8–20 mg/100 g DM),
while the iron content of the beef is 6 mg/100 g [122]. Zinc content in beef is on
average 12.5 mg/100 g, while house cricket (A. domesticus), for example, contains
29.69 mg/100 g, chapulin (Sphenarium histrio) 78 mg/100 g, and house fly pupa (M.
domestica) 85.8 mg/100 [70, 89]. Generally, edible insects are low in sodium (except
caterpillar Usta terpsichore) and calcium (except M. domestica and A. domesticus)
[70]. The composition of macro- and micronutrients largely depends on the food
consumed by the insect. The macro- and microelements can be contained in the
consumed food present in the gastrointestinal tract or in components incorporated in

the insect’s body [89, 113, 123]. The levels of these nutrients vary considerably
between species. Rumpold and Schlüter [70] concluded that edible insects generally
lack sufficient amounts of Ca and K. In contrast, Gosh et al. [102] have shown that
the calcium content in all studied insects was much higher than that in conventional
foods of animal origin except chicken eggs. Edible insects have the potential to
provide specific micronutrients such as Cu, Fe, Mg, Mn, P, Se, and Zn, and, in
addition, they can be utilized in low-sodium diets. Nowak et al. [83] compared a high
number of insect species and found that the mineral content in insects exhibited
extreme variability within and between species. According to Oonincx and
Dierenfeld [124], the high variability in microelements is a result of a small sample
size, species-specific metabolism, varying accuracy of sampling and/or analytical
techniques, and contamination.
4.6 Vitamins
Edible insects are also a source of vitamins, which are essential for normal growth
and development of a living organism by stimulating metabolic processes and
enhancing immune system functions. Due to the limited data about vitamins in
insects, it cannot be indicated which insects are a good source of vitamins. Never-
theless, Bukkens [104] has shown that the level of vitamin B1 in a whole range of
insects is 0.1–4 mg/100 g and B2 – 0.11–8.9 mg/100 g; in comparison, whole-meal
bread provides 0.16 mg and 0.19 mg/100 g of these vitamins, respectively.
Rumpold and Schlüter [70] have demonstrated that edible insects contain a whole
range of water-soluble vitamins, B1 (thiamine), B2 (riboflavin), B3 (niacin), B5
(pantothenic acid), B6 (pyridoxine), B7 (biotin), B9 (folate), and C (ascorbic acid),
and fat-soluble vitamins, A (retinol) and E (tocopherols). By comparison of the
vitamin content in insects with the vitamin requirements in human nutrition of
adults, Rumpold and Schlüter [70] noted that edible insects are generally rich in
riboflavin, pantothenic acid, and biotin. In turn, Nowak et al. [83] collected 56
literature reports about vitamin content in edible insects and found that mealworm
can be a good source of vitamin B6 (pyridoxine), B2 (riboflavin), B3 (niacin),
B9 (folate), and B12 (cobalamin). Caterpillars are especially rich in B1, B2, and B6
[82, 125].
Wakayama et al. [126] noted that many insect species were characterized by low
levels of vitamin B12 (cyanocobalamin). It is present only in animal-origin food and
is well represented in mealworm larvae (T. molitor) (0.47 μg/100 g) and house
crickets (A. domesticus) (5.4 μg/100 g in adults and 8.7 μg/100 g in nymphs).
According to van Huis [122], Williams et al. [89], and Rumpold and Schlüter [70],
insects are generally a good source of some vitamins B but not A, C, B3, and B1. The
content of vitamin A (as retinol or β-carotene) was less than 2 μg/100 g and less than
100 μg/100 g in yellow mealworm larvae, superworms, and house crickets [68, 104,
127]. Compared to commercially bred insects, those captured in the wild contain
higher levels of carotenoids, such as astaxanthin, α- and β-carotene, lutein, or
zeaxanthin; this is associated with their diet, which is a richer source of these
compounds. The high level of carotenoids in insects may be a promising source of
vitamin A in human diet [122].
There are insufficient data on the content of vitamin E in insects. According to
Bukkens [104], palm weevil larvae (Rhynchophorus ferrugineus) had 35 mg and
9 mg/100 g of α-tocopherol and βþγ tocopherol, respectively (the recommended
daily intake – 15 mg). A relatively high level of vitamin E, 9.65 mg/100 g, was
determined by Tong et al. [128] in freeze-dried silkworm powder (B. mori).
In conclusion, since the literature provides only a few data on insect vitamin
content, more research is needed to identify edible insects as a source of vitamins.
Moreover, Payene et al. [80] observed that the contents of minerals and vitamins
varied greatly between insect species; this was suggested to be due to soil contam-
ination of samples and variation in the diet of the insects.
Although significant variation was found in the data, many edible insects provide
satisfactory amounts of energy and protein; they are also rich in monounsaturated
and/or polyunsaturated fatty acids and amino acids, micronutrients (copper, iron,
magnesium, manganese, phosphorous, selenium, and zinc), vitamins B2–12, carot-
enoids, and bioactive substances, which are essential for humans.
4.7 Bioactive Substances
Insects and bioactive substances extracted from them have been used in unconven-
tional medicine by human cultures all over the world. Insects constitute an almost
inexhaustible source of compounds for scientific research. A wide range of edible
insects is a source of many bioactive components (polyphenols, enzymes, peptides/
proteins) [116, 129, 130].
Nongonierma and FitzGerald [131] focused on studies published between 2005 and
2017 and analyzed the literature on the generation of bioactive peptides (BAPs) from
edible insect proteins following enzymatic hydrolysis. Different enzyme preparations
(Alcalase™ 2.4 L, Flavourzyme™, Protamex™, papain, trypsin, and pepsin) were used
during hydrolysis of insect proteins to obtain bioactive peptides. The authors suggested
that silkworm (B. mori) is currently the most widely studied species. Generally, the
peptides obtained by enzymatic hydrolysis of edible insect proteins showed angiotensin-
converting enzyme (ACE) inhibitory activity and, hence, antioxidant and antidiabetic
properties. Moreover, selected species of edible insects studied by Zielińska et al. [12,
13] have high antiradical activity and an ability to chelate iron ions. They can inhibit
lipoxygenase and cyclooxygenase-2 activity after the digestion and absorption process.
Moreover, the heat treatment process positively affects the antioxidant properties of
peptides derived from these species.
A majority of studies were focused on insect protein-derived peptides with ACE
inhibitory activity. Vercruysse et al. [132–134] were one of the first authors pre-
senting on ACE inhibitory activity of protein hydrolyzates from insects (B. mori, B.
terrestris, S. gregaria, and S. littoralis). Dai et al. [135] investigated an angiotensin I-
converting enzyme (ACE) inhibitor peptide (Tyr-Ala-Asn) derived from T. molitor
larva protein hydrolyzate. Wang et al. [136–138] examined antihypertensive prop-

erties of a novel peptide (Ala-Ser-Leu) obtained from silkworm pupa protein on
spontaneously hypertensive rats and the effect of these peptides on the fermentation
and quality of yogurt. The peptides, Ala-Pro-Pro-Pro-Lys-Lys, Val-Glu-Ile-Ser, Lys-
His-Val, and Gly-Asn-Pro-Trp-Met, were identified in B. mori protein hydrolyzed by
Wang et al. [138], Li et al. [139], Jia [140], and Tao [141], respectively. In their study,
Wu et al. [142] investigated a silkworm larvae protein isolate (SLPI) as a source of
bioactive peptides. The SLPI hydrolyzate exhibited strong angiotensin I-converting
enzyme (ACE) inhibitory activity and antioxidant activity. The second large group
of peptides comprises antioxidant peptides obtained by proteolysis of insect proteins
with the use of different enzymes [12, 13, 132]. There are only few investigations
evaluating the α-glucosidase inhibitory activity of insect protein hydrolyzates.
However, Zhang et al. [143] used a quantitative structure-activity relationship
(QSAR) approach for prediction of α-glucosidase inhibitory activity of peptides
from B. mori proteins. Moreover, alkaloids, flavonoids, anthraquinones, tannins,
phlobatannins, steroids, triterpenoids, and cyanogen glycosides were detected in
hydrophilic and lipophilic extracts from Encosternum delegorguei (Hemiptera:
Tessaratomidae) hydrophilic and lipophilic extracts [130]. These extracts were
characterized by high levels of flavonoids and free radical scavenging activity, but
the potential trade-off from the elevated levels of cyanogen glycosides after pro-
cessing needs further investigation.
Peptides obtained from insects (Fig. 2) also have antibacterial and antifungal
properties. A few antifungal compounds have been found in insects, for example,
termicin from termites, drosomycin from Drosophila melanogaster, heliomicin
from the tobacco budworm (Heliothis virescens), and the gallerimycin peptide
from greater wax moth (G. mellonella) larvae [144]. The extract from housefly
larvae possesses broad antibacterial activity against both Gram-negative and
Gram-positive bacteria [145]. T. molitor produces antimicrobial and antifungal
tenecin 4 [146].
In turn, Chinese black ants contain compounds with anti-inflammatory, immu-
nosuppressive, and renoprotective activities [147]. Protein fractions extracted
and purified from housefly larvae have antiviral and antitumor activities [148].
Elpidina and Goptar [149] reported that proteinases from T. molitor presumably
have potential for oral administration to treat celiac disease. This insect contains a
wide spectrum of digestive proteinases operating in the midgut with a sharp pH
gradient from acid to alkaline values. Two digestive peptidases from the acidic
AM, i.e., cysteine cathepsin L and post-proline cleaving peptidase, readily
hydrolyze bonds formed by the major amino acids of cereal gluten – proline
and glutamine.
In summary, potentially bioactive peptides derived from insect proteins and
phenolic compounds have been identified (Fig. 3). Many of these peptides had
interesting activities (mainly antihypertensive) even in small animals. The bio-
active potency of edible insect protein hydrolyzates/peptides has been shown by
Nongonierma and FitzGerald [131] to be similar or higher than that of other
dietary proteins (plants and animals).
Fig. 2 Method for obtaining the bioactive peptides from edible insect proteins
Fig. 3 Edible insects as a source of bioactive peptides

Fig. 4 Insect flour and extracted insect protein (flour and extracted protein from cricket G.
sigillatus and flour and extracted protein from mealworm T. molitor, respectively)
5 Processing Edible Insects for Food
In tropical countries, whole insects are often consumed after a suitable thermal
process. However, Western consumers may be reluctant to accept this form. Some
consumers are not convinced to eat insects because of the bad associations with their
appearance. It is easier to accept products where the insect additive is not visible [41,
45]. In typical western societies, consumers find insects more appealing when used
as an ingredient of foods with familiar flavors and textures [150]. There are many
possibilities of using insects in an invisible form, for example, grinding insects to so-
called insect flour or extraction of the main insect ingredients such as protein, fat, and
chitin (Fig. 4).
5.1 Insect Flour
Cricket flour is the most popular insect product in Europe because of its versatility
[151]. The addition of the flour to traditionally eaten foods is a great way to introduce
insects into our diet without altering our habits. The powder form seems to be ideal
for use in the food industry as it can be used for all kinds of products without
changing their textural or functional properties; moreover, this additive can improve
the products [152, 153]. Furthermore, the process of flour production is not compli-
cated and, hence, cost-effective.
The easiest way to make flour from insects is to dry them first because of their
high moisture content [68]. Drying is important because it reduces not only moisture
but also water activity in the product, which is important in microbiological terms.
Insects can be dried in industrial driers or lyophilized, which is probably the best
possible method for preserving the chemical and other properties of food (Fig. 5).
Reduced water content in the finished product guarantees longer storage time, but
this is not the only advantage of producing insect flour. Milling the whole insects
retains all their components in the flour, i.e., micronutrients or chitin in addition to
proteins.
Fig. 5 The process of freeze-

drying edible insects
5.2 Extracted Insect Protein
Supplementation of food products (e.g., meat products) with legume protein is a

known practice. Extraction of insect proteins to increase the protein content in a food
product could be a useful way of increasing acceptability among wary consumers. In
this case, a small step method is a good idea. The gradual introduction of insect food
proteins will allow consumers to get acquainted with the new product that at first
sight is not different from what is known to them. However, supplementation of food
products with insect ingredients (e.g., proteins) requires extensive research of their
properties. A good understanding of the properties of proteins will allow specific
application in food production according to the potential shown. Proper use of
extracted proteins will make it possible to create the desired properties of food
without altering the taste or smell of products that are well known to consumers.
Extracted proteins may also be part of the diet of individuals with increased
protein requirements such as sportsmen. Importantly, insect protein contains a set of
essential amino acids and high content of BCAA (branched-chain amino acids) [83].
Protein isolates can be an ingredient of nutrition supplements or protein shakes – a
process already being carried out [154, 155].
5.3 Extracted Fat
Fat can be extracted from insects during flour production. This treatment prevents
unsaturated fatty acids from exposure to undesirable oxidation processes; how-
ever, fat is not an unnecessary by-product. Oils are essential to the food industry
for many uses, and oils from insects are valuable due to the composition of fatty
acids. Many species of insects are high in unsaturated fatty acids including
omega-3 fatty acids with a desirable omega-6 to omega-3 fatty acid (n6/n3)
ratio [14, 156]. Oils from different species, and species fed on different diets,
can have different fatty acids profiles. By changing insects’ diet, oils with the
desired composition of fatty acids can be received [11]. Oils obtained from
insects can be widely used in the food industry: from production of salad
dressings or supplements of traditionally consumed dishes to attempts at replace-
ment of fats used conventionally in food technology.
5.4 Chitin
Chitin seems to be a by-product of flour production, but, in fact, it is a high-value

product. Primarily, chitin acts as a dietary fiber but this is not the only function.
Chitin and chitosan (produced commercially by deacetylation of chitin) are natural
products that are compatible with plant and animal tissues, biologically functional,
biodegradable, nontoxic, and eco-friendly [7, 157]. Due to these properties, chitin
and its derivatives have many application areas including wastewater treatment,
pharmacy, medicine, cosmetics, weight loss, edible biofilm production, and reduc-
tion of low-density lipoprotein (LDL) cholesterol levels in the blood. The vast
majority of chitin currently used for these applications comes from unsustainable
harvesting of shrimp which, like much of the ocean’s other resources, is constantly
being overharvested [151]. Moreover, chitin has shown antioxidant, antimicrobial,
and antitumor potential [7, 158, 159]. The surface morphology, acetylation degree,
and molecular weight are the three main criteria determining the industrial use of
chitin and its derivatives [7, 157].
5.5 Functional Properties of Insect Protein
Supplementation of food products with insect flour or extracted ingredients requires

extensive knowledge of their properties. In the case of proteins, these properties include,
among others, solubility, thermal stability, and techno-functional properties such as
water and oil holding capacity, gelling, foaming, and emulsifying capacity. These
properties determine the strict application of insect flour or protein isolates in food
products. Furthermore, enzymatic modification of proteins is a useful mechanism to
improve the functionality compared with the native unhydrolyzed proteins [152].
Good solubility of proteins is important in many uses, mainly for formation of
emulsions, foams, and gels in designed food products. Zhao et al. [160] observed
that the lowest protein solubility for proteins from T. molitor was reached around the
isoelectric point and increased with either increasing or decreasing pH. Good
solubility of insect protein in a wide pH range ensures varied use thereof in food
industry – as a food additive in acid and alkaline food. Yi et al. [3] investigated the
techno-functional properties of proteins from five insect species: Tenebrio molitor
(larvae), Zophobas morio (larvae), Alphitobius diaperinus (larvae), Acheta
domesticus (adult), and Blaptica dubia (adult). It was found that the investigated
insect proteins had the ability to form gels depending on their concentration and on
pH and could potentially be used as gelling agents or texturizers in food. Omotoso
[153] studied the functional properties of Cirina forda (Lepidoptera: Saturniidae) –
one of the most widely eaten insects in Southern Nigeria. The water holding capacity
of dried ground insect was 300%, whereas the water holding capacity values for
silkworm (Bombyx mori) larvae and pupae were 175% and 115%, respectively.
Acid-extracted protein fractions from five different insect species, including cricket
(Acheta domesticus) protein or mealworm (Tenebrio molitor) protein, were shown to
have poor or no foam capacity, over a range of pH [3]. In turn, crickets (G. sigillatus)
heated at 50 C for 30 min were found to have a FC of 100% with a FS of 90% after
1 h [152].
Hall et al. [152] demonstrated that controlled enzymatic hydrolysis of whole
crickets successfully yielded protein hydrolyzates with improved protein function-
ality. Improved solubility of hydrolyzates makes them suitable for acidic food
systems, such as nutritional beverages and sports drinks. The authors also observed
better emulsifying and foaming properties for cricket protein hydrolyzates than the
unhydrolyzed cricket protein.
These are only a few examples of the functional properties of insects. Due to the
biodiversity of insects, these properties can vary considerably; therefore, it is
important to investigate the properties of insect proteins before using them in food
products.
6 Why Should We Look for New Sources of Nutrients?
It is estimated that around 7.6 billion people live in the world, and the urban
population has been constantly increasing by approximately 1.84% per year
between 2015 and 2020. To maintain proper health and functioning of the
organism, it is necessary to have access to safe food rich in nutrients [161]. It is
estimated that the human population in 2050 will amount to nine billion, and the
demand for meat will increase by 76%. Therefore, the issues of food quantity and
safety as well as the quality of food, feed, and fuel and environmental protection
have become a priority for policy makers [162–164]. In order to achieve sustain-
able development and stability of food markets, the impact of all human activities
on the environment should be reduced. Therefore, it is necessary to change and
improve the technology of food production and the approach of consumers and
food producers in a stepwise manner. The International Human Dimensions
Programme identified food, water, and energy as the most important targets of
quality change [165]. It should be noted that these main activities are mutually
independent, since food production is dependent on freshwater resources and
energy production [166]. Given the impact of animal production on the environ-
ment, e.g., soil erosion, deforestation, greenhouse gas emission, and water
pollution, increasing production does not seem to be a good solution for the
protein amount requirements in the future. Moreover, the prices of soybean and
fishmeal that are used in feeding due to the demand are still increasing [17].
Therefore, we are looking for alternative sources of protein and nutrients.
As edible insects are a diverse food and feed market, they can become an
alternative source of protein, and the main reasons why the insects can be an
alternative for lamb, beef, poultry, and pork are the economic, environmental,
and nutritional aspects. Nevertheless, the European Novel Food Regulation pro-
hibits the production and processing of edible insects on a commercial scale for
humans [164].
7 Environmental Impact of Entomophagy
Conversion of plant protein to animal protein is naturally inefficient (6 kg of

vegetable protein is needed to obtain 1 kg of meat protein) and responsible for the
negative influence on the environmental condition [167]. Furthermore, only 15% of
protein and energy are applied as human food, and 85% are wasted. In 2000, 942 and
617 million tons of grains were used for food and feed, respectively [168]. It is worth
noting that this is more than 500 tons of wasted food production and turned into
polluting emissions by animal metabolism. The actual conversion efficiency of
protein depends on the animal type and breed and the conditions of farming, such
as climate and feed. Poultry and pigs are more productive species of animals than
cows if they are grass-fed. Moreover, their compound stomachs are not adapted to
feeding with maize. Furthermore, the use of water, amount of feeding stuff, and
influence on the environment (water pollution or gas emission) depend on the type of
animal protein production. For example, to produce 1 kg of beef, pork, and chicken
meat, 15,400 l, 6000 l, and 3400 l of water, respectively, need to be used [169]. It
should be noted that generally 1 kg of animal protein requires about 100 times more
water than 1 kg of grain protein [167]. It should be noted that generally 1 kg of animal
protein requires about 100 times more water than 1 kg of grain protein [167]. Since
water is the main part of feed and occupies a particular place in food production, the
low energy conversion efficiency, especially in the case of cattle, is obvious. In
general, insects are characterized by a lower feed conversion rate (FCR), which is
defined as the amount of feed required to grow 1 kg of conventional livestock
animals, which corresponds to high feed efficiency. To produce 1 kg of beef, lamb,
pork, and chicken meat, 7.7, 6.3, 3.6, and 2.2 kg of feed are required [170]. In
comparison, 1.7 kg of feed is needed to obtain 1 kg of cricket meat [171]. Further,
2.2 kg of feed are used production for 1 kg of mealworm [172], 2.7 kg of feed for the
Argentinean cockroach, 1.8 kg of feed for the black soldier fly, and 2.3 kg of feed for
the house cricket [173]. This factor depends on the mode of feeding.
It should be noted that production of edible insects has a lower negative influence
on the environment than breeding conventional livestock animals. Production of
greenhouse gases (GHS), i.e., carbon dioxide (CO2), methane (CH4), and nitrous
oxide (N2O), is said to be an important cause of climate change. The latter two gases
are considered as the key global warming factors [16]. According to the life cycle
analysis (LCA) which takes into account all animal production, the contribution of
the animal sector to global greenhouse gas emissions is 9% for CO2 (on-farm energy,
animal product processing expenditures, feed and animal transport, fertilizer pro-
duction for feed crops, and land-use changes), 35–40% for CH4 (enteric fermenta-
tion in ruminants and farm animal manure), and 65% for N2O (farm manure and
urine) [174]. A relevant indicator of the environmental impact is emission of

ammonia (NH3) by animal livestock related to manure and urine production,
which causes nitrification and acidification of soil [175]. The GHS production by
five species of insects, the first three of which are considered edible: Tenebrio
molitor, Acheta domesticus, Locusta migratoria, Pachnoda marginata, and Blaptica
dubia, and comparison with the GSH production by livestock animals were the
objects of an experimental study [16]. The comparison with GHS production by
animal livestock is not easy since the differences in the size of the animals determine
differences in the metabolic rate and, hence, CO2 production. However, the CO2 eq.
(g/kg mass gain) production in the case of the aforementioned insect species was
estimated at 7.58, 1.57, 17.72, 121.86, and 37.54, respectively. This factor is in the
range of 79.59–1130 for pigs and 2850 CO2 eq. (g/kg mass gain) for beef cattle [16].
Moreover, in the case of Anabrus simplex (Orthoptera, Tettigoniidae), a production
rate of 37 g CO2/kg BM/day was reported, while 40 g CO2/kg BM/day were noted
for the locust Schistocerca americana (Orthoptera, Acrididae) [176] and 94 g of
CO2/kg BM/day for adult Tribolium castaneum (Coleoptera, Tenebrionidae) [177].
It should be noted that CO2 production is influenced by the species, temperature,
feeding status, stage of development, and activity level [16, 178, 179]. As far as the
CH4 production is concerned, Acheta domesticus and Locusta migratoria did not
produce this compound, and 4.96 CH4 (g/kg mass gain) were produced by Pachnoda
marginata, 0.16 CH4 (g/kg mass gain) by Tenebrio molitor, and 1.46 CH4 (g/kg mass
gain) by Blaptica dubia. In comparison to pigs, which produced 1.92 CH4 (g/kg mass
gain) and beef cattle, 114 CH4 (g/kg mass gain) [16], these are low values, and this
suggests that edible insects have a lower negative influence on the environment than
livestock animals. Similarly, lower values of NH3 emission were recorded in the case
of edible insects. There are many reports about NH3 emission by livestock animals.
For example, pigs emit 4.8–75 mg/kg BM/day [180–182], cattle 14–170 mg/kg BM/
day [180, 183], and poultry 72–436 mg/kg BM/day [180, 184]. The emission of NH3
in the case of insects was different, i.e., from 1.57 to 4.29 mg/kg BM/day for B.
dubia and from 2.46 to 8.01 mg/kg BM/day for A. domesticus [16]. Furthermore,
edible insects need less space than livestock animals; they are able to live in high
densities (kg biomass per m2) and exhibit low vulnerability to disease (high resis-
tance). Besides, crickets can eat a wide range of organic and waste biological
material, and the farm takes up little space (2000 insects/m2) [185]. It has been
reported that around three quarters of the world’s agricultural crop is used for
livestock production either directly or indirectly [186].
A high impact of entomophagy on the environment is exerted by the potential
decrease in application of pesticides, as collecting edible insects from crops or
wild plants can prevent pests and thus decrease the demand for insecticides.
Insects can also be used to compost manure that is produced in huge amounts
by animal livestock, especially by poultry, pigs, and beef cattle. This can be a way
to prevent soil acidification [187]. Moreover, organic material is the diet of edible
insects, and thus waste of food can be reduced [8]. The production of edible
insects has great potential not only in terms of supply of protein and nutrient for
humans but also as an economic aspect in the food sector.
8 Safety Aspect of Edible Insects as Food
One of the aspects of the lack of acceptance of edible insects by Western consumers
is the safety of insect consumption. Although insects are a good source of protein,
nutrients, and minerals, it should be kept in mind that not all insect are safe to eat.
Like other food ingredients derived from plants or animals, insects can cause
allergies (Table 4). Three allergens, arginine kinase (AK), hemocyanin (HC), and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), have been identified in the
muscle of Macrobrachium rosenbergii. Moreover, a novel specific allergen
Table 4 Main allergens identified in animals

Allergen substance Origin
Arginine kinase (AK) Black tiger prawn (Penaeus monodon) [191]
Pacific white shrimp (Litopenaeus vannamei) [192]
Giant freshwater prawn (Macrobrachium rosenbergii)
[193]
Banana shrimp (Penaeus merguiensis) [194]
Moth (Plodia interpunctella) [195]
American cockroach (Periplaneta americana) [196]
German cockroach (Blattella germanica) [197]
Silkworm (Bombyx mori) [190]
Locust (Locusta migratoria manilensis) [198]
Myxobacteria (Myxococcus xanthus) [199]
Tropomyosin (TM) Indian white prawn (Penaeus indicus) [200]
Spiny lobster (Panulirus stimpsoni) [201]
Brown shrimp (Penaeus aztecus) [202]
Pacific white shrimp (Litopenaeus vannamei) [203]
Black tiger prawn (Penaeus monodon) [204]
Carpet clam (Paphia textile) [205]
Lobster (Panulirus stimpsoni) [201]
German cockroach (Blattella germanica) [206]
American cockroach (Periplaneta Americana) [207]
Silverfish (Lepisma saccharina) [208]
Glyceraldehyde 3-phosphate Banana shrimp (Penaeus merguiensis) [194]
dehydrogenase Giant freshwater prawn (Macrobrachium rosenbergii)
[188]
Sardine (Sardinops sagax) [209]
Cricket (Gryllus bimaculatus) [188]
Hemocyanin Giant freshwater prawn (Macrobrachium rosenbergii)
[188, 210]
Lanchester’s freshwater prawn (Macrobrachium
lanchesteri) [188]
Cricket (Gryllus bimaculatus) [188]
Hexamerin1B Cricket (Gryllus bimaculatus) [188]
Firebrat (Thermobia domestica) [211]
hexamerin1B (HEX1B) was detected in Gryllus bimaculatus [188]. Allergenicity

may depend on the stage of development. Pupae of the African silkworm (Anaphe
venata) have thiaminase in their composition, which can cause thiamine deficiency
[189], and arginine kinase has been identified in B. mori (silkworm) larvae [190].
Certain insects can produce toxins as a defense mechanism. Inset specimens col-
lected in the wild may contain pesticides or other chemicals from the environment.
Insects also can directly or indirectly cause diseases. Food poisoning due to the
content of aflatoxins and cases of botulism and parasitic diseases were noted after
consumption of edible insects [212]. Edible insects can also be a source of zoonotic
agents such as bacteria, fungi, and parasites, or vectors [213]. Escherichia coli,
Klebsiella aerogenes, and Staphylococcus were identified in freshly collected palm
grubs (Rhynchophorus phoenicis) from Nigeria [214]. Following the recent scien-
tific findings, the European Food Safety Authority (EFSA) indicates that the possible
microbiologic threat from insects accepted for consumption is comparable to other
threats associated with animal protein. The biological and chemical hazard of insect-
based food and feed products mainly depends on insects’ diet as well as production
and processing methods. Moreover, experts have demonstrated that the insect
species used as a food ingredient is not the main risk, but the diet, handling, and
storage of farmed insects can pose some threat. Insects cannot be considered as
possible biological vectors or amplifiers of mammalian prions because they cannot
replicate in the insect body, provided that no ruminant or human substrates are used
as feed. Moreover, there are no data about accumulation of heavy metals by edible
insects; therefore, this aspect is largely unknown. Utilization of postconsumer food
wastes and organic sidestreams to feed insects, which is nowadays not allowed in the
European Union, should be extensively studied in the future [60, 215].
The risk for health and safety of insects as food can be prevented by consuming
insects from farms where insects are fed without pollution. It should be noted that
storage conditions, preparation methods, and proper heat treatment decrease the risk
for human health and the harmful dose-dependent effects of the compounds. Scien-
tific findings about the benefits and risk factors related to consumption of edible
insects are a valuable source of information for the EU Commission to consider the
use of insects as feed and food in a uniform fashion within the EU and beyond.
9 European Union Policy on Insects as Food
It is known that insects are not traditional food in Europe. The law governing insects
as food and feed is the European Novel Food Regulation (ENFR), which was first
established in 1997 (EC 258/97), revised in 2011 (EC 1169/2011), and repealed and
replaced in 2015 (EC 2015/2283). Within the European Union, insects have a status
of “novel foods,” which means “any food that was not used for human consumption
to a significant degree within the Union before 15 May 1997 irrespective of the dates
of accession of Member States to the Union” and which requires that a risk
assessment be performed prior to marketing according to Regulation (EU) 2015/
2283 of the European Parliament and of the Council of 25 November 2015 on novel
foods [216]. It shall apply from 1 January 2018. In this regulation, we can find short
information about insects: “The scope of this Regulation should, in principle, remain
the same as the scope of Regulation (EC) No 258/97. However, on the basis of
scientific and technological developments that have occurred since 1997, it is
appropriate to review, clarify and update the categories of food which constitute
novel foods. Those categories should cover whole insects and their parts.”
According to Regulation (EC) No 258/97, it was not totally clear that insects were
novel food. Some national food agencies (Belgium, the Netherlands, and the UK)
did not recognize that the original Novel Food Act from 1997 clearly specified this
issue. Therefore, they accepted companies dealing with insects as food. In Belgium,
FASFC (Federal Agency for the Safety of the Food Chain) has established a list of
insects that are accepted as food if they are produced in the EU, if the whole insects
are used, and if requirements for food safety have been respected [217].
Considering the standards of insect production for food, the European Council
requested the European Food Safety Authority (EFSA) for a scientific opinion to
assess the microbiological, chemical, and environmental risks arising from the
production and consumption of insects as food and feed. In October 2015, EFSA
issued a scientific opinion “Risk profile related to production and consumption of
insects as food and feed” [60]. A list of 12 insect species that were reported to have
the biggest potential to be used as food and feed in the EU was established, thereby
indicating that the list should serve as guidance in the overall assessment and should
not be considered definitive or exhaustive. In conclusion, EFSA recommended to
initiate research on the few issues mentioned such as bacteria, viruses, allergens,
prions, chemicals, processing, and environment [60].
10 Insect Business
The increase in the interest in edible insects can be seen on the basis of the number of
emerging companies in Europe and the USA. These companies deal with the mass
breeding of insects, optimization of this production, processing of insects, and
production of feed and food products, most often snacks containing insects. These
changes may revolutionize the food market but for some still remain incomprehen-
sible and surprising. In choosing food as in many other areas of life, we often follow
stereotypes. The reluctance to consume insects is most often attributed to negative
associations, i.e., the mental barrier. Improving the rank of insects can help the
catering industry, for example, by placing them in recipes and introducing them to
restaurant menus. Emerging start-ups and blogs devoted to insect consumption are
an important part of the process of overcoming barriers and stereotypes.
10.1 Insect Farming
Insect farming has the same characteristics as other animal production systems:
insects need access to water and feed (substrate) to supply energy and nutrients for
growth and excrete intestinal content (frass). Little information is available about
mass production of insects for feed and food. In European insect farms, insects are
kept in a closed environment, in boxes/cages, where the atmosphere, substrate,
water, etc., can be controlled. For assurance of economically profitable and sustain-
able insect production, an appropriate level of mechanization, cheap insect feed
production, building design, and temperature-controlled rooms have to be adapted.
One of the main elements of insect production is feeding. Most insect species
farmed for food or feed around the world are omnivores, but the productivity of
farmed insects can be improved by providing a correctly balanced diet [90]. An
integrated rearing system with mechanization, information, and automation can be
applied competitively and effectively for large-scale insect farming. Smaller farms
are not mechanized at all or only to a small extent. Climatic conditions such as
temperature or humidity and rearing area are dependent on the species of insects. The
advantage of insect farming is the use of a small area. To minimize the space needed
to farm a maximum quantity of insects, rearing boxes can be held in multilevel
shelves, which are filled with as many rearing boxes as possible to minimize the
space usage per kilogram of insect produced [218].
Conventional mealworm production is based on trays to hold larvae during their
development and adults, and the most common tray size used is a standard 65 L 50
W 15 H cm. Such a tray has sufficient depth to prevent larvae or adults from
escaping. Crickets are grown in large 36–60 cm deep boxes filled with different
materials, most commonly cardboard, such as egg cartons or packing dividers for
shipping to increase surface space. Rearing boxes can be made of different materials,
but smooth surfaces are preferred because these make it difficult for crickets to climb
the walls [218].
Moreover, traceability of edible insects, as with all agricultural products, from
farm to table is essential to maintain safety and trace any hazards or disease out-
breaks to their source. Each new production batch of insects harvested must be
properly catalogued in a way that can be traced to the raw material used to feed and
water that batch [218].
In Europe there are several professional insect farms intended for consumption by
animals and humans. “Micronutris” from France produces cricket G. sigillatus and
mealworm T. molitor, of which they produce appetizers, pralines, biscuits, and pastas
[219]. In turn, “Proti-Farm” (Netherlands) delivers high-quality insect ingredients of
the buffalo (Alphitobius diaperinus) for the food and personal care and pharmaceu-
tical industries under the EntoPure brand. Proti-Farm products are HACCP certified,
and the factories are certified with FSCC22000, ISO14000, and ISO26000 [220].
Insects farmed for human consumption in the Netherlands (Bugs Organic Food) can
be obtained from Ruig and sons at Bugs Originals [221]. A very popular insect in the
USA is the house cricket A. domesticus produced by “Cowboy Cricket Farms.” They
sell three cricket products: chocolate chirp cookie, cricket powder, and cricket flour
[222]. “AgriProtein,” operating worldwide, produces two products (a natural protein
meal and oil) which can be used as a growth facilitator in agricultural feed prepa-
rations. Some of the by-products of their larvae can also be used and sold as a soil.
And they also sell the dried larvae directly as by the pet food industry. Protix has
developed a broad range of insect-derived products such as insect protein meal,

purified insect oils and lipids, chitin and chitin derivatives, and insect fertilizer
pellets [223]. In turn, “Open Bug Farm,” a project created in the USA, establishes
a new branch of agriculture involved in insect breeding. This is an innovation
platform to stimulate interaction between farmers, researchers, and hobbyists who
want to change the world with edible insects [224].
10.2 Insect-Based Companies
It is said that the consumption of insects in Europe is new; however, analysis of the
number of entrepreneurs dealing with this topic reveals that the issue is highly
absorbing. The best example is the list of entrepreneurs around the world that was
created for the first edition of World Edible Insect Day (the 23rd of October 2015) by
Bugburger. It includes companies, organizations, and individuals who are working
on an edible-insect product, planning to deliver one, or just advocating the benefits
of eating insects [225]. At present, this list contains 180 items from Europe and the
USA, but it is probably incomplete. The list comprises such categories as insect
products, insect restaurants, insects as animal feed, online stores, professional insect
farmers, and research projects.
Sweets are a very popular category of insect products. The species mostly used
are crickets and mealworms. Examples of popular bars are “Kriket” from Belgium,
“SWARM Protein” from Germany, “Jimini’s” from France, “Crickstart” from the
USA, “Jungle Bar” from Iceland, and “Yumpa Bar” and “Sens” from the UK.
Attempts are made to create protein shakes and nutrition supplements from insects.
In turn, Hotlix, who started their business in 1970, are the pioneers of “insect candy.”
They sell classic lollipops with worms, dried grasshoppers, etc. They have relied on
the scary/yuck effect of insects. Newly established companies have a very serious
approach to insect snacks [225]. The assortment is enhanced with pralines, muesli,
cookies, and crispy insects [226]. However, insect flour is the most popular product
because it is relatively easy to produce, and it may be the basis for many products
such as cookies (“Crickelle” the cricket crackers) [227], bread, and pasta (“Aldento”)
[228]. Some European shops offer burgers and meatballs made from mealworms, for
example, supermarket chain “Coop” in Switzerland [229]. Most of the products are
sold online in shops such as “Delibugs” (Netherlands) [230], “Insectes Comestibles”
(France) [231], and “Minifood” (Belgium) [232].
Insect products are not only designed for humans but also for animals. “Wilder
Harrier” offers dog food with added cricket protein [233]; “Conscientious Cat”
proposed insect-based cat food [234]. Several companies see the potential in feeding
livestock, fish, pigs, and poultry with insects instead of other protein sources:
“Protix” (Netherlands), “Ynsect” (France), “Entomo Farm” (France), “Diptera”
(Italy), or “HiProMine” (Poland). It is not possible to mention all the companies
involved in this business due to their large number, but research projects should be
highlighted as well. The main research center in Europe that conducts extensive
research on insects is the Wageningen University [235]. On their web page, there is a
list of all edible insects in the world [58].
European and global industry organizations have also been organized. The
International Platform of Insects for Food and Feed (IPIFF) “is the voice of the
insect sector towards the European Union. IPIFF is an EU non-profit organization
which represents the interests of the insect production sector towards EU policy
makers, European stakeholders, and citizens.” They promote insects as a source of
animal proteins for both human consumption and animal feed. Members include
Ynsect (France), Protix (Netherlands), Hermetia (Germany), Koppert (Netherlands),
Proti-Farm (Netherlands), HiProMine (Poland), Micronutris (France), Jimini’s
(France), Entomo Farm (France), nextProtein, Andromeda, MealFood Europe, and
NextAlim with 26 associated members [236]. In turn, “the ASEAN Food and Feed
Insects Association – AFFIA – is a voluntary group of companies, institutions, and
natural persons which promotes and supports activities that relate to the use of
insects as food and as feed” [237]. Moreover, a journal devoted to edible insects
was established in 2015. “Journal of Insects as Food and Feed” is an online journal
issued four times a year by Wageningen Academic Publishers. The journal aims to
cover the whole chain of insect collecting or rearing to marketing edible insect
products [238].
11 Insects as a Source of Nutrients for Animals
The increased production of livestock, poultry, and fish is forced by the continuously
growing human population and the decrease in areas available for agricultural
production [17]. The increasing intensity of farming animals requires higher
amounts of feed with high-value nutrients to cover their nutritional requirements
[31, 63, 85, 239]. The nutritive needs of monogastric species include a high quality
and quantity of protein in the diet, provided proteins should have an adequate amino
acid profile, high digestibility, good palatability, and no anti-nutritional factors [240].
Nowadays, the most useful protein sources in livestock feeding are fishmeal and soy
meal [241]. The European Union currently imports 40 million tons of crop proteins
each year (mainly soya). The profitability of the EU livestock and aquaculture
sectors is at risk, taking into account the fluctuations in soybean meal prices as
well as the prices of fishmeal, which have risen fourfold over the last decade [242,
243]. These economic issues underscore the strategic importance of developing an
alternative protein source involving new protein sources in animal feed [244].
Insects might be a sustainable solution answering the urgent need for an alternative
source of feed compounds: fishmeal, fish oils, and soymeal [185, 245].
The farming of insect intended for animal feed has been the subject of studies for
years [246–248]; however, insects have not yet become a replacement for traditional
forage based on plant material [168] Both scientists and feed industrial sectors have
begun to reconsider the use of insects as feedstuff for farming animals [249] given
the economic, environmental, and nutritional aspects of entomophagy [17, 66, 85,
250–252]. Moreover, insects can be reared on organic waste, and they exhibit
favorable feed conversion efficiency [85, 253]. Therefore, it seems justified to

consider inclusion of insect proteins in commercial feed manufacturing. It is impor-
tant to elaborate intensive farming systems with selected insect species that have a
short life cycle, can be easily reared, are highly nutritious, and provide high
concentrations of proteins [239]. The use of insects as a diet compound is justified
by the fact that in nature, most insectivores (e.g., fish and birds) prey on a variety of
arthropod species [85]. The natural behavior of chicks is to pick up and eat insects
from the ground, which indicates that poultry are evolutionarily adapted to be
insectivores [254].
Among the millions of insect species living on earth, over 2000 are considered as
edible for humans, and only several species have the potential to be a new source of
nutrients for farmed animals [58, 255]. Currently, only few species of insects are
massively reared for food and feed, i.e., yellow mealworm (T. molitor), domestic
cricket (A. domestica), black soldier fly (H. illucens), the domestic house fly
(M. domestica), locusts (L. migratoria, S. gregaria, Oxya sp.), and silkworms (B.
mori) [185, 253, 256]. However, as suggested by van Huis et al. [256], when insects
are considered as feed for poultry, aquaculture, and pigs, they have to be reared on a
mass scale with stable quality. This can be achieved only by automated rearing
facilities with complete optimization and synchronization of the production pro-
cesses adjusted to the insect species that will be reared.
Regulation of European Commission EC 999/2001 and consecutive EU regulations
on processed animal protein (PAP) led to categorization of insects as ingredients that
cannot be present in the food chain or in animal feed [257]. Afterward, Regulation EC
56/2013 opened a discussion on the use of PAP obtained from non-ruminants in feed for
nonruminants, which might be considered under strict conditions. In turn, as specified
by Regulation EC 1069/2009, invertebrates are classified as “fit but not intended for
human food chains” (category 3 materials). The use of processed animal proteins
(including insects) was reauthorized for the aquaculture sector in June 2013 and is
expected to be included in poultry and pig feeding in the near future [258]. Several feed
companies have committed themselves to include insects and insect compounds in the
forage for livestock as soon as the EU legislation allows doing so [259]. Moreover, a
study conducted by Verbeke et al. [45] shows that farmers, agriculture sector stake-
holders, and citizens express a favorable attitude toward the use of insects in animal
feed, especially for fish and poultry. In contrast to Europe, poultry in West Africa is
already fed with termites collected in the wild [260].
12 Promising Opportunities of the Use of Insects
As mentioned above, the list of positive results of the consumption of insects is long.
Nevertheless, besides being food and feed, insects have other uses. Firstly, they can
be used in food-waste recycling – insects have favorable feed conversion efficiency;
waste sidestreams or other ingredients that cannot be used for feeding traditional
livestock species can be utilized as a source of feed [173, 257, 261–264]. The most
promising in this respect is the black soldier fly (Hermetia illucens). The use of
Hermetia for waste reduction has been extensively studied since the 1970s [265].
The life cycle of this species is linked with organic waste matter. Its larval stage can
be reared on a wide range of decaying materials such as rotting fruits and vegetables,
distiller grains, coffee bean pulp, fish offal, and especially on animal manure and
human excreta [17, 67, 266, 267]. This species has a capability of rapid food
processing – it is estimated that its food intake ranges from 25 to 500 mg of fresh
matter per larva per day [67]. Moreover, the study carried out by Barroso et al. [268]
showed that Hermetia meal (to be used as a livestock forage compound) could be
easily modified by dietary manipulation of larval feed.
Secondly, insects may be used for producing oils. Oil extracted from black soldier
fly larvae is a by-product of insect meal production, which makes them more useful
than the source of proteins. Hermetia-derived oil has a good fatty acid profile, which
can be optimal for feed purposes and for producing high-quality biodiesel [269].
Moreover, Zeng et al. [269] claims that the oil extracted from black soldier fly larvae
meets most of the requirements set by the international standard (EN 14214) for
biodiesel.
Thirdly, every biomaterial left with waste from insect farming can be used as a
fertilizer. Such a biofertilizer contains insect frass with addition of any matter that
falls on the floor of the rearing cage, e.g., wings, body parts, leftovers, dust, or
shredded egg carton. The use of cricket fertilizer was assumed to substitute the
application of mineral fertilizers on key crops in Thailand (e.g., rice paddies) [270].
Fourthly, insects have a mechanism of induced detoxification of plant material
with toxic compounds. This allows herbivorous insects to catabolize secondary plant
compounds before they are absorbed [271, 272]. However, a study by van
Broekhoven et al. [90] showed that longtime exposure of Z. atratus to diet with
high content of potato steam peelings caused high mortality of larvae. It should be
taken into account that chronic toxicity may result in reduced growth and lower feed
conversion efficiency [273, 274].
Finally, insects are able to process polystyrene. A study conducted by Yang et al.
[275] confirmed that mealworms (T. molitor) are the very first species of insects that
is capable to degrade and mineralize petroleum-based plastic polystyrene. Moreover,
Yang et al. [276] reported that the gut of the mealworm should be considered as an
efficient bioreactor, where the gut microbiota (especially Exiguobacterium sp. strain
YT2) plays the key role in Styrofoam degradation.
13 Conclusions
Insects are a good source of many nutrients, although the contents of most nutrients vary
widely depending on a few factors such as the insect species [10], gender [277], stage of
development [68], or diet [127]. In general, most species appear to be good sources of
proteins with essential amino acids, fatty acids (omega-3), most minerals, and most
vitamins B [10, 14, 113]. Moreover, insects are a source of biologically active sub-
stances such as antimicrobial and antifungal peptides [75, 77, 116] and angiotensin-
converting enzyme (ACE) inhibitory peptides [73], and antioxidant peptides [12, 13]. In
addition to the high nutritional value and bioactive ingredients, insects are environmen-
tally friendly. Low greenhouse gas emissions and low drinking water and biomass
consumption are just some of the factors in favor of the insect breeding [16, 172].
Furthermore, insects can be helpful in the management of by-products of the food
industry [173, 187], and their feces can be used as a fertilizer [270].
Despite the many positive sides of introducing insects to our diet, there are a
number of issues that must be met to make this happen. First of all, consumers need
to be convinced to eat insects. They should want to do it voluntarily, being certain of
their own beliefs. It is important to raise awareness about the dangers associated with
the constantly growing population which is related to, among other things, food
security and environmental pollution. The food industry can help by proposing
products containing insects in the form acceptable to consumers. Dishes with
invisible insects, e.g., only protein isolates, are a good way to expand our menu.
Research on the functional properties of the insects or components isolated from
their organisms [3, 152, 153] is a good start to gain detailed knowledge of insects as
food ingredients. There is a need for further analysis, given the multitude of edible
insect species. A better understanding of each aspect of insect consumption brings us
closer to accepting them on a par with other sources of protein. At the same time,
mass insect breeding systems should be developed by automating the production and
optimizing the breeding parameters. In addition, veterinary control should be
applied. Currently, insects cost more than they should because in Europe they are
still sold as novelties. The way insects are bred certainly has an impact on their high
price. Improving the farming system will probably be a factor in lowering production
costs. The combination of all the above factors will help the edible insects to gain a
better reputation among unconvinced consumers. However, in order to exploit the
potential of insects fully, they should become an increasing proportion of our diet
partially replacing other sources of protein such as pork, beef, and poultry.
For many years, the agricultural sector has been moving in the same direction –
maximization of plant and animal production. The use of insects as food and feed
will undoubtedly be a revolution in this sector and will change the direction of
further development of agriculture.
Entomophagy becomes an object of interest of an increasing number of researchers,
and its promotion through the media is still increasing. Although EU legislation is
cautious about the consumption of insects, the number of companies dealing with edible
insects has exponentially grown over the past few years. Start-ups are successful on the
food market and their popularity exceeds expectations. This proves that perhaps
insects will soon be acceptable and produced as feed and food on a large scale.
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Plant Proteases in Food Processing
16
Manzoor Ahmad Shah and Shabir Ahmad Mir
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
2 Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
3 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
3.1 Production from Natural Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
3.2 In Vitro Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
4.1 Cheese Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
4.2 Meat Tenderization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
4.3 Bioactive Peptide Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
4.4 Flour/Dough Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Abstract
Proteases are enzymes that hydrolyze protein molecules into peptides and amino
acids. Proteases are the most commercially important enzymes because of their
multiple applications in food and other industries. In recent decades, interest in
plant proteases has been increased rapidly. The number of industrially employed
enzymes of plant origin is still small but growing fast. Plants are an important
source of proteases as plants require proteases throughout their life cycle. These
are present in all kinds of plant tissues and, thus, can be extracted from their
natural sources or can be prepared using in vitro techniques. Plant proteases can
M. A. Shah (*)
Department of Food Science and Technology, Pondicherry University, Puducherry, India
S. A. Mir
Department of Food Technology, Islamic University of Science and Technology, Awantipora,
Jammu and Kashmir, India

444 M. A. Shah and S. A. Mir
be extracted from natural sources by aqueous maceration of various plant organs.

The crude extract thus obtained may be further purified to obtain a pure enzyme.
Production of plant proteases by in vitro techniques leads to higher enzyme yields
and minimizes the extraction procedures used in extraction from natural sources;
these techniques reduce the effects of climate and seasonal changes and also the
heterogeneity of enzymes produced from different parts of plant. Plant proteases
have the ability to coagulate milk proteins and thus have been utilized as milk
clotting enzymes in cheesemaking for centuries. These proteases are used as
crude or in purified form; they are a substitute to the calf rennet. They are used
for making different varieties of cheese in Mediterranean, West African, and
southern European countries. Proteases extracted from different plant sources
have been widely used in meat tenderization, in bioactive peptide production
from both the plant and animal sources, and in flour/dough modification in baking
industry.
Keywords
Plant protease · Papain · Bromelain · Ficin · Actinidin · Zingibain · Cardosins ·
Cheese · Milk clotting enzyme · Meat tenderization · Bioactive peptide · Dough
modification
1 Introduction
Proteases are enzymes that hydrolyze protein molecules into peptides and amino
acids. These form a very diverse and complex group of enzymes. The specificity of
these enzymes is governed by the type of the amino acid and other functional groups
close to the bond being hydrolyzed [1]. Proteases can be classified into many ways.
Based on the site of action, they can be grouped into exoproteases and endo-
proteases. Exoproteases can cleave N- or C-terminal peptide bonds, while endopep-
tidases cleave internal peptide bonds [2]. Exoproteases can be further divided into
aminopeptidases and carboxypeptidases based on the ability to cleave the N-terminal
and the C-terminal peptide bond. Endoproteases are classified on the basis of their
catalytic mechanism, i.e., enzyme active site. The MEROPS database classifies them
into seven families: aspartic, cysteine, glutamic, metallo, asparagine, serine, and
threonine. But only five types of endoproteases have been reported in plants [3].
Proteases are the most commercially important enzymes because of their multiple
applications in various industries and particularly in food, pharmaceutical, detergent,
and leather industries. In recent decades, interest in plant natural products has grown
rapidly. The number of industrially employed enzymes of plant origin is still small
but growing fast [4]. Plants are an important source of proteases as plants require
proteases throughout their life cycle [5]. The most widely used plant proteases are
papain, bromelain, ficin, actinidin, zingibain, and cardosins. Plant proteases are
being used in dairy processing, meat tenderization, bioactive peptide production,
and baking industry.
16 Plant Proteases in Food Processing 445
2 Sources
Plants are an important source of proteases as plant physiology and development

require a diverse number of proteases [6]. Proteases are required by plants through-
out their life cycle [5]. Proteases obtained from plant sources are very attractive
because they can be used over a broad temperature and pH range [7]. Proteases have
been isolated and purified from various plant sources (Table 1).
Cardosins and cyprosins are obtained from Cynara cardunculus flowers [8–24],
cynarase is from Cynara scolymus flowers [25–30], and cardosin like protease is
prepared from Cynara humilis [10].
Actinidin, bromelain, caprifig coagulant, cucumisin, dubiumin, ficin,
hieronymain, lettucine, onopordosin, oryzasin, papain, pomiferin, procirsin,
salpichroin, streblin and zingibain are obtained from Actinidia spp., Ananas
comosus, Ficus carica sylvestris, Cucumis melo, Solanum dubium, Ficus spp.,
Bromelia hieronymi, Lactuca sativa, Onopordum acanthium, Oryza sativa, Carica
papaya, Maclura pomifera, Cirsium vulgare, Salpichroa origanifolia, Streblus
asper, and Zingiber officinale, respectively. Neriifolin and neriifolin S are obtained
from Euphorbia neriifolia [43, 44]; religiosin, religiosin B [47, 48], and religiosin C
from Ficus religiosa [49]; and caricain from Carica papaya [117].
Protease extracts were obtained from Albizia lebbeck [61], Balanites aegyptiaca
[65], Centaurea calcitrapa [53–55], Foeniculum vulgare [66], Helianthus annuus
[61], Jacaratia corumbensis [62], Moringa oleifera [63], Onopordum turcicum [60],
Silybum marianum [58, 59], Solanum elaeagnifolium [64], and Withania coagulans
[56, 57]. Sun et al. [118] isolated three serine proteases from tomato (Lycopersicum
esculentum Mill.) fruit and Li et al. [119] prepared tamarillin from tamarillo fruit
(Cyphomandra betacea).
Many of the plant proteases are obtained from plants that grow in tropical or
subtropical, geographically remote areas. These plants are subjected to various
internal and external stresses, which affect the quality and quantity of proteases.
Additionally, the selection of high-yielding varieties is difficult due the long culti-
vation periods between planting and harvesting, leading to the expensive products.
Thus, the development of alternative and complimentary techniques to the extraction
of proteases from whole plant sources is quite important from socioeconomic point
of view [120]. An alternative to this is the plant cell culture using in vitro technol-
ogies. These techniques can be used to produce large quantities of proteases which
are homogenous in nature and thus can be easily commercialized [4].
3 Production
Proteases are required by plants for various physiological and developmental pro-
cesses from the beginning up to death of a plant. They are present in all kinds of plant
tissues and, thus, can be extracted from their natural sources or can be prepared using
in vitro techniques [4].
Table 1 Plant proteases in food industry

Function/
Protease Source application Reference
Cardosins and Cynara cardunculus Milk clotting [8–24]
cyprosins activity
Cynarase Cynara scolymus Milk clotting [25–30]
activity
Cardosin Cynara humilis Milk clotting [10]
activity
Onopordosin Onopordum acanthium Milk clotting [31]
activity
Oryzasin Oryza sativa Milk clotting [32]
activity
Procirsin Cirsium vulgare Milk clotting [33]
activity
Ficin Ficus racemosa Milk clotting [34]
activity
Caprifig coagulant Ficus carica sylvestris Milk clotting [35]
activity
Ginger protease, Zingiber officinale Milk clotting [36, 37]
zingibain activity
Actinidin Actinidia chinensis, Milk clotting [38–41]
Actinidia deliciosa activity
Cucumisin Cucumis melo Milk clotting [42]
activity
Neriifolin Euphorbia neriifolia Milk clotting [43]
activity
Neriifolin S Euphorbia neriifolia Milk clotting [44]
activity
Dubiumin Solanum dubium Milk clotting [45, 46]
activity
Religiosin Ficus religiosa Milk clotting [47]
activity
Religiosin B Ficus religiosa Milk clotting [48]
activity
Religiosin C Ficus religiosa Milk clotting [49]
activity
Streblin Streblus asper Milk clotting [50]
activity
Lettucine Lactuca sativa Milk clotting [51]
activity
Hieronymain Bromelia hieronymi Milk clotting [52]
activity
Protein extract Centaurea calcitrapa Milk clotting [53–55]
activity
Protein extract Withania coagulans Milk clotting [56, 57]
activity
(continued)
Table 1 (continued)
Function/
Protein extract Silybum marianum Milk clotting [58, 59]
activity
Protein extract Onopordum turcicum Milk clotting [60]
activity
Protein extract Albizia lebbeck Milk clotting [61]
activity
Protein extract Helianthus annuus Milk clotting [61]
activity
Protein extract Jacaratia corumbensis Milk clotting [62]
activity
Protein extract Moringa oleifera Milk clotting [63]
activity
Protein extract Solanum elaeagnifolium Milk clotting [64]
activity
Protein extract Balanites aegyptiaca Milk clotting [65]
activity
Protein extract Foeniculum vulgare Milk clotting [66]
activity
Actinidin Actinidia chinensis, Meat tenderizing [41, 67–71]
Actinidia deliciosa activity
Bromelain Ananas comosus Meat tenderizing [69, 72]
activity
Ficin Ficus carica Meat tenderizing [71, 73]
activity
Papain Carica papaya Meat tenderizing [69, 74–76]
activity
Zingibain Zingiber officinale Meat tenderizing [69, 75, 77]
activity
Protein extract Cucumis trigonus Meat tenderizing [77]
activity
Protein extract Asparagus officinalis Meat tenderizing [70]
activity
Protein extract Calotropis procera Meat tenderizing [78]
activity
Protein extract Pyrus pyrifolia Meat tenderizing [79]
activity [71]
Protein extract Actinidia arguta Meat tenderizing [80]
activity
Papain Carica papaya Bioactive peptide [81–97]
production
Bromelain Ananas comosus Bioactive peptide [81, 86, 90, 91, 93, 95,
production 96, 98–102]
Ficin Ficus spp. Bioactive peptide [83]
production
(continued)
Table 1 (continued)
Function/
Actinidin Actinidia spp. Bioactive peptide [103]
production
Zingibain Zingiber officinale Bioactive peptide [103]
production
Salpichroin Salpichroa origanifolia Bioactive peptide [104]
production
Pomiferin Maclura pomifera Bioactive peptide [105–107]
production
Cyprosin, Cynara cardunculus Bioactive peptide [108–110]
Cardosins, production
Cynarases
Onopordosin Onopordum acanthium Bioactive peptide [111]
production
Bromelain Ananas comosus Flour [112]
modification
Papain Carica papaya Flour/dough [113–116]
modification
Caricain Carica papaya Dough [117]
modification
3.1 Production from Natural Sources
Plant proteases can be extracted from natural sources by aqueous maceration of

various plant organs such as flowers, seeds, roots, and leaves [121]. The crude
extract thus obtained may be further purified to obtain partially purified enzyme
or pure enzyme depending upon the degree of purification. Precipitation with
ammonium sulfate is an effective way to produce substantial amounts of active
proteases [8].
A protease tamarillin was extracted from tamarillo fruit (Cyphomandra betacea
Cav.) and purified by ammonium sulfate precipitation and diethylaminoethyl-
Sepharose chromatography. Sodium dodecyl sulfate polyacrylamide gel electropho-
resis analysis showed that the peak with the highest protease activity consisted of one
protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11
and 60 C [119]. Beka et al. [65] isolated protease extract from Balanites aegyptiaca
fruit pulp. These fruits were disinfected, husked, and soaked in different buffers and
saline solutions. The extracts obtained were diafiltered, concentrated, and decolored
with active charcoal.
Gagaoua et al. [37] isolated zingibain from Zingiber officinale rhizomes.
Chopped ginger rhizomes were homogenized with 50 mM sodium phosphate buffer
(pH 7.0) containing 10 mM L-cysteine. The homogenate thus obtained was stirred
continuously for 45 min at 4 C and then filtered. The filtrate was centrifuged and
concentrated using ammonium sulfate up to 80% saturation. After an overnight
dialysis of the precipitate, the dialyzed extract was subjected to three-phase
partitioning purification system. The enzyme was found to be exclusively partitioned

in the aqueous phase. The enzyme exhibited maximal proteolytic activity at a
temperature of 60 C and pH 7.0.
3.2 In Vitro Production
The plant cells are totipotent and thus are able to produce the same chemical
compounds in vitro and in vivo. The yield of enzymes differs between the two
processes [122]. Generally, the yield of plant proteases obtained in vitro is lower than
in vivo conditions [4]. Different plant organs produce proteases with different
activities [123].
Different in vitro techniques such as callus and cell suspension cultures have been
used to produce proteases from various plant tissues by several authors. Proteases
were produced from Mirabilis jalapa culture [124], cell suspension culture of
Centaurea calcitrapa [54, 125], and callus culture of Silybum marianum [126] and
Cynara cardunculus [127]. Proteases from Hohenbergia penduliflora were produced
using micropropagation technique by Perez et al. [123]. The molecular cloning,
expression, and characterization of procirsin from the flowers of Cirsium vulgare
were studied by Lufrano et al. [33]. Feijoo-Siota et al. [128] produced recombinant
progaline B, an aspartic protease from Galium verum and expressed in the yeast
Pichia pastoris.
Callus tissue cultures were obtained from cotyledons of Cynara cardunculus.
Calli were subcultured every month using Gamborg’s B5 medium supplemented
with dichloro-phenoxyacetic acid (1 mg/l), benzyladenine (0.1 mg/l), ascorbic acid
(5 mg/l), and citric acid (5 mg/l). The cultures were maintained at 24 C and in
the dark [127]. Cell suspension of Centaurea calcitrapa were homogenized in
50 mM Tris–HCl, pH 8.1, 1 mM EDTA, and 3 mM mercaptoethanol (buffer A), at
4 C, and sonicated. The homogenate was centrifuged at 4 C, to produce the crude
extract [125].
In vitro techniques offer many advantages. Production of plant proteases by in
vitro techniques leads to higher enzyme yields and minimizes the extraction pro-
cedures used in extraction from natural sources. Furthermore, these techniques
reduce the effects of climate and seasonal changes and also the heterogeneity of
enzymes produced from different parts of plant [4].
4 Applications
4.1 Cheese Production
Plant proteases have the ability to coagulate milk proteins and thus have been
utilized as milk clotting enzymes in cheesemaking for centuries. These proteases
are used as crude or in purified form; they are a substitute to the calf rennet. Plant
proteases are a better alternative to calf rennet because of the limited availability and
high price of calf rennet, religious factors, diet, or ban on recombinant calf rennet in
some countries. Most of the proteases used as milk clotting enzymes are aspartic
proteases, but proteases belonging to cysteine and serine groups have also been
reported to have milk clotting activity under certain conditions [121].
Plant proteases are used for making different varieties of cheese in Mediterranean,
West African, and southern European countries. Spain and Portugal are the largest
producers of cheeses using plant proteases from Cynara spp. [129]. Portuguese Serra
and Serpa cheese and Spanish Los Pedroches cheese, La Serena cheese, Torta del
Casar cheese, Los Ibores cheese, and Flor de Guía cheese are prepared with pro-
teases from Cynara spp. [121]. The traditional cheeses in West African countries are
prepared using proteases from Calotropis procera [129].
A large number of plant proteases have been isolated from various plant sources
and studied for milk clotting activity (Table 1). The proteases from Cynara
cardunculus were reported to have milk clotting activity. Sanjuan et al. [11] inves-
tigated the effect of animal and plant protease (Cynara cardunculus) on the physi-
cochemical properties of Los Pedroches cheese prepared from ewes’ milk and
reported that moisture, protein, and water activity were higher, while fat and soluble
nitrogen were lower in the cheeses prepared with animal rennet compared with
cheeses prepared with plant protease. Silva et al. [12] studied the milk clotting
activity of proteases (cardosins A and B), prepared from Cynara cardunculus
flowers, and chymosin and reported that the highest specific milk clotting activity
was shown by chymosin followed by cardosin B.
Tejada et al. [15] studied the effect of animal rennet and plant coagulant obtained
from Cynara cardunculus on the sensory properties of Murcia al Vino cheese
prepared from goat milk and reported that the cheeses prepared with rennet showed
less odor and taste intensities, exhibited a bit clearer color, and were harder and
grainier but less creamy than the cheeses made with plant coagulant. In another
study, Tejad et al. [16] reported that the proteolysis was more intense, and after
60 days of ripening, it was higher in cheeses prepared with plant coagulant than with
animal rennet. The use of plant coagulant for goat cheese manufacture might
accelerate ripening, and, thus, a cheese with different sensory characteristics may
be prepared [16].
Galan et al. [17] studied the effect of two different concentrations of plant
coagulant from Cynara cardunculus in comparison with calf rennet on cheese
prepared from sheep milk and reported that higher concentration of plant coag-
ulant resulted in higher casein hydrolysis in cheese compared with lower con-
centration after 2 days of ripening. The plant coagulant improved the sensory
properties of cheese than calf rennet. Pino et al. [19] studied the effect of plant
coagulant extracted from Cynara cardunculus and calf rennet on cheese prepared
from goat milk cheese during ripening and reported that no significant differences
were found between the compositions of cheeses made with these two types of
coagulants. But the plant coagulant resulted in higher amounts of pH 4.6-soluble
nitrogen, greater breakdown of αs-casein and formation of hydrophobic peptides,
and higher ratio of hydrophobic/hydrophilic peptides throughout the ripening
than calf rennet.
Ben Amira et al. [21] prepared a protease extract from Cynara cardunculus, at
different pH, and reported that the best milk clotting properties and highest enzyme
content were shown by the extract extracted at pH 3. In another study, Ben Amira et
al. [23] studied the effect of the ripening stage and the lyophilization of Cynara
cardunculus flowers on their chemical composition, enzymatic activities of extracts,
and technological properties of cheese curds and reported that flowers harvested at
the middle of ripening stage and lyophilized produce plant coagulant with better
clotting properties, resulting in higher yield, moisture, and texture parameters of
curd.
Liburdi et al. [24] developed a technically friendly enzyme bioreactor by opti-
mizing the immobilization procedure of calf rennet and plant coagulant from Cynara
cardunculus on CLEA ® magnetic supports. The developed technique may be used
to produce soft cheeses by a continuous milk coagulation process. In another study,
Esposito et al. [30] isolated aspartic proteases from alpine thistle flowers and
immobilized it on polyacrylic support. The enzyme showed an optimal pH of 5.0,
a value very similar to the one generally used for milk clotting during cheesemaking,
and exhibited a satisfactory stability over time.
Llorente et al. [26] studied the protease activity in crude extracts from different
parts of the globe artichoke (Cynara scolymus) and reported that mature flowers are
the main source of proteolytic and milk clotting enzymes. According to Chazarra
et al. [29], the rennet strength of Cynara scolymus flower extract increased hyper-
bolically with increase in concentration of calcium, and the concentration was satu-
rated at 50 Mm. Llorente et al. [27] used Cynara scolymus flower extract and animal
rennet for the production of Gouda-type cheeses from bovine milk and reported that
the cheese yield was equal to that produced with animal rennet. Also, chemical and
sensory properties of the cheeses prepared with plant and animal coagulants were
similar. Overproteolysis and background flavor development can be prevented by
extending the brining process (40 h) of the cheeses prepared with plant coagulant.
Salvador et al. [55] isolated and purified aspartic proteases from Centaurea
calcitrapa seeds during germination and reported that the purified enzyme has an
optimum pH range of 3.5–4.5, using FTC-hemoglobin as a substrate and an opti-
mum temperature at 52 C. The clotting time of seed extracts was similar to that of
flower extracts. The extract may be used for the production of special cheeses
requiring weak coagulants. Egito et al. [61] studied the milk clotting activity of
seed extracts from albizia (Albizia lebbeck) and sunflower (Helianthus annuus) and
reported that albizia seed extract showed higher specific clotting activity than
sunflower seed extract. The albizia and sunflower extract hydrolyzed κ-casein at
the Lys116-Thr117 bond and at the Phe105-Met106 bond, respectively. Ahmed et al.
[45, 46] prepared and purified a protease dubiumin from Solanum dubium seeds. The
protease showed high milk clotting activity and coagulated skimmed milk to form a
white and firm curd. The ratio of milk clotting activity to the proteolytic activity of
dubiumin was comparable to those of calf and microbial rennet.
Vairo-Cavalli et al. [59] studied the effect of proteases isolated from flowers
of Silybum marianum (L.) on degradation of caprine and ovine caseins. The pro-
teases degraded the caprine αs1 to a lower percentage than ovine αs but the caprine
β-caseins to higher percentage than ovine β-caseins. The major cleavage site in ovine
αs1-casein was Leu99-Arg100.
Duarte et al. [62] extracted and purified a protease from the root latex of Jacaratia
corumbensis O. kuntze and reported that the purified protease showed the highest
milk clotting activity and higher optimal pH in comparison to the crude extract. Both
the crude and purified proteases showed the maximum clotting at 55 C. Faccia et al.
[35] studied the effect of plant coagulant extracted from caprifig latex on artisanal-
type Cacioricotta cheese (traditional Italian cheese) in comparison with the industrial
type prepared with calf rennet. Higher concentrations of nitrogen fractions and
peptides were observed in artisanal-type cheese prepared with the plant coagulant
than in the industrial type.
Bruno et al. [52] isolated a protease named as hieronymain from unripe fruits of
Bromelia hieronymi and reported that caseinolytic activity and milk clotting activity
was 3.3 Ucas/ml and 40 0.2 IMCU/ml, respectively, at 30 C and pH 6.5. Whey
proteins, bovine serum albumin, and α-lactalbumin were quickly degraded after
30 min, while β-lactoglobulin was considerably degraded only after 60 min at
50 C. Cheeses prepared with hieronymain and chymosin showed similar sensory
attributes as analyzed by a taste panel. Nestor et al. [64] prepared protease extracts
from ripe Solanum elaeagnifolium berries and reported that these extracts showed
lower milk clotting activities than rennin or chymosin but produced firm gels under
acidic conditions. The cheeses prepared with these extracts were softer than cheeses
manufactured with rennin or chymosin. Thus, these extracts can be suitable for the
preparation of filata-type cheeses as well as cream cheeses.
Withania coagulans fruit has traditionally been used as milk clotting agent in
cheesemaking. Protease extracts were prepared from Withania coagulans fruits [56,
57]. Pezeshki et al. [56] reported that severe proteolysis occurred in cheeses prepared
with protease from Withania coagulans in comparison to cheeses prepared with
animal and fungi coagulants. The αs1-caseins and β-caseins were absent in cheeses
prepared with Withania coagulans protease extract. The plant protease led to an
increase in soluble nitrogen in 12% trichloroacetic acid (SNTCA) during ripening of
cheeses. This may be due to higher proteolytic activity of Withania coagulans
protease extracts as compared to other coagulants. Salehi et al. [57] reported that
the protease prepared from Withania coagulans fruits showed optimal activity at
temperature 65 C and pH 5.5. The activity of the plant protease was reduced by
NaCl and CaCl2 and thus may be suitable for producing low salt content cheeses.
A milk coagulating protease was isolated and purified from ginger rhizomes [36,
37 ]. The purified enzyme showed maximum activity at pH 5.5 and at a temperature
of about 60 C. This enzyme showed higher specificity toward αs -casein followed
by β- and κ-casein, and thus the purified protease may be used as a rennet substitute
in the dairy industry [36].
Brutti et al. [31] isolated a protease onopordosin from fresh Onopordum
acanthium flowers and used it to prepare semihard-type cheese from bovine milk.
It was reported that the sensory quality of cheese prepared with onopordosin was
similar to that of commercial cheeses. Pontual et al. [63] reported caseinolytic and
milk clotting activities from Moringa oleifera flowers and reported that the
precipitated protein fraction promoted extensive cleavage of κ-casein and low level
of αs- and β-casein hydrolysis.
Grozdanovic et al. [39] reported that three kiwifruit extracts prepared from a
single fruit pulp showed significantly different levels of active actinidin, depending
on the extraction buffer used. The extract prepared at pH 5.0 showed the best milk
clotting properties, with a higher ratio of clotting activity to proteolytic activity than
purified actinidin. This extract produced a casein coagulum clearly separated from
the whey proteins and was shown to be stable at room temperature for up to
2 months. In another study Puglisi et al. [40] used aqueous solution of kiwi juice
for manufacture of mozzarella cheese and reported that the aqueous solution
exhibited high levels of milk clotting activity and a cheese yield of 10.6% was
obtained. No bitterness was found in the prepared cheeses. Zhang et al. [41] studied
the milk clotting activity and protease activity of actinidin from seven Chinese
kiwifruit cultivars and reported that the highest milk clotting activity and protease
activity were shown by actinidin from Xuxiang cultivar.
Beka et al. [65] prepared an extract from Balanites aegyptiaca fruit pulp and
reported that this extract contains two types of proteases, an aspartic protease and a
serine protease, with an optimum activity at pH 5.0 and pH 8.0, respectively, and an
optimal temperature at 50 C. The aspartic protease from the B. aegyptiaca extract
mainly involved in milk clotting conditions, like other plant proteases, is more
thermostable than bovine chymosin.
Bey et al. [66] prepared protease extracts from fennel stems and leaves and
reported that milk clotting activity of the extracts depends on coagulation tempera-
ture, pH, and calcium concentration in milk. The proteases remain active and stable
in the pH ranging from 6 to 7.5 and temperatures ranging from 40 to 60 C.
Most of the plant coagulants cause excessive proteolysis and thus result in lower
cheese yield and defects in flavor and texture [51]. Therefore, new plant coagulants
are being continuously investigated so that the increasing global demand for diver-
sified and high-quality cheese production may be fulfilled [36, 121].
4.2 Meat Tenderization
Tenderness is an important attribute of meat that affects its acceptability. There are
many methods used for tenderizing meat and may be either chemical or physical
methods. Meat tenderization by protease treatment is one of the popular methods
used in meat industry. Proteases extracted from different plant sources have been
widely used in meat tenderization [41, 68, 69, 71, 75–78, 80].
Naveena et al. [77] studied the effect of proteases from Cucumis trigonus and
Zingiber officinale rhizome on buffalo meat tenderness in comparison with papain.
The meat pieces from Biceps femoris muscles were marinated with these proteases
leading to an increase in protein solubility and reduction in shear force values
compared to control. Extensive proteolysis and reduction in the number of protein
bands were observed in enzyme-treated samples. The sensory values were higher for
enzyme-treated samples in comparison to control.
Liu et al. [68] injected kiwifruit juice in freeze–thaw abused porcine meat and
reported that shear force values increased two times after one freeze–thaw cycle but
reduced after additional freeze–thaw treatments. The kiwifruit juice injection
decreased the pH of meat from 5.6 to 5.2 and enhanced the cooking loss from
21% to 30%. The decrease in the shear force values improved tenderness of meat
more than two times after kiwifruit juice treatment. Myosin degradation was
observed in kiwifruit juice-treated meat samples.
Ha et al. [69] investigated the effect of different plant proteases (papain, brome-
lain, actinidin, and zingibain) on beef proteins and reported significant differences in
protease activity. Myofibril proteins were most effectively hydrolyzed by the
actinidin while connective tissue proteins by the zingibain. Thus, these enzymes
have the potential for specific tenderizing applications. In another study, Ha et al.
[70] reported that the protease activity of kiwifruit protease extract was more
effective at hydrolyzing beef (myofibrillar and collagen) proteins than the asparagus
protease extract. The two protease extracts hydrolyze myofibrillar and collagen
proteins differently. Thus, these proteases can be used to improve the tenderness
of specific cuts of meat, based on their intrinsic protein composition.
Enzyme extract prepared from Calotropis procera latex was applied to different
meat samples (pork, beef and chicken), and it was observed that moisture content
decreased in all the enzyme-treated samples. Firmness and toughness values of the
meat samples decreased significantly with the increased concentration of enzyme
extract. The enzyme extract had no significant effect on water holding capacity and
cooking yield of the treated samples. Protein solubility and TCA-soluble peptides
content increased in all the treated samples. The enzyme extract resulted in extensive
proteolysis in the treated samples [78].
Abdel-Naeem and Mohamed [75] investigated the effect of ginger extract,
papain, and their mixture on camel meat burger patties and reported that the ginger,
papain, and their mixture increased the collagen solubility and sensory scores and
decreased the shear force values, significantly. Ginger extract led to extensive
fragmentation of myofibrils, while papain extract resulted in the degradation of
connective tissue. Also, these extracts improved the lipid stability of treated burger
patties during storage.
Choe et al. [79] prepared a crude extract from Asian pear and reported its meat
tenderization activity. In another study, Nam et al. [71] investigated the effect of
purified pear protease on beef myofibrillar proteins, individually and in combination
with fig and kiwifruit proteases, and reported that pear protease has a good proteo-
lytic activity on beef myofibrillar proteins, especially in combination with purified
kiwifruit protease.
Wang et al. [80] investigated the effect of Actinidia arguta fruit protease on meat
tenderness and reported that the protease was effective in tenderizing beef and
decomposing actomyosin. Zhang et al. [41] studied the effect of actinidin from
seven Chinese kiwifruit cultivars on pork and rabbit meat and reported that actinidin
treatment improved the tenderness in pork and rabbit meat. The shear force was
decreased by more than half in pork and rabbit meat using the purified actinidin at a
dosage of 0.5 mg/100 g muscle.
Plant proteases in combination with other techniques can have promising effects
on meat tenderness. Barekat and Soltanizadeh [76] studied the effect of papain
enzyme alone or in combination with ultrasonication and reported that the applica-
tion of enzyme, either alone or in combination with ultrasound, significantly reduced
the filtering residue, Warner–Bratzler shear force, and textural parameters. The
highest proteolytic activity and tenderness were observed using the combined
treatment at ultrasonic power of 100 W for 20 min.
4.3 Bioactive Peptide Production
Bioactive peptides are specific protein fragments that have a positive effect on body
functions and may influence health [130]. These are short sequences of amino acids
(~2–30) with a low molecular weight that may be generated from both animal [94,
104, 131 ] and plant sources [88, 86, 103, 132]. These peptides are inactive within
the sequence of the parent protein but become active and exert a positive impact on
body functions once released. The amino acid composition and sequence of a
bioactive peptide determines its activity [133].
Bioactive peptides have high potential to be used as nutraceuticals and functional
foods to improve health and decrease the chances of disease occurrence. These
peptides have positive effect on the immune, cardiovascular, nervous, and intestinal
systems [134]. These peptides act as antimicrobials and antioxidants [135–137].
They have cholesterol-lowering effect and antihypertensive, antithrombotic, immu-
nomodulatory [138, 139], and anticariogenic properties [140].
Bioactive peptides are produced during food processing or from food by-products
by microbial fermentation or by chemical/enzymatic hydrolysis using proteolytic
enzymes derived from animals, microorganisms, or plants [141, 142]. Enzymatic
hydrolysis is the most common process used for bioactive peptide production. Plant
proteases have been used for bioactive peptide production from both the plant and
animal sources. The plant proteases such as papain, bromelain, ficin, actinidin,
zingibain, salpichroin, pomiferin, cyprosin, cardosins, cynarases, and onopordosin
have been used to produce bioactive peptides.
Bah et al. [94] reported that plant proteases (papain and bromelain) generated
hydrolysates of plasma of various animals (deer, sheep, and pig) with a lower yield
of soluble peptides compared with fungal proteases. The hydrolysates produced with
plant proteases showed lower DPPH radical scavenging, oxygen radical scavenging
capacity, and ferric reducing antioxidant power in comparison with those produced
with fungal proteases. In another study, Bah et al. [95] prepared protein hydrolysates
from red blood cell fractions separated from the blood of various animals (deer,
sheep, pig, and cattle) using plant and fungal proteases and reported that papain-
generated hydrolysates from red blood cell fractions exhibited higher ferric reducing
antioxidant power and oxygen radical absorbance capacity compared to those
generated with bromelain and fungal proteases. Bah et al. [96] reported that the
hydrolysates of cattle plasma generated with fungal protease had higher antioxidant
activities than hydrolysates generated with papain and bromelain.
Rocha et al. [104] used a protease from Salpichroa origanifolia fruits to hydrolyze
whey protein concentrates and reported that this protease hydrolyzed α-lactalbumin
protein with a higher affinity than β-lactoglobulin. The fraction containing peptides
with a molecular mass below 3 kDa demonstrated a strong DPPH (2,2-diphenyl-1-
picrylhydrazyl) radical scavenging effect.
4.4 Flour/Dough Modification
Enzymatic modification of food components is an effective means to improve their

techno-functional properties. Proteases are used in the baking industries to hydrolyze
gluten for making gluten-free products or improve various other functional proper-
ties of the flour such as modifications of dough handling properties, gluten elasticity,
and texture accompanying shorter mixing time and larger loaf volume [143]. Some
of the plant proteases such as papain, bromelain, and caricain have been used in
flour/dough modification in baking industry.
Tanabe et al. [112] used bromelain to hydrolyze wheat glutenin IgE epitope and
produce hypoallergenic wheat flour. The bread prepared from hypoallergenic wheat
flour showed higher sensory scores than the control bread made from soft flour.
Chen et al. [113] studied the effects of the papain hydrolysis on wheat flour
pasting properties and reported that papain hydrolysis significantly affected the
pasting properties of flour by changing the structural characteristics, amylase activ-
ity, and exothermic transition, especially during the early stage of hydrolysis. The
pasting parameters (peak and setback) significantly decreased with the increase in
papain concentration. Pasting temperature and pasting time increased significantly
with the increase in papain concentration. In another study, Yang et al. [114] studied
the effect of papain on fresh whole wheat dough browning index and rheology and
reported that papain had no effect on carotenoid content of dough. The higher papain
concentration (0.010%, w/w) decreased the polyphenol oxidase activity, which may
be due to either binding or hydrolysis of the specific amino acid sites of polyphenol
oxidase. Papain reduced the browning rate of whole wheat dough as compared to the
control dough. Rheological investigations revealed that papain led to decreases of
elastic (G0 ) and viscous modulus (G00 ) of doughs and this may be due to the
degradation of flour protein and liberation of amylolytic enzymes by papain.
Hatta et al. [115] reported that papain increased the specific volume of gluten-free
rice breads and decreased crumb hardness compared with control breads. Scanning
electron microscopy revealed many fine bubble cells in the crumb of the papain-
treated rice breads. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
protein in the rice batter revealed that the concentration of low molecular weight
proteins (less than 10 kDa) increased with the use of papain compared to control rice
batter. The small protein aggregates were formed through disulfide bonds, and this
network helped to retain CO2 gas during the fermentation process. This resulted in
an increase in the specific volume and a decrease in the crumb hardness of gluten-
free rice bread. Li et al. [116] optimized and evaluated the effect of different enzymes
on wheat flour and reported that alcalase and papain had greater ability to reduce
gliadin content of wheat flour than flavourzyme, pepsin, trypsin, or α-chymotrypsin.

The sequential treatment of wheat flour by alcalase–papain was more effective in
decreasing gliadin content than single enzyme treatment. The optimized conditions
of sequential enzymatic treatment resulted in complete removal of gliadin in the flour
extract, showing lowest IgE binding, and thus may be used for preparing low
allergenic wheat products.
Buddrick et al. [117] investigated the effect of enzyme caricain to reduce gliadin
content in whole wheat flour and prepare gluten-free bread. The authors reported that
the caricain was more effective in reducing the gliadin content in treated breads
compared to control breads.
5 Conclusion
Proteases hydrolyze protein molecules into peptides and amino acids. In recent
decades, interest in plant proteases has grown rapidly. Plants provide an attractive
source of plant proteases. They are present in all kinds of plant tissues and, thus, can
be extracted from their natural sources or can be prepared using various cell culture
techniques. The most widely used plant proteases are papain, bromelain, ficin,
actinidin, zingibain, and cardosins. Plant proteases are being used in dairy pro-
cessing, meat tenderization, bioactive peptide production, and baking industry.
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Part III
Lipids and Their Biological Activity
Bioactive Lipids
17
Luis Vázquez, Marta Corzo-Martínez, Pablo Arranz-Martínez,
Elvira Barroso, Guillermo Reglero, and Carlos Torres
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
2 Neutral Lipids: Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
2.1 Essential Fatty Acids and Ratio Omega-6/Omega-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
2.2 Monounsaturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
2.3 Saturated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
2.4 Medium- and Short-Chain Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
2.5 Trans-Fatty Acids and Conjugated Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
2.6 Branched-Chain Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
2.7 Edible Cold-Pressed Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
3 Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478
4 Glyceryl Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
5 Isoprenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
6 Phenolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
7 Lipid Delivery Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
L. Vázquez · M. Corzo-Martínez · P. Arranz-Martínez · E. Barroso · C. Torres (*)

Department of Production and Characterization of Novel Foods, Institute of Food Science Research
(CIAL,CSIC-UAM), Madrid, Spain
[email protected]; [email protected]
G. Reglero
Department of Production and Characterization of Novel Foods, Institute of Food Science Research
(CIAL,CSIC-UAM), Madrid, Spain
Department of Production and Development of Foods for Health, IMDEA-Food Institute, CEI
(UAM-CSIC), Madrid, Spain

468 L. Vázquez et al.
Abstract
Historically, lipids have been considered just as a source of energy for our bodies
and as the basic unit of membranes. The discovery of the platelet-activating factor
in 1979 as one of the first biologically active phospholipids was a relevant
landmark in this field. Since then, some unique biological activities have been
assigned to every single lipid class. For example, lipids, as small hydrophobic
molecules, are extraordinary as chemical messengers to send information
between organelles and to other cells. Additionally, polar lipids that contain
hydrophobic and hydrophilic regions can interact distinctly with membrane pro-
teins modulating their activities, while glycosphingolipids including their struc-
ture complex carbohydrates can play important roles in the immune system.
Therefore, in this chapter, many relevant biological functions of some lipid
classes from dietary sources will be extensively reviewed.
Keywords
Free fatty acids · Glyceryl ethers · Phospholipids · Isoprenoids · Phenolic lipids ·
Lipid delivery systems
1 Introduction
For many years, fats have been associated with dangerous substances to our health
and consequently should be minimized or avoided in our diet. Fortunately, more and
more people recognize that fats are essential ingredients for food palatability. More
interestingly, some lipids such as cholesterol have been recognized as essential for
the production of hormones, vitamin D, and bile salts, among other physiological
functions. Additionally numerous health benefits have been attributed to unsaturated
fats from seafood. However, the essential role that lipids play in life similar to
proteins and genes is not broadly understood. Very few people are aware that fat is
the most relevant part of our brain and plays an extremely important role in all other
soft tissues. Lipids are studied by nutritionists who try to find out the relationship
between fats in our diet and the composition of different organs and tissues.
Biochemists investigate synthesis and breakdown of lipids to produce energy and
building material for cells. However, lipids have not been extensively recognized as
essential molecules in cell membranes. Lipids form fascinating and intriguing
structures as a consequence of basic physical principles of self-organization that
governs interaction among many molecules. In this scenario, water plays a key role
functioning as the definite biological solvent. The unique properties of water oblige
lipid molecules to self-assemble and to produce subtle structures. Consequently,
lipids, in aqueous medium, form lipid bilayers and membranes. Lipid membranes
can be considered as extended thin layers with the thickness of only two molecules.
These structures constitute the backbone of all biological membranes which are
omnipresent in all living cells [1].
Dietary bioactive lipids, from a food science point of view, can be generically
defined as lipids with specific roles on health. Their function can be related to
17 Bioactive Lipids 469
physical or chemical properties of these molecules. Numerous evidences point out

that lipid classes provide health benefits based on mainly two mechanisms:
(1) changing the fatty acid composition of different tissues or (2) promoting
cell signaling pathways [2]. Some health benefits can be attributed to short- to
medium-chain fatty acids, although most evidences suggest that the most important
bioactive lipids are polyunsaturated fatty acids (PUFAs). PUFAs are precursors in
the biosynthesis of cellular hormones (eicosanoids) and various signaling com-
pounds with relevant roles in human health [3].
Recently, more and more interest for natural substances with beneficial activity to
humans has been observed. Among these substances, those with strong antioxidant
activity have brought a lot of attention because oxidative stress induced by multiple
factors is the main cause of many pathological conditions. However, many phenolic
compounds are mainly hydrophilic which decreases their application in oil-based
formulations and emulsions. For that reason, sources, production, and formulation of
more lipophilic derivatives of these phenolic compounds will be also discussed in
this chapter [4].
2 Neutral Lipids: Fatty Acids
The term “fatty acid” refers to any aliphatic monocarboxylic acids able to
be liberated by hydrolysis from naturally existing fats and oils. “Neutral fats”
are mono-, di-, or tri-esters of glycerol containing one, two, or three fatty acids
and are termed monoacylglycerides, diacylglycerides, and triacylglycerides, respec-
tively [5]. Fats and oils consisting of triglycerides are one of the main components of
every living organism, and they are also important constituents of the human diet.
Bioactivity and nutritional value of these fats depend on the length and number of
unsaturated bonds of the fatty acid chains that they contain. Depending on the length,
fatty acids are classified into long-chain fatty acids (LCFAs) with >12 carbons,
medium-chain fatty acids (MCFAs) with 6–12 carbons, and short-chain fatty acids
(SCFAs) which contain <6 carbon atoms [6]. Their absorption into the human
digestive system will depend to a large extent on the stereospecific distribution of
the fatty acids in the triglyceride molecule. Long-chain saturated fatty acids located
at the outer positions sn-1 and sn-3 have limited absorption, and this may influence
lipemia and serum cholesterol levels [7].
2.1 Essential Fatty Acids and Ratio Omega-6/Omega-3
Polyunsaturated fatty acids (PUFAs) have aroused great interest based on their
beneficial properties, so there is a trend toward obtaining foods rich in this type of
fatty acids. These fatty acids are generally classified into omega-3 fatty acids and
omega-6 fatty acids, depending on the position of the first double bond counting
from the methylene extreme of the hydrocarbon chain. A number of information
sources suggest that humans evolved on a diet whose ratio of omega-6 to omega-3
essential fatty acids was close to 1 [8]. Due to the modern western diets, food
processing, and agribusiness, the intake of omega-6 PUFAs has increased, and the
omega-3 PUFAs have decreased, leading to a ratio around 20:1 [9]. Although the
percentage of total calories ingested from fat is decreasing, nowadays the prevalence
of overweight and obesity has increased which represent a major public health
problem. The excessive intake of omega-6 PUFAs and very high omega-6/omega-
3 ratio contribute to the development of several diseases, including obesity; cardio-
vascular, autoimmune, and inflammatory diseases; and cancer, whereas increased
levels of omega-3 show suppressive effects. High amounts of omega-6 fatty acids,
linoleic acid (LA) and arachidonic acid (AA), compete with omega-3 PUFA absorp-
tion [10]. There is also a competition for desaturation and elongation enzymes
between LA and α-linolenic acid (ALA) [11]. Moreover, anti-inflammatory media-
tors are cleaved from omega-3 fatty acids, while omega-6 fatty acids provide pre-
cursors for pro-inflammatory metabolites [457]. Most of the health benefits
associated with omega-3 fatty acids are attributable to the long-chain polyunsatu-
rated fatty acids (LC PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic
acid (DHA) [12].
It has been established that consumption of omega-3 PUFAs ameliorates the risk
of cardiovascular disease [13], produces a decrease in blood pressure, and shows a
positive association with serum high-density lipoprotein (HDL) and inverse associ-
ation with very-low-density lipoprotein (VLDL) and triglyceride [14]. Besides, it is
associated with lower risk of glucose tolerance and non-insulin-dependent diabetes
mellitus [15].
On the other hand, EPA, DHA, and lately docosapentaenoic acid (DPA, 22:5n-3)
have shown beneficial effects in a variety of neurodegenerative and neurological
conditions [16, 17]. They exert positive effects on neuronal membrane properties
(such as membrane fluidity and permeability) and increase brain phospholipid
levels, phosphatidylserine, phosphatidylethanolamine (PE), and phosphatidy-
linositol [18]. They are also promoters of differentiation of neural stem cells via
metabolic oxidation [19, 20].
A variety of studies have investigated the relationship of neuroprotection and risk
of cognitive decline with the fatty acid composition in the blood (EPA, DPA, and
DHA). Specifically, EPA has been associated with slower cognitive decline [21],
depressive symptom risk, and dementia risk in the elderly [22]. Inflammation has an
important effect in learning and memory parameters. In this line, DHA have shown
to reduce inflammatory cytokine release, and EPA change the pro-inflammatory
profile of Alzheimer patient’s cells to a healthy cell profile, reducing the IL-6/IL-10
ratio [23, 24].
The most widely used natural source of EPA and DHA is fish oil. This has led to
overexploitation of the seas by the fishing industry, which has seriously affected a
large number of marine species. Algae aquaculture could also provide a new
important source for omega-3 PUFAs [25]. Alternatively, stearidonic acid (SDA,
18:4 omega-3) is of great interest, since its endogenous conversion to EPA and DHA
has been shown to be very efficient [26, 27]. Natural sources of SDA are the
seed oils of corn gromwell, hemp, blackcurrant, and cyanobacterium Spirulina.
Furthermore, there is a variety of plants high in SDA, including some of the

families Grossulariaceae, Caryophyllaceae, Primulaceae, and Boraginaceae (such
as echium), that could serve for oil extraction to human consumption and to
supplementation of livestock feeds, in order to increase the omega-3 PUFA content
in dairy products and meat [28]. Recently, refined oils extracted from Echium L.
species have been positively evaluated as a novel food ingredient by the EFSA
(European Food Safety Authority) because of their high concentrations of SDA and
other PUFAs such as alpha-linolenic acid (ALA, 18:3 omega-3) and gamma-
linolenic acid (GLA, 18:3 omega-6) [29]. GLA is a metabolic precursor of AA,
which exerts pro-inflammatory activities; however this conversion in humans is slow
and gives rise to the production of anti-inflammatory eicosanoids [30]. In addition,
the combination of GLA and EPA appears to enhance the anti-inflammatory activity,
for competition with the AA cascade, and it could also be useful in prostate and
breast cancer prevention or cancer-cachexia [31–33].
2.2 Monounsaturated Fatty Acids
Other fatty acids that have recently shown interesting health effects are omega-7
fatty acids such as palmitoleic acid (C16:1 n-7). Doses of 300 mg/kg in the form of
free fatty acid have shown to reduce body weight, improved hyperglycemia, hyper-
triglyceridemia, and insulin sensitivity in mice fed during 4 weeks, when compared
with the same doses of palmitic acid (16:0 n-9). Furthermore, palmitoleic acid
administration suppressed pro-inflammatory gene expressions and lipogenic genes
in the liver [34]. We can find it in animal fats, marine oils, and vegetable oils (such as
macadamia nut oil and hawthorn oil).
Moreover, extensive epidemiological studies have shown that the omega-9 fatty
acid such as oleic acid (C18:1 n-9) decreased incidence of coronary heart diseases,
metabolic syndrome, and cancer [35] and regulates many biological processes. Oleic
acid is a monounsaturated fatty acid present mostly in olive oil and other vegetable
oils such as pecan oil, macadamia oil, peanut oil, canola oil, and sunflower oil and
seed oils such as sesame oil, poppy-seed oil, and avocado oil. It is also present in
animal fats of turkey or chicken and other animals fed on grass. Oleic acid is not an
essential fatty acid; it can be formed by de novo synthesis in humans [36] and from
other fatty acids such as stearic (C18:0).
2.3 Saturated Fatty Acids
Consumption of saturated fats in Western countries is high, and their intake, espe-
cially of palmitic acid (C16:0) and myristic acid (C14:0), is related to a higher
incidence of cardiovascular diseases. After palmitic acid, stearic acid is the most
consumed saturated fatty acid in Western countries with a total of 26% of consump-
tion. Stearic acid is ingested through the consumption of animal fat derived from
pork or beef, as well as vegetable fat such as coconut or soybean oil [37]. Compared
with other saturated fatty acids, stearic acid lowers LDL cholesterol, has no effect on
HDL levels, and lowers total cholesterol [38, 39]. According to [40], this beneficial
effect of stearic on cholesterol is based on its malabsorption in the body and its
reduced solubility after being released from the triglyceride by pancreatic lipase [41].
In addition, this fatty acid is rapidly transformed to oleic acid in the liver during its
metabolism. Unlike stearic acid, palmitic acid raises total cholesterol mainly by
increasing LDL levels, which is attributed to its ability to suppress the expression of
LDL receptors. This increase in total cholesterol by palmitic acid is also attributed to
its slower passage to oleic acid, since it must be first elongated to stearic acid [40].
On the contrary, stearic acid is used to synthesize hypocaloric fats considering its low
digestibility and high excretion. An example of hypocaloric fats recognized as novel
food is Salatrim ®, which is composed of triglycerides with random positional
distribution of fatty acids; it contains LCFAs (mainly stearic) and SCFAs, such as
propionic or butyric acid [42].
On the other hand, capric acid (10:0) and lauric acid (12:0) have been shown to
possess antimicrobial and anti-inflammatory properties [43, 44]. In addition, lauric
acid has been also associated with positive effects on cardiovascular system related
to the increase of HDL levels [45] and decrease of blood pressure in rats [46].
Moreover, lauric acid has shown to prevent the prostatic hyperplasia and exert
inhibitory effects in colon cancer, endometrial cancer, and breast cancer cells [47],
and consumption of virgin coconut oil as a natural source of these two fatty acids has
shown to improve quality of life in patients during chemotherapy [48]. Lauric acid is
also present in other plant oils, seeds, and fruits and in breast milk [49, 50].
Recent studies on myristic acid have shown contradictory effects on its impact on
health. Although it has always been considered detrimental to health [51–53], a
group of authors has shown that myristic acid increases diacylglycerol kinase levels
and improves glucose uptake into cells [54]. This raises the question regarding the
potential role of myristic acid on the prevention of type 2 diabetes. These fatty acids
are presented in high concentration in high-fat dairy products which could explain
the diabetes-protective properties associated with their intake [55].
2.4 Medium- and Short-Chain Fatty Acids
Medium-chain fatty acids (MCFAs) are minor dietary components. However, their
intake provides a quick source of energy because they are easily digested and
absorbed. Triacylglycerides with three molecules of MCFAs are quickly absorbed
and transported to the mitochondria where they serve as combustible; therefore they
do not accumulate as body fat. MCFAs do not pass through the enterocyte and do not
require the formation of chylomicrons; they are directly transported by the portal
system [56].
Several studies have confirmed the potential of MCFA to reduce body fat; they
suppress fat deposition improving thermogenesis and fat oxidation in both humans
and animals [57–59]. Triglycerides enriched in MCFA are attractive for use in
low-calorie products [60]. In addition, it has been reported that MCFAs preserve
insulin sensitivity in patients with type 2 diabetes [59].
Triacylglycerides with MCFAs are used as part of liquid diet formulas suitable for
patients with digestive problems or conditions that demand fluid restriction, such as
burn injury, postoperative cancer, AIDS, respiratory distress, cystic fibrosis, and
hepatic or renal disease [61]. A relevant example of this type of fatty acid is the
case of lauric acid (C12:0). As previously mentioned, this compound as well as its
monoester monolaurin possesses numerous antibacterial and antiviral activities [62].
It is reported to be effective against Staphylococcus aureus, Enterococcus faecalis,
Mycobacterium terrae, Streptococcus agalactiae, and Listeria monocytogenes [63]
and against viruses such as herpes simplex-1, HIV, cytomegalovirus, vesicular
stomatitis, measles, and visna virus [64]. Myristic acid (C14:0), capric acid
(C10:0), and caprylic acid (C8:0) can also be virucidal and bactericidal [62].
SCFAs, mainly acetate, propionate, and butyrate, are microbial end products
generated by fermentation of fiber from the intestinal microbiota. Their presences
inside the intestine have shown a positive influence on intestinal barrier function.
Disruption of intestinal barrier function can lead to the development of many
diseases such as inflammatory bowel disease, obesity, and HIV infection. Thus
SCFAs play an important role in human physiology affecting neuroimmune func-
tion, nutrition, and protection from pathogen colonization [65–67]. SCFAs also
modulate the secretion of hormones including peptide YY and leptin and cell
differentiation and proliferation [68, 69].
The association of the plasma and colonic SCFAs with metabolic syndrome has
been extensively documented. Recently, the relation between SCFAs and regulation
of energy homeostasis have been studied [70, 71]. Many of the SCFAs produced in
the gut are used as host energy source, and this could represent 10% of our caloric
requirements per day [72]. Acetate and butyrate are involved in the synthesis of
cholesterol and palmitate in the liver and propionate in the gluconeogenesis [73]. It is
estimated that 90% of SCFAs produced in the colon are rapidly and efficiently
absorbed [74]. Furthermore, acetate and propionate are energy substrate for periph-
eral tissues and butyrate for the colonic epithelium [75].
Focusing on SCFA pathway and signaling, it has been reported that the free fatty
acid receptors, GPR43 and GPR41, can regulate host energy homeostasis in the gut
and adipose tissues [76, 77]. Mice deficient in GPR43 are obese following a normal
diet, while mice overexpressing GPR43 remain lean even with a high-fat diet [78].
Thus gut microbiota and SCFAs could affect the development of obesity, diabetes,
and insulin resistance. These disorders are characterized by the low presence of
specific microbial groups (Akkermansia muciniphila among them) and SCFAs,
leading to alteration on glucose, lipid, and energy homeostasis, gut barrier dysfunc-
tion, and low-grade inflammation [79]. Therefore SCFAs are playing significant
roles in several inflammatory mechanisms including atherosclerosis [80] and also in
cancer [81]. The anti-inflammatory effect of butyrate in the colon, which is produced
by some microbial groups such as Faecalibacterium prausnitzii [82] or
Ruminococcus [83] and other butyrate-producing bacteria, has been extensively
reported. SCFAs are involved in the pathophysiology of neuroimmune and
immune-inflammatory disease having effect on T cell differentiation, cytokine

expression, epithelial barrier function, and the recruitment of leucocytes to areas of
inflammatory activity [84]. Chronic intestinal inflammation increases the develop-
ment of colon cancer. SCFAs confer protection in carcinogenesis. Propionate
and butyrate were observed to induce apoptosis and inhibit proliferation on cancer
cells [85]. SCFAs have also reduced colitis and allergic asthma in animal models
[86–88]. Indeed treatments with SCFAs in ulcerative colitis patients have shown to
ameliorate colitis [89].
In order to drive the metabolic activity of microbiota toward fermentation
of carbohydrates, the intake of dietary fibers and prebiotics (nondigestible carbohy-
drates) is essential [90]. These nondigestible carbohydrates, such as xylans,
cellulose, inulin, fructooligosaccharides, or resistant starch, are fermented by the
anaerobic colonic bacteria to generate energy for microbial growth, moderate
concentrations of SCFAs in the colon, as well as peripheral circulation [66, 91,
92]. There are other strategies such as supplementation with specific probiotic strains
(live microbiota) to promote production of butyrate, other SCFAs, or lactate that is
also implicated in the cross-feeding mechanism between colonic bacteria [93, 94].
However, when SCFAs are produced fast and in large amounts or under special
unhealthy conditions, they could disrupt barrier function and instead exert beneficial
effects [95, 96]. Hence, it is necessary to study the appropriate concentrations of
these SCFAs to provide health benefits in each situation.
2.5 Trans-Fatty Acids and Conjugated Fatty Acids
It has been established that consumption of trans-fatty acids (TFAs) increased the
risk of heart disease [97]. But there are TFAs originated from different sources which
produce diverse effects in human health. Nonnatural or industrial TFAs (iTFAs) are
mainly formed by hydrogenation in order to achieve the conversion of liquid oils
into semisolid or plastic fats. These newly formed fats have interesting technological
characteristics but include different proportions of TFAs [98]. iTFAs are rich in
elaidic acid (trans-9-18:1) and trans-10-18:1, and their intake has shown to stimulate
atherosclerosis in humans [99]. However, some natural TFAs (nTFAs), such as
vaccenic acid (trans-11-18:1), have shown to protect against atherosclerosis [99].
Vaccenic acid is produced inside the digestive tract of ruminant animals becoming
the main trans-isomer of oleic acid (18:1) in the fat of dairy and beef products [100].
Nevertheless ruminant TFAs (rTFAs) are in low proportion in food (4–8% of total
fatty acids) [101], whereas iTFAs are present in shortenings and pastries and can
reach up to 61% of total fatty acids [102]. Several studies focus on the role of
different trans-isomers on lipid metabolism. Comparison of the effects of trans-
isomers of 18:1 in vitro has shown that trans-9 isomer upregulated the lipogenic
genes and enhanced total cell fatty acids, whereas isomers trans-11 and trans-13 did
not have this effect [103]. The same authors have observed that C18:1 trans-9 and
trans-10 increased cholesteryl ester content and triglyceride in cultured liver cells
compared to trans-isomers 11, 14, and 15 [104]. Moreover, trans-isomers 6, 9,
and 10 upregulated genes related to cholesterol and fatty acid synthesis. A meta-
analysis of studies on iTFA and nTFA did not show association between coronary
heart disease (CHD) and nTFA intake (0.5–1.9 g/day), whereas intake of iTFA
(1.3–5 g/day) showed to increase the risk of CHD [105]. In the same line, another
meta-analysis of observational studies has reported an increase in CHD (30%) and in
the risk of CHD mortality (28%), but not in type 2 diabetes, associated with 1.8% of
total energy intake of iTFA. However, rTFA intake had no effect on CHD, and
ruminant C16:1 trans-9 showed inversely association with type 2 diabetes [106].
Another review of 29 and 6 treatments with intake of equivalent range of iTFA and
rTFA did not find significant difference between the TFA from either source and the
effects on LDL/HDL ratio [107]. Further studies are needed in order to clarify these
contradictions, but it seems clear that rTFA intake does not increase the risk of CVD
and may exert certain beneficial effects on health.
The fatty acid composition of milk and dairy products is very important for its
effect on human health and depends on the cow diet, the metabolism inside the
digestive tract of ruminant animals, and the mammary metabolism [100]. Briefly,
after lipolysis of dietary lipids, bacteria inside the rumen produce, via
biohydrogenation of unsaturated fatty acids, different isomers (cis and trans) of
18:1, non-conjugated 18:2, conjugated linoleic acids (CLA), and conjugated
linolenic acids (CLnA) [108]. The two isomers of CLA (cis-9, trans-11 and trans-
10, cis-12) and their mixture have demonstrated a variety of biological activities,
among them reduction of body fat, prevention of atherosclerosis, modulation of
immune and inflammatory responses, and anticancer effects [109–111].
Conjugated fatty acids also have diverse origin. Rumenic acid (cis-9, trans-11
18:2) is the predominant CLA isomer in ruminant milk fat (75–90%) [112], followed
by trans-10, cis-12 and trans-7, cis9. Rumenic acid is basically a product of
the endogenous synthesis by the action of the enzyme γ-desaturase on the mammary
gland, which is used as a substrate vaccenic acid (trans-11 C18:1). Other
preparations of CLA are produced by chemical synthesis via alkaline isomerization
of sunflower or safflower oils (rich in 18:2n-6) containing equally proportions
of c18:2 trans-10, cis-12 and c18:2 cis-9, trans-11. Human studies have demon-
strated that C18:2 trans-10, cis-12 is implicated in lipolysis and fat oxidation
(catabolic effect) and C18:2 cis-9, trans-11 isomer has anti-inflammatory and ana-
bolic effects [113]. Supplementation with 6 g/day of CLA for 12 weeks improved
symptoms of inflammatory bowel disease in humans [114]. However, more studies
to compare the form of administration of CLA as triacylglycerols (TAG) or free fatty
acids (FFA) are necessary [115]. Studies in animal models have shown an increase of
lean body mass and reduction of fat deposition associated with the isomer trans-10,
cis-12 rather than cis-9, trans-11 [116]. However, the effects of individual isomers
on body weight have not been shown in human, although modulation of insulin
resistance [117, 118] was observed. On the other hand, effects of CLA mixture
intake during 6 months (3.2–3.4 g/day) have been reported to slightly decrease body
fat mass in humans [119]. Regarding regulation of blood pressure, the finding of a
meta-analysis of eight human studies did not report favorable effect of CLA [120]. In
addition, several studies have reported inhibitory effects of CLA on cell proliferation
of cancer and induction of apoptosis and antimutagenesis in rats and cell lines
[121–123]. However, a systemic review in humans has shown a weak inverse
relation between CLA dietary intake and breast cancer risk [124].
Conjugated linolenic acid (CLnA) is the term for the isomers of octadecatrienoic
acid (18:3) with three conjugated double bonds. CLnA are present in milk fat
and mainly in seed oils [125] such as pomegranate, which contains cis-9, trans-11,
cis-13 C18:3 (punicic acid), bitter melon with cis-9, trans-11, trans-13 C18:3
(α-eleostearic acid), or pot marigold containing trans-8, trans-10, cis-12 C18:3
(calendic acid) [126]. Studies in rat and mouse models and in human cells have
indicated that isomers of conjugated eicosapentaenoic acid, conjugated non-
adecadienoic acid, and CLnA (mostly cis9, trans11, cis15 isomer) possess
anti-adipogenic, anti-inflammatory, anticarcinogenic, and immune-modulating
properties. Several recent studies have also reported the health benefits of pome-
granate seed oil (PSO) consumption, rich in punicic acid. After 2 weeks of feeding
obese mice with PSO (2 ml/kg/day), a reduction of pro-inflammatory cytokines
(interleukin-6) and tumor necrosis factor-α in plasma was observed. The treatment
showed anti-inflammatory properties and also improved insulin sensitivity [127].
Punicic acid from PSO has also shown in vivo anti-inflammatory effects by limiting
neutrophil activation and decreasing lipid peroxidation [128]. Schubert et al. [129]
have reported that polyphenols and pomegranate seed oil, in a fermented juice, retard
prostaglandin synthesis and oxidation, promote apoptosis of breast cancer cells, and
inhibit breast cancer cell invasion and proliferation. Diet supplemented with PSO has
inhibited colonic adenocarcinomas in rats. The inhibition of colonic tumors was
related to an increase in the content of CLA in liver and colonic mucosa lipid
fraction. In the nontumor mucosa, an increase of peroxisome proliferator-activated
receptors after administration of PSO was observed [130]. Toi et al. [131] have also
reported significant potential of PSO and other pomegranate fractions for down-
regulation of angiogenesis in vitro and in vivo. Białek et al. [126] have reported that
pomegranate seed oil intake leads to an increment in the content of cis-9, trans-11
CLA in the liver of rats and decreases the activity of the enzymes Δ5- and Δ6-
desaturase more than other CLA supplements. However, other studies of lipid profile
and insulin resistance associated with exercise in healthy humans did not show
significant effect on insulin resistance and lipid profiles, except for HDL [132].
Further investigations are needed in order to demonstrate all these positive effects in
humans.
2.6 Branched-Chain Fatty Acids
Branched-chain fatty acids (BCFAs) are mostly saturated fatty acids with one or
more methyl branches on the carbon chain. Most of them are at terminal (iso) or next
to the terminal (anteiso) methyl group [133, 134]. Overall, BCFAs are found in
ruminant milk and adipose tissue of animals such as cows, sheep, and goats. They
are presumably produced and utilized as membrane lipids by microorganisms of
the rumen. The composition of BCFAs varies depending on the animal and its diet
[133, 135, 136]. Those with chains of 14–18 carbons are typically found in the fat of
beef and dairy products [137]. A study of the US retail cow milk supply has shown
that BCFA constitutes 2% w/w of the milk fat [138]. Amounts of BCFA in lanolin,
wool wax, exceed 40% [139]. Although less studied than PUFA omega-3s, BCFAs
are also present in fish. Wang et al. [140] have reported that mean BCFA content in
edible muscle across 27 freshwater fish species analyzed was 1.0 0.5% of total
fatty acids, being 1.8 0.7% in fish skin. Most of the BCFAs found were anteiso-
15:0, iso-15:0, iso-16:0, anteiso-17:0, and iso-17:0.
BCFAs are also synthesized by the skin in humans, and they constitute almost one
third of the fatty acids in the vernix (fatty film covering the fetus in utero). The
fetuses swallow the vernix suspended in the amniotic fluid, exposing their gut to
BCFA. They are also natural components of colostrum and breast milk (>1.5%w/w)
[141–143]. Moreover, BCFAs are important components of the membranes of
Lactobacillus and Bifidobacterium, bacterial genera present in the gastrointestinal
tract of newborns [144–146].
Ongoing research revel that BCFAs are bioactive compounds with positive
impact in the development of the intestinal microbiota, and anticancer effects have
been also attributed to them [147–149]. A recent study with neonatal rat pup model
has shown that BCFAs reduce necrotizing enterocolitis incidence by more than 50%,
influence microbiota composition, and are selectively incorporated into the mem-
brane lipids of the intestine [138].
2.7 Edible Cold-Pressed Oils
In recent years, a variety of edible cold-pressed oils obtained from fruits and seeds of
different plants in the market are increasing. These oils have advantages compared
to those obtained by other technologies of extraction or refining, since their charac-
teristics and flavor are not altered by the increase of temperature or the use of
solvents [150]. Thus, they present a large number of intact bioactive components
including polyunsaturated fatty acids, free and esterified sterols, tocopherols and
tocotrienols, squalene, carotenoids and chlorophylls, and various phenols and
triterpene alcohols. These compounds are characterized by possessing anti-
inflammatory properties, reducing oxidative stress, and positively modulating the
immune system, as well as chemopreventive and neuroprotective activities [150]. In
this chapter, many of the bioactive qualities of several of these compounds have been
reviewed, but further research is needed in both nutrition and pharmacology areas to
better document their functionality and contribution to health [151]. In addition,
legislative aspects regarding their identity, denomination, and certification must be
defined, as well as clarifying issues related to authenticity, safety, presence of
contaminants, rancidity, susceptibility to light and heat (for proper storage and use
in cooking), and naturally health claims [152].
Some examples of the best known bioactive-rich cold-pressed oils are virgin
olive oil, an important ingredient of the Mediterranean diet, whose health benefits
are linked to the presence of monounsaturated oleic acid, squalene, triterpenes, and
bioactive phenolic compounds. Among the bioactive phenolic compounds, those

that are raising greater attention interest are tyrosol, hydroxytyrosol, ligstroside and
oleuropein, and dialdehydic forms of elenolic acid. It is recognized for their antiox-
idant, cardiovascular, anti-inflammatory, neuroprotective, and chemotherapeutic
effects and the disease-related gene modulation; cold-pressed sesame oil, typical of
Asian cuisine, is rich in polyunsaturated fatty acids, vitamin E, and phenolic
compounds (such as gamma-tocopherol, sesamin, and sesamolin). Among their
biological activities, we find reduction of oxidative stress, liver oxidative damage
protection, and decreased cholesterol level in the blood; flaxseed oil is not classified
under the category of “cooking oils” whereby it has to be added just before serving.
This oil contains α-linolenic acid, sterols, carotenoids, and a tocochromanol
plastochromanol-8. When it is enriched with particulates, it contains lignans which
are recognized for its protective effect against colon, skin, and prostate cancer;
Camelina seed oil is suitable for cooking due to its resistance to heat. It is rich in
gamma-tocopherol, alpha-linolenic acid, and polar phenolic and possesses antioxi-
dant, anti-inflammatory, and immune system stimulation properties. Virgin argan oil
is rich in monounsaturated fatty acids and linoleic and oleic acids and also contains
squalene, tocopherols, spinasterol, and schottenol (a bioactive sterol). It is suitable
for cooking and frying and in salad dressings; it is very useful in Moroccan cooking;
cold-pressed coconut oil contains antioxidants such as phenolic compounds and
alpha-tocopherol. It is not resistant to heat due to its high content of medium-chain
triacylglycerols; walnut oil is used as uncooked condiment; it is rich in omega-6 and
omega-3 fatty acids. Rapeseed oil contains brassicasterol and plastochromanol-8.
Pumpkin seed oil is rich in linoleic and oleic acids, phenolic compounds, and low
amounts of gallic acid; Echium, black currant, and hempseed oils contain high
amounts of stearidonic acid, whose health benefits have been previously described;
evening primrose seed and borage oils are rich in gamma-tocopherol and gamma-
linolenic acid; macadamia seed oil is rich in alpha-tocopherol and monounsaturated
fatty acids. It is very resistant to heat and shelf-stable; avocado oil can be used for
frying and cooking. It is rich in monounsaturated fatty acids, chlorophylls, tocoph-
erols, and carotenoids [152–154].
At present, they are also being investigated for their anti-inflammatory activity
and high content in tocopherols, carotenoids, alpha-linolenic acid, squalene, and
phenolic antioxidants and oils from other seeds such as blackberry, amaranth, black
caraway apple, black currant, boysenberry, cardamom, cranberry, blueberry, rasp-
berry, strawberry grape, marionberry, sea buckthorn, poppy, cumin, hemp, Pistacia
atlantica, coriander, parsley, and carrot [155, 156].
3 Polar Lipids
The biological activity of phospholipids is mainly based on their amphiphilic

properties that self-assemble the polar heads and the hydrophobic hydrocarbon
chains to create bilayers which are the keystones to build cells’ and organelles’
membranes [157]. Phosphatidylcholine is the main phospholipid in the external layer
of cellular membranes, basic to warrant their function and integrity. Besides its role
in membranes, phosphatidylcholine is also implicated in the hepatic secretion
of VLDL, with an important function in cholesterol and lipid distribution to organs
and tissues. Another role of phosphatidylcholine is the formation of digestive
micelles in the intestinal lumen, as a part of the biliary secretions, promoting the
absorption of lipids. Phosphatidylethanolamine is more abundant in the internal
membrane leaflet and in mitochondria, with a relevant role in growth and stability.
Phosphatidylethanolamine is also a precursor of glycosylphosphatidylinositol and
helps protein binding to the membrane. This last activity can be explained by the
small size of PE, which confers stability to the membrane [158].
After hydrolysis of phospholipids, the formed lysophospholipids participate in
diverse biological activities related to their molecular structures. Multiple activities
have been attributed to lysophosphatidylethanolamine [159]. Lysophosphatidylinositol
has been used as a cancer biomarker [160]. Lysophosphatidylserine [161] partici-
pates in signal transduction related to activation of neutrophil, and it could also have
anti-inflammatory properties. Finally, lysophosphatidylcholine [162] could be used
in rheumatoid arthritis treatments and is also involved in DNA methylation prefer-
entially in the central nervous system [163].
Plasmalogen activities have not been fully described, but its deficiencies have
been correlated with peroxisomal disorders associated with mental retardation,
deafness, or adrenal dysfunction [164]. Moreover, plasmalogens have also potential
as biomarker in different diseases related to aging, such as Alzheimer, oxidative
stress, and inflammation [165, 166].
The role of some phospholipids in some organelles should be also considered.
In this sense, the main phospholipids in mitochondrial membrane are phosphatidyl-
ethanolamine and phosphatidylcholine. One unique property of these membranes is
the presence of cardiolipin, a tetraacyl phospholipid. One of the most important
activities of cardiolipin in mitochondria is the promotion and organization of the
respiratory chain. Besides, its role is also relevant in keeping fluidity and osmosis
[167]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the
main responsible of oxidative stress. This situation occurs when these species are
produced in excess and the cell is unable to destroy them with the help of natural
antioxidants [168–171]. In addition, ROS can also oxidize cardiolipin. This newly
restructured cardiolipin is related to mitochondria dysfunction associated with mul-
tiple pathologies such as diabetes, obesity, heart failure, hyperthyroidism,
neurodegeneration, and aging. All of these diseases are highly related to oxidative
stress, low levels of cardiolipin, and also anomalous high content of DHA in
cardiolipin [172]. Another important activity induced by fatty acid oxidation prod-
ucts is apoptosis with the participation of the mitochondrial permeability transition
pores (MPTP) [173, 174].
It is well known that many cell and membrane protein functions are related
to membrane lipids [175]. This protein function regulation by membrane lipids
involves mechanisms ranging from specific and non-specific interactions between
proteins and membrane lipids [176]. In general terms, membrane lipids can be
categorized in two broad groups [2]:
1. Lipids that bind to points of recognition of specific proteins, acting as ligands,

such as platelet-activating factor that can be grouped to the G protein-coupled
receptor family [177]. This category is based on a well-known protein-small
molecule interaction without any role on the collective membrane properties.
Such specific interaction is the case of polyphosphoinositide interactions with
specific proteins [178–181]. Another example of specific regulation takes place
with membrane lipids that are cell signaling precursors – such as arachidonic acid
in prostaglandin biosynthesis [182].
2. Lipids that, by altering their biophysical properties, have an impact on membrane
and membrane protein functions and organization [183, 184].
Membranes behave as macrostructures with a collective physical property such as

thickness, curvature, or elasticity [185–188]. An example of non-specific regulation
of membrane protein function by the membrane lipids occurs by hydrophobic
coupling between both molecules. Protein activity is modulated by the hydrophobic
thickness and intrinsic curvature of the lipids at the bilayer [188], indicating that
changes in bilayer physical properties can modify membrane protein conformation
and function [189].
Sphingolipids (SLs) are ubiquitous constituents of cell membranes in many pro-
and eukaryotic organisms. In vertebrates, they are also found in lipoproteins
(especially LDL) and other lipid-rich structures, such as epidermis. For a long
time, SLs were considered to play primarily structural roles in the membrane
formation and fluidity regulation. Likewise, SLs have long been recognized as
important structural components of the epidermis, securing the epidermal perme-
ability barrier [190]. However, over the last decades, intensive research on
SL metabolism and function has recently evidenced that SLs function as effector
molecules in several cell processes and have key roles in stimulus/agonist-mediated
signaling pathways involved in cell response modulation. Hence, sphingomyelin
participates in regulatory functions by interaction with specific proteins and serves as
a receptor for the intake of transferrin, promoting the incorporation of iron to the
cells. On the other hand, the most important property of sphingomyelin is its
implications in rafts together with cholesterol, in both cell membranes and lipopro-
teins. It has been recently proposed that metabolism of sphingomyelin and choles-
terol is closely related. Sphingomyelin could be responsible for cholesterol
distribution in cells [191, 192]. Sphingomyelin has been extensively used as a
chemopreventive agent, taking into account its activities as messenger in develop-
ment, growth, differentiation, and apoptosis of human cells.
SLs exert their effects through two general mechanisms:
1. Biophysical mechanism (based on lipid-lipid interactions), whereby SLs, mainly

sphingomyelin and glycosphingolipids, and cholesterol spontaneously self-
associate and fuse into SL-enriched platforms (also referred to as microdomains
or rafts). The formation of these microdomains has consequences to cell signaling
by altering membrane structure and, subsequently, the interaction of protein
membrane receptors with the cell membrane bilayer [193–198].
2. Biochemical mechanism (based on lipid-protein interactions), whereby SLs act as

first and second messengers by interacting with several target proteins or recep-
tors in a number of cell signaling pathways [199, 200].
In addition, a comprehensive understanding of bioactive SL function requires

further knowledge of the metabolic organization of enzymes of SL metabolism and
their interrelationships, as well as the compartmentalization of SL-mediated signal-
ing pathways, the metabolic transformation of bioactive SLs, and the integration or
coordination of overall responses. A number of studies have shown that many
pathways of SL metabolism constitute an interconnected network of specialized
and compartmentalized enzymes that not only regulates the levels of individual
biologically active molecules but also their interconversion and, ultimately, the
balance among them.
Ceramides (Cer), which are composed of sphingosine (Sph) (core of all SLs)
esterified to a fatty acyl chain via an amide linkage [201, 202], are the center of SL
biosynthesis and catabolism and precursors of complex SLs (Fig. 1a). Briefly, Cer
can be produced through the de novo pathway (from serine and palmitate by the
action of the serine palmitoyl transferase (SPT), ceramide synthases 1–6 (CerS1–6),
and desaturase (DES)) and the hydrolysis of complex SLs, especially sphingomyelin
(SM), by several sphingomyelinases (SMases). In SL biosynthetic reactions, Cer
is primarily used for the synthesis of more complex SLs, including ceramide-1-
phosphate (C1P), through phosphorylation by ceramide kinase (CK) [203];
sphingomyelin (SM), by transferring a phosphocholine head group from phospha-
tidylcholine, through the action of SM synthases (SMS) [204]; or glycosphingolipids
(GSLs), through glycosylation (mainly with glucose and galactose) by glucosyl- and
galactosyl-Cer synthases (GCS) [205]. In the catabolic pathway, glycosphingolipids
are cleaved to glucosylceramide (GlcCer) and galactosylceramide (GalCer), which
are subsequently hydrolyzed by specific β-glucosidases and galactosidases to release
Cer [206]. Cer can also be metabolized by ceramidases (CDases) [207–209] to yield
sphingosine (Sph), which in turn is phosphorylated by sphingosine kinases (SKs) to
generate sphingosine-1-phosphate (S1P). S1P can be cleared by the action of specific
phosphatases that regenerate Sph or by the action of a lyase that cleaves S1P into
ethanolamine-1-phosphate and a C16-fatty-aldehyde [201]. The many pathways of
sphingolipid metabolism constitute an interconnected network that not only regu-
lates the levels of individual biologically active molecules but also their intercon-
version and, ultimately, the balance among them.
Most enzymes of sphingolipid metabolism show specific subcellular localization(s)
(Fig. 1b). This could dictate distinct functional responses, since, coupled with the
poor solubility of SLs in cells, it imposes clear restrictions on the subcellular
localization of bioactive lipids. Thus, in the absence of specific mechanisms of SL
transport, the site of generation of a bioactive SL is likely to dictate its site of action.
Among all of these SLs, the most extensively studied so far have been sphingosine
(Sph), ceramides (Cer), and sphingosine-1-phosphate (S1P). Cer and Sph are
reported to act as tumor-suppressor lipids involved in both intracellular and extra-
cellular processes. The long-chain amino alcohol sphingosine (Sph) was the first
Fig. 1 Sphingolipid metabolism. (a) Sphingolipid biosynthesis. (b) Sphingolipid localization
SL metabolite to be identified. Sph has been connected with cellular processes

such as inducing cell cycle arrest and apoptosis by modulation of protein kinases
and other signaling pathways. It has roles in regulating the actin cytoskeleton
and endocytosis and has been shown to inhibit protein kinase C (PKC) [210]. Kinase
targets for sphingoid bases including pkh1p and pkh2p have been found in yeast,
indicating functions in regulating endocytosis, cell cycle arrest, and protein
synthesis [211]. Sphingosine is, along with other sphingoid bases (sphinganine or
dihydrosphingosine), the core of all sphingolipids. The simplest SLs, ceramides
(Cer), are composed of a sphingoid base esterified to a fatty acyl chain via an
amide linkage [201, 202]. The length of the fatty acyl chain (from C14 to C28)
conjugated to the sphingoid backbone characterizes the different species of cer-
amide, which have shown diverse biological functions.
Many different ceramide species have been observed in different human tissues
(including brain, skeletal muscle, skin, testicular, leukocyte, cardiomyocyte, and
hepatocyte tissues), differing in relative abundance, fatty acid composition, and,
hence, biological function [212, 213]. Ceramides often exert their effect via bio-
physical mechanism by forming ceramide-rich platforms that modify both receptor-
and stress-mediated cell signaling and, hence, may influence various disease states
and neurotransmission [214–217]. Thus, for example, Kolesnick and Gulbins have
recently proposed a mechanism by which FasL/Fas interaction leads to caspase-8-
dependent activation of acid sphingomyelinase, with the resultant ceramide forma-
tion capable of orchestrating raft reorganization into large cell-surface microdomains
where the proteins of the death-inducing signaling complex of Fas can oligomerize
thereby amplifying or modifying cell signaling [218]. Moreover, it is believed that
ceramide-rich platforms serve to cluster the receptors for the pathogen, and the
negative membrane curvature induced by ceramide facilitates cellular invasion of
pathogens, including several microbes, parasites, and viruses [219–223]. Often the
end result of ceramide-mediated cellular entry of pathogens is containment and/or
inactivation of the pathogen. Moreover, ceramides may also influence membrane
permeability via interactions with ion channels [224].
In addition, Cer may act as second messengers in a number of stress stimulus-
mediated signaling pathways by regulating specific protein targets, mainly phospha-
tases (as PP1A and PP2A) [225] and kinases (as protein kinase C (PKC) z [226],
raf-1 [227], and the kinase suppressor of Ras [228]). In this way, ceramides play a
role in multiple vital cell processes such as cancer cell growth, differentiation,
senescence (by telomerase inhibition and suppression of key mitogenic pathways
[229]), and apoptosis [200, 230]. Many cytokines, chemotherapeutic agents, and
other stress-causing agonists (such as heat stress, ultraviolet (UV), ionizing radia-
tion, DNA damage, and ligation of death receptors) result in an increase of endog-
enous ceramide levels through de novo synthesis and/or the hydrolysis of
sphingomyelin [231, 232] (Fig. 1a). Reciprocally, decreased levels of endogenous
ceramide caused by increased expression of glucosylceramide synthase (GCS),
which clears ceramide levels by incorporating it into glucosylceramide, result
in the development of a multidrug resistance phenotype in many cancer cells
[233]. In contrast to the actions of ceramide, S1P is emerging as a key regulator
of proliferation, inflammation, vasculogenesis, and resistance to apoptotic cell
death [234]. Many growth factors, such as epidermal growth factor and platelet-
derived growth factor, as well as the cytokines TNF-α and IL-1, activate SK1
acutely, resulting in transient elevations in the levels of S1P. S1P is then secreted
from the cell and acts either in a paracrine or autocrine manner to engage specific
transmembrane hepta-helical G-protein-coupled receptors (GRCR). In mammals,
S1P receptors are widely expressed and are thought to regulate important
physiological actions, such as immune cell trafficking, vascular development,
vascular tone control, cardiac function, and vascular permeability, among others.
In addition, S1P may participate in various pathological conditions. For example,
S1P has been implicated as an important mediator in autoimmunity, transplant
rejection, cancer, angiogenesis, vascular permeability, female infertility, and myo-
cardial infarction [235].
Sphingolipids other than ceramide and S1P are emerging as candidate bioeffector
molecules. These include C1P, which has roles in activation of phospholipase A2
[236, 237], release of arachidonic acid in response to interleukin-β (41), regulation
of vesicular trafficking, phagocytosis [238], macrophage degranulation [239],
and mitogenesis [240]. Dihydroceramide, which had been shown to be inactive
in apoptosis, has been implicated in mediating the growth inhibitory actions
of fenretinide (a retinoid analogue used in the treatment of neuroblastoma),
which has been shown to inhibit the activity of the desaturase [241, 242]. Lyso-
sphingomyelin as been shown to induce multiple cellular effects, possibly mediated
by binding to specific GPCRs [243]. Finally, glucosylceramide (GluCer) has been
implicated in resistance to chemotherapeutic agents [244] and may serve as the
endogenous cargo for the P-glycoprotein transporter MDR1 [245]. Another property
of GSLs, such as monoglycerides and gangliosides, with application in health
sciences is their protective effect against certain pathogens. As commented above,
several microorganisms, microbial toxins, and viruses link themselves to the cells
using SL-enriched platforms. Therefore, when such compounds are present in the
diet, they compete for the active union sites and help in the elimination of pathogens
from the intestine [246]. Since microbial adherence is the first step in infection [247],
site competition may have a protective effect against pathogens from food sources.
In addition to GSLs, other complex lipids that have attracted a lot of attention
because of their significant biological and technological functions are glycogly-
cerolipids [248]. They are distinguished from GSLs by their lipid moieties,
having glycans linked to the C-3 hydroxyl of diacylglycerol, and are very minor
constituents of most animal tissues, other than the testes. Several biological
activities, such as anti-algal, antiviral, antitumor, and anti-inflammatory, have been
attributed to galactolipids [248]. Monogalactosyl diacylglycerols and digalactosyl
diacylglycerols have shown inhibitory activity of DNA polymerase, antitumor
promotion, inhibition of cancer cell proliferation, and antiangiogenesis properties
[249–251]. In addition, hydrolyzed glycolipids have higher anticancer activity than
the non-hydrolyzed form [249]. The anti-inflammatory activity of monogalactosyl
diacylglycerols and digalactosyl diacylglycerols is lower as the fatty acid saturation
degree is increased [251]. Apart from that, digalactosyl diacylglycerols have also
been proposed for controlling the appetite [252].
GSLs are widely distributed in microbes and plants. Due to their amphipathic
properties, they are collectively known as biosurfactants (BS) [253]. BS offer several
advantages as compared to their surfactant homologues derived from petroleum, due
to their low toxicity, high biodegradability, environmentally friendly, high foaming
capacity, and high selectivity and specificity at extreme temperatures, pH, and saline
conditions. They are also synthesized from renewable sources and have a low critical
micellar concentration and high surface activity and biological activity, which is
important in the therapeutic and biomedical (antimicrobial) fields. Therefore the
interest in BS has increased considerably in recent years, as well as their potential
application in different industries as there is a higher concern for the protection of the
environment and sustainability [254, 255].
The oral application of dietary glycerophospholipids with a specific fatty acid
composition has the potential to cause defined alterations of the fatty acid compo-
sition of membrane phospholipids within a certain cell type. As a consequence,
cellular functions, including signaling and transport, as well as the activity of
membrane-bound enzymes, could be modulated by dietary phospholipids and
hence contribute to their health benefits [256]. Recently, lipid replacement therapy
has surged as a natural medicine approach to restore function in cell membranes and
organelles by replacing damaged membrane lipids [257]. Besides, the importance of
polar head groups from membrane lipids in protein-lipid interactions is also relevant
to the coupling of hydrophobic complementary structures, such as diacylglycerol
chains from phospholipids and proteins [258]. This coupling can be perturbed by
oxidation of the acyl chains from diacylglycerols. This oxidation and subsequent
disorder can promote enzyme activation [184, 259]. The proposed mechanism is the
change produced in the hydrocarbon chain packing and interruption of hydrophobic
bounding. This hydrophobic match can be facilitated by the conformational state of
the lipids or by selecting the optimum lipid species for that particular hydrophobic
coupling [260–262]. In order to reduce phospholipid oxidation and degradation that
take place along ingestion, digestion, and adsorption, phospholipids utilized in oral
lipid replacement therapy should be protected from acid pH of the stomach, alter-
ations by bile salts, and breakage by pancreatic enzymes and microflora. This task
can be achieved by complexation of phospholipids with certain fructooligosac-
charides, which insert in the phospholipid micelles and prevent interactions with
other molecules [263, 264]. Another important feature of phospholipids is that
enrichment of plasma membranes of the colonic mucosa with these molecules
protects from different pathogenic conditions such as ulcerative colitis and other
chronic inflammatory. The proposed mechanism for that is the hydrophobic barrier
they provide, which regulates signaling pathway involved in different conditions
such as inflammation [265]. Lipid replacement therapy has applicability in fatigue
associated with cancer and cancer therapies [266, 267]. In this sense, a nutritional
supplement called NTFactor [268] has been successfully utilized in cancer-
associated fatigue that was reduced by 30% in an 8-week treatment. The combina-
tion of lifestyle and lipid replacement therapy could be a very efficient treatment to
improve mitochondrial fusion and decrease inflammation and oxidative stress,
becoming a powerful remedy in noncommunicable prevalent diseases. As an exam-
ple, a pilot trial utilizing lipid replacement therapy has shown metabolism changes in
the direction of reducing body mass and moderating appetite [269]. In this sense,
alterations in fatty acid metabolism and glucose have an influence in insulin resis-
tance and metabolic syndrome [270]. Considering that metabolic syndrome has been
associated with the excessive presence of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) in membranes, lipid replacement therapy could

restore big oxidized membrane fragments in blood lipoproteins. New dietetic pat-
terns in conjunction with the administration of membrane phospholipids can remove
oxidized cholesterol and phospholipids from HDL and LDL [271].
4 Glyceryl Ethers
Glycerol-based ether lipids (Fig. 2) are normally minor constituents of cell mem-
branes of mammals, and its presence is major in the liver of some fish of the
Chondrichthyes class [272]. Different bioactive properties have been attributed to
1-O-alkyl-sn-glycerols (AKGs). The oral administration of AKG reduced the tumor
growth and the number of pulmonary metastases of mice inoculated with 3LL cells
[273]. AKG with hydrocarbon chains of 18:1, 14:0, and 16:1 showed the most active
antiproliferative effect, compared to 16:0 and 12:0, inducing a reduction of the tumor
blood vessel endothelial marker, suggesting an antiangiogenic effect. AKGs reduced
also the major angiogenesis stimulator, basic fibroblast growth factor (bFGF), on
endothelial cell proliferation [274] and influenced endothelial cell growth, without
showing cytotoxic effects, decreasing the cell proliferation [275].
Fig. 2 Main glyceryl ether

chemical structures
The antiproliferative effect of AKG was also tested in human mammary

carcinoma (MCF7), ovarian carcinoma (OVP10), and prostate cancer (DU45,
LnCap) cell lines, showing an increased percentage of apoptotic cells of ovarian
and prostate carcinoma and a significant reduction in the number of prostatic
cells [276, 277]. A methoxy-substituted alkylglycerol inhibited the growth
of three human colon cancer cell lines (Moser, HT29, and HCT116) [278].
AKG supplementation was able to reduce Walker 256 tumor cell growth propor-
tionately to the increase of lipid peroxidation, apoptosis, and reduction of cancer cell
proliferative capacity. Additionally, the cachexia state, associated to patients of
cancer and other progressive illnesses, was reversed with the intake of AKG [279,
280]. The prophylactic administration of AKG on patients affected by cancer can
reduce the side effects and complex injuries derived from the radiation treatments, in
particular leucopenia, thrombocytopenia [281], and fistulas [282].
Cytotoxic macrophages are activated by AKG, enhancing Fc-receptor-mediated
phagocytosis, increasing humoral immune response, and delaying hypersensitivity
reactions [283]. Regardless the carbon chain length, AKG stimulates the production
of IL-12, a cytokine involved in the activation of Th1 responses [284], and IL-2
[285], enhancing the proliferation and activation of lymphocyte B, whereas levels of
IL-4 and IL-10, involved in the activation of Th2 responses, are decreased [286].
Levels of C1q are also raised with the administration of AKG [287]. The intake of
AKG to very old people before surgical treatment induces a significant increase of
WBC, lymphocytes, IgM, and IgA in the postoperative period [288]. The adminis-
tration of AKG to gestating and lactating mammals induces a positive effect on the
immune system of the litter, with higher concentrations of IgG, erythrocytes, and
hemoglobin in their blood and a modification on the lipid profile and immune
properties of the mother’s milk [289, 290].
Short-chain AKGs have the ability to increase blood-brain barrier permeability,
allowing therapeutic molecules to cross it [291–293]. Even the penetration of high-
molecular-weight materials, such as albumin and antibodies, into the brain could be
significantly increased [292, 294]. The opening of the blood-brain barrier by the
action of AKG is short and reversible and does not affect tight junction [295].
A recent experimental study has shown that unsaturated AKGs can promote
a decrease in the high-fat-induced obesity and improve the insulin resistance in
rats [296]. Likewise, the administration of AKG in obese patients can reduce the
total cholesterol levels as well as complements 3 and 4, associated with higher risk of
metabolic syndrome [297].
The in vitro treatment of spermatozoa improves their motility and fertility, related
to PAF metabolism, resulting in a better fertilization performances when used for
artificial inseminations [298]. In vivo studies have shown similar effects in motility
and velocity of sperm after the oral intake of AKG [299].
AKG have been reported to exhibit antibacterial activity against several strains by
releasing or activating proteases, such as the enzyme autolysin [300], increasing
their activity with 1-O-chains from C8:0 to C12:0 and decreasing with longer 1-O-
chains [301].
5 Isoprenoids
Isoprenoids are considered one of the most diverse families of compounds produced
by biological systems; approximately 40,000–70,000 isoprenoid molecules are
known, including sterols, carotenoids, and quinines [302–305]. Among their bio-
logical activities are maintenance of membrane fluidity, electron transport, protein
prenylation, and cellular development. They are also utilized as fragrances and
essential oils, antibacterial and antifungal agents, and highly valuable pharmaceuti-
cals and fuel alternatives [304, 305].
According to the number of isoprene (C5) units that they contain, terpenes are
classified in hemiterpenoids (C5), such as isopentenols; monoterpenes (C10), such
as menthol and camphor; and sesquiterpenes (C15), such as zingiberene (ginger).
These are the major constituents of herbs and spices. Other sesquiterpenes and
diterpenes (C20) are pheromones, defenses, and signal transduction substances
[306–308]. The roles of isoprenoids with higher molecular weight are membrane
stabilization (such as cholesterol and other C30 compounds) and photoreception
(such as carotenoids and other C40 compounds).
Many terpenoids have been found to exhibit potent biological activity, with
several of them in development or in use therapeutically. Taxol, a diterpene extracted
from the Pacific yew, is extremely effective in the treatment of certain cancers
(ovarian, breast, lung and neck, bladder and cervix, melanoma, and Kaposi’s sar-
coma) [305, 309, 310]. A range of medicinal diterpenoid compounds (i.e., phorbol
esters and the related casbanes, lathyranes, jatrophanes, and ingenanes) are solely
produced in Euphorbiaceae and Thymelaeaceae species [311] from casbene and
neocembrene diterpene backbones [312]. These diterpenoids have gained interest
due to unique anticancer, anti-HIV, vascular relaxing, neuroprotective, anti-
inflammatory, or immunomodulatory activities [311, 313–316]. The monoterpene
limonene and related derivatives are believed to inhibit farnesylation of the growth-
promoting protein RAS, inhibiting malignant cell proliferation [317–319]. The
ability to produce terpenoid drugs in microbes could significantly reduce their
production costs, reduce pressure on unsustainable plant-derived sources, and
increase their chances of reaching clinical trials and the market.
Tocopherols and tocotrienols also known as tocochromanols are a group
of compounds with vitamin E activity with numerous biological activities essential
for human nutrition. They are comprised of different molecules: α-, β-, γ-, and
δ-tocopherol and α-, β-, γ-, and δ-tocotrienol [320]. Vitamin E is incorporated in
plasma through chylomicrons. The transformation of chylomicrons to remnant parti-
cles is the mechanism to distribute the absorbed vitamin E to circulating lipoproteins
and tissues [321]. On the other side, newly absorbed dietary lipids are incorporated
into nascent very-low-density lipoproteins in the liver. This organ controls the release
of α-tocopherol into blood plasma. This release is mediated by α-tocopherol transport
protein (TTP). When TTP is absent, tocopherol is not secreted back into the plasma,
and excess vitamin E is metabolized and excreted by the bile.
TTP is responsible of α-tocopherol transport to vital organs but is poorly effective
on tocotrienols [322]. Surprisingly, in some tissues, the level of tocotrienol is higher
than that of tocopherols pointing out the presence of an alternative tocotrienol

transport system in vivo [323].
Emulsification is a technique broadly utilized to improve absorption of hydropho-
bic drugs. One of the most well-known formulations are SEDDS (self-emulsifying
drug delivery systems) [324–326]. In addition, soft gelatin capsules (Tocovid
Suprabio™, Carotech Inc., NJ) of tocotrienol have been also developed. A study
utilizing Tocovid Suprabio™ has studied the postabsorptive fate of the different
tocotrienol isomers associated with lipoprotein subfractions in humans [327].
α-Tocotrienol concentrations in supplemented individuals are distributed among
3 μM in blood plasma, 1.7 μM in LDL, 0.9 μM in triglyceride-rich lipoprotein,
and 0.5 μM in HDL. The plasma concentration of α-tocotrienol observed in SEDDS
is two to three times higher than that previously reported in using generic supple-
ments [328, 329].
Considering structural similarities in all eight tocols, it is logical to think they
should have comparable antioxidant efficacy. However, current studies indicate that
some members of the vitamin E family possess unique biological functions not
shared by other family members. For example, α-tocopherol may inhibit platelet
adhesion apart from its antioxidant properties. In addition, α-tocopherol has shown
anti-inflammatory, antineoplastic, and natriuretic functions related to specific bind-
ing interactions [330]. Tocotrienols’ main peculiarity is the presence of three trans-
double bonds in the hydrocarbon tail. These unsaturations in the isoprenoid side
chain provide a unique conformation [331]. For this reason, α-tocotrienol is much
more flexible producing greater curvature stress on phospholipid membranes. It has
been described that α-tocotrienol possesses numerous functions that are not shared
by α-tocopherol [332]. Hence, tocotrienol inhibits the activity of 3-hydroxy-3-
methylglutaryl coenzyme A (HMG-CoA) reductase, key enzyme in cholesterol
biosynthesis [333, 334]. This activity is not shared by tocopherols [335]. In addition,
tocotrienol, but not tocopherol, reduces oxidative damage in proteins [336].
It is well known that pure and mixed isoprenoids possess anticancer activity
[337]. In strict sense, it should be indicated that tocotrienols, but no tocopherols, are
isoprenoids. Regarding the neuroprotection activity, α-tocotrienol seems to be the
most potent isoform [332, 338–340], and γ- and δ-tocotrienols have been considered
as the most potent anticancer isoform of all tocotrienols.
One special group of isoprenoid derivatives are sterols with relevant functions in
eukaryotes, including higher plants [341]. Two predominant types of sterols, cho-
lesterol and ergosterol, are found in animal and fungal cells, respectively [342]. One
of the principal functions of sterols is the structure ordering of the biological
membranes [343]. Different biophysical studies of the structure and physicochem-
ical properties of membrane sterols have indicated that cholesterol shields negative
charges reducing the net membrane surface charge [344]. It also contributes to closer
packing of hydrocarbon chains in the gel phase and increasing membrane micro-
viscosity and decreasing its permeability. Membrane permeability is affected by free
sterols at different extents. The highest permeability reduction is attained with
cholesterol followed by campesterol, β-sitosterol, and stigmasterol [345]. Some
studies have attributed to β-sitosterol and campesterol, a role in ordering fatty acid
chains at the membrane, which have an influence on water and ion permeability and
also on the activity of some membrane proteins [346].
Sterols also act as important regulatory molecules. They are hormone precursors,
regulating plant growth and development [342, 347, 348]. Moreover, sterols are
implicated in the formation of specific lipid membrane microdomains called “lipid
rafts,” with an important role in cells as transmembrane signal transduction and
enzyme anchoring [349, 350].
Sterols in plants are present in three different chemical forms: free sterols,
esterified with fatty acids (sterol esters), steryl glycosides, and acylated steryl
glycosides, also known as the carbohydrate derivatives of sterols. Much higher
proportion of free sterols is found in plants compared to sterol esters. It has been
established that the main role of sterol esterification is to maintain the physiological
level of sterols in cell membranes [351].
β-Sitosterol has been extensively utilized in drugs and dietary supplements
for restoring lipid and cholesterol level in humans. Plant sterols and saponins combined
have shown hypolipidemic and angioprotective activities [352]. Another biological
activity attributed to β-sitosterol is inhibition of cancer cell proliferation besides other
pharmacological properties such as anti-hypercholesterolemic [353], anti-inflamma-
tory, and antiangiogenic [354–356]. A proposed mechanism of β-sitosterol is the
activation of the sphingomyelin cycle which increases the ceramide production asso-
ciated with apoptosis in various cancer cells, such as human colon cancer cells [357],
human prostate cancer cells [358], human leukemic cells [359], human stomach cancer
cells [360], and human breast cancer cells [361]. It has been recently published that
stigmasterol, sitosterol, campesterol, and brassicasterol may be involved in the regu-
lation of lipid metabolism and the pathogenesis of dementia [362, 363].
The chemical form of phytosterols plays an essential role on their activity, since
it is well known that crystalline phytosterols are not soluble in the bile and,
therefore, are unable to reduce cholesterol absorption [364]. On the contrary,
development of formulations of free phytosterols with several emulsifiers has
produced significant reduction in cholesterol absorption and LDL cholesterol
[64]. Esterification of phytosterols with fatty acids increases their solubilization in
the oily phase and consequently their activity [365]. Differently, phytosterol glyco-
sides have amphipathic structure, which give rise to questions about their solubili-
zation in bile and mechanism of action. Glycosylation is very common in many cell
compounds [366], and for these compounds, bioavailability is the main concern.
For example, more than 80% of phytosterols in potatoes are found as glycosides
[367]. In a study based on a single dose, it was observed that the measured reduction
of cholesterol absorption was similar to phytosterol glycosides and phytosterol
esters [368].
A recent meta-analysis trying to elucidate if plant sterols reduce plasma concen-
trations of fat-soluble vitamins and carotenoids indicates that both plant sterols and
stanols reduce hydrocarbon carotenoid concentrations (β-carotene, α-carotene, and
lycopene), differently affect oxygenated carotenoid concentrations (not reduction in
zeaxanthin and β-cryptoxanthin but not in lutein), and have no influence on tocoph-
erol, retinol, and vitamin D plasmatic concentrations [369].
Finally, a recent study concerning to phytosterol oxidation products points out the
relevance of the assessment of their potential adverse effects [370].
6 Phenolipids
Phenolic lipids are a class of natural products composed of a phenolic head group
and an aliphatic side chain. These compounds are secondary metabolites that are not
essential for cell growth but can assist against possible stress conditions of the
producing organisms, such as infection, wounds, and UV radiation. Phenolic lipids
mainly occur in plants but also in fungi and bacteria. Originally, its production was
attributed to plant families of Anacardiaceae, Ginkgoaceae, Myristicaceae, and
Orchidaceae, but later studies showed that these compounds can be produced by
bacteria and mycobacteria pathogens. Besides, the synthesis of phenolic lipids by
several other plant families was also reported [371].
Chemically, phenolic lipids can be considered as derivatives of mono- and
dihydroxy phenols that generally contain a catechol, a resorcinol, or a hydroquinone
nucleus alkylated by a carbon chain, mostly C11–C17 (see Fig. 3) [372].
Although phenolic lipids can be toxic, causing different allergic issues, they
have been the subject of much attention due to other physiological properties.
Phenolic lipids show amphiphilic properties, caused by the simultaneous presence
of a hydrophilic phenol and a lipophilic alkyl chain in the same molecule. In aqueous
media, they can be organized in micellar structures [373]. They can also incorporate
into phospholipid membranes forming hydrogen bonds with these compounds. As
consequence, phenolic lipids have the potential to affect biophysical properties of
cell membranes, including many cellular metabolic processes, fluidity, charge and
mobility of phospholipids, and the activity of membrane-bound enzymes [372].
Stasiuk and Kozubek extensively reviewed the various biological activities of
phenolic lipids [373]. In addition, the possible participation of these compounds as
antimutagens was suggested, since they can exert protection of cells against carci-
nogenesis and serve as cytotoxic and antitumor agents by causing apoptosis
[374–376]. Moreover, due to their interaction with proteins and/or on their
membrane-disturbing properties, the ability of these compounds to inhibit bacterial,
fungal, protozoan, and parasite development besides the activity of several enzymes
has been reported [377–380].
Among short-chain alkylphenols, the phenylpropanoids are present in essential
oils and include isochavicol and derivatives, which displayed an interesting anti-
plasmodial activity [381].
The group of catechol lipids includes urushiol, which is found in plants of the
family Anacardiaceae, especially Toxicodendron spp., and it is well known by its
allergic properties. The likelihood and severity of allergic reaction to urushiol are
dependent on the degree of unsaturation of the alkyl chain [382]. Similar compounds
with a catechol nucleus esterified with a fatty acid (palmitic, stearic, and oleic acids)
have been synthesized for their lipophilic antioxidant properties. All derivatives
showed better radical-scavenging capacities than α-tocopherol and ascorbyl
Fig. 3 Structural diversity of phenolic lipids [372]
palmitate [383]. Bonediol, an alkyl catechol from the Mayan medicinal plant
Bonellia macrocarpa, showed to have antiproliferative and antiestrogenic activities.
Additionally, bonediol could induce oxidative stress and activation of detoxification
enzymes. For these reasons, it may serve as a potential chemopreventive treatment
with therapeutic potential against prostate cancer [384].
The acid form of urushiol leads to the group of anacardic acids, which also cause
an allergic skin rash on contact. These molecules consisted of a salicylic acid
substituted with an alkyl chain (saturated or unsaturated) that has 15 or 17 carbon
atoms. Moreover, anacardic acids from cashew nut shell liquid, a Brazilian natural
substance, have antimicrobial and antioxidant activities and modulate immune
responses and angiogenesis. The 15 carbon unsaturated side chain compound
found in the cashew plant is lethal to Gram-positive bacteria [385]. In a study in
mice, all doses of anacardic acids improved the antioxidant enzyme activities and
decreased vascular adhesion molecule in vessels. With doses of 50 mg/kg of
anacardic acids, animals showed decreased levels of neutrophils and tumor necrosis
factor in the lungs and bronchoalveolar lavage fluid. Thus, it was demonstrated that
this supplementation has a potential protective role on oxidative and inflammatory
mechanisms in the lungs [386]. In this field, Hamad and Mubofu extensively
reviewed the potential biological applications of bio-based anacardic acids and
their derivatives [387].
Alkylresorcinols, also known as resorcinolic lipids, are phenolic lipids composed
of long aliphatic chains and resorcinol-type phenolic rings [388]. Alkylresorcinols
are relatively rare in nature, with the main known sources being wheat, rye, barley,
triticale (cereal grasses), Ginkgo biloba, several other Anacardiaceae (Anacardium
occidentale), mango latex and peel, and some species of bacteria. In these plants,
resorcinol lipids were also related to strong allergic responses. Alkylresorcinols can
also be used for specific applications, for example, 4-hexylresorcinol is a food
additive (E-586) used as an antioxidant and color-stabilizing agent [389]. Bioactive
properties of alkylresorcinols usually based their ability to integrate into membranes
and inhibit enzymes. A wide number of in vitro studies have been carried out with
alkylresorcinols, ranging from induction of apoptosis, inhibition of lipoxygenases,
and cleavage of DNA to triglyceride reduction in adipocytes [390]. Alkylresorcinols
were shown to have anticancer activities. Hence, in vitro studies presented
inhibition of human colon, breast, lung, central nervous system, adenocarcinoma,
hepatocarcinoma, cervix squamous carcinoma, and ovarian cancer cell lines, at
micromolar alkylresorcinol concentration. Model studies suggest a high cytotoxicity
of alkylresorcinols toward cancer cells; however, intervention studies to confirm
their preventive action are needed [391].
Hydroquinone lipids are phenolic lipids with an aromatic ring belonging to the
hydroquinone group linked to a straight carbon chain. Embelin and sorgoleone and
their derivatives are included in this group of natural compounds reporting bioactive
properties. Embelin was used in traditional Asian medicine as anti-vomiting and
antidiarrhea agent [392], whereas sorgoleone can act as inhibitor of mitochondrial
respiration and photosynthesis, and it has been used as natural herbicide [393].
In addition to the aforementioned compounds, other types of phenolic esters can
also be found in nature. Phenolic compounds constitute a heterogeneous group that
consist of simple phenols and polyphenols as well as their derivatives including
phenolic acids, stilbenes, lignans, and flavonoids, by far the largest group of
phenolics. Hence, for example, alkyl esters of ferulic and/or p-coumaric acids are
present in soybean, cereals, propolis, periderm of potato, latex of sweet potato, parts
of Artemisia assoana, and leaves of Larix kaempferi. In addition, pods of

Piliostigma thonningii, roots of Tanacetum longifolium, and wax of gala apples
contain phenolic fatty acid esters of p-coumaryl alcohol, and docosyl caffeate can be
found in the halophytic plant Halocnemum strobilaceum [394].
In this regard, acylated flavonoids were reported to protect glycosides from
enzymatic degradation, to enhance pigment solubility in water, and to increase
affinity to cell membranes (resulting in better penetration), antioxidant activity,
enzyme inhibitory, and antiproliferative, cytogenetic, and antimicrobial properties.
The enhanced hydrophobicity of these molecules results in a higher degree of
penetration through the cell membrane, which permits them to interact with multiple
cellular or molecular targets leading to their various biological activities [395].
It should to be noted that apart from natural sources, phenolic lipids can also be
obtained from their corresponding phenolic compounds by modification techniques.
Hence, different strategies were intended to produce these new molecules with
potential use in technological applications or with targeted physiological properties.
One of the main purposes of these modifications is to improve the antioxidant
activity of phenolic compounds in a variety of media. Redox properties of phenolic
compounds promote their antioxidant activity, since they can act quenching singlet
and triplet oxygen, neutralizing free radicals, or decomposing peroxides [396]. The
polarity of the environment, in particular, strongly affects the antioxidant activity of
phenolic antioxidants [397]. According to the antioxidant polar paradox, hydrophilic
antioxidants tend to be more active in bulk oils or nonpolar media, whereas lipo-
philic antioxidants would inhibit lipid oxidation more efficiently in emulsions since
they have more affinity for the oil-water interfaces [398]. Because phenolic antiox-
idants are highly polar, its lipophilization becomes a crucial step in the design of new
antioxidant additives and drugs. Basically, lipophilization consists of the esterifica-
tion of a lipophilic moiety (fatty acid or fatty alcohol) to a given substrate resulting in
new more surface-active molecules [399]. Until now, numerous lipophilized antiox-
idants have been synthesized. Modification of a wide range of phenolic acids,
flavonoids, tocopherols, triacylglycerols, or phospholipids has been performed to
obtain lipophilized phenolics, also known as phenolipids [400]. As with ones found
in nature, these structured lipids possess a saturated and/or polyunsaturated hydro-
carbon chain and an aromatic ring bearing one or more hydroxyl or methoxyl
substitutes, attached via an ester bond [401].
These molecules, with an appropriate lipophilicity, have shown improved bio-
availability in vivo over their polar analogues. As reported with natural phenolic
lipids, they enhance miscibility and incorporation into lipid phases and lipocarriers
and can also be used in micellar, emulsified, and liposomal systems, so they open
potential new applications for drug delivery systems and food, nutraceutical, phar-
maceutical, and cosmetic industries [389].
It should be noted that the selection of the critical chain length of the alkyl moiety
is crucial in the design and use of lipophilic antioxidants. Although the polar paradox
states that apolar antioxidants are more active in oil-in-water emulsions than
their polar analogues, the antioxidant activity regarding alkyl chain length is actually
defined by a nonlinear trend [398]. Hence, the efficiency of hydrophobic antioxidants
a Chlorogenate alkyl esters b Rosmarinate alkyl esters

3
15
CAT Value (Trolox eq)
Antioxidant capacity
2.5
2
10
1.5
Critical chain length
1 5 Critical chain length
0.5
0 0
01 4 8 12 16 18 20 01 4 8 12 16 18 20
Alkyl chain length (carbon number)
Fig. 4 Influence of the alkyl chain length of chlorogenate (a) and rosmarinate (b) alkyl esters on
antioxidant activity in stripped tung oil-in-water emulsion [400]
increased with the alkyl chain length in emulsions and other more complex systems as
liposomes, until a maximum is reached, but from that point, the antioxidant activity
remarkably decreases, leading to a “cutoff” phenomenon [402]. Different studies
established that esters of medium-chain length (C8–C12 alkyl chains) provided the
highest activity [398]. Figure 4 shows two examples of different critical chain length
providing the best antioxidant activity for chlorogenate and rosmarinate alkyl esters.
This nonlinear behavior can be explained because antioxidants with medium alkyl
chain length tend to locate more at the oil-in-water interface, where antioxidants can
provide greater protection against oxidation. Increasing the hydrocarbon chain could
drive the antioxidant away from the interface into the emulsion droplet core, where the
phenolics would be less efficient, which may explain the cutoff effect [403].
Apart from their wide use as food additives due to their antioxidant properties,
it has been reported that, for example, propyl (E-310), octyl (E-311), and dodecyl
(E-312) gallates also possess antifungal and antibacterial activities, essentially
on Gram-positive bacteria [404]. Likewise, sapienic acid esters comprising
hydroxybenzyl alcohol, tyrosol, and coniferyl alcohol exhibited some cytotoxicity
toward different tested cell lines [389]. The effect of phenolipids on LDL oxidation
has been of interest recently. Katsoura et al. reported that methyl, ethyl, and octyl
ferulate had significantly higher antioxidant capacity toward LDL, HDL, and total
serum oxidation. In this study, the protection effectiveness increased with the
increasing length of the alkyl chain [405]. In this field, Shahidi’s group lipase-
catalyzed the synthesis of phenolipids to test the potential inhibitory effect of these
compounds on LDL oxidation as well as radical-induced DNA cleavage [389,
401]. Moreover, Danihelová et al. reviewed the biological properties of lipophilic
flavonoid derivatives (see Table 1) [406].
These functionalized antioxidants resulting from the grafting of lipid on a
phenolic moiety can be prepared by a wide range of lipophilization strategies
such as esterification, transesterification, amidation, and etherification. Normally,
lipophilization of phenolic acids can be achieved by chemical, enzymatic, or chemo-
Table 1 Biological properties of lipophilic flavonoid derivatives [406]

Biological
Flavonoid Substituent effect
Naringin, hesperidin, neohesperidin, Butyrate, decanoate, laurate "Antifungal
hesperetin glucoside
Rutin Laurate, palmitate "#Antioxidant
Chrysoeriol-7-O-β-D-(300 -E- Vinyl laurate "Antioxidant,
pcoumaroyl)-glucopyranoside, "antibacterial
chrysoeriol-7-[600 -O-acetyl-β-D-
allosyl-(1!2)-β-D-glucopyranoside]
Flavone, isoflavone Prenyl, geranyl, dimethylallyl, methyl, Antiproliferative
methoxy
Flavone Geranyl Antiproliferative
Isorhamnetin-3-O-glucoside Ethyl laurate, ethyl butyrate "Anticancer,
#antioxidant
Quercetin Tert-butylhydroxy "#Antioxidant
Isoquercitrin Butyrate, caproate, caprylate, "#Antioxidant,
decanoate, laurate, palmitate, stearate, "anticancer
oleate
Flavane, thiaflavane Methoxy "#Antifungal
Rutin, phloridzin, esculin Butyrate, caproate, caprylate, "#Antiprotease
decanoate, laurate, myristate, palmitate,
stearate, oleate, linoleate, linolenate,
arachidonate, erucate
Flavone Methylethylamine, diethylamine, Neuroprotective
piperidine, pyrrolidine
Flavone Alkyl "Anti-
inflammatory
Rutin, naringin Oleate, linolenate, linoleate "Anticancer
Quercetin Alkyl "Antioxidant
Rutin Butyrate, caproate, caprylate, "Antioxidant
decanoate, laurate, palmitate, stearate,
oleate, linoleate, linolenate
Biochanin A Alkyl #Anti-
inflammatory
Flavone Methyl, naftyl, nitro, halogen "Anticancer
Chrysin Methoxy, dodecoxy, diacetyl, "Anti-
methoxycinnamate inflammatory
Flavone, isoflavone Trifluoromethyl #Anticancer
Flavanone Methoxy, benzyl "Anticancer
Flavone Aliphatic and heterocyclic with N "#Anticancer
enzymatic esterification of (a) the carboxylic acid group (COOH) of phenolic acid
with fatty alcohols or (b) the phenolic hydroxyl group (OH) with fatty acids [403].
Although chemical esterifications are quite quick, cheap, and simple, they usually
involve drastic conditions of temperature and pH, so they can promote deoxidation of
phenolic compounds. In addition, as another drawbacks, chemical processes are
generally not selective and require many steps to remove residues and by-products
[399]. On the other hand, enzymatic syntheses offer some advantages since they provide
high selectivity, occur at mild conditions, are environmentally friendly, and require fewer
purification steps. However, it should be taken into account that enzymatic processes
may imply high costs, possible deactivation of the catalysts, and longer reaction times.
Phenolic acids have been efficiently esterified with enzymes of the carboxylic ester
hydrolase family, such as lipases, tannin acyl hydrolases, feruloyl esterases, and
cutinases, by using different organic solvents or in a solvent-free media [407].
As aforementioned, synthetic lipophilization of phenolic acids with fatty alcohols
can be used as a tool to produce these new amphiphile antioxidants. Figueroa-
Espinoza et al. report some examples to obtain phenolipids from phenolic acids
via chemical or enzymatic esterification at different conditions [407]. More partic-
ularly, Figueroa-Espinoza and Villeneuve extensively reviewed the background of
the enzymatic esterification of phenolic acids as caffeic, cinnamic, ferulic,
chlorogenic, gallic, etc. with fatty alcohols with different chain length [394].
The acylation of flavonoids, a class of phenolic compounds with a wide range
of bioactive properties, has been the subject of much investigations, since it
also permits to enhance their solubility in various media, stability, and antioxidant
activity, among other physiological properties [408]. As with phenolic acids,
acylation of flavonoids can be performed by using chemical catalysts [409].
However, apart from other advantages of enzymatic technology aforementioned,
the regioselectivity of enzymes plays a crucial role in these processes, since it
remarkably affects the functionalization of phenolic hydroxyl groups, which is
responsible for the antioxidant activity of flavonoids. Chebil et al. extensively
reviewed a multitude of investigations dealing with the regioselectivity and perfor-
mance of the enzymatic acylation of flavonoids [410]. Several factors should be
taken into account in order to enhance the performance of acylation, e.g., type,
regioselectivity, and load of enzyme, nature of the flavonoid, acyl donor, operating
conditions, etc. The reaction can be carried out using different organic solvents or
almost solvent-free systems [411]. In this regard, both ionic liquids [412] and
eutectic mixtures [413, 414] have also provided good properties as solvents, becom-
ing an alternative solution for these enzymatic transformations.
Lipophilization with different acyl donors has also been applied to other polar
antioxidants to increase its solubility and activity in nonpolar matrices. For example,
ascorbic acid (vitamin C) has traditionally been hydrophobized by esterification or
transesterification reactions with different aliphatic chains [415], mainly palmitic
acid [416], or even with polyunsaturated fatty acids [417].
Last decades, there is a growing interest in the development of structured lipids
with enhanced functional properties and health benefits. Structured lipids are orig-
inally triacylglycerols in which the composition and the distribution of fatty acids at
the glycerol backbone are modified. The use of structured triacylglycerols seemed to
be the most efficient way of delivering bioactive fatty acids, increasing their bio-
availability, and thus enhancing their physiological effects [418]. Improvements in
the solubility and miscibility of phenolic compounds in nonpolar systems can be
attained upon their incorporation into triacylglycerols [419]. Hence, the structuring
of triacylglycerols with phenolic acids could potentially result in novel structured

phenolic lipids becoming an interesting strategy for medical, nutraceutical, and food
applications [420]. These novel structured molecules may provide a great number of
beneficial properties by the combination of bioactive fatty acids (e.g., PUFA) and
phenolic compounds in the same molecule [421]. Phenolic structured lipids were
generally obtained via transesterification by acidolysis using enzyme catalysts.
Acidolysis can be used to replace the existing fatty acids in the triacylglycerol
molecule by other desired fatty acids, phenolic acids, or other organic acids [422].
It should be noted that Novozym 435 (Candida antarctica lipase) was the most
widely used and showed the best catalysis performance [423]. As examples, this
lipase was employed to effectively incorporate dihydrocaffeic acid in flaxseed
oil [424], p-coumaric acid in seal blubber oil and menhaden oil [401], dihydrocaffeic
acid and ferulic acid in trilinolein and trilinolenin [425], and cinnamic acid in
triolein [426]. The same methodology was also used for acidolysis between flaxseed
oil and selected phenolic acids (ferulic, p-coumaric, sinapic, etc.), including hydrox-
ylated and/or methoxylated derivatives of cinnamic, phenyl acetic, and benzoic
acids [419]. Besides, synthesis of phenolic lipids by transesterification of ethyl
ferulate with castor oil [423], dihydroxyphenylacetic acid and dihydrocaffeic acid
with krill oil [427], 3,4-dihydroxyphenyl acetic acid [421], and other selected
phenolic acids [428] with flaxseed oil was affected in solvent-free media, using
Novozym 435 from Candida antarctica as the biocatalyst. Supercritical carbon
dioxide is a green solvent that was also used as solvent in the reaction medium for
the acidolysis between flaxseed oil and ferulic acid [429].
The synthesis of structured phenolic lipids has great potential in the production
of lipids with health benefits. On the one hand, it permits the combination of the
antioxidant capacity of the phenolic compounds with the bioactive properties
of specific fatty acids in the triglyceride molecule. On the other hand, the
lipophilization of phenolic compounds occurs, improving the solubility, stability,
and activity of these compounds in nonaqueous systems. For these reasons, struc-
tured phenolic lipids have potential applications in functional foods, nutraceuticals,
pharmaceuticals, or cosmetics for health promotion and disease risk reduction.
7 Lipid Delivery Systems
As described above, a number of lipid-based compounds have shown in vitro

and in vivo health benefits besides their normal nutritional role [430], so that
their consumption is thought to promote human wellness and to reduce the risk of
certain chronic diseases. Moreover, some of them also provide specific functional
attributes to the product, as desirable color, flavor, or texture, and longer shelf life.
Consequently, the interest of food and beverage industries in the incorporation of
these compounds (called from now “lipophilic actives”) into commercial products is
growing [431–433]. Moreover, many of the lipophilic actives covered in this book
chapter are also applicable as nutraceuticals (therapeutic/preventive agents) and
cosmetics.
However, application of lipophilic actives is often challenging due to their

physicochemical properties, such as low chemical stability, poor water solubility,
low oil solubility, high oil-water partition coefficient, high melting point, and
crystalline state at room temperature [431, 434, 435]. Such characteristics make
them incompatible with the aqueous matrix of food and beverage products and prone
to physical, chemical, and biological degradation, either during product storage or
during gastrointestinal (GI) digestion and systemic circulation (extensive metabo-
lism and quick clearance). This may result in the generation of off-flavors or, even,
potentially toxic reaction products, a poor and variable bioavailability, and the loss
of in vivo bioactivity. All this limits industrial applicability of this kind of com-
pounds, thereby highlighting the need of appropriately formulating them before their
final application to improve these aspects.
Formulation strategies usually used by food, pharmaceutical, and cosmetic indus-
tries to overcome limitations of lipophilic actives include:
1. To mix them with other oil phase components (usually edible neutral oils) prior to
preparing the product (conventional emulsion-based products) [436]. Neutral oils
used for this purpose are usually TAG or terpene oils, which, moreover, make the
product more palatable or desirable to consumers.
2. To modify their chemical structure, mainly by esterification [437].
3. To incorporate them into a suitable nanoparticle-based and microparticle-based
delivery system before their final application [431, 432, 438].
The latter is the most commonly used strategy to efficiently incorporate lipophilic
actives into commercial products and increase their in vivo efficacy after product
ingestion or nutraceutical administration (usually by oral route). Preferably used
delivery systems are those manufactured from food-grade ingredients (such as pro-
teins, carbohydrates, lipids, and surfactants) and specifically designed for oral
administration applications.
Among them, polymer-based delivery systems (PBDS) (as polymeric micelles,
polymeric nanoparticles (PNPs), polymer-bioactive conjugates (PBCs), dendrimers,
and biopolymer-based nanocarriers) have been popularly adopted. To a lesser extent,
inclusion complexes with cyclodextrins and its derivatives as well as inorganic,
hybrid, and other novel nanocarriers (as liquid crystals and lipid nanocapsules) are
being currently used [439]. However, in recent years, an increased interest has been
focused on the incorporation of lipophilic compounds into lipid-based delivery
systems (LBDS), which has been shown to be one of the most powerful strategies
for the formulation of these kinds of compounds [440, 441]. LBDS used for oral
administration include vesicle systems (liposome and phospholipid complexes),
lipid particulate systems (solid lipid particles (SLPs) and nanostructured lipid com-
plexes (NLCs)), and emulsion-based systems (microemulsions (MEs), nano-
emulsions (NEs), and self-emulsifying delivery systems (SEDSs)) [439].
No single delivery system is suitable for every lipophilic active. Due to their
unique physicochemical characteristics, delivery systems must be carefully designed
taking into account specific physicochemical properties of the lipophilic active to be
incorporated up (including physical state, solubility, partitioning, diffusion, interac-

tions, optical characteristics, rheological properties, and stability) to overcome its
specific challenges and, thus, reach the following goals [436]:
Before ingestion:
1. To readily disperse active compound in the aqueous matrix of food and beverage
products.
2. To incorporate the active compound into food matrices without adversely affect-
ing quality attributes of the product, such as appearance, texture, flavor, or
stability.
3. To mask possible off-flavors of the lipophilic active (such as bitterness or
astringency).
4. To efficiently protect the active ingredient against chemical, physical, or biolog-
ical degradation, either during product storage, to avoid the generation of
off-flavors and the loss of bioactivity. Knowledge of the degradation mechanism
and the major factors that affect it (e.g., oxygen, light, pH, heat, enzyme activity,
etc.) will facilitate the design of a more effective delivery system.
5. To improve the product storage and handling and extend product shelf life, which
is directly related to goal 4.
6. To release the active ingredient (e.g., antimicrobial or antioxidant) at a particular
site of action during food storage. For example, the physical location of an
antioxidant within the food matrix is particularly important for determining its
effectiveness, as it should be present as the site where lipid oxidation primarily
occurs (within the oil, water, or interfacial regions). Appropriately designed
delivery systems can be used to improve the efficacy of antioxidants in food
and beverage products by delivering them to the appropriate site of action.
After ingestion of the fortified product or nutraceutical:
1. To enhance (or at least not adversely affect) the active compound bioavailability
and reduce its inter- and intra-subject variability, leading to higher in vivo efficacy
and more reproducible results in clinical assays. The Food and Drug Adminis-
tration defines bioavailability as “the rate and extent to which the active ingredi-
ent or active moiety is absorbed from a drug product, reach plasma and body
tissues and becomes available at the site-of-action in an unchanged form.” The
impact of bioavailability is especially pronounced when the bioactive compound
is intended for oral use, whereby gastrointestinal absorption constitutes the
primary barrier between the active ingredient and systemic circulation. Delivery
systems have shown to enhance oral bioavailability of lipophilic actives by
improving their dispersion in GI tract (GIT) environment (which avoids their
precipitation), protecting them against chemical (low pH in the stomach) and
biological degradation (microbiota metabolism and enzymatic degradation) in the
GIT, favoring their transportation until absorption area during GI digestion, and
increasing GI wall permeability, which significantly enhances the intestinal
absorption [439].
2. To enhance permeability of other mucosal membranes besides of GI membrane

(such as the cornea, the nasal mucosa, the respiratory mucosa, and the stratum
corneum of the epidermis) and the blood-brain barrier and increase the lipophilic
active penetration into tumoral matrices [442, 443].
3. To increase the pharmaceutical stability of the active compound, thereby length-
ening its systemic circulation time [444].
4. To release the active ingredient at a particular site of action (e.g., released in the
mouth, stomach, small intestine, colon, or targeted organ/cells), at a controlled
rate (sustained release), or in response to a specific environmental trigger (e.g.,
pH, ionic strength, temperature, or enzyme activity), which leads to targeted
effects and increased in vivo efficacy.
5. To overcome multidrug resistance [445].
6. To enhance efficiency of co-delivery of lipophilic actives and therapeutic
agents [446].
7. To reduce the effect of co-ingested food ingredients on pharmacokinetics of the
active molecule [447].
As commented above, oral route is the most commonly used for the delivery of
drugs and nutraceuticals (including lipophilic actives and other dietary bioactives).
This is generally considered as the easiest and most convenient method since it is
noninvasive, cost-effective, and less prone to side effects, such as injection-site
reactions [448]. However, to overcome limitations in the oral administration of
poor water-soluble compounds, parental (intravenous and intraperitoneal) and top-
ical (transdermal, nasal, and ocular) administration routes can be used to increase
dose precision and clinical efficacy. In recent years, topical delivery of bioactive
compounds has also drawn great attention owing to its advantages over other
administration routes and outstanding contribution in improving local action [151]
or systemic absorption, which can minimize the first-pass hepatic metabolism [152].
Moreover, we should not forget that this administration route is the one used by
cosmetic industry. These applications, however, also show several barriers that limit
their use (including low skin permeation, injection-site reactions, short biological
half-life, presystemic metabolism, or systemic toxicity [449]), and the use of nano-
carriers has demonstrated to be also an efficient formulation strategy. In case of the
parenteral route, most of the investigations have focused on utilizing nanocarriers
(as liposomes, SLNs, NCLs, PNPs, and PBCs) to enhance bioactive efficiency
through targeting of effects [450, 451], controlling drug release at site of action to
minimize side effects [452, 453], or overcoming multidrug resistance [454]. For
topical application, the incorporation of active compounds into nanocarriers
(as MEs, liposomes, ethosomes, NLCs, PNPs, and PBCs) aims to enhance skin
permeation and stability, lengthen systemic circulating time, and minimize metabolic
degradation and systemic toxicity [439].
Table 2 summarizes the major types of lipophilic actives that need to be formu-
lated, the specific challenges associated with their application, and the formulation
strategies commonly used to overcome such challenges and increase their industrial
applicability degree as therapeutics or in food fortification.
Table 2 Types of lipophilic actives commonly formulated
502
Lipophilic
active Challenge Formulation strategy Advantage of formulation
Neutral lipids
• ω-3 PUFA • Low water solubility (aqueous matrix Use of LBDS (liposomes, spray-dried • Easier incorporation into aqueous
(mainly ALA, incompatibility) emulsions, multilayer emulsions, multiple medium
EPA, and DHA) • Susceptible to lipid oxidation (problematic emulsions) and filled hydrogel particles • Easier handling and storage
• CLA long-term storage, rancid off-flavors, and (complex coacervation) • Prevention of lipid oxidation
potentially toxic reaction products)
Polar lipids
Sphingolipids • Low water solubility and in vivo • Use of LBDS (cationic pegylated • More effective at crossing the cell
(ceramides) bioavailability liposomes) membrane
• Alteration of ceramide structure (e.g., • Increased bioavailability/bioactivity
cationic pyridinium, C16-serinol, 4,6-diene- • Targeting of antitumor effects
ceramide) • No side effects or lower toxicity to
normal cells
Unsaponifiable lipids
• Terpenes
Essential oils • Low water solubility • Use of LBDS (O/W emulsions, MEs, NEs,
• Some water solubility (instability by multilayer emulsions, multiple emulsions,
Ostwald ripening) and SLNs)
• Low and variable bioavailability • Esterification (e.g., citral acetate)
• Poor chemical stability (loss of bioactivity, • Mask undesirable off-flavors
off-flavors during storage) • Easier incorporation into aqueous
• Low molecular weight and high volatility medium and handling
(ease to loss of desirable flavors) • Easier handling and storage
Carotenoids • Crystalline state at room temperature • LBDS (O/W emulsions, surfactant micelles, • Prevention of chemical degradation
• Low oil solubility MEs, NEs, NLCs), PBDS (PNPs and • Increased efficacy
Vitamin A • Low water solubility biopolymer-based nanocarriers), and CD
(retinoid) • High melting point inclusion complexes (vitamin A)
Vitamin E • Poor chemical stability • Esterification (α-tocopherol acetate)
(tocopherols) • Low and variable bioavailability
L. Vázquez et al.
17
• Sterols
Vitamin D • Low water solubility • Use of LBDS (liposomes, O/W emulsions, • Easier incorporation into aqueous
• Poor chemical stability surfactant micelles, NEs) and PBDS (PNPs medium and handling
• Low and variable bioavailability and PBCs) • Easier handling and storage
Phytosterols and • Low water solubility • Esterification with PUFAs • Prevention of chemical degradation
phytostanols • Low oil solubility • Increased efficacy
(stigmasterol,
Bioactive Lipids
• High melting point

β-sitosterol, and • Poor chemical stability
campesterol)
• Polyphenols
Puerarin LBDS (MEs, SMEDS, SLNs) and PBDS
(dendrimers)
Genistein LBDS (MEs) and PBDS (dendrimers).
Luteolin LBDS (SMEDS, SNEDS)
Silymarin LBDS (SMEDS, NLCs), PBDS (PNPs),
inorganic nanocarriers, and crystalline liquid
nanocarriers
EGCG • Low water solubility LBDS (liposomes, PL-complexes, SMEDS, • Increased bioavailability/bioactivity
• Extensive metabolism and quick clearance SNEDS) • Targeting of antitumor effects
Curcumin • Low and variable bioavailability LBDS (liposomes, S(N)EDS, NLCs) and • No side effects or lower toxicity to
PBDS (PNPs, PBCs, dendrimers) normal cells
Kaempferol LBDS (PL-complexes)
Quercetin LBDS (MEs, PL-complexesa) and folate-
modified lipid NCs
Tyrosol LBDS (phenolipid tyrosol-enriched lecithin)
Resveratrol LBDS (MEs, SLNs) and hybrid nanocarriers
EGCG ()-epigallocatechin-3-gallate, LBDS lipid-based delivery system, PBDS polymer-based delivery system, SLN solid lipid nanoparticle, NLCs nano-
structured lipid complexes, ME microemulsion, NE nanoemulsion, SMEDS self-microemulsifying delivery systems, SNEDS self-nanoemulsifying delivery
systems, PNP polymeric nanoparticle, PBC polymer-bioactive conjugate, CD cyclodextrin
a
Phospholipid (PL)-complexes include phytosomes and phenolipids (quercetin-enriched lecithin)
503
It is worth mentioning that some of the bioactive lipids that have been discussed
in the present book chapter can act, in turn, as efficient lipid-based delivery systems.
This is the case, for example, of alkylglycerols (AKGs). AKGs, previously isolated
from shark liver oil, have been used as efficient delivery systems of bioactive
substances such as butyric acid [454] and hydroxytyrosol esters [455]. Likewise,
ratfish liver oil, with an exceptionally high content in AKG, has been recently used to
obtain a bioactive lipid-based delivery system with potential self-emulsifying prop-
erties by enzymatic glycerolysis [456]. In addition to all the advantages that the
formulation of bioactive molecules with delivery systems offer, the use of bioactive
AKG to design lipid-based delivery systems provides additional benefits, since its
bioactivity could have an addition or even synergistic effect with the loaded bioac-
tive compound, giving rise to highly bioefficient formulations.
Acknowledgments This study has been funded by the Comunidad Autónoma de Madrid
(ALIBIRD, project number S2013/ABI-2728) and by Ministerio de Economia y Competitividad
(project number AGL2016-76736-C3-1-R). Pablo Arranz-Martínez and Marta Corzo-Martínez also
thank Ministerio de Economia y Competitividad and the European Social Fund: BES-2014-070395
for a predoctoral FPU grant and a Juan de la Cierva contract, respectively.
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Oxidative Stability of Edible Plant Oils
18
Terrence Madhujith and Subajiny Sivakanthan
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530
2 Mechanism of Lipid Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
2.1 Lipid Autoxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
2.2 Photooxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
2.3 Lipoxygenase Catalyzed Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
3 Harmful Effects of Oxidation of Edible Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
4 Factors Affecting the Oxidative Stability of Edible Plant Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
4.1 Fatty Acid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
4.2 Oil Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
4.3 Temperature and Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
4.4 Oxygen Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
4.5 Prooxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
4.6 Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
5 Oxidative Stability of Important Edible Plant Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
5.1 Coconut Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
5.2 Palm Oil and Palm Kernel Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
5.3 Olive Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
5.4 Canola (Rapeseed) Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
5.5 Soybean Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
5.6 Sesame Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
5.7 Sunflower Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
T. Madhujith (*)
Faculty of Agriculture, Department of Food Science and Technology, University of Peradeniya,
Peradeniya, Sri Lanka
S. Sivakanthan
Faculty of Agriculture, Department of Agricultural Chemistry, University of Jaffna, Jaffna,
Sri Lanka

530 T. Madhujith and S. Sivakanthan
6 Improving Oxidative Stability of Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541

6.1 The Use of Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
6.2 Modification of the Fatty Acid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544
6.3 Blending . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
6.4 Modifications in Oil Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
Abstract
Edible plant oils play a vital role in daily diets of people worldwide. Stability
against oxidation is the major factor limiting the application of most edible plant
oils for cooking and processing. Most native plant oils vary greatly in their
stability to oxidation depending on their composition. Oxidative stability of
edible plant oils has been extensively studied to find out the ways of improving
their stability against oxidation to widen their application. Synthetic antioxidants
are effective to improve the oxidative stability of these oils, however, recently,
following the evidences on possible toxicities of synthetic antioxidants, the use of
natural plant sources as antioxidant is gaining interest. In addition, modification
of composition of the oils through genetic modification is another successful
means to improve the oxidative stability of these oils. This chapter focuses on the
mechanism and factors of oxidation and ways of improving oxidative stability of
oils.
Keywords
Antioxidants · Autoxidation · Hydroperoxides · Photooxidation · Radicals ·
Refining
1 Introduction
Edible oils from various sources are important components of the daily diet of people
from around the world. Plant oils account for more than 75% of edible oil consump-
tion worldwide [1]. According to the US Department of Agriculture, 189.11 million
metric tons of vegetable oils have been produced globally during the season 2016/
2017. Further, the world vegetable oil production is increasing continuously, espe-
cially the production of palm, soybean and sunflower oil. Palm oil found to be the
major plant oil ranked highest (more than 60%) in the world production followed by
soybean oil [2]. In the recent past, the use of blended oils is becoming popular in
several countries such as Europe, China, Thailand, and Japan. Two or more vegeta-
ble oils are mixed at different proportion to get blended oils for different nutritional
and processing purpose [3]. Vegetable oils are the healthier choice relative to animal
fats in view of their fatty acid contents and cholesterol-free nature [4].
Studies on edible oils remain one of the prime areas of research, and nowadays
people are much health-conscious. Diet-related noncommunicable diseases,
18 Oxidative Stability of Edible Plant Oils 531
especially coronary heart diseases, are the major cause of death in developed as well
as developing countries. In this context, edible oils play a crucial role in maintaining
human well-being. Edible oils differ in their characteristics based on their composi-
tion. Oils are used as cooking oils and ingredients in variety of foods. Thus, the oils
undergo various processing, mainly, heat treatment. Therefore, the stability of these
oils for processing as well as storage conditions is the major property of oils. Major
deteriorative reaction occurring in most of the edible oils is the oxidation. Lipid
oxidation primarily depends on the fatty acid composition and the presence of minor
components. Lipid oxidation leads to the production of primary and secondary
oxidative compounds which are harmful to health. This chapter emphasizes the
mechanism and factors of oxidation of oils, effect of processing conditions and
antioxidants in oxidation, oxidative stability of some major edible oils, and mea-
surement methods.
2 Mechanism of Lipid Oxidation
Lipid oxidation is the major cause of deterioration of the quality of edible oils.
Oxidative stability of oils is defined as the resistance to oxidation during processing
and storage [5]. Lipid oxidation breaks down the fatty acids, thus, causes the loss
of nutritional quality, and produces undesirable color, flavor, and toxic components
making the food unacceptable or less acceptable by consumers; thus it is an indicator
to determine the quality and shelf life [6]. Different mechanisms have been proposed
as responsible for the oxidation of edible oils during processing and storage such as
autoxidation and photosensitized oxidation. The kind of mechanism depends on the
kind of oxygen available. Triplet oxygen (3O2) and singlet oxygen (1O2) can react
with oxygen to cause autoxidation and photosensitized oxidation, respectively [7].
2.1 Lipid Autoxidation
Unsaturated fatty acids are prone to autoxidation which proceeds via free-radical
chain mechanism. Rate of oxidation depends on the degree of unsaturation and
increases with increase in the number of double bond [8]. Free-radical mechanism of
lipid autoxidation involves the attack of oxygen at the allylic position leading to the
formation of hydroperoxides. These primary oxidation products further decompose
into secondary oxidation products [9].
Lipid autoxidation involves three steps: initiation phase (induction period), prop-
agation phase (exponential phase), and termination stage [10]. In the initiation step,
when lipid (LH) is exposed to an initiator (heat, light or metal ions), hydrogen atom
of double bond is abstracted, and alkyl radical (L•) is formed [11]. This free radical
abstracts hydrogen from other lipid molecules and reacts with the hydrogen to form
hydroperoxide (LOOH) (primary oxidation product) and another alkyl radical. Alkyl
radical also reacts with molecular oxygen (3O2) to form peroxy radical (LOO•),
which abstracts hydrogen from other lipid molecules and reacts with hydrogen to
form hydroperoxide and another alkyl radical. These radicals catalyze the oxidation
reaction, and autoxidation is called the free-radical chain reaction [7]. The rate of
formation of peroxy radical and hydroperoxide depends only on oxygen availability
and temperature [12]. Radicals react with each other to form non-radical species, and
the reaction is terminated [7]. The most labile hydrogen atom in monounsaturated
acids is on the carbon atoms adjacent to the double bond, whereas, in polyunsatu-
rated acids, the most labile hydrogens are on methylene groups between two double
bonds. The abstraction of hydrogen is followed by electron rearrangement to form
conjugated dienes since the radicals formed are unstable [13].
Hydroperoxides are nonvolatile, odorless, and very unstable and further
break down via several steps into secondary oxidation products called carbonyl
compounds such as aldehyde, ketones, acids, alcohols, acids, esters, and short-
chain hydrocarbons via monomolecular and bimolecular reactions [7, 13–15].
Hydroperoxides begin to decompose as soon as they are formed. During the
first stages of autoxidation, their rate of formation exceeds their rate of decom-
position, and the reverse is true at later stages of autoxidation [16]. Most of the
secondary oxidative products are responsible for the off-flavor in the oxidized
edible oil. Aliphatic carbonyl compounds (alkanals, 2-alkenals, and trans,
trans-2,4-alkadienals) have more influence on the oxidized oil flavor because
of their low threshold values [7]. Breakdown of hydroperoxides also produces
nonvolatile monomeric compounds, including di- and tri-oxygenated esters
derived from the corresponding keto-, hydroxy-, hydroperoxy-, and epoxide
esters. During induction period, only very little changes occur in lipids,
followed by fast deterioration of lipids, and off-flavors become noticeable and
the lipid is no longer edible [13].
Autoxidation of oleate (C18:1, n9) produces four allylic hydroperoxides
containing OOH groups on carbon 8,11 (cis-8-OOH, cis-11-OOH) and 9,10,
(trans-9-OOH, trans10-OOH). Autoxidation of linoleate (C18:2, n6) produces a
mixture of cis, trans and trans, trans (9-OOH and 13-OOH) conjugated diene
hydroperoxides. Autoxidation of linolenate (C18:3, n3) produces a mixture of
cis, trans, trans, trans, and cis (9-OOH, 12-OOH, 13-OOH and 16-OOH) conju-
gated diene hydroperoxides [13, 16].
Initiation:
LH ! L∙ + H∙
Propagation:
L∙ + O2 ! LOO∙
LOO∙ + LH ! LOOH + L∙
Termination:
LOO∙ + LOO∙ ! non-radical product
LOO∙ + L∙ ! non-radical product
L∙ + L∙ ! non-radical product
Hydroperoxides initiate new chain reaction after reacting with free radicals [17].
2.2 Photooxidation
Oxidation of edible oils is accelerated by light, especially in the presence of

sensitizers [7]. In the presence of light, photosensitizers such as chlorophyll and
porphyrin convert triplet oxygen into singlet oxygen, which is a highly reactive non-
radical molecule [15]. If wavelength of solar light is less than 220 nm, unsaturated
fatty acids cannot absorb light; however photosensitizers absorb light energy and
convert triplet state sensitizer to singlet-state sensitizer. Ground-state singlet sensi-
tizers absorb light energy very rapidly and become excited singlet sensitizer which
can return to their ground state via emission of light, internal conversion, or
intersystem crossing. Fluorescence, heat, and excited triplet state of sensitizers are
produced by emission of light, internal conversion, and intersystem crossing, respec-
tively [7]. Photooxidation can proceed via two types of mechanism. An electron or a
hydrogen atom transfers between an excited triplet sensitizer and a substrate,
producing free radicals or radical ions (Type I). Excited triplet sensitizers react
with triplet oxygen to produce superoxide anion by electron transfer. Superoxide
anion produces hydrogen peroxide which reacts with superoxides and forms singlet
oxygen in the presence of transition metals (iron or copper). Excitation energy of
triplet sensitizers can be transferred to triplet oxygen by triplet–triplet annihilation to
produce singlet oxygen, which reacts with the double bond of unsaturated fatty
acids, producing an allylic hydroperoxide (Type II). The rate of type I and type II
process may differ depending on the kinds of sensitizers, substrates, and concentra-
tions of substrates and oxygen [7, 13, 15, 18–20]. Hydroperoxides formed by
photooxidation are decomposed by the same mechanisms for the hydroperoxides
formed by autoxidation. The mechanism of photooxidation is explained in detail by
Choe and Min [7].
2.3 Lipoxygenase Catalyzed Oxidation
Lipoxygenase is an enzyme found in plants. It is another reason for the deterioration

of edible plant oils, especially during oil extraction. Lipoxygenase reacts with oils
containing1,4-cis, cis-pentadiene system producing corresponding hydroperoxy
derivatives and cis-trans conjugated hydroperoxide [21]. Reaction of this enzyme
produces hydroperoxides, which decompose to form secondary oxidation products
with strong undesirable flavors. Lipoxygenase produces similar volatile compounds
to those produced by autoxidation [22]. Lipoxygenase contains a ferrous atom as
inactive Fe (II) and is oxidized to active Fe (III) by fatty acid hydroperoxides
or hydrogen peroxide. The active enzyme abstracts a hydrogen atom from the
methylene group of a polyunsaturated fatty acid with the iron being reduced to
Fe (II) producing conjugated dienes followed by reaction with oxygen generating
peroxyl radical and hydroperoxide [13, 20]. Rice bran and soybean are the two major
plant sources containing lipoxygenase; thus the oils extracted from these sources can
easily undergo lipoxygenase-catalyzed oxidation during processing.
3 Harmful Effects of Oxidation of Edible Oils
Lipid oxidation reduces the shelf life and nutritive value of food by limiting the
content of essential polyunsaturated fatty acids [23]. Lipid oxidation products are
considered to be harmful for health as they consist of compounds with mutagenic,
carcinogenic, and cytotoxic properties [24]. These compounds cause health prob-
lems such as growth of tumor cells through lipid peroxidation as hydroxides of fatty
acids are cytotoxic. Oxidation of long chain fatty acids causes neuromyopathic
disease both in infant and adults [15, 25].
Consumption of lipids containing excessive free radicals may cause alterations in
the redox state of human body, leading to lipid peroxidation [4]. Unstable free
radicals tend to stabilize themselves by abstracting electrons from membrane lipids
to start a self-propagating chain reaction causing structural rearrangement of the
lipids. The rate of bond cleavage increases until the molecule gets stabilized.
Oxidative damage to lipid structures eventually leads to disorganization and dys-
function of, as well as damage to membranes, enzymes and proteins [4, 26].
4 Factors Affecting the Oxidative Stability of Edible Plant

Oils
The oxidative stability of oil is influenced by the fatty acid composition of the oil;
processing, heat or light; the concentration and type of oxygen; free fatty acids,
mono-, and diacylglycerol concentration; transition metals; peroxides; thermally
oxidized compounds; pigments; and antioxidants. These factors interactively affect
the oxidation of oil, and it is not easy to differentiate the individual effect of the
factors [7].
4.1 Fatty Acid Composition
The natural fatty acid composition of oil and the positions of the fatty acids in the
glycerol backbone determine the susceptibility and resistance to oxidation [27]. Oils
containing more unsaturated fatty acids are oxidized more quickly than the oils
containing less unsaturated fatty acids [28]. As the degree of unsaturation increases,
the rate of oxidation increases [7] producing more complex mixtures of hydroper-
oxides. In addition, as the degree of unsaturation of fatty acids increases, the rate of
formation and the amount of primary oxidation products are accumulated [29]. The
relative rate of autoxidation of oleate/linoleate/linolenate was reported to be in the
order of 1:40–50:100 on the basis of the oxygen uptake and 1:12:25 on the basis of
peroxide development [13]. Soybean, sunflower, corn and rapeseed oils are often
considered unsuitable for continuous frying due to their high content of polyunsat-
urated fatty acids [30]. Xu and others [31] studied the stability of camellia oil after
frying potatoes by comparing with palm oil and peanut oil and reported that camellia
oil possesses highest oxidative stabilities followed by palm oil, while peanut oil
showed the least stability. They have concluded that the fatty acid composition and
tocopherol contents of oils are the important factors of oxidative stability of the oils.
Camellia oil contains higher proportions of monounsaturated fatty acid compared to
saturated and polyunsaturated fatty acid than other two oils, and the peanut oil
contains higher proportion of polyunsaturated fatty acid. The monounsaturated
fatty acid is relatively stable toward oxidation, while polyunsaturated fatty acids
are highly susceptible to oxidation [32]. Even though the fatty acid composition of
the oils is the major factor determining the oxidative stability of the oil, oxidative
stability of oils is a function of several other factors [30].
4.2 Oil Processing
The oil processing affects the oxidative stability of an oil. All crude oils after
extraction from their sources contain nontriglyceride components such as free fatty
acids, partial acylglycerols, sterols, tocopherols, hydrocarbons, pigments (gossypol,
chlorophyll), vitamins, phosphatides, protein fragments, trace metals, and resinous
and mucilaginous materials. The objectionable nontriglycerides cause unwanted
effects during processing such as darkening and formation of foam, smoke, and
precipitate and develop off-flavors. Thus, crude oils are refined to remove the
objectionable constituents [33]. Typical refining process consists of neutralization,
bleaching, winterization, and deodorization steps. Even though the objective of the
refining is to remove impurities with minimum damage to the oil, unfortunately,
these processing steps may result in losses in naturally occurring bioactives such as
tocopherols, tocotrienols, sterols, and phenols, and the extent of loss depends on the
processing parameters and nature of the oil [33, 34]. Thus, the refined oils may have
less oxidative stability than their unrefined counterparts [30].
4.3 Temperature and Light
Autoxidation of oils and the decomposition of hydroperoxides increase as the

temperature increases. As temperature increases, the solubility of oxygen decreases
drastically although all the oxidation reactions are accelerated [35]. At temperatures
above 150 C, hydroperoxides are reduced to very low level or become absent, and
the formation of new compounds is very rapid, indicating that the rate of peroxide
decomposition is higher than that of their formation [36].
The formation of autoxidation products during the induction period of autoxida-
tion is slow at low temperature, and the content of polymerized compounds increases
significantly at the end of the induction period. Light of shorter wavelengths has
more detrimental effects on the oils than longer wavelengths, and the effect of light
on oil oxidation becomes less as temperature increases [7, 12]. However, as tem-
perature increases, the influence of light on oxidation becomes less important [35].
The effect of temperature on photooxidation is less prominent than on autoxidation.
Light has more influence than temperature in photooxidation [7].
The packaging of oils is very important to minimize the photooxidation. Trans-

parent plastic bottles can increase oil oxidation. The incorporation of UV absorbers
such as Tinuvin 234 (2-(2-hydroxy-3,5-di (1,1-dimethylbenzyl)phenyl)
benzotriazole) or Tinuvin 326 (2-(30 -tert-butyl-20 -hydroxy-50 -methylphenyl)-5-
chlorobenzo-triazole) into the transparent plastic bottles can be effective to improve
the oxidative and sensory stability of edible plant oils stored under light [7, 37, 38].
4.3.1 Frying
Frying is one of the commonly used culinary methods. In recent years, frying oils
became an important component of daily diet due to rising demand for deep-fried
products. However, much concern has been raised on the biological effects of
consumption of fried foods containing oxidized lipids. During the frying process,
oil is exposed to an extremely high temperature in the presence of air and moisture.
It may result in a high rate of production and decomposition of peroxides. Deep-fat
frying decreases the unsaturated fatty acids and increases polar material [39]. Thus,
the quality of the frying oil is of paramount importance.
The chemistry of oxidation during frying is very complex since both thermal and
oxidative reactions take place during frying at high temperatures. Reaction mecha-
nism of thermal oxidation is principally the same as the autoxidation mechanism;
however, the thermal oxidation takes place at faster rate than the rate of autoxidation
[39–41]. These oxidative reactions are mainly influenced by the phenolic com-
pounds, tocopherols, and fatty acid composition of the oil and frying temperature
[4, 16, 42, 43]. Repeated frying accelerates the oxidation of oil leading to formation
of primary and secondary oxidative products which are absorbed by the fried food
and eventually get into the gastrointestinal tract and the circulation system after
ingestion leading to health problems such as high blood pressure [4].
Juárez and others [44] investigated the discontinuous deep frying of churros
in soybean oil, sunflower oil, and partially hydrogenated fats and found that, after
80.5 h of deep frying, all the oils exceeded 25% of total polar compounds except
partially hydrogenated fat and losses of tocopherols during frying reached 76.0%.
Marinova and others [39] investigated the oxidative stabilities of refined sunflower,
grape seed, soybean, corn, and olive oils at frying temperature. Their results dem-
onstrated that olive oil possesses better stability against thermal oxidation when
compared to polyunsaturated oils. Further, it is shown that corn and soybean oils
are most resistant to oxidation at frying temperature among unsaturated oils.
4.4 Oxygen Concentration
The oxidation of oil takes place when oxygen and catalysts are in contact with the
oil. Both concentration and type of oxygen affect oxidation of oils. The oxygen
concentration in the oil depends on the oxygen partial pressure in the headspace of
the oil [45]. If the partial pressure of oxygen in the headspace is high, invariably high
amount of oxygen becomes dissolved in the oil. The amount of dissolved oxygen is
positively associated with enhanced oxidation of the oil. Prooxidants such as
transition metals and light accelerate the effect of oxygen concentration on the
oxidation of oil [7, 45]. For example, addition of 70 ppm copper to rapeseed oil
exposed to air for 35 days resulted in 70 times higher hexanal concentration
compared to the sample devoid of copper [45]. If the oxygen concentration is
sufficiently high, the rate of oxidation of oil is independent on oxygen concentrations
and vice versa. Ratio of surface to volume of the oil also has influence on the
oxidative stability. When the surface to volume ratio is increasing, oil can react more
efficiently with oxygen; thus, the relative rate of oxidation is less oxygen-dependent
with a low oxygen content [7, 45, 46].
4.5 Prooxidants
Crude oils contain nontriglycerides which can act as prooxidants such as free fatty
acids, mono- and diacylglycerols, metals, phospholipids, peroxides, and chloro-
phylls. Trace amounts of metals such as copper, iron, manganese, and nickel can
be absorbed by plants during the growth and during fat and oil processing. These
metals substantially reduce the oxidative stability of oils [47]. Transition metal ions
are involved in redox reaction, which leads to hydroperoxide decomposition [13].
These metals’ effects can be diminished by the use of chelating agents such as citric
and phosphoric acids [47].
Free radicals produced during hydroperoxide decomposition act as initiators
of autoxidation. Radicals generated from food contaminants may also participate
in oxidation process acting as catalysts. Some molecules absorb UV light energy
and are converted into an excited singlet state. Pigments such as riboflavin and
porphyrins (chlorophyll, hemoglobin, myoglobin) and some synthetic dyes can act
as initiators of lipid oxidation [13].
4.6 Antioxidants
Antioxidants are the compounds which inhibit the oxidation of fats and oils. They
extend the induction period or slow down the rate of oxidation [7]. The presence of
antioxidants (naturally present or intentionally added) is one of the most important
factors determining the stability of oils against oxidation.
Tocopherols, tocotrienols, carotenoids, phenolic compounds, and sterols are the
naturally occurring antioxidants in plant oils. Among these, tocopherols are the most
important antioxidants, especially in soybean, canola, sesame, sunflower, and corn
oils. Palm oil contains a high amount of tocotrienols. β-Carotene is one of the
important natural antioxidants present in unrefined plant oils. β-Carotene acts as
antioxidant by light filtering, singlet oxygen quenching, sensitizer inactivation,
and free-radical scavenging [7]. In addition to tocopherols, lignans are important
antioxidants in sesame oils, which is well known to possess high stability against
oxidation, even though it contains high level of unsaturation [46].
Non-refined oils have a better stability at elevated temperature than refined

oils [30] as the refining can lead to reduction in the natural antioxidants.
Szydłowska-Czerniak and Łaszewska [48] reported that the refining process of
rapeseed oils decreased the antioxidant capacity by about 60% and total phenolic
content by above 80%.
5 Oxidative Stability of Important Edible Plant Oils
Edible plant oils can be categorized into three groups based on their fatty acid
composition such as oils containing high levels of saturated fatty acids (lauric
acid, myristic acid, palmitic acid, and stearic acid), oils with high levels of mono-
unsaturated fatty acids (oleic acid), and oils containing high amount of polyunsat-
urated fatty acids (linoleic and linolenic acid). Examples for first category include
coconut oil and palm kernel oil. Examples for oils with high levels of monounsat-
urated fatty acid include olive, canola, rapeseed, peanut, hazelnut, avocado, and
sesame oils. Corn, soybean, sunflower, cottonseed, grape seed, and safflower are
examples for the third category [49]. Table 1 shows the range of fatty acid found
in important plant oils.
5.1 Coconut Oil
Coconut (Cocos nucifera) oil remains an important edible oil for the food industry
for many years. It contains more than 90% of saturated fatty acids [51]. Coconut oil
being a highly saturated oil is extremely stable against oxidation, therefore suitable
for frying [52]. Lauric acid is the major saturate fatty acid present in coconut oil.
The generation of trans-fatty acids is also very minimal during frying operations.
5.2 Palm Oil and Palm Kernel Oil
Palm oil and palm kernel oil are obtained from oil palm (Elaeis guineensis).
In addition to palm oil and palm kernel oil, their fractions are also produced globally
to be used for edible purposes. Palm oil is edible oil derived from the fleshy
mesocarp of the oil palm fruit, and palm kernel oil is derived from the kernel of
the fruit of the oil palm. Palm oil contains almost equal portions of saturated and
unsaturated fatty acids, mainly as palmitic acid (42–47%) and oleic acid (37–41%).
Palm oil is highly stable against oxidation [33, 53]. Palm kernel oil contains high
amount of lauric acid (45–55%), thus known as lauric oil. Fractionation of palm oil
into palm stearin and palm olein and palm kernel oil into palm kernel stearin and
palm kernel olein further enhances their applications in foods with different stabil-
ities [54].
18
Table 1 Fatty acid composition of important edible plant oils

Fatty acid C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3
Coconut oil ND-0.7 4.6–10.0 5.0–8.0 45.1–53.2 16.8–21.0 7.5–10.2 ND ND ND 2.0–4.0 5.0–10.0 1.0–2.5 ND-0.2
Palm oil ND ND ND ND-0.5 0.5–2.0 39.3–47.5 ND-0.6 ND-0.2 ND 3.5–6.0 36.0–44.0 9.0–12.0 ND-0.5
Palm kernel oil ND-0.8 2.4–6.2 2.6–5.0 45.0–55.0 14.0–18.0 6.5–10.0 ND-0.2 ND ND 1.0–3.0 12.0–19.0 1.0–3.5 ND-0.2
Palm olein ND ND ND 0.1–0.5 0.5–1.5 38.0–43.5 ND-0.6 ND-0.2 ND-0.1 3.5–5.0 39.8–46.0 10.0–13.5 ND-0.6
Palm kernel olein ND-0.7 2.9–6.3 2.7–4.5 39.7–47.0 11.5–15.5 6.2–10.6 ND-0.1 ND ND 1.7–3.0 14.4–24.6 2.4–4.3 ND-0.3
Palm kernel ND-0.2 1.3–3.0 2.4–3.3 52.0–59.7 20.0–25.0 6.7–10.0 ND ND ND 1.0–3.0 4.1–8.0 0.5–1.5 ND-0.1
stearin
Palm stearin ND ND ND 0.1–0.5 1.0–2.0 48.0–74.0 ND-0.2 ND-0.2 ND-0.1 3.9–6.0 15.5–36.0 3.0–10.0 ND-0.5
Palm superolein ND ND ND 0.1–0.5 0.5–1.5 30.0–39.0 ND-0.5 ND-0.1 ND 2.8–4.5 43.0–49.5 10.5–15.0 0.2–1.0
Oxidative Stability of Edible Plant Oils
Rapeseed oil ND ND ND ND ND-0.2 1.5–6.0 ND-3.0 ND-0.1 ND-0.1 0.5–3.1 8.0–60.0 11.0–23.0 5.0–13.0
Rapeseed oil (low ND ND ND ND ND-0.2 2.5–7.0 ND-0.6 ND-0.3 ND-0.3 0.8–3.0 51.0–70.0 15.0–30.0 5.0–14.0
erucic acid)
Safflower seed oil ND ND ND ND ND-0.2 5.3–8.0 ND-0.2 ND-0.1 ND-0.1 1.9–2.9 8.4–21.3 67.8–83.2 ND-0.1
Safflower seed oil ND ND ND ND-0.2 ND-0.2 3.6–6.0 ND-0.2 ND-0.1 ND-0.1 1.5–2.4 70.0–83.7 9.0–19.9 ND-1.2
(high-oleic acid)
Sesame seed oil ND ND ND ND ND-0.1 7.9–12.0 ND-0.2 ND-0.2 ND-0.1 4.5–6.7 34.4–45.5 36.9–47.9 0.2–1.0
Soybean oil ND ND ND ND-0.1 ND-0.2 8.0–13.5 ND-0.2 ND-0.1 ND-0.1 2.0–5.4 17–30 48.0–59.0 4.5–11.0
Sunflower seed oil ND ND ND ND-0.1 ND-0.2 5.0–7.6 ND-0.3 ND-0.2 ND-0.1 2.7–6.5 14.0–39.4 48.3–74.0 ND-0.3
Sunflower seed oil ND ND ND ND ND-0.1 2.6–5.0 ND-0.1 ND-0.1 ND-0.1 2.9–6.2 75–90.7 2.1–17 ND-0.3
(high-oleic acid)
Sunflower seed oil ND ND ND ND ND-1 4.0–5.5 ND-0.05 ND-0.05 ND-0.06 2.1–5.0 43.1–71.8 18.7–45.3 ND-0.5
(mid-oleic acid)
Source: Codex standard for named vegetable oils (CODEX-STAN 210 – 1999) [50]
ND Non-detectable
539
5.3 Olive Oil
Olive (Olea europaea) oil is best suited for food applications involving high
temperatures such as frying. Fatty acid composition of the oils determines the
suitability of oils for various applications. In this context, the fatty acid composition
of the olive oil complies with the criteria of the stable healthy frying oils, that is,
olive oil contains high ratio of monounsaturated-to-polyunsaturated fatty acid, low
in saturated and polyunsaturated fatty acids, and most importantly very low in
linolenic acid [35, 55].
Further, olive oil is considered to be a good-quality frying oil owing to its
relatively low melting point; thus, the oil can drain easily from the fried food
resulting in low content of trapped oil in the fried food [56].
Virgin olive oil has a higher resistance to oxidative deterioration compared to
refined oils because of the presence of phenolic antioxidants such as polyphenols
and tocopherols and low polyunsaturation. Polyphenols are eliminated or reduced
drastically during the refining process [35].
5.4 Canola (Rapeseed) Oil
The edible canola/rapeseed oil is obtained from low erucic and low glucosinolate
species such as Brassica campestris, Brassica napus, and Brassica juncea.
Canola oil carries excellent nutritional value because of its unique fatty acid
composition with a high amount of oleic acid (50–66%) and also contains linoleic
acid (18–30%) and linolenic acid (8–12%) [33, 57]. However, canola oil has less
oxidative stability because of the high amount of polyunsaturated fatty acids.
Thus, canola oil is subjected to various modifications to improve their oxidative
stability such as hydrogenation, fractionation, interesterification, and physical
blending [57].
5.5 Soybean Oil
Soybean (Glycine max L.) oil is an important plant oil in terms of nutritional quality
attributed to its high content of polyunsaturated fatty acids (n6 and n3) and
tocopherols; however, it is highly susceptible to oxidation [58]. Soybean oil is
mainly composed of polyunsaturated fatty acids such as linoleic acid (n6) (56%)
and α-linolenic acid (n3) (8%) which are considered essential fatty acids as they
are necessary for growth and development of the human body. They act as precursors
of prostaglandins and hormones which play an important role in the regulation of
some physiological and biochemical functions of the human body [58]. However the
high amount of linolenic acid reduces oxidative stability of the oil, leading to
decreased shelf life [59].
Soybean oil contains relatively high amount of tocopherol compared to other
major polyunsaturated oils such as rapeseed, corn, and sunflower [60]. Research
studies have shown that refined soybean oil is highly susceptible to oxidation
because of its high content of unsaturated fatty acids and the absence or less amount
of natural minor compounds with antioxidant effect [58].
5.6 Sesame Oil
Sesame (Sesamum indicum) oil contains mainly monounsaturated fatty acids

(45–49%) and polyunsaturated fatty acid (37–41%). It contains thermally stable
natural antioxidants as lignans; however, its high content of polyunsaturated fatty
acids makes sesame oil highly prone to autoxidation [51].
5.7 Sunflower Oil
Sunflower (Helianthus annuus L.) oil contains linoleic (48–74%) and oleic acid
(14–39%) as the major fatty acids [33]. High content of unsaturation makes the
sunflower oil highly susceptible for autoxidation and less suitable for deep frying. As
explained later in this chapter, new phenotypes with modified fatty acid composition,
mainly with increased oleic acid content, have been developed through genetic and
breeding techniques in order to make the sunflower oil suitable for thermal applica-
tions such as deep frying.
6 Improving Oxidative Stability of Oils
6.1 The Use of Antioxidants
Antioxidants are substances that when introduced into substrate at low concentration
compared to that of an oxidizable substrate significantly inhibit oxidation of that
substrate by inhibiting formation of free radicals or by interrupting propagation of
the free radical [61, 62]. Addition of antioxidant during oil processing is one of the
most effective means to retard fat oxidation. Antioxidants are of two types based on
mechanism of action: primary antioxidants and secondary antioxidants. This can be
further classified into natural and synthetic.
6.1.1 Primary Antioxidants

Primary antioxidants are chain-breaking antioxidants, that is, they are capable of
neutralizing lipid free radicals by stopping their radical state by donating hydrogen.
Butylated hydroxy anisole (BHA), butylated hydroxyl toluene (BHT), tertiary
butylhydroquinone (TBHQ), tocopherols, and flavonoids are examples for primary
antioxidants [62, 63].
The reaction mechanism can be explained schematically as follows (antioxidant
molecule is denoted by AH).
ROO• + AH ! ROOH + A•
R• + AH ! RH + A•
RO• + AH ! ROH + A•
ROO• + A• ! ROOA
RO• + A• ! ROA
A• + A• ! A – A
Some primary antioxidants such as propyl gallate, proanthocyanidins, and

ascorbic acid act as antioxidants via more than one mechanisms such as free-radical
scavenging, oxygen sequestering, metal chelation, and light energy absorption [64].
6.1.2 Secondary Antioxidants

The secondary antioxidants (preventive antioxidant) retard the rate of oxidation
through the removal of the substrate or singlet oxygen quenching mechanism
[65–67]. Mechanisms of secondary antioxidants include metal chelating, singlet
oxygen quenching and inactivation of photosensitizers and lipoxygenase [7]. Metals
act as prooxidants by reducing the activation energy of the oxidation, especially in
the initiation step, to accelerate oil oxidation. Metal chelators form insoluble metal
complexes or provide steric hindrance between metals and food components. Exam-
ples include citric acid, EDTA, polyphenols, lignans, and ascorbic acid [7, 45, 64].
The mechanism of action of singlet oxygen quenchers involves deactivation of
singlet oxygen to the ground-state triplet oxygen or getting oxidized themselves by
singlet oxygen [7, 68]. Tocopherols, carotenoids, phenolics, and ascorbic acid retard
oxidation of lipids through quenching singlet oxygen [69]. Photosensitized com-
pounds such as chlorophyll and riboflavin transfer the energy to triplet oxygen to
form singlet oxygen, or transfer an electron to the triplet oxygen to form a superoxide
anion radical, which react with lipid to produce free radicals [7]. Energy of the
photosensitizers is transferred to the singlet state of antioxidant to become a triplet
state of antioxidant, which is changed to singlet state by transferring the energy to
the surrounding or emitting phosphorescence. Carotenoids retard lipid oxidation
through inactivating photosensitizers [70].
Synergism
Addition of combinations of primary and secondary antioxidants is often reported
to have synergistic effect, that is, combined action is more effective to retard lipid
oxidation than the sum of their single effect and increases the length of induction
period [71, 72].
6.1.3 Natural and Synthetic Antioxidants

Natural antioxidants are found in several plant sources such as grain, seeds, cereals,
nuts, fruits, vegetables, and spices [73]. Plant extracts contain various antioxidants
such as flavonoids (quercetin, kaempferol, myricetin), catechins or phenols
(carnosol, rosmanol, rosamaridiphenol), and phenolic acids (carnosic acid,
rosmarinic acid) [74–76]. Tocols are the natural antioxidants found in plant-based
oils, which include four tocopherol and four tocotrienol isomers, each designated as
alpha, beta, gamma, and delta on the basis of the chromanol ring. The α-tocopherol
has been reported to be the most active isomer biologically, whereas the γ-tocopherol
is perceived as the best antioxidant [47]. The vitamin E, most importantly
α-tocopherol, is the well-known antioxidant found in vegetable oil. They are
known to give protection against peroxidation of polyunsaturated fatty acids [77].
Among all types of tocopherols, α-tocopherol is the most unstable, thus easily
destroyed at high temperatures [31]. Juárez and others [44] reported that there
were significant losses of α-tocopherol during frying because α-tocopherol degrades
more quickly at high temperatures than at room temperature [78].
Vegetable oils containing high amounts of unsaturated fatty acids such as soy-
bean, sunflower, sesame, corn, and peanut oils are very unstable for continuous
frying due to their content of polyunsaturated fatty acids. However, the presence
of natural substances such as tocopherols, oryzanol, sterol fraction, squalene, among
others enhances their stability at higher temperatures [30].
Several studies attempted to study the effect of addition of synthetic antioxidants
to the edible plant oils. Azeez and others [67] investigated the effect of direct
incorporation of TBHQ, BHT, and mixed (TBHQ and BHT) on the oxidative
stability of palm olein, soybean oil, and linseed oil at room temperature and 70 C
for 168 h and reported that TBHQ had significant effect on the oxidative stability
of palm olein at 70 C, while TBHQ and BHT had synergetic effect on stability of
soybean oil at room temperature and Linseed oil at 70 C.
Gertz and others [30] investigated the efficacy of some antioxidants on stability of
refined sunflower and rapeseed oils and reported that α-tocopherol, tocopherol
esters, and BHA exhibit low antioxidant effects at frying temperature, while ascorbic
acid 6-palmitate and some phytosterol fractions were found to possess the greatest
antioxidant activity.
Synthetic antioxidants, such as BHT, BHA, and TBHQ, are very effective in
edible plant oils to protect against oxidation and, thus, widely used in many oils.
However, recently, their use has been discouraged following findings related to
possible toxicity and carcinogenicity of these synthetic antioxidants as evidenced
by animal studies [79]. For example, BHA and BHT have been shown to possess
carcinogenic activity in rodents [80–82]. This has drawn considerable attention to
the use of natural antioxidants from plant sources as replacement for synthetic
antioxidants for improving the oxidative stability of oils [58, 74].
During the past two decades, research studies have been focused extensively
toward the use of natural plant extracts as sources of antioxidants to replace the
synthetic antioxidants [79]. In recent years, tendency toward the use of agro-indus-
trial by-products as sources of antioxidant is increasing [73, 74, 83, 84]. Compared
to synthetic antioxidants, natural plant extracts have been shown to exhibit higher
antioxidant activity and thermal stability which are the most important criteria for an
antioxidant to be used for fats and oils [62, 79]. Some examples for such sources
include α-tocopherol, pomegranate peel [85, 86], green tea [87], olive waste [88],
sesame cake [74], sesame seed [89], rosemary (Rosmarinus officinalis L.) [85, 90],
Eucalyptus citriodora leaf [91], celery [92], oregano (Origanum vulgare) [85],
cinnamon, and other spices and herbs [62]. In recent years, ascorbyl palmitate is
gaining popularity to be used in edible oils. Ascorbyl palmitate is “generally

recognized as safe” (GRAS) according to the Food Drug Administration without
limitation on levels to be used in food [70].
Antioxidative effect of α-tocopherol (vitamin E), fat-soluble carotenoid, has been
extensively studied [62]. Hraš et al. [93] studied the antioxidative activities of four
natural antioxidants such as rosemary extract, α-tocopherol, ascorbyl palmitate, and
citric acid in sunflower oil stored at 60 C. Among them, rosemary extract had the
best antioxidative activity. Rosemary extract exhibited additive antioxidative effect
when combined with citric acid and ascorbyl palmitate. Further they have reported
that α-tocopherol exhibited prooxidative effect. Choe and Min [7] explained that at
high concentration, tocopherol radical may abstract hydrogen from lipids with very
low concentration of peroxy radical and produces tocopherol and lipid radical, which
may increase the lipid oxidation; thus, tocopherol act as prooxidant instead of
antioxidant. Thus, the natural antioxidants may not be always preventive against
oxidation [94].
Abdelazim and others [74] found that sesame cake extract possesses stronger
antioxidant activity than BHT and BHA, however less than that of TBHQ. Hassanien
and Abdel-Razek [95] have found that addition of roasted sesame seed as a source of
natural antioxidant can improve the stability of edible oils. Sesame seeds contain a
myriad of natural antioxidant components including lignans such as sesamin,
sesamolin, and sesaminol. In addition to enhancing the stability of oils against
oxidation, these compounds play a vital role in maintaining good health. Green tea
extracts at higher concentrations than 200 ppm showed excellent antioxidant activity
in both oils, and its efficacy was higher than that of BHA, BHT, and α-tocopherol but
less than that of TBHQ. Sayyad et al. [90] studied the effect of rosemary extract on
thermoxidative stability of soybean oil and reported the higher stability of the
soybean oil added with rosemary extract (3000 ppm) than the oil added with
TBHQ (50 ppm).
Gertz et al. [30] reported that natural substances such as squalene, sterol fraction,
quercetin, oryzanol, and ferulic acid are more efficient than synthetic antioxidants to
enhance the stability of vegetable oils at higher temperatures. Further, ascorbyl
palmitate is more efficient than BHA or α-tocopherols to increase the stability of
vegetable oils during frying at high temperature [30]. Alavi and Golmakani [66]
reported that spirulina can improve the oxidative stability of olive oil.
Recently, a study has been reported on the antioxidant activity of encapsulated
olive leaf extract in soybean oil. And their results indicated that nano-encapsulation
of olive leaf could be a suitable novel technique to improve the antioxidant activity
of natural sources [96].
6.2 Modification of the Fatty Acid Composition
Modification of fatty acid composition through natural plant breeding or genetic

modification is another way of improving the oxidative stability of vegetable oils
[49]. Refined edible oils such as soybean, rapeseed, sunflower, or peanut oils have
high contents of polyunsaturated fatty acids, linoleic, and linolenic acids; thus, they
are not suitable for repeated deep fat frying. Even though palm kernel and coconut
oils are more stable, their high content of saturated fatty acid limits their applications
[97]. Genetic and breeding techniques are employed mainly to alter the composition
of saturates, oleic acid, and linolenic acid. High and mid-oleic acid content can
increase the oxidative stability at high cooking temperatures [98], especially during
deep frying. In recent years, high and mid-oleic acid oil crops developed through
breeding techniques by private companies are commercially available [49, 99].
Some examples are Nexera™ (Omega-9 canola and Omega-9 sunflower oils),
Plenish™ high-oleic soybeans by E. I. du Pont de Nemours (Wilmington, DE,
USA), and Vistive-Gold™ low-saturated high-oleic soybeans by Monsanto Co.
(St. Louis, MO, USA) [49, 100, 101]. Some examples for oil crops developed by
genetic means to contain higher oleic acid levels than normal include soybean (from
24% to 84%), palm (from 36% to 59%), canola (from 57% to 89%), sunflower (from
29% to 84%), peanut (from 55% to 76%), cottonseed (from 13% to 78%), and
safflower (from 10% to 81%) [49]. Soybean phenotypes with linolenic acid less than
4% (low linolenic) and less than 2% (ultralow linolenic acid) have been developed
through mutation [59].
High content of linolenic acid is the important factor responsible for the poor
oxidative stability of some oils such as soybean oil. Partial hydrogenation of highly
unsaturated oils can increase the oxidative stability significantly. However, the use
of partially hydrogenated oils is discouraged because partial hydrogenation leads to
formation of trans fats. Therefore, modification of fatty acid composition by breed-
ing and genetic methods is effective [49]. New phenotypes of soybean, sunflower,
corn, safflower, and rapeseed have been produced to have improved oxidative
stability as well as nutritional value. High-oleic peanut oil developed through
breeding has much greater autoxidation stability as compared to normal oleic peanut
oil [102].
6.3 Blending
Blended oils are prepared by blending of different oils together. Blending is a way
of modifying the fatty acid composition, physicochemical properties, and functional
properties of edible oils without changing their chemical composition [89].
Okogeri [103] studied the frying stability of peanut oil blended with palm kernel
oil at different ratios (90:10, 80:20, 70:30 and 60:40) and reported that all blends
after used for frying contained less polar compounds than control.
6.4 Modifications in Oil Processing
The heat applied during the conventional oil processing technique accounts for
the major loss of natural antioxidants present in edible oils. Cold-pressing oils
may help retain higher levels of natural antioxidants to have acceptable shelf life
without added synthetic antioxidants [95]. Oils obtained by cold pressing are known
as virgin oils, which are very popular due to their typical color, taste, and flavor.
Since there is no heat treatment, most of the natural components are present in the oil
[104]. It is reported that cold-pressed olive oil has a stronger antioxidant activity
attributed to the presence of natural phenolic compounds [89]. Wroniak et al. [105]
have reported that oil flushing with nitrogen was a very effective way to reduce the
changes caused by oxidation in cold-pressed rapeseed and sunflower oil.
7 Conclusion
Autoxidation and photosensitized oxidation are the main deteriorative processes that
occur in edible plant oils during processing and storage. The oxidation of edible oil
leads to the production of off-flavor and toxic compounds and diminishes the oil
quality and shelf life. Oxidative stability of edible plant oils is thus the determining
factor of the selection of suitable oil for different processing and storage methods.
Oxidative stability of oils differs depending on fatty acid composition, the presence
of minor components (antioxidants or prooxidants), and the processing or storage
conditions, mainly, temperature, light, and oxygen. Oxidative stability of the oils
can be improved by modifying the processing conditions such as the use of low
temperature, exclusion of light and oxygen, removal of prooxidants, and the use of
antioxidants.
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The Anti-inflammatory Properties of Food
Polar Lipids 19
Ronan Lordan, Constantina Nasopoulou, Alexandros Tsoupras, and
Ioannis Zabetakis
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
2 Inflammation and Platelet-Activating Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
2.1 Inflammation and Cardiovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
2.2 The Role of Platelet-Activating Factor in Inflammation and Atherosclerosis . . . . . . . 558
2.3 PAF Metabolic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
2.4 Polar Lipids, Inflammation, and Cardioprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
2.5 Diet and Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
3 Food Polar Lipids with Anti-inflammatory and Cardioprotective Properties . . . . . . . . . . . . . . 564
3.1 Fish Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
3.2 Dairy Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
3.3 Wine Lipid Microconstituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
3.4 Polar Lipids of Olive Oil and By-Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
3.5 Various Foods of the Mediterranean Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
4 Future Research and Applications of Food Polar Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Abstract
Cardiovascular diseases (CVD) are the leading cause of death globally. Inflam-
mation is central to the pathology of CVD and is present throughout the athero-
sclerotic process. Unresolved inflammation can lead to atherosclerosis and the
subsequent development of CVD that can cause a major cardiovascular event.
Lifestyle and nutrition are modifiable risk factors for the prevention of CVD.
R. Lordan · A. Tsoupras · I. Zabetakis (*)

Department of Biological Sciences, University of Limerick, Limerick, Ireland
C. Nasopoulou
Department of Food Science and Nutrition, School of the Environment, University of the Aegean,
Myrina, Lemnos, Greece

554 R. Lordan et al.
Research shows that some dietary patterns, such as the Mediterranean diet,
are associated with a decreased risk of CVD. Polar lipids, which are found in
abundance in foods of the Mediterranean diet, are lipids that possess potent anti-
inflammatory and antithrombotic effects against the actions of platelet-activating
factor (PAF). PAF is potent phospholipid mediator of inflammation that plays a
significant role in all stages of atherosclerosis. Bioactive lipids present in various
foods can inhibit the pro-inflammatory activities of PAF via their effects on the
PAF/PAF receptor (PAF-R) signaling but also via modulating PAF metabolism
toward homeostasis. This chapter reviews the relevant research pertaining to the
anti-inflammatory and cardioprotective properties of polar lipids in various foods.
Keywords
Polar lipids · Inflammation · Platelet-activating factor · Cardiovascular disease ·
Mediterranean diet
Abbreviations
CHD Coronary heart disease
CRP C-Reactive protein
CVD Cardiovascular disease
FA Fatty acids
HDL-C High-density lipoprotein cholesterol
IHD Ischemic heart disease
IL-6 Interleukin-6
LDL-C Low-density lipoprotein cholesterol
Lyso-PAF AT Lyso-PAF acetyltransferases
MFGM Milk fat globule membrane
MI Myocardial infarction
PAF Platelet-activating factor
PAF-AH PAF acetylhydrolase
PAF-CPT PAF-cholinephosphotransferase
PAF-R Platelet-activating factor receptor
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PI Phosphatidylinositol
PS Phosphatidylserine
SFA Saturated fatty acids
SM Sphingomyelin
1 Introduction
Cardiovascular diseases (CVD) are the leading cause of death in Europe accounting
for 45% of all deaths; however, with increased success in lifestyle alteration, devel-
oping new treatments, and advancements in medicine, CVD mortality is modestly
19 The Anti-inflammatory Properties of Food Polar Lipids 555
falling in most European countries. CVD is a collective term used to describe diseases
affecting the heart and/or blood vessels and includes coronary heart disease (CHD)
also referred to as ischemic heart disease (IHD), cerebrovascular disease, peripheral
artery disease, congenital heart disease, hypertension, heart failure, and stroke [1].
However, CHD is still the leading single cause of mortality accounting for 19% of
all deaths among men and 20% of all deaths among women each year. Stroke is the
second most common single cause of death in Europe, accounting for 9% in men and
13% of all deaths in women each year [2]. Atherosclerosis is a slow progressive
vascular disease, which underpins CVD. Atherosclerotic lesions or plaques develop
in large- and medium-sized arteries, through the formation of foam cells that leads
to a plaque covering a necrotic core in the vessel wall that can fissure, erode, and
rupture, resulting in a major cardiovascular event such as a myocardial infarction
(MI) [3, 4]. Systemic and unresolved inflammation not only participates in all stages
of atherosclerosis but is also proposed as one of the major causes for the develop-
ment of this disorder and its subsequent CVD manifestations [5].
Increasing evidence suggests that lifestyle and nutrition play a pivotal role, either
in the development or in the prevention of chronic diseases such as CVD [6, 7]. It is
well-established that several risk factors for CVD are modifiable through dietary and
lifestyle changes. Thus, healthy nutrition plays a key role in the primary prevention
of CVD, particularly in relation to the attenuation and prevention of systemic
inflammation [8]. Over the last 70 years, evidence suggests that the Mediterranean
diet may be a particularly healthy dietary pattern, which if adopted can reduce
cardiovascular disease risk.
In the last decade, there has been a surge of reviews and meta-analyses referring
to the beneficial outcome of the adoption of Mediterranean diet in a plethora of
chronic diseases that are either directly or indirectly related to inflammation and
chronic diseases such as heart failure and CVD [5, 9–14]. It is thought that this
dietary pattern is full of bioactive nutrients that work synergistically to reduce the
inflammatory milieu and prevent the onset of chronic disease.
In particular, platelet-activating factor (PAF) seems to play a key role in inflamma-
tory signaling and manifestations associated with all stages of atherosclerosis devel-
opment and its subsequent cardiovascular disorders. However, specific micronutrients
of the Mediterranean diet, belonging to the food-derived polar lipids family, benefi-
cially affect the activities of this potent inflammatory mediator [5]. This chapter reviews
the recent literature surrounding the relationship between PAF, inflammation, athero-
sclerosis, CVD, and the anti-inflammatory properties of food-derived polar lipids.
2 Inflammation and Platelet-Activating Factor
2.1 Inflammation and Cardiovascular Diseases
The human body encounters a large number of stimuli, many of which lead to injury
or infections, damaging cells and tissues. Inflammation is a necessary and protective
physiological response of the innate immune system designed to overcome such
damage. Upon an inflammatory response, activated immune cells, such as blood

leukocytes, produce various agents including reactive oxygen species (ROS), elas-
tases, cathepsins, proteinases, eicosanoids, and other lipid mediators such as PAF
that either promote or suppress inflammation. If the injury or infection is unresolved,
the inflammatory response becomes chronic, leading to oxidative damage of plasma
lipoproteins that results in the inappropriate recruitment of immune cells to the site of
the inflammatory response, thus further exacerbating the inflammatory response [15].
Previously, atherosclerosis and CVD were referred to as simply lipid-related
disorders due to dyslipidemia (hypercholesterolemia or hyperlipidemia) in humans
and various evidence from animal studies. It has long been believed that atheroscle-
rosis is merely involved in the passive accumulation of cholesterol into the arterial
walls as part of the formation of foam cells, which was recognized as the hallmark of
atherosclerotic lesions and subsequent CVD. However, recent systematic reviews
and meta-analyses have begun to question the validity of the lipid hypothesis since
they have revealed that there is lack of an association or an inverse association
between low-density lipoprotein cholesterol (LDL-C) and both all-cause and CVD
mortality in the elderly [16]. As such, these studies underpin the rationale for more
research in relation to the cause (and not only the risk factors) of chronic diseases
such as atherosclerosis and CVD, but also it may be time to re-evaluate the
guidelines for cardiovascular prevention [5].
Even though atherosclerosis and CVD were previously viewed as lipid storage
disorders, thanks to recent research advancements, we now recognize that inflam-
mation plays a key role in the initiation and progression of atherosclerosis [5, 17];
chronic and unresolved inflammatory manifestations seem to be the key causative
underlying mechanistic players at the molecular and cellular level that are respon-
sible for the onset and development of subsequent inflammation-related chronic
disorders such as atherosclerosis and CVD. In particular, endothelial dysfunction
plays a significant role [5].
Evidence from epidemiological studies also supports the hypothesis that
inflammation is the underlying cause of atherosclerosis and the development of
CVD [18, 19]. For instance, it has been demonstrated that elevated levels of
C-reactive protein (CRP) and inflammatory markers in the blood are associated
with an increased risk of future CVD events, even when classical risk factors have
been taken into account [18–22]. Furthermore, several systematic reviews and
randomized trials targeting a number of cytokines suggest that low-grade or
systemic inflammation precedes incident cardiovascular events, and as such that
also implies that inflammation might cause vascular diseases that lead to major
CVD events [19].
Inflammation plays a key role in all stages of the formation of vascular lesions
maintained and exacerbated by several risk factors such as an unhealthy diet
and lifestyle, smoking, hyperlipidemia/hypercholesterolemia, hypertension, autoim-
mune diseases, etc. [5]. The consequence of chronic inflammation is endothelial
dysfunction. Endothelial dysfunction is usually characterized by an inflammatory
milieu acting on leukocytes and endothelial cells, through an interplay with other
immune cells such as T lymphocytes, neutrophils, mast cells, dendritic cells, and
platelets, which is orchestrated by the overexpression and increased production

of pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor
(TNF) and its receptor, CRP, type I interferons (IFN-α, IFN-β), adhesion molecules,
chemokines, lipid inflammatory mediators such as PAF and eicosanoids, increased
generation of ROS, increased oxidation of LDL-cholesterol, and a reduction of the
bioavailability and levels of protective nitric oxide [5]. Inflammation is governed by
the crosstalk between all of these molecules and their cellular interactions [5].
Therefore, deciphering the mechanistic pathways implicated in the inflammatory
crosstalk in the onset, development, and progression of atherosclerosis is of great
importance, in order to unravel possible preventive and therapeutic approaches
to CVD, with less side effects [5]. Common junctions in the mechanistic crosstalk
of inflammatory mediators, signaling pathways, and cellular interactions that occur
during chronic and unresolved inflammation seem to be a promising therapeutic
target for the prevention and treatment of inflammation-related chronic diseases.
Drug-based therapeutic interventions targeting inflammatory mediators such as cyto-
kines (i.e., by using specific antibodies against pro-inflammatory cytokines and their
receptors) and eicosanoids (i.e., by using specific inhibitors of COX-1 and COX-2)
have also been proposed, and relevant trials such as CANTOS and CIRT are still in
progress. However, such approaches can sometimes lead to undesirable effects and
may leave the individual immunocompromised and at greater risk of infections, since
disruption of the physiological balance of the immune system seems to be a risky
strategy [23, 24]. These observations are clearly due to the multifaceted effects of
such mediators in normal physiology.
On the other hand, it is also important to elucidate the mechanistic pathways
of inflammation in order to timely and beneficially resolve inflammatory process.
Several lipid mediators play specific roles in resolving systemic inflammation [25].
Characteristic examples of such lipid mediators are metabolites derived from
omega-3 (ω3) or omega-6 (ω6) polyunsaturated fatty acids (PUFA), including
linoleic acid (18:2 ω6), arachidonic acid (20:4 ω6), eicosapentaenoic acid (EPA,
20:5 ω3), and docosahexaenoic acid (DHA, 22:6 ω3). Generally, ω6 PUFA are
described as pro-inflammatory, whereas ω3 are described as anti-inflammatory,
while the ratio of ω6/ω3 also gives an insight into the correlation between the
inflammatory status related to these lipid mediators and several pathologies [26].
Furthermore, Serhan et al. have proposed that the resolution of inflammation is an
active process orchestrated by distinct cellular events and endogenous lipid media-
tors, including lipoxins, resolvins, protectins, and maresins [27–29]. These novel
lipid mediators are derived from ω3 and ω6 fatty acids, which may explain some of
the mechanistic beneficial effects of dietary PUFA; thus research in this field is
ongoing. For instance, ω6 PUFA supplementation from fish oil has been associated
with anti-inflammatory and cardioprotective properties, due to their interactions with
the eicosanoid pathways [30]. However, recent reviews suggest that the protective
properties of ω3 and ω6 may be exaggerated somewhat and have come under scrutiny
in recent years. In particular, administration of fish oil supplements high in ω3 may
not have any significant beneficial effect against CVD [31]. This will be discussed
further in Sect. 3.1.
Since PAF-related inflammatory cascades belong to the most vital joint

mechanistic pathways of inflammation-related chronic disorders, these inflammatory
pathways have been identified as promising targets for the attenuation of the
inflammatory response, with respect to dietary interventions studies, especially to
those with foods containing bioactive polar lipids [5, 32]. The inverse effects of the
Mediterranean diet on chronic diseases are mostly related to the pleiotropic effects of
its food constituents on several of the inflammation-related pathways. For example,
following a Mediterranean dietary pattern leads to the reduction of several inflam-
matory mediators and biomarkers related to the endothelial functionality, such as
decreases in plasma CRP, IL-6, and intracellular adhesion molecule-1 (ICAM-1)
[33]. Moreover, the Mediterranean diet beneficially affects the PAF pathway and
metabolism toward homeostasis. These beneficial effects may be due to the presence
of polar lipids with anti-inflammatory potential [5].
2.2 The Role of Platelet-Activating Factor in Inflammation and

Atherosclerosis
PAF, 1-O-alkyl-2-sn-acetyl-glycero-3-phosphocholine [34], is a potent phospholipid

mediator, which is characterized by an alkyl ether linkage at the sn-1 position, an
acetyl group at the sn-2 position, and a phosphocholine group at the sn-3 position
(Fig. 1). These are important structurally distinguishing features that are critical
to the biological activity of PAF, which is mediated by stereospecific binding to its
specific receptor (PAF-R) [35, 36]. PAF plays a role in several chronic diseases, in
particular CVD [5, 37]. The structure of PAF is distinctive due to the position of
an ether linkage at the sn-1 position; as such moieties are not common in animals.
Similarly it is unusual for an acetic acid to be esterified directly to the sn-2 position of
glycerol. PAF was the first phospholipid known to possess messenger functions by
binding to its specific receptor on the cell membrane, rather than by physicochemical
effects on the plasma membrane of the target cell [38]. Interestingly, there are several
proposed phospholipid structures that are considered part of the “PAF family,” a
newly coined term to describe these PAF-like molecules that not only share similar
Fig. 1 The structure of the classic platelet-activating factor molecule [34]

structures but can also exhibit similar bioactivities [37]. However, these molecules
do not seem to possess the same level of activity as the classical PAF structure, as
increasing the chain length beyond three carbons at the sn-2 position decreases its
biological potency. For instance, there are several compounds synthesized by
numerous cells that are similar to PAF but bear a fatty acid instead of a fatty alcohol
at sn-1 position. These moieties have less than 1% of the potency of PAF. Similarly,
modifying the polar group at the sn-3 position decreases the potency of the PAF-like
molecules [39, 40].
PAF and PAF-like molecules act through their binding to a unique G-protein-
coupled seven-transmembrane receptor (PAF-R), which triggers multiple intracellu-
lar signaling pathways depending on the target cell and the levels of PAF in the
tissue or blood [41]. As a result, PAF is involved in several physiological processes
including the modulation of the normal inflammatory response, regulation of blood
pressure and the coagulation response, fetal implantation, lung maturation, initiation
of parturition, and exocrine gland functions [32]. PAF is most known for its role in
anaphylaxis [42] and atherosclerosis [5, 43].
PAF is produced and released in large amounts by inflammatory cells in response
to certain stimuli including upstream regulators like IL-1, IL-6, endothelin, TNF-α,
and PAF itself [37, 44, 45]. Increased PAF levels at the site of inflammation can
activate several cells that can lead to a broad spectrum of effects as a result of PAF
production, depending on the type of cell or tissue. This occurs due to the effects of
various downstream mediators enhancing production and release of PAF and several
other inflammatory mediators, including eicosanoids, TNF-α, IL-1α, IL-6, IL-8,
growth factor, and ROS, and the expression of integrins and selectins in the
membranes of activated cells [32, 43, 44]. The crosstalk between PAF and various
upstream and downstream mediators that affect PAF production seems to be
interconnected during inflammatory manifestations [5, 32]. In particular, PAF can
induce leukocyte adhesion, leukocyte degranulation, chemotaxis, respiratory burst,
and increased vascular permeability [46, 47]. Recently it has been demonstrated
that the activation of mast cell PAF-R promotes the immunosuppressive effects of
PAF in part through histamine and prostaglandin E2-dependent mechanisms [48].
The PAF pathways serve as one of the main junctions between many inflamma-
tory cascades that can lead to inflammatory-related disorders such as CVD and
cancer [5]. As a result the PAF pathways have been identified as a possible
therapeutic target for a number of inflammation-related diseases. Research in this
field initially were focused on inhibiting the PAF/PAF-R interactions, thus pre-
venting the initiation of the complex PAF inflammatory pathways [32, 37, 49–52].
Several molecules of synthetic and natural origin [3, 53] have been have been
identified, which have the capacity to competitively or noncompetitively displace
PAF from its binding sites [54]. Although specific PAF antagonists have exhibited
promising results in various studies, the most beneficial effects have been identified
in polar lipid extracts of several foods [5]. These food extracts exhibit anti-
thrombotic, antioxidant, and anti-inflammatory activities through inhibiting PAF
activities and beneficially modulating PAF levels by altering its metabolism [32].
These molecules are discussed further in Sect. 3.
2.3 PAF Metabolic Enzymes
PAF is biosynthesized at sub-picomolar concentrations [55] and is strictly

controlled by two enzymatic pathways known as the remodeling pathway and the
de novo pathway [56]. The de novo pathway is catalyzed by a specific dithiothreitol-
insensitive CDP-choline, 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase
(PAF-cholinephosphotransferase [PAF-CPT], EC 2.7.8.2) [57], which converts
1-O-alkyl-2-acetyl-glycerol to PAF. The remodeling pathway is catalyzed by lyso-
PAF acetyltransferases (lyso-PAF ATs, EC 2.3.1.67) [57], which acetylates lyso-PAF.
The de novo pathway is known as the minor endogenous PAF synthesis pathway [58],
which maintains hemostatic levels of PAF during normal cellular functions [59]. The
remodeling pathway is considered the primary enzymatic pathway for PAF synthesis
in various inflammatory and allergic disorders [60].
Interestingly, apart from the remodeling pathway which is always activated in both
acute and chronic inflammation, the key enzyme of the de novo pathway, PAF-CPT,
seems to be more active during chronic inflammatory manifestations, thus contribut-
ing to an increase of basal levels of PAF that seem to be related to the continuous
activation of inflammatory cascades during the development of inflammation-related
chronic disorders [5, 44, 61, 62]. Thus, the regulation of the biosynthetic pathways of
PAF seems to be more complicated than was initially thought. In addition, both PAF
biosynthetic routes correlate with well-established inflammatory and immunological
biomarkers (i.e., several cytokines, viral load, CD40L, etc.) in several chronic
diseases [5, 38, 44, 61–66].
Apart from its enzymatic biosynthetic pathways, PAF and PAF-like lipids can also
be produced through nonenzymatic synthesis by oxidative transformation of other
lipids during oxidative stress or inflammation [55, 67]. When lipids on lipoproteins
such as LDL are oxidized, Ox-LDL is produced where bonded PAF-like molecules
can mimic the actions of PAF affecting PAF-related inflammation [5]. These path-
ways are not regulated enzymatically. Vice versa, PAF and PAF-like lipids can also
stimulate the production of ROS and nitrogenous species such as reactive nitrogen
species (RNS) during oxidative and nitrosative stress in inflammation-induced endo-
thelial dysfunction and atherosclerosis [37].
PAF is catabolized to its biologically inactive form, lyso-PAF, by a specific PAF-
acetylhydrolase (PAF-AH, EC 3.1.1.47). The plasma isoform of PAF-AH is known
as lipoprotein-associated phospholipase A2 (Lp-PLA2) [68], since it circulates in
blood in association with plasma lipoprotein particles such as LDL-C and high-
density lipoprotein cholesterol (HDL-C), or PLA2 group 7 [68–71]. PAH-AH also
has the capacity to cleave short-chain acyl chains at the sn-2 position of oxidized
phospholipids, PAF-like phospholipids, and PAF [43].
The amount of PAF present in biological tissue is controlled by the balance
of the anabolic and catabolic PAF pathways [72]. Enzymatic biosynthesis of PAF
contributes to basal PAF levels and a periodic increase of PAF levels during normal
inflammatory responses, while during unresolved and chronic inflammatory mani-
festations, the enzymatic biosynthesis of PAF is responsible for pathologically
increased PAF levels through a continuous induction of the PAF cycle [5]. Oxidative
stress and inflammatory conditions also favor nonenzymatic synthesis of both PAF
and PAF-like molecules, such as oxidized phospholipids and oxidized lipids bonded
to Ox-LDL. These molecules can amplify the PAF cycle-related inflammatory
cascades [5]. On the other hand, PAF catabolism is activated during both acute
and chronic inflammatory manifestations and inactivates both PAF and PAF-like
molecules, as a homeostatic response against PAF-related inflammatory manifesta-
tions. Thus, the PAF metabolic enzymes are implicated in a number of inflammatory
manifestations, including heart failure [73, 74], inflammatory bowel disease [72],
HIV [62], cancer [44], and atherosclerosis [5].
2.4 Polar Lipids, Inflammation, and Cardioprotection
Polar lipids, such as phospholipids, glycolipids, sphingolipids, etc., are a lipid class
that structurally contain hydrophobic hydrocarbon residues and a hydrophilic group
such as a carbohydrate group, or a phosphate head group. Phospholipids consist of
a glycerol, usually esterified to a saturated long-chain fatty acid at sn-1 position, to
an unsaturated long-chain fatty acid at sn-2 position, and to a phosphorylated head
group at sn-3 position [8]. Phospholipid head groups are generally cytidine mono-
phosphate, hydroxyl, choline, ethanolamine, serine, or inositols [75]. The general
structure of phospholipids is outlined in Fig. 2.
Even though the main function of phospholipids is to support the formation,
integrity, and functionality of cell membranes, there are various phospholipid species
that possess a plethora of additional functions, in cell signaling, inflammation, and
digestion [32]. For example, phosphatidylcholine (PC) and phosphatidylethanol-
amine (PE) are concentrated in the plasma membrane of macrophages [76] and are
major phospholipids in human plasma, where 76% and 17% of the total glyceropho-
spholipids present are PC and PE, respectively [77]. PC and PE are also the most
abundant phospholipids in erythrocytes [78]. Dietary phospholipids are usually
digested in the lumen, while almost 20% of intestinal phospholipids are absorbed
passively and without hydrolyzation and preferentially incorporated directly into
plasma lipoproteins. These phospholipids travel through the blood stream and may
be incorporated into several cell membranes [32]. The same processes occur for
phospholipids baring PUFA within their structures, which are mainly of marine
origin. Thus, PC and PE are the largest phospholipid reservoirs of dietary ω3 and
ω6 PUFA in the cells and biological fluids involved in inflammatory processes [15].
Other less abundant phospholipids can affect inflammatory responses independent of
the production of lipid mediators; cardiolipin, which contains two phosphatidic acids
esterified to glycerol, is the most abundant phospholipid in the inner membrane of
mitochondria [79]; it can mediate apoptosis [80] and compromise cellular respiration
[81] under conditions of oxidative stress. Other phospholipids also seem to contribute
directly and/or indirectly to several inflammatory cascades and thus are involved in
the onset, progression, and often remediation of inflammatory diseases. These phos-
pholipids include plasmalogens, PAF, oxidized phospholipids, and phospholipids
that are carriers of fatty acids precursors of eicosanoids [32].
GPLs Structure Sphingomyelin Alkyl-GPLs Structure

• Choline • Choline
Hydrophilic head-group X • Ethanolamine Hydrophilic head-group X Choline Hydrophilic head-group X • Ethanolamine
• Serine • Serine
O • Inositol O O • Inositol
Phosphate O=P–O– • Glycerol
Phosphate O=P–O– Phosphate O=P–O–
• Glycerol
O O O
Glycerol CH2–CH–CH2 HO–CH2–CH–CH2 Glycerol CH2–CH–CH2

backbone
backbone
Sphingosine backbone
O O CH NH O
O
ether-bond
O=C C=O CH C=O C=O
Fatty chain
Fatty chain
Fatty acid
Fatty acid
Fatty acid
Fatty acid
Fig. 2 Phospholipids are generally composed of glycerol, two fatty acids esterified to glycerol
at the sn-1 and sn-2 positions, and a phosphorylated head group esterified at the sn-3. The most
common structures of various phospholipids are presented. Phospholipids possess a glycerol
backbone (GPLs). Sphingomyelin is characterized by a sphingosine backbone (SPLs). Alkyl-
phospholipids (Alkyl-GPLs) have a fatty chain linked with an ether bond at the sn-1 position of
the glycerol backbone. (Reproduced with permission from Lordan et al. [32])
On the other hand, several foods contain bioactive polar lipid compounds
that have well-established modes of anti-inflammatory action, whose pleiotropic
therapeutic effectiveness and lack of toxicity ensure clinical safety [5, 32]. Dietary
polar lipids have been found to exert anti-inflammatory actions in various models of
inflammation [3]. Research has shown that there are specific structures of polar lipids
that possess antithrombotic and anti-inflammatory effects against PAF [5, 82].
For example, specific PC and PE lipid species of marine origin as well as specific
glycolipids in wine and olive oil have exhibited potent bioactivity against PAF-
induced inflammation, with beneficial effects against atherosclerosis and CVD [5].
Some of these bioactive structures are presented in Fig. 3. These structures will be
further discussed in Sect. 3.
2.5 Diet and Inflammation
Research has established that there is a clear link between an individual’s diet and
systemic inflammation [84–86]. A maladaptive diet and lifestyle are the dominant
underlying causes of low-grade inflammation [87, 88]. Processed and energy-rich
Fig. 3 (a) The typical structure of PAF. (b) A representative diagram of the bioactive marine
phospholipids as elucidated previously by Sioriki et al. and Nasopoulou et al. [82, 83]. Generally,
PC and PE derivatives exhibit the greatest biological activity in marine sources. (Reproduced with
permission from Tsoupras et al. [5]). PAF platelet-activating factor, PLs polar lipids
foods and beverages lead to exaggerated postprandial elevations in plasma glucose

and triglycerides. As a consequence of our increased intake of these foods in Western
societies, postprandial hyperglycemia and hyperlipemia are common [3, 87]. Fur-
thermore, postprandial lipemia is now considered an independent risk factor
for CVD, obesity, type 2 diabetes mellitus, and metabolic syndrome [3]. Spikes in
glucose and triglycerides can lead to the production of excess plasma ROS that can
initiate pro-inflammatory reactions [87–89]. Furthermore, it has been demonstrated
that excessive intake of macronutrients such as glucose and saturated fat promotes
various inflammatory responses, including an increase in cytokine activity and
inflammatory transcription factors [87, 90, 91]. Activated immune cells can either
promote or suppress inflammatory processes. It is postulated that prolonged expo-
sure to such inflammatory responses on a daily basis can contribute to the patho-
genesis of chronic diseases by establishing a perpetual low-grade inflammatory state
[92]; therefore, reducing inflammation to homeostatic levels is essential to avoid
long-lasting damage to host tissue [93].
Various dietary patterns can resolve the inflammatory process due to the presence
of various bioactive molecules in foods, such as bioactive lipids [3] and peptides [94].
The Mediterranean and Nordic dietary patterns seem to be two such diets that
evidence suggests may affect health due to anti-inflammatory properties [95].
Recently, a significant number of studies refer to the beneficial outcomes observed
due to the adoption of a Mediterranean dietary pattern in a plethora of several chronic
diseases that are either directly or indirectly related to inflammation [5]. In addition,
the Mediterranean diet has also been associated with beneficial outcomes in second-
ary CVD prevention [12]. When patients suffering from CVD or diabetes adhere to
the Mediterranean dietary pattern, the incidence of recurrent myocardial infarction
and cerebrovascular events is reduced. The protective effect of the Mediterranean
dietary pattern can be maintained for up to 4 years after the first myocardial infarction
(Lyon Diet Heart Study) [96]. Moreover, in contrast to the contradictions of lipid
hypothesis and mortality in elderly people [16], the HALE project has also shown that
individuals aged 70–90 years adhering to the Mediterranean diet and a healthy
lifestyle have a 50% lower rate of all-cause and cause-specific mortality [97].
Followers of the Mediterranean diet are also less likely to suffer sudden cardiac
death and age-related cognitive decline [14].
The inverse association between Mediterranean diet and all-cause mortality
and cardiovascular mortality has been attributed to several of its pleiotropic protec-
tive effects. The Mediterranean diet can beneficially influence several risk factors
including lowering BMI, blood pressure, and lipid levels (i.e., the ratio of LDL-C/
HDL-C), reducing insulin resistance, and improving HDL-C functionality. However,
the main beneficial impact of Mediterranean diet seems to be associated with the
improvement of endothelial function and a decrease of the inflammatory milieu.
It seems that the reduction of inflammation-related mediators and biomarkers such as
PAF and several cytokines and of oxidative stress (with lower concentrations
of oxidized LDL and improved apolipoprotein profiles) and platelet aggregation
and blood coagulation are responsible for the observed beneficial effects of the
Mediterranean diet [5, 8, 32].
Even though the beneficial outcomes of healthy dietary patterns such as the
Mediterranean diet are profound and well documented, in Westernized and develop-
ing countries, there is an increase in the consumption of ultra-processed foods, which
is associated with systemic inflammation [87, 98] and may contribute to the rise in
noncommunicable diseases. It is suggested that reducing the dietary share of ultra-
processed foods by increasing the consumption of unprocessed or minimally pro-
cessed foods and freshly prepared meals made from these foods can be an effective
way to substantially improve the substantial and nutritional quality of Westernized
societies. However, given the ubiquity of ultra-processed foods in general, radical
whole population strategies are needed to achieve the necessary reductions in ultra-
processed food consumption [99].
3 Food Polar Lipids with Anti-inflammatory and

Cardioprotective Properties
Polar lipids from various foods, particularly from foods of the Mediterranean diet,
seem to exhibit biological activities against the PAF pathway [5]. Some polar lipids
act as PAF antagonists that block the binding of PAF with its receptor, and others act
as PAF-R agonists but, as aforementioned, possess far less potency than PAF itself.
Polar lipids are also thought to directly or indirectly interrupt the binding of PAF to
its receptor by altering the lipid raft microenvironment of the PAF-R in cell mem-
branes and thus its subsequent intercellular pathways [32]. In addition, polar lipids
can modulate the activities of the regulatory metabolic enzymes of PAF. Several
studies show that these lipid constituents can downregulate the metabolic enzymes
that control PAF biosynthesis and upregulate the enzymes responsible for PAF
degradation [61, 100]. Thus, dietary patterns based on foods rich in such bioactive
polar lipids seem to possess beneficial effects through the modulation of PAF
metabolism toward homeostatic PAF levels and activities [5]. Bioactive polar lipids
are present in various foods of the Mediterranean diet and seem to exhibit specific
structures (Fig. 3). These foods include fish, dairy, wine, olive oil, and various other
foods. Interestingly, the anti-inflammatory properties of various types of polar lipids
seem to be as a result of a synergistic effect between the different polar lipids [5]. The
various sources of polar lipids and their bioactivities are discussed.
3.1 Fish Polar Lipids
Epidemiological studies and clinical trials have demonstrated the protective role of
fish and fish oil consumption against CVD [101], while their nutritional benefits
have mainly been associated with their omega-3 PUFA content [102, 103]. However,
several recent reviews and meta-analyses have concluded that insufficient evidence
exists to suggest a beneficial effect of ω3 PUFA supplementation in adults with
chronic diseases such as atherosclerosis and CVD [31, 32]. It is now well-established
that more complex mechanisms underlie the beneficial effects of fish and fish oil
consumption and administration of marine products that go far beyond the ω3
PUFA-/eicosanoid-related mechanisms [32]. Other lipid constituents are also present
in fish and fish oils that have different metabolic effects after absorption with distinct
biological activities not only limited to their superior incorporation to plasma
lipoproteins and cell membranes and bioavailability of their omega fatty acids but
also to their reported anti-inflammatory activities through also other mechanisms
than the ARA/eicosanoid pathway, such as the inhibition of the PAF pathway and the
modulation of PAF metabolism [32].
Fish contains between 1% and 1.5% phospholipid by weight, where PC
derivatives are generally the predominant phospholipids followed by PE, phosphati-
dylinositol (PI), phosphatidylserine (PS), lyso-PC, and sphingomyelin (SM) [32].
Polar lipids of fish exhibit both in vitro antithrombotic [82, 83, 104–109] and in
vivo antiatherogenic properties [110]. In 1996, Rementzis et al. [104] reported the
presence of polar lipids in Scomber scombrus (mackerel) that exhibited inhibitory
activity against PAF and thrombin-induced platelet aggregation. This leads to a series
of other studies that identified polar lipids in various fish species that exhibited effects
against PAF in vitro and in vivo [32].
Notably, Nasopoulou et al. have explored the anti-PAF and antiatherogenic
properties of marine polar lipids in a series of studies that examined the polar lipid
extracts from wild and cultured sea bass (Dicentrarchus labrax) and sea bream
(Sparus aurata). These polar lipids seem to exhibit strong agonistic and antagonistic
effects against PAF-induced platelet aggregation [106, 110, 111], and they seem to
inhibit the activities of the basic biosynthetic enzymes of PAF [61]. Furthermore, an
in vivo study separated 12 healthy male New Zealand rabbits of specific weight and
age into 2 even groups: A and B [110]. Group A was given an atherogenic diet for
45 days as was group B; however, the group B diet was also supplemented with
marine polar lipid extracts from Sparus aurata. After 45 days the HDL-C levels were
significantly increased in the rabbits that were supplemented with marine polar lipids
in comparison with the control group. In addition, PAF catabolic enzyme activity
(plasma PAF-AH) was significantly increased, and platelet aggregation efficiency
was reduced in the rabbits fed marine PL in comparison with the control group. Of
greater significance was the fact that hypercholesterolemic rabbits supplemented
with polar lipids developed early atherosclerotic lesions that were of a statistically
significant lower degree ( p < 0.017) than that of the control group as demonstrated
in Fig. 4. It seems the overall anti-PAF effects exhibited by the marine polar lipids
led to a reduction in the formation of foam cells indicating that these lipids possess
antiatherogenic properties and thus may explain some of the beneficial effects
observed due to fish consumption.
A follow-up study was published that utilized plasma from the same rabbits
fed the same fish polar lipids, which investigated the effects of the polar lipids
on PAF biosynthesis and PAF catabolism [64]. The specific activity of PAF-AH
was decreased in rabbits’ platelets of both groups A and B and in rabbits’ leukocytes
of group A ( p < 0.05). On the other hand, the specific activity of Lp-PLA2
was increased in both groups A and B in both leukocytes and platelets ( p < 0.05).
PAF-CPT demonstrated an increased specific activity only in rabbits’ leukocytes of
group A ( p < 0.05). Neither of the two groups showed any change in lyso-PAF-AT-
specific activity ( p > 0.05). Free and bound PAF levels increased in group A while
decreased in group B ( p < 0.05). These results indicate the fish polar lipids modulate
PAF metabolism upon atherosclerotic conditions in rabbits leading to lower PAF
levels and PAF activity in the blood of rabbits with reduced early atherosclerotic
Fig. 4 Representative optic micrographs x100 of aortic wall sections stained with hematoxylin and
eosin from the two experimental groups, where atherosclerotic lesions appear as foam cells between
the arrows. (a) Group A (atherogenic diet); (b) group B (atherogenic diet enriched with marine polar
lipids)
lesions compared to the control group. Therefore, it can be said that the joint
inhibitory effects of these lipids against PAF/PAF-R interactions and the ability to
modulate the catabolic and biosynthetic enzymes of PAF are due to the intake of the
polar lipids in this investigation. Further studies are required to see if these effects are
demonstrated in other animal models and in human studies.
Notably, recent studies have revealed the existence of bioactive polar lipids in
several oily fish species, such as sardines. Polar lipids were also present in cod liver
oil, which is the main source of marine-derived health [112]. Sardine polar lipid
extracts possessed a higher content of bioactive polar lipids that also demonstrated
higher antithrombotic activities against platelet aggregation when compared to that
of fish oil derived from cod liver oil [112]. Similar studies in Irish organic farmed
salmon (Salmo salar), another oily fish species, revealed that Atlantic salmon is also
a rich source of bioactive polar lipids with potent antithrombotic activities against
platelet aggregation [113]. Moreover, it was revealed that the antithrombotic effects
of salmon polar lipids were attributed to their anti-PAF effects, since they exhibited
potent inhibitory effects against PAF-induced human platelet aggregation and much
less potent effect toward thrombin-induced platelet aggregation. It was thus pro-
posed that these bioactive polar lipids seem to act synergistically against platelet
aggregation either by inhibiting PAF-induced activities or by very low agonistic
effects to its receptor [113]. Structural elucidation of these salmon-derived polar
lipids revealed that they also belong to the alkyl-acyl PC and alkyl-acyl PE deriv-
atives that bare ω3 PUFA such as the EPA and DHA at the sn-2 position of their
glycerol backbone [113]. These results suggest that salmon is a source of bioactive
polar lipids rich in ω3 PUFA and that the physiological and pleiotropic beneficial
effects of marine polar lipids can be attributed to the coexistence of both important
structural elements such as their polar head groups and the constituting PUFA within
their structures.
3.2 Dairy Polar Lipids
The negative perception of dairy fats stems from the effort to reduce dietary saturated
fatty acid (SFA) intake due to their association with increased cholesterol levels
upon consumption and thus an increased risk of CVD development [3, 114].
However, recent research and meta-analyses have demonstrated the benefits of
full-fat dairy consumption, based on greater bioavailability of high-value nutrients
(such as vitamin D) and anti-inflammatory properties [114]. Dairy products contain
a complex lipid profile and are distinguished by the fact that they are the most natural
source of short-chain fatty acids (C4–C8, 4–13 wt% total FA), which are generally
esterified on the sn-3 position of the triglyceride [115]. The nonpolar lipids or neutral
lipids (triglycerides or TG; 96–97% of milk lipids), the polar lipids (glyceropho-
spholipids, sphingolipids, glycosphingolipids, glycolipids; 0.2–2% of milk lipids),
and cholesterol create an oil in water emulsion to form milk. These lipids form
spherical milk fat globules that contain triacylglycerides (0.1–15 μm) that are
surrounded in a complex trilaminar membrane (4–12 nm) composed of proteins,
phospholipids, and sphingolipids, suspended in an aqueous liquid phase, which is

derived from mammary endothelial cells [116]. This unique structure is the milk fat
globule membrane (MFGM), which mainly consists of lipids (40%), proteins (60%),
and cholesterol [117]. The membrane of the outer leaflet consists of phospholipids
and cholesterol, which stabilizes the triglyceride-rich milk fat globule against coa-
lescence and protects the core from lipolytic degradation and oxidation [32]. The
MFGM structure is depicted in Fig. 5.
Other sources of phospholipids in dairy products include MFGM fragments and
lipoprotein particles, which are believed to be remnants of the mammary secretory cell
membranes. Similar to the MFGM, phospholipids originate from the apical plasma
membrane of the mammary gland secretory cell [116–122]. The phospholipids present
Fig. 5 Illustration of the milk fat globule membrane (MFGM). The sizes in this schematic are not
in proportion. A phospholipid monolayer surrounds the triacylglycerol core, followed by a protein-
aceous coat connecting the monolayer to the outer phospholipid bilayer. Adipophilin (ADPH) is
located in the inner layer polar lipid layer, while xanthine dehydrogenase/oxidase (XDH/XO) is
located between both layers. PE, PS, and PI are generally concentrated on the inner surface of the
membrane, whereas PC, SM, glycolipids (G), cerebrosides, and gangliosides are mainly located in
the external membrane. SM and cholesterol (C) can form rigid domains in the cellular membrane
known as lipid rafts. Glycoproteins are distributed over the external membrane surface; these
include butyrophilin (BTN), mucin 1 (MUC1), PAS 6/7, and CD36. (Reproduced with permission
from Lordan et al. [32])
Table 1 Typical phospholipid composition of bovine, caprine, and ovine milk

Dairy polar lipids TPLa PCb PEb PIb PSb SMb
Bovine milk [118, 130–132] 0.3–1.1 20–40 20–42 0.6–12 2–11 18–35
Ovine milk [133] 0.2–1.0 26–28 26–40 4–7 4–11 22–30
Caprine milk [133] 0.2–1.0 27–32 20–42 4–10 3–14 16–30
Abbreviations: PC phosphatidylcholine, PE phosphatidylethanolamine, PI phosphatidylinositol,
PS phosphatidylserine, SM sphingomyelin, TPL total polar lipids
a
Mean values expressed as % of total lipid composition
b
Expressed as % of total phospholipids
in bovine, caprine, and ovine milks are quantitatively minor lipid constituents of milk;
however they possess several beneficial techno-functional properties and are involved
in various physiological processes making them nutritionally valuable [32].
Research by our group has demonstrated that bovine, ovine, and caprine dairy
products possess polar lipids with potent anti-inflammatory activities against PAF as
demonstrated in a series of in vitro experiments on washed rabbit platelets [123–125].
Research has shown that as milk is fermented to yoghurt and then to cheese, the
bioactivity of the PAF inhibitors seems to increase [3]. This indicates that the processes
of fermentation and lipolysis are crucial to altering the bioactivity of the polar lipid
fractions of milk, and this bioactivity increases the further fermentation proceeds [3,
114]. It is thought that the fermentation process alters the fatty acid composition of the
polar lipids, thus leading to variation in the biological activity, depending on the
microorganisms used. Therefore, yoghurt and cheese seem to exert greater biological
activity against PAF than just milk polar lipids. In particular, these effects have been
attributed to microorganisms such as Lactobacillus delbrueckii ssp. bulgaricus and
Streptococcus thermophilus, which are commonly used in yoghurt production [3,
126]. However, further research is required to identify whether these microorganism
may also play a role in the biosynthesis of these bioactive lipids or if they interact with
the MFGM. It is also imperative to identify other fermented foods that may contain
bioactive polar lipids such as wine, beer, and kefir in order to elucidate and establish
how fermentation impacts the health effects of various foods.
Research also indicates that polar lipids of caprine and ovine milk and dairy
products possess greater bioactivity against PAF than those of bovine milk and dairy
products [125, 127]. This may be due to their varied phospholipid content (Table 1).
Therefore, further research is required to confirm these observations. Furthermore,
polar lipids seem to be bioavailable in in vivo and clinical studies and seem to be able
to exert their biological activities [110, 128, 129]; thus whether dairy lipids share the
same bioavailability remains to be seen.
3.3 Wine Lipid Microconstituents
Associations between wine consumption and the “French paradox” motivated inten-
sive research into the health benefits of moderate wine consumption. The nutritional
superiority of wine over other alcoholic beverages is thought to be attributed to

microconstituents within wine that contain bioactive compounds whose mechanistic
pathways are currently under investigation [134]. Alcohol consumption has a com-
plex relationship with human health, and the harmful effects of alcohol are well-
known and in contrast to the beneficial effects [135]. Various studies have linked
excessive alcohol consumption to various chronic diseases and conditions including
cancers and liver cirrhosis [136]. Despite these negative associations, moderate
alcohol consumption is associated with a number of health benefits [135]. Research
indicates that moderate alcohol consumption (1–2 drinks/day) is associated with
positive effects against a number of cardiovascular risk factors [134, 137, 138].
In addition, large epidemiological studies have demonstrated that moderate alcohol
consumption significantly reduces cardiovascular risk factors, morbidity, and
mortality through a dose-effect relationship that is characterized by a J-shaped
curve [138].
Generally, wine contains between 12% and 15% (v/v) ethanol. The ethanol
content of wine is associated with a number of beneficial effects including alterations
of plasma lipoproteins and modifications of blood platelet function and coagulation
[139]. However the benefits of moderate ethanol intake due to alcoholic beverage
consumption can only partly explain the protective effects of wine [140]. Mecha-
nistically, lipid and phenolic microconstituents in wine may play a role in attenuating
inflammatory processes associated with CVD. Phenolic compounds include pheno-
lic acids (p-coumaric, cinnamic, caffeic, gentisic, ferulic, and vanillic acids), trihy-
droxy stilbenes (polydatin and resveratrol), and flavonoids (catechin, epicatechin,
and quercetin), while their polymerization gives rise to the viniferins and pro-
cyanidins. The phenolic content of white wines is considerably lower than red
wines due to their manufacturing process. Red wines are usually produced with
the grape skins, whereas white wine is generally produced from free-running juices
that do not have contact with the skin of the grapes [140, 141]. Initially, the
biological activity of wine was attributed to their antioxidant properties; however,
currently it is well-established that wine consumption is associated with anti-inflam-
matory activities [142], and these cardioprotective effects are induced irrespective of
their antioxidant properties.
In fact, these anti-inflammatory effects may be attributed to the anti-PAF effects
of various lipid and phenolic microconstituents within the wine [140]. Studies show
that polar lipids in wine can inhibit the biological actions of PAF [143–146] and
modulate enzymes involved in PAF metabolism in vitro [147]. Interestingly, within
these studies it was also unexpectedly revealed that white wines, musts, and the
specific grapes that produced the white wines possessed polar lipids with potent anti-
PAF activities, while structural elucidation of these lipids revealed that they were
mostly glycoglycerolipids or glycol-glycerophospholipids and not phenolic com-
pounds [143, 144, 146], supporting the notion that their beneficial effects can be
attributed to their anti-PAF effects and not to any putative antioxidant effects.
Clinical studies have also been conducted, which demonstrate that the beneficial
effects of wine consumption may be due to bioactive components in wine that affect
platelet function. Most notably, in the pursuit for answers to the observed French
paradox, researchers saw a reduction of platelet aggregation due to moderate alcohol

consumption [148, 149].
Clinical studies have been conducted that demonstrate that wine consumption
may exert its anti-inflammatory effects through the modulation of PAF interaction
with the PAF-R and PAF metabolism. A recent study took ten healthy men who
participated in four daily trials on separate days. They consumed a standardized
meal along with white wine, red wine, an ethanol solution, or water. Blood samples
were collected before and after meal consumption and at several time points over a
6-h period, and various hemorheological values were determined. It was found that
wine consumption improved platelet sensitivity independently of the alcohol,
triglyceride levels were lowered during postprandial elevations, and plasminogen
activator inhibitor-1 levels were not impacted negatively by the alcohol consump-
tion. These effects are attributed to the presence of PAF inhibitors in wine [129]. A
similar clinical study by the same group also examined the postprandial effects of
wine consumption on the PAF metabolic enzymes. The same trial methodology,
standardized meal, wines, and controls were used. It was found that consumption
of wine, but not alcohol alone, reduced the activity of specific PAF biosynthetic
enzymes (PAF-CPT and lyso-PAF AT) during postprandial elevation, thus attenu-
ating postprandial inflammation [150]. Again these effects have been attributed to
microconstituents within wine that exhibit potent anti-PAF effects. It seems that the
attenuation of the inflammatory may be as a consequence of the attenuation of PAF
levels by lipid microconstituents and/or polar lipids that act as PAF inhibitors.
Further research is required to further our understanding of this phenomenon and
to discern whether these effects are seen in other fermented alcoholic beverages
such as beer or spirits.
3.4 Polar Lipids of Olive Oil and By-Products
Virgin olive oil is the main source of dietary lipid in the Mediterranean diet, and
it has been linked to numerous health benefits including reduced systolic blood
pressure, an enhancing effect of T-cell-mediated function, and an increase in HDL-C
[151, 152]. The beneficial effects of olive oil consumption have been attributed to
its high concentration of monounsaturated fatty acids (MUFA), namely, oleic acid
[153, 154]. However, other seed oils have similar levels of MUFA but do not seem to
possess the same biological activity. Therefore, research has turned its attention
to other compounds such as polar lipids or phenolic compounds [155]. Polar lipids of
virgin olive have demonstrated antagonistic effects against PAF, and following
various chromatographic separations, the most active fraction was identified as a
glycerol ether glycolipid [156].
A subsequent in vivo study aimed to investigate the effects of virgin olive
oil consumption in 40 male hypercholesterolemic white New Zealand rabbits
[128]. The rabbits were randomly distributed into four groups. Group A were fed
an atherogenic diet containing cholesterol; group B were fed the same atherogenic
diet supplemented with 15% (w/w) virgin olive oil; group C were fed atherogenic
diet supplemented with olive oil polar lipids isolated from the same volume of
olive oil supplemented in group B; finally, group D were fed the atherogenic
diet supplemented with olive oil neutral lipids isolated from the same volume of
olive oil supplemented in group B. Baseline measurements were taken for a
number of parameters, and the rabbits were fed their respective diets for
45 days before testing and prior to being euthanized. Rabbits that received the
polar lipid extract had a significant reduction of early atherosclerotic lesions
compared to group A that received only an atherogenic diet, and they also
retained the elasticity of the aortic wall. Rabbits that received neutral lipids of
olive oil did not exhibit a reduction in early atherosclerotic lesions compared to
the control group. PAF levels in the blood were higher for group A and decreased
in group D by day 45. In group A, along with increased PAF levels, PAF-AH was
also increased, atherosclerotic lesions formed, and vessel wall elasticity was
decreased. In groups B and C, blood PAF-AH increased, platelet aggregation
was attenuated, less oxidation occurred in the plasma, lesion thickness was
reduced, and vessel wall elasticity was retained. However, most of these effects
were not observed in animals fed neutral lipids (group D), although blood PAF
and plasma oxidation were lower [128]. This study demonstrated for the first time
that it was the polar lipid composition of the virgin olive oil that was responsible
for the antiatherogenic activities observed [155].
Interestingly, studies have also shown that polar lipids present in the by-products
of the olive oil industry possess potent antithrombotic, anti-inflammatory, and anti-
PAF activity in vitro and in vivo [157–159]. It has also been shown that these lipids
may also beneficially modulate PAF metabolism toward the reduction of PAF levels
to homeostatic levels, thus explaining the rationale for their observed in vivo effects
on reducing PAF levels [100].
Olive pomace is one of two major by-products of the olive oil industry, the other
being olive mill wastewater. Unfortunately, for every 100 kg of olive oil produced,
there is 35 kg of olive pomace produced that is generally an underutilized waste
product, which has been proposed as a viable alternative to fish oil in the aquaculture
industry [160]. In a study by Nasopoulou et al. [107], cultured fish (Dicentrarchus
labrax and Sparus aurata) were fed olive pomace as a substitute for fish oil
in fish feed. These fish demonstrated satisfactory growth performance and statisti-
cally decreased levels of fatty acids while also possessing potent inhibition of PAF-
induced platelet aggregation. A follow-up study demonstrated that the most active
polar lipid fractions were specific PC and PE derivatives [83]. Overall, studies
carrying out structural elucidation of fish polar lipids from fish fed a diet enriched
with olive pomace indicate that the most biologically active lipid fractions contain
various diacyl-glycerophospholipid species that seemed to mainly consist of 18:0
or 18:1 fatty acid in the sn-1 position and either 22:6 or 20:2 fatty acids in the sn-2
position [82, 83]. The proposed structures of these novel bioactive phospholipid
species are demonstrated in Fig. 3. Therefore, replacing fish oil in the diet of sea
bream and sea bass may improve their cardioprotective effects upon consumption by
humans while also tackling the problems associated with by-products of the olive oil
industry.
3.5 Various Foods of the Mediterranean Diet
It is well-accepted that lifestyle plays a crucial role in the prevention of CVD [161].
Evidence suggests the most important behavioral risk factors of CVD are an
unhealthy diet, physical activity, and harmful use of alcohol and tobacco [162].
Previous research has focused on targeting specific nutrients, particularly the reduc-
tion of SFA in an individual’s diet; however, recent research indicates that this
approach was short-sighted and ineffective and that nutritional interventions in
CVD have proved to be an effective strategy with tangible evidence, indicating
that the synergistic effects of food combined into a dietary pattern provide the
maximum benefit obtainable from nutrition [5, 9, 114, 163]. It seems that most
are in agreement that the optimal dietary pattern to reduce CVD includes high
consumption of fruits, vegetables, legumes, whole grains, nuts, fish, and poultry,
moderate dairy and heart-healthy vegetable oil intake, low red meat intake, and
minimal consumption of refined grain products, added sugars, industrial trans
fats, sugar-sweetened beverages, and processed meat [163, 164]. The Dietary
Approaches to Stop Hypertension (DASH) diet and Mediterranean diet reflect
these dietary patterns and are palatable, relatively easy to adhere to, and continually
recommended for good cardiovascular health [5, 9, 163, 165, 166]. Certainly, while
nutritional research should move away from simply focusing on single nutrients as
a panacea to prevent CVD, it is important to recognize that it is most likely these
nutrients working synergistically together in the diet that leads to the beneficial
effects observed against CVD.
There have been several studies published that have identified various food
sources containing anti-inflammatory polar lipids. Many of these foods are found
in the Mediterranean diet. The traditional Mediterranean diet originates in the olive-
growing regions of the Mediterranean and has strong cultural associations with these
areas [95]. Various definitions of the Mediterranean diet have been proposed, and the
diet has been adopted beyond the Mediterranean region and becomes increasingly
medicalized as both a treatment and prevention intervention [167]. Despite these
variations, the Mediterranean diet remains based on fresh, seasonal, and local food
[166]. The Mediterranean diet is generally characterized by the high consumption
of fruit, vegetables, legumes, cereal foods (preferably whole grains), breads, tree
nuts, and olive oil, including moderate servings of milk, dairy products, eggs, fish,
shellfish, and white meat, and low consumption of processed foods or red meats.
Wine is consumed in moderation in non-Islamic countries, and coffee is considered
the hot beverage of choice, which nowadays is consumed with sugar [168–170].
In addition, the Mediterranean diet has qualitative cultural and lifestyle elements,
such as conviviality, culinary activities, adequate rest, and physical activity [171],
which seem to be key for good health and longevity as witnessed in the Blue
Zones [5]. However, whether these cultural aspects are vital to the success of the
Mediterranean diet or they simply improve the health effects observed through a
healthier habitual lifestyle approach requires further in-depth research.
The Mediterranean diet is rich in beneficial fatty acids with a characteristically
high content of MUFA and a higher MUFA/SFA ratio than other diets [172, 173]. Due
to the low glycemic index and glycemic loads [174], high dietary fiber [175], and anti-
inflammatory effects [176], the Mediterranean diet is synonymous with favorable
effects on health status. Evidence suggests that Mediterranean diet is associated with
a number of beneficial health effects, including lower incidences of several chronic
diseases such as CVD [9, 14], cancer [177, 178], diabetes [179], and neurodegener-
ative diseases [180, 181]. It has been proposed that these associations are mainly due
to the anti-inflammatory nature of the Mediterranean diet [5, 182].
In particular, evidence suggests that these effects may in part be due to the
presence of bioactive polar lipids in foods of the Mediterranean diet, which possess
inhibitory properties against PAF activities [5, 183]. The polar lipids found in foods
of the Mediterranean diet exhibit in vitro and in vivo anti-inflammatory activities
through either directly or indirectly inhibiting the PAF/PAF-R pathways and thus
PAF activities but also by downregulating its levels through modulating the activities
of key metabolic enzymes of PAF by either upregulation of the PAF catabolic
enzymes or the downregulation of the basic PAF biosynthetic enzymes [44, 61, 64,
100, 147, 150]. These polar lipid microconstituents in association with various other
beneficial components of the Mediterranean diet may be responsible for the observed
preventative effects of the Mediterranean diet against the development of CVD.
Several of the foods that have demonstrated anti-PAF effects are presented in
Table 2 along with other foods and components of the Mediterranean diet that exhibit
inhibition of PAF-related inflammatory pathways and manifestations.
Notably, the uptake of dietary polar lipids seems to beneficially affect the func-
tionality of HDL lipoproteins especially in atherosclerotic conditions [5]. HDL-C is
generally described as the “good” cholesterol, since it removes excess cholesterol
from the blood stream and from atherosclerotic plaques, and it has exhibited anti-
inflammatory and antioxidative properties through a plethora of cardioprotective
enzymes bonded to HDL, including the aforementioned PAF-AH enzyme activity,
which is the main catabolic enzyme of PAF [71]. These HDL-associated activities
contribute to the maintenance of endothelial cell homeostasis, which protects the
cardiovascular system [191]. Plasma PAF-AH is also found in atherosclerotic lesions,
since it comigrates there along with the lipoproteins (i.e., LDL-C), where it is
incorporated. Plasma PAF-AH (Lp-PLA2) mainly plays an anti-inflammatory role
in leukocyte/platelet/endothelium activation and seems to suppress atherogenic
changes in plasma lipoproteins (such as LDL-C) by promoting the catabolism of
PAF and by removing oxidized phospholipids present in Ox-LDL. These oxidized
phospholipids mimic the actions of PAF and are generated by oxidative modifications
of lipoproteins such as LDL during pro-atherogenic and atherosclerotic events [5, 68,
70, 71 ]. Thus, during inflammatory cascades that increase PAF levels, this isoform
of PAF-AH (LpPLA2) seems to be activated as a homeostatic mechanism to down-
regulate these events, by downregulating the levels of PAF and oxidized phospho-
lipids, as a terminator signal [192].
Thus, HDL and its enzymes, including PAF-AH, seem to protect against these
manifestations. Dietary intake of bioactive polar lipids, particularly those baring ω3
PUFA, increases HDL-C levels and the incorporation of such anti-inflammatory and
antioxidant dietary polar lipids to HDL, thus providing an additional protective
Table 2 Studies on the beneficial impact of microconstituents from foods of the Mediterranean
diet, such as polar lipids and vitamins, toward inflammation-related disorders, through their effects
on the PAF pathways and metabolism. (Modified with permission from Tsoupras et al. [5])
Studied food and
components Type of study Results
PL of goat and sheep Ex vivo studies in hPRP Inhibition of PAF-induced
meat platelet aggregation [193]
PL of red and white In vitro studies in WRP and in Inhibition of PAF-induced
wine, musts, grape U937 macrophages platelet aggregation and
skins, and yeast In vivo postprandial dietary modulation of PAF metabolism
interventions studies in humans toward reduced PAF levels [129,
134, 143, 144, 146, 147, 150]
PL of fish (sea bass, sea In vitro studies in WRP, hPRP, Inhibition of PAF-induced
bream, salmon, etc.) and HMC platelet aggregation, modulation
Ex vivo studies in hPRP of PAF metabolism toward
In vivo studies in hyperlipidemic reduced PAF levels, and
rabbits reduction of the thickness of
atherosclerotic lesions in
hypercholesterolemic rabbits
[61, 64, 82, 83, 104–107,
109–111, 113, 184]
PL of olive oil and olive In vitro studies in WRP and in Inhibition of PAF-induced
pomace HMC platelet aggregation, modulation
In vivo study in hyperlipidemic of PAF metabolism toward
rabbits reduced PAF levels, reduction of
the thickness of atherosclerotic
lesions in hypercholesterolemic
rabbits, and regression of the
already formed atherosclerotic
lesions [100, 128, 156, 157]
PL of seed oils In vitro studies in WRP Inhibition of PAF-induced
(soybean, corn, platelet aggregation [156]
sunflower, and sesame
oil)
PL of hen egg In vitro studies in WRP Inhibition of PAF-induced
platelet aggregation [185]
PL of dairy products In vitro studies in WRP and ex Inhibition of PAF-induced
(milk, yoghurt, cheese, vivo studies in hPRP platelet aggregation [114,
etc.) 123–125]
Lipid extracts from Ex vivo studies in hPRP Inhibition of PAF-induced
garlic platelet aggregation and de-
aggregation of aggregated
platelets [186]
Vitamin D and its In vitro studies in WRP and Inhibition of PAF-induced
analogues human leukocytes, ex vivo platelet aggregation and
studies in hPRP, and in vivo modulation of PAF metabolism
studies in hemodialysis patients toward reduced PAF levels and
reduced levels of several
cytokines [38]
Vitamin E Ex vivo studies in hPRP and Inhibition of PAF-induced
whole blood platelet aggregation [187, 188]
(continued)
Table 2 (continued)
Studied food and
components Type of study Results
Mediterranean-based In vivo studies in humans Reduction of PAF-induced
meals and diets, rich in platelet activity in patients with
PL with anti-PAF type 2 diabetes mellitus and
effects metabolic syndrome and healthy
subjects
Eugenol in cinnamon In vivo studies in male Wister Reduction of PAF-induced
and basil rats gastric ulcers [189]
Dietary gangliosides In vivo studies in male Sprague- Reduced PAF synthesis and PAF
Dawley rats levels [190]
Curcumin In vitro studies in hPRP Inhibition of PAF-induced
platelet aggregation
Abbreviations: HMC human mesangial cells, hPRP human platelet-rich plasma, PAF platelet-
activating factor, PL polar lipids, WRP washed rabbit platelets
mechanism by increasing plasma PAF-AH activity and by protecting the HDL

enzymes (such as PAF-AH) from oxidation-related inactivation. This is in agreement
with the beneficial in vitro and in vivo effects of several dietary polar lipids,
especially on PAF metabolism and HDL biofunctionality [32].
Overall, the protective outcomes of the adoption of Mediterranean diet toward
chronic diseases seem to be associated with the pleiotropic beneficial effects of its
bioactive microconstituents that are not only limited to increasing plasma HDL-C
levels and functionality and providing better stability against oxidation but mainly
on their effects on the levels, activities, and metabolism of key inflammatory
mediators such as PAF [5, 32, 44]. However, more in vivo results are required
in several chronic disorders and their inflammation-related manifestations in order
to further support these findings. In particular, clinical trials implementing dietary
patterns such as the Mediterranean diet that are rich in bioactive polar lipids
interacting with the PAF/PAF-R pathways and metabolism are required to gain
further insight into the role of PAF in chronic diseases.
4 Future Research and Applications of Food Polar Lipids
Systemic inflammation leads to chronic diseases such as CVD; therefore it is

imperative to research and understand the mechanisms that govern inflammation.
Inflammation is a multifactorial process that leads to occurrence of complex condi-
tions and diseases. Due to the complex etiology of CVD, there is not one single
solution or ‘panacea’ to prevent its occurrence; hence the array of pharmaceutical
treatments such as statins and antihypertensives that are available to patients to
tackle the various risk factors associated with CVD. Successful dietary and lifestyle
interventions are credible alternatives to pharmaceutical treatments that have the
potential to tackle multiple risk factors at a time [103], albeit difficult for the
individual that undertakes these changes. Adherence to a healthy dietary pattern such
as the Mediterranean diet has demonstrated favorable effects for the primary pre-
vention of CVD. Bioactive components of the foods of the Mediterranean diet play a
role in the attenuation of systemic inflammation [5]. PAF plays a critical role in the
inflammatory process that seems to be underappreciated. Polar lipids have the ability
to disrupt the binding of PAF to its receptor and modulate the PAF metabolic
enzymes; therefore these lipids may prove important in the modulation of the
inflammatory response. Polar lipids are found in abundance in the Mediterranean
diet; thus further studies are required in order to discern their mechanisms and
potential in the prevention of CVD and other chronic disease. As CVD is still the
leading cause of death worldwide, scientific research on healthy dietary patterns such
as the Mediterranean diet and the DASH diet are imperative. Furthermore, the
development of novel nutraceutical and pharmaceutical products targeting inflam-
matory pathways such as the PAF pathway or targeting numerous inflammatory
pathways at once will play an integral role in the primary prevention and treatment of
CVD in the future.
Furthermore, polar lipids such as phosphatidylcholine (lecithin) are functional
food ingredients and are currently used as food additives in a broad range of
products including dairy products, instant drinks, baked goods, chocolate, and food
supplements. Polar lipids are widely utilized as emulsification agents in the food
manufacturing and pharmaceutical industry. The increased accessibility of high-
quality polar lipids derived from animal and marine products or by-products will
open the field of polar lipid research to new opportunities in food, both for their future
use as a superior nutritional source of bioactive molecules with beneficial effects and
for use in the food, pharmaceutical, nutraceutical, and cosmetic industries. Further
research and development in these areas have the potential to induce significant
social, commercial, health, and environmental benefits [32].
Acknowledgments The authors would like to thank the Department of Biological Sciences,
University of Limerick, Limerick, Ireland, for their continued support.
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Neuroprotective and Antiaging Essential
Oils and Lipids in Plants 20
Mamali Das and Kasi Pandima Devi
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
2 Chemistry of Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
3 Chemistry of Plant Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
4 Are Aging and Neurodegenerative Diseases Related? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
5 Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
6 Neuroprotective Effects of Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
6.1 Effect on Cholinesterase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593
6.2 Effect on Learning and Memory (Cognition) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
6.3 Effect on Aβ Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
6.4 Effect on Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
7 Neuroprotective Effects of Plant Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
7.1 Contribution of Long Chain ω-3 PUFAs to Brain Function and Its Molecular
Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
7.2 DHA Depletion and Cognitive Impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Abstract
Age-related neurological disorders, such as Alzheimer’s and Parkinson’s disease,
have a huge medical and economical impact in both the industrialized and
nonindustrialized countries. Neurodegenerative diseases alone affect 74 million
people worldwide and among them, 6.8 million die every year. Essential oils
(EOs) and plant lipids (PLs) are used since long time in traditional medicine for
their ability to manage a wide range of diseases. There are numerous reports on
the neuroprotective and antiaging potentials and mechanism of PLs and EOs.
M. Das · K. Pandima Devi (*)

Department of Biotechnology, Alagappa University [Science Campus], Karaikudi, Tamil Nadu,
India

588 M. Das and K. Pandima Devi
Several clinically important EOs and their components from Mentha piperita,
Eucalyptus globulus, Nigella sativa, Jasminum sambac, Rosmarinus officinalis,
and plant-derived lipids like stearidonic acid (SDA) from Echium oil, stigmas-
terol, β-sitosterol, from Datura innoxa, palmitic acid, linoleic acid from Celastrus
paniculatus, and many more plants are reported for their neuroprotective and
antiaging effects. This chapter aims to emphasize on the current finding on EOs
and PLs tested against aging-associated neurodegenerative disorders like
Alzheimer disease (AD) and possible molecular mechanism of their
neuroprotective effects.
Keywords
Essential oils · Plant lipids · Alzheimer’s disease · Cholinesterase inhibitors ·
Antioxidants · Amyloid-β · NFTs · Dementia · BACE1
1 Introduction
Aging is a time-dependent natural process that affects almost all living organisms.
The oldest definition by Harman defines aging as the progressive accumulation of
changes with time that are associated with the ever-increasing susceptibility to
disease and death [1]. Though aging is one of the most popular topics among
mankind from the historical time period, inauguration of aging research could be
addressed back to only 30 years with the discovery of first long-lived strains in
Caenorhabditis elegans (C. elegans) [2]. Currently, age is regarded as the main risk
factor for prevalent diseases, including cardiovascular diseases, neurodegenerative
disorders, and cancer [3]. Aging leads to accumulation of genetic damage which
promotes aging-associated diseases [4, 5]. The epigenetic changes like alteration in
DNA methylation, posttranslational histone modification, and chromatin remodeling
also augment aging process [6]. Additionally, chronic expression of unfolded or
misfolded proteins contributes to the development of some specific and fatal age-
related neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s dis-
ease, and cataracts (Fig. 1) [7]. Aging also reduces efficacy of the respiratory chain
by dysfunctioning mitochondrial membrane potential thus increasing electron leak-
age and reducing ATP generation mediated by activation of inflammasomes [8],
therefore mitochondrial dysfunction and aging process has been suspected to be
linked.
Essential oils (EOs) represent a mixture of highly complex, naturally occurring
plant secondary metabolites abundantly present in flowers, leaves, seeds, rhizomes,
and barks and are usually isolated via hydrodistillation and cold pressing methods
[9]. They are used as spiritual, mental, and physical healing agents from very ancient
time period [10] and nowadays are used as traditional medicines, aromatherapy,
massage therapies, as well as in cosmetics, perfumes, and food industries [11, 12]. In
Egypt, Greece, and Rome, EOs extracted from aromatic plants were employed for
the prevention and treatment of various diseases [13]. EOs act as natural safeguards
in plants with antimicrobial, herbivores repellent, and insect attractants and thus are
20 Neuroprotective and Antiaging Essential Oils and Lipids in Plants 589
Fig. 1 Aging as a cause of neurodegenerative diseases like AD: Alzheimer’s disease, HD:
Huntington’s disease, PD: Parkinson’s disease, and ALS: amyotrophic lateral sclerosis
hugely used as antibacterial, insecticidal, and antifungal agents. Recently, the use of
EOs has dragged much scientific attention for the management of several neurolog-
ical diseases due to improved knowledge on their structure and other biological
activities.
Lipids which are basically fatty acids (straight carbon chain, with hydrogen atoms
at one end and a carboxyl group (–COOH) at the other end) are important component
of fat-soluble part of plants, animals, and microorganisms. Some naturally synthe-
sized fatty acids like omega-9 (ω-9), which need not to be obtained through diets, are
termed as nonessential fatty acids [14], while some fatty acids like omega-3 (ω-3)
and omega-6 (ω-6) fatty acids (Fig. 2) which form a major part of neuronal tissue
Essential Fatty acids

ω-3 Fatty acids ω-6 Fatty acids
11 12
9 13 11 12
10
7 17 18
14 9 19 CH3
8 13
HO 5 10 20
6 19
1 15 16 7 14 17
ALA H3C 8 LA 18
2 4 HO 5
20
O 1 6 15 16
3 8 14 20
15 2 4
7 9 13 19 21
O
6 10 12 16 18 3
5 22
11 17
HO CH3
1 4 21 22
2
EPA 14
8 20
O 7 9 13 15 19
3
O 2 OH
3 1 6 10 12 16 18
5
10 16 22 11 17
4 HO AA
5 9 11 15 17 21 CH3 4
1
23 2
6 8 12 14 18 20
O
7 13 19 3
DHA
Fig. 2 Major essential fatty acids. ALA: alpha linoleic acid, EPA: eicosapentanoic acid, DHA:
docosahexanoic acid, LA: linoleic acid, and AA: arachidonic acid
cannot be synthesized by the body itself and need to be obtained through plant and
marine fish oil are considered as essential. In recent years, these ω-3 fatty acids have
been found to be critical for the development of human nervous system particularly
during neonatal phase [15]. At the same time, its deficiency in adults lead to severe
abnormalities in learning, neural, and visual systems and also may cause obesity,
cardiovascular disease, inflammation, and cancer. Therefore, investigation of health
benefits of dietary supplementation of ω-3 fatty acids has become a promising area
for drug developing bodies.
2 Chemistry of Essential Oils
Essential oils are concentrated hydrophobic liquids with several volatile aromatic
compounds and have many synonyms like volatile oils, ethereal oils, or aetherolea.
Usually, they consist of low molecular weight compounds with terpenes and ali-
phatic compounds in variable concentrations [16]. Some EOs have usually higher
concentrations (20–95%) of two or three components, whereas, other components
are present in minor amounts. For instance, linalool constitutes 68% of the
Coriandrum sativum EO, whereas, α/β thuyone and camphor constitute 57% and
24% of Artemisia Herbaalba EO, respectively. Similarly, D-limonene constitutes
>80% of citrus peel oils, α-phellandrene and limonene make 36% and 31% of
Anethum graveolens leaf EO, respectively. The chemical composition of EOs vary
both numerically and stereochemically depending on several factors like extraction

techniques, climate, plant origin, soil composition, vegetative cycle stage, and age
[17]; therefore, they are needed to be extracted from the same plant part and
harvested under the same growing conditions and in most efficient season. Essential
oils combat wide range of health issues that are listed in Table 1.
3 Chemistry of Plant Lipids
Lipids including fatty acids, fats, oils, steroids (sterols), waxes, cutin, suberin,
glycerophospholipids (phospholipids), glyceroglycolipids (glycosylglycerides), ter-
penes, and tochopherols are ubiquitous compounds in plants. Several groups of
lipids have been shown to provide health benefits either through modification of
tissue fatty acid composition or induction of cell signaling pathways. While some
health benefits are derived from consumption of short- to medium-chain fatty acids,
evidence suggests that the polyunsaturated fatty acids (PUFAs) are the most impor-
tant bioactive lipids. Among the essential PUFAs, the most important are ω-3 and
ω-36 fatty acids. PUFAs are found mostly in plant seed oils and are important
substrates for the biosynthesis of cellular hormones (eicosanoids) and other signaling
compounds that modulate human health. They are essential for storing metabolic
energy, provide protection against pathogens and dehydration, they carry electrons,
and absorb light. Gas-liquid chromatography (GLC) and normal-phase high-perfor-
mance liquid chromatography (HPLC) analysis of fatty acids and fat-soluble
Table 1 Common essential oils and their health benefits

Name of essential oil Effective for
Lemon Toning, circulation, and respiration
Lavender Acne and relaxation
Orange Respiration and skin moisturizer
Pine Anxiety, dump skin, and muscle ache
Rose Obstruct menses and dry skin
Rosemary Dandruff, digestion, and pain relief
Sandalwood Insomnia and respiratory infection
Sage Joint pain, fever, and appetite loss
Tea tree Fungal infection in skin
Patchouli Sexuality problem and damaged skin
Peppermint Poor digestion and sore feet
Cinnamon Poor digestion and nausea
Ginger Stomach upset and muscle ache
Clove Respiratory and skin infections
Fennel Menstrual dysfunction and constipation
Jasmine Depression
Grapefruit Depression
Cedarwood Eczema and acne
bioactives from Datura and Hyoscyamus seeds showed major fatty acid as linoleic
acid followed by oleic, palmitic, and stearic acids. Also, the crude seed extract was
rich in phytosterols, like stigmasterol, β-sitosterol, lanosterol, D5-avenasterol, and
sitostanol. Datura seed extracts showed stronger radical scavenging activity than
Hyoscyamus species [18]. Similarly, studies on the lipid profile of Indian Celastrus
paniculatus seed oil revealed oleic, palmitic, and linoleic as the major fatty acids
followed by glycolipids and phospholipids in its oil. Phytosterol like β-sitosterol
were also found in high amount with campesterol and stigmasterol in low amount.
Celastrus paniculatus oil exhibited stronger radical scavenging activity [19]. Plants
especially leafy vegetables and nuts are rich in ω-3 PUFAs, and these are currently
the main source to obtain α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and
docosahexaenoic acid (DHA) [20]. ALA is commonly present in plant sources
walnuts, flax seeds, butternuts, red and black currant seeds, pumpkin seeds, wheat
germ, soy and canola oil, and leafy green plants like Purslane in high concentrations.
Perilla frutescens seed oil is also a rich source of ω-3 linolenic acid [21]. Among the
plant sources, flax seeds have the highest concentration of ALA.
4 Are Aging and Neurodegenerative Diseases Related?
As we age, along with some visible changes like hair and skin, our brain and central
nervous system also undergo the aging process. This is one of the facts that people
are more likely to suffer from many neurological problems after the age of 65. Aging
makes the patients more vulnerable to irreversible diseases like Alzheimer’s disease,
Parkinson’s disease, and stroke and thus has been considered as a major risk factor of
the above said diseases. With advances in molecular biology, many age-related
signaling pathways like rapamycin (TOR) [22], and insulin/IGF-1 signaling, have
been found to be connected with neurodegeneration [23]. Most of the age-related
neurodegenerative diseases like AD are characterized by accumulation of disease-
specific misfolded proteins in the central nervous system which leads to
neurodegeneration, synaptic dysfunction, and neuroinflammation in central nervous
system, causing several pathological effects [24] (Table 2).
5 Alzheimer’s Disease
Alzheimer’s disease (AD) is the fifth leading age-related neurological disorders in

the world and is characterized by cognitive dysfunction, memory loss, psychological
changes, and disability to perform day-to-day activities due to oxidative stress-
induced scarcity in cholinergic neurotransmission, accumulation of amyloid plaques
(amyloid-β, Aβ), and neurofibrillary tangles (NFTs) in the brain [25, 26]. The risk of
AD considerably increases with aging, affecting 7–10% of 65, and 40% of people
over 80 years of age. It is a multifactorial dementia; therefore, exploration of
pathway-based inhibitors is the most helpful way in the anti-AD drug development.
In this context, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
Table 2 Some of the common age-related neurological disorders and their pathological effects
Age-associated
S. neurodegenerative
No. diseases Pathological hallmarks Disease consequences
1 Alzheimer’s Aggregated β-amyloid and tau Mental decline, difficulty in
disease in cerebral cortex and thinking and understanding,
hippocampus, oxidative stress, delusion, disorientation,
activated microglia forgetfulness, inability to create
new memories, depression, and
hallucination
2 Parkinson’s Aggregated α-synuclein in Bradykinesia, rigidity and rest
disease substantia nigra areas of the tremor, sleep disturbance,
midbrain and basal forebrain executive dysfunction,
depression, psychosis, and
confusion
3 Amyotrophic Aggregated superoxide Cramping, muscle weakness,
lateral sclerosis or dismutase-1 (SOD1) in spinal problems with coordination,
Lou Gehrig’s cord and motor cortex fatigue or feeling faint,
disease shortness of breath vocal cord
spasm, apathy, difficulty raising
the foot, difficulty swallowing,
drooling, lack of restraint,
severe constipation, and tremor
4 Huntington’s Aggregated huntingtin (Htt) Jerky body movements, mood
disease protein swinging, reduced mental
abilities, coordination
inhibitor compounds such as donepezil, galanthamine, rivastigmine, tacrine, etc.

represent majority of drugs approved for clinical use in the symptomatic AD relief,
while EOs target several pathways and thus provide better healing.
6 Neuroprotective Effects of Essential Oils
6.1 Effect on Cholinesterase Activity
Cholinesterases are one of the potential AD targets and a variety of EOs show
promising anticholinesterase activity. EOs from the leaves and flowers of Polygo-
num hydropiper are recently reported to show free radicals scavenging and AChE,
BChE inhibitory activity [27]. Extensive GC-MS analysis of the leaf and plant
extract showed caryophylene oxide (41.42%) as the major EO in leaf and
decahydronaphthalene (38.29%) as major EO of the flower. Also, EO from Rumex
hastatus has reported anticholinesterase and antiradical potentials with IC50 values
of 32.54 and 97.38 mg/ml for AChE and BChE, respectively, and IC50 values of
3.71 and 6.29 mg/ml for antioxidant assays [28]. Narcissus poeticus L. flower oil
also show in vitro AChE, BChE inhibitory activity [29]. EOs like thymol and
p-cymene (19% and 16.1% concentration, respectively) from Origanum ehrenbergii

showed potential antioxidant and AChE, BChE inhibitory activity [30]. Similarly,
the anti-AChE activity of spathulenol from Marlierea racemosa Vell. were evaluated
by de Souza et al. [31]. Cistaceae family (Cistus creticus, Cistus monspeliensis,
Cistus salvifolius, Cistus villosus, and Cistus libanotis) were investigated for anti-
oxidant and cholinesterase inhibitory potentials [32]. EOs from C. monspeliensis
and C. libanotis were found to be most potent antioxidants. C. salvifolius exhibited
AChE inhibitory, while, C. libanotis, C. creticus, and C. salvifolius showed BChE
inhibitory activity.
6.2 Effect on Learning and Memory (Cognition)
Learning is the ability to acquire new and modify existing information, while,
memory refers to the storing and retrieving ability of this information, and cognition
is the combination of these two vital processes. In AD, deterioration of cholinergic
neurons lead to cognitive deficits [33], and therefore, a cholinergic boost may
potentially revert the cognitive dysfunction [34]. Based on this idea, several nicotinic
and muscarinic agonists were tested, but the process failed due to limited efficacy,
toxicity, and bioavailability problems [35]. EOs are reported for their beneficial
effects in Alzheimer and dementia by several research groups. Shimizu et al.
reported the effect of EOs from Eucalyptus globulus Labill. and Lavandula
angustifolia Mill. on persistent attention [36, 37]. Rosmarinus officinalis L. of family
Lamiaceae which is frequently used in diet formulations was also found to be a great
source of EOs with strong antiradical, antibacterial, antifungal, and anticancer
properties [38]. Rosemary EOs are also reported as neurostimulants, moderate
AChE inhibitors, locomotor activity enhancers, vigor motivators, and cerebral cortex
stimulators [39].
6.3 Effect on Ab Aggregation
Formation of senile Aβ plaques and NFTs are important hallmarks of AD [40]. Aβ

originates from enzymatic hydrolysis of the amyloid precursor protein (APP). The
APP is cleaved by α, β, and γ secretase enzymes in a sequential manner, leading to
the formation of Aβ17–42 by α- and γ-secretases and neurotoxic Aβ1–42 by β and γ
secretases [41]. An imbalance in the generation and removal of Aβ results in its
neuronal accumulation and becomes the starting point for neurodegeneration and
neuroinflammation in AD. As a result, inhibition of β-amyloid cleaving enzyme 1
(BACE1) or β-secretase becomes an important target in management of AD.
6-gingerol, a predominant constituent of ginger EO, showed the neuroprotective
potential against Aβ25–35-mediated oxidative and nitrosative cell death in SH-SY5Y
cells via inhibition of DNA fragmentation, interruption in mitochondrial membrane
potential (MMP), activation of caspase-3 and increased Bax/Bcl-2 ratio, suppression
of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [42].
6-gingerol also upregulate the expression of mRNA and proteins responsible for the
synthesis of c-glutamylcysteine ligase, implicated in the biosynthesis of glutathione.
Similarly, the EO from SuHeXiang Wan (SHXW), a Chinese traditional medicinal
prescription consisting of 15 crude herbs whose essential oil has been shown to have
anticonvulsant and antioxidative activity used as traditional medicine to treat sei-
zures, infantile convulsions, AD, and other neurodegenerative disorders [43]. Pre-
inhalation of SHXW EO revitalized the memory of AD animal model injected with
Aβ1–42, suppressed Aβ1–42 mediated c-jun N-terminal kinase (JNK), protein kinases
(p38), and tau phosphorylation in the hippocampus of animals. Also, SHXW EO
decreases Aβ-mediated apoptosis and ROS production by upregulation of HO1 and
Nrf2 expression in SH-SY5Y cells. Consecutively, thymol and carvacrol on cogni-
tive function, it was found that both the EOs improved cognitive functions in animal
models [44]. Most importantly, both samples were safe at their highest concentra-
tions. Also, EO from Coriandrum sativum var. microcarpum (coriander) had anti-
depressant, anxiolytic, and antioxidant potentials upon inhalation in Aβ1–42 rat
models of AD [45]. Similarly, EOs of Zataria multiflora Boiss. (Lamiaceae) showed
profound cognitive and neuroprotective effects in AD animal models by improving
their cognitive abilities in a concentration-dependent manner [46].
6.4 Effect on Oxidative Stress
Generation of free radicals during aerobic respiration is an essential part of living

beings. These free radicals readily attack DNA, proteins, fatty acids, and other
essential molecules and therefore are implicated in a variety of disorders including
AD, cancer, aging, and inflammation, and several plant extracts has been found to
have potential radical scavenging activities [47–52]. Free radicals are neutralized to
nonradical forms by various enzymes including CAT, SOD, and hydroperoxidase.
But, when free radical generation is abnormally high, then administration of free
radical scavengers from outside becomes necessary. Furthermore, in aging brain and
AD patients, mitochondrial dysfunction lead to excessive production of free radicals
and subsequently result in pathological abnormalities. Aβ is a potent initiator of ROS
and RNS production which rapidly cause oxidative damage of neural, microglial,
and cerebrovascular cells and tissues [53]. Investigation of the neuroprotective
effects of lavender (Lavandula angustifolia ssp. Angustifolia Mill. and Lavandula
hybrida Rev) revealed that its EO showed antioxidant and antiapoptotic potentials
[54]. Lavender EO mediates its protective effects via strong antioxidant activities. In
addition, there are numerous examples of EOs from chamomile, clove, thyme,
eucalyptus, juniper, basil, cumin, cinnamon, and coriander to possess considerable
antioxidative potentials [55–57]. Similarly, dietary intake of oregano oil has been
reported to delay lipid oxidation in animal models [58]. EOs from Achillea
millefolium were reported to scavenge hydroxyl free radicals via inhibition of lipid
peroxidation [59]. Studies on EOs from Salvia multicaulis and Salvia cryptantha
showed antioxidant activities higher than the standard drugs [60]. Many aroma
components of EO like linalool, α-terpinene, 1,8- cineole, β-terpinolene, β-terpinene,
menthon, thymol, eugenol, and isomenthone have reported antioxidant potentials

[61]. Similarly, the EO of Melissa officinalis L. rich in menthone, geranial, iso-
menthone, and citronellal has reported antioxidative properties [62]. A comprehen-
sive account of possible neuroprotective mechanism of EOs is given in Fig. 3.
7 Neuroprotective Effects of Plant Lipids
7.1 Contribution of Long Chain v-3 PUFAs to Brain Function and

Its Molecular Mechanism
Brain is a repository of DHA and AA that are highly susceptible to free radical-
induced damage [63]. Oxidative damage of the brain is characterized by increased
lipid peroxidation, which deteriorates neuronal functions [64]. Recent studies shows
participation of DHA in brain pathological activities and involvement in regulation
of many metabolic and inflammatory pathways, and postmortem samples from AD
brains show gradual decline of DHA in patient’s brain [65]. Also, studies reveal that
higher dietary intake of DHA can reduce the risk of cognitive impairment or AD
[66–68]. It has also been found that ω-3 PUFAs cannot benefit patients with already
diagnosed AD progression but can enhance learning and memory function only in
age-related cognitive decline in healthy elderly populations [69]. Recently, through a
study carried out in rats, it has been found that DHA deficiency activates caspases in
AD models and intensify the age-related decline of glutamatergic transmission in
learning and memory functions [70]. On the other hand, DHA supplementation
diminish oxidative stress or lipid peroxidation and protect against memory loss in
AD rat models [71] by reducing the accumulation of neuronal Aβ and tau protein
[72]. This information may help researchers to design application of ω-3 PUFAs for
better relief from age-related cognitive consequences.
DHA that is transferred from the maternal circulation to fetal brain plays a crucial
role in synaptogenesis. DHA is incorporated into fetal brain through fatty acid
transport protein-4 (FATP 4) [73]. However, the amount of maternal DHA in the
synapses and neural membranes depends on the dietary intake [74–78]. Studies have
shown that DHA acts as an endogenous ligand for retinoic acid receptors (RAR) and
retinoid x receptors (RXR) [79] which decrease with age and correlated with age-
related memory deficits. Dyall and colleagues suggest that DHA supplementation
reverse the decrease of RAR and RXR which alleviate the memory deficits and
increase neurogenesis [80]. Patients diagnosed with AD show lower DHA levels in
plasma and brain [81, 82] which not only could be due to lower dietary intake of ω-3
fatty acids but also due to increased oxidation of PUFAs [83, 84]. Preclinical
evidence suggests that a DHA-enriched diet reduces amyloid formation [85, 86].
Akbar and coworkers provided additional evidence that DHA is highly enriched in
neuronal membranes and provides neuroprotection via PI3, Akt pathway which is a
critical signaling pathway for neuronal survival [87]. Dietary supplementation of
DHA has been shown to increase the levels of hippocampal BDNF (brain-derived
20
Neuroprotective and Antiaging Essential Oils and Lipids in Plants
Fig. 3 Overview of possible molecular mechanism of essential oils executing multifactorial neuroprotective effects in Alzheimer’s disease condition. EOs
mitigate AChE, BACE1 activity; Aβ oligomer formation; τ, JNK, and p38 phosphorylation; Bax/Bcl2 ratio; and ROS and RNS production, thus enhancing
memory and cognitive power and simultaneously reducing oxidative stress and neuronal apoptosis
597
Fig. 4 Outline of possible molecular mechanism of DHA executing multifarious neuroprotective

effects in Alzheimer’s disease condition. DHA reduce overall Aβ-induced neurodegeneration and
apoptosis by increasing APP membrane fluidity, thus reducing Aβ release, activating Ca2+/calmod-
ulin-dependent kinases, and increasing brain-derived neurotrophic factor and reducing oxidative
stress and lipid peroxidation
neurotrophic factor which is essential for maintaining synaptic plasticity and cell
survival [88], and activation of Akt is known to be linked to increase in BDNF. Also,
DHA-rich diet activates Ca2+/calmodulin-dependent protein kinase (CaMKII) which
is critical for learning and memory and plays a crucial role in induction and
maintenance of long-term potentiation in hippocampus [89, 90]. Literature survey
also propose that DHA modulates multiple cellular functions like enhanced mem-
brane fluidity of amyloid precursor protein (APP) and reduced amyloid-β release by
shifting the mechanism towards nonamyloidogenic pathway [91]. DHA is also
suggested to assist N-methyl-D aspartate (NMDA) responses [92] and block K+
channels [93], thus directly affecting memory and learning [94]. Most recently, DHA
supplementation has also been shown to amend gene expression at the transcrip-
tional level, by activating members of peroxisome proliferator-activated receptor
(PPAR) family [95] and stabilizing mRNA of several enzymes associated with
glucose and lipid metabolism [96]. Figure 4 gives a broad outline of DHA imparting
neuroprotective effect through various mechanisms.
7.2 DHA Depletion and Cognitive Impairment
Studies in AD animal models suggest that DHA deficiency in neural tissue leads to
behavioral changes similar to that in patients with AD. Furthermore, experimental
evidence suggests that DHA decreases with age, particularly in regions of the
hippocampus which are crucial for higher brain functions such as memory formation
and cognition [97, 98]. Decreased DHA levels are reported to detrimentally affect
the major excitatory neurotransmitter, glutamate, which contributes to the integrity
of brain function in learning memory performance [99]. Recently, it has been
demonstrated that DHA protects rat cortical neurons from soluble Aβ oligomer-
induced neurodegeneration and apoptosis [100, 101].
8 Conclusions
In recent times, plant-based therapies have got considerable attention owing to their
comparative safety, potency, and efficacy on multiple targets. Among the natural
products, EOs from aromatic and medicinal plants is of great interest because of their
antioxidant, cholinesterase inhibitory, anti-amyloid properties, and high availability
at the target due to their lipophilic character. Thus, EOs can be very effective
alternative for the management of AD in comparison to synthetic drugs which are
associated with severe side effects. Also, numerous EOs have been reported to
possess strong antioxidant potentials and can be effectively used in free radical-
induced disorders including neurological diseases and aging. This chapter also
highlighted the potentiality of long chain ω-3 fatty acids to reduce low-grade
inflammation in the early stages of age-related neurodegenerative diseases like AD.
Acknowledgment The authors gladly acknowledge the bioinformatics infrastructure facility,

Alagappa University, funded by the Department of Biotechnology, Ministry of Science and
Technology, Government of India (No. BT/BI/25/015/2012). Mamali Das acknowledges
DST–PURSE for offering Research Fellowship.
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Protective Effect of Omega 3 Fatty Acids
EPA and DHA in the Neurodegenerative 21
Disease
Edwin E. Martínez Leo, Rafael A. Rojas Herrera, and

Maira R. Segura Campos
Contents
1 Neurodegenerative Disease: Epidemic of the Twenty-First Century . . . . . . . . . . . . . . . . . . . . . . . 606
2 The Nervous System as an Immuno-Privileged Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
3 Neurodegenerative Disease and Neuroinflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
3.1 Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
3.2 Parkinson’s Disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
4 Advances in the Nutritional Treatment of ND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
4.1 Anti-inflammatory Characteristics of Omega 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
4.2 Potential Neuroprotective Effect of Omega 3 (ω-3) in ND . . . . . . . . . . . . . . . . . . . . . . . . . . 616
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
Abstract
Neurodegenerative diseases (ND) are characterized by the death of neurons in
different regions of the nervous system, followed by functional deterioration. The
most frequent pathologies are the group of dementias such as Parkinson’s disease
(PD) and Alzheimer’s disease (AD), which represent an important impact on
society and quality of life of people, mainly in the elderly group. Omega 3 is a
polyunsaturated fatty acid, whose anti-inflammatory effect has been related to
benefits on the neuroinflammatory processes characteristic of ND. Although the
clinical evidence is unclear, epidemiological studies report improvement in
cognitive performance and provide evidence on its neuroprotective effect in
specific regions of the nervous system. The objective of this review is to deter-
mine the neuroprotective effects of omega 3 fatty acids on neurodegenerative
disease.
E. E. Martínez Leo · R. A. Rojas Herrera · M. R. Segura Campos (*)

Facultad de Ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico

606 E. E. Martínez Leo et al.
Keywords
Neuroinflammation · Neuroregenery · Functional nutrition · Neuroprotection
1 Neurodegenerative Disease: Epidemic of the Twenty-First

Century
The ND integrate a heterogeneous group of diseases with involvement of the central

nervous system (CNS) characterized by a progressive neuronal loss in specific brain
areas or anatomic systems. This progressive loss of nerve cells is what causes the
neurological and neuropsychological signs and symptoms characteristic of this group
of diseases [52]. ND affect several activities of the organism, such as balance, move-
ment, speech, respiration, and functions of the heart. Some ND are Alzheimer’s disease
(AD), amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia (AF), Huntington’s
disease (HD), Parkinson’s disease (PD), and spinal muscular atrophy [40].
Currently, intervention in issues related to ND faces a complex context. There are
multiple sociological factors such as increase of the life expectancy, increase of the
population and aging, greater difficulty of scientific-technical advances, and predict-
able chronification of the diseases. Likewise, the presence of socioeconomic factors
(increased job insecurity, impoverishment of the middle classes, increased healthcare
costs, and weakening public social and health investment) has been circumstances
that, in their combination, have increased the prevalence of ND [16].
According to the report of the World Health Organization (WHO), the ND affect
around the world about one billion people. The report Neurological Disorders: Public
Health Challenges shows that around a billion people are affected worldwide; 50
million suffer from epilepsy and 24 million suffer from Alzheimer’s and other demen-
tias. Neurological disorders affect people from all countries, without distinction of sex,
educational level, and economic status. Given the above, the WHO advocates that
neurological care be integrated into primary healthcare, in order to prevent ND, through
strategies that may also directly affect the aspects of metabolic alteration [59, 60].
As the global aging rate increases, the impact of ND will be felt in both developed
and developing countries. According to Rita Levi-Montalcini, Nobel Prize in Med-
icine, “the burden of neurological disorders is reaching significant proportions in
countries where the percentage of people over 65 years of age increases” [59]. Until
2015 and because of its prevalence, the three main ND were Alzheimer’s and other
dementias, reporting 35 billion global cases, Parkinson’s with 23 billon worldwide
cases, and multiple sclerosis with 2 billion [61].
2 The Nervous System as an Immuno-Privileged Site
The nervous system (NS) is one of the smallest and at the same time most complex of
the organism. It consists of an intricate, specialized, and highly organized network of
cells called neurons and glial cells. The NS carries out a complex set of activities that
21 Protective Effect of Omega 3 Fatty Acids EPA and DHA in the. . . 607
allow a human being to feel different smells, produce speech, and remember past
events. It also provides signals that control body movements and regulate the
functioning of tissues and organs [47].
The NS is subdivided into CNS, consisting of encephalon and spinal cord, and the
peripheral nervous system (PNS), which includes all nerve tissues located in the
periphery. The brain is the main constituent organ of the CNS, whose cognitive
function involving networks of cells including neurons, one of the most important
cell populations in the NS, is involved. Neurons are the main functional units of the
CNS and have the ability to communicate with each other quickly and efficiently
through electrical or chemical signals that are translated as action potentials [2]. The
CNS is protected from damage and injury, by the blood-brain barrier (BBB), which
is a physical barrier located between blood vessels and brain tissue, with a very high
selective permeability that limits the access of molecules to protect the brain from
any type of pathogen or toxic substance. The foregoing allows this system to be
immunologically specialized [10].
The CNS is a specialized and essential system in the survival and defense of
injuries to the organism, for which reason it has been classified as immuno-
privileged. The anatomical sites that present this condition are able to tolerate the
introduction of antigens without initiating an inflammatory immune response [22].
Although there is still debate between the homeostatic maintenance of the CNS and
the immune system, it is well known that immune cells (lymphocytes and macro-
phages) play an important role in neurodegeneration [10]. In the particular case of
the CNS, the lymphocytes have the capacity to enter through the cerebrospinal fluid
in the choroid plexus or the cerebral parenchyma. Said extravasation of immune cells
is mediated by the adhesion molecules ICAM (intercellular adhesion molecules) and
VCAM (vascular cell adhesion molecules), which are present in the endothelium of
the blood-brain barrier. This allows the CNS to develop a more regulated immune
response, compared to other sites in the body [47].
Conversely, not all nerve cells produce action potentials; such is the case of glia.
Glial cells differ from neurons, since they do not have synaptic contacts and have the
ability to divide throughout life. The main functions of glial cells are to (1) maintain
an ionic medium in nerve cells, (2) modulate the speed of propagation of nerve
signals, (3) modulate the synaptic action by controlling the uptake of neurotrans-
mitters, (4) provide a foundation for neural development, and (5) contribute (or
prevent, in some cases) the survival of a neuronal lesion [35].
Glial cells are classified mainly into two groups: (1) macroglia, which include
astrocytes and oligodendrocytes, of ectodermal origin, and (2) microglia, of meso-
dermal origin, which invade the CNS during embryonic development at the time of
vascularization [15]. Microglia constitute part of the macrophages and constitute
between 5% and 10% of the neuronal cell population [36].
Under normal conditions, the microglia is at rest, but if there is an injury or
infection, mechanisms that promote neurotoxic activities are activated, producing
reactive oxygen species (ROS) and reactive nitrogen species (RNS) and secreting
prostaglandins, chemokines, and cytokines. They favor an inflammation status. If
the activation of the microglia remains until it becomes chronic, it leads to an
imbalance in the inflammatory response, generating a cycle of inflammation and

tissue damage [35]. Inflammation is defined as the response of vascularized living
tissue to the lesion, and in this sense, it is a complex cascade of physiological
responses to harmful stimuli from the environment [12].
Another factor that triggers inflammation is oxidative stress (OXS). Several
studies show that a state of chronic-systemic oxidation plays a crucial role in the
pathogenesis of ND [9, 44]. OXS is defined as the electrochemical imbalance
between prooxidant substances (ROS and ROS) and antioxidant substances, in
favor of the former. The body has antioxidant defense mechanisms, both enzymatic
(superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)),
and nonenzymatic (glutathione), for protection against prooxidants. However, before
the chronic development of the disease, such as obesity and diabetes, there is
a depletion of the antioxidant systems, which favors the perpetuation of OXS [33].
Particularly, the brain is susceptible to OXS lesions because it is an organ with high
energy use and metabolic demand, so minimal imbalances of the redox state favor
tissue damage and activation of neuroinflammatory mechanisms [8].
The inflammation present in ND, both acute and chronic, occurs in response to
injury or alteration in the CNS. The chronic and unbalanced inflammatory response
is a source of damage to the integrity and function of the CNS cells, since the neural
tissues have a restricted cell regeneration and are extremely vulnerable to immune
and inflammatory processes [46]. Inflammation determines the progression of neu-
ronal death in ND. In this sense, the excessive and destructive immune response
becomes a chronic and persistent process that leads to progressive and irreversible
neurodegeneration inducing a vicious circle that causes cognitive deterioration,
giving rise to the progression of chronic ND such as AD and PD [12].
3 Neurodegenerative Disease and Neuroinflammation
The term neuroinflammation describes an extensive process of physiopathological

mechanisms and phenomena mediated by alterations in the glial cell morphology.
Neuroinflammation is a reactive state of the immunological component of the CNS.
This concept of neuroinflammation encompasses the response or set of responses
that occur in the CNS to any damage occurring in the tissue [6].
As previously described, microglia play an important role in the development of
the neuroinflammatory response. This cell has toll-like receptors, involved in the
recognition of damage to the CNS by chemical agents, such as ROS and RNS, and
production of proinflammatory cytokines. Given the exposure to inflammation
molecules, the cells of the microglia are activated, and this results in a series of
processes that involve the morphological transformation of an inactive branched
state to an active phagocytic ameboid and the proliferation and migration toward
sites of injury by chemotaxis [32].
The activation of the microglia causes a release of products that may have
neurotoxic effects within which they are the proinflammatory cytokines interleukin
1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), interferon gamma
Fig. 1 Microglial activation model conducive to neurodegeneration
(IFN), oxidizing compounds such as ROS and RNs, as well as excitatory amino
acids such as glutamate, all act together to modulate the inflammatory processes that
affect the permeability of the BBB [12, 30] (Fig. 1).
IL-1β is one of the most important cytokines in the progression of the
neuroinflammatory process, playing an important role in the development of acute
neuronal injuries. Likewise, it has been demonstrated that the administration of the
IL-1 receptor antagonist (IL-1ra) prior to brain injury significantly reduces the death
of bark neurons in rats. The use of transgenic animals with specific modifications of
the genes that express IL-1β has shown that their deficiency exhibits a reduction of
necrosis despite the fact that the animals are subjected to different neuronal aggres-
sions [15].
Also, IL-1β induces the expression of inducible nitric oxide synthase (iNOS),
responsible for triggering the release of nitric oxide (NO-) in glial cells and causing
nitrosative stress. The RNS are oxidative molecules with special attachment to lipid
peroxidation, which favors mainly the rupture of cell membranes in CNS. Lipid
peroxidation is the most frequent oxidative damage at the cellular level, produced
particularly by the hydroxyl radicals (OH-) and peroxynitrite (ONOO-), on the
polyunsaturated fatty acids of cell membranes. Initially, a fatty acid is oxidized by
leaving a hydrogen atom of a methylene group to the OH- that acts as an oxidizing
agent, producing peroxyl radical. This, in turn, repeats the chain process until
irreversible tissue damage occurs [18].
A second type of glia cell involved in neuroinflammatory processes are the
astrocytes that reside in the CNS. Its activation leads to a condition called
astrocytosis, where the number and size of the glial fibrillary acidic protein
(GFAP), a cytoskeletal protein that contributes to hypertrophy and hyperplasia of
astrocytes, increases. This leads to the generation of a glial scar in the area of tissue
necrosis, excluding non-neuronal cells, and serves as a complement in spaces of
neuronal loss (Zhang et al. 2010a).
For its part, among the main functions of astrocytes is its participation in the CNS
immune response. Active astrocytes produce a variety of molecules, which are
involved in the initiation, progression, and regulation of the inflammatory response.
In addition to the production of pro- and anti-inflammatory cytokines, there is a
greater expression of the enzyme genes cyclooxygenase 2 (COX-2) and iNOS as
well as higher production of NO-, an important molecule in the development of
oxidative stress and neuronal necrosis (Zhang et al. 2010). Table 1 lists the main
substances released by glial cells that participate in neuroinflammatory processes.
The constant and prolonged activation of glial cells and astrocytes, as described
previously, leads to a chronic state of inflammation, which perpetuates the release of
proinflammatory molecules. The above contributes to the development of neurode-
generative processes, for example, AD, Parkinson’s, and Huntington’s, which are
characterized by slow and progressive neuronal loss in certain areas of the brain such
as the hippocampus, the striatum, the black substance of compact part (SNpc), and
the cerebral cortex [23].
It is important to point out that neuroinflammation is only the mechanism of
immunological protection against CNS damage, so its activation is of benefit for the
protection of the integrity of the NS. However, chronic inflammation is characterized
by the prolonged activation of the microglia and the perpetuation of mediators of
inflammation, which increases the cellular oxidative and nitrosative state. In view of
the above, a response chain is generated between oxidative stress-inflammation-
Table 1 Molecules released by microglia and astrocytes during the neuroinflammatory process
Microglia Astrocytes
Complement proteins Complement proteins
Neurotrophic factors Neurotrophic factors (NGF, NFDB, CNF,
Proinflammatory cytokines (IL-1, IL-6, IL-17, GDFI-1)
TNF-α) Proinflammatory cytokines (IL-1, IL-6, IL-17,
ROS (OH-, O2, H2O2) TNF-α)
RNS (NO, ONOO) ROS (OH, O2, H2O2)
iNOS RNS (NO, ONOO)
iNOS
COX-2
NGF neuronal growth factor
NFDB neurotrophic factor derived from the brain
CNF niliary neurotrophic factor
GDFI-1 growth factor derived from insulin-1
Fig. 2 Schematic representation of the neurodegeneration process through inflammation
oxidative stress, which favors necrosis, increasing cognitive decline and the devel-
opment of NS, as described in Fig. 2 [7].
3.1 Alzheimer’s Disease
The greatest evidence of the role of chronic neuroinflammation of the CNS as a

trigger for neurodegenerative disease is in studies of AD. Considered as the most
common neurodegenerative disease and the main cause of dementia in older adults,
approximately 10% of all people over 65 and up to 50% of those over 85 are
diagnosed with this disease [50].
AD is an irreversible and progressive disorder characterized by the loss of
neurons mainly in the cerebral cortex and hippocampus, which leads to memory
loss, cognitive deterioration, and changes in personality. Although its etiology is
unclear, it is suggested that the accumulation of β-amyloid proteins in the neuritic
plaques and tau protein residues are responsible for the attraction and activation
of glial cells, triggering reactive changes in the microglia, and release of pro-
inflammatory mediators and ROS that contribute to the necrosis of neuronal cells,
leading to chronic and progressive neurodegeneration [49].
It should be noted that genetic susceptibility, trauma, diet rich in fats and simple
sugars, alterations in cholesterol homeostasis, deficiency of cyanocobalamin, and
iron overload are among the risk factors described. However, although none of the
aforementioned appears to be a direct etiological factor, they do act as signaling
Fig. 3 Microglial activation model responsible for neuroinflammation in AD
devices at the CNS level, triggering alterations in the redox state, activation of glial
cells, and protein abnormalities. Therefore, the constant and prolonged exposure of
any of these factors could be an important trigger that favors a chronic
neuroinflammation response [31].
One of the main approaches about the development of AD is the tau protein,
which plays a fundamental role in the maintenance of microtubules of neurons and
as a component of axonal transport. Currently, it is known that tau hyper-
phosphorylation leads to the self-aggregation of this protein, triggering the disartic-
ulation of microtubules, disorders in neuronal activity, and loss in its ability to
transmit messages. The above is translated into a cellular lesion that leads to the
activation of microglia, release of proinflammatory cytokines, and the development
of neuroinflammation (Fig. 3). The persistent alteration of the signaling mechanism
produces necrosis of nerve cells and cognitive deterioration, eventually causing the
process of neurodegeneration [49, 57].
Currently, evidence of the importance of inflammation in neuronal damage comes
from immunohistochemical and molecular biology studies done in the brain tissues
of patients with AD, which revealed the distinctive features of inflammation, includ-
ing the activation of microglia and astrocytes, the expression of cytokines, and the
invasion of immune cells. Studies performed in patients with AD reveal high
concentrations of proinflammatory cytokines in the blood, mainly IL-6, TNF-α,
IL-1β, IL-12, and IL-18. For its part, it has also been shown that the chronic
treatment of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and
ibuprofen, significantly reduces the risk of developing AD [50].
3.2 Parkinson’s Disease (PD)
PD is the second most common neurodegenerative disorder associated with age and
of unknown origin, affecting 4.1 to 4.6 million people over 50 years of age
worldwide, and it is predicted that by 2030 this figure will double due to the increase
in hope of life [60].
PD is characterized by the presence of disorders in involuntary movements,
associated with the selective loss of dopaminergic neurons in the SNpc and by the
presence of intraneuronal cytoplasmic protein inclusions (Lewy bodies) in the
surviving neurons, as well as in other central regions and peripheral of the CNS.
The accumulation of Lewy bodies in neurons is associated with the activation of
microglia, the increase of proinflammatory cytokines, and the decrease of some
neurotrophic products, which together lead to degenerative changes in the SNpc
and locus coeruleus, specifically in dopaminergic neurons. In this process pro-
inflammatory, it is believed that the major histocompatibility class II complex
(MHC-II), half of the neurodegeneration process, since an increase in its expres-
sion due to the proinflammatory activation of the microglia has been related to a
greater neuronal death in these regions [25]. Although several causes of PD are
described, recent studies show that neuroinflammation and microglial activation
play an important role in its pathogenesis, derived from a microenvironment of
neurological damage that breaks down the homeostatic mechanisms that promote
cell survival [51].
The degeneration of dopaminergic neurons produces a decrease in dopamine
levels that causes a loss of synaptic regulation in basal ganglia. The basal ganglia are
collections of nerve cell bodies located subcortically near the base of the brain. They
include the striatum (caudate and putamen), subthalamic nucleus (NST), pale globe
external (Gpe) and internal (Gpi), ventral nucleus of the thalamus, and the SNpc and
SN pars reticulata (SNpr). These nuclei are responsible for motor control, since they
are interconnected with the cortex and the brain stem and the thalamus [41].
Dopaminergic neurons are very sensitive to any pathological change that occurs
in the brain. In response to the damage, the microglia increases the secretion of
neurotrophic factors and proinflammatory cytokines and the activation of nitric
oxide synthase, NADPH oxidase, and cyclooxygenase, which cause damage to the
neuron and the reactivity of astrocytes; therefore a progression of neurodegeneration
occurs in a self-powered way [51].
Activated microglia produces large amounts of superoxide radicals and is the
main source of oxidative stress responsible for the death of dopaminergic cells in PD
(Wang et al. 2015) (Fig. 4). In postmortem brains, high levels of proinflammatory
cytokines have been observed and in animal models active microglia from early
stages of neuronal degeneration [26, 39].
The in vivo correlation between microglial activation and the corresponding loss
of dopaminergic neurons in early PD was studied by Ouchi in 2005. This correlation
supports the hypothesis of neuroinflammatory response mediated by microglia that
contributes significantly to the degenerative process and suggests the importance of
Fig. 4 Representation of the inflammatory mechanisms involved in the development of PD
early therapeutic intervention with neuroprotective drugs [42]. Currently, research is

aimed at finding effective and preventive treatments for ND, paying special attention
to the search for methods to normalize this phenomenon of excessive reactivity in the
cells of the microglia, without inhibiting their elementary functions or causing a
reduction in total microglia [58].
4 Advances in the Nutritional Treatment of ND
Scientific and technological advances in nutrition and health make it necessary to

have effective interventions in the nutritional management of ND. Functional nutri-
tion is an area of nutriology whose axis is the use of bioactive compounds in food, at
specific doses, for the restoration of homeostasis lost in the disease. In the particular
case of ND, its mechanisms of biochemical and metabolic alteration are associated
with neuroinflammation process, which is why the use of bioactive compounds with
anti-inflammatory effect is a potential neuroprotective agent in ND [34].
The most widely studied anti-inflammatory and used in clinical practice is
omega 3. Its anti-inflammatory activity lies in being a substrate of cyclooxygenase
(COX) and lipoxygenase (LOX), for the synthesis of prostaglandins, thromboxanes,
and leukotrienes of the anti-inflammatory series [34].
4.1 Anti-inflammatory Characteristics of Omega 3
The fatty acids of the omega 3 family are long-chain polyunsaturated fatty acids
(PUFA) that present their first unsaturation in the third carbon. Among the main
representatives of the family of omega 3 fatty acids is α-linolenic acid, present in
foods of plant origin and which is metabolized into eicosapentaenoic acid (EPA) and
subsequently into docosahexaenoic acid (DHA) in animal organisms [54].
EPA and DHA are important structural components of the phospholipids of
cell membranes. Diets rich in EPA and DHA increase the proportion of these fatty
acids in cell membranes, particularly in lymphocytes, which in addition to
reducing the content of arachidonic acid (AA) in the membranes of these cells,
by a competitive effect, decreases the generation of proinflammatory products
derived from ω-6 PUFA. EPA is also a substrate for COX (1 and 2) and
lipoxygenase-5, which competes with AA for the generation of eicosanoids, but
in the case of EPA, these have anti-inflammatory properties. Due to the above,
dietary supplementation with EPA can reduce the formation of prostaglandin E2
(PGE2), thromboxane A2 (TXA2), and leukotriene B4 (LTB4) and maintain the
levels of prostaglandin I2, potent vasodilator, thromboxane A3, aggregation
inhibitor platelet, and leukotriene B5 anti-inflammatory non-chemotactic that
inhibits cell adhesion (Table 2) [55].
While the products of AA metabolism (PGE2, TXA2, and LTB4) have pro-
inflammatory properties, the products of EPA conversion have antagonistic proper-
ties that decrease inflammation, vasoconstriction, and platelet aggregation and may
even antagonize the effects, typically proinflammatory effects of eicosanoids derived
from AA [1].
Table 2 Metabolic effects of eicosanoids derived from arachidonic acid (AA) and
eicosapentaenoic acid (EPA)
Cyclooxygenase pathway Lipoxygenase pathway
Endothelial cells Platelets Leukocytes
AA EPA AA EPA AA EPA
Prostacyclin I2 Platelet Thromboxane Thromboxane Leukotriene B4 Leukotriene B5
Vasoconstrictor antiaggregation A2 A3 Proinflammatory Anti-inflammatory
Platelet Vasodilator Platelet Vasodilator Chemotactic Non-chemotactic
aggregation aggregation Cell adhesion Inhibits cell
Vasoconstrictor adhesion
The production of proinflammatory cytokines is regulated by the availability of

eicosanoids derived from AA, which can be modulated by the intake of ω-3 PUFA,
which act at the gene level, since the expression of the genes for cytokines and cell
adhesion molecules it is reduced in response to the ω-3 PUFA exposure. In addition,
ω-3 PUFA directly affect the intracellular signaling pathways associated with the
activation of transcription factors, such as nuclear factor κβ (NF-κβ) and peroxisome
proliferator-activated receptors (PPARs) that regulate the expression of a series of
genes whose products are proinflammatory [1].
Supplementation with EPA and DHA is also able to reduce the production of
proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, which are released when
macrophages and monocytes are activated. Although these cytokines are potent
activators of immune function, the excess activity of these substances contributes
to pathological inflammation, a situation observed in metabolic diseases of inflam-
matory origin [17].
4.2 Potential Neuroprotective Effect of Omega 3 (v-3) in ND
Several studies have shown the neuroprotective role of ω-3 fatty acids in various
circumstances such as the ND to exhibit benefits in the modulation of neuronal
activity, the regulation of immune cell response, and also the production of lipid
mediators pro-resolution and more potent anti-inflammatories, such as resolvins [20,
28, 29]. Also, neuroprotective effect is observed in lesions induced by ischemia and
by excitotoxicity produced by neurotransmitters (glutamate, mainly) [21].
The neuroprotective effects of ω-3 are due to multiple factors and may be related
to a series of molecular effects at the neuronal level, especially in the CNS. Studies in
rodents indicate that long-chain ω-3 fatty acids play a role in cognitive and behav-
ioral functions. Although ω-3, due to its chemical structure (with numerous double
bonds), are more vulnerable to oxidative stress (characteristic of metabolic and
neurodegenerative diseases), in cells in general and especially neurons, they can
reduce the damage caused by oxidative stress through neuroprotectins (docosanoids
derived from DHA) [5]. In addition, ω-3 regulate the expression of neuroprotective
genes, such as the expression of the antiapoptotic Bcl2 gene [4].
According to Chen et al. [13], ω-3 supplementation inhibits microglial activation
and the subsequent inflammatory response by regulating the translocation and
nuclear secretion of HMGB1 and its activation in the signaling pathway TLR4/
NF-κβ, pathways responsible for neuroinflammation after injury neuronal. The
above is an important evidence that leads to the neuroprotective effects exhibited
by ω-3 fatty acids.
Chronic ω-3 fatty acid deficiency increases anxiety in rodents, particularly in
stressful conditions [19]. Also, chronic DHA deficiency is accompanied by anxiety
and learning and memory impairments that have been associated with changes in the
processes of neurotransmission [11].
Specifically, the diet deficient in alpha-linolenic acid reduces the dopaminergic
neurotransmission of the nucleus accumbens of murine models. Through in vivo
microdialysis experiments, elevated basal levels of dopamine and its metabolites

(DOPAC and HVA) were observed in rats subjected to a diet deficient in fatty acids,
in comparison with controls, indicating alterations in dopaminergic neurotransmis-
sion in the nucleus accumbens [67].
Other investigations focused on the neuroprotective effects of ω-3 on AD reveal
the presence of low levels of plasma DHA. In an animal model (mouse with
Alzheimer’s disease), when administering a diet enriched with ω-3 (EPA þ DHA),
a reduction in the accumulation of β-amyloid peptide (peptide with neurotoxic
actions) was observed in more than 70% of the cases [27].
The administration of 15 mg of EPA and DHA per g of intake per day in female
rats, treated on the second day of pregnancy up to 14 days after calving, significantly
reduced brain damage and improved long-term neurological outcomes up to 5 weeks
after a neonatal hypoxic ischemic injury. The ω-3 polyunsaturated fatty acids exerted
an anti-inflammatory effect in microglia both in the in vivo model of ischemic-
hypoxic and in vitro microglial cultures subjected to inflammatory stimuli by
inhibiting the activation of NF-κβ and the subsequent release of inflammatory
mediators (Zhang et al. 2010).
In more recent studies, mice induced to neuropathic pain by partial ligation of the
sciatic nerve received oral treatment of a fish oil concentrate of 2.3 g/Kg of weight,
and reduced levels of TNF-α in the spinal cord, from the myeloperoxidase activity
(MPO) sciatica and the expression of activation transcription factor 3 (ATF-3), an
important marker of neuronal injury, were observed. Likewise, the diet high in ω-3
improved the sciatic functional index (SCI), as well as the electrophysiological
record, which corroborates the increase in the expression of GAP43 and the total
number of myelin fibers observed in the sciatic nerve. These results point to the
regenerative and possibly protective properties of a combined oral administration of
EPA and DHA after a peripheral nerve injury, as well as its anti-neuroinflammatory
activity, evidencing the ω-3 fatty acids that promise therapeutic results for the
treatment of neurodegeneration [48].
4.2.1 Alzheimer’s
The consumption of ω-3 fatty acids is associated with a reduction of
neuroinflammation markers in AD [24, 53]. Limited evidence in human models
shows that ω-3 improve the metabolic neuroinflammatory alterations present in AD.
An intervention study reveals a decrease in the loss of gray matter volume after the
combined treatment of DHA/EPA [62]. Yassine et al. [63] showed significant
associations between low serum concentrations of DHA with increased cerebral
amyloid load, smaller brain volume (SBV), and alteration in nonverbal memory, in
volunteers without cognitive or mild impairment.
Correlative epidemiological studies suggest that a high intake of foods rich in ω-3
is associated with greater cognitive performance and possibly less neuronal deteri-
oration and risk of AD [38]. Results recently presented at the Alzheimer’s Associ-
ation International Conference in Toronto indicate that blood DHA levels are
significantly associated with superior cognitive ability in two large population-
based studies, totaling more than 5000 individuals [56].
It should be noted that although clinical trials of ω-3 supplementation following

the diagnosis of AD do not show significant effects, studies have shown its potential
preventive effect, associated with its anti-inflammatory effects [43, 45, 64]. Recently
published data from the MAPT (Multidomain Alzheimer Preventive Trial) report no
significant benefit in elderly participants who received 800 mg/day of DHA supple-
ments for more than 3 years [3].
4.2.2 Parkinson’s
Evidence from clinical studies about ω-3 fatty acid supplementation in PD is still
very limited. In animal models, it has been observed that DHA induces a recovery of
the dopaminergic system after an extensive Parkinson’s injury. After the induction of
dopaminergic denervation with 6-hydroxydopamine (6-OHDA), a high intake of
DHA led to a) increased levels of dopamine in the striatum, b) greater dopaminergic
terminals in striated body, and c) increase of the soma area of dopaminergic neurons.
Although the cell count remained unchanged, such improvement of key components
in the dopaminergic system suggests that DHA activated the compensatory mecha-
nisms that contribute to the functionality and recovery of CNS [14]. Therefore, these
data suggest that DHA induced neuroregeneration and could be used after diagnosis
of PD.
Recently Mori et al. [37] demonstrated that supplementation of 3 g/Kg of weight
(18% EPA and 12% DHA) in Wistar rats mitigates the loss of SNpc neurons and
nerve terminals in the striatum, after an induction of damage with 6-OHDA. This
protective effect was associated with reductions in iNOS-immunoreactive cell den-
sity and reactivity of microglia and astrocytes, thereby reducing dopaminergic
damage in PD.
5 Conclusion
The scientific evidence shows the potential neuroprotective effect of omega 3 fatty
acids on ND, associated with the reduction of neuroinflammation. Although more
clinical studies are necessary to obtain ω-3 dosages in the prevention and treatment
of ND, the trials in animal and epidemiological models are an important reference for
its recommendation as part of a nutritional treatment.
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Application of Lipid Nanocarriers for the
Food Industry 22
Zahra Rafiee and Seid Mahdi Jafari
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
2 Nanoencapsulation by Lipid-Based Nanocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
2.1 Nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
2.2 Nanoliposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627
2.3 Nano-Phytosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
2.4 Lipid Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
3 Nanoencapsulation of Food Bioactive Compounds by Lipid-Based Nanocarriers . . . . . . . . 637
3.1 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
3.2 Natural Food Colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642
3.3 Bioactive Oils and Essential Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643
3.4 Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
3.5 Natural Antimicrobial Agents and Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
3.6 Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
3.7 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
4 Conclusion and Future Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 654
Abstract
Bioactive compounds effectively contribute to human health, and hence, they
have recently attracted great attention in order to fortify food products and
develop novel functional foods. However, employment of bioactive compounds
in food matrices has some limitations. Most of the bioactive substances are
readily decomposed in food as well as within the gastrointestinal tract that
cause remarkable losses in their efficiency. Furthermore, they display low water
solubility, poor bioavailability, and insufficient dispersibility. Other problems are
Z. Rafiee · S. M. Jafari (*)

Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural
Sciences and Natural Resources, Gorgan, Iran
e-mail: zahra_rafi[email protected]; [email protected]

624 Z. Rafiee and S. M. Jafari
related to their interaction with food ingredients and unfavorable effects on

sensory attributes of food products. Lipid-formulation nanoencapsulation tech-
nologies including nanoliposomes, nanoemulsions, lipid nanoparticles (SLNs,
solid lipid nanoparticles, and NLCs, nanostructured lipid carriers), and nano-
phytosomes potentially help to solve these issues. These nanodelivery systems
provide more stability, solubility in different media, functionality, bioavailability,
targeting properties, and the ability of controlled release in food and pharmaceu-
tical practices. This chapter reviews lipid-based nanocarriers in terms of produc-
tion methods, types, characteristics, and composition for incorporation of
different bioactive compounds. Also, food applications of various bioactive
compounds incorporated in the commonly used lipid-based nanocarriers are
highlighted. In this sense, the relevant recent studies have been discussed.
Keywords
Bioactive compounds · Nanoencapsualtion · Lipid-based nanocarriers ·
Functional foods · Food applications
1 Introduction
Health concerns have recently increased the tendency for development of functional
foods and novel food products fortified with bioactive compounds and nutraceuticals
especially phytochemicals [14, 205]. However, there are some limitations for direct
utilization of bioactive compounds in food matrices. Most of the bioactive sub-
stances suffer from low stability and decomposition when exposed to unfavorable
conditions during food processing and storage (e.g., light, oxygen, and moisture) as
well as within the gastrointestinal tract (GIT) which reduces their efficiency and
bioactivity [1, 139]. Moreover, they exhibit low water solubility, poor bioavailabil-
ity, and insufficient dispersibility in food systems, interact with food ingredients, and
negatively influence sensory properties of food systems [39, 46, 51, 66].
Food-grade delivery systems for nanoencapsulation of bioactive compounds
represent an efficient way to solve these drawbacks. Among different nanodelivery
techniques, lipid-based nanocarriers (Fig. 1) including nanoliposomes, nano-
emulsions, lipid nanoparticles (SLNs, solid lipid nanoparticles, and NLCs, nano-
structured lipid carriers) and nano-phytosomes have received a great attention owing
to their beneficial characteristics [6, 64]. They have great potentials to accommodate
and release various bioactive compounds (hydrophilic, lipophilic, and amphiphilic
compounds) in a sustained and controlled manner, improve solubility and encapsu-
lation efficiency of hydrophobic bioactive compounds, decrease their volatility, and
enhance their target specificity. Lipid-based nanocarriers also promote effectiveness
and bioavailability by enhancing stability of entrapped compounds in food mediums
and during digestion [43, 79, 192]. Furthermore, these systems are biocompatible
and can be fabricated by inexpensive and safe components for large-scale industrial
practices via simple and available production technologies without the use of organic
solvents [79, 98]. This chapter will review lipid-based nanocarriers in terms of
22 Application of Lipid Nanocarriers for the Food Industry 625
Fig. 1 Schematic illustration of three important nanostructured lipid-based encapsulation formu-

lations: nanoliposomes (a), nanoemulsions (b) plus NLCs and SLNs (c). (Reprinted with permis-
sion from Ref. [6])
production methods, types, characteristics, and composition. Also, food applications

of different bioactive compounds incorporated in the commonly used lipid-based
nanocarriers are highlighted. Future trends will also be discussed in the last section.
2 Nanoencapsulation by Lipid-Based Nanocarriers
Encapsulation is described as an effective and practical technology for incorporating

different unstable compounds within carrier materials which can occur in micro- and
nanoscale. According to nanotechnology definition, an average size under 1000 nm
is referred to as nanoparticle; although for therapeutic and pharmaceutical delivery
systems, it should be lower than 100 nm [54, 194]. Many studies have indicated
superiority of nanocarriers to micro ones. Compared to microencapsulation systems,
nanosized carriers offer more stability, solubility in different media, functionality,
bioavailability, absorption, homogeneity, targeting properties, and the ability of
controlled release in food and pharmaceutical practices which are associated with
their greater surface area and larger reactivity [48, 52]. Moreover, food formulations
containing nanoparticles with sizes smaller than 100 nm have acceptable sensory
attributes due to their high optical transparency [114, 115, 160].
There are several methods for preparing nanoencapsulated bioactive compounds
which can be categorized into five different classes including lipid-based techniques,
specialized-equipment techniques, nature-inspired techniques, biopolymer-based
techniques, and disparate techniques [79]. Nanoencapsulation technology should
be selected on the basis of various factors such as release pattern, cost, the nature and
physicochemical features of the wall and core materials, purpose of encapsulation,
etc. [6, 167].
Diverse types of wall materials can be applied to incorporate bioactives including
proteins, lipids, and carbohydrates. Carbohydrate- and protein-based nanocapsules
cannot be applied in industrial applications because of occurring complex chemical
or heat treatments which make it difficult to control processing conditions. In
contrast, lipid-based nanocarriers not only have scaling-up potentials and low
toxic effects but also offer greater encapsulation potency [54, 199]. The presence
of emulsifiers is mostly essential to incorporate hydrophobic bioactive substances (e.
g., flavonoids, fatty acids, aromas, phytosterols, lipophilic vitamins, and caroten-
oids) due to their non-solubility nature. Furthermore, utilization of digestible lipids
provides sufficient blended micelles for solubilization and carrying lipophilic com-
pounds which consequently facilitate their intestinal absorption [78, 164]. Accord-
ingly, lipid-formulation nanoencapsulation technologies have received remarkable
attention as novel and promising vehicles for bioactive ingredients in food and
pharmaceutical industries. Nanoliposomes, nanoemulsions, SLNs, NLCs, and
nano-phytosomes are basic kinds of lipid-based nanocarriers which are described
in the following sections [50, 64, 79].
2.1 Nanoemulsions
The emulsion is defined as a system composed of two immiscible liquids (mostly

water and oil) where one is dispersed as the droplet form (the dispersed or internal
phase) in other one (the continuous or external phase). Emulsions can be classified
into three main types in terms of particle size of droplets: microemulsion
(10–100 nm), macroemulsion (0.5–100 μm), and nanoemulsion (known as mini-
emulsion, 100–500 nm) [80, 83].
The nanoemulsions are formulated in well-optimized mixtures consisting of at
least three components being oil, aqueous phases, and surfactant(s). The oil phase of
oil-in-water (O/W) nanoemulsions and water phase of water-in-oil (W/O) nano-
emulsions are generally hosting the lipophilic and hydrophilic bioactive ingredients,
respectively [131, 132]. The bioactive compounds are usually solubilized in the
related phase prior to the formation of nanoemulsions. The lipophilic phase can be
comprised of triacylglycerols (long-chain, medium-chain, and short-chain triglycer-
ides), essential oils, mineral oils, fat substitutes, waxes, or combination of them. In
addition to the oil and water phases, the preparation of nanoemulsions requires the
use of surfactant(s) which is adsorbed on newly formed droplet surfaces and
facilitates droplet disruption and protects droplets toward aggregation [128].
As illustrated in Table 1, nanoemulsions can be generally produced by two
different techniques: high-energy (e.g., ultrasonication, electrified coaxial liquid
jets, microfluidization, high-pressure homogenization, and high shear mixing) or
low-energy emulsification approaches [81, 82]. High-energy techniques need high
levels of mechanical energy to provide intense disruptive forces for generating fine
droplets. In contrast, low-energy procedures are based on utilizing the internal
chemical energy of the oil-water-surfactant mixtures, and emulsification is merely
carried out via simple stirring [79]. It is possible to fabricate nanoemulsions by
modifying the system conditions. For instance, large amounts of emulsifiers, a high
volume fraction of dispersed phase, and changes in the hydrophilic-lipophilic
balance of matrix through modifying parameters (e.g., composition or temperature)
lead to generation of nanoemulsions by microemulsification, catastrophic inversion,
and phase inversion methods (inversion temperature and phase inversion composi-
tion), respectively [9, 183]. Low-energy methods are not suitable for scaling up
and industrial purposes [11, 12]. High-energy techniques are highly favorable for
producing ultra-fine droplets, but they may degrade susceptible compounds [84].
Furthermore, nanoemulsions can be manufactured by cubosomes and colloidosomes
as well as microfluidic channels [6].
Nanoemulsions possess distinct features such as a small droplet size, optical
transparency, high surface area, and high kinetic stability which make them appro-
priate candidates for the delivery of bioactive compounds [137, 138]. They display
good potentials to improve solubility, absorption, and bioavailability of incorporated
bioactive compounds [182, 186]. However, some adverse phenomena may take
place in nanoemulsion systems like Ostwald ripening, flocculation, creaming, coa-
lescence, and gelling, which negatively influence their storage stability. On the other
hand, they require high levels of emulsifiers and particular devices for nano-
encapsulation [6, 127, 172].
2.2 Nanoliposomes
Liposomes are self-assembled spherical vesicles consisting of at least one concentric

lipid bilayer that encloses an aqueous phase [145]. Dispersing phospholipids in
aqueous solutions results in orientation of hydrophobic groups toward the inside
of the sphere with the hydrophilic heads toward outside. Therefore, a spherical
bilayer membrane is formed which surrounds the aqueous core [47, 54]. Liposomes
are categorized into different classes of large unilamellar vesicles (LUVs), small
unilamellar vesicles (SUVs), giant unilamellar vesicles (GUVs), multivesicular
Table 1 Overview of nanoemulsion preparation techniques (Reprinted with permission from

Ref. [6])
Technology Process steps
Hot homogenization 1. Melting the lipid and dissolving/dispersing the bioactive
technique compound in the lipid
2. Dispersing the bioactive-loaded lipid in hot aqueous surfactant
mixture
3. Premixing using a stirrer to form a coarse pre emulsion
4. High-pressure homogenization at temperatures above lipid
melting point
5. Hot O/W nanoemulsion
6. Solidification of the nanoemulsion by cooling down to room
temperature
Cold homogenization 1. Melting the lipid and dissolving/dispersing the bioactive
technique compound in the lipid
2. Solidification of the bioactive-loaded lipid in liquid nitrogen or
dry ice
3. Grinding in a powder mill (50–100 μm)
4. Dispersing the powder in an aqueous surfactant dispersion
medium (premix)
5. High-pressure homogenization at room temperature or below
High-pressure 1. The lipid is pushed with high pressure (100–2000 bars)
homogenization through a very high shear stress
2. Disruption of particles down to the submicrometer or
nanometer range
Solvent emulsification- 1. Lipids and bioactive compounds are dissolved in a water-
evaporation method immiscible organic solvent with low boiling point
2. The solution is then emulsified in the aqueous emulsifier
solution
3. Evaporate in rotary evaporation at 50–60 C
Solvent emulsification- 1. Both the solvent and water are mutually saturated in order to
diffusion technique ensure the initial thermodynamic equilibrium of both liquids
2. Lipid and bioactive compound are dissolved in water-saturated
solvent, and this organic phase is stirred using mechanical stirrer
3. After formulation of O/W emulsion, water in typical ratios
from 1:5 to 1:10 is added to the system in order to allow solvent
diffusion into the continuous phase, thus leading to the
aggregation of the lipid in the nanoparticles
Microemulsion technique 1. Lipids are melted, and bioactive compound is incorporated into
molten lipid
2. Mixture of water, co-surfactant, and surfactant is heated to the
same temperature as lipids and added under mild stirring to the
lipid melt
3. A transparent, thermodynamically stable system is formed
when the compounds are mixed in correct ratios for
microemulsion formation
4. This microemulsion is then dispersed in a cold aqueous
medium under mild mechanical mixing of hot microemulsion
with water in a ratio in the range 1:25–1:50
Melting dispersion method 1. Bioactive compound and solid lipid are melted in an organic
solvent regarded as oil phase. Simultaneously water phase is also
(continued)
Table 1 (continued)
heated to the same temperature as oil phase
2. The oil phase is added to a small volume of water phase, and
the resulting emulsion is stirred at high speed for few hours
3. The emulsion is cooled down to room temperature to yield
nanoparticles
Ultrasonication technique 1. The core material is melted
2. Addition of phospholipids along with an aqueous medium
3. Dispersing the melted material at increased temperatures by
ultrasonication
Solvent injection 1. Lipids are dissolved in a water-miscible solvent (e.g., acetone,
isopropanol, and methanol) or water-miscible solvent mixture
2. Quickly injected into an aqueous solution of surfactants
through an injection needle
Double emulsion technique 1. Bioactive compound (mainly hydrophilic ones) is dissolved in
aqueous solution emulsified in melted lipid
2. The primary emulsion is stabilized by adding stabilizer that is
dispersed in aqueous phase containing hydrophilic emulsifier
3. Emulsion is stirred and filtered
vesicles (MVVs), and multilamellar vesicles (MLVs) regarding their size and
lamellarity [68].
A wide variety of methods have been employed for producing nanoliposomes.
These techniques are generally divided into mechanical (extrusion, ultrasonication
by probe or bath sonicator, microfluidization, and high-pressure homogenization)
and nonmechanical methods (reversed-phase evaporation, freeze-drying-rehydra-
tion, freeze-thawing, depletion of mixed detergent-lipid micelles, and injection
methods) [203]. Mozafari [141] proposed a method for liposome preparation
based on heating treatment. Today, novel methods such as dense gas techniques,
supercritical fluid technology, dual asymmetric centrifugation, membrane contactor
technology, cross-flow filtration technology, and freeze-drying of double emulsions
method have been developed for preparing nanoliposomal formulations [76, 121,
207]. Table 2 presents advantages and disadvantages of various preparation methods
of nanoliposomes.
Nanoliposomes are mostly used in food, bioprocessing, and pharmaceutical
industries for incorporation of bioactive compounds to enhance their solubility,
bioavailability, and functionality, provide controlled and sustained release, and
protect them against adverse conditions. Also, nanoliposomes can be manufactured
from natural and safe components (e.g., soy, egg, or milk) and are biocompatible and
targetable. Additionally, liposomal ingredients such as phospholipids and
sphingolipids have potential health benefits for humans [51, 171]. Owing to amphi-
philic nature, liposomes are capable of encapsulating bioactive compounds with
different degrees of solubility. Hydrophobic compounds are distributed within the
lipid bilayers, while hydrophilic substances are enclosed inside aqueous space.
Compounds with intermediate polarity also can be located between aqueous
Table 2 Overview of conventional and novel methods for nanoliposome production (Reprinted
with permission from Ref. [6])
Technology Advantages Disadvantages
Conventional Thin-film Rapidly formed; low Usually yields large and
methods dispersion energy input to form. multilamellar vesicles with
(Bangham Highest stability and low encapsulation
method) transition cooperatively efficiencies (EEs)
Ethanol/ether Organic solvent residue, Highly heterogeneous
injection ready to nozzle blockage in dispersion; lower
ether system, time- encapsulation efficiency
consuming, sterilization vs. LUVs. Nearly
issue impossible to determine
mass
Ultrasonication: Can be performed directly Probe sonication can
probe on hydrated MLVs. degrade sample/localized
Preferred method to form overheating
SUVs Requires constant cooling.
Excellent for reduction of Liposomes are metastable
large MLVs to more (low storage stability)
homogenous dispersion of Low volume required for
SUVs effective treatment
Ultrasonication: Less destructive to Requires extensive
bath liposomes, more sonication to obtain
homogenous product, and minimum size limit of
greater reproducibility vs. SUV; not always possible.
probe sonicator. Increased Nonhomogenous product;
sample volume capability. requires removal of large
Increased control over vesicles by
sample temperature chromatographic/
centrifugal methods
Reverse-phase Higher entrapment Heterogeneous distribution
evaporation efficiency reported for of MLVs and unilamellar
variety of molecules with vesicles; additional
MLVs (proteins, nucleic homogenization required.
acids, etc.) Solvent exposure may
inhibit protein activity.
Incomplete removal of
organic solvent
Freeze-dried High entrapment Dehydration best
rehydration efficiency. Improved controlled by freeze-
vesicles homogeneity in mixing of drying; long time to
lipid species/liposome. process
Useful for forming antigen-
entrapping vesicles
Microfluidic Control of particle size, Not suitable for bulk
channel production of vesicles with production, organic solvent
diameter up to 29 nm use, with agitation over
treatment, damage of the
liposome structure, and the
leakage of encapsulated
components
(continued)
Table 2 (continued)
Technology Advantages Disadvantages
Detergent No solvent used; protein Limited number of useful
depletion activity retained. No detergents. Dialysis is very
mechanical energy input. slow process, and some
Homogenous distribution detergent is likely to
of liposomes. Useful for remain in the sample.
multiple lipids and Detergent may negatively
molecules for interact with molecule of
encapsulation. Best for interest
entrapping membrane-
associated proteins
Membrane Homogenization of Liposomes must be held
extrusion liposomes improved over above TM to facilitate
other methods; rapid extrusion. Manual
preparation of unilamellar extruders handle only
vesicles from MLVs small volumes (1 ml).
Solute leakage can occur
during extrusion. High salt
concentration
Novel Heating method Absence of potentially Long time to process
methods toxic solvents and using
very low shear forces;
applied to large-scale
production
Freeze-drying of Relatively high Long time to process
double encapsulation efficiency
emulsions and excellent stability
during long-term storage
High-pressure Can process high lipid Solvent ionic strength must
homogenization, concentration (150 mg/ be carefully controlled to
microfluidization ml). Lab data is easily control liposome size.
inferred to processing Complete homogenization
plant. High reproducibility. is time-consuming in
Most useful for large-scale microfluidizer
production of liposomes
Supercritical Control of particle size, High cost, low yield, high
fluid injection possible in situ pressure up to 350 bar used
and sterilization, low organic
decompression solvent consumption
Dual asymmetric Simple method, Not suitable for bulk
centrifugation homogenous liposome production, high pressure,
production with 60 nm with agitation
size, high trapping
efficiency
Dense gas Possible in situ Need multiple stages to
techniques sterilization, producing achieve the final size of
stable and homogenous liposome, high pressure up
iposome, low organic to 200–300 bar, readily
solvent consumption block nozzles
compartment and the lipid moieties. Furthermore, nanoliposomes provide simulta-

neous encapsulation, release, and delivery of compounds with different polarities
[4]. For instance, this possibility has been revealed for α-tocopherol with ascorbic
acid and glutathione [141].
Despite mentioned advantageous properties, some drawbacks restrict potential
applications of liposomes. They are unstable systems and easily undergo oxidation,
hydrolysis, fusion, and aggregation, decreasing loading capacity of bioactive com-
pounds [37, 203]. Also, liposomes exhibit a short release time and inadequate half-
life circulation. Using cholesterol, hydrogenated soy phosphatidylcholine, and neu-
tral long-chain saturated phospholipids (e.g., disteroylphosphatidylcholine) can
increase the stability and rigidity of bilayers. Phytosterols have been recently
introduced as a suitable substitute of cholesterol in liposomal formulations especially
for patients with hypercholesterolemia [47, 121]. Moreover, several techniques such
as freezing, lyophilization (freeze-drying), supercritical fluid technology, and spray-
drying have been evaluated for extending the shelf life of liposome structures [54].
Overall, lyophilization is considered as the best stabilization method for thermolabile
substances loaded within liposomes [28]. Furthermore, polymer coating is a suitable
and efficient stabilization method for liposomal systems. Coating with chitosan can
improve stability, bioavailability, and sustained release of liposomes containing
bioactive compounds [37]. Also, it was demonstrated that polyethylene glycol
coating could effectively promote circulation half-life, functionality, and stability
of bioactives [125].
2.3 Nano-Phytosomes
The term phytosome comes from two words of “phyto” and “some” which mean
plant and cell-like, respectively [19, 184]. Phytosome, also called as herbosome and
phytolipid delivery system, is resulted from the complex of plant extracts or phyto-
active compounds (i.e., flavonoids and terpenoids) with phospholipids, which are
formed via hydrogen bonds between their polar moieties [94, 95].
Basically, main components of phytosome formulations include phytochemicals,
phospholipids, and solvents. The ratio of phospholipid to phyto-actives plays an
important role in phytosome systems, and 1:1 or 2:1 ratios are often preferred. The
common phospholipids for producing phytosomes are phosphatidylcholine, phos-
phatidylethanolamine, and phosphatidylserine. The former is most widely used for
producing phytosomes. Aprotic solvents like acetone, ethyl acetate, dioxane, and
methylene chloride can be successfully utilized in phytosome systems, but ethanol as
a safe solvent is a good candidate for food purposes [59, 64, 205]. In order to
separate organic solvents from obtained phytosomes, various methods can be used
including spray-drying, freeze-drying, vacuum evaporation, and precipitation via
n-hexane as an aliphatic hydrocarbon. Different techniques have been applied to
develop phytosomes such as precipitation, anhydrous cosolvent lyophilization, anti-
solvent precipitation, and solvent evaporation [95, 206].
The phytosome and liposomes are similar in the terms of structure. However,
phytosomes possess a higher ability to enhance stability, absorption, and bioavail-
ability of incorporated ingredients than liposomes which is associated with presence
of H-bonds between phospholipids and the loaded compounds in phytosome struc-
tures. In contrast, there is no chemical interaction in liposomes, and the bioactives
are merely surrounded by phospholipid bilayer. Intensifying bioavailability and
absorption of lipid-soluble compounds by phytosomes as a delivery system is
more evident, which reduces required amount to exhibit their advantageous effects
and boost functionality [134, 188, 193].
It has been demonstrated that antioxidant activity of phenolic compounds in
phytosomal systems is more than their free ones. In addition to food applica-
tions, there is a growing trend for usage of phytosomes in cosmetics owing to
improving skin penetration of bioactive compounds. They can easily pass
through the membrane and release bioactives into the cells, prevent degradation
of bioactives by gut bacteria as well as digestive secretions, and provide target
specificity [59, 134, 188]. Phytosomal products are commercially available in
the market [7].
In spite of the advantages, phytosomes are sensitive to pH variations, and it is
better to use them in food products with the neutral pH values such as milk [64].
2.4 Lipid Nanoparticles
2.4.1 Solid Lipid Nanoparticles (SLNs)

SLNs are nanoparticles with an organized crystalline structure and can entrap
different bioactive components within their lipid network [212]. SLNs are basically
derived from O/W emulsions, but they use solid lipids instead of the liquid one (oil),
and therefore, they remain in solid form at ambient as well as human body temper-
atures [211].
Main ingredients utilized within SLN formulations are solid lipids, surface
active materials (emulsifiers), and water. Lipid matrices are fabricated using a
wide variety of natural or synthetic digestible lipids. Some of the most common
lipids include triglycerides (tripalmitin, tristearin, and trimyristin), glyceryl mono-
stearate (Imwitor), glyceryl palmitostearate (Precirol ATO 5), glyceryl behenate
(Compritol 888 ATO), fatty acids (palmitic acid, stearic acid, and decanoic acid),
cholesterol, waxes (carnauba wax, cetyl palmitate, and beeswax), and different
types of hard fats [49, 63, 214]. Phospholipids as neutral surfactants (lecithin),
ionic surfactants (sodium lauryl sulfate, sodium deoxycholate, sodium dodecyl
sulfate, sodium cholate, sodium taurodeoxycholate, sodium oleate, tri-
methylammonium bromide, and stearylamine), nonionic surfactants (Poloxamer,
Span, Pluronic, Tween, Solutol, Tego Care, and Brij), polyvinyl alcohol, and
polyethylene glycol are the most important emulsifiers or surfactants which
improve stability of lipid matrices [49, 110, 190]. Furthermore, mono- and diglyc-
erides as solubilizing agents can enhance solubility and loading efficiency of
bioactive compounds [54].
Water insoluble phenolic

Water insoluble phenolic
Solid lipid
Solid lipid
(Bioactive-enriched matrix) (Bioactive-enriched shell) (Bioactive-enriched core)
Solid lipid nanoparticles (SLN), typical size<100 nm

Liquid Lipid
Water insoluble phenolic Water insoluble phenolic
Solid Lipid with Solid Lipid
different dimension
Solid Lipid
Amorphous Lipid
Liquid Lipid
(Imperfect Type) (Amorphous type) (Multiple Type)
Nanosturcture lipid carrier (NLC) typical size<100 nm
Fig. 2 Classification of SLNs and NLCs. (Reprinted with permission from Ref. [97])
Encapsulation of bioactive compounds within SLNs is usually carried out via

three models including homogeneous matrix (solid solution) model, bioactive-
enriched shell model, and bioactive-enriched core model (Fig. 2). In homogeneous
matrix model, the dispersion of bioactive compounds entirely occurred in the lipid
matrix. This type is mainly achieved by cold homogenization technique or when
highly lipophilic compounds are entrapped inside SLNs applying hot homogeniza-
tion. No surfactant is used for fabrication in this model. Bioactive-enriched shell
model is formed as high concentration of surfactants is used or when phase separa-
tion from the liquid oil droplet takes place during the cooling process. This model
has a burst release pattern. When the inverse phenomenon of bioactive-enriched
shell model occurs, bioactive-enriched core type can be achieved. In this model,
bioactive compounds precipitate first, and subsequently the shell entraps lower
amounts of incorporated ingredients. This model results in controlled release of
bioactive compounds [60, 97].
SLNs were developed to overcome the defects of other lipid-based nanocarriers
such as nanoliposomes and nanoemulsions. They can be prepared without use of
organic solvents; provide prolonged release, target specificity, and biocompatibility;
protect incorporated compounds against adverse external conditions as well as
chemical reactions (e.g., oxidation); improve stability, solubility, dispersibility, and
bioavailability; facilitate large-scale production; and be utilized in different food
products [148]. However, SLNs have limited space for encapsulating bioactive
compounds. They may also undergo crystallization, gelation, aggregation, and

expulsion of core materials during storage period which negatively influences their
characteristics and release behavior [40, 60].
2.4.2 Nanostructured Lipid Carriers (NLCs)

To overcome deficiencies of SLNs, NLCs were introduced by [221] as an efficient
strategy for nanoencapsulation of bioactive compounds. NLCs are originated from
O/W emulsions like SLNs. However, inner core of NLCs is obtained from a mixture
of solid lipid (70%) and liquid lipid (30%), while lipid matrix of SLNs entirely
consists of solid lipids (100%) [6, 43].
Basically, both saturated oils (e.g., paraffin oil and medium-chain triglycerides)
and unsaturated oils (e.g., oleic acid, seed and vegetable oils) are employed for
developing NLCs as liquid lipids [49]. Most common type of liquid lipid for
producing NLCs is medium-chain triglycerides (MCT) such as Miglyol ® 812.
However, healthy unsaturated fatty acids (e.g., oleic acid and linoleic acid) can be
considered as a substitute for saturated oils [44, 143, 199, 219].
NLCs are structurally divided into three diverse types: imperfect crystal,
amorphous, and multiple models by considering type of bioactive compounds,
the purpose of nanoencapsulation, preparation techniques, and makeup (Fig. 2).
In the first type, NLCs are fabricated by diverse lipids with specific dimensions
which increase their capacity for incorporating bioactive compounds. In the
second group called amorphous model, noncrystalline structures are produced
by a mixture of solid lipids with some other lipids (isopropyl myristate,
hydroxyoctacosanyl, hydroxystearate, or MCT). In turn, the mixture can effec-
tively decrease expulsion and release rate by forming an amorphous matrix. This
model can prevent degradation of bioactive compounds which are highly soluble
within liquid lipids. In the latter group called multiple model, employing certain
liquid lipids (e.g., hydroxyl stearate and isopropyl myristate) leads to reduction
of crystallization and therefore expulsion. The model is particularly suitable for
enclosing and modulated release of bioactive compounds which exhibit great
solubility in liquid lipids [60, 97, 143].
Preparation method of NLCs and SLNs are the same. Diverse approaches have
been applied to fabricate these nanocarriers as shown in Table 3 which could be
categorized into two major groups. The first group includes methods which require
large amounts of energy like high-speed homogenization, ultrasound, and high-
pressure homogenization, and the second group are methods with low energy
consumption like microemulsions, solvent-based methods, and phase inversion
temperature [143, 144]. It could be concluded that hot, cold, and the high-pressure
homogenization techniques are practically more favorable for making these nano-
carriers compared with other methods [6]. NLCs have attracted increasing attention
because they possess good capability to optimize penetration, absorption, bioavail-
ability, and release rate of loaded bioactives in food, cosmetic, and pharmaceutical
applications [30].
Table 3 Overview of production methods for SLNs and NLCs (Reprinted with permission from
Ref. [97])
Hot homogenization 1. Melting of the lipid and dissolving/dispersing of the bioactive
technique in the lipid
2. Dispersing of the bioactive-loaded lipid in hot aqueous
surfactant mixture
3. Premix using a stirrer to form a coarse pre-emulsion
4. High-pressure homogenization at temperature above lipid
melting point
5. Hot O/W nanoemulsion
6. Solidification of the nanoemulsion by cooling down to room
temperature
Cold homogenization 1. Melting the lipid and dissolving/dispersing of the bioactive in
technique the lipid
2. Solidification of the bioactive-loaded lipid in liquid nitrogen or
dry ice
3. Grinding in a powder mill (50e100 mm)
4. Dispersing the powder in an aqueous surfactant dispersion
medium (premix)
5. High-pressure homogenization at room temperature or below
High-pressure 1. The lipid is pushed with high pressure (100e2000 bars)
homogenization through a very high shear stress
2. Disruption of particles down to the submicrometer or
nanometer range
Solvent emulsification- 1. Lipids and bioactive compound are dissolved in a water-
evaporation method immiscible organic solvent with low boiling point
2. The solution is then emulsified in the aqueous emulsifier
solution
3. Evaporation in a rotary evaporator at 50–60 C
Solvent emulsification- 1. Both the solvent and water are mutually saturated in order to
diffusion technique ensure the initial thermodynamic equilibrium of both liquids
2. Lipid and bioactive are dissolved in water-saturated solvent,
and this organic phase is stirred using mechanical stirrer
3. After the formulation of O/W emulsion, water in typical ratio
from 1:5 to 1:10 is added to the system in order to allow solvent
diffusion into the continuous phase, thus leading to the
aggregation of the lipid in the nanoparticles
Microemulsion technique 1. Lipids are melted, and bioactive is incorporated in molten lipid
2. A mixture of water, co-surfactant(s), and the surfactant is
heated to the same temperature as the lipids and added under mild
stirring to the lipid melt
3. A transparent, thermodynamically stable system is formed
when the compounds are mixed in the correct ratios for
microemulsion formation. Thus the microemulsion is the basis
for the formation of nanoparticles of a requisite size
4. This microemulsion is then dispersed in a cold aqueous
medium under mild mechanical mixing of hot microemulsion
with water in a ratio in the range 1:25–1:50.
(continued)
Table 3 (continued)
Melting dispersion method 1. Bioactive and solid lipid are melted in an organic solvent
regarded as oil phase. Simultaneously water phase is also heated
to the same temperature as oil phase
2. The oil phase is added to a small volume of water phase, and
the resulting emulsion is stirred at high speed for few hours
3. The emulsion is cooled down to room temperature to yield
nanoparticles
Ultrasonication technique 1. The core material is melted
2. Addition of phospholipids along with an aqueous medium
3. Dispersing the melted material at increased temperature by
ultrasonication
Solvent injection 1. Lipids are dissolved in a water-miscible solvent (e.g., acetone,
isopropanol, and methanol) or water-miscible solvent mixture
2. Quickly injected into an aqueous solution of surfactants
through an injection needle
Double emulsion technique 1. Bioactive (mainly hydrophilic ones) is dissolved in aqueous
solution emulsified in melted lipid
2. The primary emulsion is stabilized by adding stabilizer that is
dispersed in aqueous phase containing hydrophilic emulsifier
3. Emulsion is stirred and filtered
3 Nanoencapsulation of Food Bioactive Compounds by

Lipid-Based Nanocarriers
3.1 Phenolic Compounds
Phenolic compounds are regarded as a main group of bioactive compounds with

many unique features (such as antioxidant, antimicrobial, antiviral, anti-inflamma-
tory, anticancer, antithrombotic, and anti-allergic effects) that make them a qualified
choice for food and pharmaceutical sectors [13]. Enrichment/fortification of foods by
phenolic compounds not only improves their chemical and microbial stability but
also can be effective in prevention and cure of some disease such as cardiovascular
and neurodegenerative diseases [145, 148].
In spite of several beneficial properties, employment of phenolic compounds in
food matrices has some limitations. Generally, phenolic compounds are readily
decomposed in food as well as GIT systems. Nonpolar phenolic compounds are
poorly dissolved in aqueous foods due to their lipophilic character. Highly polar ones
do not pass through the biological membranes because of low hydrophobicity.
Moreover, phenolic compounds with multiple phenolic rings are insufficiently
absorbed as a result of forming massive molecular structures [64, 117]. Other
problems are related to their bitter and stringent taste, creation of hazy appearance
in beverages, and unfavorable effects on sensory attributes. Moreover, phenolic
compounds participate in browning reactions as well as oxidation, leading to
produce brown color and undesirable odors along with nutritional losses in fortified
foods [145, 148]. Interaction between phenolic compounds and food components
not only leads to aggregated and precipitated proteins but also can adversely affect
their bioactivity [173].
In contrast to in vitro experiments, phenolic compounds exhibit low bioactivity
within body system because they have conditioned solubility, low gastric retention
time, and weak penetrability and stability. Thus, higher amounts of these compounds
will be required for exerting their biological activities which result in further effects
on food products [51].
Lipid-based nanocarriers potentially help to solve these issues. They provide the
stability and controlled release for phenolic compounds in food matrices as well as
GIT system, consequently causing a longer shelf life and enhancing bioaccessibility.
The nanodelivery systems facilitate passage of polar phenolics from aqueous parts
toward lipophilic mediums and then bloodstream which enhances their in vivo
efficiency and bioavailability [101]. They promote antioxidant and antimicrobial
activity of phenolic compounds. Solubility of lipophilic phenolic compounds can be
also improved in beverages and aqueous foods without any significant changes in
sensorial properties of final products by lipid-based nanocarriers [53].
Many studies have been carried out on incorporation of phenolic compounds into
lipid-based nanocarriers as displayed in Table 4. For instance, Ni et al. [147]
encapsulated quercetin in nanoemulsions via high-pressure homogenization tech-
nology. Quercetin-loaded nanoemulsions displayed good phytochemical properties
by considering an average particle size, zeta potential, and encapsulation efficiency.
The prepared nanoemulsions exhibited similar antioxidant activity to free quercetin.
Furthermore, they found that nanoencapsulation improved release behavior and
bioavailability of quercetin in simulated GIT conditions. Also, they added the loaded
quercetin into a beverage formulation and observed that it caused no negative effects
on texture and appearance of the beverage during storage [147].
Epigallocatechin gallate (EGCG) and catechin loaded in soy lecithin nano-
liposomes was prepared by Rashidinejad et al. [174] and then added to ripened
low-fat cheese. The results showed that addition of the loaded EGCG and catechin
increased antioxidant activity and phenol content of cheese after simulated GIT
digestion without any notable effects on pH, chemical composition, and cheese
production yield. Moreover, EGCG and catechin completely remained in cheese
structure and were not detected in whey [174].
Gaber et al. [58] fabricated SLNs containing myricetin and evaluated effect of
some additives, pH variation, and heat treatments on their stability. Based on their
results, lipid-soluble antioxidants (vitamin E and BHT) could improve thermal
stability of incorporated compounds. Beside antioxidants, presence of Poloxamer
407 and Tween 80 as stabilizer was essential to form stable systems. Loaded
myricetin exhibited a lower decomposition rate and longer half-time in buffer
solutions compared to its free counterpart. Nanoencapsulation also influenced
release pattern of loaded phenolic compounds.
Babazadeh et al. [15] prepared NLCs as carriers of rutin using food-grade
components (oleic acid, cacao butter, and Tween) and added nanoencapsulated
Table 4 Some selected studies on incorporation of phenolic compounds into lipid-based

nanocarriers
Phenolic
Nanocarrier compound Results Reference
Nanoliposomes Luteolin Enhancement of bioavailability upon [213]
nanoencapsulation
Curcumin - High stability against alkaline pH and [31]
metal ions
- Good storage stability
- Sustained release
- Maintenance of cellular antioxidant
activity after nanoencapsulation
Quercetin and - Higher antioxidant activity than free [25]
resveratrol flavonoids
- Suitable stability
Curcumin Nanoliposomes obtained from ethanol [191]
injection method were superior to those
produced by dry thin-film method
regarding encapsulation efficiency and
storage stability
Quercetin Better physicochemical and sensory [57]
properties along with higher stability of
whey drink fortified with whey protein
isolate-coated liposomes in comparison
with uncoated ones
Resveratrol - Maintenance of antioxidant capability [77]
after nanoencapsulation
- Effect of preparation method on
physiochemical properties of
nanoliposomes
Resveratrol Improved stability against UV-B light [24]
Catechin and - Retention of phenolic compounds in [174]
epigallocatechin cheese matrix without any significant
gallate change in its physiochemical properties
- Improved antioxidant activity
- Protection against digestion
Curcumin Enhanced bioavailability and plasma [198]
antioxidant activity
Epigallocatechin - Increase of stability in simulated [220]
gallate intestinal conditions
- Retention of antioxidant capacity during
in vitro digestion
Ellagic acid Sustained release [118]
Phytosome Curcumin Enhancement of bioavailability and ([38];
health-promoting properties [122])
Hesperetin Promoted bioavailability and antioxidant [142]
activity
Curcumin Improvement of oral absorption and [217]
stability by combining chitosan and
phytosome
(continued)
Table 4 (continued)
Phenolic
Rutin - Maintenance of antioxidant activity upon [16]
nanoencapsulation
- Good storage stability
- Desirable sensory attributes of beverages
containing phytosomal formulation
Nanoemulsion Curcumin - Significant effect of emulsifier charge on [161]
phytochemical properties, release pattern,
and performance of nanoemulsions during
digestion
Curcumin - Good stability against ionic strengths, [185]
pH, and pasteurization
- Gradual release in simulated
gastrointestinal condition
Quercetin - Enhanced bioaccessibility [147]
- Maintenance of antioxidant activity upon
nanoencapsulation
- High physical and chemical stability
- Good sensorial properties of beverage
formulation enriched with
nanoencapsulated quercetin
Quercetin Enhanced stability [92]
Quercetin Good physical stability and sustained [21]
release
Resveratrol Enhancement of chemical stability against [42]
UV light
Resveratrol - Sustained release [189]
- Improved stability and bioavailability
Solid lipid Resveratrol Improved oral bioavailability [153]
nanoparticles Curcumin Increase of chemical stability, [197]
bioavailability, dispersibility, cellular
uptake, and functionality
Myricetin Using stabilizers (tween 80 and Poloxamer [58]
407): prolonged half-life time, decrease of
degradation rate of flavonoid and sustained
release in buffer solutions
Epigallocatechin Increase of stability and anticancer activity [170]
gallate
Resveratrol NLCs had smaller particle size and higher [67]
encapsulation efficiency than SLNs
Quercetin NLCs and nanoemulsions had higher [2]
bioaccessibility as compared to SLNs and
free phenolics
Curcumin Reinforced the permeation under in situ [90]
intestinal conditions
(continued)
Table 4 (continued)
Phenolic
Curcumin - Improved oral bioavailability [178]
- Increase of permeation after
nanoencapsulation
Quercetin Increase of oral absorption [107]
Nanostructured Quercetin Sustained release and improved [112]
lipids carriers bioaccessibility
Ferulic acid - Controlled release profile [73]
- Increased the pharmacological activities
Hesperetin - Enhanced stability upon coating with [55]
different biopolymers
- Better release properties
- Improvement of solubility and positive
effect on sensory attributes of fortified
milk regarding color and taste
Quercetin - Promoted solubility [75]
- Sustained release
- Good storage and oxidative stability
Rutin - Good stability during processing and [15]
storage period
- Acceptable sensory attributes of fortified
beverages
Curcumin and Increment of solubility [3]
genistein
Curcumin - Protective role against gastrointestinal ([156];
digestion [213])
- Suitable release behavior in simulated
intestinal fluid
Curcumin Enhancement in intestinal absorption, [227]
solubility, and stability
Luteolin Promotion of oral absorption and [112]
bioavailability
rutin into beverages (orange juice, milk, apple juice). The NLCs had good
physiochemical and morphological properties. Fortified beverages not only were
physically and thermally stable but also presented an acceptable sensorial quality
[15]. These researchers in another study applied thin-film hydration method to
incorporate rutin into phytosomal systems produced by soybean phosphatidylcho-
line [16]. Optimized formulations remained highly stable during storage and gener-
ated fine particles. Phytosomes preserved antioxidant activity of rutin, while inverse
trend was observed for free one. No evident changes were found in sensory attributes
and pH values of fortified beverages with loaded phenolic compounds. Furthermore,
some studies have demonstrated high potential of phytosomes for improving absorp-
tion, bioavailability, and health-promoting properties of curcumin [38, 122, 217].
3.2 Natural Food Colorants
Colorants are extensively applied in the food industry to increase the product
attractiveness and consumer satisfaction. Food colorants can be classified into
three main groups: natural, synthetic, and inorganic. Consumption of synthetic
colorants negatively affects human health and can cause cancer and allergic reac-
tions. Hence, there has been recently growing tendency to natural types [5, 119].
Among natural food colorants, carotenoids and flavonoids are the most common
types for food applications. In addition to exhibiting antioxidant activity, some
flavonoids like curcumin, quercetin, luteolin, and anthocyanins can be used as
coloring agents [79, 124]. Natural colorants are not only a safe alternative to
synthetic ones but also can provide numerous health benefits. They seem to be
effective in prevention and reducing the risk of many diseases such as cardiovascular
diseases, age-associated diseases, cancer, neurodegenerative disorders, and eye
diseases [91, 111, 196]. Nonetheless, most of natural colorants display high sensi-
tivity to oxidation and are degraded when exposed to external factors such as light,
pH, and heat. On the other hand, food components, enzymes, and other nutrients
affect their stability. They also exhibit low bioavailability and limited solubility.
Thus, it is necessary to retain structural integrity of natural food colorants in order to
exploit them in food products and formulate novel functional foods [111, 120, 176].
In this regard, lipid-based nanocarriers are suitable options, because they have an
excellent ability to enhance solubility, absorption, sustained release properties,
effectiveness, and stability of natural colorants [70, 200].
Several reports considering the incorporation of different kinds of natural food
colorants into lipid-based carriers have been recently published, and some of them
have been reviewed in the study published by Akhavan and Jafari [5]. As an
example, Tan et al. [202] investigated efficiency of nanoliposomes achieved by
thin-film evaporation technique for incorporating diverse carotenoids including
β-carotene, canthaxanthin, lycopene, and lutein. They proved that physiochemical
properties, release pattern, and bioaccessibility were directly associated with carot-
enoid type. Indeed, their results indicated a gradual and prolonged release of β-
carotene and lutein under GIT tract conditions. By contrast, release of canthaxanthin
and lycopene occurred rapidly. Also, bioaccessibility followed the order of lutein>
b-carotene> lycopene> canthaxanthin [202]. This group in another study used
chitosan to cover liposomal surfaces through layer self-assembly deposition method
[201]. Coated nanovesicles could retain lutein and β-carotene better than canthaxan-
thin and lycopene. Besides, biopolymer coating modified arranged structure of lipid
bilayers and increased their rigidity. At the same time, chitosan positively affected
release behavior of carotenoids within simulated GIT fluids. Round shape of lipo-
somes was also kept after coating. These authors suggested that coating can be
considered as an efficacious way to generate stable nanoliposomal systems.
In a recent work, Rabelo et al. [169] accommodated Açaí berry extract as a rich
source of anthocyanins into W/O nanoemulsions via high-pressure homogenizer to
boost their stability and functionality. MCT and CR-310 (tetraglycerin monolaurate
condensed ricinoleic acid esters) were main components of nanoemulsions and acted
as the oil phase and emulsifying agent, respectively. Formed nanoemulsions dem-
onstrated a good storage stability and no signs of phase separation observed over
time. Nanoencapsulation caused a decrease in droplet size. Besides, all formulations
were able to preserve phenolic compounds plus antioxidant potency of extracts when
stored for up to 1 month at 4 C [169]. In another research, Li et al. [108] applied
high-pressure homogenization approach to formulate astaxanthin-loaded SLNs with
three kinds of solid lipids including glycerin monostearate, glycerol distearates, and
stearic acid plus Tween 20 as an efficient emulsifier. SLNs were highly stable to
degradation at both 4 and 25 C, and no significant increase was observed in particle
size during storage. Indeed, loaded astaxanthin was released slowly over time into
simulated intestinal and gastric fluids [108].
Mehrad et al. [130] investigated protective impact of SLNs consisted of corn oil,
palmitic acid, and whey protein isolate (as a stabilizer) on β-carotene. They proved a
remarkable enhancement in chemical and oxidative stability of this pigment upon
nanoencapsulation. However, SLNs were susceptible to increment of ionic strength,
elevated temperatures and acidic conditions [130]. Nazemiyeh et al. [222] found that
SLNs enabled a prolonged stability for lycopene and there was no evident decrease
in encapsulation efficiency after 3 months storage at 4 C. In another work, Oliveira
et al. [150] fabricated NLCs using a mixture of tristearin and high oleic sunflower
oil, and their efficiency in incorporation of β-carotene was compared with tristearin
SLNs. All samples showed nanometric sizes and a broad particle size distribution
(PDI). High oleic sunflower oil created no significant changes in particle size.
However, PDI of samples decreased after enclosing β-carotene. NLCs were superior
to SLNs in terms of loading efficiency, although both NLCs and SLNs could equally
protect β-carotene from deterioration. Better performance of nanodelivery systems
was achieved by increasing high oleic sunflower oil levels. Overall, NLCs were
more advantageous than SLNs [150].
3.3 Bioactive Oils and Essential Fatty Acids
Main types of omega-3 fatty acids include eicosapentaenoic acid (EPA),

docosahexaenoic acid (DHA), and α-linolenic acid (ALA). Fish oil is regarded as
the best source of EPA and DHA [165]. In addition to fish oil, omega-3 fatty acids
are found in algae, krill, and land plants [29, 103]. Omega-3 fatty acids possess
several health benefits and could effectively prevent and reduce the risk of different
diseases such as cardiovascular diseases, inflammation, diabetes, autoimmune dis-
orders, and cancer [27, 61, 146]. Since, the human body does not have the ability to
synthesize omega-3 fatty acids; therefore, they are named as essential fatty acids and
must be obtained through foods. As most of diets contain insufficient amounts of
omega-3 fatty acids, a great interest has been focused on increase of their intake
through fortification of different foods. Nonetheless, oxidative, chemical, and ther-
mal instability, poor bioavailability and water solubility, and unfavorable odor and
flavor make direct usage of omega-3 fatty acids in food matrices difficult. Lipid-
based nanocarriers allow to take the advantages of omega-3 fatty acids and also offer
a wide range of potential applications for these bioactives in food sector [65, 96].
Many nanoemulsion researches have been centered on optimizing preparation
methods, composition (regarding oil type, surfactant, and co-surfactant), and differ-
ent parameters influencing phytochemical characteristics in order to promote stabil-
ity and bioavailability of fish oil and omega-3 fatty acids. For instance, Gulotta et al.
[69] produced fish oil-loaded nanoemulsions by self-emulsification technique and
revealed that various parameters including fish oil concentration, oil composition
(lemon oil and medium-chain triglycerides), surfactant-to-oil proportion, and
cosolvent type (polypropylene glycol and ethanol and glycerol) have a great impact
on their characteristics and stability. Moreover, optimized formulations provided
stable nanoemulsion systems, and there were no significant changes in the droplet
size after storage for 1 month at refrigerated and room temperatures. Nevertheless,
particle size increased at higher storage temperatures [69].
Walker et al. [210] indicated that particle size of droplets significantly influences
the oxidative stability of fish oil-loaded nanoemulsions, while physical stability of
system was dependent on the surfactant-to-oil ratio. On the other hand, micro-
fluidized nanoemulsions contained larger amounts of secondary oxidation products
(TBARS) after storage for 2 weeks at 55 C as compared to those formed with
spontaneous emulsification [210]. According to the study of Lane et al. [105], it is
better to apply algal oil and mixture of soy lecithin and Tween 40 instead of flaxseed
oil and soy lecithin alone for generation of nanoemulsions with fine droplet sizes
[105]. Nejadmansouri et al. [146] demonstrated that using whey protein isolate in
nanoemulsion formulations leads to a suitable storage stability regarding particle
size, but viscosity is slightly increased during storage for 28 days [146]. Polymer
coating has a positive effect on the stability of nanoemulsions bearing fish oil.
Esquerdo et al. [223] revealed that using chitosan as a coating agent prevents
creaming and breaks down of fish oil-loaded nanoemulsions.
Different approaches have been employed for incorporating essential fatty acids
and fish oils into nanoliposomes. Hadian et al. [72] produced nanoliposomes
entrapping EPA and DHA by sonication (bath and probe types) and extrusion
methods. Nanoliposomes produced by sonication contained higher amounts of
secondary oxidation products (heptanal propanal, hexanal, and pentanal) than
extruded ones. The highest loading capacity was also related to probe sonicated
nanoliposomes [72].
Moreover, some food systems have been fortified with incorporated form of these
bioactives. In a recent work, Ojagh and Hasani [149] investigated the effect of
adding different concentrations of fish oil incorporated in nanoliposomes on the
sensory and technological attributes of fortified bread during storage in the refriger-
ator for 25 days. They confirmed desirable physiochemical properties of nano-
vesicles regarding particle size, PDI, and encapsulation efficiency. Indeed, addition
of the fish oil-loaded nanoliposomes into breads increased nutritional value and loaf
volume of the breads without notable adverse effects on their sensory and texture
characteristics [149]. Similarly, bread and milk samples enriched with fish oil-loaded
nanoliposomes obtained acceptable sensory scores in contrast to free and
microencapsulated ones [175]. In another study, Ghorbanzade et al. [65] used

nanoliposome systems to encapsulate fish oil and fortified yogurt with the loaded
fish oil. They stated that the fortifying yogurt with the loaded fish oil led to higher
EPA and DHA contents as well as a lower peroxide value, acidity, and syneresis than
samples treated with its free form during 21 days of storage at 4 C. Also, they found
that the sensory characteristics of the yogurt fortified with nanoencapsulated fish oil
was significantly similar to control sample, containing no fish oil [65].
There are few works on fish oil and essential fatty acids loaded within SLNs.
Salminen et al. [181] incorporated fish oil into SLNs composed of tristearin (as the
carrier) and quillaja extract alone or blended with lecithin (low or high melting point)
as a surfactant. They discovered that all formulations showed statistically constant
PDI and particle sizes during storage at a dark place for 51 days except for samples
containing quillaja/low-melting lecithin. Moreover, the SLNs containing quillaja/
high-melting lecithin had a more chemical stability than those containing quillaja
alone or quillaja/low-melting lecithin [181].
NLCs have been also successfully used for loading krill oil. Zhu et al. [218]
incorporated krill oil into NLCs by hot homogenization technique and used palm
stearin and lecithin as oil and emulsifier, respectively. The smallest particle size and
the highest homogeneity were obtained from optimized formulation (65% krill oil
and 1.1% lecithin). Prepared NLCs showed protective effect against UV light-
induced photooxidation. They also had a suitable physical and long-term stability
at low temperatures [218]. Salminen et al. [180] utilized NLCs and nanoemulsion
systems with similar formulations (except tristearin applied in production of NLCs)
for nanoencapsulation of fish oil. Based on their results, NLCs yielded more stable
(in the terms of size and aggregation stability) and smaller particles than nano-
emulsions. Furthermore, the NLCs showed higher protective effects on fish oil
against lipid oxidation compared to the nanoemulsions [180].
3.4 Vitamins
Vitamins are bioactive compounds with outstanding health-promoting properties

which effectively contribute to human growth and development. Vitamin deficien-
cies play a key role in development of some diseases such as cancer and cardiovas-
cular problems [99]. As human body is not able to produce many vitamins, they must
be obtained via diet. However, it is not always possible to meet recommended daily
intake of vitamins. Thus, fortification of food products with vitamins may be needed
to ensure adequate intake of vitamins [20, 157]. Some issues should be considered
for successful fortification. Many vitamins are readily degraded under storage and
processing conditions. They also exhibit a low stability in GIT system. Depending
on type, vitamins show different extents of sensitivity to oxidation, heat treatment,
and pH variations. Poor bioavailability is also regarded as an important limitation in
direct addition of vitamins into food formulations. Moreover, unit operations of food
processing such as blanching cause a considerable decline in the content of water-
soluble vitamins [151, 155]. On the other hand, hydrophobic vitamins are not a
suitable choice for fortification of aqueous-based food products due to their low
water solubility and insufficient dispersibility [135].
Lipid-based nanocarriers can be applied as a proper alternative in order to
protect vitamins against multiple processes and environmental stresses, improve
solubility and absorption, and release them in the determined sites [47, 98]. Many
researchers have encapsulated different kinds of vitamins into lipid-based nano-
carriers as shown in Table 5. For example, Liu et al. [113] evaluated deposition of
chitosan and sodium alginate on the surface of nanoliposomes loaded with vitamin
C as core material. These nanocarriers showed a slower release and higher rate of
oxidation of vitamin C when stored for 90 days at 4 C than uncoated ones. They
concluded that the shell of chitosan and sodium alginate could effectively cover the
surface of anionic nanovesicles and enhance their stability toward hydrolysis and
oxidation. They also fortified mandarin juice with coated nanoliposomes and
observed that microbial stability of the fortified mandarin juice was higher than
samples treated with uncoated carriers with a negligible effect on sensory proper-
ties of juice [113].
Bochicchio et al. [20] evaluated incorporation of different vitamins (B12, D2,
and E) into nanoliposomes through an ultrasound-assisted method and the thin-
film hydration technique. They reported that SUVs and MLVs had a high encap-
sulation efficiency. Increasing the hydrophobicity of vitamins led to higher encap-
sulation efficiencies. Moreover, the entrapment of vitamins into lipid nanovesicles
protected them against chemical degradation during 10 days of storage at simu-
lated extracellular environment conditions [20]. In another study, Couto et al. [35]
loaded vitamin B2 into the SLN systems through a modified PGSS process using
hydrogenated canola oil as lipid phase, polyethylene glycol as stabilizer, and
sodium lauryl sulfate as surfactant. They investigated effects of different parame-
ters such as vitamin and stabilizer concentration, pressure, and molecular weight
on loading and encapsulation efficiencies and reported that the optimum SLN
preparation conditions with respect to loading and encapsulation efficiency were
as follow: vitamin concentration of 2%, pressure of 15 MPa, and 5% polyethylene
glycol [35].
Walia et al. [209] prepared a fish oil-in-water nanoemulsion via ultrasonication
technique to load vitamin D with a high encapsulation efficiency. Their results
revealed that nanoencapsulation could preserve antioxidant capacity and antimicro-
bial activity of vitamin D during storage. Also, the fish oil-based nanoemulsions
improved the vitamin D bioavailability in stimulated GIT conditions [209]. Pinto et
al. [163] developed different NLCs by using vegetable oils enriched with α-tocoph-
erol and demonstrated that olive, sunflower, coconut, and sweet almond oils could be
successfully applied to prepare free and loaded NLCs with negative surface charges
and a nanometric size. Also, they found that the formulated NLCs had a high
encapsulation efficiency and released α-tocopherol in a controlled manner. However,
the DSC analysis has showed that incorporation of α-tocopherol resulted in reduced
crystallinity of NLCs but could improve their antioxidant activity. Accordingly,
these authors suggested that the formulated NLCs could be successfully used for
preparing long-term stable products [163].
Table 5 Incorporation of different vitamins into lipid-based nanocarriers

Nanocarrier Vitamin type Results Reference
Nanoliposomes Vitamins E and C - Maintenance of antioxidant activity [123]
of vitamins in orange juice before and
after pasteurization
- Liposomes provided good microbial
stability for orange juice during
storage without any significant
change in sensory properties
Vitamin A - Suitable characteristics [160]
- High concentration of cholesterol
led to lower encapsulation efficiency
Vitamin C - Higher chemical and microbial [113]
stability of mandarin juice and lower
release rate after coating of
nanoliposomes with chitosan and
sodium alginate
- Good protection against oxidation
without any significant effect of
sensory properties of mandarin juice
Vitamin C - Better storage stability and more [109]
encapsulation efficiency in
nanoliposomes obtained from the
double emulsion-dynamic high-
pressure microfluidization method
Vitamin B1 High thermal stability during storage [56]
Vitamins E, B12, Multilamellar large vesicles resulted [20]
and D2 in the maximum encapsulation
efficiency
Vitamin C Enhanced stability during long-term [215]
storage
Vitamin A Decreased degradation of vitamin [102]
Nanoemulsions Vitamin E High bioaccessibility in O/W [152]
emulsions produced by long-chain
triglycerides
Vitamin B2 Improved stability [22]
Vitamin E - Effective protection during storage [74]
- Good physical stability
Vitamin E - High thermal stability [179]
- The smallest droplets and highest
transparency were observed for
formulations containing 30%
propylene glycol or 20% ethanol
Vitamin E Increase in release rate of vitamin into [140]
the buffer solution caused by micellar
solubilization
Vitamin D - Increase in thermal stability by [71]
using sodium dodecyl sulfate as co-
surfactant
- Effect of surfactant type, surfactant-
(continued)
Table 5 (continued)
to-oil proportion, and stirring
conditions on characteristics of
droplets
Vitamin K1 - Good permeation ability [26]
- Good storage stability over time
under (at) different temperatures
Folic acid (Vitamin The optimum conditions for (Assadpour
B9) preparing maltodextrin-whey protein et al. 2016)
double emulsions: 3 mg/ml folic acid
in the 12% dispersed phase and water
to span 80 ratio of 0.9
Vitamin E - Enhanced antimicrobial effect, shelf [41]
life, and bioavailability of vitamin in
fruit juice
- High encapsulation efficiency by
using mustard oil and tween 80
Vitamin E The optimum condition for [129]
incorporating vitamin: 1% vitamin E
acetate concentration, 6.18% oil
contents, 135 MPa homogenization
pressure, and 6.39% surfactant
concentration
Vitamin D Enhanced bioavailability acquired by [209]
fish oil-based nanoemulsion
A type of vitamin B: Inhibitory effect on the spore [32]
Thiamine dilauryl germination of Fusarium oxypansum
sulfate (TDS) f. sp. as well as mycelial growth
Vitamin D3 Good stability against UV irradiation [106]
Vitamin E Production of nanoemulsions bearing [216]
vitamin by natural surfactant (Q-
®
Naturale ) for using in food
formulations
Solid lipid Vitamin B2 Encapsulation efficiency and loading [35]
nanoparticles capacity influenced by different
(SLNs) parameters such as molecular weight
and concentration of stabilizer,
vitamin concentration, and pressure
Vitamin B12 Improved anticarcinogenic activity [62]
Vitamin D2 Protective effect against oxygen and [158]
light
Vitamin A - Controlled release during 6 h [89]
- Higher release rate compared to
nanoemulsions in longer storage
times
(continued)
Table 5 (continued)
Nanostructured Vitamin A Good storage stability [159]
lipids carriers Vitamin E The highest stability achieved by [208]
(NLCs) adding 5% (w/w) of tween 20 to
NLCs formulation
Vitamin D3 - Good physiochemical properties [155]
and suitable storage stability
- Controlled release
Vitamin D3 Improvement of intestinal absorption [135]
Production of stable NLCs by using
Precirol as solid lipid and Poloxamer
407 as surfactants
Vitamin E - NLC formulations-based vegetable [163]
oils (sunflower, sweet almond, olive,
and coconut oils) led to good
phytochemical properties and high
physical stability
- Improved scavenging activity
3.5 Natural Antimicrobial Agents and Essential Oils
Recently, the tendency for utilizing natural antimicrobial agents has been increased
due to hazardous and harmful impacts of synthetic additives on human health and
emergence of antibiotic-resistant bacteria [46, 126]. Among natural antimicrobial
agents, essential oils and bacteriocins especially nisin have attracted much consid-
eration to inhibit microbial growth in food systems. In addition to antimicrobial
effect, essential oils possess numerous beneficial and health-promoting properties
such as antioxidant, anti-allergic, anticancer, anti-inflammatory, and antiviral activ-
ities. Besides, essential oils and their components act as natural flavoring agents in
food, pharmaceutical, and cosmetics industries [45]. In spite of remarkable advan-
tages, there are serious challenges in development of food products containing
essential oils. They are extremely reactive, volatile, and chemically unstable; readily
oxidized when exposed to heat, oxygen, or light; and decomposed during processing
stages and storage as well as within GIT conditions that reduces their antimicrobial
potency. Furthermore, essential oils display poor solubility and dispersibility
in aqueous food systems and influence sensory attributes of food products [10, 43,
204].
Nisin is a polypeptide composed of 34 amino acids obtained from certain strains
of Lactococcus lactis and found in two main forms, namely, nisin A and nisin Z.
Nisin Z shows a higher stability and dispersibility in food formulations than A type
[34]. Nisin as the only GRAS (generally recognized as safe) bacteriocin can inhibit
growth of several species of pathogenic and nonpathogenic Gram-positive bacteria
such as Listeria monocytogenes, Bacillus spp., and Staphylococcus aureus, protect
food products from microbial spoilage, prolong their shelf life, and improve food
safety [88, 166]. Nevertheless, there are some problems that restrict direct usage of
nisin and cause remarkable losses in its antimicrobial activity such as low stability
against adverse environmental factors as well as proteolytic inactivation, unwanted
interaction with food ingredients, and inadequate dispersion in food systems [34,
166]. Moreover, utilizing free form of nisin may create some issues within foods. For
example, nisin may negatively influence starter culture activity in cheese matrix and
consequently decrease consumer acceptance [23].
Lipid-based nanodelivery systems can solve these problems because they provide
targeted and prolonged release, inhibit undesirable interactions and degradation, and
minimize appearance of resistant strains of bacteria. These carriers also facilitate
passage of antimicrobials through cell membrane, enhance bioactivity of essential
oils, and thus reduce concentrations required for exerting antimicrobial activity
which leads to preserving sensorial properties and quality of fortified foods [34,
100, 116].
Several studies have demonstrated the beneficial effects of lipid-based nano-
carriers on antimicrobial activity, stability, and flavor retention of essential oils and
their constituents for food applications. For instance, Balta et al. [18] evaluated
antimicrobial potential of nanoemulsions containing geraniol and linalool against
some foodborne bacteria (E. coli, Listeria innocua, and Pseudomonas lundensis) by
a simulated medium of meat. Both loaded compounds could control the microbial
growth. However, they showed a lower efficiency for declining Pseudomonas
lundensis counts compared to others [18]. In another research by Lu et al. [116],
citral essential oil-loaded nanoemulsions exhibited an inhibitory effect on bacterial
strains (S. aureus, E. coli, Pseudomonas aeruginosa, Enterococcus faecalis, Salmo-
nella typhimurium, and L. monocytogenes). Nevertheless, they had a similar inhibi-
tion zone diameter and antimicrobial efficiency against both Gram-positive and
Gram-negative species [116].
In the study conducted by Artiga-Artigas et al. [10], four essential oils obtained
from lemongrass, mandarin, thyme, and oregano oil were incorporated into nano-
emulsion systems via microfluidization with the aid of Tween 80 and pectin as
emulsifying agents [10]. Chuesiang et al. [33] produced nanoemulsions loaded with
cinnamon essential oils by phase inversion temperature technique using MCT and
Tween 80. They revealed that droplet size and stability of nanoemulsions were
affected by concentration of essential oils, oil phase, and surfactant [33]. In another
work, Mendes et al. [133] fabricated nanoemulsion systems for loading Eugenia
brejoensis Mazine essential oil as an antimicrobial agent by homogenization. Based
on their results, nanoemulsions had good characteristics, and physical stability
resulted from appropriate emulsifiers and increasing rotational speed. Besides,
bactericidal activity was dependent on droplet size, and nanoemulsions with smaller
particle sizes were more efficient against Pseudomonas fluorescens. When loaded
essential oils were also applied in sliced ham, their antimicrobial capability was
reduced [133].
The dual incorporation of nisin and garlic extract into nanoliposomes was carried
out by Pinilla and Brandelli [162], and antimicrobial effectiveness of loaded nano-
liposomes was investigated against some bacterial species (Salmonella enteritidis, L.
monocytogenes, and S. aureus and E. coli) in whole milk when stored at 37 C.
Nanoliposomes were equally effective on bacteria, while free form of nisin and
garlic extract could not individually inhibit microbial growth. Natural antimicrobial
agents also presented a higher ability to reduce L. monocytogenes counts [162]. Cui
et al. [36] employed soy lecithin-derived nanoliposomes for boosting the stability
and antimicrobial activity of clove oil against E. coli and S. aureus. Loaded essential
oils could prevent growth of bacteria. However, as S. aureus increased the release
rate of clove oil by secreting pore-forming toxins, nanoliposomes showed a higher
antimicrobial effect. Moreover, adding loaded nanoliposomes into tofu formulation
led to an efficacious inhibitory activity toward S. aureus [36].
Sebaaly et al. [187] found that nanoliposomes prepared by ethanol injection
method promoted stability of eugenol against UV radiation. Additionally, antioxi-
dant activity of eugenol was preserved after nanoencapsulation [187]. Tian et al.
[204] fabricated SLNs bearing citral through high-pressure homogenization
approach by a blend of Span 80 and Tween 80 (1:1 ratio) and glycerol monostearate
as surfactant and solid lipid, respectively. According to their results, larger amounts
of incorporated citral were retained during storage at 37 C for 12 days in compar-
ison with its free counterpart [204]. Zhao et al. [224] revealed that SLNs were able to
promote bioavailability and sustained release properties of Yuxingcao essential oil.
Prombutara et al. [166] nanoencapsulated nisin within SLNs via high-pressure
homogenization technique by Imwitor 900 as solid lipid with aid of surfactant and
co-surfactant that were Poloxamer 188 and sodium deoxycholate, respectively. Nisin
concentration significantly influenced physiochemical properties of SLNs. Release
rate of nisin was dependent on medium conditions in terms of pH and salt concen-
tration. Nanoencapsulation of nisin led to a prolonged antimicrobial activity toward
Lactobacillus plantarum and L. monocytogenes [166]. Keivani Nahr et al. [100]
entrapped cardamom essential oil into NLCs produced by food-grade ingredients
including cocoa butter and Tween 80 and assessed impact of nanoencapsulation on
its antimicrobial capacity. Obtained NLCs displayed desirable physiochemical char-
acteristics and a good stability over 30 days of storage. Loaded essential oil was also
superior to free form regarding antimicrobial potentials [100]. Lewies et al. [225]
proved the efficient bactericidal activity of nisin-loaded NLCs against S. aureus and
S. epidermidis.
3.6 Phytosterols
Phytosterols are one of the most popular bioactive compounds with a high hydro-
phobicity and can be divided into two main groups including stanols and plant
sterols (i.e., β-sitosterol, campesterol, and stigmasterol). Phytosterols possess numer-
ous health benefits such as anticancer, inflammatory, antioxidant, and antidiabetic
activities. They can also prevent cardiovascular diseases and arteriosclerosis by
decreasing low-density lipoprotein (LDL) cholesterol in serum [17, 39, 195].
Phytosterols especially β-sitosterol have recently attracted great attention in order
to develop novel functional foods [154], although low bioavailability, high melting
point, and poor aqueous solubility make it difficult to incorporate β-sitosterol
into food systems without a suitable carrier. Besides, they show a high oxida-
tion and crystallization tendency, reducing their functionality. Phytosterols are
often applied in esterified forms which can enhance their solubility in fat-rich
foods such as spreads without any adverse influence on other food matrices [79,
195]. Nanoencapsulation presents an efficient solution for these obstacles.
There are few studies on incorporation of phytosterols into lipid-based nano-
carriers and their applications in the food industry. Bagherpour et al. [17]
fortified butter with NLCs bearing β-sitosterol prepared by hot homogenization
approach, and physicochemical characteristics plus oxidative stability of sam-
ples were assessed. Obtained NLCs had a nanometric size, negative surface
charge, desirable homogeneity, and high encapsulation efficiency. Also, they
were physically stable during long-term storage. No evident change was
observed at peroxide and acid values of butter samples containing NLCs and
they showed a good oxidative stability. Antioxidant activity of loaded β-sitos-
terol was maintained in butter matrix over 3 months of storage at refrigerated
temperature, while its free form experienced a significant loss [17].
Alexander et al. [8] added plant sterols instead of cholesterol into liposomal
formulations. Characteristics of nanoliposomes are influenced by phospholipid and
sterol concentrations. Their results revealed that plant sterols significantly increased
the particle size and encapsulation efficiency, although nanoliposomes exhibited a
low physical stability in presence of plant sterols and undergo phase separation
during storage. These authors suggested that optimization of plant sterol to phos-
pholipid ratios and selection of suitable preparation methods can improve storage
stability of nanoliposomes containing plant sterols [8]. Panpipat et al. [154]
employed different sterol to phospholipid ratios plus various types of β-sitosteryl
fatty acid esters with the aim of producing nanodispersions. On the basis of their
results, β-sitosterol was less effective in producing homogeneity of nanodispersions
compared to its esterified forms. Moreover, nanoparticles obtained from esters of
β-sitosterol were smaller and more stable. On the other hand, β-sitosteryl unsaturated
fatty acid esters preferred to make nanoemulsions with desirable characteristics
instead of nanodispersions which can be satisfactory for pharmaceutical and food
sectors [154].
Ribeiro et al. [177] formulated and characterized novel lipid-based colloidal
nanodispersions for enclosing phytosterols using various types of triacylglycerols
and Tween 20. They found that supercooled emulsions and amorphous particles
were created as a result of applying Myritol and trilaurin as liquid and solid
triacylglycerols, respectively. These systems could entirely prevent the crystalliza-
tion of phytosterols as well as lipids. Furthermore, they presented good stability
during long-term storage [177]. In a recent study by Soleimanian et al. [195],
β-sitosterol-loaded NLCs were fabricated by three different types of lipids including
glyceryl behenate, pomegranate seed oil, and propolis wax. They demonstrated a
notable impact of type and content of lipids on physiochemical properties of NLCs.
Increase in phytosterol content and decrease of lipid content led to larger size and
lower values for encapsulation efficiency. Crystallinity of β-sitosterol also dimin-
ished upon nanoencapsulation [195].
In the study of Lacatusu et al. [104], a mixture of natural oils including fish oil,
grape seed oil, and squalene was used to make NLCs comprising β-sitosterol alone
and simultaneously β-sitosterol and green tea extract. Obtained NLCs had a great
encapsulation efficiency and surface charge less than 30 mV, indicating stable
systems. Dual nanoencapsulation of β-sitosterol and green tea extract not only
caused an evident decrease in particle size but also promoted antioxidant efficiency.
Loaded β-sitosterol was able to entrap free radicals better than unencapsulated
sample. Additionally, NLCs enabled gradual and controlled release of β-sitosterol
[104].
3.7 Enzymes
Among the lipid-based nanocarriers, nanoliposomes are the most commonly used
system for delivering enzymes in food matrices particularly cheese to shorten the
ripening time and reduce production costs. High amount of enzymes are lost by
whey when directly utilized in milk to accelerate ripening and consequently raise
manufacturing cost of cheese. Moreover, adding free form of enzymes leads to low
cheese yield, uneven distribution in curd, and undesirable sensory attributes of final
product [47, 85].
Nanoliposomes enable gradual release, more stability, appropriate partitioning
into food matrix, and enhanced performance for enzymes and also positively
influence taste, flavor, and texture of cheese [121, 136]. Flavourzyme is a mixture
of fungal proteases which has desirable effects on flavor and ripening of cheese.
Jahadi et al. [86] prepared nanoliposomal formulations for accommodating
Flavourzyme by heating method. Then, nanoencapsualted form of Flavourzyme
was added into cow milk and produced Iranian white brined cheese. Nanoliposomes
did not significantly affect the curd and whey composition as well as cheese yield as
compared to control [86]. These researchers in another study found that ripening
period had the greatest effect on protein decomposition of cheese samples followed
by concentration of loaded Flavourzyme and brine treatment time (brining time).
Moreover, 0.3% w/w of loaded enzyme, 1 month of ripening, and brining time for
8 h processed samples with the most proteolytic activities and the highest level of
acceptance in sensory attributes. Also, the optimum concentration of enzyme-loaded
nanoliposome led to defeat drawbacks related to texture and taste in cheese samples
treated with free form [87].
On the other hand, stability and functionality of enzymes dramatically are
affected by temperature and pH values within food products, and they are easily
denatured and inactivated under harsh environmental conditions. Karami et al. [93]
fabricated SLNs entrapping superoxide dismutase as an antioxidant enzyme by cold
homogenization technique. Loaded SLNs had desirable properties concerning par-
ticle size and encapsulation efficiency. Moreover, nanoencapsulation provided
remarkable improvement in stability, enzymatic potency, release behavior, and
permeability of superoxide dismutase [93]. In the study conducted by Qi et al.
[226], catalase as a hydrogen peroxide-scavenging enzyme was nanoencapsulated
into SLNs. Main components of SLNs were Poloxamer 188, tripalmitin, and soy-
bean phosphatidylcholine which acted as surfactant, oil phase, and stabilizers,
respectively. SLNs were highly stable owing to bear a surface charge above
30 mV. Indeed, nanodelivery systems demonstrated protective effects against
proteolysis and released catalase sustainably [168].
4 Conclusion and Future Trends
Lipid-formulation nanoencapsulation technologies could be considered as an effi-

cient alternative to overcome the limitations related to direct usage of bioactive
compounds especially hydrophobic ones within food systems and also applied to
design novel functional foods. They can be prepared without use of organic solvents;
provide prolonged release, target specificity, and biocompatibility; protect incorpo-
rated compounds against adverse external conditions, chemical reactions, and diges-
tion; improve their stability, solubility, dispersibility, efficiency, and bioavailability
which consequently facilitate intestinal absorption; and enhance biological activity
of bioactives. However, there are still some challenges in development of nano-
formulations regarding toxicological safety, effects on human body, and commer-
cialization. Moreover, it is essential to fabricate lipid-based nanocarriers by using
food-grade materials and healthy lipids for food and pharmaceutical practices.
Besides, more investigations need to be carried out on stability, release behavior
and added level of nanoparticles to preserve sensory attributes especially appearance
in food products, promote functionality of bioactive compounds, and ensure safety
for in vivo applications. Therefore, these issues must be taken into account for
applying nanoencapsulated bioactive compounds in food, pharmaceutical, and cos-
metics industries.
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CLAs in Animal Source Foods: Healthy
Benefits for Consumers 23
Paolo Polidori, Silvia Vincenzetti, Stefania Pucciarelli, and
Valeria Polzonetti
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 668
2 Lipids in Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670
3 Effects of Animal Nutrition on Meat Lipidic Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672
4 Conjugated Linoleic Acid (CLA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673
4.1 Biosynthesis of CLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
4.2 CLA Synthesis in the Rumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
4.3 CLA Endogenous Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677
5 CLA in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
5.1 CLA in Meats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
5.2 CLA in Chicken, Turkey, and Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
5.3 CLA in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
5.4 CLA in Milk and Dairy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
6 Human Dietary Intake of CLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
7 Cancer and CLA Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
7.1 Colorectal Cancer (CRC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
7.2 Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
7.3 Skin Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686
7.4 Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
8 CLA and Cardiovascular Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
9 CLA and Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
10 CLA and Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
11 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693
P. Polidori (*)
School of Pharmacy, University of Camerino, Camerino (MC), Italy
S. Vincenzetti · S. Pucciarelli · V. Polzonetti
School of Biosciences and Veterinary Medicine, University of Camerino, Camerino (MC), Italy

668 P. Polidori et al.
Abstract
Conjugated linoleic acid (CLA) is a group of polyunsaturated fatty acids that exist
as positional and stereo-isomers of octadecadienoate (18:2). Among these iso-
mers, the most studied two isomers are cis 9, trans 11-CLA and trans 10, cis
12-CLA due to their biological effects. CLA can be naturally synthesized in
the rumen of ruminant animals by bacteria Butyrivibrio fibrisolvens via the
Δ-9-desaturase of trans 11 octadecanoic acid pathway. The major dietary sources
of CLA are represented by meat and milk from ruminant animals. Although
references to CLA can be traced back to the 1950s, current interest in the health
benefits of CLA started in the late 1980s, after it was identified as the anti-
carcinogenic component present in fried ground beef. Since then, an extensive
literature has documented the anticarcinogenic effects of CLA. In addition, there
is some evidence that CLA is also anti-atherosclerotic, has beneficial effects on
type 2 diabetes, and may play a key role in helping to regulate body fat. The fact
that the richest natural sources of CLA, meat and dairy products, are consumed by
people worldwide has very interesting implications for public health.
Keywords
Conjugated linoleic acid · Fatty acids · Animal source foods · CLA and cancer ·
CLA and human diet · CLA and human health
CLA Conjugated Linoleic Acid
DHA Docosahexaenoic acid
EPA Eicosapentaenoic acid
FAME Fatty Acid Methyl Esters
MUFA Monounsaturated Fatty Acids
PUFA Polyunsaturated Fatty Acids
SFA Saturated Fatty Acids
TFA Trans Fatty Acids
1 Introduction
Anthropology has previously recognized the importance of food and diet variations
among time periods. It is possible to identify the profile of meat consumption during
human evolution in four periods: the first could be characterized by an opportunist
hunting; while in the second, hunting had grown to a bigger scale and lasted 2 to 3
million years; in the third period, men started to domesticate animals and plants, which
had began 10,000 years ago; during the fourth and last period studies determined that
meat contained compounds which could increase disease risk [1]. Anthropological
data have also suggested an important influence of meat consumption in human erect
posture. Bipedalism is probably the first and most important characteristic which
distinguished humans from their ancestors as it allowed a more efficient locomotion
and load carrying, which are important advantages in hunting [2]. Cranial–dental
23 CLAs in Animal Source Foods: Healthy Benefits for Consumers 669
changes are quite visible when analyzing hominids fossils. Molar teeth size has
decreased and the jaws and front teeth have become stronger. Also shearing crests
have grown. These changes could be explained through the urgent need of tearing and
chewing meat rather than grinding leaves, fruits, seeds, and cereals [3].
Gastrointestinal tract features can also aid in determining dietary preferences
considering that the gut of herbivores and pure carnivores suffered different phys-
iological and metabolic adaptations. On one hand, plant-based diets are associated
with a sacculated stomach and well-developed caecum and colon, which increased
with plant fiber content. On the other hand, a carnivore’s stomach is well developed
and acidic with a large small intestine. Humans are omnivorous thus fitting in neither
category. They have a simple stomach and a relatively long small intestine but also a
reduced caecum and colon [4]. The fact that the small intestine is the most prominent
organ in the human gastrointestinal tract is due to the need for adaptation to a varied
diet, including nutritionally dense foods, with great volume and conducive to being
digested in the small intestine.
Meat is an important source of high-value animal protein in many regions of the
world. Around the globe, the diets of relatively more urbanized populations are
characterized by a higher content of meat, poultry, and other animal products than
the less diversified diets of rural communities [5]. Contrary to popular perception,
average red meat intakes appear to be moderate and in line with current recommen-
dations in developed countries [6]. Red meat intakes in these countries range from
the lowest consumption in Greece at 31 g per day for women and 55 g per day for
men to the highest consumption in The Netherlands at 79 g per day for women and
136 g per day for men (see Table 1).
Essentially, meat is basically composed of water, protein, lipids, minerals, and
carbohydrates. Lean muscle tissue contains approximately 72–74% moisture, 20–22%
protein, 3–5% fat, 1% ash, and 0.5% of carbohydrate. These proportions are largely
variable, especially in the lipid content, which depends on species, amount of fatten-
ing, inclusion of the adipose tissue, and so on. There is an inverse relationship between
the percentages of protein and moisture and the percentage of fat so that meats with
high content of fat have lower content of moisture and proteins [7].
Skeletal muscle contains a variable amount of lipids, between 1% and 13%. Lipid
content mainly depends on the degree of fattening and the amount of adipose tissue.
Lipids can be found within the muscle (intramuscular), between muscles
Table 1 Mean daily intake (g/d) of total red meat (fresh and processed) in selected countries
Countries Women Men
Spain 67 127
Australia 55 110
Canada 55 101
Italy 60 91
United Kingdom 47 78
Greece 31 55
Source: Adapted from [6]
(intermuscular), and in adipose tissue. Intramuscular lipids are mainly composed of

triacylglycerols, which are stored in fat cells, and phospholipids, which are located in
cell membranes. The amount of cholesterol in lean meat is around 50–70 mg/100 g.
Intermuscular and adipose tissue lipids are mainly composed of triacylglycerols and
small amounts of cholesterol, around 40–60 mg/100 g [8].
Triacylglycerols are the major constituents of fat. The fatty acid content mainly
depends on age, production system, type of feed, and environment [7]. Monogastric
animals such as swine and poultry tend to reflect the fatty acid composition of the feed
in their fat. In the case of ruminants, the nutrients and fatty acid composition are
somehow standardized due to biohydrogenation by the microbial population of the
rumen [9]. The properties of the fat will depend on its fatty acid composition. A great
percentage of the triacylglycerols are esterified to saturated and monounsaturated fatty
acids. When triacylglycerols are rich in polyunsaturated fatty acids (PUFA) such as
linoleic and linolenic acids, fats tend to be softer and prone to oxidation. These fats
may even have an oily appearance when kept at room temperature. Phospholipids are
present in cell membranes, and although present in minor amounts, they have a strong
relevance to flavor development due to their relatively high proportion of PUFA.
Major constituents are phosphatidylcholine (lecithin) and phosphatidylethanolamine.
The phospholipid content may vary depending on the genetic type of the animal and
the anatomical location of the muscle [10]. For instance, red oxidative muscles have a
higher amount of phospholipids than white glycolytic muscles.
2 Lipids in Meat
Lipid contributes substantially to the caloric content of meat. It also has marked
effects on mouth-feel and flavor of meat. There are three sources of lipid in meat: the
muscle fibers, subcutaneous adipose tissue, and intramuscular (interfascicular,
marbling) adipose tissue [11]. Once subcutaneous adipose tissue has been removed,
the primary contributor to lipid content of meat is intramuscular adipose tissue.
Pioneering researchers from the seventeenth and eighteenth century such as
Robert Boyle, Poulletier de la Salle, Antoine François de Fourcroy, and others
began the study of modern lipid chemistry. During the nineteenth century the
chemist, Chevreul, identified several fatty acids, suggested the name “cholesterine”
for the fatty substance in gallstones, coined the word “glycerine” and showed that
fats are comprised of glycerol and fatty acids [12]. During the twentieth century
many advances have been made in terms of the understanding of lipid structure and
function. Lipids include waxes, oils, fats, steroids, and related compounds ranging
from soaps to petrochemicals [13]. Triacylglycerol, which is a neutral lipid, are
made up of three fatty acids attached to a molecule of glycerol, and they vary in
their physical properties according to the chemical structure of the fatty acid.
A phospholipid is a lipid containing phosphoric acid as mono- or diester and is the
main building block for cell membranes. Fatty acids are “amphiphilic,” i.e., has a
carboxyl group (hydrophilic) at the polar end and a hydrocarbon chain at the
nonpolar tail (hydrophobic). Fatty acids consisting of only single bonds are termed
“saturated.” If there are carbon–carbon double bonds in a fatty acid chain, the fatty
acid is termed “unsaturated.” Oleic acid (C18:1) contains one double bond and is
termed” monounsaturated.” A fatty acid with more than one double bond is termed
“polyunsaturated.” The polyunsaturated fatty acids (PUFA) predominate in vegeta-
ble oils. The two determinants of the consistency of a lipid is the length of the
predominant fatty acid chains and the presence or the absence of double bonds [13].
Fatty acids with twelve or more carbon atoms are referred to as long-chain fatty acids
and are typical of fats from animal origin.
Meat fat comprises mostly monounsaturated fatty acids (MUFA) and saturated
fatty acids (SFA). The most ubiquitous fatty acids are oleic (C18:1), palmitic
(C16:0), and stearic (C18:0) acids. Poultry and pork contain somewhat more unsat-
urated fatty acids (10–15% of total fatty acids) than beef and lamb, and also a notable
amount of PUFAs. Linoleic acid (C18:2) is the predominant PUFA (0.5–7%),
followed by alpha-linolenic acid (up to 0.5%) [14]. Trans-fatty acids (TFA) comprise
about 1–2% of total fatty acids across all types of meat; in ruminant meats they
represent 2–4%. Pigs have much higher proportions of the major polyunsaturated
fatty acid (PUFA) linoleic acid (18:2 n-6) than cattle and sheep [15]. Linoleic acid is
derived entirely from the diet. It passes through the pig’s stomach unchanged and is
then absorbed into the blood stream in the small intestine and incorporated from
there into tissues. In ruminants, the fatty acid, which is at high levels in concentrate
feedstuffs (grains and oilseeds), is degraded into monounsaturated and saturated
fatty acids in the rumen by microbial biohydrogenation and only a small proportion,
around 10% of dietary 18:2 n-6, is available for incorporation into tissue lipids.
The second most important PUFA is α-linolenic acid (18:3 n-3), which is present
in many concentrate feed ingredients but at lower levels than 18:2 n-6. This is a
major dietary fatty acid for ruminants since it constitutes over 50% of total fatty acids
in grass and grass products. Again, a high proportion is biohydrogenated to saturated
fatty acids in the rumen. A variable proportion of dietary 18:3 n-3 is biohydrogenated
(85–100%) but this is more than for 18:2 n-6 (70–95%), so less is available for
incorporation into tissues [16]. As with 18:2n-6, proportions in ruminants are higher
in muscle than adipose tissue. Muscle contains significant proportions of long chain
(C20–22) PUFA which are formed from 18:2 n-6 and 18:3 n-3 by the action of Δ5
and Δ6 desaturase and elongase enzymes. Important products are arachidonic acid
(20:4 n-6) and eicosapentaenoic acid (EPA, 20:5 n-3) which have various metabolic
roles including eicosanoid production.
The n-3 and n-6 fatty acids are required in the diets of humans as well as other
monogastric mammals as they cannot be synthesized de novo. These fatty acids
function as carriers of the fat-soluble vitamins (Vitamin A, D, E and K) and play a
crucial role in the immune response of both man and animal. Essential fatty acids
with 18-carbon molecules include linolenic acid (9-cis-, 12-cis-,15-cis-octa-
decatrienoic acid; C18:3 n-3) and linoleic acid (9-cis, 12-cis-octadecadienoic acid;
C18:2n-6). The two most important 20-carbon essential fatty acids are arachidonic
acid (C20:4 n-6), which is formed by desaturation and elongation of linoleic acid,
and EPA (C20:5 n-3), which is formed by desaturation and elongation of α-linolenic
acid [17]. Meat, fish, and fish oil are the only significant dietary sources of C20:4 n-6
(arachidonic acid) and C22:6 n-3 docosahexaenoic acid (DHA). Meat contains lower
concentrations of these polyunsaturated fatty acids when compared with oily fish
[18]. The two most readily found n-3 fish oil fatty acids are EPA and DHA.
Cholesterol in meat exists in two forms: as free cholesterol and as cholesterol
ester [19]. Free cholesterol is associated primarily with cellular and subcellular
membranes of muscle and intramuscular adipocytes. Cholesterol ester, located
within the triacylglycerol-rich central lipid vacuole, comprises about 75% of the
total cholesterol in adipose tissue. Muscle fibres, which are rich in membranes but
contain comparatively little lipid, have approximately 75% of their total cholesterol
associated with membranes and the other 25% associated with their neutral lipids.
There are two reasons for the minimal effect of intramuscular adipose tissue on the
concentration of cholesterol in meat:
1. Each gram of intramuscular adipose tissue contributes only 1.2 mg of cholesterol.

2. Any increase in intramuscular adipose tissue replaces an equal volume of muscle,
which contributes as much as 0.65 mg of cholesterol per gram.
Thus, any contribution of intramuscular adipose tissue to total cholesterol is

diluted by the amount of muscle it displaces. For this reason, different cuts of
meat may vary substantially in the number of calories from fat but will differ only
slightly in their cholesterol content [19].
3 Effects of Animal Nutrition on Meat Lipidic Profile
A great research effort has been exerted since the 1980s for the manipulation of the
fatty acid composition of meat, to achieve nutritional recommendations, especially
an increase in the ratio between PUFA and saturated fatty acids (SFA). More
recently, nutritionists recommend that PUFA composition should be manipulated
toward a lower n-6:n-3 ratio. Fats with a higher content of PUFA have lower melting
points that affect the fat firmness. Softer fats may raise important problems during
processing if the integrity of the muscle is disrupted by any mechanical treatment
(chopping, mincing, stuffing, etc.). The major troubles are related to oxidation and
generation of off-flavors (rancid aromas) and color deterioration, specifically a trend
toward yellowness in the fat [8].
Pigs and poultry are monogastric animals that incorporate part of the dietary fatty
acids practically unchanged into the adipose tissue and cellular membranes, where
desaturation and chain elongation processes may occur [7]. The extent of incorpo-
ration may vary depending on the specific fatty acid and the type of feed. Different
types of cereals as well as dietary oils and their effects on the proportions in fatty acid
composition have been studied. The use of canola or linseed oils produces a
substantial increase in the content of linolenic acid (C 18:3), which is an n-3 fatty
acid. In this way, the n-6:n-3 ratio can be reduced from 9 to 5 [20]. Other dietary oils
such as soy, peanut, corn, and sunflower increase the content of linoleic acid (C18:2),
an n-6 fatty acid. Although it increases the total PUFA content, this fatty acid does
not contribute to decrease the n-6:n-3 ratio, just the reverse. A similar trend is
observed in the case of poultry, where the feeds with a high content of linoleic
acid such as grain, corn, plant seeds, or oils also increase the n-6/n-3 ratio. As in the
case of pork, the use of feeds containing fish oils or algae, enriched in n-3 fatty acids
such as EPA (C 22:5 n-3) and DHA (C 22:6 n-3) acids, can enrich the poultry meat in
n-3 fatty acids and reduce the n-6/n-3 ratio from around 8.4 to 1.7 [15]. The main
problem arises from oxidation during heating, because some volatile compounds
such as hexanal are typically generated, producing rancid aromas. The rate and
extent of oxidation of muscle foods mainly depends on the level of PUFA, but they
are also influenced by early postmortem events such as pH drop, carcass tempera-
ture, aging, and other factors. Feeds rich in saturated fats such as tallow yield the
highest levels of palmitic, palmitoleic, stearic, and oleic acids in pork loin [21].
Linoleic and linolenic acid content may vary as much as 40% between the leanest
and the fattest animals [22]. The PUFA content is especially high in phospholipids,
located in subcellular membranes such as mitochondria, microsomes, and so on,
making them vulnerable to peroxidation because of the proximity of a range of pro-
oxidants such as myoglobin, cytochromes, nonheme iron, and trace elements [23].
Muscle contains several antioxidant systems, for example, those of superoxide
dismutase and glutathione peroxidase, and ceruloplasmin and transferrin, although
they are weakened during postmortem storage.
The fatty acid profile in ruminants is more saturated than in pigs, and thus the fat
is firmer [14]. The manipulation of fatty acids in beef is more difficult due to the
rumen biohydrogenation. More than 90% of the PUFA are hydrogenated, leaving a
low margin for action to increase the PUFA/SFA ratio above 0.1. However, meats
from ruminants are rich in conjugated linoleic acid (CLA), mainly 9-cis,11-trans-
octadecadienoic acid, which exerts important health-promoting biological activity
[24]. In general, a good level of nutrition increases the amount of intramuscular fat.
On the other hand, food deprivation may result in an induced lipolysis that can be
rapidly detected (in just 72 h) through a higher content of free fatty acids and
monoacylglycerols, especially in glycolytic muscles [25].
4 Conjugated Linoleic Acid (CLA)
During the 1930s, biological chemists began to realize that the cow could convert
dietary nonconjugated fatty acids to a conjugated component. However, the mech-
anism for this transformation and the location at which it was effected were
unknown. By 1950, it was established that there was a species-to-species difference
in the unsaturated fatty acid content of body fat from pasture-fed animals. Linolenic
acid is the predominant pasture fatty acid. When ruminant animals such as cows and
sheep consume this acid, only trace amounts appear in body tissues or milk. On the
other hand, the horse, a nonruminant, transfers a considerable proportion of dietary
linolenate to its depot fat [26].
Observations that rumen contents could hydrogenate polyunsaturated fatty acids
to produce acids with trans-unsaturation provided a biological explanation for the
presence of trans-fatty acids in the depot fat of sheep and oxen, noted as early as
1928 [27]. Later, scientists from New Zealand [28] examined the trans-unsaturated
fatty acid content of fat from a selection of ruminant and nonruminant animals. As
expected, the depot fats from ruminants (Sambur, fallow deer, ox, and sheep)
contained conjugated diene at around the 0.5% level. An unexpected finding was
the presence of considerable conjugated diene in the body fat of the nonruminant
marsupials, wallaby (3.5%), and quokka (2.9%). Depot fat trans-monounsaturated
fatty acid content from these marsupials was also exceptionally high, 19% and 21%,
respectively. The high conjugated diene and transmonoene content is explained by
their possession of a ruminant-like digestion.
The isomer cis-9,trans-11-18:2 was identified in the depot fat of pasture-fed
lambs using Gas Liquid Chromatography analysis [29]. Trivial names for the natural
cis-9, trans-11-isomer have been suggested: firstly [30] was proposed bovinic acid,
but this name was considered too restrictive because the isomer is also produced in
the rumen of a number of other species of commercial importance. For this reason
was later suggested rumenic acid, a name that is now accepted and used [31].
During the early 1980s, Michael Pariza and his colleagues at the University of
Wisconsin found that an isolate from grilled minced beef could inhibit carcinogen-
esis. The anticarcinogenic isolate was shown to consist of isomers of conjugated
octadecadienoic acid in which the constituent double bonds are separated by a single
carbon-to-carbon bond instead of a methylene group. The isomers were referred to
collectively as conjugated linoleic acid for which the acronym CLA is now used [27].
Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids
that exist as positional and stereo-isomers of conjugated dienoic octadecadienoate
(18:2). The predominant geometric isomer in foods is the c9t11-CLA isomer, also
called “rumenic acid” [24], followed by t7,c9-CLA, 11,13-CLA (c/t), 8,10-CLA (c/
t), and the t10c12-CLA isomer. The three-dimensional stereo-isomeric configuration
of CLA may be in combinations of cis and/or trans configurations (Fig. 1).
CLA is found in foods such as beef and lamb, as well as dairy foods derived from
these ruminant sources [32]. Numerous physiological properties have been attributed
to CLA including action as an antiadipogenic, antidiabetogenic, anticarcinogenic,
and antiatherosclerotic agent (Table 2). In addition, CLA has effects on bone
Fig. 1 Main isomers conjugated linoleic acid. Top: C18:2Δ9c,11 t (rumenic acid). Bottom:
C18:2Δ10t,12c
Table 2 Physiological properties of CLA

Major function Physiological model
Body Decrease adiposity in chicks, mice, rats;
composition Decrease adiposity in human subjects.
Diabetes Decrease the onset of diabetes in male rats;
Aids in the management of metabolic parameters in human subjects with
type 2 diabetes;
Decrease insulin sensitivity in mice.
Carcinogenesis Decrease chemically induced mammary carcinogenesis in rats;
Decrease growth of transplantable breast cancer tumor cells in nude mice;
Decrease growth of transplantable prostate cancer tumor cells in nude mice;
Decrease chemically induced colon carcinogenesis in rats;
Decrease chemically induced forestomach.
Atherosclerosis Decrease atherosclerotic plaque formation in hamsters.
Immune system Decrease eicosanoid and histamine production;
Increase onset of lupus in mouse model.
Source: Adapted from [24]
formation and the immune system as well as fatty acid and lipid metabolism and
gene expression in numerous tissues [24].
The primary dietary sources of CLA for humans are food products derived from
ruminant animals, mostly cattle, including meat fat, milk, cheese, yoghurt, and butter
[33]. There are two biosynthetic processes responsible for the formation of CLA.
These processes are carried out primarily in ruminant animals but also to a lesser
extent in nonruminant animals. The first process is the incomplete biohydrogenation
of linoleic acid and linolenic acid in the rumen; the second biosynthetic process is the
endogenous conversion of transvaccenic acid, an intermediate of biohydrogenation,
to CLA in tissues [34]. The main dietary source of linoleic acid for ruminant animals
is concentrated feed consisting mainly of grains and seed oils, whereas the main
dietary source of linolenic acid is pasture grasses [35].
4.1 Biosynthesis of CLA
Because potential health benefits have been associated with dietary consumption of
CLA, enhancement of CLA concentrations in meat and milk has become an impor-
tant objective in animal nutrition research. It is generally accepted that CLA in
ruminant meat and milk originates from incomplete biohydrogenation of linoleic
acid in the rumen [36]. A study performed in 1951 [37] noticed that the body fats of
ruminants possessed less linolenic acid than horses on the same high linolenic acid
diet and suspected something to have happened in the rumen. Linolenic acid was
incubated in rumen fluid, and it was demonstrated the formation of TFA in the
rumen. Utilizing rumen contents from fistulated sheep grazing pasture [38], it was
confirmed the existence of TFA as a result of rumen biohydrogenation. Later [39] it
was also demonstrated that conjugated dienoic acids accumulated when linoleic acid
was incubated with rumen contents, but not when linolenic acid was incubated with it.
4.2 CLA Synthesis in the Rumen
A diverse range of rumen bacterial species have been isolated; they demonstrate a
capacity to isomerize cis-double bonds of unsaturated fatty acids to form conjugated
cis/trans double bond systems and further hydrogenate these conjugated acids [36].
A study performed in 1967 [40] showed the production of c-9, t-11 CLA from
linoleic acid by Butyrivibrio fibrisolvens. When the same bacteria were incubated
using linolenic acid as the substrate, c-9, t-11, c-15 C18:3 were produced, in which
18:3 later was found to be hydrogenated to trans vaccenic acid [41]. No c-9, t-11
CLA was formed from linolenic acid.
The biohydrogenation of linoleic acid and linolenic acid in the rumen occurs in a
similar manner. The first reaction in linoleic acid biohydrogenation is the isomeri-
zation where the double bond at carbon-12 position is transferred to carbon-11
position forming c-9, t-11 CLA. It is followed by the rapid hydrogenation of cis-9
bond leaving trans vaccenic acid. Both these steps are carried out by group A
bacteria, while the last step of biohydrogenation of oleic to stearic acid is carried
out by group B bacteria [42]. The enzyme responsible for the conjugation of cis-9,
cis-12 double bonds was identified as linoleic acid isomerase (EC 5.3.1.5). It is a
particulate enzyme bound to the bacterial cell membrane [36] and demonstrates an
absolute substrate requirement for a cis-9, cis-12 diene system and a free carboxyl
group [43], found in both linoleic acid and linolenic acid. Similarly, the linolenic
acid is first isomerized at cis-12 position to form c-9, t-11, c-15 C18:3, which were
then reduced at both the cis bonds to produce trans vaccenic acid. The final step is
similar to that of linoleic acid. A common intermediate during the biohydrogenation
of linoleic acid and linolenic acid was found to be trans vaccenic acid [42]. Its
reduction appears to be rate limiting in the complete biohydrogenation of unsatu-
rated C18 fatty acids resulting in the accumulation of trans vaccenic acid in the
rumen [44]. This is the predominant pathway of rumen biohydrogenation of linoleic
acid and linolenic acid. Subsequently, a product precursor relationship between trans
vaccenic acid and CLA was observed both in vitro and in vivo (sheep) with
increasing concentrations of LA in the diet [45].
With a low-fiber diet, a change in trans octadecenoic acid profile of milk occurred
and trans-10 octadecenoic acid became the predominant trans octadecenoic acid in
milk fat [46]. This suggested the hypothesis of another pathway for the ruminal
synthesis of t-10, c-12 CLA involving bacterial c-9, t-10 isomerase with the forma-
tion of a t-10, c-12 double bond as the first step in the process [36]. The c-12, t-11
isomerase from Butyrivibrio fibrisolvens can hydrogenate t-10, c-12 octadecadienoic
acid [41], thus producing t-10 octadecenoic acid. It has been shown that more than
50% of the linoleic acid was converted to t-10, c-12 isomer of CLA and only 10%
was converted to t-10 octadecenoic acid by anaerobic Propionibacterium isolated
from mouse cecum [47]. Another rumen bacteria Megasphaera elsdenii YJ-4 have
also been shown to produce t-10, c-12 isomer of CLA [48]. The t-10, c-12 isomer
was formed from linoleic acid but not from either of the linolenic acid as was the case
with c-9, t-11 isomer of CLA. It is not clear whether t-10 octadecenoic acid is
desaturated at cis-12 position to produce t-10, c-12 isomer in the rumen or in some
other tissues endogenously.
Rumen pH has an important role in maintaining a viable rumen environment
suitable for Butyrivibrio fibrisolvens involved in the biohydrogenation of linoleic
acid and linolenic acid. It has been shown that ruminal pH at 6.0 or above has a
positive effect on trans vaccenic acid and CLA contents in rumen cultures [49]. It is
of higher importance in high yielding dairy and beef animal diets where large
amounts of grain are included in the diet and thus decrease the rumen pH below
6.0. Other than its positive effects on Butyrivibrio fibrisolvens, how rumen pH
affects overall biohydrogenation of unsaturated fatty acids of 18, 20, or 22 carbons
in the rumen in relation to CLA and trans vaccenic acid has not been investigated in
detail. It has been shown that CLA could be increased by supplementing diets with
feed sources such as fish oil or marine algae (Schizochytrium sp.), which are rich in
20- or 22-carbon fatty acids [50] that do not yield either CLA or trans vaccenic acid
during biohydrogenation in the rumen. The mechanism by which supplementation of
fish oil or marine algae increases concentration of milk fat CLA and trans vaccenic
acid is not clear. It has been proposed that the longer chain polyunsaturated fatty
acids from fish oil inhibit the complete biohydrogenation of C in the rumen by
inhibiting the 18:2 growth of bacteria responsible for hydrogenating trans vaccenic
acid or through the inhibition of their hydrogenases [36] leading to an increased
escape of trans vaccenic acid from the rumen. Further research is needed on the
pathway for rumen biohydrogenation of longer chain poly-unsaturated fatty acids
from fish oils and other fatty acids sources of marine origin to clearly define the
mechanisms involved in enhancing CLA and trans vaccenic acid contents in food
products from ruminants.
4.3 CLA Endogenous Synthesis
While the origin of CLA from linoleic acid by Butyrivibrio fibrisolvens in the rumen
was accepted, it was not sufficient to account for all the CLA present in milk or meat.
The levels of CLA in meat and milk from ruminants compared with nonruminants
suggest a close association between rumen function and CLA levels in tissue and
milk fat.
A study performed in 1996 [51] found that lactating sheep grazing pastures with
no supplemental linoleic acid produced a high level of c-9, t-11 CLA in milk fat. The
puzzle as to why the milk fat showed greatly increased absorption in the ultraviolet
region when cows were turned out to pasture, even though the pastures are high in
LNA and not LA, continued to intrigue the scientists. Two years later another study
demonstrated that supplementation of fish oil, which is high in PUFA of 20 or more
carbons did not produce c-9, t-11 CLA or trans vaccenic acid as the intermediate
during biohydrogenation, and also increased c-9, t-11 CLA content in the milk of
cows [52]. Basing their research on these preliminary results, it was possible to get to
the conclusion that ruminal synthesis of CLA was only marginal and could not
account for the amount of CLA present in milk and meat from ruminants [36].
Overall these findings suggested that the CLA formed during the biohydrogenation
of linoleic acid in the rumen was not the only source and that another source needed
to be explored.
Initially it was proposed that CLA could be synthesized endogenously from trans
vaccenic acid by Δ9-desaturase. A recent study [53] provides several lines of
evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate
both from ruminal biohydrogenation and from direct Δ13-desaturation of vaccenic
acid in mammary tissue.
An experiment [54] was conducted to examine whether increased CLA in milk of
dairy cows fed fresh pasture compared with alfalfa and corn silages was because of
ruminal or endogenous synthesis. Eight Holsteins were fed a total mixed ration using
alfalfa and corn silages as the forage source in confinement or grazed in a replicated
crossover design. The proportion of total fatty acids as CLA (primarily c9, t11-18:2)
in g/100 g was 0.44 v. 0.28 in ruminal digesta, 0.89 v. 0.53 in omasal digesta and
0.71 v. 1.06 in milk during confinement feeding and grazing, respectively. Blood
plasma CLA was 0.54 v. 1.05 mg/l for the two treatments, respectively. The
increased concentration of CLA in milk with grazing likely resulted from increased
synthesis through desaturation of t11-18:1 in the mammary gland. Other authors [55]
estimated rumen output of CLA in nonlactating cows and then extrapolated the
results to lactating cows on the basis of feed intake. They estimated the endogenous
synthesis of CLA to be >80% of the total. Another study [56] even estimated 100%
of CLA to be derived from endogenous synthesis. It is not surprising, considering the
fact that the amount of CLA detected in the blood serum of cows is none or very
small [57].
It is possible that a higher proportion of CLA is synthesized endogenously in
cows fed all pasture diets compared with cows fed grains, oil seeds, or oils, because
LNA, which is high in fresh pastures, does not produce CLA as the intermediate
product during its biohydrogenation in the rumen. However, all these studies used an
indirect approach to estimate the CLA synthesized endogenously and it may be over-
or underestimated under different feeding regimens. A direct approach, which may
be difficult, is probably needed to measure the actual uptake of CLA and its
incorporation into milk fat by the mammary gland to estimate the mammary
synthesis of CLA more accurately.
In other ruminants, information on the proportion of rumen and endogenous
origin of CLA is limited. However, the fact that increased CLA content in lamb
meat [58] and goat milk fat [59] was associated with high TVA contents indicates
that post ruminal synthesis could be the predominant one. A very high correlation
(r = 0.99) of CLA with TVA in goat milk fat [59] suggests that mammary synthesis
predominates over rumen synthesis of CLA.
Several other trans-C 18:2 isomers are found in rumen digesta, and milk and
tissue lipids [41]. Unlike c-9, t-11 CLA, no detailed studies have been conducted to
study the endogenous synthesis of other isomers of CLA, including t-10, c-12,
probably because many of them contribute 0.05% or less in milk or meat fat and their
biological significance has not been established.
5 CLA in Foods
CLAs are widely distributed in various foods, primarily in dairy products and meat
from ruminants. In general, ruminant products have the highest amounts of CLA,
while vegetable products and some seafood contain only trace amounts [60].
The wide variation of the CLA content in dairy products reflects the differences in
CLA contents of the milk fat. The CLA content in milk fat is markedly influenced by
the cows’ diet [46], the PUFA content of the feed [61], dietary regimen [62], and the
presence of ionophores [63]. The influences of the processing parameters and starter
cultures on the preparation of cheese are contradictory [64], and further studies are
necessary to evaluate whether the conditions increase the CLA content and change
the isomer distribution in cheese. CLA contents in long-ripened propionic-acid-
fermented cheeses (e.g., old Emmenthal, or yogurt) with added probiotic cultures
appear to be slightly elevated [60]. The content of CLA in dairy products ranged
from 0.63% in condensed milk to 1.16% in cow’s milk, and from 0.40% in Gouda to
1.70% in Jurassic cheese [60]. These values should not be viewed as representative
of one product. The wide variation of the CLA content in dairy products reflects the
differences in CLA contents of the milk fat.
Meat from animals such as pigs contains low amounts (0.12%) of CLA because
such animals lack a rumen. The CLA in pork may originate from feedstuffs that
contain CLA such as meat meal or tallow.
5.1 CLA in Meats
Food sources originating from ruminants are known to have markedly higher CLA
concentration than those from monogastric animals. Table 3 shows the CLA con-
centration of meat from different animal species usually used in the human diet. The
Table 3 CLA content in different meat and meat products (mg/g FAME)
Lamb 5.6
Beef 2.9–4.3
Pork 0.6
Chicken 0.9
Turkey 2.5
Salami 4.2
Cooked ham 2.7
Smoked bacon 0.8–2.7
Smoked ham 2.9
Source: Adapted by [36]
highest CLA concentrations were found in lamb (4.3–19.0 mg/g lipid) and with
slightly lower concentrations in beef (1.2–10.0 mg/g lipid). The CLA content of
pork, chicken, and meat from horses is usually lower than 1 mg/g lipid. Interestingly,
turkey seems to have a relatively high CLA content (2–2.5 mg/g lipid), but reasons
for this are unclear [32]. Some data on CLA meat content of animals less common in
human diets like meat from elk (1.3–2.1 mg CLA per gram fatty acids), bison
(2.9–4.8 mg/g fatty acids), water buffalo (1.83 mg/g fatty acids), and zebu-type
cattle (1.47 mg/g fatty acids) are also available [65]. The highest CLA concentration
(38 mg/g fatty acids) of all animals was found in adipose tissue of kangaroos [66].
Large variations in the CLA content are not only reported between animal species
but also within muscles of the same species.
The CLA concentrations reported in various studies may be underestimated
because only the c9,t11–18:2 isomer was determined, and not the total CLA content
[67]; however, this isomer accounts for more than 80% of the total CLA, even if a
study found that between 76% and 92% (depending on the species) of the total CLA
was c9,t11–18:2, while other studies [68] determined a value of 78% in lamb rib
loins, and a value of about 59% in beef [69].
A trial performed with the aim to evaluate the effect of the slaughter weight on the
CLA isomer content of intramuscular fat in three different kind of lamb muscles,
specifically Longissimus dorsi, Triceps brachii, and Semimembranosus, showed that
the body weight at slaughter significantly affected the amount of rumenic acid in
intramuscular fat of Semimembranosus and Triceps brachii in Massese lambs, but
not in the Longissimus dorsi. In particular, the heaviest animals showed the highest
amount of rumenic acid [70]. The different behavior shown by LD muscle did not
seem to be related either to differences in the stearoyl Co-A desaturase enzyme
activity, or to differences in the rumen activity, or to differences in fat deposition.
Thus, further studies are needed to verify whether this different behavior among
muscles is related to their different metabolism and/or to a different tissue utilization
of fatty acids [71].
Grilling rib-eye, sirloin, T-bone, and ground beef to an internal temperature of
80 C increases both the c9,t11-isomer (mg/g fat) and total CLA (mg/g fat) content
of these meats [69], but a significant increase was observed only in the case of the
grilled T-bone steak. Results obtained in the same study [69] showed that frying,
grilling, microwaving, and baking ground beef patties did not produce any major
changes in the CLA content. With the exception of baking, the higher internal
temperature (80 C) generally resulted in higher CLA concentrations. During refrig-
erated storage of ground beef, oxidative damage occurs but this does not influence
the CLA content. Results obtained till now demonstrated that cooking and storage of
meat does not negatively alter its CLA content [72].
CLA can be produced with very limited amount by gastric bacterial
biohydrogenation in pig resulting in low amount of CLA in pork [73]. However,
pork is an ideal candidate for CLA enrichment by feeding chemically synthesized
CLA because CLA cannot be further saturated and can be deposited in tissues with
relatively high efficiency [73]. The cis 9, trans 11 isomer of CLA could be incorpo-
rated by 46.4% in subcutaneous adipose tissue and the cis 11 and trans 13 was
incorporated by 0.74% in intramuscular fat. Feeding pigs with 1% CLA for 47 days
significantly increased the CLA content including the cis 9, trans 11 and the trans 10,
cis 12 in belly fat [74]. However, results on pig growth performance in response to
CLA supplementation are not homogeneous, as reported by [75]. This may be due to
a deficit of nutrients in diets containing CLA isomers, as these fatty acids act as
repartitioning agents, promoting lean tissue deposition. The high lean tissue depo-
sition and the low body fat deposition of CLA-treated pigs [76] may require
an increase in dietary protein content and/or protein quality to maintain protein
synthesis and growth in finishing swine. In fact, the utilization of substances with
repartitioning effects such as ractopamine required an increase in lysine content in
finishing pigs [77]. Consequently dietary CLA supplementation might induce an
insufficient nutritional level of an essential amino acid like lysine, the first limiting
amino acid in pig diet used for protein deposition.
In a study performed on rabbit meat, it was demonstrated that dietary CLA
improved the oxidative stability of rabbit muscle, increasing shelf life, decreasing
lipogenic enzyme activity, and reducing plasma triglycerides and total cholesterol
[78]. The same authors also found that addition of CLA isomers to rabbit diet
modified lipid content and fatty acid composition and reduced lipid oxidation in
LL [79]. The increase in CLA content, from about 1.3 to 10.4 mg/100 g of edible
meat, suggests that the nutritional quality of rabbit meat for human consumption may
be improved.
CLA content very similar to those found in ruminants has been determined in
meat obtained from foals slaughtered at 24 months of age [80]; equid meat is a
typical red meat, even if it is produced by a monogastric hind-gut fermenter [81].
However, considering another equid such as donkey, in one of the few studies
recently performed on donkey meat lipid profile characterization, it was not possible
to determine CLA [82].
5.2 CLA in Chicken, Turkey, and Eggs
The content of CLA may be increased by 40 times in breast and thigh meat, by
feeding CLA enriched diets to broilers [83]. Despite this high increase in the CLA
content in breast and thigh meat, the recommended daily intake for CLA in humans
may only be covered by roughly 10% [84]. Furthermore, CLA enriched poultry meat
shows deviations in meat quality, increasing toughness and showing also a darker
color [85]. This is also confirmed by sensorial evaluation: color and flavor of CLA
enriched meat received positive results from the test panel, whereas, texture and
juiciness were negatively scored. The toughness of the meat may be explained by the
higher proportion of SFA in CLA enriched muscle tissues and the consequent
decrease in UFA, which increase the fat melting point [85].
In egg yolk lipids, CLA was not even detected when laying hens were fed a
normal concentrate diet [35]. The effects of dietary CLA supplementation on eggs
fat profile have been recently reviewed [86]; an interesting result showed that the egg
yolk surface from hens fed CLA diets sometimes had relatively dark color with light
spots. Dietary CLA increased the firmness of hard-cooked egg yolk. The texture of
yolks from hard-cooked CLA eggs was rubbery and elastic, and the yolks were more
difficult to break. It was speculated that the quality changes of CLA eggs were
related to the increase of yolk water content, the movement of ions between yolk and
albumen through yolk membrane, and the changes of egg yolk pH during storage.
Dietary supplementation of CLA significantly influenced fatty acid profile in egg
yolk lipids: myristic, palmitic, stearic, CLA (9-cis, 11-trans CLA and 10-trans,
12-cis CLA) were increased by dietary CLA, while palmitoleic, oleic, linoleic,
linolenic, arachidonic, and DHA acids were decreased. Total CLA concentration
observed when 5% CLA was fed ranged between 8.5 to 8.6% (Ahn et al. 1999). The
decrease in the concentrations of linoleic and linolenic acids in yolk lipids of hens
fed CLA probably reflects the relatively low concentration of these fatty acids in the
CLA source as compared with soybean oil. Decreases in arachidonic acid and DHA
acid in yolk lipids from hens fed CLA also could be related to the low concentration
of dietary linoleic and linolenic acids, which serve as precursors to the formation of
arachidonic and DHA acids. Another possibility is that CLA may compete with
linoleic or linolenic acid for Δ6-desaturase, the rate-limiting step for the conversion
of these fatty acids into arachidonic acids or DHA acid in liver microsomes [87].
5.3 CLA in Fish
Several studies demonstrated the cardiac benefits of a regular intake of fish, a source
of the long-chain n-3 fatty acids EPA and DHA [88]. A sharp demarcation in the
linoleic acid content of fish showed that most marine fish typically had only 1–2%
of linoleic acid. This content is so low as to continue to preclude speculation
concerning whether the fatty acid of the muscle of this food would or would not
contain CLA [89]. The situation changes completely when considering the new
developments in farmed fish that accumulate fatty acids from a variety of dietary
ingredients in their growth period. The reason is that the diets represent a balance
between the cost and efficacy of the ingredients. The need is to provide good quality
protein for growth and meet minimal specific needs for “essential” fatty acids,
especially in salmonids, plus vitamins and minerals. For salmonids, replacement of
the most common dietary fat, fish oil, with vegetable oils has become common
lately.
Seafoods are not considered to be important sources of CLA, either, but the term
seafood is so loosely used that it is often difficult to learn from brief references
whether the source of most of the fat or lipid in the reference is from cold-water or
warm-water fish, or from the fish alone [89].
5.4 CLA in Milk and Dairy Products
The CLA content in dairy cows milk fat can be affected by a cow’s diet, breed, age,
non-nutritive feed additives, such as ionophores, and by the use of synthetic mixtures
of CLA supplements [90]. Among these factors, the diet is known to strongly
influence the CLA content of milk and includes feedstuffs such as pasture, conserved
forages, plant seed oils, cereal grains, and animal fat.
The positive effect of pasture-based diets on the CLA content of milk fat has been
demonstrated in several experiments [90]. Cows grazing pasture had 500% higher
CLA content in milk fat (2.21% of total fatty acids) compared to cows fed a diet
containing 50% conserved forage (hay and silages) and 50% grain (0.38% of total
fatty acids). Other researchers have also demonstrated that the CLA content of milk
increased linearly as the proportion of fresh grass from pasture in the diet was
increased.
About 48% to 56% of the total fatty acids in fresh forages consist of C18:3. Fresh
grass supplies C18:3 as a substrate for ruminal biohydrogenation. However, the
abundant supply of C18:3 from fresh grass only partly explains the large increases in
CLA and trans vaccenic acid contents of milk fat from pasture-fed cows. Besides
this, the high concentrations of soluble fiber and fermentable sugars present in fresh
grass may create an environment in the rumen without lowering the ruminal pH that
is favorable to the growth of the microbes responsible for CLA and trans vaccenic
acid production. Ruminal pH is generally relatively high in cows grazing pasture
compared to cows fed a combination of conserved forage and grain.
The effects of several feeding strategies on CLA concentration in dairy milk have
been recently described; most experiments were conducted on Holstein or Friesian
cows. The most common results indicated that CLA in milk is related to the
availability of its precursors (oleic acid, linoleic acid and linolenic acid) from
ruminal andendogenous synthesis [91]. The average difference in CLA content of
milk fat among Brown Swiss, Holstein-Friesian, and Jersey breeds is 15% to 20%
when fed similar diets. Brown Swiss cows have inherently higher CLA in milk fat,
followed by the Holstein-Friesian and Jersey breeds; data on other breeds are too
limited to form any firm conclusions [90].
Microbial fermentation can contribute to increases in CLA content in dairy
products through isomerase and reductase reactions in the biohydrogenation path-
way. Moreover, the process temperature can also influence the CLA content in dairy
products. Dairy products, such as cheese, are the richest sources of CLA, and CLAs
are formed in cheese during processing and storage [92]. When the animals were
pasture fed, the contents of CLA in milk fat were much higher than that when a
fodder mixture was supplied to cows. These data indicate that linoleic acid-enriched
feed can increase the CLA contents in milk and cheese through fermentation by
ruminal bacteria. The influence of cheese processing, e.g., changes in temperature,
microbial fermentation, or ageing, on CLA production are proportional [93].
Existing literature suggests that CLA in milk fat is a stable compound under normal
processing and storage conditions, while processing dairy products at >80 C may
slightly elevate the CLA content.
Lactic acid bacteria, bifidobacteria, and some propionibacteria are used as starter
cultures in cheese and are able to efficiently convert linoleic acid to CLA. Thus,
CLA-enriched cheeses typically originate from CLA enriched milk, and starter
cultures also have a great influence on the CLA content [93].
6 Human Dietary Intake of CLA
It is not easy to determine how much CLA humans are currently consuming, and
how much should be consumed in order to obtain the health benefits and anticancer
effects that have been observed from adding proper levels of CLA to the diet.
Rigorous documentation of dietary CLA intake in any population is not available
currently. However, several investigators have utilized indirect methodologies to
estimate both typical and extreme intakes of CLA in a limited number of
populations. Using 3-d dietary records in conjunction with published CLA contents
of foods, typical CLA intake has been estimated to be between 52 and 137 mg/day
for young men and women in the United States and 430 and 350 mg/day for German
men and women, respectively [30]. The use of a semiquantitative food-frequency
questionnaire to estimate typical CLA intake in lactating women suggests that CLA
intake in this population is 227 mg/day [30].
The CLA and TFA dietary intake in Germany is shown in Table 4; the estimated
daily CLA intake was found to be 0.36 g/day for women and 0.44 g/day for men. The
CLA intake compares to approximately one-fifth the daily intake of TFA and may
reach physiologically active levels.
To date, statements about health promoting effects of CLA are mainly based on
animal trials and remain to be proven in humans [36]. In human trials synthetic CLA
supplements are usually used and these do not reflect natural isomer composition in
foodstuffs. Whether natural CLA sources (meat and milk from ruminants) have a
similar impact on human health warrants further research.
Furthermore, estimations of the dietary intakes of CLA isomeric forms by infants,
children, and adolescents have not been documented. The dietary CLA intake
reflects individual, dietary, and ethnic consumption habits. Finally, examination of
the relationships among human dietary intake of CLA isomers, their concentrations
in adipose tissue and plasma, and risk of various chronic degenerative diseases (e.g.,
cancer, diabetes, and obesity) is essential for scientists to better understand the
importance of dietary CLA in human health. Thus, enhancing our knowledge
concerning CLA intake in various populations must remain a primary focus for
research in this area.
Table 4 Estimated CLA and TFA intake in Germany

Women Men
CLA (g/d) TFA (g/d) CLA (g/d) TFA (g/d)
Milk and dairy products 0.24 0.90 0.28 1.1
Meat and meat products 0.08 0.2 0.11 0.3
Fish <0.01 <0.01 <0.01 <0.01
Margarines <0.01 0.1 <0.01 0.2
Cakes and pastries 0.03 0.2 0.03 0.2
Chocolate and sweets <0.01 0.1 0.01 0.1
Source: Adapted by [60]
7 Cancer and CLA Consumption
The initial observations arousing interest in the biological effects of CLA showed
their ability to reduce chemically induced cancers in animal models. In contrast to
the many hundreds of phytochemicals that possess varying degrees of anti-
carcinogenic activity, CLA is unique because it is a group of fatty acids isomers
found in highest amounts in animal products [94]. Furthermore, CLA was found to
possess anticarcinogenic activity that turned out be quite potent. The following
sections present the published experimental literature that indicates an effect of
CLA on cancer inhibition, primarily in animal models of chemically induced cancer.
7.1 Colorectal Cancer (CRC)
The publication of the World Cancer Research Fund/American Institute for Cancer
Research [95] raised considerable alarms about the cancer risks associated with red
and processed meats, in concluding that they are a convincing cause of colorectal
cancer (CRC). There are a number of possible mechanisms for a link between meat
consumption and CRC. These include the promotion of carcinogenesis by high-fat
intake, the production of carcinogenic heterocyclic amines (HCAs), and/or polycy-
clic aromatic hydrocarbons (PAHs) during cooking, the formation of carcinogenic
N-nitroso compounds (NOCs) either within meat per se or as a result of endogenous
processes, and the promotion of carcinogenesis by hem iron [96]. It is also suggested
that the high energy density of meat increases the likelihood of obesity, itself a major
risk factor for cancer [95]. For each of the mechanisms implicated in cancer
formation, there is an approach to reducing any cancer threat. The selection of
meat will affect its fat content. It seems that wild animals are overall lower in fat,
with a lower proportion of saturated fatty acids and higher proportion of polyunsat-
urated fatty acids as compared with farmed animals. In general, the selection of
animals according to genotype, and diet they are fed on will, profoundly influence
not only the total fat concentration in the meat but also the nature of the fats. Obvious
modulations include dietary fat intake, but controlling the intake of other nutrients
such as vitamin A will also have an influence.
Meat and meat products vary greatly in their fat content according to the animal
species, age of the animal, and part of the carcass used. The fat content and fat
composition is also affected by animal feeding, a fact that is exploited for modifi-
cation of the meat fatty acid composition, with the relatively best results in single-
stomached pigs and poultry [97]. Data on the average fat content and fatty acid
composition of meats and meat products are published as part of food composition
tables throughout the world. The fat content of meat has decreased in recent years,
with new breeds coming onto the market, and different trimming processes. Of the
nutrients considered to have detrimental effects, saturated fatty acids (SFA) provide
around 50% of the fatty acids found in meat, while trans fatty acids (TFAs) make up
only a small component. The rest is mainly made up of monounsaturated fats, with
small amounts of polyunsaturated fatty acids (PUFA), including small amounts of

n-3 PUFA, which are likely to be beneficial in cancer prevention [95].
7.2 Breast Cancer
Breast cancer is the clinical expression of the development of tumor tissue initially
within a breast, then within different other sites during the metastatic evolution
period. This tumor tissue results from a multistep carcinogenic process that probably
extends over several decades. This process consists of an accumulation of genetic
alterations acquired during a woman’s life span, which leads to the neoplastic
transformation of normal epithelial cells. Dietary lipids can modulate mammary
carcinogenesis at several stages; some of them may contribute to the prevention of
acquired genetic alterations through their antimutagenic activity and therefore act at
the very early stages of the initiation process. Lipids can also influence tumor
promotion [98]. Fatty acids, the main components of dietary lipids, have several
properties that make them potential targets for use in a dietary prevention of breast
cancer. For a few years, growing interest has focused on particular fatty acids,
geometrical and positional isomers of linoleic acid, i.e., conjugated linoleic acids
(CLA). These dietary conjugated dienes present interesting anticarcinogenic prop-
erties in animal studies of mammary carcinogenesis; furthermore, these properties
are unique in that CLA can act at very low concentration, close to that observed in
the diet [99].
Whether CLA may be considered as potential targets for use in a nutritional
prevention of breast cancer remains an attractive issue. The amount of CLA needed,
the duration of intervention, as well as the proper stage in life for such an interven-
tion are not known at present. In humans, only epidemiological studies have been
conducted and results are inconclusive. Only 1 study found a negative association
between the intake of CLA and risk of breast cancer in postmenopausal women,
while other studies reported no effect of CLA intake on protection against breast
cancer [100].
7.3 Skin Cancer
Although not nearly equal in extent to the investigations on mammary cancer

inhibition, the effect of CLA on skin carcinogenesis has received a fair amount of
examination and was the tumorigenic site of the first study to demonstrate an
anticancer effect of CLA [94]. From the many hundreds of chemicals in a partially
purified extract from fried ground beef, a substance that exhibited anticarcinogenic
activity has been identified [101]. Using the classic two-stage mouse epidermal
carcinogenesis model, this substance was identified as CLA. In this experiment,
synthetically prepared CLA was topically applied 7 d (20 ng/mouse), 3 d (20 ng/
mouse), and 5 min (10 ng/mouse) before dermal treatment with 50 nmol DMBA per
mouse (n = 20/group). One week later, the female CD-1 mice received twice weekly
topical treatments with 6 μg 12-O-tetradecanoyle-13-acetate (TPA) until study

termination at 16 weeks post-DMBA. Compared with control mice (n = 30),
CLA-treated mice had an ~15% lower papilloma incidence and 50% fewer papillo-
mas per mouse.
Some years later, results obtained by other researchers [102] demonstrated that
CLA has less biopotency against mouse skin carcinogenesis and perhaps a less steep
dose-response curve than for mammary cancer inhibition.
7.4 Prostate Cancer
In contrast to the research on breast cancer cell lines, the investigation of CLA on
prostate cell lines is limited. A study was carried out in order to examine the anti-
proliferative effects of different concentrations of a commercial preparation of
conjugated linoleic acids (CLA) mixture of isomers [cis -9, trans -11 CLA (c9,t11
CLA): trans -10, cis -12 CLA (50:50)] and their constituent isomers on PC-3, a
human prostatic carcinoma cell line, and to study their effects on gene expression
(mRNA and protein levels) of different enzymes and oncoproteins involved in
oncogenesis and progression of prostate cancer [103]. The trans -10, cis -12 CLA
was the most effective isomer (55% inhibition); this isomer seems to work prefer-
entially through modulation of apoptosis and cell cycle control, while c9,t11 CLA
isomer affects arachidonic acid metabolism.
8 CLA and Cardiovascular Function
The intake of fat, particularly of saturated fat, is directly associated with the
concentration of cholesterol in the blood, and the cholesterol in the blood is directly
associated with the risk of ischemic heart disease [104]. A little more than two-thirds
of the cholesterol is transported by low-density lipoprotein (LDL) and one-third by
high-density lipoprotein (HDL). High concentrations of total cholesterol in the blood
will almost always mean that LDL cholesterol is high. High concentrations of LDL
cholesterol and low concentrations of HDL cholesterol increase the risk of arterio-
sclerosis and ischemic heart disease. Many epidemiological studies have found that a
high content of meat in the diet is associated with increased risk of ischemic heart
disease. The higher risk of ischemic heart disease has early on been ascribed to the
contribution of SFA from the meat. PUFA increase HDL cholesterol; if SFA in the
diet are replaced by MUFA or PUFA, a fall in total cholesterol and in LDL
cholesterol is seen. SFA with a carbon chain length of 12–16 increase total choles-
terol and LDL cholesterol compared to the monounsaturated fatty acid oleic acid
(C18:1), while stearic acid (C18:0) has a neutral effect on concentrations of total
cholesterol and LDL cholesterol [105].
Some studies [106] investigated on the effects of dietary CLA on atherosclerosis.
In a study, rabbits were fed semisynthetic atherogenic diets (14% fat and 1%
cholesterol) with or without CLA (0.5 g/day per rabbit) for 22 weeks. CLA feeding
was associated with significant reductions in total cholesterol, LDL-cholesterol, and

plasma triacylglycerol concentrations. CLA did not affect HDL-cholesterol concen-
trations per se; accordingly the decrease in LDL-cholesterol resulted in a significant
reduction in the LDL-cholesterol/HDL-cholesterol ratio.
Feeding 1% CLA as part of a semipurified atherogenic diet (0.2% cholesterol)
had a significant beneficial impact on both the progression and regression of
atherosclerosis [107]. The effects on atherosclerosis were observed, even though
plasma lipids were unaffected. All levels of CLA significantly reduced total
cholesterol and VLDL plus LDL-cholesterol concentrations, whereas HDL-choles-
terol was not affected.
Different studies found that CLA reduces atherosclerosis, is hypocholesterolemic,
reduces adipose tissue mass; however, epidemiological studies and controlled trials
in humans are inconclusive and the methodology used is often questionable [108].
The studies evaluating CLA and atherogenesis, carried out till today in different
animal models, are thus inconclusive in predicting how CLA will behave in man;
there is at present no evidence in support of the anti-atherogenic effect of CLA. In no
species has a consistent, reproducible, dose-dependent effect of CLA been
established. Until such data are published, the debate on the involvement of CLA
in cardiovascular function and blood lipids will continue.
9 CLA and Obesity
Meat fat has a relatively high content of stearic acid and, hence, is less cholesterol-
increasing compared with fats with a lower ratio between stearic acid and SFA with a
lower carbon chain length (e.g., dairy fat). The high fat content in the diets of
industrialized populations, in connection with low level of physical activity, is
directly related to the high and increasing occurrence of obesity and ischemic
heart disease [104]. Prevention and treatment of obesity, therefore, is based on a
reduced fat intake by exchanging high-fat meat with lean meat. There is some
evidence that lean meat can be advantageous in a slimming diet, primarily since a
high protein intake seems to increase satiety. Consequently, meat, especially lean
meat, does not increase the risk of ischemic heart disease or obesity.
CLA is marketed in the United States as a dietary supplement. The focus of
marketing efforts is directed toward the implications that CLA will reduce fat mass
and increase lean body mass in humans. The basis for these clinical impressions
arises from animal studies that demonstrate remarkable alterations in body fat and
lean body mass [109]. There are numerous theories on the mechanisms of alterations
in body composition seen in animals ingesting CLA, but few studies have been
carried out to address this question. Table 5 lists some of the potential mechanisms
by which CLA may reduce fat mass and/or increase lean body mass. Alteration of
membrane structure is an overarching potential mechanism that might affect a
number of activities such as enzyme and hormonal response, permeability, mem-
brane fluidity, and receptor number and/or function. CLA has a different spatial
configuration than the parent linoleic acid molecule, which could produce an altered
Table 5 Potential mechanisms of action of CLA to alter body composition

1. Alter membrane structure or function
2. Alter eicosanoid, arachidonic, and/or prostaglandin metabolism
3. Alter cytokine production or response
4. Alter peroxisome proliferator–activated receptor (PPAR) activity
5. Alter sympathetic nervous system activity
6. Alter growth hormone or growth factors
Source: [109]
membrane structure. CLA may be metabolized to different compounds in the

eicosanoid pathway, thus affecting the amounts of arachidonic acid and
prostaglandins.
Although there is evidence in animals of the ability of trans-10,cis-12 CLA to
reduce adiposity and increase lean mass, such unequivocal data supporting the
efficacy of either of the CLA isomers in humans are not available [110]. Before
CLA supplementation is recommended, further research on human effectiveness and
safety of the different isomers of CLA is required.
CLA is known to alter cytokines [111] and tumor necrosis factor (TNF)-a and
other cytokines may play major roles in fat metabolism and the etiology of at least
some types of obesity. Increased amounts of CLA in the diet reduce fat mass and
increase lean body mass. This is the picture seen with administration of β-3 adren-
ergic agonists to animals [112] and is postulated to occur in humans [113]. β-3
agonists increase both the metabolic rate and the proportion of energy derived from
fat oxidation. CLA has been shown to increase metabolic rate and specifically to
increase fat oxidation. Finally, it is possible that CLA might alter the secretion or
action of growth hormone, insulin-like growth factor, or other growth factors.
Administration of growth hormone results in an increase in accumulation of lean
body mass and a decrease in fat mass due to increased lipolysis [114].
A recent study [115] demonstrated that dietary supplementation with CLA iso-
mers to dairy cows resulted in a higher plasma IGF-I and leptin level, but in the
conclusions section it was stathed that further investigations are necessary to define
the exact mode of action of CLA as well as to understand its role in the regulation of
the somatotropic axis regulating liver IGF-I secretion.
The first study performed in humans to evaluate body composition used 20
subjects who were of normal weight to mildly overweight [109]. This was a random-
ized, double-blind, placebo-controlled trial of CLA, 1.8 g/day, versus placebo for a 3-
month period. Body composition measurements were performed using a near infrared
apparatus. The initial body weights and percentage body fat measurements for the
CLA and placebo subjects were 70.9 kg versus 71.8 kg and 21.3% versus 22.0%,
respectively. After 3 months, the placebo group had a body weight of 72.4 kg and
body fat of 22.4%. The CLA group had a nonsignificant decrease in body weight to
70.2 kg, but the percentage of body fat significantly decreased by 4.3–17.0%.
Another study [116] evaluated 23 experienced resistance-trained males who were
matched into two groups for body weight and total training volume. In a randomized
study design, the CLA group was given six capsules per day of a 60% pure CLA
preparation and the placebo group was given olive oil capsules for a total period of
28 days. Subjects also were tested for strength performance with bench press and leg
press exercises at the beginning and end of the 28-day period. There were no
significant differences in body mass, percentage of body fat, or lean body mass
between placebo and CLA subjects. Also, there were no significant changes in
strength performance on the exercise tests. However, the trends for both strength
exercises favored an improved performance in the CLA group. The length of time
that the CLA preparation was given was short compared with the time it was
administered in animals, and the doses of CLA given to the humans were much
lower on a per kilogram basis.
In a clinical trial performed with the aim to test CLA for the treatment of obesity
[109], 80 obese subjects were randomized to CLA versus placebo in a double-blind
fashion over 6 months. All subjects were asked to follow a standardized diet and
exercise regimen with a modest reduction in calories and a modest increase in
exercise. Subjects took 2.7 g of CLA daily and body composition was assessed by
underwater weights. Mean body weight at baseline in the CLA and placebo groups
was 97.1 kg and 96.9 kg, respectively. The percentage of body fat was 38.8% and
35.7%, respectively. There were no significant changes in either body weight or
body fat after 6 months. Both groups lost ~2.5 kg of weight and ~1 kg of body fat.
Post-hoc analyses of the data revealed a subpopulation of individuals who gained
lean body mass. There were twice as many subjects in the CLA group than in the
placebo group who gained lean body mass, but the CLA subjects lost body fat on
average, whereas the placebo group gained. The difference in body fat change was
statistically significant. There were no major side effects or adverse events noted in
either group, with the exception of one person in the CLA group who exhibited
significant edema and weight gain for a period of several weeks. This resolved when
CLA was discontinued, but the subject restarted CLA without the knowledge of the
investigators and had no further problems over the last 3 months of the study.
CLA has been demonstrated to affect a wide variety of enzymes and hormones in
the body, multiple mechanisms may come into play. Priority areas for future research
include attention to the changes CLA produces in membrane structure and function,
the effects on prostaglandin and cytokine function, and the effects on sympathetic
nervous system activity. Human studies of CLA did not demonstrate a clear effect of
CLA on body composition. Because of the contradictory results, raise concern about
the possibility of the deleterious effects of trans-10,cis-12 CLA on the lipid profile,
glucose metabolism and insulin sensitivity, it is advisable to consider with caution
the use of CLA supplements containing high quantities of trans-10,cis-12 CLA,
especially in obese patients with type 2 diabetes or metabolic syndrome,
which constitutes a considerable proportion of the whole overweight/obese popula-
tion [117].
However, uncertainty about the appropriate dose for humans, coupled with the
possibility that the effects may be best demonstrated during a weight accumulation
phase, suggests that dose-response studies are critical. Studies of the usefulness of
CLA in the prevention of weight gain would be of major interest. In addition, studies
in growing children may be considered in the future when questions of dose are
answered and more information on safety is available.
10 CLA and Diabetes
Diabetes mellitus type 2 is the most prevalent form of diabetes, comprising 90–95%
of the diagnosed cases [118]. Individuals who have insulin resistance and a relative
insulin deficiency exhibit the elevated postprandial blood glucose levels that typify
this disease. Insulin resistance may be improved with weight reduction, dietary
management, and/or pharmacological treatments. There are numerous risk factors
for the development of type 2 diabetes including the presence of impaired glucose
tolerance, ethnicity, age, gender, and genetics; central to all of these risk factors is
obesity.
Based on the fact that CLA can reduce adiposity in experimental animals, a study
to elucidate the role of CLA in the development of type 2 diabetes in male rats has
been performed [24]. Male rats were fed semipurified diets containing no CLA
(control), 1.5% CLA, or the antidiabetic thiazolidinedione drug, troglitazone
(0.02%) for 2 weeks. Rats fed the CLA or thiazolidinedione diet exhibited signifi-
cantly reduced ( p < 0.05) fasting glucose, insulinemia, triglyceridemia, free fatty
acid levels, and leptinemia compared with control rats.
Whereas CLA reduces fasting insulin in diabetic animals, it modestly increases
fasting serum insulin in nondiabetic swine [119], mice [120], and humans [121].
Because fasting insulin may be used as a surrogate marker for insulin resistance,
these data suggest that CLA reduces insulin sensitivity under a normoglycemic state.
In agreement, after long-term feeding (8 months) of a CLA-diet, an induction of
insulin resistance was observed in male mice; CLA-induced insulin resistance was
associated with lipodystrophy. The impact and significance of CLA for reducing
insulin sensitivity and/or altering lipodystrophy for people who are normoglycemic
is unknown.
Because CLA was able to delay the onset of diabetes in the rat model, CLA as an
aid in the management of type 2 diabetes in humans was examined [24]. A double
blind, randomized study to determine the effect of daily supplementation with CLA
or placebo (safflower oil) on metabolic parameters of diabetes was conducted.
Subjects with type 2 diabetes were provided with supplements with CLA or placebo,
instructed to maintain a healthy diet using the Food Guide Pyramid and asked not to
change their diet or activity habits for the 8-week intervention period. CLA supple-
mentation (6.0 g CLA/day) significantly decreased fasting blood glucose, plasma
leptin, body mass index, and weight. Low density lipoprotein levels significantly
increased, but less in the CLA-supplemented group than in the placebo group. In
addition, body fat (%) was modestly decreased ( p < 0.08) in subjects supplemented
with CLA. Fasting insulin, triglycerides, cholesterol, and high-density lipoprotein
were not significantly affected by CLA. According to 3-day diet records, energy
intake was not significantly different between groups at baseline or throughout the
study. Supplementation with CLA for 8 weeks could be associated with favorable
alterations of several metabolic parameters of subjects with type 2 diabetes. Further

work is needed to determine the therapeutic potential of CLA in the management of
type 2 diabetes; basing on the available results, it is possible to say that CLA may
represent an important agent for the treatment of type 2 diabetes.
11 Conclusions
It is well known that meat contains a large amount of proteins with high biological
value. However, in recent years, the bioactivities of certain compounds that are
present in meat, even though in minor amounts, have received increased attention
because they may have an important nutritional role. CLA is a group of isomers of
octadecadienoic acid that is abundant in the fat of ruminants like cattle and sheep. The
content may change depending on the breed, feed, and age. CLA has been reported to
reduce the risk for certain types of cancer like the colorectal cancer as well as having
other activities like antiartherioesclerotic, antioxidative, and also playing certain role
in controlling obesity. It has also been found to favorably modulate immune function
in humans. Although the consumption of ruminant products contributes to blood
concentrations of CLA in humans, it is largely unknown whether these physiological
doses might have biological effects in humans.
Because small amounts of CLA (0.5% of diet) have been shown to alter the
expression of genes and impact conditions such as carcinogenesis, obesity, diabetes,
and atherosclerosis in experimental animals, it is possible that small amounts
consumed over a prolonged period of time may exert similar beneficial effects in
human beings. Understanding the role of CLA in modulating events associated with
macronutrient metabolism suggests that CLA may be a healthy dietary component
with the potential for impacting human health in the areas of cancer, obesity,
diabetes, and cardiovascular disease. However, more work is needed to fully eluci-
date the safety and efficacy of isomers and doses that are required for exerting this
breadth of potential beneficial effects. It is hoped that with improved understanding
of the doses and isomers required, improvements in recommendations may be made
to people regarding the intakes of CLA to improve health.
In this context, the role of dietary CLA in human cancer risk is probably the most
interesting one. There are some data showing a favorable effect of CLA on human
cancer cells. More extensive evidence from human studies is generally lacking,
although there are a few epidemiologic studies that have focused on evaluating the
role of dairy products in relation to various cancers, and at least one has found an
inverse relationship between milk intake and breast cancer.
Among anticarcinogens, CLA is unique for the following reasons:
• It is an animal-derived product with its highest levels found in ruminant meat and
dairy products.
• It exerts a potent action through dietary intervention under a wide range of
conditions.
• It possesses multiple modes of action.
The time has come to conduct clinical studies with CLA in order to judge its
potential as a chemopreventative agent for humans.
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Part IV
Food Carbohydrates and Derived Compounds
Total Dietary Fiber Intake, Whole Grain
Consumption, and Their Biological Effects 24
Semih Otles and Emine Nakilcioglu-Tas
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702
2 Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
2.1 Definitions of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
2.2 Classification, Sources, and Physiological Effects of Dietary Fiber . . . . . . . . . . . . . . . . . 704
2.3 Recommendations for Dietary Fiber Intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
3 Whole Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
3.1 Definition and Intake of Whole Grains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
3.2 Biochemical Constituents of Whole Grains and Their Roles in a Healthful Diet . . . 708
4 Biological Effects of Dietary Fiber Intake with Consumption of Whole Grains . . . . . . . . . . 711
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718
Abstract
Unlike refined grains, whole grains, which consist of entire grain, contain high
micronutrients and dietary fiber in their bran and seed. In the literature many
studies showed that high-fiber diets may reduce the incidence of chronic diseases
such as diverticulitis, diabetes, obesity, heart diseases, and some cancer types.
Once upon a time, whole grains were neglected by researchers. Following the
determination that dietary fibers are present in the whole grains together with
micronutrients and phytochemicals, the focus of the studies has shifted towards
observational studies related to whole grains intake. Cereal fibers have proven to
have stronger health effects as a result of synergistic effects with phytochemicals
and micronutrients in whole grains. This chapter describes the dietary fiber-
related health effects of whole grain consumption after mentioning characteristics
of dietary fiber and whole grains.
S. Otles (*) · E. Nakilcioglu-Tas

Department of Food Engineering, Faculty of Engineering, Ege University, Bornova, Izmir, Turkey

702 S. Otles and E. Nakilcioglu-Tas
Keywords
Cardiovascular diseases · Cereal fibers · Diabetes · Dietary fiber · Whole grains
Abbreviations
AACCI American Association of Cereal Chemists International
BMI Body mass index
CAD Coronary artery disease
CCNFSDU Codex Committee on Nutrition and Foods for Special Dietary Uses
DRI Dietary Reference Intakes
FAO/WHO Food and Agriculture Organization/World Health Organization
FDA Food and Drug Administration
FOS Fructooligosaccharide
GM Galactomannan
IDF Insoluble dietary fiber
MCAD Minimum coronary artery diameter
NTD Neural tube defects
OD Odds ratio
RDA Recommended Dietary Allowance
RR The effect size across studies
SDF Soluble dietary fiber
US United States
1 Introduction
Nutrition makes a significant contribution to the etiology, prevention, and progres-

sion of disease [1–4]. Diets such as the Mediterranean-type dietary patterns, which
have high in fruits, vegetables, whole grains, and proteins from plant sources and
lower in meat and meat products have been shown to have long-term beneficial
effects on health [4–7]. It is not just happenstance that longevity is also characterized
by similar dietary patterns, even though the influence of other lifestyle factors cannot
be ignored [4, 8]. High levels of dietary fiber, unsaturated fatty acids, antioxidants,
and foods that have low energy density are some of the protective components of the
diet [4, 9]. The all bioactive components of the diet work together for promoting
health.
One of the most important constituents in foods that have beneficial physiological
effects in humans is dietary fiber. The consumption of dietary fiber has decreased
considerably as a result of the fact that the foods have begun to be processed and
refined more in the last two centuries [10, 11]. It has been determined that increased
consumption of low-fiber foods in the diet as a result of the transition from high-fiber
diets to low-fiber diets in human nutrition may lead to an increase in the prevalence
24 Total Dietary Fiber Intake, Whole Grain Consumption, and Their Biological. . . 703
of chronic diseases such as diverticulitis, diabetes, obesity, heart diseases, and some
cancer types [11, 12]. After this hypothesis, there has been an increase in interest and
researches related with dietary fiber and foods which are thought to be important
sources of dietary fiber [11].
Whole grains are rich sources of vitamins, minerals, and phytochemicals as well
as fiber. The phytochemicals of whole grains contain phenolics, carotenoids, vitamin
E, lignans, β-glucan, inulin, resistant starch, sterols, phytates, etc. Plant-based foods
like fruits, vegetables, and whole grains, which include significant amounts of
bioactive compounds, can also meet desirable health benefits beyond basic nutrition
to decrease the risk of some diseases [13–15]. Epidemiological studies ensure
evidence that whole foods, including whole grains, are protective against a broad
range of diseases, and this protectiveness is usually greater than that seen with any
individual ingredient [16, 17]. Dietary guidance suggests consumption of whole
grains to decrease the risk of chronic diseases such as cancer and cardiovascular
disease (CVD) [18]. Epidemiologic studies declare the opinions that whole grains
are protective against diabetes, obesity, CVD, and cancers, especially gastrointesti-
nal cancers including gastric and colonic [16, 18]. According to the results of
research among a large group of women aged 55 to 69 years, eating at least one
serving a day of whole grain foods significantly decreased the risk of mortality from
all causes compared with women who ate almost no whole grain products [19, 20].
The protective effect of dietary fiber taken from whole grain foods on diseases is
much greater than the individual effect of dietary fiber. It is thought that this is caused
by the synergistic effect of phytochemicals with dietary fiber in whole grain foods.
Therefore, it is more beneficial that dietary fiber intake by consuming complex food
sources such as whole grains for human health.
In this chapter, we describe the definitions and intakes of dietary fiber and whole
grains and health effects of dietary fiber intake by consuming whole grains.
2 Dietary Fiber
2.1 Definitions of Dietary Fiber
The definition of dietary fiber was used in 1953 by Hipsley to show the nondigestible
constituents which consist of the plant cell wall [21–23]. Since then, the term has
evolved and the Codex Committee on Nutrition and Foods for Special Dietary Uses
(CCNFSDU) established an internationally has approved the legal definition of
dietary fiber in 2009 [23–25]. According to CCNFSDU, dietary fiber is defined as
carbohydrate polymers with ten or more monomeric units, which are not hydrolyzed
by the endogenous enzymes in the small intestine of humans and belong to the
following three categories:
(1) Edible carbohydrate polymers that are naturally present in the food as it is
consumed.
(2) Carbohydrate polymers that have been obtained physically, enzymatically, or

chemically from food raw material and that have been shown to have beneficial
physiological effects on health as proved by scientific evidence are accepted by
competent authorities.
(3) Synthetic carbohydrate polymers that have a physiological effect on the health
benefit as indicated by generally accepted scientific evidence by the competent
authorities [24].
While the expression “dietary fiber” describes the nondigestible carbohydrates

and lignin that are intrinsic and intact in plants, the term “functional fiber” expresses
the isolated nondigestible carbohydrates that have beneficial physiological effects in
human body [26]. When inulin is formed naturally in consumable plants such as
onions and chicory, it is defined as a dietary fiber; if it is synthetically produced and
added to yogurt, it is categorized as a functional fiber [21–23]. Total fiber is also the
sum of dietary fiber and functional fiber, and dietary fiber on food labels includes
both dietary and functional fiber [23]. The expression “nondigestible” means both
not digested and not absorbed in the human small intestine [26].
2.2 Classification, Sources, and Physiological Effects of Dietary

Fiber
According to the solubility of dietary fiber, it is classified into two categories:

insoluble dietary fiber (IDF) such as cellulose, part of hemicelluloses, resistant
starch, and lignin; and soluble dietary fiber (SDF) such as oligosaccharides, includ-
ing fructooligosaccharide (FOS), pectins, β-glucans, galactomannan (GM) gums,
alginate, and psyllium [23, 24, 27, 28].
2.2.1 Soluble Dietary Fiber

SDF is present in the form of a sticky or viscous, rather than a hard tissue in the food
[11, 29]. Although SDF are found in fruits and vegetables, the best sources of SDF
are oats and dried beans [30]. FOS, also known as oligofructose and inulin, are
collectively entitled as fructans [24, 27]. They are found in plants such as agave,
artichokes, asparagus, leeks, garlic, onions, yacon, jicama, and wheat.
Pectin is found in most primary cell walls and is particularly rich in the nonwoody
parts of terrestrial plants. It is a linear polysaccharide mainly consisted of about 300
to 1000 D – galacturonic acid monosaccharide units [24, 31]. Although fruits are the
major source, pectins represent 15–20% of the fiber in vegetables, legumes, and nuts
[24, 32].
The β-glucans are polysaccharides of d-glucose monomers linked by β-glycosidic
bonds, which occur most commonly as cellulose in plants, the bran of cereal grains,
the cell wall of baker’s yeast, certain fungi, mushrooms, and bacteria [24, 31].
GMs are also polysaccharides comprising of a mannose backbone with galactose
side groups. They are commonly used in foods as stabilizers owing to their high
water binding capacity and their emulsification and viscosity increasing properties.
GM gums, such as fenugreek gum (mannose:galactose; 1:1), guar gum (mannose:

galactose; 2:1), tara gum (mannose:galactose; 3:1), and locust bean gum (mannose:
galactose; 4:1), change according to their ratios of mannose and galactose [24, 33].
Alginates are unbranched polysaccharides consisting of 1–4 linked β-d-
mannuronic acid and α-guluronic acid. Alginate is distributed widely in the cell
walls of algae. It is also an exopolysaccharide of bacteria including Pseudomonas
aeruginosa and commercially available alginates now come only from algae. It is
binding with water and it forms which use as thickening agent. These viscous
hydrogels are also useful for numerous biomedical applications [24, 34].
Psyllium is the common name used for several members of the plant genus
Plantago. The seeds of this plant are used commercially for the production of
mucilage. The phrase of psyllium is used for the seed husk, the seed, and the entire
plant. The seed husk of Psyllium is a rich source of SDF that is known as psyllium
hydrophilic mucilloid, psyllium hydrocolloid, and psyllium seed gum [24, 31].
Some SDF components including oat bran, pectin, and guar gum stimulate fecal
excretion of bile acids. On the other hand, wheat bran has no such effect; it promotes
a different composition of bile acids than does pectin [35–38]. Some SDFs also
reduce serum cholesterol level [38, 39]. Although short-term studies (a single meal
or a few days) show that SDFs enhanced glucose tolerance and increased insulin
sensitivity in healthy subjects, the results of long-term studies do not support them
[38, 40]. Dietary fiber influences colonic function and activities of the microflora.
High fiber intakes increase bacterial mass but do not change the microflora compo-
sition [38, 41–43]. Colonic bacteria attack fermentable fiber components and then at
least a portion of them degrades to short-chain fatty acids and gases [38]. These
short-chain fatty acids decrease the colon cancer risks by suppressing toxigenic
bacterial reactions [44, 45]. Some studies also express that SDFs prevent type II
diabetes owing to the viscosity of the fibers [24, 46].
2.2.2 Insoluble Dietary Fiber

IDFs include cell wall components like cellulose, lignin, and hemicellulose that exist
mainly in wheat, most grain products, and vegetables [30]. Cellulose is the best
known, most widely distributed, and only truly fibrous component of the plant cell
wall. It forms about 25% of the fiber in grains and fruit and about a third of the fiber
in vegetables and nuts [24, 32]. It is a polymer that consists of glucose and the β-
glucoside linkages. The β-linkages in cellulose are not hydrolyzed by the human
intestinal enzymes, so cellulose is accepted as a dietary fiber. Cellulose also has the
property of absorbing water (0.4 g water per gram of cellulose), and this explains its
ability to increase fecal weight when added to the diet [47].
Hemicelluloses are polysaccharides containing sugars other than glucose, which
are associated with cellulose in cell walls. They present in both water soluble and
insoluble forms in cell walls. Hemicellulose constitutes about a third of the fiber in
vegetables, fruits, legumes, and nuts. The primary dietary sources of hemicellulose
are cereal grains [24, 32].
Lignin is a lipophilic phenolic polymer that can absorb bile acids [48]. It is
composed of phenylpropane units which are thought to be chemically bound to
the carbohydrates in woody structure [49]. It presents only in small amounts in the
diet [26]. Good sources of lignin are foods with a woody component such as celery
and the outer layers of cereal grains [24].
Resistant starch (the sum of starch and starch-degradation products that do not
digest in the small intestine) performs a function as dietary fiber, after it reaches the
large intestine [26, 50]. Legumes are main sources of resistant starch and 35% of
legumes starch is escaping digestion [26, 51]. Small amounts of resistant starch are
formed by processing and baking of cereal and grain products. Actually, many new
functional fibers are resistant starches which are added to processed foods [26].
The function of IDFs is to shorten bowel transit time, to increase fecal bulk, and to
render feces softer [30]. The reasons for these are their high water-holding capacities
and having insoluble characteristics [48]. Addition of some IDF sources in the diet
may also cause a decrease in mineral retention [52].
Although about 75% of the dietary fiber in foods is found as the insoluble
fraction, products known as soluble or insoluble fiber sources are often sources in
both of these fiber types [30]. The energy value of dietary fiber has detected as 0 kcal
g-1 if it is insoluble and 4 kcal g-1 if it is soluble [53].
2.3 Recommendations for Dietary Fiber Intake
Consuming foods with dietary fiber is very important for enhancing bowel function
and reducing symptoms of chronic constipation, diverticular disease, and hemor-
rhoids. There are increasing proofs that a diet rich in these foods can reduce not only
high blood cholesterol risk but also hypertension, stroke, and cancer risks [54]. On
the other hand, people who consume low dietary fiber and complex carbohydrates
and high fat, especially saturated fat, in diets incline to have more heart disease,
obesity, and some cancers [30].
General recommendations for dietary fiber intake range between 25 and 35 g per
day (g d-1) for adults. The data found in guidelines for dietary fiber consumption of
children are the extrapolated data from studies in adults. According to the Dietary
Reference Intakes (DRI), the average intake of people of all aged should be 14 g per
1000 kcal; for children 1 to 3 years of age, average intake should be 19 g d-1; and for
children 4 to 8 years of age, average intake should be 25 g d-1. The American
Academy of Pediatrics suggests dietary fiber intake of 0.5 g per kg body weight for
children older than 2 years. In 1995, American Health Foundation developed the rule
of “age plus five” which indicates that intake of dietary fiber should be greater than
or equal to the age plus 5 g d-1 for children over 2 years [22, 23, 55]. Despite the
recommendations for dietary fiber intake, the average dietary fiber intake among
people is lower than it should be. It changes from 9.4 4.0 g d-1 to 10.7 3.6 g d-1
in females, and 10.7 4.2 g d-1 to 12.0 4.5 g d-1 in males [23].
Even though many benefits of high fiber diets are also naturally found in the
nondietary fiber components such as natural antioxidants, it is best to take dietary
fiber from foods in high amounts. But dietary fiber consumption with food is almost
half the recommended level, as can be seen. For this reason, it is thought that the
consumption of fiber supplements may be beneficial when used moderately [30].
3 Whole Grains
3.1 Definition and Intake of Whole Grains
According to the guidelines of Food and Drug Administration (FDA), “whole grain”
contains “cereal grains that consist of the intact and unrefined, ground, cracked or
flaked fruit of the grains whose principal components – the starchy endosperm, germ
and bran – present in the same relative proportions as they exist in the intact grain”
[56]. The more common whole grain cereals are whole wheat, whole oats/oatmeal,
whole grain cornmeal, popcorn, brown rice, whole rye, whole-grain barley, wild rice,
triticale, millet, and sorghum, while emmer, farro, spelt, and wheat berries are less
common whole grain cereals consumed worldwide [57].
Although American Association of Cereal Chemists International (AACCI) had
identified “whole grains” in a similar way with FDA, in 2006 the AACCI expanded
this definition to include pseudocereals. Pseudocereals such as amaranth (Amarantus
caudatus), quinoa (Chenopodium quinoa Willd.), and buckwheat (Fagopyrum spp.)
were included in the definition of “whole grains” because of their macronutrient
composition and consumption trends similar to that of cereals. Also, some tradition-
ally and minimally processed forms of whole grains including lightly pearled barley
or wheat, bulgur (cracked wheat), and nixtamalized corn are considered whole grains
[58, 59].
In April 2013, the AACCI further described a whole-grain product as being one
which must include 8 g or more of whole grain per 30 g of product [4, 59]. The FDA
acknowledged the whole grain definition created by AACCI but stated that at least
51% of the total weight of the product must be whole grains in order to qualify for
whole grain health claims [4, 60]. Also, whole grain food must contain 1–7 g dietary
fiber [61].
Whole grains can be consumed in whole, cracked, split, flaked, or ground. They
are often milled into flour that is used to make breads, cereals, pasta, crackers, and
other grain-based foods. Regardless of how the whole grain is processed, the pro-
portions of bran, germ, and endosperm in a whole grain product must be approxi-
mately the same as an original grain [57]. A whole grain can be a complete food such
as brown rice or can be also a food ingredient such as whole wheat flour in bread or
cereal.
The 2005 Dietary Guidelines for Americans and Healthy People 2010 advises the
consumption of at least three servings of whole grain (each equivalent to three
ounces) per day [15]. Additionally, dietary guidelines expresses that the
recommended grains should come from enriched or whole grain products. Generally,
at least half the consumed grains should come from whole grains. Moreover, their
consumption is low in the United States (US), mostly owing to taste, cost, and poor
eating habits [56]. It is noteworthy that grains intake in Europe and North America is
even lower than in less developed countries, although grains ensure approximately
two thirds of the calorie and protein intake in the world [62, 63]. Ninety percent of
Americans do not meet the recommendation related with whole grain consumption,
and the mean intake of whole grains in the United States is less than one serving per
day [15, 64]. Even in Denmark, which has one of the highest whole grain intake in
the world, only about 6% of people consume seven or more servings of whole grains
[65].
3.2 Biochemical Constituents of Whole Grains and Their Roles in

a Healthful Diet
Whole grains are composed of three main parts as starchy endosperm, germ, and
bran. In order for a grain to be able to be characterized as a whole grain, it must
contain all three parts mentioned above. The bran and germ contain the dietary fiber
and most of the biologically active components, whereas endosperm is mainly
structure of starch and protein [66]. About 50–75% of the endosperm is starch
which is the major energy supply for the embryo during germination of the kernel.
About 8–18% of the endosperm is storage proteins along with cell-wall polymers.
Relatively few vitamins, minerals, fiber, or phytochemicals are found in the endo-
sperm fraction [61]. The bran contains the aleurone that protects the grain from
bacteria, molds, insects, and severe weather [66]. The germ contributes quite small to
the dry weight of most grains, for example, 4–5% in wheat and barley. The germ of
maize provides a much higher contributor to the total grain structure than that of
wheat, barley, or oats [61].
Whole grains, except for rice, are high in dietary fiber and amino acids (arginine,
lysine, etc.), low in fat, concentrated sources of starch, high in vitamins, particularly
vitamins B (thiamin, niacin, riboflavin, pantothenic acid, etc.), and good sources of
minerals (Ca, Mg, K, P, Na, Fe, etc.) [18, 61, 67]. Other components in whole grains
associated with improved health status include tocotrienols, lignans, phytoestrogens,
phenolic compounds (predominantly ferulic acid and diferulates), phytic acid, and
some unique compounds to grain products (avenanthramides, avenalumic acid) [15,
18, 61, 67–69]. They are responsible for the high antioxidant activity of whole grains
[61, 67]. The types and varieties of grains effect the concentration of phytochemicals
in whole grains [15, 70]. Table 1 shows a comprehensive list of biochemical
constituents in some whole grains products.
During the milling process, the bran and germ are separated from the starchy
endosperm and then the starchy endosperm is ground to flour. Refined flours have
lower nutrients than whole grains because of the higher concentrations of nutrients in
the outer part of the grains [62, 63]. Milling process may open up the food matrix,
thereby let the release of firmly bound phytochemicals from the grain structure [61].
Milling of grains results in significant nutrient loss [18, 63].
Grains are usually subjected to some types of processing such as cooking and
parboiling before consumption in developed countries. Commercial cereals are
generally extruded, puffed, flaked to make a desirable product. Most researchers
24
Table 1 Energy, macronutrient, fiber, some vitamin and mineral contents of common whole grain products [71]
Whole grain products Serving size Energy Carbohydrate Protein Fat (g) Fiber (g) Vitamin E Folate
(kcal) (g) (g) (mg) (μg)
Bread, whole wheat 1 slice 81 13.67 3.98 1.12 1.9 0.850 13
English muffin, whole wheat 1/2 muffin 67 13.33 2.90 0.69 2.2 0.140 16
Bread pita, whole wheat 1,4 in. 74 15.40 2.74 0.73 2.1 0.170 10
Diameter
Crackers, whole wheat 6 crackers 118 19.20 2.92 3.90 2.8 0.390 8
Oats, regular or quick, cooked 1/2 cup 83 14.04 2.97 1.78 2.0 0.090 7
with water
Ready-to-eat cereal, all bran 1/2 cup 81 23.01 4.07 1.52 9.1 0.380 406
Rice, brown, medium grain, 1/2 cup 109 22.92 2.26 0.81 1.8 – 4
cooked
Spaghetti, whole wheat, cooked 1/2 cup 87 18.58 3.73 0.38 3.2 0.210 4
Popcorn, air popped 3.5 cups 108 21.78 3.62 1.29 4.1 0.080 9
Thiamin Riboflavin Niacin Pantothenic acid Pyridoxine Ca (mg) Fe (mg)
(mg) (mg) (mg) (mg) (mg)
Bread, whole wheat 1 slice 0.126 0.053 1.420 0.207 0.069 52 0.79
English muffin, whole wheat 1/2 muffin 0.099 0.046 1.125 0.229 0.054 87 0.81
Bread pita, whole wheat 1,4 in. 0.095 0.022 0.795 0.233 0.074 15 0.86
Diameter
Crackers, whole wheat 6 crackers 0.050 0.006 1.278 0.230 0.051 10 0.92
Oats, regular or quick, cooked 1/2 cup 0.089 0.019 0.262 0.363 0.006 11 1.05
with water
Total Dietary Fiber Intake, Whole Grain Consumption, and Their Biological. . .
Ready-to-eat cereal, all bran 1/2 cup 0.704 0.840 4.588 0.329 3.720 121 5.46
Rice, brown, medium grain, 1/2 cup 0.099 0.012 1.297 0.382 0.145 10 0.52
cooked
(continued)
709
710
Table 1 (continued)
Spaghetti, whole wheat, cooked 1/2 cup 0.076 0.032 0.495 0.293 0.055 10 0.74
Popcorn, air popped 3.5 cups 0.029 0.023 0.646 0.143 0.044 2 0.89
Mg (mg) P (mg) K (mg) Na (mg) Zn (mg) Cu (mg) Mn
(mg)
Bread, whole wheat 1 slice 24 68 81 146 0.57 0.073 0.696
English muffin, whole wheat 1/2 muffin 23 93 69 120 0.53 0.070 0.591
Bread pita, whole wheat 1,4 in. 19 50 48 124 0.43 0.081 0.487
Diameter
Crackers, whole wheat 6 crackers 30 91 95 194 0.73 0.016 0.594
Oats, regular or quick, cooked 1/2 cup 32 90 82 5 1.17 0.087 0.679
with water
Ready-to-eat cereal, all bran 1/2 cup 112 356 316 80 3.84 0.322 2.297
Rice, brown, medium grain, 1/2 cup 43 75 77 1 0.6 0.079 1.070
cooked
Spaghetti, whole wheat, cooked 1/2 cup 21 62 31 2 0.57 0.117 0.965
Popcorn, air popped 3.5 cups 40 100 92 2 0.86 0.073 0.312
It is adopted from “USDA National nutrient database for Standard Reference 26. Nutrient Data Laboratory, https://www.ars.usda.gov/northeast-area/beltsville-
md-bhnrc/beltsville-human-nutrition-research-center/nutrient-data-laboratory/”
S. Otles and E. Nakilcioglu-Tas
determined that biologically important compounds, like antioxidants, in the pro-

cessed whole grains are not removed [61, 72]. According to the results of study with
rye, many of the bioactive compounds are stable during food processing, and there
can be an increase in their ratios with appropriate processing [61, 73].
Whole grains include significant amounts of bioactive phytochemicals that can
provide decreasing the risk of chronic diseases [14, 15, 64, 74]. The phytochemicals
in whole grains can be protective or can act synergistically to exhibit these protective
effects [19]. The recent evidence shows that the complex mixture of bioactives in
whole foods can be more healthful than individual isolated components [15, 74]. The
beneficial effects related with whole grain consumption originate due to the presence
of the unique phytochemicals in whole grains [15].
According to 2005 Dietary Guidelines, refined and whole grains are important
sources of carbohydrates in particular. Carbohydrates provide energy to the body in
the form of glucose. Glucose is the only energy source for red blood cells and the
preferential energy source for the brain and central nervous system, and preferred
energy source for the placenta and fetus during pregnancy. Carbohydrates in grains
are mainly found in the form of starches and some fibers. Carbohydrates in grains are
mainly found in the form of starches and some fibers. The US Recommended
Dietary Allowance (RDA)’s recommendation for daily carbohydrate consumption
is 130 g per day for adults and children. If glucose is not present in the diet or
glycogen is depleted, which is the body’s storage form of glucose, the body will
transform protein to glucose for supplying the essential fuel to the brain and protect
blood glucose levels [57]. Protein destruction will imperil the life and vital activities.
Compared to refined grains, whole grains are more abundant in nutrients. This is
very important in the intake of some components such as folic acid [57]. Refined
grain products can be fortified with folic acid and the consumption of folic acid is
associated with decreased risk of birth defects, including neural tube defects (NTD),
and heart disease [57, 75, 76]. Most whole grain foods, the exception of both hot and
cold breakfast cereals, do not need to be fortified with folic acid or other vitamins
and minerals. Consumptions of whole grains are more beneficial due to these and
similar situations [57].
4 Biological Effects of Dietary Fiber Intake with

Consumption of Whole Grains
Epidemiological evidences show that the consumption of whole grains is correlated

with reduced risk of chronic diseases such as CVD, diabetes, the metabolic syn-
drome, and certain cancers. Also, whole grain consumption can play a positive role
in weight management [4]. Some examples of similar studies are given below.
Relevant evidence was assessed by Montonen et al. (2003) [77], which high fiber
intake with whole grain foods reduced the risk of type 2 diabetes. A cohort consisted
of 4316 Finnish men and women aged 40–69 years were followed for 10 years and
an inverse association between the intake of whole grain foods and the risk of type 2
diabetes was found. A similar relation was not found for the intake of fiber from
vegetables and fruit. The risk reduction of type 2 diabetes as a result of whole grain
intake was approximately 35%.
Newby et al. (2007) [78] examined associations between the consumptions of
whole grains, refined grains, and cereal fiber with weight management. It had been
observed that compared with subjects in the lowest quintile of whole grain intake,
subjects in the highest quintile had lower body mass index and weight and smaller
waist circumference. It was determined that whole grains and grain fiber had similar
and collective effects on weight, body mass index (BMI), waist circumference
changes. It was thought that the cereal fiber and its constituents could in part mediate
these relations. In the same study, the associations between the consumptions of
whole grains, refined grains, and cereal fiber with both cholesterol and blood glucose
level changes were also evaluated. According to the results of study, the consump-
tion of whole grains was inversely related with total cholesterol and LDL cholesterol
levels and 2-h glucose amount. Although the effects of cereal fiber on cholesterol
and glucose levels were similar with whole grains, the refined grains were increased
fasting insulin among women but not men.
Intakes of whole grains and cereal fiber were inversely associated with the risk of
chronic diseases. But their relation with total and disease-specific mortality remains
unclear. In the study of Huang et al. (2015) [79], it was aimed to evaluate the
association of whole cereals and cereal fiber intake with all causes and cause-specific
mortality. The study included 367,442 participants that followed from 1995 to 2009.
Participants with cancer, heart disease, stroke, diabetes, and self-reported end-stage
renal disease at baseline were not accepted to the study. A total of 46,067 deaths were
documented over an average of 14 years of follow-up. At the end of the study, it was
observed that consumption of whole grains were inversely related with risk of all-
cause mortality and death from CVD, cancer, diabetes, infections, respiratory dis-
ease, and other causes. When individuals were compared according to their whole
grain and cereal fiber intakes, the lowest all-cause mortality risk was found to be
17% for those who had the highest intake of whole grains and 19% for those who had
the highest intake of cereal fiber. The lowest disease-specific mortality risk was also
determined to be 11–48% for those who had the highest intake of cereal fiber and
15–34% for those who had the highest intake of whole grains. The associations
between whole grains consumption with the deaths from CVD, respiratory disease,
and infections were not found significant, but the associations between all-cause
mortality with deaths from cancer and diabetes were significant ( p < 0.029). In the
result of study, it was determined that consumption of whole grains and cereal fiber
could inversely associate with reduced total and cause-specific mortality and cereal
fiber was one potentially protective component in whole grains.
The effect of fermented whole grain cereal kernels with a high content of amylose
(40%) and/or beta-glucan (4.6%) on postprandial glucose and insulin responses in
healthy adults was evaluated by Alminger and Eklund-Jonsson (2008) [80]. For this
purpose, a glucose solution or tempe fermented whole-grain barley or tempe
fermented whole grain containing 25 g carbohydrates randomly was given to 13
healthy volunteers between the ages of 20 and 75. Blood samples were collected
immediately before meal and at 15, 30, 45, 60, 90, and 120 minutes after meal
started. For each individual according to Food and Agriculture Organization/World

Health Organization (FAO/WHO) standards, the glycemic index and insulin index of
the meals were calculated. It was detected that the lowest peak of insulin response
occurred after the high β-glucan oat tempe meal, while the peak of glucose was
lowest after the high-amylose/high β-glucan barley tempe meal. During the first
60 minutes after meal, the average blood glucose and insulin responses for both
barley and oatmeal dishes were significantly lower than for the reference glucose
load ( p < 0.05). The glycemic indexes calculated for the barley and oat tempe were
30 and 63, respectively. The obtained data suggested that whole grain products that
reduce postprandial glucose and insulin responses could be got by fermentation and
by using grains with an increased amylose and/or β-glucan content.
Systematic review and meta-analysis of prospective observational studies were
performed by Aune et al. (2011) [81] for determining the association between intake
of dietary fiber and whole grains with risk of colorectal cancer. For these reasons,
PubMed and several other databases up to December 2010 and the reference lists of
studies included in the analysis as well as those listed in published meta-analyses
were used as data sources. Among them, prospective cohort and nested case-control
studies for dietary fiber or whole grain intake and incidence of colorectal cancer were
examined. According to the investigated 25 prospective studies, the relative risk
of developing colorectal cancer for 10 g daily consumption of total dietary fiber
(16 studies) was 0.90 ( p < 0.05), for fruit fiber (9 studies) was 0.93, for vegetable
fiber (9 studies) was 0.98, for legume fiber (4 studies) was 0.62, and for cereal fiber
(8 studies) was 0.90. The relative risk of increase in colorectal cancer in the case of
three servings of daily whole grains consumption (six studies) was 0.83. In the study,
a high intake of dietary fiber, especially cereal fiber and whole grains, resulted in a
reduced risk of colorectal cancer.
Chen et al. (2016) [82] who believed that the association between whole and
refined grain consumptions and stroke risk remained unclear conducted a study
using the MEDLINE and EMBASE databases until February 29, 2016. Seven
prospective studies with a total of 446,451 individuals and 5892 stroke cases were
examined. Compared to low consumption, the relative risk of stroke in the high
consumption was 0.95 for total grains, 0.92 for whole grains, and 0.99 for refined
grains ( p < 0.05). Diets rich in whole grains were inversely associated with ischemic
stroke risk whose relative risk was determined as 0.75 ( p < 0.05). Meta-analysis
expressed that the consumptions of whole and refined grain were not associated with
total stroke risk, but whole grain consumption was associated with decreased
ischemic stroke risk. It was clear that this was caused by the high dietary fiber
contents of whole grains.
Although Hollænder et al. (2015) [83] acknowledged the potential role of whole
grains in the prevention of CVD, they believed that the results of randomized
controlled studies on blood lipids were inconsistent due to the compositional differ-
ences of some foods containing dietary fiber. They evaluated the influence of whole
grains on changes in levels of total cholesterol (TC), LDL cholesterol, HDL choles-
terol, and triglycerides compared with non–whole grain foods by using a meta-
analytic approach. In the study which contained systematic literature review of
selected databases, randomized controlled comparisons were performed between

whole-grain foods and a non–whole grain control in adults. A total of 6069 articles
were screened and data were extracted from most suitable 24 studies. Generally,
whole-grain intake reduced LDL cholesterol (weighted mean difference:
0.09 mmol L-1) and TC levels (weighted mean difference: 0.12 mmol L-1)
compared to the control ( p < 0.05). The greatest effect on TC (weighted mean
difference: 0.17 mmol L-1) was observed in the whole grain oat consumption.
Whole grain foods tended to lower triglycerides compared with the control
(weighted mean difference: 0.04), whereas no effect of whole grain foods on
HDL cholesterol was observed. Also, no relation was found between whole-grain
dose or baseline TC concentration and any of the outcomes, while study duration
was positively related with changes of TC and LDL cholesterol levels. Researchers
proved that the consumption of whole grain diets lowered LDL cholesterol and TC
but did not lower HDL cholesterol or triglycerides. They also indicated that whole
grain oats were the most effective grain to lower cholesterol.
Chen et al. (2016) [84] assessed the associations between whole-grain intake and
risk of dying from any cause such as CVD, and cancer with a meta-analytic
approach. Relevant works on subjects were determined by researching PubMed
and EMBASE databases and bibliographies of full publications. In the study, 13
studies about total mortality (104,061 deaths), 12 studies about CVD mortality
(26,352 deaths), and 8 studies about cancer mortality (34,797 deaths) were used.
Three of these studies were related to whole-grain intake, while the remaining
studies were related to whole-grain products intake. Summary RRs were also
calculated using the random effect model at the 95% confidence interval and were
used as the effect size across studies. In the dose-response analysis which converted
the intake of whole-grain products to the amount of whole grain, the summary RRs
for an increment in whole-grain intake of 50 g d-1 were 0.78 for total mortality, 0.70
for CVD mortality, and 0.82 for cancer mortality. A similar decrease in the mortality
from ischemic heart disease (RR: 0.68) was also observed, but the same case was not
observed for stroke (RR: 0.93). Nonlinear relationships between whole grain intake
and total ( p < 0.001) and CVD mortalities ( p < 0.001) was proven. The relation
about cancer mortality ( p = 0.12) was not nonlinear because these relationships
appeared slightly more steep at lower ranges (<35 g d-1) of the intake compared to
higher ranges. The findings supporting the significant inverse relations between
whole grain intake and mortality related to any cause, CVD or cancer, suggested
that whole grain consumption could be increased to improve public health.
Whole grains had attracted attention due to their potential role in weight regula-
tion. A high intake of whole grains was related with smaller weight gain according to
prospective cohort studies, while the results from randomized controlled studies
were less consistent. Using a meta-analytic approach, Pol et al. (2013) [85] evaluated
the influences of whole grains on changes in body weight, percentage of body fat,
and waist circumference compared with non–whole grain foods. A systematic
literature search was conducted in selected databases and 26 studies comparing
whole grains and non–whole grain control consumptions in adults were defined.
According to data from 2060 participants, it was found that whole grain intake had
no effect on body weight (weighted mean difference: 0.06 kg) but had a little effect
on body fat percentage (weighted mean difference: 0.48%) compared to a control
( p < 0.05). Whole grain consumption did not reduce body weight compared to
control consumption, but it could create a small beneficial effect on body fat. It was
thought that the significant differences in body weight and fat were not determined
because of the relatively short duration of intervention studies (16 weeks).
Behall et al. (2006) [86] studied the effect of a whole grain diet containing brown
rice/whole wheat and/or barley on blood pressure. For this purpose after a 2-week
controlled diet, 16 participants consumed a diet which had been replaced by 20% of
its energy with whole wheat/brown rice, barley, or half whole wheat-brown rice/half
barley during 5 weeks each. Participants were selected from those with systolic
blood pressure <140 mmHg, diastolic blood pressure <90 mmHg, and cholesterol
levels between 200 and 240 mg dL-1. While the measurements of blood pressure
were performed weekly, their weights were determined daily before breakfast. As a
result of the study, it was seen that systolic blood pressure decreased by 2.2 mmHg
during the control diet and an extra 1.4 to 6.7 mmHg in the whole grain diets.
Diastolic blood pressure also decreased by 2 mmHg in the control diet and an extra
2.9 to 3.7 mmHg in the whole grain diets. The replacement of white rice with brown
rice, white bread with whole grain bread, and low fiber cereals with barley or whole
wheat cereals resulted in reduced systolic and diastolic blood pressure in mildly
hypercholesterolemic men and women, and whole grain diets helped to lower blood
pressure.
Pancreatic cancer was known as the most fatal cancer type in the United States.
The intake of grains and cereals within a large population-based case-control study
of pancreatic cancer was evaluated by Chan et al. (2007) [87]. A 131-item semi-
quantitative food frequency questionnaire was applied to 532 cases and 1701
controls. Participants were asked to inform their frequency of individual food
items intake in the 1 year prior to cancer diagnosis (for cases) or 1 year before
interview (for controls). In this study, consumption of whole grain, refined grains,
mixed grains, and sweetened refined grains as well as individual grain items were
studied. Odds ratio (OR) and 95% confidence intervals were calculated as predic-
tions of relative risk. It was observed that participants who consumed > or =2
servings of whole grains daily had a lower risk of pancreatic cancer as compared to
participants who consumed <1 serving per day (OR = 0.60, p < 0.05). Similar
results were obtained for brown rice (OR = 0.72, p < 0.05) and tortillas (OR = 0.56,
p < 0.05). If the consumption of doughnuts and cooked breakfast cereals were > or
=2 servings per week as compared with <1 serving per month, the risk of cancer
increased (OR = 1.8 for doughnuts, p < 0.05; OR = 1.3 for oatmeal/oat bran,
p < 0.05; OR = 2.1 for other cooked breakfast cereals, p < 0.05). Dietary fiber was
inversely related with cancer risk (for highest quartile vs. lowest, OR = 0.65,
p < 0.05). The data obtained from the study supported the hypothesis that higher
consumption of whole-grain or high fiber foods could decrease pancreatic cancer
risk. Refined grains consumption was not also associated with risk.
One of the recommended mechanisms for the protective effects of whole grains
against chronic disease risk was their gut microbiota effect. Fermentation of soluble
fibers in whole grains could possess a prebiotic effect by altering the composition
and/or activity in the gastrointestinal microbiota. To prove this, Costabile et al.
(2008) [88] compared the effectiveness of whole grain wheat and wheat bran to
modulate the gastrointestinal microbiota by using double blind crossover studies.
Thirty-one healthy individuals were chosen randomly to either consume a whole
grain wheat breakfast cereal (48 g day-1) or wheat bran breakfast cereal (48 g day-1)
daily during 3 weeks. After a period of 2 weeks, the participants consumed the other
cereal type for another 3 weeks. It was found that the numbers of fecal bifidobacteria
and lactobacilli, which are beneficial for human health, were significantly higher
after consuming the whole grain cereal as compared to the wheat bran cereal
(9.3 0.4 vs. 8.8 0.4 log10cells g-1 feces, p < 0.001 and 8.7 0.2 vs.
8.4 0.2 log10cells g-1 feces, p < 0.05, respectively). The 2.5-fold increase in the
concentration of ferulic acid (an antioxidant in plant foods) in the blood compared to
the baseline was also observed as a result of consuming both cereals.
Erkkilä et al. (2005) [89] examined the relations between dietary fiber and whole
grain intakes with progression of coronary atherosclerosis in women due to the fact
that increased dietary fiber and whole grain intake was associated with lower risks of
coronary artery disease morbidity and mortality. Regular dietary intake was evalu-
ated at baseline with a validated semi-quantitative food frequency questionnaire.
Angiographic evaluations were made in a cohort of postmenopausal women with
coronary artery disease (CAD) in the Estrogen Replacement and Atherosclerosis
Trial (n = 229) at baseline and after 3 years. The relations between baseline and total,
cereal, fruit, and vegetable fiber intake and changes in coronary artery diameter and
appearance of new lesions were determined. Changes in minimum coronary artery
diameter (MCAD) and mean percent stenosis of women who consumed >6 servings
of whole grains per week as compared to women who consumed <6 servings per
week tended to be smaller after multivariate adjustment ( p = 0.04 and p = 0.07,
respectively). Higher cereal fiber consumption than fruit and vegetable-derived fiber
was associated with a smaller change in MCAD compared to lower cereal fiber
consumption ( p = 0.05). These findings showed that CAD risk was decreased with a
high intake of cereal fiber but not with fruit and vegetable fiber. The results supported
dietary recommendations regarding the consumption of foods containing rich fiber,
especially whole grain products, among postmenopausal women.
Karmally et al. (2005) [90], who believed that Hispanic Americans had higher
risk scores for coronary heart disease than non-Hispanic whites, conducted a study
about the efficacy of fiber-rich foods in management of hypercholesterolemia in
Hispanics. A total of 152 Hispanic Americans aged 30–70 with baseline low-density
lipoprotein cholesterol (LDL-C) levels between 120 and 190 mg dL-1 and triglyc-
erides <400 mg dL-1 joined the study. Participants were randomly chosen to
consume 90 g of corn (no brand specified) or oat (Cheerios) cereal per day for the
next 6 weeks after consuming a National Cholesterol Education Program Step 1 diet
during 5 weeks. The compliance rate among the participants was 100% in the corn
cereal group and 99% in the oat cereal group and their body weight was stable during
the study. While adding the oat cereal in the diet resulted in significant reductions in
total cholesterol ( 10.9 mg dL-1; 4.5%) and LDL-C ( 9.4 mg dL-1; 5.3%),
there were no significant changes in total cholesterol (1.2 mg dL-1) and LDL-C
(1.2 mg dL-1) in the corn cereal group. Also, it was found that every cereal did not
have a significant effect on high-density lipoprotein cholesterol (HDL-C), triglycer-
ides, or apo A-1 plasma levels. According to obtained data, the ready-to-eat oat
cereal was effective in lowering cholesterol levels of men and women with mild to
moderate hypercholesterolemia. An oat cereal can be evaluated as a therapeutic
option together with a cholesterol-lowering diet for cure of mild to moderate
hypercholesterolemia.
It was thought that whole grains could help in preventing overweight and obesity
by increasing satiety and by slowing down starch digestion and absorption. For the
purpose of reaching more exact opinions in this topic, van de Vijver et al. (2007) [91]
assessed the association between consumption of whole grain foods and dietary fiber
with BMI, overweight, and obesity among participants in the Netherlands Cohort
Study in a cross-sectional (n = 4237) and in a prospective (n = 1257) setting. A
validated semi-quantitative food frequency questionnaire was used for assessing the
dietary intake of participants. As the result of the study, the intake of whole grain was
found to be inversely associated with BMI in both men and women after multivariate
adjustment. Compared to normal weight, the risk of obesity in women and men is
lower by 10% (2–16%) and 4% (1–7%), respectively, for each additional gram of dry
whole grain consumption ( p < 0.05). It was estimated that a decrease of 1 unit BMI
was associated with a 33 g day-1 increase in dry whole grain intake, and there was a
slight inverse relationship between baseline fiber intake and weight gain over
20 years. The results of the study with healthy middle-aged participants showed
that men and women with a high whole grain intake had lower BMI, overweight, and
obesity risks as compared to men and women with a low whole grain intake.
5 Conclusion
Cereals are a major source of energy in many countries, and surprisingly less
attention has been paid to the quality of cereals such as “refined” or “whole” in the
different dietary recommendations of different countries. Whereas whole grains have
beneficial effects on glucose-insulin homeostasis, certain types of cancers, blood
lipids, and gastrointestinal health, especially due to their high dietary fiber contents.
When the literature is searched, it is seen that a large mass of evidence on the health
outcomes of whole grains has accumulated in the last 10 or 15 years. The associa-
tions between whole grain intake and a lower risk of major diseases and death can be
explained by several mechanisms. Meta-analysis, which combines the results of
many scientific studies, has some weaknesses due to the fact that there are few
studies for some endpoints, such as mortality from diabetes and infectious diseases,
and it has poor information on the assessment of whole grain intake. Future studies
should improve the evaluation of whole grain intake, which is similarly reported,
using biomarkers to track compliance in randomized trials, and using validated
assessment methods in observational studies. There is a need to further studies
related to the biological mechanisms of health effects of whole grains and the
contribution to health of different whole grain types. For example, the numbers of
studies suggesting that whole grain oats may be more beneficial in CDVs as
comparing to whole grain wheat should be increased, and further research should
be conducted in similar fields. For healthy life, whole grain consumption on a daily
diet should not be forgotten.
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Eur J Clin Nutr 63(1):31–38
Inulin-Type Fructans Application in
Gluten-Free Products: Functionality 25
and Health Benefits
Natalia Drabińska, Cristina M. Rosell, and Urszula Krupa-Kozak
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
2 Structures, Sources, and Functionality of Inulin-Type Fructans . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
3 Health-Related Properties of Inulin-Type Fructans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
3.1 Prebiotic Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
3.2 Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
3.3 Intestinal Morphology and Gut Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
3.4 Body Mass Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
3.5 Lipid Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
3.6 Mineral Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
4 Gluten and the Application of Gluten-Free Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
4.1 Gluten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
4.2 Gluten-Related Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
4.3 Gluten-Free Diet as a Treatment of Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
4.4 Gluten-Free Diet as a Dietary Trend . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739
4.5 Controversy Concerning a Gluten-Free Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739
5 Technological Importance of Gluten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740
6 State of Art of Application of Inulin-Type Fructans in Gluten-Free Products . . . . . . . . . . . . . 742
6.1 Inulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
6.2 FOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
6.3 Synergy Between Inulin and FOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
N. Drabińska (*) · U. Krupa-Kozak

Department of Chemistry and Biodynamics of Food, Institute of Animal Reproduction and Food
Research of Polish Academy of Sciences, Olsztyn, Poland
C. M. Rosell
Food Science Department, Institute of Agrochemistry and Food Technology (IATA-CSIC), Paterna,
Valencia, Spain

724 N. Drabińska et al.
Abstract
The increasing demand on a high-quality gluten-free (GF) products and an
increasing prevalence of GF consumers favors the development of research
aimed to improve the overall quality of GF products. To obtain a functional GF
product providing the additional health benefits, the fortification of GF food is
applied. Recently, inulin-type fructans (ITFs) were proposed as multi-task ingre-
dients of GF products, improving their nutritional and health-related properties.
In this chapter, the most recent studies on GF products in which ITFs were
applied as valuable ingredients affecting the rheological and technological param-
eters of GF products are presented. The literature data with the successful
applications of ITFs in the GF products and with their health beneficial properties,
as presented in this chapter, points to a great potential of ITFs in the GF
technology. The promising evidences of beneficial impact of ITFs on character-
istic of GF goods may contribute to further development and intensified research
on new GF products of superior quality that will be dedicated to people suffering
from gluten-related disorders.
Keywords
Inulin-type fructans · Inulin · Fructooligosaccharides · Gluten-free diet · Gluten-
free products · Prebiotic
AGA Anti-gliadin
ASD Autistic spectrum disorders
ATI Amylase and trypsin inhibitor
BMI Body mass index
CD Celiac disease
DGP Anti-deaminated gliadin
DP Degree of polymerization
EMA Anti-endomysium
FODMAPs Fermentable oligo-, di-, and mono-saccharides and polyols
FOS Fructooligosaccharides
GF Gluten-free
GFD Gluten-free diet
HLA Human leucocyte antigen
IBD Irritable bowel disease
IBS Irritable bowel syndrome
IgG Immunoglobulin G
ISAPP The International Scientific Association of Probitics and Prebiotics
ITFs Inulin-type fructans
JAR Just-about-right
mRNA Messenger RNA
MW Molecular weight
NCGS Non-celiac gluten sensitivity
QDA Quantitative descriptive analysis
25 Inulin-Type Fructans Application in Gluten-Free Products: Functionality and. . . 725
RDA Recommended daily allowances

SCFA Short-chain fatty acids
TLR Toll-like receptor
tTG Anti-tissue transglutaminase
WA Wheat allergy
WDEIA Wheat-dependent, exercise-induced anaphylaxis
1 Introduction
The term “functional food” is defined as a group of products, which may improve the
general condition of the body and decrease the risk of specific diseases [1]. To obtain
functional food products that will provide the additional health benefits above those
arising from its basic composition, a fortification involving the addition of one or
more biologically active compounds or nutrients is applied. Fortification is described
as one of the most effective strategies available to improve micronutrient status [2].
Recently, due to the westernization of diet, increasing prevalence of food intoler-
ances, including gluten intolerance, are noted [3]. In gluten-related disorders, nutri-
tional deficiencies of vitamins, minerals, proteins, and fiber are frequently observed
[4, 5]. The nutritional deficiencies are mainly associated with a lower nutritional
value of the gluten-free (GF) products as compared to the gluten-containing coun-
terparts and because they are rarely fortified [4, 6]. To meet the growing demands of
GF food consumers, the enrichment of GF products aimed to improve their palat-
ability and nutritional value seems to be important and reasonable [7, 8]. Recently,
inulin-type fructans (ITFs) were proposed as multi-task ingredients of GF products,
improving their nutritional and health-related properties [9].
2 Structures, Sources, and Functionality of Inulin-Type

Fructans
Generally, fructans are water-soluble mixtures of oligo- or polysaccharides

containing at least two adjoining fructose units. The molecule of glucose can be
linked by α-(1–2) linkages, similar as in sucrose molecule, at the beginning of chain
but it is not obligatory. Fructans can vary based on the degree of polymerization
(DP) and chemical structure. In higher plants, five types of fructans can be distin-
guished based of their structure: inulin-type fructans, inulin neoseries-type fructans,
levan-type fructans, levan neoseries-type fructans and mixed levan-type fructans [9]
(Fig. 1). ITFs and levan-type fructans are ketose-based fructans with mostly or
exclusively β-(2–1) and β-(2–6) fructosyl-fructose linkages, respectively. Therefore,
both groups of fructans are mostly linear; however, a small amount of branched units
with β-(2–6) and β-(2–1) configuration for ITFs and levan-type fructans, respec-
tively, can occur [10]. Neoketose is a base for both inulin neoseries-type fructans and
levan neoseries-type fructans, which are characterized by the presence of glycosidic
Fig. 1 Representation of fructans’ types with their precursors and type of fructofuranosyl linkages
bonds like inulin- and levan-type fructans, respectively, and glucose on both ends of
the molecule [10]. Elongation of inulin neoseries-type fructans starts from the C1 of
the fructose group and/or from the C6 of the glucose unit of the initial sucrose, while
elongation of levan neoseries-type fructans comprises molecules for which the
elongation starts from the C6 of the glucose moiety. Mixed levan-type fructans
include both types of fructofuranosyl linkages: β-(2–1) and β-(2–6) leading to
formation of branched molecules [10].
In plants, ITFs composed of β-D-fructosyl units linked by (2–1) glycosidic bonds
consist from 2 to 200 monomers, while ITFs synthesized by bacteria can be much
longer reaching even 100,000 units [11]. Based on the length of the chain (DP), ITFs
can be divided into long-chain inulin (DP > 10) and short-chain oligofructose
(DP < 10) [9, 12]. DP determines the physicochemical properties of ITFs. Among
commercially available ITFs, Synergy 1 (Raftilose ®, Orafti, Tienen, Belgium) that is
a mixture of long- and short-chain fructans have the features of both types of
polymers.
The prevalence of fructans in the plant kingdom is very common, and they can be
found in approximately 36,000 species [11]. The main taxonomic orders containing
the largest amounts of ITFs are Asteraceae represented by chicory (Cichorium
intybus) and Jerusalem artichoke (Helianthus tuberosus) – the main sources of
ITFs; as well as Liliales, including leek, onion, asparagus, and garlic [13]. Tubers
and roots of Asteraceae are the richest source of ITFs. Plant genotype, cultivar
conditions tissue, the developmental stage as well as the conditions of post-harvest
storage are factors influencing the chemical structure and yield of ITFs [14, 15]. The
majority of ITFs used by food industry is obtained from chicory, containing even
15–20% of inulin. Non-fractioned inulin extracted from chicory roots consists of
oligomers and polymers ranged from 3 to 70 DP. The contents of ITFs in different
Table 1 Content (% of fresh weight) and chain length of inulin in various plant
Source Inulin content Chain length
Chicory (root) 15–20 DPa < 40 = 83%
DP 40 = 17%
Jerusalem artichoke (tuber) 14–20.5 DP < 40 = 94%
DP 40 = 6%
Globe artichoke 2–7 DP 40 = 87%
DP 5 = 95%
Dandelion (leaves) 12–15
Garlic (bulb) 9–16 DP 5 = 75%
Leek (bulb) 3–10 DP = 12 – majority
Onion (bulb) 1.1–7.1 DP 2–12
Asparagus (modified stem) 2–3
Wheat (bran) 1–4 DP 5 = 50%
Barley (cereal) 0.5–1.5
Rye (cereal) 0.5–1
Banana (fruit) 0.3–1 DP < 5 = 100%
a
DP degree of polymerization
plant sources are summarized in Table 1. Inulin can also be produced enzymatically
from sucrose following the transglycosilation and hydrolysis catalyzed by fructosyl-
transferase [16]. Synthetic inulin is characterized by a lower polydispersity as
compared to plant-derived one [17].
Main role of fructans in plants is related with storing the energy, but they also take
part in the osmoregulation and in the response for abiotic-stress [18, 19]. Inulin
obtained from chicory is present in a form of white fine powder with a slightly sweet
taste due to the presence of both long- and short-chain fractions. The unique
chemical structure of ITFs having the furanose conformation and the backbone
with the polyethylene oxide determines its higher molecule flexibility as compared
to pyranose configuration [17]. The properties of individual fraction depend on DP,
determining the versatility of ITFs applications. The short-chain oligofructose is
characterized by sweet taste and its sweetness constitutes approximately 35% of
sucrose. The technological properties of oligofructose are similar to features of
glucose syrup, therefore it can be used to improve the mouth-feel of light-type
food products with lower caloric value without compromising sweet taste [20].
Long-chain inulin has neutral taste and it is not sweet but is able to form the gel
network when it is mixed at high concentration with water. The aqueous liquid
emulsion can be used to replace fat in food production, what is commonly applied in
the production of low-fat dairy products with smooth, creamy texture [21]. Fat
resembling features are also used in the meat and bakery industries to formulate
low caloric products [22–25]. ITFs can be applied not only in the food industry but
also for pharma. They can be used as targeted colon deliverer, to stabilize the
protein-based pharmaceuticals, to enhance the dissolution rate, and to diagnose
kidney failure by urine secretion rate [17, 26, 27].
3 Health-Related Properties of Inulin-Type Fructans
3.1 Prebiotic Effect
Β-configuration of ITFs is responsible for their unique properties. ITFs are not
hydrolyzed by the enzymes of the upper intestinal digestive tract as they only have
the ability to hydrolyze linkages in α-configuration, therefore ITFs reach the colon
unchanged [28]. This specific property of ITFs is one of the crucial features which
allow considering ITFs as prebiotics. Nevertheless, the definition of prebiotic has
evolved many times since 1995, when Gibson and Robertfroid [29] explained
prebiotic as “a nondigestible food ingredient that selectively stimulates growth
and/or activity of one or a limited number of bacteria in the colon, thereby improving
host health” to the final, the most recent definition developed in 2017 by The
International Scientific Association of Probiotics and Prebiotics (ISAPP) that sim-
plified it to “a substrate that is selectively utilized by host microorganism conferring
a health benefit” [30]. To specify this definition, three criteria should be accepted:
prebiotic must be selectively utilized by the commensal microorganisms; have
adequate evidence of health benefit for the target host; and must not be degraded
by the target host enzymes. ITFs meet all the abovementioned criteria [30]. The
fermentation of prebiotics causes changes in the gut microbiota composition. ITFs
are preferably degraded by β-fructanoidase synthesized by bifidobacteria [30]. The
presence of carbohydrates stimulates the fermentation process leading to an increase
in bacterial quantity and fecal mass. The final products of ITFs metabolism are
hydrogen, methane, carbon dioxide, lactate, and short-chain fatty acids (SCFA) [31].
SCFA are considered to have beneficial direct and indirect effects on human health.
Higher amount of SCFA decrease pH in the intestines inhibiting growth of patho-
genic bacteria. Butyrate is the energy substrate for colonic epithelial cells, and it is a
key factor in the colonocytes proliferation and development. Water-soluble SCFA
can easily pass the intestinal barrier getting into bloodstream, and then spread in the
body. Acetate is used by the brain, muscles, and tissue, while propionate is metab-
olized by liver, reducing the hepatic production of cholesterol [31].
Inulin has been found to stimulate the development of Lactobacillus and
Bifidobacterium, with simultaneous inhibiting of amount of harmful bacteria like
Escherichia coli and Clostridium [32]. The products of fermentation of
Bifidobacterium are mainly lactate and acetate. The in vitro chemostat study
conducted by Langlands and co-workers [33], aimed to evaluate the effect of the
prebiotic carbohydrates oligofructose and inulin on the mucosal microorganisms,
showed a significant increase (P < 0.05) in the number of Bifidobacterium and
Lactobacillus in biopsies taken from the caecum, transverse and descending colon,
and rectum. Authors reported a significant increase of eubacteria in subjects con-
suming fructooligosaccharides (FOS) and inulin but no changes in the total anaer-
obes clostridia, bacteroides, or coliforms, neither in their proliferation indices [33].
However, the in vitro models do not allow analyzing the complexity of
the intestinal ecosystem; therefore to confirm the probiotic effect of ITFs, the in
vivo studies and human trial were necessary. Recent in vivo study on inulin
administration in rats’ diet of reduced amount of calcium showed a stimulation of

bifidobacteria, particularly Bifidobacterium animalis, and an increase in SCFA in the
groups fed with ITFs [34]. The stimulation of microbiota was dependent on calcium
intake, and the positive changes were observed only in animals fed with a
recommended amount of calcium. The DP-dependent influence of ITFs on gut
microbiota was determined in the study on C57BL/6 J mice [35]. Authors reported
that ITFs with lower DP stimulated the intestinal microbiota more efficiently than
long-chain inulin. However, in both groups, the growth stimulation of the butyric-
producing bacteria was observed. In the group receiving FOS, the dominant bacteria
in Verrucomicrobia phylum was Akkermansia muciniphila, belonging to mucin-
degrading species [35].
Numerous human studies, varying the applied dose of prebiotic, the DP, and the
duration of experiment confirmed the prebiotic effect of ITFs [33, 36–39]. Random-
ized, double-blind placebo-controlled study on obese women receiving 16 g of
oligofructose-enriched inulin or placebo (maltodextrin) was designed by Salazar
and co-authors [40]. After 3 months supplementation, the number of
Bifidobacterium longum, Bifidobacterium pseudocatenulatum, and Bifidobacterium
adolescentis increased in the ITFs-consuming group. In general, in the obese and
overweight human, the SCFA concentration is elevated [41]. In the study by Salazar
and co-workers [40], the normalization of the SCFA concentration, with the signif-
icant decrease in acetate, propionate and total SCFA content, was noted in the
prebiotic group. The increase in Bifidobacterium counts was determined in the
randomized trial with 4-week intake of 8 g of FOS by 12 healthy elderly volunteers
[42]. Prebiotic effect of inulin was also reported in the study of Tuohy and co-
workers [39]. Authors reported the significant increase in Bifidobacterium in pooled
fecal samples collected from ten healthy volunteer after 14-days intake of 8 g of
inulin. Small but significant increase was observed also in clostridium group. Inulin
intake had no effect on the total bacteria number, Bacteroides spp., Lactobacillus sp.,
and Enterococcus sp. The authors noticed that the more prominent increase in
Bifidobacterium was observed in volunteers with a lower initial number of these
bacteria [39]. Fecal microbiota composition was weekly monitored in the study with
Jerusalem artichoke and chicory inulin-supplemented snacks introduced to 45 vol-
unteers [43]. The increase in the count of Bifidobacterium with simultaneous
reduction in Bacteroides – Prevotella ratio and Clostridium histolyticum/Clostridium
lituseburense was observed in both experimental groups fed with ITFs [43].
3.2 Dietary Fiber
According to the definition of Institute of Medicine of The National Academies,

dietary fiber consists of nondigestible carbohydrates and lignin that are intrinsic and
intact in plants [44]. Dietary fibers should be resistant to the enzymes of early
sections of intestinal track and should be metabolized in the colon by intestinal
microbiota [45]. The interest in dietary fibers is related with a wide spectrum of
health beneficial properties, including reducing the risk of type 2 diabetes,
hypertension, cardiovascular disease and helping in the body mass management

[46]. The consumption of fibers should consist of 25–38 g per day [47]. ITFs are
characterized by the low caloric value (1–2 kcal/g) as well as by β-configuration,
allowing to reach large intestine in the intact form in almost 90% determines that
ITFs belong to the dietary fiber group, thus they are considered as dietary fiber [20,
48, 49]. Precisely, based on their physiochemical characteristic, ITFs belong to the
group of soluble dietary fibers, being better fermentable and viscous [31]. Therefore,
ITFs-containing products are labeled as a source of dietary fiber.
3.3 Intestinal Morphology and Gut Function
In general, it is well known that intestinal microbiota has a crucial role in the whole
body homeostasis and maintenance of the gut health. Prebiotics, including ITFs,
stimulating the development of the beneficial strains of bacteria are considered to
improve the gut health in terms of morphological changes, maintenance of intestinal
integrity, and alleviating the symptoms of intestinal diseases.
ITFs were applied as a supplement in a study aimed to compare the morphometry
of germ-free rats and rats with human fecal microbiota [50]. The height of villi and
depth of goblet cells was improved in groups receiving ITFs. Moreover, ITFs had
beneficial effect of epithelial mucus layer, especially in a group with fecal microbiota
transplantation [50]. The fragility and spontaneous bleeding are considered as a
symptom of low-fiber diet due to the reduced amount of SCFA [51]. In other study,
10% of inulin, FOS, and cellulose were applied in the diet of obese mice for 4 weeks
to evaluate the effect of fibers on gastroenterology physiology [52]. The authors
noticed that in groups fed ITFs, the cecal crypt depth was improved as compared to
cellulose-fed and control groups. Additionally, inulin-fed mice had better cecal
transmutal resistance. Moreover, a lower expression of colonic mRNA of genes
encoding occludin, zonulin 1, AMP kinase, and monocarboxylic transporter 1 was
observed in inulin-fed mice, pointing the negative changes in the gut barrier func-
tioning. However, as authors underlined, because the deteriorating in the transmutal
resistance and morphological failures were not observed in inulin-fed mice, this
mechanism may be more complex and require further studies [52]. Unfavorable
effect of ITFs on gut barrier was reported in other studies with animal model
[53–55]. However, in the human studies no effect of ITFs on gut barrier was
observed [56–58]. On the contrary, Russo and co-workers [59] reported improve-
ment in the circulating level of intestinal permeability markers, such as zonulin and
glucagon-like peptide 2 after administration of an inulin-enriched pasta, suggesting
the potential role of inulin in the prevention of intestinal disorders [59].
The randomized, controlled trial evaluated the effect of oligofructose on the
prevalence of diarrhea in 282 infants aged 6–12 month [60]. Diet supplemented
with prebiotics did not affect the prevalence of diarrhea; however, the mean number
of days with diarrhea was smaller in the oligofructose group than in the control
group. Nonetheless, authors concluded that a prebiotic supplementation cannot be
substitutive of breast-feeding [60]. Klessen co-workers [61] analyzed the influence
of inulin on the constipation in ten elderly women and found that in most cases,
prebiotic increased the stool frequency from 1–2 to 8–9 per week and modified a
stool consistency. In the initial part of this study, the daily dose of 20 g of inulin was
applied and after increased gradually up to 40 g per day. In most of women, the
inulin dose has no impact on the stool frequency, except of one [61]. Similar findings
were reported in the studies conducted on 56 healthy children (age 16–46 weeks)
[62]. The authors reported that FOS in a dose of 0.74 g per day taken for 28 days
resulted in improved stool frequency and consistency as compared to placebo-fed
children. Contrary results were obtained by Bouhnik and co-workers [42] who
reported no influence after 4 weeks’ supplementation with 8 g FOS on the stool
weight, water percentage and stool frequency in six elderly persons.
3.4 Body Mass Management
The increasing evidence of obesity and overweight became a significant problem in

the well-developed countries. It is speculated that the increased intake of dietary
fibers can help in the body weight management. Thus, a growing number of studies
have attempted to develop products with higher content of dietary fibers as well as to
answer the question if dietary fibers are actually able to reduce obesity.
Single-blinded, crossover, placebo-controlled pilot study in ten healthy persons
taking 8 g of oligofructose or placebo twice a day for 2 weeks was conducted by
Cani and co-workers [63]. Morning consumption of ITF significantly improved
satiety (P = 0.04), while after evening intake, significantly improved satiety and
reduced hunger. The energy intake at breakfast and lunch and total daily energy
intake were reduced in the group receiving oligofructose as compared to placebo
group. Therefore, oligofructose was proposed as dietary supplement in obese people
[63]. Similar findings were reported by Genta and co-authors [64], who stated that
FOS can help in the obesity management. In their study, 0.29 g or 0.14 g FOS per kg
per day in the form of yacon syrup, being a rich source of FOS, were taken for
120 days by 35 obese and slightly dyslipidemic premenopausal women divided into
subgroups. The intake of FOS, resulted in reduced body weight, waist circumfer-
ence, and body mass index (BMI). Moreover, women receiving yacon syrup had
lower fasting serum insulin and serum LDL-cholesterol levels and reduced Homeo-
stasis Model Assessment index [64]. Parnell and Reimer [65] examined the effect of
oligofructose supplementation on body weight and the concentration of satiety
hormones in overweight and obese adults. Forty-eight obese subjects, without any
other health problems, ingested 21 g oligofructose or a placebo (maltodextrin) per
day for 12 weeks. Supplementation of diet with oligofructose significantly reduced
(P = 0.01) the body weight, while in the placebo group, the body weight increased
up to 0.5 kg. The level of glucose and insulin decreased in the ITFs group and
increased in placebo group. The profile of satiety hormone secretion changed after
prebiotic intake with increased level of peptide YY and reduced ghrelin, suggesting
the contribution in the reduction in energy intake. On the other hand, oligofructose
intake did not affect the secretion of glucagon-like peptide 1 [65]. A randomized
double-blind, cross-over intervention with oligofructose (10 g/d or 16 g/d) or

placebo for 13 days conducted in healthy adults with average BMI of 24.8 kg/m3
have evidenced a 11% reduction in the energy intake in the 13th day as compared to
the baseline in a group receiving oligofructose [66]. The difference in the energy
intake was also noticed between both groups receiving different doses of
oligofructose with 2801 kJ and 3177 kJ for 10 g and 16 g of oligofructose,
respectively. The reduction of energy intake may be explained by higher secretions
of peptide YY and glucagon-like peptide 1 [66]. The most recent randomized
double-blind, placebo-controlled trial in obese and overweight children where Syn-
ergy 1 (8 g/d) was administered for 16 weeks, showed a significant increase in the
feeling of fullness and lower prospective food consumption during breakfast in ITFs
group at the end of experiment as compared to the baseline [67]. The energy intake
was dependent on age, and 11–12 years old children receiving prebiotic had signif-
icantly reduced (P = 0.04) energy intake as compared to placebo group, while in
younger children (7–10 years old) the difference was not detected. Moreover, the
concentration of adiponectin and ghrelin was higher in the group receiving ITFs as
compared to placebo [67].
Contrary to that, few studies reported no effect of ITFs supplementation of
appetite control, and body weight management. Archer and co-authors [68]
substituted fat in sausage patty with inulin and lupin-kernel fiber and evaluated
their effect on palatability, perceptions of satiety, and food intake in 33 men. Inulin-
enriched patty had no impact on satiety, while lupin-kernel patty was more satiating.
However, the total fat intake was 18 g lower in group consuming inulin-enriched
patty [68]. Another study with two doses of FOS per day (5 g at breakfast and 8 g 2 h
before dinner) where applied to measure satiety [69]. Low morning dose did not
affect satiety or food intake during a lunch, while a higher dose influenced satiety in
the gender-dependent manner: in women the food intake decreased, however in men
increased [69]. Another study also reported that bars enriched with 10 g of different
fibers, including inulin and FOS, did not change satiety and food intake in women
[70]. These divergent results could be explained by the differences in the DP and/or
doses of applied ITFs. Therefore, this issue requires further studies.
3.5 Lipid Profile
Inadequate diet, rich in fat and sugar, with insufficient amount of dietary fiber is
suggested to be a factor of cardiovascular diseases, in particular of hypertension,
stroke, and heart failure [71]. Therefore, several studies were conducted to assess if
changing of dietary habits or dietary supplements could help in reducing the risk of
cardiologic conditions. So far, the studies directly evaluating the effect of ITFs on
cardiovascular diseases have not been performed; however, their influences on the
serum lipid profile have been examined widely.
The study of Letexier and co-workers [72] showed that 10 g of inulin per day
significantly reduced the level of serum triglyceride and liver liponeogenesis, but at
the same time, it did not affect total cholesterol, HDL- and LDL-cholesterol
concentrations [72]. Other results were obtained by Balcazar-Munoz and co-workers

[73] in a 4-weeks randomized placebo-controlled trial in 12 obese individuals who
received 7 g of inulin per day. Authors showed a significant decrease in total
cholesterol, LDL-cholesterol, VLDL-cholesterol, and triglyceride levels as com-
pared to placebo [73]. The study on healthy men (22 subjects) fed for 5 weeks
with inulin-enriched pasta were performed by Russo and co-workers [74] and
indicated an increase in HDL-cholesterol by 36% in the group fed with inulin-
pasta. At the same time, total cholesterol to HDL cholesterol ratio, concentrations
of triglycerides and lipoprotein (a) were reduced by 22%, 23%, and 17%, respec-
tively. In other clinical study, 10% decrease in LDL-cholesterol was reported in type-
2 diabetes patients with elevated lipid level after 14 days intake of 8 g/d of FOS [75].
On the other hand, no effect of ITFs on lipid profile was determined in many
studies in healthy people [76–78]. The meta-analysis of randomized controlled trial
showed that ITFs are able to reduce LDL-cholesterol across all study population,
while HDL-cholesterol improvement and reduced fasting glucose trend was noticed
only in the type-2 diabetes mellitus subgroup [79]. Therefore, further study on the
effect of ITFs on lipid profile, especially studies explaining their dose- and DP-
dependent influence are necessary for a definitive conclusion.
3.6 Mineral Absorption
Several in vitro and in vivo studies as well as in human trials have attempted to
determine the effect of the intake of ITFs on mineral absorption. Recent in vitro
study conducted by Krupa-Kozak and co-authors [80] evaluated the effect of inulin
and FOS on calcium uptake and absorption from the calcium-enriched GF bread.
The results showed that short-chain FOS improved the cellular calcium uptake from
calcium-fortified GF bread. The authors concluded that the absorption of calcium is
dependent of DP [80].
Rat model was used to assess the effect of ITFs with different chain length and
their combinations on the calcium and magnesium absorption in a 50 male Wistar
rats [81]. A significant improvement of magnesium absorption was reported for all
ITFs formulations. In case of calcium, only the mixture of short-chain and long-
chain ITFs increased absorption significantly; however, the increasing trend was
observed also in single formulations [81]. Recent 6-week nutritional experiment on
rat model was conduct by Krupa-Kozak and co-workers [34], who aimed to evaluate
the effect of inulin of calcium absorption depending on the amount of this mineral in
diet. The authors reported that inulin stimulated the absorption of calcium especially
in rats fed a diet of reduced calcium level [34]. The results of this study are somehow
in agreement with the results of a human trial on adolescent girls consuming a diet
supplemented with oligofructose-enriched inulin [82, 83]. Synergy 1 intake in a dose
of 8 g per day resulted in the improvement of calcium absorption in girls with
normalized calcium intake and in girls with calcium deficiency; however, the
beneficial effect was more prominent in the second group [82, 83].
Although the majority of in vivo studies are focused on the evaluation of the
impact of ITFs on calcium and magnesium absorption, in one study the effect of
5 week supplementation of mixture of ITFs with different DP (Synergy 1) on iron
utilization in piglets fed corn and soybean meal diet was assessed [84]. Final blood
hemoglobin concentrations and the overall hemoglobin repletion efficiency of pigs
increased significantly (P < 0.01) by 28% and 15%, respectively, in pigs
supplemented with 4% of ITFs. Changes in the iron utilization were positively
(r = 0.55 and 0.69, P < 0.01) correlated with dietary ITFs concentrations. Also
iron concentration in the digesta of proximal, middle, and distal colon were signif-
icantly (P < 0.05) higher in pig supplemented with 4% ITFs [84].
The effect of ITFs on mineral absorption was evaluated in numerous human trials.
Yap and co-workers [85] reported that absorption of iron, magnesium, and zinc were
significantly improved in infants fed with inulin-enriched formulations. On the other
hand, the retention of calcium and copper were not affected by inulin intake [85].
However, early studies showed that intake of 15 g of oligofructose per day
improve the calcium absorption in adolescent girls [86]; numerous studies indicated
that the most prominent effect on mineral absorption is observed with oligofructose-
enriched inulin as compared to individual ITFs [81, 87, 88]. Supplementation for
6 week with 10 g of Synergy 1 per day resulted in the increase of fractional
absorption of calcium and magnesium in 15 postmenopausal women as compared
to control [89]. Moreover, the beneficial effect of ITFs was observed also in terms of
bone turnover biomarkers, including serum osteocalcin and urinary
deoxypyridinoline cross-link [89]. One year interventional randomized, placebo-
controlled study showed that 8 g of Synergy 1 significantly improved calcium
absorption in a group of 100 adolescents (9–13 years old) [87]. Authors emphasized
that the significant increase in true calcium absorption was observed just after
8 weeks of trial, and it was maintained till the end of study in Synergy 1 group. At
the end of the experiment, the bone condition parameters were evaluated and showed
an improvement in bone mineral content and bone mineral density in ITFs group as
compared to control [87].
4 Gluten and the Application of Gluten-Free Diet
4.1 Gluten
Gluten is a mixture of storage proteins found in the endosperm of the mature grain of
wheat and some other cereals within the grasses family, including rye and barley. It
consists mainly of protein (75–85%) and lipids (5–10%), while most of the remain-
der is starch and nonstarch carbohydrates, being the residue occurring after washed
out the starch granules and other soluble substances from the wheat flour [90].
Gluten proteins are the main source of nitrogen for a growing seedling [91]. Mixture
of gluten proteins is composed by monomeric gliadins and polymeric glutenins [92].
Gliadins and glutenins differ in their alcohol solubility. Gliadins are soluble in
ethanol-water mixtures, while glutenins are insoluble. Gliadins belong to prolamins
of wheat. The prolamins present in rye, barley, and oats are secalins, hordeins, and
avenins, respectively. Gliadins can be subdivided into α/β, γ, and ω-gliadins based
on their electrophoretic mobility. α/β and γ-gliadins are low molecular weight pro-
teins (MW 28–35 kDa) with six and eight cysteine residues respectively, whereas ω-
gliadins (MW 40–75 kDa) are sulfur poor [90]. The structure of gliadins consists of
approximately 300 amino acids with the high percentage of proline and glutamine
and low amount of lysine and methionine [90]. The amino acid characteristic is
responsible for the harmful effect of gliadins with 13- and 33-mer oligopeptides
involved in the alleviated immunological response in celiac patients [93]. The
glutenin fraction comprises aggregated proteins linked by inter-chain disulfide
bonds; they have varying size ranging from about 500,000 to more than 10 million
[94]. Amino acid characteristic of glutenins is similar to gliadins with high content of
glutamine and proline [95]. The amount of gluten in wheat grain has changed in
time. Genetic modification aimed to improve yielding of crops, result in the arriving
of new varieties with multiplied genes also coding the gluten proteins. These
changes in the wheat sensitivity are widely reviewed [91, 96].
4.2 Gluten-Related Disorders
A growing popularity of a gluten-free diet (GFD) is related with increasing incidence

of gluten-related disorders [97]. In certain group of individuals, the intake of gluten
can lead to the development of diseases, which can be divided into two main groups
based on the pathogenesis: allergic and autoimmunological. Allergic diseases
include wheat allergy (WA), while celiac disease (CD), Dermatitis herpetiformis,
and ataxia belong to the second group. Nowadays, many cases of patients suffer
from hard to diagnose disease, non-fitting to none of defined gluten-related disorders
but for who treatment with GFD helps and are enclosed in the so-called non-celiac
gluten sensitivity (NCGS).
4.2.1 Celiac Disease

Celiac disease is autoimmunological disease, observed in genetically predisposed
individuals, resulting in the villous atrophy as a consequence of presence of gluten in
diet. The diagnosed CD patients constitute approx. 1% of general population [98];
however, in many cases CD remain unrecognized or incorrectly diagnosed [99]. CD
is observed mainly in highly developed countries due to the westernization of a diet
[100]. CD can be diagnosed at every age, not as erroneously thought, only in
children. The prevalence of CD is twice more frequent in women as compared to
men [101].
Pathogenesis of CD is complex and includes genetic, environmental, immuno-
logical, metabolic, and infectious factors [102]. The genetic predisposition is
inherited dominantly. Most of person diagnosed with CD contain genes of human
leukocyte antigen (HLA): HLA DQ2 and HLA DQ8, placed on a chromosome 6,
encoding proteins presenting gluten to lymphocyte CD4þ [103]. Approximately
90–95% of individuals with CD inherit alleles coding HLA-DQ2, while in majority
of the rest, HLA-DQ8 is detected [104]. It is suspected that also other genes may be
involved in the pathogenesis of CD. The expression of HLA-DQ is necessary but not
sufficient for the development of CD. Breast feeding, time of gluten introduction into
the diet of the child, a history of infectious, certain medicals and smoking can be
environmental factors contributing to the CD development [105–108]. Groups with
higher risk of the incidence of CD include: relatives of CD patients and people
suffering from type-1 diabetis mellitus, Hashimoto’s thyroiditis, IgA deficiency, and
genetic disorders such as Down’s syndrome and Turner’s syndrome [92].
The immune response to ingested gluten depends on the presentation of gluten
peptides for lymphocytes T. In colon, the gluten protein undergoes deamidation by
enzyme, tissue transglutaminase, with formation of strongly immunogenic peptides.
Intraepithelial lymphocyte infiltrates and immune system stimulation manifested by
increased production of interleukin 15 is observed in genetically predisposed indi-
viduals [109]. Activation of lymphocytes T, following the gluten peptide recogni-
tion, results in damage to the intestinal mucosa by direct action of inflammatory
mediators and extracellular matrix metalloproteinases secreted by stimulated fibro-
blasts, leading to the development of inflammation of the endothelium and lamina
propria of small intestine, cryptic hypertrophy, and the atrophy of intestinal villi
[103, 110]. One of the consequences of morphological intestinal changes in CD is a
limitation of the absorption process. Malabsorption is related to deficiencies of
important alimentary nutrients, leading to growth deficit, anemia, vitamin deficien-
cies, thyroid diseases, mental disorders, fatigue, and infertility.
A wide range of manifestations of CD complicates its diagnosis. Based on the
clinical picture, three main types can be distinguished: classic, atypical, and silent
[111]. Some of researchers suggest also the addition of a term latent CD and a
genetic susceptibility for CD to this classification. Classic CD is manifested by
gastrointestinal symptoms such as diarrhea, fatty stools with bad odor, constipations,
abdominal pain, loss of appetite, and weight loss. This CD type is diagnosed mainly
in children. As compared to classic form, the atypical CD is more common, and it is
diagnosed in older children, teenagers, and adults. Except of typical gastrointestinal
symptoms, atypical CD can be manifested with verity of extraintestinal symptoms,
including anemia, mental disorders, and bone alterations [112–114]. Silent CD is
diagnosed accidently in relatives of CD patients or during screening studies [115].
The diagnosis of CD is confirmed by clinical picture, duodenal biopsy, genetic
tests, and serological tests. Serological tests include measurements of several anti-
bodies: anti-tissue transglutaminase (tTG), anti-endomisium (EMA), anti-gliadin
(AGA) and anti-deaminated gliadin (DGP) [92].
4.2.2 Wheat Allergy

Wheat allergy is a classic form of food allergy being a defense response to a contact
with wheat proteins exclusively. Proteins present in other cereals do not cause an
immunological response in WA patients. The incidence of WA is estimated at 0.4%
of worldwide population, wherein the most frequently is diagnosed in children [91,
116]. The frequency of the WA is age-dependent, in fact in 2 years old children WA
incidence was estimated in 2%, while in older children it increased up to 9% [117].
Main signs of WA are skin lesions, typical for food allergies, and gastrointestinal
symptoms such as diarrhea, abdominal pain, and constipation [118]. It is possible to
distinguish few types of WA depending on the way of exposure to the allergen and
the mechanism of immune response. First type is classic WA manifested by the
gastrointestinal, respiratory, and skin symptoms; second is wheat-dependent, exer-
cise-induced anaphylaxis (WDEIA), which appears after physical activity; and the
last type is the baker’s asthma, commonly appearing in the workers of bakeries and
mills because of the exposition of the wheat particles irritating the respiratory track.
The incidence of WA is the smallest among gluten-related disorders but the conse-
quences of WA can be the most serious ones because of the possible anaphylactic
shock, which can be fatal.
Gluten proteins are not the only triggers responsible for allergic reactions in WA.
Another wheat proteins belonging to albumin and globulin group have similar
ability. Gliadin ω-5 is the main allergen in WDEIA and skin allergies [119], while
gliadin α- and γ- as well as protein ATI CM3 (amylase trypsin inhibitors), belonging
to albumin group, are responsible for atopic dermatitis [120]. Baker’s asthma is a
response to the presence of low mass glutenins (α-, ω-gliadins), but the main role in
the pathogenesis of Baker’s asthma is played by several albumins and globulins,
including ATI, lipid transporting proteins, and serpins [121].
Diagnosis of WA is based on the analysis of serum IgE concentration and skin
tests. Because of the similarities of immunological epitopes in phylogenetically close
varieties, the presence of cross-allergies with allergens of grass and weeds is
possible. So far, the most reliable method of food allergies diagnosis is the challenge
with allergy-causing food, and then attempt of its elimination from a diet [122].
Therefore, in the diagnosis of WA, gluten-challenge is applied.
4.2.3 Non-Celiac Gluten Sensitivity

Non-celiac gluten sensitivity belongs to gluten-related disorders; however, its char-
acteristic and course does not fit CD or WA [97]. It is suspected that the main cause
of NCGS is an imbalanced diet containing foods of high gluten content [123]. NCGS
is diagnosed mainly in adults, particularly in female [124]. Individuals with irritable
bowel syndrome (IBS) and allergy are particularly vulnerable to incidence of NCGS
[125, 126]. High rate of NCGS has been reported in relatives of CD patients,
amounting 13% [127]. NCGS patients suffer from symptoms similar to classic
CD, including abdominal pain, diarrhea, as well as extraintestinal symptoms such
as depression, fatigue, and musculoskeletal pain. In NCGS, symptoms appear in
short time after gluten intake. Contrary to CD, NCGS patients do not have elevated
concentration of antibodies characteristic for CD (anti-tTG, EMA). There are also no
changes in the allergic tests including the measurement of immunoglobulins levels
and skin tests. Therefore, the diagnosis of NCGS is based only on the elimination of
CD and WA.
Pathogenesis of NCGS is not fully known; however, in its development an
important role of the activation of innate immune response, changes in the intestinal
barrier functioning and intake of food containing inhibitors of amylases was indi-
cated [100, 127, 128]. It is also suspected that NCGS can be related with a high
intake of fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs)

[124]. Persons suffering from NCGS show markedly increased expression of toll-
like receptors (TLR) involved in the innate gastrointestinal immune response and
lower expression of FOXP3 Treg markers associating with the development of
autoimmune diseases such as CD [100].
4.3 Gluten-Free Diet as a Treatment of Other Diseases
Nowadays, a GFD is considered as a treatment alleviating several symptoms and

diseases. The attempts have been made to check the possibility of application of
GFD in other diseases, in particular a GFD was proposed as a potential treatment
for the selected symptoms of irritable bowel disease (IBD). A controlled trial of
application of a GFD in patients with IBS-diarrhea indicated that a GFD can
reduce the frequency and change the consistency of stool, improve the intestinal
barrier functions, and alter rectosigmoid messenger RNA expression of tight
junction proteins [129]. Gluten changed the level of various cytokines in periph-
eral blood mononuclear cells in IBD, and it was concluded that the extent of these
alterations were associated with HLA-DQ2 or HLA-DQ8 genotype [129]. To
identify the subgroup of IBD patients who potentially respond to a GFD, another
approach proposed the evaluation of serum IgG antibodies against gliadin or
tissue-transglutaminase in combination with HLA-DQ2 expression [130]. More-
over, an internet-based cross-sectional study utilizing a GFD questionnaire in
1647 patients with IBD reported that among the analyzed group 0.6% and 4.9% of
participants were diagnosed with CD and NCGS, respectively [131]. Amongst the
respondents, 19.1% had previously tried a GFD, while only 8.2% admitted
currently following of GFD. What was interesting, 65.6% of participants reported
an improvement of gastrointestinal symptoms after the elimination of gluten from
the diet, and 38.3% described symptoms of disease as less frequent and severe
[131]. Studies conducted on patients with fibromyalgia both with IBS or
CD indicated that a GFD can slightly alleviate the symptoms of those diseases
[132, 133].
Emerging evidence suggested the importance of insufficient enzymatic activity,
increased gastrointestinal permeability, and the absorption of toxic byproducts of
incompletely digested proteins from dairy (casein) and cereals (gluten) in the
development of autistic spectrum disorders (ASD) [134]. Therefore, gluten-free
and casein-free diets are proposed as a treatment of ASD. The results of the
randomized single-blind trials indicated the improvement in the communication,
social isolation, repetitive and challenging behavior, and better development in
children on a GFD, casein-free diet as compared to those on control diet [135,
136]. On the other hand, another studies reported no significant (P < 0.05) differ-
ences in verbal and nonverbal communication and behavioral indexes in children
following a GFD as compared to controls [137, 138]. The efficiency of a GFD in
ASD was widely reviewed [134, 139].
4.4 Gluten-Free Diet as a Dietary Trend
The total occurrence of gluten-related disorders is observed in low percent of the

whole population, meanwhile the increasing trend in the GF products consumption
and thus production is noticed, projecting that the GF food market will reach 4.89
billion dollars in 2021 [140]. Surprisingly, an emerging number of healthy people
begin a GFD, believing that gluten can have harmful health effects [141]. The
prevalence of a GFD consumers in US society, who were not diagnosed in any of
gluten-related disorders consists at least 0.5% [142]. Another study evaluating this
trend showed that the prevalence of CD remained constant (0.58–0.77% of general
population), while the amount of a GFD followers increase from 0.52% in
2009–2010 up to 1.69% in 2013–2014 [141]. Authors explained this rising GFD
popularity phenomena by the popular opinion that gluten carries negative health
effects, therefore a GFD is healthier than a conventional gluten-containing diet,
easier access to GF products, and the increasing prevalence of self-diagnosed
gluten-related disorders and following of diet without the consultation with physi-
cian [141]. Recent prospective cohort study performed in 64,714 women and 45,303
men responding for food frequency questionnaires for 26 years showed no relation
between presence of gluten in food and the development of coronary heart disease
[143]. On the contrary, authors suggest that a non-necessary GFD application is
related with the avoidance of whole grains having beneficial effects on heart
conditions, more likely may result in the coronary heart disease development. The
authors underline that the promotion of a GFD among people without diagnosed
gluten-related disorders should not be recommended [143].
4.5 Controversy Concerning a Gluten-Free Diet
GFD is based on the elimination of gluten-containing foodstuff from a daily regime.

The commercially available GF products have higher content of fat and sugar as
compared to corresponding gluten-containing counterparts [6]. Studies on children
indicated significantly higher energy intakes in CD patients as compared to controls
[144]. Additionally, GF products are poor in several important nutrients, especially
proteins and mineral components, as well as non-nutritional but physiologically
important components, like dietary fiber [6]. For that reason, the efforts have been
made to improve the nutritional value of GF products by addition of non-gluten
natural additives. Highly nutritional grains, like quinoa, teff, buckwheat, and ama-
ranth, dietary ingredients, and mineral supplements were applied in the development
of new gluten-free bakery goods [145–148]. These procedures resulted in improving
the nutritional quality as well as augmented the technological properties of GF
products.
The difficulty in the proper nutrients balance in the GFD is not the only problem.
Recent studies indicated that a GFD can increase the exposure to certain toxins as
compared to conventional diet. Fourfold increase in the serum levels of mercury in
CD patients following a GFD as compared to the healthy controls following regular

diet was reported by Elli and co-workers [149]. The source of mercury in a GFD is
not known; however, these results are worrisome because of the harmful effect
caused by mercury, especially in the nervous system [150]. Recently, emerging
number of evidence underlie the higher content of arsenic in rice [151]. Rice, rice
flour, and other rice products are common replacement of wheat in a GFD; therefore
they might elevate the arsenic levels in CD patients, but this must be studied.
Because of all abovementioned controversy of GFD, the proper dietary balance
should be established with an assistance of a dietician to keep diversity of the diet
[152] and the development of new, high-nutrition, and safe GF products should be
explored.
5 Technological Importance of Gluten
Gluten plays an important role in the technology of the cereal-based food products.
Due to its unique properties, gluten contributes to batter emulsification and visco-
elasticity, provides cohesiveness to dough during processing, retains leavening
gases, sets the crumb structure, and imparts elasticity to the bread texture [90,
153]. Gluten properties result of the ability of gliadins and glutenins to aggregate
and form a network, the structure of which result from the presence of noncovalent
(ionic, hydrogen, and hydrophobic) bonds [90]. The unique physiochemical prop-
erties of gluten derive from the presence of both protein fractions, equally affecting
the quality of dough. Dough strength and elasticity is related with insoluble glutenin,
being more elastic and cohesive. Properties of glutenins are correlated with the
presence of disulfide bonds. When these bonds are reduced, glutenins become
soluble in aqueous solutions of alcohols, such as gliadins, and they lose the strength
features [90]. The soluble gliadin is mainly responsible for the viscosity and exten-
sibility of the dough, because they are inflexible and less cohesive than glutenins.
The viscosity is a consequence of high content of proline, allowing the dough to flow
and rice [91]. Under conditions providing sufficient dough hydration and when the
dough is optimally developed by the mixing process, the disruption of the initially
spherical protein particles takes place, together with the stretching and alignment of
proteins, which leads to the formation of a three-dimensional structure. By holding
gases produced during dough proofing or fermentation, a gluten network allows
bread to rise. Finally, during the baking, gluten changes its characteristics from
elastic to semi rigid, contributing to the change from dough to crumb. The crumb
structure is then formed. These functions of the gluten network give bread its chewy
and elastic texture.
Many studies on gluten-free baking products have focused on the design of a GF
matrix to overcome the negative impacts of the absence of the gluten network. The
replacement of gluten is yet a technological challenge, as it is an essential structure-
building protein which is necessary for formulating high-quality cereal-based goods,
thus the production of such products is difficult.
A technology of GF baked products is different from the traditional processing of

gluten-containing bakery foods. GF dough itself varies considerably from wheat
dough, being closer to batter [154, 155]. The consistency of GF dough is greatly
dependent on the amount of water or hydration, showing very low consistency
during mixing when water adsorption is higher than 90% [156]. For that reason,
GF dough does not require kneading, like conventional bread dough, and instead it is
blended in a mixer [154]. Therefore, this dough is more like a batter of cake than
bread dough [155]. Despite the intensive development of the GF production, many
baked GF breads and confectionary products currently on sale are still of inferior
quality. The lack of gluten makes it very difficult to obtain an acceptable texture and
sufficient volume of baked goods because of the absence of a proper protein network
necessary to hold the carbon dioxide produced during proofing [7]. The obtained GF
products have a crumbling texture and pale crust [157, 158]. Another problem with
GF-baked goods is the reduced volume of obtained loaves due to low carbon
dioxide-binding activity during rising. Furthermore, baked GF products, mainly
composed of different starches, stale very quickly and are characterized by reduced
sensory qualities compared with their gluten-containing counterparts. Gluten is also
responsible for the structure of dry pasta and its texture during cooking. A gluten
network coagulates during cooking and creates strengthened network, preventing the
starch granules to leach [159]. Lack of gluten network results in sticky pasta with
poor consistency [159].
The imitation of viscoelastic properties of gluten is a key technological task, and
numerous studies have therefore been conducted to overcome this problem. Hydro-
colloids and gums, due to their water-binding capacity and ability to form a gel
network, were proposed as substitutes for a gluten network to improve of the
rheological properties, structure, mouth-feel, acceptability, and shelf life of GF-
baked products [160, 161]. Enzymes were applied to improve the quality and extend
the shelf-life of GF products [156, 162]. Positive effects were also obtained by using
sourdough fermentation as an effective way to improve the texture, flavor, shelf-life,
and nutritional value of bread [163].
Recently, Padalino and others [164] studied a range of vegetable flours in the
formulation of GF spaghetti and indicated that yellow pepper pasta was the most
desirable vegetable flour due to its orange color, homogeneity, and pleasant taste;
additionally, yellow pepper pasta decreased the hardness of the pasta, compared to
the control. Susanna and Prabhasankar [165] analyzed the inclusion of high protein
flours (soya, channa, and sorghum flours) for the production of GF pasta and showed
that formulations containing the highest level of soya flour and channa flour, along
with hydrocolloids and whey protein concentrate, produced the best cooked pasta
characteristics, having the lowest cooking loss, a soft texture, and a pasta with higher
protein content compared to the control of Triticum durum flour.
Poor structure and texture are not the only problems of GF products. They are also
characterized by inferior nutritional qualities. Therefore, to increase their palatability
and dietary quality, aside from the typically used corn and rice, a wide range
of naturally GF grains (oat, sorghum, and millet) and pseudocereals (buckwheat,
amaranth, quinoa, and teff), rich in valuable proteins, minerals, dietary fibers,
and bioactive compounds, were proposed as GF substitutes [8, 145, 146, 166, 167].
Legumes, nut and carob germ, and chia containing important nutrients have been
described as valuable components of GF baked products [168, 169]. Another
important approach of the GF production is the addition of animal (dairy and egg
proteins) and non-gluten plant proteins (zein), as those substances have both a
nutritional and a technological role [156, 170–173]. Nowadays, a number of research
have been conducted to evaluate the effect of ITFs on the quality and nutritional
properties of GF products.
6 State of Art of Application of Inulin-Type Fructans in

Gluten-Free Products
6.1 Inulin
The application of inulin and Jerusalem artichoke in the traditional bread of

improved technological properties without compromised sensory acceptance [174,
175] has attracted scientists to explore widely this topic. This interest resulted in a
number of researches aimed to utilize inulin in traditional bread making [176–179],
as well as in other bakery products such as quick breads (scones) [180], pasta [181,
182], different types of cakes [183–185], muffins [24], biscuits [186], and extruded
snacks [187].
Although the application of inulin in baked products resulted in decrease tech-
nological parameters (lower volume, darkening of the crust, increased crumb hard-
ness, higher rate of staling), but it improved the palatability and flavor, therefore 5%
inulin addition was assumed as a compromise between the improvement in nutri-
tional and sensory quality and decreased technological features [177, 188]. How-
ever, a recent study indicated that it is possible to optimize the formulation with even
30% incorporation of fibers without compromising the quality of bread [179]. As a
sweetener and fat replacement, ITFs were proposed as additive to gluten-containing
confectionery products. Muffins with up to 50% fat substitution with inulin were
characterized by increased fiber content, moisture, as well as crumb density and
springiness [24]. In cakes, even 70% substitution of fat by inulin was possible,
resulting in a product with acceptable sensory properties and reduced caloric value
[189]. Incorporation of inulin in wheat pasta allowed to obtain pasta with improved
nutritional value, without compromising the sensory and cooking quality [182, 190].
On the other hand, Bustos and co-workers [181] showed that inulin is completely
lost during cooking and its incorporation into pasta affects negatively its technolog-
ical properties.
The successful experience of inulin utilization in the traditional cereal products
technology contributed to the development of studies on GF foodstuff. As bread is a
staple food, consumed daily by the majority of population, thus the majority of
research on GF product refers to it. The supplementation of GF bread with different
percent of inulin showed that the amount of incorporated inulin plays the main role
in changing the properties of GF bread [191]. Volume of loaves increased with the
increasing share of inulin, reaching even 9% increase in bread with 8% inulin

addition. This phenomenon can be explained by high water binding capacity of
inulin. Unfortunately, bread fortified with higher amount of inulin (8%) was char-
acterized by reduced crumb cohesiveness and springiness and wrinkled crust clas-
sifying inulin-rich GF bread as second-grade quality product [191]. The enrichment
of GF bread with inulin significantly (P < 0.05) reduced the crumb hardening rate as
compared to the control non-fortified GF bread. The lower hardening can be
attributed to the slower migration of water from crumb to crust. The results of this
study showed that the GF bread preparation enriched with 5% of inulin allows to
obtain the product with the highest quality.
Hager and co-workers [192] demonstrated that native inulin from chicory deter-
mined the rheological properties of fortified GF batter and consequently the quality
of GF bread. Moisture content was 5% higher in the GF formulation fortified with
inulin as compared to unfortified controls, suggesting that inulin microcrystals led to
formation of a gel structure enclosing large amounts of water. During the formation
of gel, water is immobilized in inulin macromolecular zones, enclosing large
amounts of water, leading to less elastic and more viscous dough [192]. Rheological
and viscoelastic properties of GF bread enriched with inulin were a focus of research
of Juszczak and co-authors [193]. In their study, the increasing amount of inulin
resulted in lower consistency and paste viscosity of the GF batter. The increase of the
viscoelastic compliance values and gelatinization temperatures were also reported.
Changes in the rheological properties can be associated with the reduced volume of
starch granules and limited friction. Water binding capacity of inulin may reduce the
swelling of starch granules affecting their size [193].
Chicory-fortified GF bread was characterized by darker crust being a conse-
quence of Maillard reaction, attributed to a larger number of reducing ends formed
due to degradation of inulin [192]. On the other hand, as compared to control, the
obtained bread had an increased rate of staling and harder crumb, what can play
critical role in the consumer acceptance [192]. Contrary results were obtained by
Juszczak and co-workers [193], who reported reduction in crumb hardness in
parallel with the increasing amount of the inulin addition, suggesting that it can be
a method of extension of shelf-life of GF bread.
The addition of inulin to GF bread fortified with bovine plasma proteins were
analyzed by Rodriguez Furlán, and co-workers [194]. The enrichment of GF bread
with inulin resulted in decreased moisture loss what was attributed to greater number
of hydrophilic groups in the inulin molecule and consequently higher water retention
[195]. Reduced moisture loss contributed to delay the staling rate of GF bread.
Additionally, the incorporation of inulin improved the quality of GF bread crumb by
reducing its hardness [194]. It can be explained by uniformity and stabilization of the
air bubbles diameter and decreasing the thickness of the walls surrounding the small
air cells. The cracked surface of the crust of GF bread disappeared after inulin
addition, making it smoother and the volume of loaves increased [194].
Recent studies of Scarini and co-authors [196] aimed to compare the effect of
soluble and insoluble fibers addition on the technological and nutritional properties
of GF bread, indicated that inulin used at 5% and 10% substitution level decreased
the dough firmness. Addition of inulin at 10% level resulted in GF bread with less
compact crumb and the highest cell area of crumb as compared to control GF and GF
breads supplemented with other fibers. The texture of loaves was also influenced by
inulin addition with lower firmness and increased specific volume of bread in both
inulin-enriched formulation. The proteins digestibility increased in GF bread with
5% substitution and then decreased in bread with 10% inulin level [196].
The popularity of confectionary products and their high amount of sugar moti-
vated scientist to develop modified sweets with higher nutritional quality, including
dietary fiber. In GF products, as gluten is replaced with fat and sugar to maintain the
texture, the problem of unhealthy sweets is especially important. ITFs as fat and
sugar substitute were proposed as additives to GF confectionary products.
Maghaydah and co-workers [197] demonstrated that different levels of inulin
increased moisture of GF cookies in parallel with increasing share of inulin. More-
over, inulin increased the dietary fiber content in fortified GF cookies. Cookies are
considered as a good deliverer of nutritional components, especially for children,
because of sweet taste, long shelf-life, and low cost. Additionally, the results showed
no impact of inulin addition on sensory quality, including flavor, texture, aroma, and
softness as well as the technological properties, especially width, thickness, and
spread factor [197].
The effectiveness of application of Jerusalem artichoke powder, containing even
50–60% of inulin in dry mass, as a replacement of both fat and sugar in GF biscuits
formulation have been reported by Sharoba and co-workers [198]. Jerusalem
artichoke powder was used as 25%, 50%, 75%, and 100% substitution of sugar
and corn oil in the GF formulation separately, indicating that inulin enriched product
can be successfully replaced control biscuits. The inulin-enriched biscuits were
characterized by 29% increase in protein and even fourfold increase in fiber contents
than control unfortified GF biscuits. Additionally, the caloric value reduced with the
increasing amount of inulin. The mineral content, including calcium, magnesium,
and iron was 26, 54% and even twofold higher in inulin-rich biscuits as compared
to control. The substitution of oil or sugar lower than 75% did not cause significant
changes in both physical and sensory properties of biscuits but allowed them to
deliver the recommended daily allowances (RDA) of protein, fiber, and minerals
[198].
Gularte and co-workers [199] examined the effect of incorporation of both
soluble or insoluble fibers and their mixes into a GF layer cake. The addition of
blended soluble (inulin) and insoluble (oat fiber) fibers improved the technological
properties, particularly specific volume of the fortified cake. Addition of inulin alone
resulted in decreased properties of dough, including density and springiness, and
reduced the amount of resistant starch [199]. It is well known that the amount of
resistant starch is a parameter of concern in carbohydrate-based products [200].
Recent studies aimed to analyze the rheological parameters of chickpea flour-
based GF muffins substituted with different concentrations of different biopolymers,
including inulin [201]. Authors reported that an inulin-enriched batter was similar to
batter supplemented with wheat protein in terms of heating pattern. The value of loss
tangent during non-isothermal heating increased, resulting in more clear decreases in
elastic modulus as compared to dissipative modulus at early stage of heating. Higher

values of loss tangent are associated with reduced viscoelasticity of batter [201].
Pasta is a staple food based on starches, being a good source of carbohydrates in
human diet. Simplicity of cooking, storing, and handling supports the high popular-
ity of this product. Majority of GF pasta is produced of corn and rice starch. Like
other GF products, technological properties and nutritional value of GF pasta are
poorer as compared to semolina-based product [9]. Therefore, many researchers
have attempted to improve the properties of pasta by incorporation of additives
having beneficial effect on texture with simultaneous revised impact of human
health.
First, the use of inulin was proposed as a supplement of semolina-based pasta.
The nutritional quality of inulin-enriched pasta improved by inhibiting the release of
sugar during in vitro starch digestion is what reduced the predicted glycemic index
by even 15% in formulation with 10% inulin concentration [202]. Inulin influenced
the swelling index but did not affect the cooking loss of pasta, adhesiveness, and
elasticity. Moreover, the incorporation of inulin reduced firmness of pasta, owing to
the ability of inulin to compete with starch for available water, limiting the swelling
and gelatinization of starch granules [202].
Contrary results were obtained in term of GF pasta. Mastromatteo and co-workers
[203] prepared GF pasta based on maize starch with addition of inulin up to 20%,
and they found that enrichment with 5% and 7.5% of inulin resulted in pasta with the
highest values of elongation and shear viscosity. Authors stated that the incorpora-
tion of inulin over 12.5% increase the firmness of the pasta dough, and finally, too
high share of inulin, amounting 15% and 20%, resulted in pasta with such high
firmness that made impossible to conduct rheological analysis. However, the dough
preparation might affect the rheological properties. The authors applied a pre-
gelatinization process [203]. Consecutive heating and cooling of starch in water
condition results in the breaking of amylose and amylopectin chains, and conse-
quently their rearrangement into a crystalline form. It contributes to the retrograda-
tion of starch and starts physical changes in the dough including higher viscosity,
improved gel formation and increased degree of crystallinity and exudation of water
[204]. The main difference in the starch retrogradation between GF and gluten-
containing pasta can be related with the properties of proteins. Gluten proteins have
been shown to have no impact on amylopectin; however, glutenin fraction was found
to inhibit the starch retrogradation [205]. In contrast, another fractions of wheat
protein, including gliadin, albumin and globulin have the ability to improve the
retrogradation process [205]. The sensory analysis of dry and cooked gluten-free
pasta enriched with inulin showed that these products had an improved quality,
wherein the pasta fortified with 5% inulin was characterized by the best sensorial
properties, being the most similar to control pasta [203]; however, the higher amount
of inulin in spaghetti was associated with a decrease in sensory score, but these
products were still acceptable.
Traditional white sauce is based on the wheat flour; however, people suffering
from gluten-related disorders can replace it with non-gluten counterparts, such as
rice and corn starches [206, 207]. Authors reported that inulin added to the gluten-
and lactose-free, fat-reduced white sauce based on rice starch improved gelatiniza-
tion of the starch and reduced the viscosity [206]. Additionally, the high water-
binding capacity of inulin inhibited starch degradation and hindered the leaching of
amylose. Inulin added to sauce limited the formation of bonds between proteins and
starch, and consequently, the sauce was more homogeneous. Another study of the
same group of authors showed that also a corn starch-based white sauce can be
successfully supplemented with inulin [207]. Inulin-enriched white starch-based
sauces were characterized by acceptable sensory quality and stability during refrig-
erating, therefore they were proposed as a suitable substitute for people suffering
from CD and lactose intolerance [206].
Tárrega and co-authors [20] studied the effect of fat substitution with different
DP inulin on the quality of custard – creamy dessert based on milk or cream and egg
yolk which can include also starch or gelatin. The rheological properties of analyzed
custard based on hydroxypropyl-distarch phosphate from Tapioca starch and fortified
with inulin significantly differed from whole-fat dessert. However, the inulin-enriched
custard was characterized by higher thickness, sweetness, and vanilla flavor perception.
The formulation with higher amount of inulin as compared to short-chain FOS (ratio
75:25) gave a product with the best texture and pseudoplasticity [20]. Another studies
conducted on custard confirmed that inulin can positively influence texture of dessert,
due to the water binding capacity of inulin allowing to maintain the thermally stable
complexes [207, 208]. The inulin-enriched desserts were characterized by increase of
creaminess and sensation of roughness, the improvement of consistency, and simulta-
neously the decrement of smoothness [20, 208].
In general, dairy products are naturally GF products consumed all over the
world. The beneficial effects of dairy fat on human health as well as on the
physiochemical and technological properties of dairy products are commonly
known [210]. Recently, consumers are looking for low-fat and low-caloric products
without compromising the texture and sensory properties of products. Long-chain
inulin, having the ability to form crystals, is used as a texture-creator in low-fat
dairy products. This ability was used in the studies conducted on acid casein-
processed cheese analogs resulting in improved meltability, density, cohesiveness,
and viscosity with simultaneous reduction in hardness and adhesiveness [211].
Another study confirmed that substitution of 10% of fat in cream cheese with inulin
allow to obtain the product with chemical features similar to control high-fat cheese
but with reduced caloric value [212]. Additionally, inulin incorporated into cheese
reduced the rate of syneresis in spreads and fresh cheese [212]. Dave and co-
workers [213] reported that the inulin percent of substitution of fat can affect the
rheological properties of cheese spreads. They showed that 6% fat replacement
result in nonacceptable cheese with a mushy texture. However, increasing concen-
tration of inulin (7% and 8% of fat substitution) caused improvement of the
spreadability, reaching the parameters close to a control full-fat cheese [213].
Moreover, inulin was applied to improve the nutritional properties of petit-suisse
cheese due to the prebiotic effect [214]. Authors reported that inulin added simul-
taneously with probiotics resulted in the most promising formulation having the
potential health benefit.
The stability of inulin in moderate pH condition was used in studies on fermented

milk resulting in the increase in the total solid contents leading to increase of the
acidity of fermented product. The modified fermented milk was characterized by
higher viscosity as compared to control unfortified milk. Additionally, thixotropy of
inulin-rich fermented milk increased in parallel with increasing temperature, while
hysteresis decreased [215].
6.2 FOS
Short-chain ITFs are characterized by completely different properties as compared to

long-chain inulin; therefore, the effect of their incorporation into GF products is
diverse. The influence of the addition of ITFs with different DP into GF bread on the
physiochemical properties and staling rate was studied by Ziobro and co-workers
[216]. The application of FOS significantly (P < 0.05) increased GF bread volume in
parallel with the increasing amount of the ITF addition. Authors explained it by the
competition for unbound water between short-chain ITFs, consisting of mono- and
oligosaccharides, and starch, resulting in the delay of starch gelatinization, and
consequently the increase of loaves volume. FOS improved porosity and uniformity
of bread crumb and reduced staling rate during storage [216]. The authors concluded
that FOS can be successfully used to improve the technological quality of GF bread,
while the inulin with high DP can be used in bakery technology only to improve the
nutritional value, when the improvement in the structure and mechanical properties
in not necessary.
Short-chain FOS is characterized by a higher sweetness which can influence the
sensory perception of GF products with their application. The study conducted by
Morais and co-workers [217] evaluated the effect of different ITFs on the sensory
properties of GF bread, using a quantitative descriptive analysis (QDA) performed
by a trained sensory panel or consumer tests of CD patients. FOS-enriched GF bread
was characterized by better taste and aroma as well as improved appearance param-
eters such as porosity, texture, and crust color [217].
Cassava starch is used for making special cheese GF bread, popular in the
countries of South America [218]. FOS was proposed as a supplement of this
special type of GF bread influencing water absorption index and starch solubility.
The pasting viscosity decreased with increasing concentration of FOS in GF bread
formulation due to higher solubility of FOS as compared to starch. Authors
reported changes in GF cheese bread with 9% FOS addition; however, still this
amount was not sufficient to consider FOS-enriched bread as a good source of
dietary fiber [218].
Addition of 6% of short-chain FOS into custard improved consistency, viscosity,
and elasticity of dessert [20, 208, 209]. However, authors stated that these effects
were strongly dependent on the presence of carrageenan. High water-binding capac-
ity of water-soluble FOS contributes to increased viscosity [20]. Sensory analysis
showed that FOS-enriched custard is characterized by higher sweetness and inten-
sive flavor [20, 208].
FOS can be also used as sugar substitute in low-caloric yoghurt [219]. The
increasing concentration of FOS (up to 8%) affects the physicochemical and
rheological properties of yoghurt, increasing thixotropy, viscosity, as well as storage
and loss moduli. Texture of FOS-enriched yoghurt has been characterized as a weak
gel. The authors evaluated also post-acidification of FOS-supplemented yoghurts
showing no changes in acidity, what is desirable in modern yoghurts and concluded
that FOS was not utilized by applied bacteria (Streptococcus thermofilus and
Lactobacillus bulgaricus) as a carbon source. Survival analysis of consumer accep-
tance showed that increasing amount of FOS reduced the sensory quality of
yoghurt. The addition of approx. 2.6% of FOS was reported as the most accurate
for yoghurts [219].
6.3 Synergy Between Inulin and FOS
As short-chain FOS and long-chain inulin differ in their properties, studies were
performed aiming to evaluate the effect of a mixture of both fractions to obtain
product with improved properties. Commercially available oligofructose-enriched
inulin (Synergy 1) was used in a study on GF bread [220]. In general, the techno-
logical properties of GF bread supplemented with Synergy 1 were improved.
Irrespective of the amount of fructans added to a formulation, breads have higher
specific volume which could be attributed to higher CO2 retention capacity. Addi-
tionally, ITFs containing reducing sugars positively affected the crust color of
gluten-free bread, what resulted from the formation of brown nitrogenous polymers
and melanoidins as the ends products of Maillard reaction during baking [220].
Moisture of Synergy 1-enriched batter and bread was reduced, leading to faster
staling. The authors also confirmed the improvement of the sensory qualities
of gluten-free breads fortified with ITFs. Irrespective of the amount of fructans
added, the enriched breads received higher scores in terms of appearance, color,
texture, and taste. The nutritional quality of GF bread was improved after addition of
Synergy 1 in term of higher concentration of dietary fiber and reduced glycemic
index (from 71 to 48) and glycemic load (from 12 to 8) in formulation with 12%
addition of ITFs [220].
Synergistic effect of FOS and inulin was used also to improve the quality of
chocolate dairy dessert [221]. Sensory analysis showed that formulation with 7.5%
of ITFs and 0.2% guar gum had the best scores in terms of appearance, aroma, taste,
texture, and overall acceptability of chocolate dairy desserts. On the contrary,
addition of 10% of ITFs decreased dramatically the sensory acceptance of that
product. The study indicated that the moderate concentration of ITFs (7.5%) and
guar gum (0.2%) was the most appropriate in term of creaminess of chocolate
evaluated using a 9-point just-about-right (JAR) scale [221].
Table 2 Summarized effect of the addition of ITFs with diversified DP on the quality parameters
of gluten-free products.
Product Percent of additive Effect Reference
Inulin
Bread 3–8 Increased loaves volume [190]
Reduced crumb cohesiveness and
springiness
Wrinkled crust
Reduced hardening
Bread 9 Increased moisture [191]
Increased specific volume
Darker crust
Reduced hardness
Bread 4–12 Lower dough consistency and paste [192]
viscosity
Increased the viscoelastic compliance
values
Increased gelatinization temperatures
Reduced crumb hardness
Bread 0.5–3.5 PUFIa Reduced moisture loss [193]
Reduced staling
Increased loaves volume
Improved crumb color and porosity
Bread 5, 10 Reduced dough firmness [195]
Improved porosity
Increased specific volume
Reduced firmness
Cookies 3–4.5 Increased moisture [196]
Increased content of dietary fiber
No influence on sensorial quality
Biscuits 25–100% sugar and Improved protein and dietary fiber content [197]
oil substitution Reduced caloric value
Improved mineral content
No influence of physical and sensorial
quality
Layer cake 20 or mixed with Decreased density and springiness (inulin [198]
oat fiber (1:3 ratio) alone)
Reduced starch resistance (inulin)
Improved specific volume (inulin þ oat
fiber)
Muffins 5–15 Heating pattern similar to wheat protein- [200]
containing dough
Reduced viscoelasticity of batter
Pasta 2.5–10 Reduced predicted glycemic index [201]
Reduced firmness
Reduced starch swelling and gelatinization
No effect on cooking loss, adhesiveness and
elasticity
(continued)
Table 2 (continued)
Pasta 5–20 5–7.5% – improved elongation and shear [202]
viscosity
Over 12.5% – increased firmness
Improved sensory quality
White sauce 2.5 Improved gelatinization [205, 206]
Reduced viscosity
Reduced starch degradation
Improved homogeneity
Custard 7.5 Increased thickness, sweetness and vanilla [20, 207,
flavor perception 208]
Improved texture and pseudoplasticity
Increased creaminess and smoothness
Cheese 1–3 Improved meltability, density, cohesiveness, [210]
analogs viscosity
Reduced hardness and adhesiveness
Cream 8–12 Cheese with reduced caloric value but with [211]
cheese properties similar to full-fat cheese
Reduced syneresis rate
Cheese 6–8 Mushy texture (6%) [212]
Improved spreadability (7–8%)
Petit-suisse 10 Stimulation of growth of probiotic bacteria [213]
cheese
Fermented 5 Improved viscosity [214]
milk Increasing thixotropy and decreasing
hysteresis parallel with increasing
temperature
FOS
Bread 3–8 Increased loaves volume (5–8%) [190]
Increased hardness (5–8%)
Decreased volume and hardness (3%)
Bread 4–12 Increased loaves volume [215]
Delayed starch gelatinization
Improved porosity and uniformity
Reduced staling
Bread 0.75 Improved sensory characteristic in terms of [216]
taste and aroma
Improved porosity, texture and crust color
Cheese 9–29 Increased starch solubility and decreased [217]
bread water absorption index
Decreased specific volume
Custard 7.5 Improved consistency, viscosity and [20, 207]
elasticity
Higher sweetness and intensified flavor
Yoghurt 2–8 Increased thiotrophy and viscosity [218]
Reduced texture
Reduced sensory perception
(continued)
Table 2 (continued)
Synergy 1
Bread 4–12 Reduced moisture [219]
Increased volume and specific volume of
loaves
Darker crumb and crust color
Reduced glycemic index and increased
dietary fiber content
Improved sensory perception
Chocolate 5–10 Acceptable sensory properties and [220]
dairy creaminess
dessert
a
A formulation of ultrafiltred and freeze-dried bovine plasma with the addition of inulin (63%)
7 Conclusions
The increasing demand on high-quality GF products and an increasing prevalence of

GF consumers favors the development of research aimed to improve the overall
quality of GF products. The literature data on the application of ITFs in the GF
products and on their health beneficial properties, as presented in this chapter, points
to a great potential of ITFs in the GF living. In this chapter, we have presented the
most recent studies on GF products in which ITFs were applied as valuable ingre-
dients affecting the rheological and technological parameters of GF products (Table 2).
ITFs added to GF products interact with other ingredients and additives, but, in
general, they improved the sensory perception of obtained GF products. The evi-
dences of beneficial impact of ITFs on characteristic of GF goods presented in this
chapter are promising and therefore, could contribute to further development and
intensified research on new GF products of superior quality that will be dedicated to
people suffering from gluten-related disorders.
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Development of Dietary Fiber-Rich Meat
Products: Technological Advancements 26
and Functional Significance
Nitin Mehta, Manish Kumar Chatli, Pavan Kumar, Om Prakash Malav,

Akhilesh Kumar Verma, Yogesh Kumar, and Dinesh Kumar
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
2 Dietary Fiber: Definition and Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
3 Analysis of Dietary Fiber: Methodologies and Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
4 Physiological Benefits and Health Implications of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . 770
5 Technological Functionality of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
6 Effect of Fiber Addition on Physicochemical Properties of Meat Products . . . . . . . . . . . . . . 774
7 Fiber Incorporation and Proximate Composition of Meat Products . . . . . . . . . . . . . . . . . . . . . . 785
8 Dietary Fiber and Textural Properties of Meat Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
9 Sensory Properties of Meat Products in Relation with Added Fiber Sources . . . . . . . . . . . . 786
10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
N. Mehta (*) · M. K. Chatli · P. Kumar · O. P. Malav · D. Kumar

Department of Livestock Products Technology, College of Veterinary Science, Guru Angad Dev
Veterinary and Animal Sciences University, Ludhiana, Punjab, India
[email protected]; [email protected]
A. K. Verma
Department of Livestock Products Technology, College of Veterinary Science, Pandit Deen Dayal
Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya evam Go Anusandhan Sansthan (DUVASU),
Mathura, Uttar Pradesh, India
Y. Kumar
ICAR – Central Institute of Post-Harvest Engineering and Technology (CIPHET), Ludhiana,
Punjab, India

764 N. Mehta et al.
Abstract
As a source of proteins having high biological value, essential nutrients like
minerals, vitamins, and surfeit of bioactive compounds, meat is considered as
an ideal food for masses. With the advancement of technology and change in
socioeconomic status, the consumers have housed high preference for processed
and value-added meat products. These products generally lack nutrients like
complex carbohydrates that include dietary fiber. Fiber is considered as an
important component in diet owing to its multifarious utilities as cardioprotective,
weight reducer, management of diabetes, antioxidant, and stress reliever. Its
inclusion in diet has been stressed in many studies and incorporation in products
particularly high energy-dense products like meat is imperative. For adults, the
recommended acceptable intakes of dietary fiber are 28–36 g/day and out of that,
70–80% must be insoluble fiber. Apart from acting as an integral fraction of diet,
dietary fiber performs many functions in meat products, viz., improvement in
yield, desirable processing attributes, fat reduction, texture modification, etc.
Being a by-product of agriculture, these fiber sources are comparatively cheaper
and its inclusion on meat products helps in reducing its overall cost of production.
Thus, the inclusion of fiber in meat products helps in improving processing and
technological functionality with proven health benefits for consumers.
Keywords
Dietary fiber · Meat · Technological · Processing · Cooking yield · Sensory
1 Introduction
An increasing awareness among people regarding their health and well-being has
propelled the research in food science to a new level. The inclination to natural sources
has been increased in recent times owing to many nutritional diseases of affluence that
have posed a challenge to both processors as well as medical professionals [1].
However, rapid urbanization and globalization has introduced ready-to-eat, ready-to-
cook, and packaged processed products on consumer’s platter. These products are rich
in fat, salt, and calories but are highly deficient in dietary fiber. Earlier, unrefined
grains and vegetables which contain a significantly high amount of fiber were an
essential dietary component but the industrial revolution brought a dynamic change in
lifestyle resulting in removal of key components from diet, particularly fiber [2]. The
extensive study of dietary fiber in recent times could be directly attributed to its
multifunctional roles. An improvement in technological and processing characteristics
of products along with beneficial physiological effects like lowering blood sugar,
cholesterol, improving cardiac health, etc., led the researchers to search new sources of
fiber which can be readily incorporated in food products [3].
Meat is considered as a nutrient-dense food item with a high amount of protein,
fat, minerals, and vitamins that have a comparatively better bioavailability than other
26 Development of Dietary Fiber-Rich Meat Products: Technological Advancements. . . 765
food stuffs [4]. However, a damaging crusade against meat as a culprit for many
health hazards has blemished its image to great extent. The deficiency of complex
carbohydrates like dietary fiber in meat has been implicated with an increase in
number of diseases like colon cancer and cardiovascular diseases [5]. A solution that
has been proposed is enrichment of meat products with various fiber sources. It will
not only lead in designing meat product with desired nutritional attribute but also a
superior technological advantage attributed to characteristic features of fiber like
water- and oil-binding capacity in addition to antioxidant function [6]. A number of
studies have already been carried out by using fiber alone or in combinations for
formulation of various categories of meat products [7, 8]. This chapter highlights
about the functionality of dietary fiber, its physiological and technological signifi-
cance with a major emphasis on its application in meat products.
2 Dietary Fiber: Definition and Classification
Since 1953 when Eben Hipsely first coined the term dietary fiber, its definition has
been revised many a times and till date it is evolving. Initially referred to as
unavailable carbohydrate content in foods that lowered the rates of pregnancy
toxaemia [9], it included lignin, cellulose, and hemicellulose. Later, investigations
regarding correlation of disease incidence and fiber intake led the researchers to
recommend increased stool volume and softness through higher dietary fiber intake
and hence a new definition was proposed [10]. The most unswerving definition was
proposed by Trowell [11] that dietary fiber consists of plant components resistant to
digestion by enzymes in alimentary canal of humans, and it includes cellulose,
hemicellulose, lignin, oligosaccharides, pectin, gums, and waxes. So thereafter, a
categorization of carbohydrates into two basic groups depending upon their digest-
ibility in GIT was done. The first group included starch, fructans, and simple sugars
that are easily hydrolyzed and absorbed in small intestine. They are sometimes
referred to as nonstructural or nonfibrous polysaccharides. The second group
included cellulose, hemicellulose, lignin, and pectin, that are resistant to hydrolysis
in small intestine, and are referred to as nonstarch polysaccharides or structural
carbohydrates. Contemporary definition of dietary fiber has been proposed by
American Association of Cereal Chemists (AACC) and Codex Alimentarius Com-
mission (CAC). According to AACC, 2001, “Dietary fiber refers to the edible part or
analogous carbohydrates that are resistant to digestion and absorption in the human
small intestine with complete or partial fermentation in the large intestine. According
to this definition dietary fiber includes polysaccharides, oligosaccharides, lignin, and
associated plant substances. Dietary fibers promote beneficial physiological effects
including laxation, and/or blood cholesterol attenuation, and/or blood glucose atten-
uation” [12]. The latest definition proposed by CAC states that dietary fiber are
carbohydrate polymers with degree of polymerization not lesser than 3 and are
resistant to digestion as well as absorption in small intestine. Further, it possesses
properties like capability to increase stool bulk by decreasing intestinal transit time,
766 N. Mehta et al.
reduction of cholesterol, and postprandial glucose levels and fermentable by intes-

tinal microbiota [13].
Dietary fiber can be classified in a number of ways such as on the basis of source
from which they are derived (plant, animal, or synthetic), structure (linear, nonlinear,
or branched), or solubility. The most acceptable classification is on basis of enzy-
matic fermentation behavior in a simulated system of that of human alimentary
canal. It can be divided into two broad categories, viz., soluble dietary fiber (SDF)
and insoluble dietary fiber (IDF). Insoluble fiber comprises cellulose, part of hemi-
celluloses, and lignin, whereas, soluble fiber constitutes pectins, gums, pentosans,
and mucilage [16, 17].
Various components of dietary fiber are presented in Table 1 and classification of
fiber on basis of solubility is presented in Table 2.
Cellulose: Cellulose is an unbranched chain of β(1!4) linked glucose monomers
and is the most abundant polysaccharide found in nature which forms major
constituent of cell walls in green plants and vegetables. The close packing of linear
polymers renders it mechanically strong, water insoluble, and resistant to digestive
enzymes in human gut. However, a partial microbial degradation in the large
intestine may produce beneficial short chain fatty acids. It has an ability to bind
water, thereby, increase fecal bulk, and relieve constipation. Two different compo-
nents of cellulose had been studied by Aspinall [19] on basis of relative hydrolysis.
The major portion of cellulose is crystalline component that has noncovalent hydro-
gen bonds responsible for its immense mechanical strength, resistance to degrada-
tion by gut microbes, and water insolubility. Whereas, the amorphous component
constitutes a minor portion, i.e., 10–15% and is readily hydrolysable.
Hemicellulose: Similar to cellulose, these are also integral component of plant
cell wall. It consists of chain of β(1!4) linked glucose monomers which are smaller
in size than cellulose, usually branched and contain a number of sugar moieties like
pentose (xylose and arabinose) and hexose units (mannose, galactose, rhamnose) [3,
20]. The main function in human system is binding of cholesterol and promotion of
smooth bowel movements.
Lignin: It is a complex polymer that consist about 40 oxygenated phenylpropane
units including coniferyl, sinapyl, and p-coumaryl alcohols that have undergone a
complex dehydrogenative polymerization [21, 22]. Variable molecular weight and
inertness owing to strong intamolecular bonding are characteristic features of this
polymer.
Pectin: Composed of galacturonic acid chains connected with α(1!4) bonds,
this linear polymer forms important structural component of plant cell wall and outer
skin of fruits and vegetables. It is highly water soluble polysaccharide which is easily
degraded by the microflora of the colon. A unique gel-forming capability of pectin
makes it an ideal constituent of health foods as it decreases the rate of gastric
emptying, improves cholesterol and lipid metabolism [23], and prevents and control
diabetes in human beings [24]. Further, technologically it is applied in food as a
gelling and thickening agent.
Hydrocolloids: It includes a wide range of polysaccharides that are viscous in
nature. Majorly formed in secretory cells of plants, these are highly branched and
Table 1 Various components of dietary fiber [1, 14, 15]

Fiber Principal
components groupings Description Fiber sources
Nonstarch Cellulose Principal structural Cellulose plants (vegetable,
polysaccharides component of cell wall in sugarbeet, various brans)
and plants; β-(1, 4) glucose
oligosaccharides polymer chain; soluble in
concentrated acids
Hemicellulose Polysaccharides in cell Arabinogalactans,β-glucans,
wall with β-(1, 4) arabinoxylans,
glycosidic linkage glucuronoxylans,
xyloglucans, galactomannans,
pectic substances
Polyfructoses A polysaccharide of Inulin, oligofructans
fructose that may contain
some other sugars too,
e.g., β-(2–1)-D-
fructosyl-fructose
Gums and Secreted by plants at site Seed extracts
mucilages of injury; may contain (galactomannans – guar and
rahmnose, arabinose, locust bean gum), tree
xylose, galactouronic exudates (gum acacia, gum
acid, mannuronic acid, karaya, gum tragacanth), algal
etc. polysaccharides (alginates,
agar, carrageenan), psyllium
Carbohydrate Pectins Cell wall components Fruits, vegetables, legumes,
analogues with D-galactouronic potato, sugarbeets
acid polymer chains;
homogalactouronan,
rhamnogalactouronan,
xylogalactouronan, etc.
Resistant Starch and starch Various plant such as maize,
starches and degradation products; pea, potato
maltodextrins contain α-(1,4)-D-
glucose polymer chain
with α-(1,6)-D- glucose
branching
Chemical Synthetic in nature Polydextrose, lactulose,
synthesis cellulose derivatives
Enzymatic Produced by enzymatic Neo sugar or short chain, guar
synthesis hydrolysis; may possess hydrolysate
prebiotic properties fructooligosaccharides, levan,
xanthan gum,
transgalactooligosaccharides,
oligofructose,
xylooligosaccharide, curdlan
Lignin Lignin Noncarbohydrate cell Woody plants
wall component that
resists bacterial
degradation;
polyphenols like
(continued)
768 N. Mehta et al.
Table 1 (continued)
Fiber Principal
components groupings Description Fiber sources
syringyl alcohol,
guaiacyl alcohol as main
chain
Substances Waxes, cutin, Protective in nature too Plant fibers
associated with suberin
nonstarch
polysaccharides
Animal-origin Chitin, Glucosamine linkages Fungi, yeasts, invertebrates
fibers chitosan, may be present
collagen,
chondroitin
Table 2 Classification of dietary fiber on basis of water solubility [18]

Class Examples
Insoluble Cellulose
Soluble (only in hot water) Agars, amylose, algins, kappa-type carrageenans (in the
presence of K+ or Ca2+), gelan, konjac, mannan, locust
bean gum, low-methoxyl pectins, granular starches, and
starch derivatives
Soluble (in water at room temperature Curdlan, hydroxylpropyl celluloses, hydroxylpropyl
but insoluble in hot water) methylcelluloses, and methylcelluloses
Soluble (in water at room temperature Alginates, amylopectins, carboxymethyl celluloses,
and hot water) dextrins, iota-type carrageenan, guar gum, gum Arabic,
high-methoxyl pectins, polydextrose, and xanthan gum
bind with water to form gels. This property is utilized in food systems as a gelling
agent and thickener, e.g., gums, mucilage, and seaweed extracts (agar, alginate,
carrageenan, etc.). The physiological effect of hydrocolloids is exerted by its enzy-
matic hydrolysis owing to higher soluble fraction of dietary fiber. It is widely used as
cholesterol lowering and weight reducing component in diets.
Resistant starches (RS): Starches that escape the enzymatic degradation in small
intestine are referred to as resistant starch. Divided into four categories, it has a wide
application in food matrix. Type 1 (RS1) is made up of starch granules that are
encapsulated by an indigestible plant matrix rendering it physically inaccessible and
heat stable [25]. Type 2 (RS2) occurs in its native form such as in an uncooked potato
and are resistant to enzymatic hydrolysis due to its compact structure. Type 3 (RS3)
represents the most resistant form and are crystallized starches made by unique
cooking and cooling of gelatinized starch. Type 4 (RS4) is a starch consisting of
chemical bonds other than α-(1,4) and α-(1,6). The chemical modification is induced
by esterification, crosslinking, or transglycosylation and is responsible for its limited
digestibility in human gut [26].
Nondigestible oligosaccharides: They are found naturally in plants, fruits, and
cereals and are synthesized by polymerization of monosaccharides or disaccharides.
They perform similar physiological functions as that of dietary fiber and are highly
fermentable in intestine, e.g., fructo-oligosaccharides and galacto-oligosaccharides.
Due to their prebiotic properties, its application in food systems is quite promising.
3 Analysis of Dietary Fiber: Methodologies and

Modifications
The analytical methods of dietary fiber estimation is varied and have been modified
with time. Weende system started way back in nineteenth century and is still adopted
in many industries to determine fiber content. The proximate system of analysis
relies on determining carbohydrate content by difference. Further through successive
acid and alkaline digestion, the extraction of crude fiber was done, but the crude fiber
makes up only very small part of whole dietary fiber. During acid–base treatment,
hemicellulose and lignin loss occurs, and hence the total fiber is underestimated.
Later, with the advancement of chemical analytical protocols, different solvent
methods were employed for extraction and measurement of dietary fiber from
foods [27]. The protein and starch are removed with various solvents, leaving behind
undigested fiber. These methods are divided into many subtypes based on type of
solvent used e.g., alcohol insoluble fiber extraction, neutral detergent method, and
acid detergent method. The neutral detergent method measuring insoluble fractions
and lignin provided a reliable methodology for estimating fiber fractions [28].
However, the relative disadvantage of solvent extraction methods is that there can
be damage to the fiber during processing by solvent at many stages, leading to
underestimation of total fiber. Currently, the estimation of dietary fiber in foods is
carried out by three different methods, viz., nonenzymatic-gravimetric, enzymatic-
gravimetric, and enzymatic-chemical. However, enzymatic-gravimetric Association
of Official Analytical Chemists (AOAC) method and enzymatic-chemical method
are predominately used [29, 30]. Enzymatic-gravimetric method was first introduced
by Schaller [31] wherein he presented amylase treatment in preexisting methodology
of Van Soest [32]. Later, Prosky et al. [29] adapted the method for insoluble and
soluble fractions which is still in use. This method determines mainly a group of
polysaccharides, lignin, and some of associated compounds like waxes. The enzy-
matic treatment removes starch and protein along with precipitation of soluble fiber
components by organic solvents. The main disadvantage of this method is that
oligosaccharides and some of resistant starches are not quantified, which has been
rectified by McCleary et al. [33] in their modified method of fiber determination.
The enzymatic-chemical methods also involve removal of starch and protein as a
first step. Thereafter, precipitation with ethanol or dialysis ensures separation of SDF
fraction from hydrolyzed starch and sugars [34]. The neutral sugar contents are
determined by GLC or HPLC, whereas, total sugars are computed spectrophotomet-
rically. Filtration is carried out to get the residue after hydrolysis of total poly-
saccharides and quantified as Klason lignin. The nonstarch polysaccharide and
Klason lignin in combination gives total dietary fiber value in addition to separate
quantification of SDF and IDF contents.
770 N. Mehta et al.
Additionally, mixed treatment methodology for determination of total dietary

fiber content yields better results than any single treatment. It involves chemical,
mechanical, enzymatic, and microbial fermentation methods in proper combina-
tions. Zong Cai et al. [35] reported that microbial fermentation improved the content
of SDF by 15% when used alone, whereas, the yield increased to 35% when used
in combination with microfluidization. So, the hybrid methodologies perform
better in extraction and separation of dietary fiber constituents than a single
treatment process [27].
4 Physiological Benefits and Health Implications of Dietary

Fiber
A strong link has been established between dietary fiber consumption and human
health through a number of studies carried out on human subjects. A correlational
analysis through independent observational studies helped in zeroing down on
concept of dietary fiber’s role as a potential disease-preventing nutrient [36, 37].
However, the mechanism through which fiber exerts its effect depends mainly on
type and composition of that fiber.
(a) Dietary fiber and cardiovascular health: Cardiovascular diseases are reported
to be the most common cause of death around the globe [38]. Basically, it is a
group of etiologies that includes coronary heart disease, hypertension, arrhyth-
mias, strokes, and heart failure [39]. The role of fiber in preventing cardiovas-
cular disease is multifaceted. It acts as reducing agent of hyperlipidemia and
hypocholesterolemia through interfering with lipid or bile acid metabolism [2,
40]. Babio et al. [41] reviewed the different sources of dietary fiber on lipid
profile and concluded that intake of highly viscous dietary fiber results in
decreasing LDL-cholesterol. Highly fermentable dietary fiber like pectin,
gums, mucilage, etc., produce short-chain fatty acids (butyric, acetic, propionic),
and it further results in rejuvenation of microbiota and reduction in intestinal
inflammation which leads to further lowering down of cholesterol in body. On
similar lines, Zhang et al. [42] established cholesterol lowering effect of oat bran
in ileostomized hypercholesterolemic individuals. Lowering of serum total cho-
lesterol and LDL-cholesterol by 4% and 6%, respectively has been reported by
supplementing diet with psyllium [43]. Other mechanisms that have been
suggested include inhibition of hepatic lipoprotein production and cholesterol
synthesis by fermentation products of dietary fiber. This may lead to increased
insulin sensitivity [44]. The dietary fiber may also directly interact with lipase
enzyme resulting in reduction of its activity [45], bind with bile salts, thereby
preventing lipid emulsification and its absorption in small intestine [46], may act
as a barrier between lipase enzyme and fat droplet by forming a protective
covering around it [47] or by increasing aqueous phase viscosity leading to
disturbance in lipid droplet coalescence and disruption in stomach and intestine
[48]. Dietary fiber may also interact with bile acids in small intestine resulting in
lower reabsorption and higher excretion of fat including cholesterol [49]. Fur-
ther, it may also alter micelle formation and simultaneously limit incorporation
of cholesterol in that micelle [50].
(b) Maintenance of gut health: Major carbohydrates that reach the intestine and
exert their beneficial action are nonstarch polysaccharides, some polyols, resis-
tant starch, and nondigestible oligosaccharides [51]. Dietary fiber undergo
fermentation in the large intestine particularly, colon resulting in production of
favorable short-chain fatty acids (SCFA), primarily, acetate, propionate, and
butyrate. They play a key role in maintaining gut health. Butyrate is preferential
fuel for colonocytes that forms a protective lining in colon [52]. Also, SCFA
result in lowering pH of colon leading to inhibition of growth of pathogenic
microorganisms [53, 54]. The fermentation mass in colon further results in
increase in fecal volume and output. Further, absorption of water by dietary
fiber adds to fecal buildup [55, 56]. Increased bulk of feces decreases colonic
transition time and prevents constipation and production of carcinogenic com-
pounds [57]. Some of the fiber exhibit prebiotic properties, e.g., inulin and
fructo-oligosaccharides (FOS) that encourage and stimulate growth of beneficial
bacteria like lactobacilli and bifidobacteria in the gut [58]. Dietary fiber also
plays a key role in the maintenance of gastrointestinal immunity through
increasing T-cell mitogen response and stimulating gut-associated lymphoid
tissue (GALT) [59].
(c) Dietary fiber and its role in prevention and management of diabetes:
Prospective cohort epidemiological studies have concluded a direct link between
consumption of fiber in diet and reduction in prevalence of diabetes. Manage-
ment of type 2 diabetes majorly revolves around glycemic control. Intake of low-
glycemic index (GI) diets will control hyperglycemia, and all the fiber-rich diets
are low GI in nature. Several events leading to decrease in blood glucose level
after consumption of fiber are explained by a number of workers. Lowering of
postprandial glucose peak, which leads to decreased insulin demand, and over-
exhaustion of the pancreas is facilitated through consumption of fiber. Delayed
gastric emptying after consumption of soluble fiber results in lower postprandial
glucose and insulin levels and higher consumption of insoluble fiber reduces
absorption of carbohydrates in GI tract by increasing passage rate of foodstuffs
in gut [60]. Kelley and Mandarino [61] reported that increase in free fatty acids
in blood may result in alteration of glucose metabolism through inhibition of
GLUT-4 transporters. However, the SCFA produced by dietary fiber in gut
results in decrease in serum free fatty acid and may further reduce blood glucose
levels. Dietary fiber could limit the transportation of glucose to intestinal absorp-
tive surface by producing contractions and thus result in decreasing postprandial
glucose peak [62]. Another mechanism through which dietary fiber can result in
preventing diabetes is by stimulating postprandial insulin release via accelerating
secretion of incretin hormones, i.e., GLP-1 and GIP and by increasing insulin
sensitivity in the tissues [63, 64].
(d) Dietary fiber and cancer: Anticarcinogenic and antitumorigenic effects of
dietary fiber are well-documented and supported by research on human subjects.
772 N. Mehta et al.
Fermentation of fiber by colonic bacteria results in the production of SCFA

which are recognized to be potential players in slowing growth and increasing
apoptosis in colon cells. Further, they have a role in decreasing mutations and
cancer risk by activation of different drug-metabolizing enzymes [65]. Tang et
al. [66] ascertained the relationship between dietary fiber intake and reduction in
risk of esophageal cancer in Xingjiang, China, through case-control study. The
regular intake of fiber was found to be inversely associated with esophageal
cancer risk. Another mechanism proposed for role of fiber in countering cancer
risk is that fiber adds to fecal bulk and thereby decreases the interaction between
intestinal mucosa and cancer-risk agents present in feces [67]. Also, decrease in
intestinal transit time will reduce the production of pathogenic bacteria in colon
and eventually limit the production of carcinogens [68].
(e) Dietary fiber and weight management: A major predisposing factor for CVD
and diabetes is excess weight and obesity. A number of studies have established
a strong negative correlation between fiber intake practices and weight control in
human subjects. Miller et al. [69] established effect of diet on fat content in body
and reported that lean subjects had a higher fiber intake than their obese
counterparts. The mechanism that have been proposed for role of fiber in
decreasing weight is that it absorbs water and adds bulk resulting in satiety
[70]. Hyperinsulinemia that leads to lipogenesis and increased incidence of
obesity may also explain the role of fiber in weight management [2]. Fiber-rich
diets delay gastric emptying, resulting in slower rate of absorption, and subse-
quently lesser gain of weight. Sakata [71] proposed that diets rich in fiber may
increase chewing time and efforts resulting in various cephalic- and gastric-
phase signals and early satiation. It is an established fact that fibers help in
controlling and managing weight and subsequently root out the chances of
associated ailments like CVD and diabetes.
(f) Dietary fiber and mineral bioavailability: Some fiber sources regulate the
absorption of minerals in the gut which further affects overall ionic balance in
the body. Soluble dietary fibers like pectin, certain gums, etc., helps in the
absorption of minerals like calcium, iron, and magnesium [72, 73] by producing
SCFA and decreasing intestinal luminal pH [74] and proliferation of epithelial
cells in the caeco-colon [75]. Both these factors may lead to enhanced dissolution
of insoluble minerals salts in colon. Further, fiber absorbs water and increases its
content in colon, solubilize the minerals, and enhance absorption through per-
meable gut membrane [76].
5 Technological Functionality of Dietary Fiber
For the development of dietary fiber enriched meat products, the technological
properties of incorporated fiber play a key role. Apart from providing all the possible
health benefits discussed in previous section, fiber sources possess attributes that
help in modifying the physicochemical characteristics of meat products. This may
pose a new way for designing and developing meat products for enhanced consumer
acceptability.
(a) Hydration properties and oil-binding capacity: Water absorption, water-hold-

ing capacity (WHC), and swelling are the parameters that encompasses hydra-
tion properties of dietary fiber. Water absorption is dependent on substrate pore
volume [77] and is determined by water uptake as measured by Baumann
apparatus [78]. The amount of water retained by known weight of dietary fiber
at specified time and temperature conditions refers to water-holding capacity of
that fiber. Due to hydrophilic nature of polysaccharides, fiber holds water
molecules in void spaces. This property of dietary fiber is largely dependent
on its source, e.g., fiber from cereal by-products have lowest affinity whereas
soluble fibers from algae have highest affinity. Other than this, the structure of
dietary fiber, percentage of soluble fraction, and presence or absence of charged
groups are major determinants of hydration properties. Also, fiber possesses
capacity to hold oil that may help in designing novel functional meat products.
Oil-holding capacity (OHC) is dependent on surface properties of fiber, overall
charge density, and hydrophillicity [79, 80]. Dietary fibers with high OHC may
be useful in stabilizing meat emulsion, whereas, superior WHC ensures higher
product yield, juiciness, and calorie and salt control in meat products. Further, it
may be used as a texture and viscosity modifier in emulsion-based meat
products.
(b) Solubility: On the basis of degree of solubility in water, fiber may be catego-
rized into insoluble and soluble dietary fractions. The regularity in the structure
of polysaccharides decides its fate for enzymatic degradation. Higher degree of
irregularity, e.g., β- glucan renders weak linkages in structure and fiber is prone
to enzymatic degradation resulting in easy solubilization whereas, regular
structure leads to insolubility of fiber, e.g., cellulose. The technological func-
tionality differs for both fractions. Soluble fiber tends to increase viscosity, gel
strength, and emulsification as compared to insoluble fiber. As a result, meat
product incorporated with SDF is juicy, has a higher cooking yield, and
emulsion stability with soft texture. Insoluble fraction tends to increase hard-
ness in meat products with a concurrent reduced gel strength. So, as per the
product attributes and demands, the type of fiber to be incorporated may be
decided by processors.
(c) Viscosity: Ability of dietary fiber particularly SDF to form gels and resist flow
behavior determines its viscosity. It is one of the most important technological
functionality of fiber that determines its application in various categories of meat
products. Water soluble fiber increases viscosity of solution and vice versa [81].
(d) Antioxidant Properties: Countering free radicals and delaying lipid peroxida-
tion is one of the most important functionality of dietary fiber, particularly,
nonstarch polysaccharides (NSP). It has been reported that several fractions of
rice bran possess superior antioxidant properties [82] which are equally effective
as ascorbate. This property may be utilized for the extension of storage life of
meat products without any addition of synthetic antioxidants.
774 N. Mehta et al.
6 Effect of Fiber Addition on Physicochemical Properties of

Meat Products
Effect of addition of dietary fiber in meat products can be visualized through series of
change in their functional properties. Different sources of fibers and their possible
effects on meat products is summarized in tabulated form (Table 3). Various phys-
icochemical properties, viz., pH, water-holding capacity, cooking yield, etc., are
profoundly affected by incorporation of dietary fiber. This in turn affects the
processing characteristics of meat products, e.g., an increase in emulsion stability
by addition of fiber sources in meat holds immense importance from technological
and economic perspective. Further, the change in pH due to fiber source addition
affects the overall quality characteristics.
The change in pH of the meat product after addition of fiber is largely dependent
on the pH of fiber source. The citrus by-products have comparatively lower pH and
when incorporated in meat products, the pH drops significantly. Alesson-Carbonell
et al. [83] reported that addition of lemon albdeo in nonfermented dry cured
sausages resulted in a decrease in pH owing to the organic acids present in raw
albedo. Similarly, with the addition of peach dietary fiber suspensions in low-fat
frankfurters, viscosity of meat batter increased. Further, a decline in pH was
observed and the decrease was prominent as the level of inclusion of peach dietary
fiber increased [84]. Incorporation of kinnow rind powder in goat meat patties
resulted in lower pH of products as compared to control [85]. On the contrary,
Fernandez-Lopez et al. [86] found that there was no decrease in pH in Spanish dry-
fermented sausages incorporated with orange dietary fiber. Apart from citrus by-
products as a source of fiber in meat products, many other sources have resulted in
pH change in different classes of meat products. Yilmaz [87, 88] added different
levels (5, 10, 15, and 20%) of rye and wheat bran in low-fat meatballs and observed
an increase in pH value as compared to control. Similar findings have been
reported by Rao and Reddy [89] and Talukdar and Sharma [90] in chicken loaves
and chicken meat patties incorporated with gram flour and wheat and oat bran,
respectively. However, Mehta et al. [91, 92] reported that addition of psyllium husk
in chicken meat patties and rolls had no significant effect on pH. Similarly, Caceres
et al. [93] and Wu and Lin [94] reported that soluble fiber didn’t affect the pH of
meat products and it was similar to control.
Water-holding capacity is another parameter that is technologically significant in
the development of functional meat products. Ability to retain its own or added water
ensues increase in emulsion stability and cooking yield. Tunisian beef sausages
incorporated with three dietary fibers, namely, VITACEL LC 200, barley beta-
glucan concentrate, and VITACEL KF500 potato fiber were developed and its
physicochemical properties were studied [95]. The fiber displayed high water and
oil-binding capacity resulting in an overall increase in water-holding capacity
(WHC) of beef sausages. However, no significant effects (P > 0.05) on aw values
was observed. A significant decrease in cooking loss was also observed with an
increase in amount of fiber in beef sausages. The reduction in diameter of sausages in
Table 3 Various fiber sources in formulation of meat products and their effects
Type of meat
Type/source of fiber product Major effects on meat products References
Highly soluble fiber sources
Fructo- Fermented 40% reduction in fat content [104]
oligosaccherides cooked sausages Improvement in technological and
sensory properties of sausages
Sausages Cooked sausages with 35% less [93]
energy value and high amount of
soluble dietary fiber without adverse
impact on sensory properties
Fat reduction close to 40% was
obtained
Dry-fermented Fiber had no effect on [105]
sausages physicochemical parameters and
microbiota evolution during ripening
Decrease in lightness value for color
and hardness value for texture was
observed
Psyllium husk Chicken patties Significant increase in dietary fiber [91]
content
Increase in emulsion stability and
cooking yield
Cholesterol content of meat product
was decreased significantly
Chicken meat 4% level of incorporation of [92]
rolls psyllium husk was found to be
optimum
Decrease in moisture, protein, and fat
content of chicken rolls with fiber
Fall in texture and tenderness scores
on sensory evaluation
Inulin Low-fat bologna With addition of fiber, increase in [106]
sausage moisture and decrease in fat content
Significant change in textural
attributes than control
Pork loaves Improvement in processing quality [107]
and functionality of loaves
Significant increase in cooking yield
and emulsion stability
Crude fiber increased by 250% in
treated products
Sausages Addition of inulin did not increase [108]
crude fiber and textural properties in
sausages
Mortadella Textural properties revealed [109]
increased hardness values even at
lowest concentration of 2.5%
Improvement in sensory scores as
compared to control
(continued)
776 N. Mehta et al.
Table 3 (continued)
Type of meat
Veal meatballs Lower concentration of fat and total [110]
trans fatty acids as compared to
control
Meatballs with 20% inulin had
lowest moisture, salt, and redness
value but highest ash and protein
contents
Water-boiled Reduction of fat from 43 to 52% [111]
sausage without any loss to sensory quality
Cooked meat Better emulsion stabilization with [112]
batters creamy and softer texture
Low-fat dry- Decrease in total calorie counts [113]
fermented without any change in sensory
sausages acceptability
Pectin Cooked ham Increase in sensory acceptability [114]
with addition of fiber
Alginate Sausages Reduction in fat without [115]
compromising sensory attributes
Carrageenan Mutton kofta Reduction in fat content with an [116]
increase in protein, ash, and
carbohydrate content
Soft texture with low free fatty acid
content
Konjac flour Frankfurters Treated products were better on [117]
sensory acceptability scores
Better shelf life than high fat control
Cereals and bran
Wheat bran Meatballs Product with lighter color than [88]
control
High ratio of unsaturated to saturated
fatty acid content
Beef patties Increase in hardness and gumminess [118]
of product but decreased springiness
and cohesiveness
Decrease in protein and increased fat
content of cooked beef patties
Chicken patties Increase in water-holding capacity [90]
and emulsion stability
Higher cooking yield, firmness, total
dietary fiber, and unsaturated fatty
acid content, whereas, lower sensory
attributes, moisture, protein, fat, and
cholesterol contents
Rice bran Kung-wan: an Decrease in protein and fat content, [119]
emulsified pork however, an increase in carbohydrate
meatball content
(continued)
Table 3 (continued)
Type of meat
Decrease in textural parameters of
hardness, gumminess, and chewiness
Sensory acceptability up to 10%
level of inclusion
Meat batter Increase in emulsion pH, emulsion [120]
stability, and cooking yield
Viscosity of batters containing fiber
was higher than control
Hardness, cohesiveness, gumminess,
and chewiness increased in fiber-
added meat batter
Emulsion-type Increase in moisture, fat, and pH [121]
sausages Sausages with 3% rice bran had
lowest cooking loss and 4% rice bran
were hardest in textural analysis
Pork meat batter Increase in emulsion stability and [122]
cooking yield
Increase in moisture, protein, ash, pH
values, yellowness value, textural
parameters, viz., hardness,
gumminess, chewiness,
cohesiveness, and viscosity of meat
batter
Meat emulsion Increase in moisture and ash content [123]
and yellowness value, cohesiveness,
gumminess, chewiness, and
sarcoplasmic protein solubility
Reduction in total fat content in
product
Low-fat Increase in moisture and ash content [124]
frankfurters Reduction in fat, cholesterol, energy,
and trans-fat levels
Increased pH, cooking yield, and
TBA values
Pork protein gel Addition of rice bran fiber at 1% [125]
level increased moisture,
myofibrillar protein solubility, and
water-holding capacity
Decrease in lightness, redness, and
textural values
No change in protein on SDS gel
electrophoresis
Chicken rolls Decrease in sensory scores of meat [126]
and patties rolls and patties with addition of rice
bran and psyllium husk combination
Rye bran Meatballs Decrease in total fat, total trans fatty [87]
acids, moisture, salt content, weight
losses, and redness value
(continued)
778 N. Mehta et al.
Table 3 (continued)
Type of meat
Increase in protein, ash, lightness,
and yellowness values
Meatballs Due to particulate nature, rye bran [127]
was found to be the best fiber source
in meatballs as compared to oat bran
and barley fiber
No significant difference in firmness
as compared to reference after pan
frying
Oat bran Low-fat beef Decrease in shear force values with [128]
burgers increase in level of fiber
Chevon patties With the increasing level of oat bran, [129]
decrease in moisture, protein, and fat
content but an increase in ash and
total dietary fiber content
Increase in concentration of
unsaturated fatty acids and decrease
in cholesterol, cooking loss, and
shear force
Light bologna Increase in product yield, lightness, [130]
and fat-free and hardness value
frankfurters Decrease in redness value and purge
at 3% concentration
Meatballs Substitution of fat by fiber source [131]
and it decreased concentration of
total fat and trans fatty acids
With the increase in amount of oat
bran (5%–20%), moisture and fat
content decreased and protein and
ash content increased
No significant effect on sensory
properties by fiber addition
Beef patties Significant improvement in cooking [132]
yield, fat retention, and moisture
content
Higher juiciness perception as
compared to control
Oat flour Beef patties Decrease in moisture content of raw [133]
patties but increased after cooking
No effect on protein, ash, and fat
content
Improvement in cooking
characteristics with simultaneous
decrease in lightness value
No adverse effects on sensory
properties
Mutton kofta Decrease in fat content whereas, an [134]
increase in moisture, protein, ash,
and carbohydrates.
(continued)
Table 3 (continued)
Type of meat
Significant increase in cooking yield
No significant detrimental effect on
physicochemical and sensory
attributes at 8% oat flour inclusion
level
Chicken kofta Increase in product yield and [135]
decrease in fat
Increase in hardness, gumminess,
cohesiveness, and chewiness
Ragi millet/finger Chicken patties No significant effect on fat and [136]
millet protein content
Reduction in shrinkage of patties
after cooking
Decrease in lightness and yellowness
values
Chevon patties Increase in emulsion stability and [137]
cooking yield
Increase in pH, moisture, water
activity, ash, and fat retention
whereas, decrease in protein and fat
content
Significant increase in fiber and
calcium content
Decrease in instrumental texture
profile attributes
Barley Pork bologna Firmer texture and better sensory [138]
sausages properties
Better purge control and water-
holding capacity during storage
Corn bran Chevon rolls Decrease in moisture and protein [139]
content while no significant effect on
fat and ash content
Increase in crude fiber, water-
holding capacity, and emulsion
stability
Increase in polyphenolic content in
fiber-enriched rolls
Fruits, vegetables, and their by-products
Apple pulp Fermented Reduction in energy value of [140]
sausages products to 35% and fiber content
Sensory and textural properties
decreased with inclusion of fiber
Chicken nuggets Decrease in batter stability and [100]
cooking yield
Reduction in texture and overall
acceptability scores
Decrease in pH, moisture, dietary
fiber, and color parameters
(continued)
780 N. Mehta et al.
Table 3 (continued)
Type of meat
Grape fiber Minced fish Delay in oxidation in minced horse [141]
muscle mackerel muscle during the first
3 months of frozen storage
Minced fish Direct correlation of bound water [142]
muscle after thawing and cooking with
amount of fiber used
Increase in softness and loss of
cohesiveness
Delay in lipid oxidation parameters
Chicken Significant decrease in redness and [6]
hamburgers yellowness values
Improvement in oxidative stability
and radical scavenging activity
Sensory acceptability was not
affected by addition of fiber
Frankfurters Increase in protein, dietary fiber, and [143]
water-holding capacity
Decrease in color values and
oxidation levels
Frankfurters up to 2% level of
inclusion were sensory acceptable
Tomato fiber Chopped Reduction in pH, increase in water- [144]
cooked chicken holding capacity, and increase in
products product hardness proportional to fiber
addition
Reduction in lipid oxidation
Chicken Decrease in moisture and an increase [145]
sausages in ash content
Increase in emulsion stability,
cooking yield, and crude fiber content
Orange Fermented Reduction in energy value of [139]
sausages products
Decrease in sensory and textural
properties with inclusion of fiber
Best organoleptic quality at 10%
pork back fat ad 1.5% orange fiber
Dry-cured Decrease in lipid oxidation [146]
sausage parameter, i.e., TBARS value in
samples with orange fiber
Significant reduction in residual
nitrite level
Salachichon pH, water activity, residual nitrite [147]
(Spanish dry- level, and Micrococcus count were
fermented affected during dry curing
sausage)
Bologna Decrease in TBARS values along [148]
with aerobic and lactic acid bacteria
counts
(continued)
Table 3 (continued)
Type of meat
Decrease in juiciness and increase in
hardness perception on sensory
analysis
Sucuk (Turkish Significant effect on lactic acid [149]
dry-fermented bacteria, Micrococcus, and pH values
sausage) Decrease in residual nitrite level and
increase in TBARS value
Decrease in cooking loss and increase
in lightness and yellowness values
No statistical difference in sensory
scores of texture, color, odor, taste, and
general acceptability scores than
control
Carrot dietary fiber Sobrasaada Decline in all sensory attributes with [150]
(dry-fermented an inclusion above level of 3%
sausage) Significant effect on pH and
hardness value
Chicken nuggets Improvement in cooking yield and [151]
emulsion stability
Lower protein, fat, and ash content but
higher moisture and fiber than control
Hunter color values were higher in
treated products, whereas, no change
in textural parameters as compared to
control
Low-salt pork Improvement in water-binding [152]
sausages capacity and marked effects on
textural properties
Marked increase in whitening with
increasing content of fiber
Peach dietary fiber Frankfurters Increase in viscosity of meat batters [84]
and water retention
Reduction in pH values and no
change in protein and collagen
content
Fermented Sensory and textural properties [140]
sausages decreased with inclusion of fiber
Lemon albedo Bologna Increase in moisture, protein, and [153]
sausages fiber content whereas, decrease in fat
content
Increase in hardness and decrease in
juiciness perception
Breakfast Decrease in shrinkage and cooking [154]
sausages loss
Increased value for lightness
Citrus fiber Bologna Enhancement of oxidative stability, [155]
sausages color, and dietary fiber content
Negative effect on textural
(continued)
782 N. Mehta et al.
Table 3 (continued)
Type of meat
properties, whereas, no effect on
sensory parameters below 2% level
Sugarbeet fiber Frankfurters Slight lowering of sensory scores [156]
Frankfurters Increase in water-holding capacity [157]
and total dietary fiber content
No significant effect on sensory
scores
Turkish-type Increase in water-holding capacity [158]
salami and total dietary fiber content with
incorporation of fiber
No significant changes in
appearance, color, texture, or any
other sensory scores
Sugarcane dietary Low-fat meat Replacement of fat up to 10% level [159]
fiber batter Improved texture and sensory scores
Increase in water- and fat-binding
properties, dynamic rheology, and
reduction of cholesterol contents
Oyster mushroom Chicken patties Decrease in hardness, cohesiveness, [160]
gumminess, and chewiness but
increase in springiness value as
compared to control
Legume hulls and flour
Peanut and cowpea Chicken nuggets Decrease in moisture loss, fat gain, [161]
flour and protein content
Decrease in shear force values and
energy as compared to control
Unacceptable flavor scores at 20%
level
Soy hull Camel meat Reduction in thickness, fat, water [162]
patties retention, and shear force values
Significant effect on chemical
composition, cooking yield, flavor,
and cooking yield
Inner pea fiber Beef patties Increase in cooking yield and [163]
tenderness without adverse effect on
flavor
Bengal gram and Chicken loaves Reduction in cooking loss and [89]
black gram flour extract release volume and increase
in emulsion stability and pH values
Soybean, Bengal Buffalo meat Increase in yield and protein content [164]
gram, green gram, burger with simultaneous decrease in
and black gram flours shrinkage and fat absorption
Roasted flours registered lower TBA
values, i.e., enhancement of
oxidative stability during storage
(continued)
Table 3 (continued)
Type of meat
Blackeye bean, Meatballs Improvement in cooking yield, fat [98]
chickpea, lentil retention, and moisture retention
flours, and rusk Lower penetration values and higher
sensory acceptability scores
Hazelnut pellicle Low-fat beef Increase in cooking yield, diameter, [97]
burger and thickness of beef burgers
Decreased sensory scores of
appearance, flavor, and juiciness as
compared to control
Roasted pea flour Chicken nuggets Nonsignificant increase in cooking [165]
yield up to 10% level
Pea fiber Beef burgers Improvement in water-holding [166]
capacity, cooking yield, and decrease
in shrinkage
Minimization of production cost
without compromising sensory
attributes
Soy flour and moong Buffalo meat Reduction of moisture and fat [167]
bean powder patties content and increase in protein and
fiber content
Increase in water-holding capacity
and decrease in cooking loss
Pea flour Low-fat bologna Improvement in texture profile [168]
values with lower cooking and purge
loss
Increase in water-holding capacity
without compromising sensory
attributes
Bambara groundnut Beef patties No significant effect on proximate [169]
seed flour composition of raw patties but
moisture, protein, and carbohydrate
content differed significantly in
cooked products
Reduction in shrinkage and increase
in cooking yield, moisture, and fat
retention
Tiger nut fiber Pork burgers Increase in cooking yield, fiber [170]
content, fat, and moisture retention
Increase in textural parameters like
chewiness but negative effects on
color attributes
Chickpea hull flour Chicken nuggets Decrease in moisture, protein, ash, [101]
color values, total cholesterol,
emulsion stability, and cooking yield
Improvement in dietary fiber content
and textural parameters
Black gram hull flour Chicken rolls Fall in organoleptic quality with an [171]
and patties increasing level of black gram hull
784 N. Mehta et al.
control was reported to be higher as compared to fiber-treated products. Addition of

carrageenan or oat fiber increased cooking yield by increasing emulsion stability and
water-holding capacity in low-fat frankfurters [96]. Decreasing fat content from
30% to 5% resulted in increased loss during cooking but addition of 2% oat
fiber led to reduction in total expressible fluid from 9.6% to 7.6% and from 4.8
to 4.3% in frankfurters with 5 and 12% targeted fat, respectively. Turhan et al.
[97] observed improvement in reduction of cooking loss and thickness of beef
burgers by addition of hazel nut pellicle and on similar lines increase in
cooking yield was observed on addition of bean and lentil flour in meatballs
by Serdaroglu et al. [98]. Carrot fiber and dietary fiber suspensions from olive
mill wastewater was tried as dietary fiber source in low-fat meatballs by
Galanakis et al. [99] and they too were found to be in agreement with other
authors for improvement in cooking properties and restriction of oil uptake
leading to sustained reduced fat content. But on the contrary, a decrease in
emulsion stability and cooking yield was recorded in low-salt and low-fat
chicken nuggets incorporated with apple pulp and chickpea hull flour which
could be explained by replacement of lean by fiber sources [100, 101]. Simi-
larly, Morin et al. [102] also found an increase in cooking loss in breakfast
sausages on addition of carboxymethyl cellulose.
Various other sources of fibers like psyllium husk have been used for increas-
ing fiber content in meat products and a concurrent increase in emulsion stability
and cooking yield had been observed. It was attributed due to the presence of
soluble dietary fiber in husk that might have entrapped moisture and lead to the
formation of a gel during cooking [91, 92]. Similar finding have been reported
by Luruena-Martınez et al. [103] who recorded the effect of addition of locust
bean/xanthan gum on various quality characteristics of low-fat frankfurters. They
observed a significant increase in emulsion stability, cooking yield, and lower
jelly and fat separation. Lemon albedo as a source of dietary fiber was used in
emulsions by Saricoban et al. [172]. Two types of albedo at five different
concentrations, viz., 0, 2.5, 5, 7.5, and 10% were added in emulsions and their
functional properties were studied. An increase in emulsion capacity with highest
value at 5% level was observed. Similar increasing trend was observed in
emulsion stability and viscosity values; however, no change in flow behavior
of emulsions was observed other than pseudoplasticity. Addition of fiber as a fat
replacer was tried by Pinero et al. [132] and compared with high-fat control
(20% fat). They found an increase in cooking yield, fat retention, and moisture
content in fiber-added meat products as compared to high-fat control. A decrease
in shrinkage of cooked patties from 9.13 to 6.76% with addition of Bambara
groundnut seed flour (BGSF) and an increase in cooking yield, moisture reten-
tion, and fat retention from 79.1 to 87.2%, 67.5 to 78.05%, and 73.51 to 88.34%,
respectively in beef patties was observed [169]. Thus, addition of fiber in various
meat products is generally associated with improvement of physicochemical
properties, viz., emulsion stability, cooking yield, pH, water-holding capacity,
reduction of shrinkage, etc. The change is largely dependent on fiber source and
processing characteristics.
7 Fiber Incorporation and Proximate Composition of Meat

Products
The overall composition of meat products can be changed with incorporation of fiber.
Fiber generally reduces fat content and increases carbohydrate fraction along with
dietary fiber component. This has somehow generated a new class of functional meat
products with enhanced health benefits. Hydrated oat bran was used by Chang and
Carpenter [173] in frankfurters and an increase in moisture and carbohydrate content and
a decrease in total fat had been reported by them. This can be attributed to increase
water-binding capacity of soluble fiber in oats. On the contrary, formulation of chevon
patties with oat bran at different levels, viz., 15, 20, 35, and 50%, resulted in decrease in
moisture, protein, and fat content [129]. Soluble and insoluble fiber content in product
increased and authors concluded that nutritional value of chevon patties could be
enhanced with 15 or 20% oat bran addition. Similar studies were carried out by Yilmaz
and Daglioglu [131] and Yilmaz [87] who reported an increase in protein and ash
content but a decrease in moisture and fat content with addition of oat bran and rye bran,
respectively in meatballs. Rice bran as a source of dietary fiber was used in emulsified
meatballs known as Kung-wan in Taiwan by Huang et al. [119]. They reported a
significant decline in protein and fat content of meatballs with increasing level of rice
bran above 5%. However, an increase in fiber and carbohydrate content was also
observed. Similarly, Choi et al. [123] estimated values for protein, moisture, fat, and
ash content in meat batter formulations designed with varying levels of grape seed oil
and rice bran fiber and found that protein, fat, and ash content in treatments with 2% rice
bran fiber were higher than control. The rice bran incorporation in chicken meat rolls
and patties at three different levels, viz., 5, 10, and 15% was tried by Mehta [174]. A
significant decrease in moisture and protein level was observed in treated products,
whereas, an increase in fat, ash, and dietary fiber content was recorded. Utilization of oat
flour at three different concentrations in beef patties resulted in decrease in moisture
content of raw products and an increase was observed in cooked ones. However, no
change in protein, fat, or ash content with addition of oat flour was reported irrespective
of cooking or not [133]. Bilek and Turhan [175] used flaxseed flour at 3–15% levels in
beef patties and reported an increase in fat and ash content but a decrease in moisture and
protein percentage in products. Cereal brans at 5–20% levels in meatballs were utilized
as a fiber source by Yasarlar et al. [176] and recorded a gradual decrease in moisture and
fat percent while an increase in protein, ash, and dietary fiber with increasing levels of
wheat and oat bran. An increase in protein and fiber content of various meat products
with the use of legume flours have been reported. Kenawi et al. [167] studied the effect
of incorporation of mung bean powder and/or low fat soy flour on proximate compo-
sition of buffalo meat patties. They observed that at a level of 10%, both the fiber sources
led to reduction in moisture and fat content whereas an increase in the fiber and protein
contents. However, a lower protein content in beef patties incorporated with common
bean flour [177] and Bambara groundnut seed flour [169] was observed. The change in
proximate composition is dependent on the type of fiber incorporated in meat products.
Soluble fibers like psyllium husk, oat bran, etc. tend to increase moisture content due to
enhanced water-binding capacity, whereas, protein-rich legume flours result in increase
786 N. Mehta et al.
of protein content in final product. A modification can be designed as per product and
consumer requirement by the processor.
8 Dietary Fiber and Textural Properties of Meat Products
Texture of meat product is an important attribute that holds technological as well as

sensorial importance. Human perception is a key factor in deciding optimum textural
quality of meat products; however, to objectify, various instruments are used to measure
texture of meat products, viz., Instron Texture Analyzer, Warner Bratzler shear press,
and Kramer Shear apparatus. The fiber addition in meat products modifies the texture to
a great extent and ultimately affects chewability. There can be both negative as well as
positive influence of fiber addition on textural parameters. Addition of tapioca starch, oat
fiber, and whey protein in low-fat beef burgers resulted in decrease in all textural
parameters [128]. Garcia et al. [109] reported an increase in hardness value of morta-
della, a Spanish cooked meat product incorporated with inulin when added at 2.5%
level; however, in form of gel, it produced same effect at 7.5%. Similarly, an increase in
hardness value was reported in pig liver pate developed using three enzymatically
extracted fibers along with dry potato pulp and commercial potato fiber (potex) as
reference [178]. A gradual decrease in hardness, gumminess, and chewiness of pork
sausage with incorporation of hydrated oatmeal was observed by Yang et al. [179]. On
the other hand, addition of 2% kimchi powder in breakfast sausages resulted in increased
value of harness, gumminess, and chewiness as compared to control [180]. Similar
trend has been observed in a number of meat products due to the addition of soy fiber,
plasma protein, and various kinds of fiber sources [181, 182]. Incorporation of fiber has
resulted in modified textural properties in fish-based products too. A low-fat fish sausage
added with swelite (a dietary fiber obtained from inner pea) and fibruline (a dietary fiber
obtained from chicory root) was developed by Cardoso et al. [183] and addition of
swelite resulted in improved gel strength and hardness whereas, the products from
fibruline were poorer in texture than control in terms of cohesiveness and chewability.
Wheat bran as a source of dietary fiber was utilized in the formulation of cooked beef
patties by adoption of a Box-Behnken design wherein three different variables were
studied simultaneously. Wheat bran resulted in higher hardness and gumminess value
but a lower springiness, resilience, and cohesiveness without any influence on adhe-
siveness [118]. Nonconventional fiber sources like grey oyster mushroom at 0, 25, or
50% level were used in chicken patties by Wan Rosli et al. [160] and they found that
hardness, cohesiveness, gumminess, and chewiness of chicken patties decreased with
incorporation of oyster mushroom whereas, the value for springiness increased.
9 Sensory Properties of Meat Products in Relation with

Added Fiber Sources
The consumer acceptability is center to any product formulation and development. It

is a complex phenomenon which is judged by sensory evaluation. The different
subsets for meat products are generally color and appearance, flavor, texture,
juiciness, and overall acceptability. Tender and juicy meat products attract focus of
consumers. Utilization of various fibers has been associated with a change in sensory
properties, particularly juiciness and texture. An increase in hardness and decrease in
juiciness by the addition of lemon albedo in bologna sausages has been reported by
Fernandez-Gines et al. [153]. Similarly, addition of chickpea flour at 2.5 and 5%
levels in low-fat pork bologna had no adverse effects on sensory perception.
However, textural properties of cohesiveness and graininess increased but juiciness
was rated much lower scores than control [184]. However, many fiber sources
produced much better sensory perception than control itself, viz., hull-less waxy
barley and normal starch barley in pork bologna sausages [138]. According to
Kenawi et al. [167], 5% of both low-fat soy flour and mung bean powder resulted
in the product with highest values for color, taste, odor, juiciness, and overall
acceptability as compared to control. The extent of change in organoleptic quality
is largely dependent on the concentration of fiber used in the meat product, e.g.,
carrot dietary fiber, if added in a concentration above 3% in sobrassada resulted in
poorer sensory scores than control [150]. In concordance with these observations,
cooked beef patties formulated with Bambara groundnut flour at 5% level had no
significant effect on sensory attributes but at higher level, i.e., 7.5%, a significant fall
in sensory scores for appearance, flavor, and overall acceptability was observed by
Alakali et al. [169].
10 Conclusions
With the advancement of technology and reliance on processed products, meat

industry is expanding its horizon with introduction of novel value-added products
in market. Lack of essential components like dietary fiber can be a great hazard for
health of consumers. Thus, its inclusion is needed for portraying meat as a complete
food. Further, numerous functional properties like water retention, viscosity, etc.
help in formulating the products with higher yield and better sensory acceptability.
The technological advancements in developing low-fat, low-salt, high-fiber, and
functional meat products can be realized by integrating fiber sources in meat. It is
a novel area wherein lot of unconventional fiber sources can be tried for gelling up in
meat products.
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Chemistry, Biological, and Pharmacological
Properties of Gum Arabic 27
Hassan Hussein Musa, Abdelkareem Abdall Ahmed, and
Taha Hussein Musa
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
2 Structure of Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
3 Chemical Properties of Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
4 Physical Properties of Gums Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
5 Biological Properties of Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803
5.1 Antioxidant Properties of Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803
5.2 Effect of GA on Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
5.3 Effect of GA on Blood Glucose Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
5.4 Effect of GA on Intestinal Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
5.5 Degradation of GA in the Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
5.6 Effects of GA on Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
5.7 The Effect of GA on Tooth Mineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
5.8 Effect of GA on Hepatic Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
6 Pharmaceutical Properties of Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
H. H. Musa (*)
Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of
Khartoum, Khartoum, Sudan
A. A. Ahmed
Department of Physiology and Biochemistry, Faculty of Veterinary Sciences, University of Nyala,
Nyala, Sudan
T. H. Musa
Key Laboratory of Environmental Medicine, Ministry of Education, School of Public Health,
Southeast University, Nanjing, Jiangsu, China

798 H. H. Musa et al.
7 Food and Cosmetic Properties of Gum Arabic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807

8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809
Abstract
Gum Arabic (GA) is a natural branched-chain multifunctional hydrocolloid with
a highly neutral or slightly acidic, arabino-galactan-protein complex containing
calcium, magnesium, and potassium. Gum Arabic is dried exudate obtained from
the stem and branches of Acacia trees manly Acacia senegal and Acacia seyal.
GA was used by the Ancient Egyptians as an adhesive when wrapping mummies
and in mineral paints when making hieroglyphs since the second millennium BC.
In modern times, GA is used in foods, pharmaceutical, and many other industries.
In this chapter, we describe the structure, chemical, and physical properties of
Gum Arabic. In addition, biological properties include antioxidant properties of
Gum Arabic, an effect of GA on renal function, blood glucose concentration,
intestinal absorption, degradation of GA in the intestine, lipid metabolism, tooth
mineralization, and hepatic macrophages. Similarly, pharmaceutical, food, and
cosmetic properties of Gum Arabic are discussed.
Keywords
Gum Arabic · Chemical · Biological · Pharmacological · Food · Cosmetic ·
Properties
Abbreviations
AG Arabinogalactan
AGP Arabinogalactan protein
ATGL Adipose triglyceride lipase
CAT Catalase
CDC Chenodeoxycholic acid
CRF Chronic renal failure
GA Gum Arabic
GP Glycoprotein
GPx Glutathione peroxidase
HDL High-density lipoprotein
HSL Hormone-sensitive lipase
LDL Low-density lipoprotein
MDA Malondialdehyde
MGL Monoacylglycerol lipase
TAP 2,4,6-Triaminopyrimidine
TC Total cholesterol
VLDL Very low density lipoprotein
27 Chemistry, Biological, and Pharmacological Properties of Gum Arabic 799
1 Introduction
Gum Arabic is a natural branched-chain multifunctional hydrocolloid with a highly

neutral or slightly acidic, arabino-galactan-protein complex containing calcium,
magnesium, and potassium [1]. According to the definition of the Joint Expert
Committee for Food Additives (JECFA), Gum Arabic is a dried exudate obtained
from the stem and branches of Acacia trees (Fig. 1) [2]. There is more than 1000
species of the genus Acacia Gums; only two are significant for commercial purposes
Acacia senegal and Acacia seyal [3]. Acacia senegal is considered the best in quality
due to a low quantity of tannins and comprises the majority of global trade [4],
whereas Acacia seyal produces a lower grade of Gum [5]. Acacia trees are abundant
in central Sudan, central and West Africa, and tropical and semitropical areas of the
world [6, 7]. Sudan is the leading producer of Acacia Gums worldwide, followed by
Nigeria, Chad, Mali, and Senegal [8]. Europe and USA are the most important GA
markets, while Japan is the largest Asian consumer.
The use of GA dates back to the second millennium BC when the Ancient
Egyptians used Gum Arabic as an adhesive when wrapping mummies and in mineral
paints when making hieroglyphs [9]. In modern times, they are used in foods,
pharmaceutical, and many other industries [10–13].
2 Structure of Gum Arabic
The chemical composition of GA is complex, and numerous papers have been

published on this subject [14]. The backbone of GA is composed of 1,3-linked
β-D-galactopyranosyl units. The side chains are composed of two to five 1,3-linked
β-D-galactopyranosyl units, joined to the main chain by 1,6-linkages. Both the main
and the side chains contain units of α-L-arabinofuranos, α-L-rhamnopyranosyl, β-D-
glucopyranosyl, and 4-O-methyl-β-D-glucopyranosyl, the last two mostly as end
units (Fig. 2).
Gum Arabic consists mainly of high-molecular weight polysaccharides and their
calcium, magnesium, and potassium salts, which on hydrolysis, yield three main
Fig. 1 Gum Arabic formed

on a wounded branch of
A. senegal
Fig. 2 Structure chemic of

the Acacia Gum
fractions of polysaccharides and proteins, including arabinogalactan (AG),

arabinogalactan protein (AGP), and glycoprotein (GP), which differ from their
molecular weight and chemical composition [15]. The arabinogalactan fraction
represents 88% of the total Gum weight has a low molecular weight (Mw, ~300
KDa) and associated little protein content below 1%. The arabinogalactan protein
fraction (~10% of the total Gum) has a high molecular weight (Mw) (~1500 KDa)
and protein content (~10%). The glycoprotein fraction (<2% of the total Gum)
has the lowest molecular weight (Mw, ~250 KDa) and the highest protein content
(~20%e50%). Among these fractions, AGP is the most interfacially active compo-
nent [16, 17 ], and primarily responsible for the emulsifying properties of GA [18,
19 ]. This fraction can be adsorbed on the oil–water interface to form a viscoelastic
film and reduce the interfacial tension between oil and water because of its amphi-
philic characteristics, which is conferred by the hydrophobic protein chains com-
bined to the hydrophilic polysaccharide fragments [17].
3 Chemical Properties of Gums
Chemically, GA is a complex mixture of macromolecules of different size and

composition characterized by a high proportion of carbohydrates (D-galactose and
L-arabinose) (~97%), and a low proportion of proteins (<3%) [14]. The chemical
composition of GA varies slightly depending on its origin, climate, harvest season,
tree age, and processing conditions, such as spray dying [20–23]. Many studies have
shown some differences between the chemical composition of the GA from Acacia
senegal and Acacia seyal [24–26], the most recent study was conducted by Lopez-
Torrez et al. [29]. Both Acacia Gums contained the same amino acids with a higher
content of protein in A. senegal (2.7%) than in A. seyal (1.0%) (Table 1).
Hydroxyproline, serine, leucine, and proline were the most abundant residues
and represented more than 55% of the total amino acids in each variety (Table 2).
Similar amino acid profiles were identified in previous studies on Acacia Gums
from different origins [27–29]. The A. senegal Gum samples shown to contain approx-
imately twice the amount of protein compared to the Gum obtained from A. seyal [28].
Table 1 Biochemical composition of A. senegal and A. seyal Gums in dry basis (mean standard
deviation)
Component (mg g1) A. senegal A. seyal
Total dry matter 889.0 0.27 893.0 0.02
Sugarsa 940.0 950.0
Galactose (%)b 35.8 1.20 36.9 1.05
Arabinose (%)b 30.3 2.50 47.6 0.60
Rhamnose (%)b 15.5 0.35 3.0 0.30
Glucuronic acid (%)b 17.4 1.15 6.7 0.40
4-O-Me-glucuronic acid (%)b 1.0 0.05 5.8 0.55
Proteins 27.0 0.01 10.0 0.04
Minerals 33.0 0.24 40.0 0.07
a
Total content of sugars was calculated by the difference of proteins and minerals from 1000 mg g1
in dry basis
b
Sugar composition was determined by GC-MS
Source: Lopez-Torrez et al. [29]
Table 2 Amino acid composition for A. senegal and A. seyal Gums in dry basis (mean standard
deviation)
Amino acids (mg g1) Abbreviations A. senegal A. seyal
Alanine Ala 0.49 0.04 0.22 0.01
Arginine Arg 0.31 0.05 0.12 0.00
Aspartic acid Asp 1.24 0.04 0.49 0.04
Glutamic acid Glu 0.92 0.01 0.28 0.003
Glycine Gly 0.79 0.004 0.25 0.07
Histidine His 1.37 0.01 0.27 0.01
Hydroxyproline Hyp 6.26 0.52 2.13 0.19
Isoleucine Ile 0.31 0.02 0.13 0.01
Leucine Leu 1.83 0.04 0.60 0.0
Lysine Lys 0.63 0.02 0.12 0.03
Phenylalanine Phe 0.82 0.05 0.21 0.01
Proline Pro 1.61 0.14 0.56 0.001
Serine Ser 2.50 0.05 0.95 0.03
Threonine Thr 1.42 0.02 0.34 0.02
Tyrosine Tyr 0.31 0.04 0.14 0.02
Valine Val 0.71 0.0001 0.31 0.07
Total amino acids 21.5 0.47 7.1 0.16
Source: Lopez-Torrez et al. [29]
4 Physical Properties of Gums Arabic
The physical properties of GA may vary depending on the origin and age of trees,
the exudation time, and climate. Treatment of Gums after collection such as
washing, drying, and bleaching in the sun and storage conditions effected the
physical properties of Gums [19]. Gum Arabic of excellent quality is tear-shaped,
Table 3 International Property Value

specifications of Gum
Moisture (%) 13–15
Arabic quality FAO [32]
Ash content (%) 2–4
Internal energy (%) 30–39
Volatile matter (%) 51–65
Optical rotation (degrees) (26)–(34)
Nitrogen content (%) 0.26–0.39
Cationic composition of total ash at 550 C
Copper (ppm) 52–66
Iron (ppm) 730–2490
Manganese (ppm) 69–117
Zinc (ppm) 45–111
Source: Dauqan and Abdullah [35]
round, with an orange-brown color. After it is crushed or shattered, the pieces are
paler in color and have a vitreous appearance. GA has high water solubility and a
relatively low viscosity compared with other Gums. GA can get dissolved at water in
a concentration of 50% w/v, forming a fluid solution with acidic properties (pH
~4.5). The resulting solution is colorless, tasteless, and does not interact easily with
other chemical compounds [30, 31]. The viscosity of GA solutions can be modified
by the addition of acids or bases as these change the electrostatic charge on the
macromolecule.
The physical properties of Gum Arabic established as quality parameters include
moisture, total ash, volatile matter, and internal energy, which were regarding with
reference to Gums taken from Acacia senegal species in Sudan (Table 3). These
parameters can be used to identify raw Gums mostly used as food additives [32, 34, 33].
Gum Arabic is a natural product complex mixture of hydrophilic carbohydrate
and hydrophobic protein components [32]. Hydrophobic protein component functions
as an emulsifier which adsorbs onto surface of oil droplets, while hydrophilic
carbohydrate component inhibits flocculation and coalescence of molecules through
electrostatic and steric repulsions in food additives [36, 37].
The moisture content facilitates the solubility of GA carbohydrate hydrophilic
and hydrophobic proteins [38]. The total ash content is used to determine the critical
levels of foreign matter, insoluble matter in acid, calcium, potassium, and magne-
sium [39]. The compositions of cations in the ash residue are used to determine the
specific levels of heavy metals in the Gum Arabic quality [32, 40]. The volatile
matter determines the nature and degree of polymerization of the compositions
contained in sugar (arabinose, galactose, and rhamnose) which exhibits strong
binding properties to act as emulsifiers and stabilizers in the manufacture of cough
syrups in the pharmaceutical industry [2]. The GA internal energy is the required
energy to produce an amount of carbon by heating at 500 C to release carbon
dioxide. Optical rotation is used to determine the nature of GA sugars as well as to
identify the source of production. Nitrogen content in Gum Arabic determines the
number of amino acid compositions with the range of 0.26–0.39% [32].
5 Biological Properties of Gum Arabic
5.1 Antioxidant Properties of Gum Arabic
Oxidative stress is largely occurred through the deregulation of oxidants/antioxi-

dants injury or breakdown of macromolecules of biological nature such as proteins,
carbohydrates, lipids, and nucleic acids, ensuing in the imbalances of intracellular
homeostasis and consequently the production of several types of reactive oxygen
species (ROS) which finally induce more oxidative injury or damage [41].
The antioxidant enzymes activity plays a vital role in the damage of hyper-
glycemia-related tissue [42]. Previous studies indicated that production of ROS in
diabetes might commence the chronic diabetic lesions development on many
tissues such as blood vessels [43], eye retina [44], kidneys [45], and neurode-
generative disorders [46].
Superoxide dismutase (SOD) [47], catalase (CAT) [48], and glutathione peroxi-
dase (GPx) [49] are considered the main vital defense tools against reactive oxygen
molecules, which are involved in the oxidative damage [50, 51]. In the diabetic
patients, the oxidative stress was found to induce various unfavorable effects at the
molecular levels in cell physiology [52]. The oxidative stress reduced the concen-
trations of glutathione (GSH) in patients with type 2 diabetes [52], decreased the
activity of CAT [53], reduced the concentrations of renal SOD [54], and increased
the level of heat shock protein 70 (HSP70) [55]. The reduction in antioxidant
components can cause diabetic complications [56]. The administration of Alloxan
by GA was cause a significant reduction of antioxidant enzymes including GPx,
CAT, and SOD. The reduction of antioxidant enzymes activities was associated with
observable augment in production of malondialdehyde (MDA). The elevation MDA
levels ultimately cause oxidative stress, and consequently it mirrors a decreased
antioxidant defense potentiality [50, 57].
Several studies indicated that GA is exert a nephron-protective effect against
gentamicin (antibiotic) and cisplatin-induced nephrotoxicity in rat [58, 59], and
doxorubicin-induced cardio toxicity in rat [60]. Trommer and Neubert [61] indi-
cated that the treatment of GA together with polysaccharides reduced lipid perox-
idation. In contrast, Ali [62] revealed that administration of GA to rats at
concentrations of 2.5%, 5.0%, and 10.0% in the form of drinking water for eight
successive days did not significantly change the levels of free radical scavenger’s
GSH, acid vitamin C (ascorbic acid, and SOD, or lipid peroxidation). The mech-
anism of action through which GA improves the antioxidant capacity may be due
to the fact that GA contains several types of amino acid residues such as lysine,
tyrosine, and histidine, which are commonly considered as antioxidants biomole-
cules [63, 64]. Moreover, the antioxidant properties of GA in biological systems
require a more direct knowledge of the antioxidant capacity [65, 66]. Conse-
quently, the antioxidant activity of GA in biological systems is still an unresolved
issue, and therefore it requires a more direct knowledge of the antioxidant capacity
of GA that can be obtained by in vitro experiments against different types of
oxidant species.
5.2 Effect of GA on Renal Function
The administration of GA in the form of drinking water (15%, w/v) reduces the
concentrations of plasma urea and creatinine in adenine-induced chronic renal
failure (CRF) in rats. It also decreased the clearance of creatinine and induced
significant increases in the inflammatory mediator’s concentrations. Further, the
treatment with GA significantly ameliorated all adverse effect that induced by
adenine. The mechanism underlying the salutary effect of GA in adenine-induced
CRF may associate with mitigation of the adenine-induced inflammation and gen-
eration of free radicals [67].
Pretreatment of GA (7.5 g/kg/day per oral administration), starting 5 days before
mercuric chloride injection, resulted in a complete reversal of Hg-induced increase
in blood urea nitrogen, creatinine, thiobarbituric acid reactive substances, and total
nitrate/nitrite to control values. Pretreatment of GA prevented Hg-induced degener-
ative changes of kidney tissues. Thus indicated that GA is an efficient cytoprotective
agent against Hg-induced nephrotoxicity [68]. The protective effect of GA on renal
function was also confirmed to significantly reduce blood creatinine and urea
nitrogen concentrations in diabetic nephropathy patients [69]. Recent studies have
shown that GA significantly reduced serum urea and urinary protein. In addition,
GA significantly decreased α-SAM and TGF-β1 gene expression in kidney and
increased kidney cytokeratin19 and E-cadherin gene expression when compared to
STZ-treated group. Therefore, GA may attenuate the development of nephropathy in
type I diabetes rat [70].
5.3 Effect of GA on Blood Glucose Concentration
In India, GA is one of the indigenous medicines, and it has many medicinal uses. In
Tunisia, the use of GA as hypoglycemic medicinal plants to treat diabetic patients
was found to reach more than 70% compared to other medicinal plants [71].
A clinical trial used 40 participants with a daily supplement of powdered GA
(10 g/day) for 16 weeks in healthy individuals, prediabetics patients, type 2 DM
patients, and diabetic nephropathy patients. The results showed that supplementation
of GA significantly decreased fasting blood glucose levels and glycosylated hemo-
globin (HbAc1) together with significant reduction of blood uric acid and total
protein concentrations [72]. Moreover, supplementation of GA in different forms
to the normal or diabetic or fed-high fat diet rat/mice significantly reduced blood
glucose levels [83, 85]. The blood glucose lowering propriety of GA may be because
GA inhibits absorption of glucose in the intestine via interaction with membrane
abundance of sodium-glucose transporter 1 (SGLT1) in experimental mice [69].
5.4 Effect of GA on Intestinal Absorption
The small intestine is a major part of the gastrointestinal tract (GIT) where almost all
organic nonelectrolytes and electrolytes absorbed in it different mechanisms which
are operating at the cellular and molecular levels [73]. Concurrent intestinal secretion
is a physiological occurrence which is tightly controlled by various mechanisms.
This process maintains within the intestinal lumen a condition of variability, dilution,
and solubilization that is indispensable to the usual intestinal function of digestion
and absorption. The net effects of those mechanisms play a significant role to
maintain the normal mammalian small intestine in an absorptive manner. On the
other hand, under a certain condition, secretion forces exceed absorption, and a net
secretory condition ensues resulting in diarrhea and dehydration. It has been reported
that GA enhances small intestinal absorption of sodium in normal experimental rats
[74, 75]. GA is also found to increase the absorption of sodium and water in two
animal models of diarrheal disease [76]. In normal young rats, the addition of 5 and
10 g/L of GA increased sodium removal rates from the intestinal lumen perfused
with oral rehydration solutions containing either 60 or 90 mM sodium. Although GA
tended to ease bidirectional fluid movement in those experiments, the net absorption
of water was not influenced [77]. At high concentration, GA was associated with
increases and expansion of the basolateral intercellular space. Experimental diarrhea
was induced in rats by either 1 week of drinking cathartic (magnesium citrate-
phenolphthalein) solution to produce chronic osmotic-secretory effects or by jejunal
perfusion of theophylline to induce jejunal secretion. The positive influences of the
GA on electrolyte and fluids absorption were shown in jejunal perfusion studies in
experimental rats that were recovering from chronic osmotic diarrhea induced by
cathartic agents [73]. In free living rats, the administration of GA in the form of
drinking supplemented ad libitum revealed accelerated recovery when compared to
those receiving either water or oral rehydration solutions (ORS) without GA [73].
GA was reported to increase water and electrolytes movement, e.g., water and
sodium, from the intestinal lumen to the blood stream [78].
5.5 Degradation of GA in the Intestine
Gum Arabic is mostly indigestible to both animals and humans as it is not degraded
in the small intestine. However, it fermented in the large intestine particularly in
colon due to the enzymatic action of microorganisms [79]. Several studies have
reported that intestinal bacteria can ferment GA to short-chain fatty acid, mainly
propionate [80].
5.6 Effects of GA on Lipid Metabolism
Gum Arabic is considered as a dietary supplement that reduces the deposition of

body fat. Ingestion of GA was revealed to decrease body mass index (BMI) and
percentage of body fat in healthy adult human females [81]. GA has many anti-
obesity properties as a dietary supplement in both humans and experimental animals.
GA serves as a dietary fiber which helps to reduce body weight and fat deposition.
The property of lowering caloric density of the diet of GA is the most potential
mechanism involved in the reduction of body weight [82]. In addition, our recent
reports suggest that other complex mechanisms might be involved [83]. The prop-
erty of lowering glucose and fat absorption of GA is an additional proposed
mechanism; nevertheless, this mechanism was debated in earlier reports [84]. GA
suppressed diet-induced obesity by altering the expression of mRNA levels of genes
involved in lipid metabolism in mouse liver [83]. In addition, GA consumption in the
form of drinking water decreased visceral adipose tissue (VAT) associated with
downregulation of 11β-hydroxysteroid dehydrogenase type I in liver and muscle
of mice [85].
Administration of GA reduced plasma total cholesterol, triglyceride, and low
density lipoprotein (LDL) concentrations in human [86] and mice [85]. In agreement
with these findings, we reported that GA supplementation decreased plasma LDL,
very low density lipoprotein (VLDL), and total cholesterol concentrations, whereas
increased HDL concentrations. Numerous mechanisms have been proposed to reveal
the hypocholesterolemic effects of dietary fiber [87]. One potential clarification is
that dietary fiber increases the viscosity of the intestinal contents, and therefore,
interfering nutrient with absorption and micelle formation, which, sequentially,
decreases intestinal lipid absorption [88]. Another mechanism suggested that soluble
fibers act by disrupting the enterohepatic circulation of bile acids, consequential to
increased bile acid excretion, and subsequently reduces plasma cholesterol concen-
trations [89]. Moreover, the viscosity of fermentable dietary fibers is found to
contribute substantially to the lipid lowering effects in rat [90].
Supplementation of GA significantly downregulated hepatic adipose triglyceride
lipase (ATGL) mRNA and conserved receptor expressed in brain 2 (SREB2) mRNA
expression in mice fed with high-fat diet (HFD) [83, 91]. HMG-CoA reductase
(HMGR), the rate-limiting enzyme in cholesterol biosynthesis mRNA expression,
was significantly lowered in the GA-treated mice [83]. The dietary fibers have
pertained to the hepatic mRNA expression of HMGR in rat [92, 93]. Thus, these
results provided insight into how dietary fibers affect lipid metabolism at the gene
level. Adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and
mono-acyl-glycerol lipase (MGL) are tightly regulated by a nutritional factor.
However, under a condition of energy intake, imbalance leads to failure to effi-
ciently control their activity. Consequently, serious metabolic disorders will occur
and is believed to be a key mechanism in the development of type 2 diabetes in
obesity [94].
5.7 The Effect of GA on Tooth Mineralization
Dental caries is found to occur when the tooth enamel is lost due to an imbalance of
the demineralization and demineralization phases, and prevention can be accom-
plished if the demineralization phase is enhanced [95]. A number of agents have
been used for that purpose, including fluoride [96]. Lately, using histopathological
methods, it is found that GA can promote demineralization possibly by sustaining
other demineralization activities [97]. This supporting function was approved to the
rich content of Mg2þ, Ca2þ, and Kþ salts of polysaccharides in GA, and to the
influence of the Gum on Ca2þ metabolism and possibly phosphate. It is also

reported that GA contains cyanogenetic glycosides and other numerous types of
enzymes such as peroxidases, oxidases, and pectinases that exhibit antimicrobial
properties against certain microorganisms such as Prevotella intermedia and Pro-
phyromonas gingivalis [98].
5.8 Effect of GA on Hepatic Macrophages
Macrophages are known to play a vital role in the regulation of immunological

process in all vertebrates. It was reported that the GA activates macrophage by its
ability to produce superoxide anions in vitro [99]. While other studies report that GA
was competent to blocking the macrophage function completely [100, 101]. There-
fore, the scientist inferred that such influences of GA would promise the consider-
ation in the treatment of chronic liver disease, as a disturbed function of Kupffer cells
and hepatic macrophages occurs in this kind of disease and is involved in its
complications, such as end toxemia [102].
6 Pharmaceutical Properties of Gum Arabic
In recent years, plant-derived polymers have evoked tremendous interest due to their
diverse pharmaceutical applications such as diluents, binders, disintegrants in tab-
lets, thickeners in oral liquids, protective colloids in suspensions, gelling agents in
gels, and bases in suppository [103].
In the pharmaceutical industry, GA is used as a carrier of drugs since it is
considered a physiologically harmless substance. GA has some biological properties
as an antioxidant [61, 104, 105] on the metabolism of lipids [106, 107] and in
treating many diseases such as kidney [102, 108], cardiovascular [109], and gastro-
intestinal diseases [110].
Gum Arabic reduces glucose absorption, increases fecal mass and bile acids, and
has the potential to beneficially modify the physiological state of humans [111]. GA
is slowly fermented by the bacterial flora of the large intestine producing short-chain
fatty acids [112]. In addition, GA is able to selectively increase the proportion of
lactic acid bacteria and bifidus bacteria in healthy subjects. Previous studies have
shown that a daily intake of 25–30 g of GA for 21–30 days reduced total cholesterol
by 6% and 10.4%, respectively. The decrease was limited only to LDL and VLDL,
with no effect on HDL and triglycerides [113, 114].
7 Food and Cosmetic Properties of Gum Arabic
Gum Arabic is used in textiles, ceramics, lithography, cosmetic, paints, and paper-
making [9, 116]. In the food industry, GA is primarily used in confectionery, bakery,
dairy, beverage, and as a microencapsulating agent. Acacia Gums are unique among
the various hydrocolloids; they are used notably in food industry because they
modify and control the rheological properties of aqueous food systems acting as
thickeners, stabilizers, film formers, suspending agents, flocculants, and emulsifiers.
A. senegal Gum is most widely used in food applications mainly because of its better
emulsifying properties than A. seyal Gum [115]. In addition, Gum solutions of A.
senegal are generally less colorful than A. seyal. These properties explain differences
in the higher price of A. senegal Gum compared to A. seyal in the international
market. Gum Arabic is well recognized as emulsifier used in essential oil and flavor
industries such as production of citrus and cola flavor oils for soft drinks [31, 34]. In
dairy products, Gum Arabicis is used as a stabilizer in frozen products like ice and
ice cream, absorbing water and producing a finer texture. In cosmetic industry, Gum
Arabic is used as smoothener in lotions and protective creams, and adhesive in facial
masks or face powders [9].
8 Conclusion
Although there are more than 1000 species of the genus Acacia Gums, only two are
significant for commercial purposes: Acacia senegal and Acacia seyal. The chemical
composition of GA varies slightly depending on its origin, climate, harvest season,
tree age, and processing conditions, while the physical properties of Gum Arabic
established as quality parameters including moisture, total ash, volatile matter, and
internal energy.
GA improves the antioxidant capacity because it contains several types of amino
acid residues such as lysine, tyrosine, and histidine, which are commonly considered
as antioxidants biomolecules. GA effects renal function through reduction in blood
creatinine and urea nitrogen concentrations in diabetic nephropathy patients. GA
significantly decreased fasting blood glucose levels and glycosylated hemoglobin
(HbAc1) together with a significant reduction in blood uric acid and total protein
concentrations. GA increases water and electrolytes movement from the intestinal
lumen to the blood stream. GA can be fermented by intestinal bacteria to short-chain
fatty acid, mainly propionate. GA, as a dietary fiber, helps to reduce body weight and
fat deposition.
In the pharmaceutical industry, GA is used as a carrier of drugs since it is
considered a physiologically harmless substance. GA has some biological properties
as antioxidant on the metabolism of lipids, and in treating many diseases such as
kidney, cardiovascular, and gastrointestinal diseases.
In the food industry, GA is used in confectionery, bakery, dairy, beverage, and as a
microencapsulating agent. In dairy products, it is used as the stabilizer in frozen
products like ice and ice cream, absorbing water and producing a finer texture. In
cosmetic industry, Gum Arabic is used as smoothener in lotions and protective
creams, and adhesive in facial masks or face powders.
Acknowledgment Authors acknowledge all researchers whom conducted studies on Gum Arabic.
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Resistant Starch in Food
28
Pinky Raigond, Som Dutt, and Brajesh Singh
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
2 Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
3 Classification of Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
3.1 Rapidly Digestible Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
3.2 Slowly Digestible Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
3.3 Resistant Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
4 Relationship between RDS, SDS, and RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
5 Classification and Structure of Resistant Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
6 Properties of RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823
7 RS Production Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824
7.1 Heat Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824
7.2 Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827
7.3 Chemical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 828
7.4 Hydrostatic Pressure Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
8 Improving RS Production in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
9 Commercially Available RS Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
10 Applications of RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
11 Health Benefits of RS Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
11.1 RS: A Type of Dietary Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
11.2 RS Improves Probiotic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
11.3 Hypoglycemic Effect of RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
11.4 Hypocholesterolemic Effect of RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
11.5 RS Plays a Role in Energy and Weight Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
11.6 Reduction of Gallstone Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
11.7 Enhanced Absorption of Minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
11.8 Other Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
P. Raigond (*) · S. Dutt · B. Singh

Division of Crop Physiology, Biochemistry and Post Harvest Technology, ICAR-Central Potato
Research Institute, Shimla, Himachal Pradesh, India

816 P. Raigond et al.
12 Future Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838

13 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
Abstract
Consumers’ awareness on benefits of intake of low carbohydrate foods is increas-
ing day by day. Carbohydrate-rich foods are generally blamed for causing/worsen-
ing diabetes and weight gain. However, the presence of resistant starch in
carbohydrate-rich foods makes them somewhat suitable for health conscious per-
sons. Resistant starch is of utmost importance to nutritionists and food processors.
Resistant starch has assumed great importance due to its unique functional proper-
ties and health benefits. Resistant starch provides health benefits such as glycemic
control, control of fasting plasma triglyceride and cholesterol levels, and absorption
of minerals. Native quality of starch, processing techniques, and storage tempera-
tures affect the resistant starch content in food. Commercial preparations of resis-
tant starch are available in the market. These RS preparations are being used by
food industries as an ingredient to lower the caloric value of the food products and
also to improve textural and organoleptic characteristics of food. This book chapter
will give insights into classification, structure, properties, production, applications,
and health benefits of resistant starch.
Keywords
Resistant starch · Classification · Properties · Production technology ·
Applications · Health benefits
Abbreviations
ANN Annealing treatment
GI Glycemic index
HAS High amylose starch
HHP High hydrostatic pressure
HMT Heat moisture treatment
HPT High pressure treatment
NSP Non-starch polysaccharides
RDS Rapidly digestible starch
RS Resistant starch
SCFA Short chain fatty acids
SDS Slowly digestible starch
1 Introduction
Carbohydrates are the main energy source for our body and their energy is used
first in the body before protein and fat. Starch is the main form of carbohydrate
present in most of the foods. Starch is made up of two major components,
28 Resistant Starch in Food 817
i.e., amylose and amylopectin. These components are present in different ratios
in different plants/foods. Quality of starch depends on the ratio and organiza-
tion of these two molecules in starch granules [1]. Starch is indigestible in its
raw form and its digestibility improves to great extent during cooking. Most of
the part of starch becomes digestible after cooking; however, some parts remain
resistant to digestion. A part of starch present in the diet that escapes digestion
and absorption in the small intestine and is fermented in the large intestine of
humans, with the production of short chain fatty acids (SCFA), is termed as
“resistant starch” (RS) [2]. Resistant starch is present in most of the starchy
foods. Resistant nature of the starch depends upon the quality of native starch
such as its amylose: amylopectin ratio, compactness, crystallinity, etc. Along
with these, behavior and nature of food, cooking methods, temperatures, stor-
age after cooking, and interaction of starch with proteins, lipids, and other
carbohydrates are also known to influence the digestibility of starch [3].
American Association of Cereals Chemists and the Food Nutrition Board of
Institute of Medicine of the National Academies have defined RS as a type of
dietary fiber. There are five different types of RS based on their origin and
characteristics. RS is known to positively influence the functioning of digestive
tract, gut microbial flora, blood cholesterol, and glycemic index (GI) level and
assists in the control of diabetes, protects from colon cancer, diverticulitis, and
hemorrhoids through production of SCFA [4, 5].
Due to increasing awareness about healthy and nutritious foods, consumers are
now concerned with supplementary health merits of a food and are ready to pay more
for healthy food products. International food industry is working on development of
innovative functional food products with additional health benefits to fulfill the
growing demands for functional foods. Most of carbohydrate-rich foods are
known to be high glycemic. Glycemic index (GI) ranks food according to their
effect on blood glucose level and high GI food cause fatal health problems such as
diabetes and obesity. Development of carbohydrates-based functional foods with
low GI is need of the hour. Due to the inverse relation between GI and RS, food
nutritionists are looking for use of RS as food fortificant to lower the GI of food. For
development of functional foods, fortification is an economically feasible way to
deliver the desired component such as RS, in targeted population group. RS is
naturally available in different botanical sources, which provides opportunity of its
use as a functional ingredient. Food products fortified with RS are becoming popular
among consumers. RS is already being incorporated in variety of products such as
breads, cookies, muffins, pizza crust, tortillas, breakfast cereals, and snack products.
For production of RS at a large scale, various techniques/treatments such as heat,
enzymatic, heat with enzyme and chemical treatments are being practiced. Litera-
ture/reports on RS have appeared in the past but there is continuous accumulation of
newer information as resistant starch continues to attract the attention of researchers.
Therefore, it became necessary to update the available information on RS, and this
chapter attempts to analyze the information published, especially in the recent past
on classification, structure, properties, production technologies, applications, and
health benefits of resistant starch.
2 Starch
Starch is the most abundant storage polysaccharide present in form of granules in

cereals or legume seed endosperm, tubers (potato and sweet potato), unripe fruits
(banana and mango), and in many other plant reserve organs. Depending on the
botanical source starch is present in diverse shapes such as round, oval, lenticular,
and angular and the granule size generally range between 1 and 100 μm. A starch
granule is made up of a number of monosaccharide or glucose molecules that are
linked together with α 1–4 and α 1–6 linkages [6]. Starch is made up of two
components namely amylose and amylopectin. Amylose is a linear chain of
glucose with 6000 degree of polymerization, whereas, amylopectin is highly
branched and its degree of polymerization is up to two million. Carbohydrates
are stored in insoluble and tightly packed manner in starch granules [7]. Based on
the X-ray diffraction spectrum, starches are divided into two forms, i.e., “A type”
and “B type.” These two types differ in the granule crystalline structures. A-type
starches are present in cereals, whereas, B-type starches are present in tubers and
other amylose-rich starches. In case of legumes, mixture of both A-type and B-
type crystalline structures are present and this mixture is termed as “C-type”
structure [8].
Enzymes such as α-amylase, glucoamylase, and sucrose-isoamylase can hydro-
lyze the gelatinized/digestible starches in small intestine. B-type starches resist
enzymatic digestion, whereas A-type starches are slowly digested [9, 10]. Digest-
ibility of starch is affected by a number of factors such as supramolecular structure
(packing of crystallites inside starch granules), ratio of amylose to amylopectin,
fine structure of amylose, as well as surface characteristics of starch granule [11].
Starch granules being compact structures are insoluble in cold water. Starch
gelatinizes at high temperatures (generally above 40 C) and starches from differ-
ent sources have different gelatinization temperature. Gelatinized starch contains
dissolved carbohydrates, amylose, and low polymerized chains of amylopectin that
binds considerable amount of water upon cooling. Amylose and amylopectin
chains in gelatinized starch realign upon cooling of starch paste and this process
is termed as “retrogradation.” Retrogradation occurs at higher rate if the starch
paste is stored at lower temperatures. Retrograded starches contain both crystalline
and semi-crystalline structures and are hence semi-crystalline in nature. Products
of amylopectin retrogradation are less thermostable compared to amylose retro-
gradation products and their rehydration occurs at the temperatures over 60 C and
120 C, respectively.
3 Classification of Starch
Starch is classified based on its rate and extent of digestion and physiological
properties. There are three types of starches viz. rapidly digestible starch (RDS),
slowly digestible starch (SDS), and resistant starch (Table 1) [12, 13].
Table 1 Classification of starch [13]

Starch
fraction RDS SDS RS (Type 1–4)
Digestion Within 20–120 min, small >120 min, not in the small
timeline (in 20 min, intestine intestine, main action in colon
vitro)/place mouth and
small intestine
Examples Freshly Native waxy maize Raw potato, stale bread
cooked food starch, millet, legumes
Amount (g/ Boiled hot Boiled millet: 28 Raw potato starch: 75
100 g dry potato: 65
matter)
Main Rapid source Slow and sustained Effects on gut health (e.g.,
physiological of energy source of energy and prebiotic, fermentation to butyrate
property sustained blood with hypothesized
glucose anticarcinogenic effects)
Structure Mainly Amorphous/crystalline Depending on the type, mainly
amorphous crystalline
3.1 Rapidly Digestible Starch
Rapidly digestible starch is defined as a “type of starch which is rapidly (within 20

minutes) converted to glucose molecules by the enzymatic digestion” [13]. High
levels of RDS are present in starchy foods cooked under moist heat such as potatoes
and breads. Major portion of dietary starch is rapidly digestible. More RDS in food is
detrimental to health, as it releases glucose to blood at a fast rate which elevates
blood glucose level and insulin response [14]. RDS is significantly correlated to GI
in literature [15–17].
3.2 Slowly Digestible Starch
Slowly digestible starch is defined as a “type of starch which is converted to

glucose after 120 minutes of enzymatic digestion” [13]. SDS is a physically
inaccessible amorphous starch that takes long time for digestion and is
completely digested in the small intestine. Englyst’s in vitro test revealed that
raw cereal starches are rich in SDS. However, human testing of normal corn
starch (A type) revealed the slow and prolonged postprandial glucose release
profile [18]. Slow digestion property of native starches is generally lost during
processing of starchy food under moisture [19]. SDS-rich foods as well as low
GI foods confer similar health benefits. Such foods delay the occurrence of
metabolic syndrome, diabetes, and cardiovascular diseases [20–22]. Retrogra-
dation of partially debranched amylopectin after isoamylase treatment generates
SDS [23, 24].
3.3 Resistant Starch
The term “resistant starch” was coined by Englyst et al. to describe a small fraction
of starch that resist hydrolysis by α-amylase and pullulanase treatment in vitro [12].
RS resists digestion and absorption in the small intestine and is fermented in the
colon. When compared to RDS and SDS, RS is not hydrolyzed to glucose in the
small intestine within 120 minutes of being consumed. Amylose amylopectin ratio
influences the resistant nature of starch. Digestion of amylose is slow and that of
amylopectin is fast after retrogradation. RS is a linear molecule of α 1, 4 D-glucan
and is mainly derived from retrograded amylose in cooked starchy food. Large
number of factors influences the rate and extent of starch digestion and all of these
factors are interlinked which complicate the understanding of resistant nature of
starch. With some exceptions such as pea starch, RS content of granular starch is
directly correlated with its amylose content [25–27]. Due to this positive correlation
as well as other functional and nutritional properties of amylose, agricultural
researchers started working on development of crops containing high amylose
starches (HAS) [28]. High amylose potato, barley, and wheat have already been
developed by some researchers [29–32].
4 Relationship between RDS, SDS, and RS
All the three starch fractions are related to each other. RDS shows inverse relation-
ship with SDS and RS. SDS and RS are structurally more similar in terms of
retrogradation and coexist in processed foods [33]. SDS can be made from RDS
by several physical, chemical, and physiological methods [34].
5 Classification and Structure of Resistant Starch
Resistant starch is classified into five sub-types viz. RS1, RS2, RS3, RS4, and RS5
(Table 2). Earlier reports described about only three types of RS, i.e., RS1, RS2,
RS3, however, over the period of time two more types, i.e., RS4 and RS5, were
added to the classification [13, 35–38].
RS1: This type of starch is physically inaccessible to digestion due to its
entrapment within whole or partly milled grains or seeds and due to presence of
intact cell walls in grains, seeds, or tubers. This type of RS escapes digestion due to
inaccessibility of amylolytic and digestive enzymes and passes to small intestine as
such [39]. Only proper grinding/milling can make this RS to digest completely in
small intestine. Its heat stable nature does not allow it to break down or open up
during normal cooking.
RS2: Native starch granules which are protected from digestion due to their
conformation/structure are termed as RS2. Raw potato and green banana contain
this type of starch. RS2 being crystalline and compact in nature has no access to
digestive enzymes and amylases and hence is poorly susceptible to hydrolysis [40].
Table 2 Classification of types of resistant starch (RS), food sources, and factors affecting their
resistance to digestion in the colon [59]
Resistance
RS minimized Digestion in small
type Description Food sources by intestine
RS1 Physically protected Whole- or partly milled Milling, Slow rate, partial
grains and seeds, chewing degree, totally
legumes digested if properly
milled
RS2 Ungelatinized Raw potatoes, green Food Very slow rate, little
resistant granules bananas, some processing degree, totally
with type B legumes, high amylose and cooking digested when
crystallinity, slowly corn freshly cooked
hydrolyzed by α-
amylase
RS3 Retrograded starch Cooked and cooled Processing Slow rate, partial
potatoes, bread, conditions degree, reversible
cornflakes, food digestion,
products with repeated digestibility
moist heat treatment improved by
reheating
RS4 Chemically modified Foods in which Less A result of
starches due to cross- modified starches have susceptible chemical
linking with chemical been used (e.g., breads, to modification, can
reagents cakes) digestibility resist hydrolysis
in vitro
RS5 Amylose-lipid Foods with high Not Can resist digestion
complexes amylose content susceptible
to
hydrolysis
by α-
amylase
Both RS1 and RS2 show slow and incomplete digestion in small intestine. Nowotny
observed the resistant nature of raw potato starch in 1937 while working on the
enzymatic hydrolysis of raw starch from numerous plant species [41]. He observed
the small extent of enzymatic hydrolysis of raw potato starch. Later his results were
confirmed by various researchers [42, 43]. Fine-grain high amylose maize starch and
coarse grain potato starch showed similar resistant nature. The structure as well as
resistance of high amylose maize starch is retained even during processing/prepara-
tion of food. Starches from different sources vary in the size of granule. Granule size
affects the extent of enzyme adsorption on its surface. However, no relationship was
found between the extent of enzyme adsorption and degree of starch hydrolysis [44].
Later the extent of enzymatic hydrolysis was linked to degree of starch crystalliza-
tion. Starch hydrolytic enzymes first degrade the amorphous region, therefore,
resistant nature was attributed to the crystalline region of starch; however, resistant
nature of starch is not always linked to its degree of crystallinity. High amylose
starches as well as B-type starches are more resistant to enzymatic hydrolysis [43, 45].
In B-type starches only surface of granule is hydrolyzed, whereas in A-type starches

deep enzymatic hydrolysis takes place. Starch resistance depends upon the size of
blocklets in the granule. Potato starch contains large blocklets formed due to the
double helices and are incorporated in crystalline layers of granules and hence potato
starch is more resistant compared to cereal starches which contains small blocklets
[46–48]. Resistant nature of starch is not dependent on any one factor, but a large
number of factors such as granule size, shape, amylose content, crystallinity, and also
size of pores/holes in the granule affects the starch resistance [39].
RS3: Physically modified starches are termed as RS3. RS3 is formed during
moist-heat processing of food and is basically a retrograded starch or more precisely
a retrograded or recrystallized amylose found during cooking of gelatinized starch
and in cooked starchy foods that are stored at low or room temperature. RS3 is highly
thermostable and is used as an ingredient in a wide variety of conventional foods
[40]. Most common examples of RS3 are cooked and cooled potatoes and corn
flakes [49]. RS3 is nutritionally important type of RS and has good quality due to
high water holding capacity compared to granular starch [50]. During storage of
gelatinized starch at low temperature for some time, amylose double helices realign
and aggregate to form a highly thermostable B-type crystalline structure. Amylose
retrogradation renders α-1 ! 4 glucosidic linkages inaccessible to amylase which is
responsible for resistant nature of RS3. Being thermostable these aggregates can be
rehydrated only at high temperatures, i.e., above 150 C. Formation of RS3 is highly
influenced by storage temperature and duration. Storage of starch paste at low
temperature results in more RS3 formation compared to storage at high temperature
[51]. Starch crystallinity is also affected by storage temperature. Storage of
gelatinized starch at low temperature results in B-type crystallinity, whereas storage
at boiling temperature results in A-type crystallinity [52]. Both amorphous as well as
crystalline fractions are present in starch gels, wherein amorphous fraction is hydro-
lyzed by amylolytic enzymes and crystalline fraction remains resistant [53]. Forma-
tion of RS3 is hampered by formation of amylose-lipid complexes, which results in
less production of RS3 [54]. When starch gels are stored at low temperatures,
amylopectin also participates in formation of partly crystallized gels. However,
crystallization by amylopectin is quite slow and amylopectin crystallization struc-
tures are less thermostable compared to amylose crystallization structures and can be
rehydrated at 55–70 C temperature [36]. Amylopectin crystallization structures are
also resistant to amylolytic enzyme activity. Formation of RS3 depends on the
botanical origin of starch, amylose content, and procedure used for its preparation.
Preparation of amylopectin retrogradation products is a lengthy process; however,
this process can be hastened with repeated heating and cooling of starch paste.
RS4: Chemically modified starches are known as RS4 [55]. RS4 is a group of
starches in which digestibility is decreased with chemical procedures such as etheri-
zation, esterification, or cross-bonding with chemicals. Based on water solubility
of RS4 as well as analysis method, RS4 is further divided into four sub-categories
[4]. Chemical modification mainly involves conversion, substitution, and cross-
linking. During chemical modification enzyme access to starch is blocked by forma-
tion of atypical linkages which decrease its digestibility by hydrolytic enzymes [56].
The resistance of acetylated and hydroxypropylated starches is directly proportional

to degree of substitution [57]. When compared to native starches, hydroxypropyl
distarch phosphate and acetylated distarch phosphate are more resistant to enzyme
hydrolysis [58]. The level of resistance in RS4 is directly proportional to the degree
of chemical modification. For example, higher the degree of substitution with
phosphoric acid, more will be the resistance of monostarch phosphate. The structure
as well as composition of starch granules is changed during chemical modification,
which in turn increase their resistance to amylolytic enzymes.
RS5: Amylose chains are penetrated by lipids in starch from many plant sources.
RS5 is a type of RS which is formed due to amylose-lipid complexes. Generally,
these complexes are formed during food processing; however, they can also be
prepared under controlled conditions. These type of complexes appeared in high
amylose starches, hence formation of amylose-lipid complexes is influenced by the
amylose-amylopectin ratio of starch and botanical source. RS5 comprises polysac-
charides of water insoluble linear poly α-1,4 glucan that are resistant to hydrolysis by
α-amylase [38]. These polysaccharides promote the formation of SCFA, mainly
butyrate, the most important SCFA [59].
6 Properties of RS
RS has gained importance because of its nutritional aspects as well as functional

properties. RS can be used in a variety of foods due to numerous desirable properties
such as swelling, viscosity increase, gel formation, and water-binding capacity
(Table 3) [60]. Due to these properties RS can replace flour on one-to-one basis
without affecting dough rheology significantly. Other properties of RS include its
bland flavor, which upon incorporation in some other food does not change taste of
Table 3 Functional properties and advantages of commercial sources of RS2 and RS3 [59]
Natural sources
Bland in flavor
White in color
High gelatinization temperature
Fine particle size (which causes less interference with texture)
Good extrusion and film-forming qualities
Lower water properties than traditional fiber products
Lowering the calorific value of foods
Increase coating crispness of products
Increase bowl life of breakfast cereals
Functional food ingredients
Useful in products for celiacs as bulk laxatives and in products for oral rehydration therapy
Allow the formation of low-bulk high-fiber products with improved texture, appearance,
And mouth feel (such as better organoleptic qualities) compared with traditional high-fiber
products
food. RS appear white in color and have fine particle size. Like taste, texture of food
also remains unaltered due to its fine particle size. Due to low calorie content of RS
(1.6–2.8 Kcal g1) it can complement reduced fat and low sugar food formulations
and can impart special characteristics along with dietary fiber fortification in high
fiber food [61]. Low water holding capacity of RS provides good handling during
processing, improves crispiness, expansion, and texture in the end product. Other
qualities of RS include high gelatinization temperature, good extrusion, and film
forming property. Incorporation of RS improves coating crispiness of the product
and also enhances the bowl life of breakfast cereals. Food industry is successfully
using RS in a range of baked and extruded products. RS is highly suitable in grain-
based, low moisture to moderate moisture food systems [59].
7 RS Production Technologies
To fulfill the growing demands for functional foods, food industry is investigating
ways to produce innovative functional food products with additional health benefits.
With increasing awareness about health and nutrition among consumers, researchers
and producers are aiming to develop functional foods with supplementary health
merits [62]. Researchers and nutritionists are working together for development of
functional foods with low GI. Due to various health benefits such as positive
influence on digestive tract functioning, gut microbial flora, blood cholesterol, GI
as well as on diabetes control, and being low calorie ingredient RS can be used for
fortification of high GI foods to convert them to low GI [63]. Presence of natural
sources of RS makes it suitable functional ingredient for fortification purposes [64].
In order to increase the intake of dietary fiber, consumers are ready to pay more for
RS-fortified food products. Several techniques are available to alter the GI as well as
rate of starch digestion. These techniques involve modification of key functional
ingredients using low or no calorie sugars, formation of starch lipid-complexes, or
through processing techniques such as heat moisture treatment and extrusion. RS is
included in the food to combine physical characteristics of the food such as texture,
water holding capacity, processing stability, and nutritional functionality. To pre-
serve the nutritional functionality of RS-containing food, stability of RS during
processing is of utmost importance [65]. Various techniques such as heat, enzymatic,
heat with enzyme, and chemical treatment are available for manufacture of RS.
7.1 Heat Treatment
7.1.1 Heating Cooling Cycles

Repeated heating and cooling cycles for RS production are being used since long.
Another method for RS production involves starch gelatinization followed by
enzymatic debranching of gelatinized polymer, deactivation of the debranching
enzyme, and isolation of the resultant product by drying/extrusion/cocrystallization.
RS produced by using heating-cooling cycles is RS3. Various researchers have
reported the increase in RS3 content by using different combinations of storage

duration and heating and cooling temperatures. More RS3 yield is obtained with
gelatinization in high pressure autoclave as compared to water bath (90 C) [66].
High RS3 can be prepared by autoclaving of starch (30% w/v) at 121 C for 1 h and
cooling at room temperature followed by storage at 48 C for 1 day. Same steps are
repeated several times to obtain high RS3 yield. Heating of wheat starch suspension
(20% w/w) at 100 C for 16 h, followed by air drying results in 12.9% RS3 as
compared to 10.4% tested as dietary fiber [67]. Boiling and cooling of potatoes at
4 C for overnight results in 2.8-fold increase in RS yield [68]. RS content increased
up to 63% after boiling and cooling of potatoes at 4 C for 48 h compared to boiled
hot potatoes [49]. Gelatinization of starch at 120 C for 20 min followed by cooling
to room temperature yielded high amount of RS [69]. Starch gelatinization at 110 C,
121 C, 127 C, 134 C, and 148 C for 30 min to 1 h can increase the RS3 yield
even in starches containing normal amylose level [70–72]. Gelatinization of water-
starch suspension in 1:3.5 ratio at 134 C with four repeated heating cooling cycles
results in good yield of RS3 [73, 74].
7.1.2 Hydrothermal Treatment

Hydrothermal treatment involves physical modifications that change the physico-
chemical properties of starch without altering its granular structure. Annealing
(ANN) and heat moisture treatment (HMT) are the main hydrothermal treatments
where the temperature and heating time need to be controlled. These treatments are
based on the starch to moisture ratio. In ANN treatment, a combination of excess of
water (>40%) and temperature below gelatinization is used, whereas HMT takes
place under controlled moisture content (10–30%) with high temperature
(90–120 C) [75]. Partial acid hydrolysis enhances the effect of hydrothermal
treatment and can result in heat stable granular RS [76]. HMT is natural physical
modification technique that is safer than chemical modification of starch. In ANN
treatment temperature must be held below gelatinization temperature to retain the
initial granular structure of starch. Granular structure is lost due to combination of
gelatinization and melting at 40–60% moisture level. Enhanced granular stability
due to hydrothermal treatment results in high RS content [65].
ANN treatment is known to enhance the degree of starch crystallinity, strengthen
crystalline form of granules, and order starch chains in crystalline as well as
amorphous layers. All of this increases granule stability with decreased solubility
and swelling capability and hence, in turn, increase resistance of starch granules to
amylolytic enzyme [77]. Lee et al. [78] used different combinations of moisture
(20–25%) treatment, duration (1, 5, and 9 h), and temperatures (110 C, 130 C, and
150 C) to treat waxy potato starch (0% amylose) and achieved maximum RS yield,
i.e., 66.8% with the combination of 20% moisture, 110 C temperature, and 5-h
treatment.
Starch treated with HMT has increased resistance to amylolytic enzymes. HMT of
high amylose maize starch at 100 C temperature and moisture content below 35%
leads to crystallite formation along with ordering and binding of amylose chains in
the amorphous region which results in lower degree of swelling and hence results in
resistant nature of starch [79]. Chung et al. [80] reported 7.7%, 20.3%, and 5.6%
increase in RS yield after HMT of corn, pea, and lentil starches, respectively. Chung
et al. [81] compared the effect of ANN and HMT on RS yield and observed that
HMT results in higher RS yield compared to ANN. ANN increased RS to 0.3%,
1.3%, and 1.6% whereas increase with HMT was 1.7, 4.7, and 5% in starches of pea,
lentil, and navy bean, respectively. An extensive range of botanical sources and a
range of HMT conditions were studied by various researchers and they observed
numerous changes on granular surface, swelling factor, amylose leaching, gelatini-
zation temperature, X-ray diffraction pattern, crystallization, and gelatinization
parameters of starch [82–85].
Most widely used methods to increase RS3 yield are ANN, acid hydrolysis of
amylomaize starch, and repeated freeze-thawings [65, 86]. Generally high amylose
corn starch is used for preparation of RS3 [87]. ANN treatment (50 C for 24 h at
50% moisture) increased RS content in pinto bean and black bean to 39.7% and
19.7%, respectively. Jiranuntakul et al. [88] observed that different starches respond
differently to HMT. They observed that HMT (25% moisture, 100 C temperature,
and 16 h) increases the RS from 27–40.3% in waxy corn starch, whereas same
conditions decrease the RS from 4.7% to 29.7% in normal corn, rice, and
potato starch. Zero to seven cycles of boiling and freezing at 18 C for 23 h to
whole wheat flour in water (1:15% w/v) increased the RS content from 1.03% to
8.07% [89].
7.1.3 Extrusion
Extrusion technique is widely used in food processing industry to prepare food
products with different shapes. This is a high temperature short time process
(HTST) and has the potential to increase the RS content of food product up to
certain extent. During extrusion high shear force causes depolymerization
followed by thermal cleavage of the starch molecule. As a result straight chains
are produced which are more prone for retrogradation into RS3 [90]. RS content of
normal corn starch increased from 11% to 20% after acid hydrolysis followed by
low or high shear extrusion [91]. Extrusion conditions such as barrel temperature,
screw speed, and shear force have more impact on RS yield as compared to starch
moisture content [92]. Extrusion of maize starch with 12–18% moisture did not
show significant increase in RS content, whereas at 20% moisture level, RS content
increased significantly. Origin of starch affects the RS yield during extrusion.
Because of high gelatinization temperature and amylose content banana starch is
most efficient for production of RS through extrusion, compared to other starches
[64]. RS content of wheat starch increase from 0.8% to 2.8%, normal maize starch
from 1.5% to 2.1% [93], barley from 2% to 3% [94] after extrusion. Reports on
increase in RS after extrusion are conflicting as many studies reported decrease in
RS content after extrusion and attributed this to degradation of starch granules
under high temperature, pressure, and shear forces [95]. Possible reason for this
conflict could be the interaction of starch with other components available in
various food matrix.
7.1.4 Heat and Enzyme Treatment

RS production can be increased by using a combination of heat and enzymatic or
chemical and enzymatic modification. Enzymes or chemicals can be used to remove
amorphous region of retrograded starch. Example of simultaneous heat and enzyme
treatment is pullulanase treatment of gelatinized starch and isolation of the product
by drying/extrusion. RS production is also achieved by controlled heat treatment of
starch, followed by debranching with the help of enzymes, annealing, and drying
[96]. Treatment of gelatinized starch with debranching enzyme such as isoamylase
or pullulanase results in debranched amylopectin starch. Debranched amylopectin
starch is used in formation of reduced-fat food products and can be prepared from
any starch containing amylopectin such as common corn and waxy maize starch. In
high amylose maize starch, RS content is increased by enzymatic debranching
followed by extrusion or drying, where the addition of an inorganic salt or
debranched starch before the isolation further increases the RS content [74, 97].
Debranching of potato amylopectin with pullulanase before repeated heating-
cooling cycles as well as maize starch debranching increase the RS3 yield [70,
98]. However, to get high yield of RS3 from maize starch autoclaving of starch at
121 C for 1 h before debranching is required.
7.2 Enzymatic Treatment
Lower molecular mass of starch and amylopectin debranching play a role in

enhanced RS production and enzymes are used for both of these purposes [99].
Debranching enzymes such as pullulanase and isoamylase act only on α 1,6 glyco-
sidic bonds at branch points of amylopectin and break these bonds. As a result
amylose content of the starch increases which in turn forms tightly packed crystalline
structures which is responsible for the resistant nature of starch. Enzymes such as α-
and β-amylase can also be used to break the α-1,4 glycosidic bonds. Both
enzymes act on different areas of starch molecules, where α- amylase breaks all
α-1,4 glycosidic bonds leaving those near the branch points and releases glucose
monomers. Whereas β-amylase breaks every other α-1,4 glycosidic bond from
nonreducing end of amylopectin or amylose and releases maltose units. In
practice, pullulanase and isoamylase are commonly used for RS production
compared to α- and β-amylase. As α-amylase breaks almost all the α-1 ! 4
glycosidic bonds of starch, it lowers the starch paste viscosity and as a result
adversely affects crystal formation. In low viscosity starch pastes fast movement
of linear chains causes difficulty in crystal formation [100]. Optimization of α-
amylase is required to obtain sufficient yield of RS. Measurement of total dietary
fiber content of RS samples through enzyme treatment revealed that the concen-
tration of α-amylase is more critical compared to amyloglucosidase [101]. The α-
amylase activity showed inverse relationship with RS content. Poly 1,4-α-D-
glucan can also be used for production of readily fermentable heat stable RS of
optimal chain length [102]. Pullulanase enzyme can also be used to produce RS
with the same cooking quality as that of untreated rice starch or flour. Starches
from potato, oat, barley, sago, corn, wheat, tapioca, and arrow root can be used to
produce RS through pullulanase treatment [74].
7.3 Chemical Treatment
Chemical reagents are used to block the enzyme access and as a result modified
starch escapes digestion after chemical treatment. Chemical treatment changes the
molecular structure of starch which results in high RS production. Acidification,
esterification, and cross-linking are the major forms of chemical modification of
starch.
7.3.1 Acidification
The purpose of acidification is to first hydrolyze the amorphous parts of starch
granules followed by hydrolysis of crystalline region. This process generates short
chains of amylopectin which are disorganized by autoclaving followed by acidifi-
cation. During retrogradation, these chains reorient to form more ordered double
helix structures which resist enzyme hydrolysis [103]. Acid modification followed
by autoclaving and retrogradation to increase RS yield is reported by several
researchers [104, 105]. Acids such as hydrochloric acid, orthophosphoric acid, and
sulfuric acid can be used for starch modification [106]. Tester et al. [107] obtained
49.5% RS by treatment of lima bean (Phaseolus lunatus) with hydrochloric acid at
1/60 parts of native starch at 90 C for 1 h. Xie and Liu [108] obtained 68.3% RS
yield with chemical treatment of normal corn starch with citric acid followed by dry
heating at 140 C for 7 h and heating at 100 C in boiling water bath. During
acidification by citric acid, citric anhydride substitutes the hydroxyl glucans of starch
chains that resist digestion by amylolytic enzymes. Thermal-acid treatment of starch
for production of RS needs optimization, as high intensity of this treatment may
destroy the resistant structure of starch. Heat and citric acid treatment of normal
maize starch increased RS yield to 35.62% [109].
7.3.2 Cross-Linking
Food industry is using cross-linking to improve functional property, freeze thaw stability,
and cold storage stability of starch pastes. Cross-linking stabilizes and strengthens starch
by randomly adding inter- and intramolecular bonds [110, 111]. Seib and Woo [112] and
Woo and Seib [113] have described the use of various cross-linking techniques for
increasing RS yield in normal starches derived from several botanical sources. Starches
are chemically modified by treating with multifunctional reagents that form ether or ester
linkage between hydroxyl groups of starch molecule [111]. Chemically modified starches
show resistance to enzyme hydrolysis in both raw as well as gelatinized form [114].
During chemical modification, starch resistance is improved by substitution of starch
hydroxyl group with citryl, acetyl, octenylsuccinyl, and hydroxypropyl [108, 115–118].
Cross-linking of starch with phosphate showed contradictory results as some authors
[113, 119] reported decrease in digestibility of starch and some authors [58, 118, 120]
reported either slight or no change in the digestibility of starch. These contradictions
could be due to the difference in the origin and property of starch and the conditions used
to modify starch. For production of cross-linked starches bi-or-polyfunctional reagents
such as sodium trimetaphosphate, phosphorous oxychloride, or mixed anhydrides of
acetic acid and dicarboxylic acid like adipic acid are used. Kahraman et al. [121] reported
use of reagents such as sodium triphosphate or its mixture with sodium tripolyphosphate
for cross-linking glucans for RS production. Factors such as source of starch, reaction
conditions like time, temperature, pH, and type and concentration of cross-linking
reagent affect the chemical and functional properties of cross-linked starches [111,
122]. Woo and Seib [113] reported positive correlation between RS content and reaction
time. Temperature and pH both increase the RS content of cross-linked corn and wheat
starch, where cross-linking is more subjective to pH than temperature-induced modifi-
cations. Cross-linking of corn and wheat starch yield 80.4% and 83.9% RS, respectively
[123]. Wheat and corn starch has different optimum conditions for production of cross-
linked RS. For production of cross-linked wheat starch 38 C temperature and pH 12 and
for cross-linked corn starch 70 C temperature and pH 12 are optimum treatment
conditions [121]. Carlos-Amaya et al. [124] reported increase in RS from 21.49% to
29.14% in banana starch with dual modification using cross-linking and esterification.
Starch may appear as swollen granules, combination of starch fragments, as well as
dispersed starch molecules or completely dispersed starch molecules based on the degree
of gelatinization. Ratio of starch fractions viz. RDS, SDS, and RS is determined by the
degree of starch gelatinization and the structural changes in starch caused by physical or
chemical treatment [125].
Chemically modified starches have been utilized as food additives, thickening or
gelling agents, and fat replacers. Hydrothermal treatments increase the accessibility
of chemically modified starches to the amylolytic enzymes. However, the level of
digestion is determined by the origin of starch and degree of substitution with
chemical groups [120]. Di-starches are modified RS with high dietary fiber content
( 70% w/w) and their resistance to the activity of amylase show direct correlation
with degree of chemical substitution [113]. Acetylated retrograded starches also
come under chemically modified starches and their quality is influenced by degree of
substitution and raw material used for esterification [126]. Hydroxypropylation,
roasting with glycine, and cross-linking with epichlorohydrin also increase starch
resistance to amylolytic enzymes [125].
7.4 Hydrostatic Pressure Treatment
Hydrostatic pressure treatment (HPT) is a nonthermal food processing method where

food is processed at high hydrostatic pressure (HHP) ranging from 200 to 600 MPa.
Water is used as a pressure transmitting medium in this process [127]. During HPT,
starch microstructure is influenced by factors such as pressure level, method of
pressure application, time, temperature, constitution of food, and phase state of
food [128–132]. High pressure treatment at 120 to 600 MPa for 30 min converted
the C-type X pattern of starch to B-type in mung bean [131]. Potato starch being
more resistant to pressure than rice, corn, or tapioca starch yields low RS at equal
HHP level [133]. Among the continuous and two-cycle (200 and 600 MPa) HPT,
HHP at 600 MPa significantly alters the microstructure and lowers the RS content of
rice starch in comparison to HHP level 200 MPa [134]. Two 15-min cycle of high
hydrostatic pressure treatment at 200 MPa could be beneficial to increase RS
content. Zhang et al. [135] reported 11.7% RS yield of high amylose maize starch
(50% amylose content) gelatinized at high pressure and temperature (10.3 MPa,
110 C) and retrograded for 24 h.
8 Improving RS Production in Plants
In view of the industrial application and the nutritional benefits of resistant starch,
researchers around the globe have been working to increase the RS content of the
plants. The approaches for increasing the RS content in plants includes natural
selection, conventional breeding, as well as transgenic. All these approaches are
based on biosynthetic pathways of starch metabolism. The key enzymes for starch
biosynthesis are AGPase, starch synthases, and branching enzymes. Generation of
the sugar nucleotide ADP-glucose is catalyzed by AGPase. Starch synthases cata-
lyze the polymerization of glucose residues resulting in formation of α-1,4 glucans.
Branching enzymes cleave α-1,4 glucans and reattach the cleaved chain to an α-1,4
glucan chain by an α-1,6 glycosidic linkage thereby forming a branch.
In 2000, Gerhard et al. developed very-high-amylose potato starch by manipulating
starch branching enzymes through genetic engineering [29]. They simultaneously
inhibited two isoforms of starch branching enzyme to below 1% of the wild-type
activities which resulted in altered starch granule morphology and composition. In
these potatoes amylopectin was found to be absent, whereas the amylose content
increased to levels comparable to the highest commercially available maize starches.
Slade et al. used TILLING (Targeting Induced Local Lesions in Genomes) approach
and identified mutations in the form of single nucleotide polymorphisms (SNPs) in
starch branching enzyme IIa genes (SBEIIa) [136]. They combined these new alleles of
SBEIIa through breeding which resulted in the development of high amylose durum
and bread wheat varieties containing 47–55% amylose and having elevated resistant
starch levels compared to wild-type wheat. Recently in 2014, Sparla et al. applied the
TILLING approach on barley and identified 29 new alleles in five genes related to
starch metabolism known to be expressed in the endosperm during grain filling: BMY1
(Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase
1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). A line having a nonsense
mutation in SSIIa exhibited a twofold increased amylose/amylopectin ratio [137].
9 Commercially Available RS Products
Starch Australia Ltd. introduced the first commercial RS, i.e., Hi-maize. Later other
companies introduced new commercial starches to market using different prepara-
tion technologies. The commercial RS prepared by different companies vary in
percent RS content. Other commercial RS are CrystaLean ® (RS3), Novelose ®240

(RS2), Novelose ®260 (RS2), Novelose ®330 (RS3), Eurylon ® (RS2), Amylomaize
VII (RS2), and Neo-amylose (RS3) (Table 4). CrystaLean is RS3 produced by starch
retrogradation of high amylose maize starch ae-VII hybrid. National Starch and
Chemical Co. (USA) introduced Hylon-VII, a natural high amylose maize starch.
Most of the above RS3 products are prepared by amylose retrogradation of high
amylose corn starch using repeated heating and cooling cycles under controlled
moisture and temperature conditions. These processes lead to manufacture of gran-
ular form of concentrated RS containing up to 47–60% RS content. A highly
crystalline RS3 namely Actistar Act*-RS3 has also been prepared using
maltodextrins as starting material. Due to the starting material and process used
for the production, Act*-RS3 tastes very natural. High amylose corn starch is also
being used for the production of Fibersym HA, which is being used in a wide array of
lower-net-carbohydrate food products. Fibersym HA provides more than 70% die-
tary fiber and is used in the preparation of food products such as pizza crust, breads,
tortillas, cookies, muffins, breakfast cereals, snack products, and nutritional bars.
Potato starch is used for the production of Fibersym 80ST. Fibersym 80ST has
slightly higher water holding property which influences the properties of finished
food products like cookie spread and muffin volume. Nutriose FB06 and Fibersol-2
also contain high RS content and provide 85% and 90% fiber content, respectively.
Fibersym 80ST, Fibersym RW, Fibersym HA, and Fibersol-2 are all RS4 prepara-
tions and are available in the market. RS preparations without altering the organo-
leptic properties of food products reduce the availability of some saccharides. RS
fortification does not alter the quality of product and organoleptic properties of
extruded, baked products and confectionary remains unchanged [59, 138].
10 Applications of RS
Potential physiological benefits and unique functional properties of RS have

attracted the attention of nutritionists and food processors. With ever increasing
awareness of consumers towards health and nutritious food, they are concerned with
supplementary health merits derived from regular ingestion of RS along with
traditional nutritional aspects of food [139]. Looking into this, food processors,
researchers, and producers are working on development of improved foods with
slow digestion and additional health benefits. RS is one such ingredient which is
being used for fortification of food products to enhance the nutritional value and
health merits of food. RS being naturally present in broad range of starchy foods
makes it convenient to use it as a functional ingredient for fortification purposes. RS-
fortified food products are gaining popularity among consumers and consumers are
even ready to pay more for such food products to increase their dietary fiber intake
[140]. At commercial level, RS containing starch ingredients are available in name
of “resistant starch.” Most of these RS-enriched products are fully digestible and act
as RS supplier [108]. In mid-1990s the first ever commercially available RS product
was reported. However, nowadays RS-rich powders are prepared by large number of
Table 4 Commercially manufactured resistant starches commonly used in various foods [59].
Brand name
of commercial RS/TDF% Physiological and/or health
RS Type content benefits Manufacturer
Hi-maize RS2 30–60% Prebiotic properties. Lowers fecal National Starch
TDF pH. Increases the level of SCFA and
(in particular butyrate which may Chemicals co.,
reduce cancer risk). Increases USA
bowel action with its mild
laxative effect. Increases the
bowels’ beneficial microflora
CrystaLean RS3 19.2–41% Prebiotic effect. Increases Opta food
RS proportion of butyrate. Increases ingredients Inc.,
cell proliferation in proximal USA
colon (in rats). Provides soluble
dietary fiber and prebiotic effects.
Low glycemic index
Novelose 240 RS2 47% RS Lowers glycemic response when National Starch
used as a substitute for flour and and chemicals
other rapidly digested co., USA
carbohydrates
Novelose 260 RS2 60% RS Lowers glycemic response when National Starch
used as a substitute for flour and and chemicals
other rapidly digested co., USA.
carbohydrates
Novelose 300 RS3 <30% Lowers glycemic response when National Starch
TDF used as a substitute for flour and and chemicals
other rapidly digested co., USA
carbohydrates
C*Actistar RS3 53% RS Health benefit potential. Prebiotic Cerestar (Cargill
effect. Source of butyrate. company)
Supports the immune system.
Reduced glycemic response. Low
calorific value. Easily
fermentable RS. Very well
tolerated.
Fibersym™ RS4 >70% Acts as prebiotic, reduces the MGP ingredients,
HA TDF glycemic and insulin response of Inc. (Atchison,
healthy individuals as well as Kans.) and
type 2 diabetics Cargill
Fibersym™ RS4 80% TDF Acts as prebiotic, reduces the MGP ingredients,
80ST glycemic and insulin response of Inc. (Atchison,
healthy individuals as well as Kans.) and
type 2 diabetics Cargill
Nutriose FB – 85% TDF Low calorific value Roquette, Freres,
France
Fibersol 2 – 90% TDF Probiotics effect, intestinal ADM/Matsutani
regularity, and blood sugar
regulation
(continued)
Table 4 (continued)
Brand name
of commercial RS/TDF% Physiological and/or health
RS Type content benefits Manufacturer
HylonR VII RS2 23% TDF Increases level of SCFA National Starch
and chemicals
co., USA.
Neo-amylose RS3 87 or 95% Prebiotic. Protects against Protos-biotech.
RS inflammatory intestinal disease. (Celanese
May protect against colorectal ventures GmbH)
cancer. May help control blood
glucose levels in diabetics
companies employing various technologies. RS cannot be replaced by traditional

insoluble fiber in a RS-fortified food product, due to the high quality of RS-enriched
final product [141]. RS is being used in the preparation of moisture-free food
products. Cross-linked RS prepared from starches of maize, tapioca, and potato are
being used for formulation requiring pulpy texture, smoothness, flowability, and low
pH and high temperature storage [142]. Baked products, pasta products, as well as
beverages are fortified with RS to improve their textural properties and nutritional
quality. Most of the fat in imitation cheese was successfully replaced with RS,
without adversely affecting the meltability or hardness of RS. In such cases, RS
provides dual benefits, one being reduction of fat in a food product and another being
health benefits conferred by RS itself. Nowadays large number of RS/fiber-fortified
products such as high fiber breads, biscuits, and breakfast cereals are available in the
market. The availability of techniques to prepare RS tolerant to processing made it
possible to prepare RS-rich food products. Dry pasta products containing up to 15%
RS can be prepared, without affecting dough rheology during extrusion. In compar-
ison to unfortified pasta, RS-fortified pasta appears light in color and has a firm
texture in the same time as that of unfortified pasta [74]. RS added opacity to the
beverages. It is being used in thickened opaque health drinks in which insoluble fiber
is desirable. RS is superior to other fibers, due to its bland taste, which imparts less
gritty mouthfeel and masks flavor to much lesser extent. However, other fibers have
a strong flavor, coarse texture, and poor and dry mouth feel.
11 Health Benefits of RS Consumption
Health awareness is increasing at a fast rate among consumers. To meet the growing
demand of consumers for functional foods, international food industry is investigat-
ing ways to produce innovative food products with additional health benefits.
Carbohydrate-rich foods are main part of our diet and most of the carbohydrate-
rich foods are high glycemic. Therefore, development of carbohydrate-rich foods
with low GI is need of the hour. High GI foods adversely affect health by spiking
blood glucose level and cause insulin disturbance. Carbohydrate products with low
GI can improve the control of obesity and diabetes and subsequently can reduce the
risk of cardiovascular diseases [62]. However, all RS types are not effective to
control cholesterol level. The composition and properties of RS determine the extent
of production of SCFA by bacterial fermentation of RS in the large intestine [143].
RS confers physiological benefits of soluble fiber and has a positive impact on
colonic health by increasing crypt cell production rate or decreasing colonic epithe-
lial atrophy in comparison with no fiber diet. Consumption of carbohydrate-rich
foods was highly recommended, until recently. However, due to change in the
viewpoint on nutrition, in developed countries, nutritional quality of food is
expressed by the tendency of reducing calorific value of food. Civilizational changes
also contributed to increase in dietary fiber intake that is required for proper
functioning of body. RS being a natural food component, attracted much attention.
Dietary fiber including RS with little calorific value confers several health benefits
like laxation, blood cholesterol attenuation, and blood glucose attenuation.
11.1 RS: A Type of Dietary Fiber
Dietary fibers are “the edible part of plants or analogous carbohydrates that are
resistant to digestion and absorption in the human small intestine with complete or
partial fermentation in the large intestine” [144]. Champ et al. [145] defined dietary
fiber as “a part of plant present in the form of a chemical substance which has
indigestibility in the small intestine and/or has beneficial digestive and physiological
effects and metabolic fate.” Dietary fiber comprises of non-starch polysaccharides
(NSP), lignin, RS, and nondigestible oligosaccharides. Dietary fiber majorly consists
of NSP and lignin. NSPs include majorly cellulose and hemicelluloses (glucan,
gums, and pectin). NSP are completely resistant to amylolytic enzymes. In the
small intestine, soluble dietary fiber such as β-glucan and arabinoxylan leads to
the formation of viscous solution and hence, increase viscosity in the intestine,
which in turn slows intestinal transit, delays gastric emptying leading to slow
glucose and sterol absorption [146]. Lignin, cellulose, and hemicelluloses are insol-
uble dietary fibers with high water-holding capacity which contributes to increased
fecal bulk. Dietary fibers that are not fermented in the body are excreted in the feces.
Dietary fibers are essential for body and they help in regular bowel movement, blood
cholesterol attenuation, and blood glucose attenuation.
11.2 RS Improves Probiotic Bacteria
Prebiotic property of RS is of great interest. Prebiotics are nondigestible food

ingredients that beneficially affect the host by selectively stimulating the growth as
well as activity of one or more number of bacterial species residing in the colon and
as a result improve host health. Prebiotic and probiotic have a symbiotic relationship.
One of the best example of prebiotic is fructo-oligosaccharide, others being inulin,
oligofructose, RS, and inulin type-fructans [140, 148]. RS plays a role as prebiotic as
well as symbiotic [148]. Probiotics enhance the epithelial barrier, increase adhesion
to intestinal mucosa, and concomitantly inhibit the pathogen adhesion, competitive
exclusion of pathogenic microorganisms, production of anti-microorganism sub-
stances, as well as modulation of the immune system [149]. RS is known to promote
the growth and activity of probiotic bacteria in the colon and also can interact with
other prebiotic dietary fibers such as β glucan [145, 150, 151]. RS ingestion helps in
extending the viability of some probiotic organisms in the colon. RS acts by
protecting some of the ingested organisms on their path to colon and increase the
initial levels of bacterial species once they reach the colon. RS acts as a substrate for
probiotics in the colon [147]. High amylose starch (HAS) being source of RS2 also
acts as prebiotic and prevents the development of nonreversible insulin resistance,
lower plasma cholesterol, and triglyceride concentration compared to diet rich in
amylopectin starch [152]. RS affects fecal bulk and SCFA metabolism, hence has
direct impact on colonic health [74]. RS3 has a high rate of fermentation by intestinal
microflora which leads to production of SCFA containing high concentration of
butyrate, which has positive impact on colon health [153]. In comparison to RS3,
RS4 is inaccessible to enzymes due to chemical modification of starch. Large
number of microorganisms and enzymes residing in the colon digest and metabolize
most of the biopolymers. RS is hydrolyzed to glucose by bacterial amylase, which is
further metabolized into organic acid (e.g., lactic acid) and gases (CO2, H2, CH4)
[65, 154]. Among the SCFA, butyrate acts as a main nutrient for colonocytes and
deficiency/lack of butyrate increase the risk of colonic diseases such as colon cancer.
Compared to all other dietary fibers, RS produces high concentration of butyrate,
hence is very much important for human health. RS being source of butyrate in colon
prevents the risk of colorectal cancer. Butyrate acts as a source of energy for
epithelial cells and inhibits the malignant transformation of such cells in vitro. The
rate of fermentation of RS in the colon determines the site of production of SCFA
[144]. RS can also be beneficial to adults suffering from colonic lesions. Overall RS
has the potential to lower the risk of many diet-related diseases and improve the
human health [98, 155].
11.3 Hypoglycemic Effect of RS
FAO recommended increased intake of low GI foods with emphasis on diabetics and
subjects with impaired glucose tolerance [156]. GI is a ranking of food/food products
with respect to their influence on postprandial glycemia [1]. Hypoglycemia occurs as
a side effect in diabetes mellitus patients and can occur in other diseases as well. RS-
rich foods have slow digestion rate. This property of RS is of much importance to
type II diabetic patients, due to its slow rate of glucose release as well as the time it
takes to metabolize. The starch inaccessibility to digestive enzymes (such as α-
amylase, isoamylase, and pullulanase) in starchy foods is responsible for reduced
postprandial blood glucose and insulin response. Along with RS, diets rich in SDS
are also good for health of diabetics as well as nondiabetic persons [13, 157]. Whole
grains containing high concentration of dietary fibers are a good example of low GI
food, as they release glucose at slow rate [158]. Processing technique can lower the
starch digestibility and can increase the SDS and RS content in cereal grains and can
hence enhance the nutritional value of cereal grains. The outer layer and germ of
wheat, barley, rye, and oat grains is rich in many bioactive compounds such as
dietary fiber, antioxidants, phenolics, lignin, vitamin, and minerals [159]. Such
compounds are effective in reduction of risk of cardiovascular diseases, cancer,
diabetes, obesity, coronary heart disease, and other chronic diseases [160]. Food
industry is focusing on producing functional foods with low GI using these cereal
whole grains.
11.4 Hypocholesterolemic Effect of RS
Asp et al. [161] investigated the role of RS in relation to its hypocholesterolemic

effect and protective effect against colorectal cancer. RS affects lipid metabolism,
where total lipid, total cholesterol, low-density lipoprotein (LDL), high-density
lipoprotein (HDL), very-low-density lipoprotein (VLDL), intermediate density lipo-
protein (IDL), triglycerides, and triglyceride-rich lipoprotein are all affected by RS
[4]. Feeding of rat with RS diets containing up to 25% raw potato raises the cecal
size, cecal pool of SCFA, absorption of SCFA in colon and lowers the plasma
cholesterol and triglycerides [74]. Martinez-Flores et al. [162] observed hypo-
cholesterolemic properties of cassava starch extruded with 9.7% RS. Such starches
can be used in food to improve overall cardiovascular health. RS ingestion decreased
the serum cholesterol level in rats fed with cholesterol-free diet [163]. Some con-
tradictory reports on the role of RS in altering triglycerides and cholesterol levels are
available, which emphasize the need of more research in this particular area to obtain
a clear picture on effect of RS on lipid metabolism in humans.
11.5 RS Plays a Role in Energy and Weight Management
RS releases energy partially in small intestine as glucose and partially in large

intestine as fermentation by-product such as acetate, which in turn provide balanced
energy for long time after consumption. Energy value of RS is quite low and is
calculated to be approximately 8 kJ g1 (2 kcal g1) in comparison to energy
provided by completely digestible starch, i.e., approximately 15 kJ g1 [164]. RS
has been reported to play a role in modification of fat oxidation, as satiety agent, and
in weight management [4, 165, 166]. Mobilization and utilization of fat stored as an
indirect result of reduction in insulin secretion is enhanced with RS-rich diets [167].
Compared to fully digestible carbohydrates, RS-enriched foods provide few calories
along with lower glucose response. RS reduces the total and regional body fat
accumulation. Diet enrichment with RS is a natural, endogenous way to increase
gut hormones which are effective in reducing energy intake [168]. RS fortification
may be a very effective natural approach to treat obesity.
11.6 Reduction of Gallstone Formation
High secretion of insulin due to consumption of digestible starch-based food is

leading to gallstone formation [74]. The occurrence of gallstone formation is quite
less in Southern India where whole grains are preferred over flour, compared to
North India, where majorly flour is used [169]. The lesser occurrence could be due to
the high intake of dietary fibers and RS in South India. The dietary fiber intake in the
USA, Europe, and Australia is two- to fourfold lower compared to India and China,
where high starch diets are consumed. These results are correlated with the differ-
ence in the number of gall stone cases in these countries [74, 170].
11.7 Enhanced Absorption of Minerals
Studies on rat as well as human have shown that RS increase ileal absorption of a
number of minerals. RS consumption enhances absorption of only calcium in
humans, whereas in rats fed with RS-rich diets, increase in absorption of calcium,
magnesium, zinc, iron, and copper was observed [151]. Consumption of diet
containing 16.4% RS increases calcium and iron absorption in infant pigs [171].
Schulz et al. [172] reported increase in calcium and magnesium absorption by
enhancing mineral solubility in the cecum and/or large intestine in rats by RS2
consumption [172]. Feeding raw potato starch to rat enhanced the fermentation in the
distal part of digestive tract, resulting in increased absorption of calcium, magne-
sium, zinc, and copper owing to hypertrophy of the cecal wall and cecal acidification
[173].
11.8 Other Health Benefits
RS reduces inflammatory bowel diseases such as ulcerative colitis and other large
bowel problems such as diverticulitis and constipation [4]. As RS is involved in
the production of SCFA, particularly, butyrate in vivo, it may prove a useful
adjunct to traditional treatments of ulcerative colitis. Diets rich in RS2 as well as
RS3 normalize the cell function-like activation of colonic cell proliferation,
restoration of apoptotic response and uptake of SCFA, increased cecal level of
butyrate, improved cecal and distal macroscopic and histological observations in
rats with chemically induced colitis [174]. RS affects immune function through
production of pro-inflammatory cytokines and expression of number of receptors
on T- and B-lymphocytes that are required for initiation of immune response
[175, 176].
12 Future Trends
The increased number of research papers that have appeared on RS is testimony to

the importance of RS. RS in food is an active area of research. Research is being
carried out on various aspects of RS, such as its increase in food through processing
techniques, production of RS-fortified food, studies on health benefits of RS in vitro
and in vivo, etc. Due to keen interest of researchers and nutritionists, several
commercial preparations of RS are available which can be used to increase fiber
content of food. More efforts are required for production of RS resistant to thermal
processes during processing. Although several health benefits of consuming RS are
reported, further studies are required for development of RS preparation with
optional characteristics to claim specific health benefits such as improved blood
health and lowering the GI. Although some figures have been proposed for specific
health benefits, in general, it is very difficult to suggest a figure for RS consumption
for general health benefit. Overall RS consumption has been decreased due to
modern food processing practices. However, due to desirable functional and phys-
iological properties of RS, there is an increasing trend to incorporate commercial
preparation of RS in processed foods. In developed countries, where processed food
daily intake is considerable, RS will provide dual benefit, one being dietary fiber and
another being bioactive functional food component to increase gut hormones that
reduce energy intake and hence treat obesity. As the demand for healthier food
increases owing to its physiological benefits, RS can form essential ingredient of
innovative foods to be developed in future.
Future may witness the more tailor made starch derivatives with multiple mod-
ifications. Buttriss and Stokes [140] reported development of insoluble resistant
maltodextrins with similar functionality as that of RS. More such resistant compo-
nents may appear in future. Nondigestible chemically modified starch derivatives
may have increased application in food formulations [16]. As extrusion also
enhances RS concentration of native starch, more extruded products with high RS
can be expected in future [177].
13 Conclusions
Due to research on its different aspects (physical, chemical, and physiological) good
understanding of RS has been achieved. Reports show that the consumption of RS
has declined over the last few decades, possibly due to intake of low fiber foods and
fast foods. Looking into this trend, food scientists have developed number of RS/
dietary fiber-rich products easily available in the market. RS is ideal for fortification
of ready to eat cereals, snacks, pasta, noodles, baked foods, and fried foods. These
products can be labeled simply as “starch conferring additional nutraceutical bene-
fits.” Processing conditions can be altered to increase its content in food. Alterations
in number of heating-cooling cycles, pH, temperature, time, freezing, and drying,
etc., alter the RS content. Products prepared with RS incorporation have better
crispness, mouthfeel, color, and flavor compared to those prepared with traditional
fibers. RS has assumed great importance due to its physiological properties that can
reduce the risk of several diseases, including colon cancer and diabetes and also is
useful in controlling obesity and diabetes. RS-fortified products have better con-
sumer acceptability because of its unique physiochemical properties.
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Gluten-Free Cereals and Pseudocereals:
Nutrition and Health 29
Mario Fernández de Frutos, Bartosz Fotschki,
Ricardo Fernández Musoles, and José Moisés Laparra Llopis
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848
2 Gluten-Free Cereals and Pseudocereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849
2.1 Food Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 849
2.2 General Features of Most Commonly Consumed Pseudocereals . . . . . . . . . . . . . . . . . . . . 850
2.3 Health Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853
3 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 858
Abstract
Cereals constitute a staple food for large groups of population worldwide.
However, the protein fraction of cereals continues receiving increasing attention
from the clinical community because of its close involvement in both the devel-
opment of immunological processes and intestinal disorders. Thus, together with
constant technological innovations to the increasing demands of the consumer,
makes necessary to optimize food formulations promoting health outcomes. In
this context, beneficial health implications are being reported based on the
advantageous nutritional profile of gluten-free cereals, but mostly pseudocereals.
The latter represent a good source of proteins (albumins/globulins) reducing the
M. F. de Frutos · J. M. L. Llopis (*)

Madrid Institute for Advanced Studies in Food, Madrid, Spain
B. Fotschki
Department of Biological function of food, Institute of Animal Reproduction and Food Research of
the Polish Academy of Sciences, Olsztyn, Poland
R. F. Musoles
Valencian International University, Valencia, Spain

848 M. F. de Frutos et al.
intake of prolamins. Additionally, pseudocereals provide an optimal lipid profile

(ratio of saturated versus unsaturated fatty acids) and bioactive compounds with a
potential significant impact on the consumer’s health. Currently, the underlying
mechanisms by which these beneficial health effects occur still remain unsolved.
Moreover, some recent data point to metabolic effects beyond their nutritional
value. These could have an important impact on immunological processes,
although studies on these aspects result inferential. Future research should
approach epidemiologic studies and toward consolidating the mechanisms of
action, especially in the human body.
Keywords
Cereals · Gluten-free · Health benefits · Metainflammation · Nutrition ·
Pseudocereals
1 Introduction
At present, continuous social development and commercial globalization are moti-

vating the increase in food production, where constant technological innovations are
needed to respond to the increasing demands of the consumer of high quality food,
which is the closest thing to a fresh product with a processed minimum, healthy and
nutritious. The important socioeconomic changes in most countries have led to a
substantial change in dietary habits favoring an increase in the consumption of
animal fats and proteins. This tendency is reflected in an increase in the incidence
of diseases, directly or indirectly, associated with alterations of the hepatic homeo-
stasis that give rise to a wide spectrum of pathologies related to the metabolic
syndrome, obesity, and type 2 diabetes (T2D).
For the past two decades, the interest of researchers, the food industry, and
consumers has focused on those foods that, in addition to containing essential
nutrients, have bioactive components that promote health and/or prevent chronic
diseases. Here, we can find cereals that have been cultivated worldwide from ancient
times [1, 2]. In the case of cereals, in addition to playing an important nutritional
function, they are responsible for multiple beneficial effects for health [3]. This has
led to propose health claims for the consumption of whole grains in USA (1999) and,
as of 2002, in England and Sweden [4]. The rich composition of whole grains or their
fractions, along with their high dietary fiber content, have motivated many nutri-
tional interventions that have focused on highlighting their potential for healthier,
more nutritious foods [5, 6]. Thus, recent studies support the relationship between
regular intakes of whole grains and the lower risk of diseases such as T2D, obesity,
cardiovascular disorders, metabolic syndrome, and cancer [7–9].
The protein fraction of cereals has received much attention from the clinical
community because of its close involvement in both the development of immuno-
logical processes and intestinal disorders. With the increasing development of
immune function measuring systems and better understanding of the potential role
of cereals in immunometabolic (i.e., obesity, T2D, nonalcoholic fatty liver disease)
29 Gluten-Free Cereals and Pseudocereals: Nutrition and Health 849
[10–12] diseases, people became aware and more selective about food supply. Here,
gluten-free cereals and pseudocereals emerged as advantageous alternatives to wheat
in innovative healthier and nutritive goods [6, 13–15].
A healthy diet helps protect us from malnutrition as well as immune-metabolic
disorders. Cereals are recognized as an essential food within a healthy diet and are
recommended for frequent consumption. However, despite the recognized health
benefits of cereal consumption, cereals are often considered as grains that provide a
great deal of nutrients, forgetting to mention specific health benefits.
This chapter summarizes recent research on pseudocereals providing comprehen-
sive information to better estimate their potential contribution to human health.
Beyond its economic, nutritional, and environmental importance, cereals have
played a key role in the history of mankind. Animal and human trials support the
beneficial effects derived from whole grains consumption, mostly referring to wheat
and its derivatives, rice, and maize. Otherwise, pseudocereals belong to a group of
nongrasses, which can be consumed as seeds or flours; even kernels can be used to
produce diverse food products. The consumption of these grains has experienced a
significant increase in recent years worldwide. Thus, general features of those
gluten-free cereals and their specific characteristics in comparison to most common
cereals are presented.
2 Gluten-Free Cereals and Pseudocereals
According to recent statistics, world cereal trade in 2017–18 has been lifted by
8 million tons to a record 403 million tons, implying an 8.7 million ton (2.2%)
expansion from 2016–17 [16]. The world’s most important cereals in the diet are
corn, wheat, and rice. Barley, oats, and rye are cereals whose consumption is not so
widespread [17]. Other cereals such as sorghum and millet are mainly used for
animal consumption, intended for human consumption in some regions [18]. Nota-
bly, pseudocereals (amaranth, quinoa, and buckwheat) receive increasing attention
and are consumed with great acceptance due to their advantageous nutritional
profile [3].
2.1 Food Uses
Good nutrition is our source of energy to live and be active, as well as the first
defense against disease. Here, vegetal kingdom satisfies a considerable part of the
nutritional needs for the human being; in this context, cereals have been part of the
diet of most cultures and civilizations for millennia. Cereals constitute an agronom-
ically important crop from the environmental point of view and an economic source
of protein, both in human and animal feed. Pseudocereals show out to substitute
wheat in different respects, but mainly due to their high protein concentration,
minerals, vitamins, and fatty acids [3]. These grains have been part of human food
for centuries, although as they have been neglected by food industries and nutrition,
Table 1 Several types of products prepared with most commonly used gluten-free cereals [15, 21, 24]
Gluten-
free
cereal Amaranth Quinoa Buckwheat
Grain Flour Whole Flour Grain Flour Kernels
Product Toasted, Bread, rolls, Broths, Porridge, Unpeeled, Biscuits, Tea
popped, cakes, muffins, soups, coarse semolina, cookies,
extruded, pancakes, stews, bread, omelets bread,
milk cookies, rice-like pasta, cakes
noodles products cookies
current knowledge is still limited. The advantageous nutritional features of these

grains make them good candidates as part of strategies, both conventional and
modern breeding approaches, in order to provide a public health benefit with the
ultimate goal of overcoming deficiencies worldwide [6, 19]. Currently, it is assumed
the high nutritional value of pseudocereals; however, the study to what extent these
grains impact nutritional status resulted inferential.
Gluten-free cereals are incorporated into wheat-based products up to a certain
proportion to improve the nutritional value of the resulting product [6, 20]; however,
it still constitutes a great technological challenge. Traditional processing methods for
these cereals include hand-operated wooden or stone pestle and mortar, which still
are used today in certain regions. Today, processing occurs mainly based on applying
the dry- and wet-milling processes. The latter are decided according to the objective
to obtain flours at a high extent or fractions where there can be found other defined
components. Gluten-free cereals can represent a good source of other ingredients of
technofunctional importance (i.e., kernels, saponins, oils). There are numerous
scientific publications that define the physicochemical and functional properties of
the major components of pseudocereals [21–23].
Gluten-free cereals are being used in a wide variety of foods, devoted to human,
pets, and animal feed. Commonly, they are used in several ways, either as seeds,
whole or plain grain, and flour (Table 1). The transformations that occur in the
distinct components of the flours derived from these cereals during the processing
and cooking of foods improve their nutritional value, favor the reduction of
“antinutritive” components, and, in some cases, the formation of bioactive com-
pounds that can contribute significantly to the reduction of metainflammatory
diseases [15, 25–28].
2.2 General Features of Most Commonly Consumed

Pseudocereals
2.2.1 Quinoa
Chenopodium quinoa is a highly nutritious grain that has been cultivated from
ancient years in South America. The origin of quinoa appears to be in the Andean
mountains. During recent times, there has been an increased interest worldwide for
this grain in the United States, Europe, and Asia. Quinoa has been selected by the
Food and Agriculture Organization of the United Nations (FAO) as one of the crops
destined to significantly contribute to food security in the next century.
Quinoa seeds contain a higher percentage of bran than common cereals that
makes feasible the carriage of higher levels of protein and fats in these [29]. One
of the aspects that could limit the widespread utilization of quinoa is the relatively
high amount of saponins that appear in the outer layer of the seeds and are
considered as “antinutritional” factors. These are also rich in carbohydrates
representing the most part of total fraction of nutrients. There can be found mono-
saccharides such as xylose, arabinose, fructose, and glucose, and disaccharides such
as maltose and mainly sucrose [30]. Starch also represents an important source of
carbohydrates in the grain. Dietary fiber in quinoa exhibits a composition where it
composed mainly of cellulose, hemicellulose, beta-glucans, and lignin. Notably,
quinoa carries a significant higher protein fraction compared to other cereals. Here,
there can be found protease inhibitors affecting gastrointestinal proteolytic enzymes
[25]. The genetic background is identified as a major cause for the variations in
content and quality of these proteins. Additionally, environmental conditions and
cultivation methods also contribute significantly to these variations [31, 32]. Lipid
content in quinoa is composed mainly of unsaturated fatty acids (85%), particularly
linoleic (~52%), oleic acid (~25%), as well as saturated palmitic acid (~10%) [33,
34]. In a global perspective, quinoa presents a favorable profile of polar lipids
(phospholipids) (25.2 g/100 g total lipids) with potential benefits on inflammatory
processes, cancer, cardiovascular diseases, neurological disorders, liver diseases,
and antioxidant carrier [35]. This phospholipid profile is particularly important
since quinoa constitutes a good source of bioactive compounds such as phytosterols,
flavonoids, and tocopherols [36, 37].
2.2.2 Amaranth
There can be found up to 26 Amaranthus species, but the three grain species known
as amaranths include Amaranthus hypochondriacus L., Amaranthus cruentus L.,
and Amaranthus caudatus L. These were domesticated in Central America
(Tehuacan, Mexico) over 4000 years BC.
Amaranth seeds are of high nutritional value, the carbohydrates representing the
major part of the total nutrient fraction in the seed. Starch is the most abundant
component in the seeds accounting for up to 65% to 75%, while dietary fiber
accounts for only 5% [38]. One of the most interesting advantages of amaranth
starch is the production of very small granules (0.75–3 μm) that exhibit extremely
high water-absorption capacity [39] providing unique functional properties to food.
Numerous studies have shown that amaranth starch exerts important physiological
functional properties due to its high digestibility [40]. The nutritional value of
amaranth is closely connected to its high protein content and amino acid profile.
They display a characteristic protein distribution of about 40% albumins, 20%
globulins, 25–30% glutelins, and 2–3% prolamins [41, 42]. Amaranth is considered
an essential candidate in special diets for patient with coeliac disease; however, the
Table 2 Fatty acid profile in gluten-free cereal and pseudocereals

Gluten-free cereal and pseudocerealsa
Fatty acids Rice
(g/100 g) Amaranth Barley Buckwheat Quinoa bran Wheat
Palmitic (16:0) 18.5–23.8 18.5–21.0 18.2–19.5 9.7–11.4 18.6 15.4
Stearic (18:0) 3.2–4 – 2.18 0.6–0.79 1.75 –
Oleic (18:1) 22–33.3 15.2–15.6 36.4–37.1 24.8–25.6 42.4 22.3
Linoleic (18:2) 38.2–44.8 24.0–52.4 34.8–35.5 52.3–52.8 34.8 54.2
Linolenic (18:3) 0.2–1.0 2.6–5.5 1.93 3.9–7.0 1.1 3.5
Other 7.08–11.3 5.4–6.8 3.8–10.6 2.44–6.8 1.23 4.6
a
References: [47, 66–69]
recently identified participation of defined globulins in immunological processes

[10] warrants further investigation. It has been reported that cooking and popping of
seeds decreases the fraction of albumins and globulins [43]. But the immunomod-
ulatory potential of the resulting peptides and other structures has not yet been
clarified. For amaranth, the lipid fraction is mainly localized in the germ and seed
coat. The oil content of amaranth is commonly estimated between 5% and 9% [44,
45], although a study reported values up to 19.3% [46]. These discrepancies could be
caused by differences in the extraction and purification methods employed as well as
by environmental factors such as temperature and latitude that influence the com-
position of the oil of amaranth seed [44]. Amaranth constitutes a good source of
linoleic (C18:2), oleic (C18:1), palmitic (C16:0), and stearic (C18:0) acids (Table 2).
Amaranths are also of high nutraceutical value as they are important sources of
phytochemicals and antioxidants [47–49]. Origin, accession, and location constitute
key aspects determining the content of, for example, tocopherols [47, 48]. Otherwise,
after different abiotic stresses, the expression of amaranthin-like genes results
enhanced. Amaranthin is a homodimeric lectin that was first discovered in the
seeds of Amaranthus caudatus and serves as a model for the family of
amaranthin-like lectins. Molecular modeling studies have unraveled the structure
of amaranthin-like proteins and their carbohydrate-binding sites. Their particular
structure allows to interact with the proliferative region of normal human colonic
epithelium and neoplastic lesions of the colon. However, a causal association of
these lectins to physiological proliferation processes still results inferential.
2.2.3 Buckwheat
Despite the name, buckwheat (Fagopyrum esculentum Moench) is not related to
wheat, but to sorrel, knotweed, and rhubarb. Native to the steppes of Central Asia
and Siberia, it was spread to West being introduced to Turkey and Poland and later
expanded to Central and South Europe. Today, the expansion of buckwheat reached
Great Britain, Canada, and EE.UU.
Considerable pharmacological evidences both in vitro and in vivo support pos-
itive effects of Fagopyrum buckwheats such as antitumor, antioxidant, anti-
inflammatory, hepatoprotective, and antidiabetic [50]. These activities are mostly

associated to their particular nutrient composition. Carbohydrates represent (~70%)
the major proportion of buckwheat’s endosperm, among these the main component
is starch that comprises 55% of the total fraction [51, 52]. The starch present in
buckwheat seeds has a different composition to the rest of cereals, with a higher
concentration of amylose and lower of amylopectin and also a significant fraction
resistant (35%) to digestion considered as a dietary fiber [53]. The buckwheat’s
starch capacity to retain water has been established as higher than that of wheat and
corn starch [54]. Factors associated to low digestibility of buckwheat starch are small
granule size, amylose content, starch-protein interactions, and lipid complexes.
Buckwheat proteins exhibit an amino acid profile more nutritionally favorable than
wheat. Notably, a more balanced concentration of amino acids is found, except for
glutamine and proline [55]. The established higher composition of individual protein
fractions such as albumins and globulins in buckwheat in comparison to wheat [55]
supposes a significant difference in their potential immunomodulatory features.
Meanwhile, globulins from wheat exert a clear inflammatory effect; the beneficial
pharmacological effects described for buckwheat suggest different bioactive effects
derived from buckwheat’s globulins. Nowadays, the very low concentration of
glutelins and prolamins in buckwheat makes it a good crop to be part of gluten-
free diets [42]. In relation to other gluten-free cereals and pseudocereals, buckwheat
provides lower oil content (~2–3 g/100 g) [44]. Otherwise, buckwheat shares with
quinoa and amaranth a lipid profile rich in ϖ-3 (linoleic) and ϖ-6 (linolenic) acids as
well as oleic acid. Notably, fatty acid content in buckwheat is influenced by seeding
time in addition to environmental factors and variety [56]. For buckwheat, neutral
lipids represent 85% of the total content, while polar lipids are found in proportions
up to 15%. Similarly to other grains, buckwheat seeds are a good source of anti-
oxidants such as tocopherols, phenols, and flavonoids, specially quercetin and rutin.
This characteristic favored the interest and use of buckwheat hull by food industries.
In addition, groats have also been used for porridges. Other bioactive compounds
identified in buckwheat seeds are D-chiro-inositol, myo-inositol, and fagopyritols,
which play an essential role in insulin sensitivity lowering glucose blood levels and
blood pressure.
2.3 Health Implications
The nutrient content and profile alone does not describe the quality or physiological
and health implications sufficiently (Fig. 1). Digestibility, available lysine, net
nutrient utilization, and efficiency ratio are some other parameters that help to better
estimate the nutritional value of the gluten-free cereals and pseudocereals. In gen-
eral, the protein quality of pseudocereals is close to that of casein. Significant
differences in starch digestibility for pseudocereals are found [25]. Rice and maize
are low in protein, fiber, and folate that contrast those concentrations in pseudo-
cereals [3, 57].
Fig. 1 Health-related
outcomes associated to
gluten-free cereals intake Intestinal
disorders
Immune Prebiotic
disorders effects on gut
microbiota
Health
implications of
gluten-free
cereals intake
Control of
Cancer
glycaemia
Control of
colesterol
levels
2.3.1 Digestibility
The importance of proteins in gluten-free grains relies on their quality. The amino
acid composition in pseudocereals provides to them a significant advantageous
nutritional profile due to the high content of essential amino acids. Particularly,
methionine, lysine, arginine, tryptophan, and several sulfur-containing amino acids
are found at higher concentrations than in other gluten-free cereals. Because of the
high proportion of lysine in globulins, pseudocereals provide about twofold higher
amount of this amino acid than wheat or maize. Thus, they represent an excellent
tool to fulfill the nutritional requirements for populations at risk such as children
[58, 59]. There can be found several studies in relation to the impact of processing
methods on protein quality. A literature search reveals that pseudocereals proteins
remain stable, preserving their high quality and digestibility, after washing, cooking
[60], and popping [43]. However, a recent study reported a decreased digestibility
of quinoa proteins when using an extraction medium with pH values ranging from
8 to 11 [27]. These conditions could have an important impact during preliminary
treatments to eliminate of saponins from quinoa by using a “washing” step.
2.3.2 Immunity
Pseudocereals, contrary to common grains such as wheat, contain low or no storage
prolamin proteins [61], which are the main storage proteins in cereals and the toxic
proteins in celiac disease (CD). Thus, these grains received increasing attention as
safe and suitable for CD patients as part of the gluten-free diet. Otherwise, high
proportions of pseudocereals proteins are constituted by albumins (40%) and glob-
ulins (20%) [59]. Two main classes of globulins can be differentiated in amaranth:
7S (conamaranthin) and 11S (amaranthin) storage globulins [62]. The 11S globulins
(salt-soluble proteins) are one of the major storage protein fractions in amaranth.
Amaranth 11S globulin has a molecular mass around 398 kDa, and under reduced
conditions, it shows characteristic bands of the acidic (3632 kDa) and basic
(2422 kDa) subunits of 11S proteins. Additionally, a 5955 kDa band corresponds
to the nonprocessed proglobulin with structural characteristics similar to other 11S
globulins [63]. Similarly, in quinoa, the 11S (chenopodin) and the 2S globulin
account for 37% and 35% of the total protein content, respectively [64].
Among the different buckwheat proteins, there has been identified a 13S globulin seed
storage protein with significant allergenic potential (Fagag1, 61.2 kDa, accession number
AF152003, Genbank) usually present as a hexamer linked by disulfide bond [65].
The broad umbrella of heterogeneous conditions identified for reactions to gluten
have revealed the need to establish an international consensus and new nomenclature
and classification for these; including CD, nonceliac gluten sensitivity, and allergy
affecting up to 10% of the general population [70]. Gluten or the epitopes triggering
these disorders are found in all Triticum species; wheat, spelt wheat, einkorn wheat,
emmer, durum wheat as well as rye, barley, triticale, and oat. Nowadays, cereal
grains that are considered safe to CD patients are rice, maize, millet, and sorghum.
However, recent immunological studies have identified several immunogenic com-
ponents of the globulin fraction, α-amylase/trypsin inhibitors [10, 11]. The different
downstream signaling pathways triggered by these globulins engage the chemokine
receptor CXCR3 and either MyD88-dependent or independent signals associated to
the Toll-like receptor (TLR)-4. The potential physiological benefit derived from an
inhibited amylase activity favored the use of these globulins for type 2 diabetes and
obese patients. Notably, these pathologies constitute important risk factors for
nonalcoholic fatty liver disease (NAFLD) that today has become the most common
liver pathology worldwide affecting an estimated 15–30% of most populations.
Thus, 10–20% of subjects with NAFLD develop the severe variant of nonalcoholic
steatohepatitis (NASH), hepatic inflammation, and the development of liver fibrosis
with high liver-related morbidity and mortality, part of which is due to the develop-
ment of hepatocellular carcinoma. However, current evidence for a causal associa-
tion as well as definition of the potential role of globulins from pseudocereals within
these disorders has largely been inferential. Buckwheat leaf and flowers are source of
α and β amylase inhibitor activity [71, 72]; however, extracts from buckwheat inhibit
proinflammatory signals in macrophages (264.7 cells) stimulated with the prototyp-
ical TLR4 agonist; lipopolysaccharide (LPS) from Gram negative bacteria [73]. Die-
tary buckwheat has been associated to prevention of lymphocyte immunosenescence
in aged mice [74]. Taken together these studies, there could be hypothesized the
more preserved prevention of reactive oxygen species derived from TLR4 stimula-
tion. In this sense, inclusion of flours from pseudocereals into bread formulations as
substitute of wheat improved the nutritional iron status [6]. Systemic iron homeo-
stasis is tightly regulated by the liver through synthesis of hepcidin, which associates
to inflammatory stimuli regulated by immunological transcription factors such
as the proliferator-activated receptor-γ coactivator-1α (PGC1α) that participates in
the regulation of metabolic, but also immune activation. Thus, scarce existing
studies suggest innate immune-mediated alleviating effects on iron homeostasis
dysregulation.
This effect is important not only from a nutritional point of view, but also because
nutritional iron is an effective modulator of the TLR4 signaling. For example, it has
to be kept in mind that activation of aryl hydrocarbon receptor mediates suppression
of hypoxia-inducible factor-dependent erythropoietin expression. The flavonoids
content of buckwheat has been indicated to interact with the aryl hydrocarbon
receptor at intestinal level increasing its expression [75], thus also influencing
TLR4-mediated response(s).
2.3.3 Metainflammation
Inflammation constitutes a physiological process necessary to keep functional
appropriate cellular response(s). The innate branch of immune system has a crucial
role in determining proper inflammatory homeostasis. Notably, a low-grade inflam-
mation represents a common feature to many metainflammatory diseases such as
NAFLD and associated comorbidities such as T2D and obesity. The scarce studies
existing show the positive effects reducing inflammatory conditions within the
gut-liver axis [6]. Notably, pseudocereals were used with lower proportions
(5–25%) in relation to wheat flour. It was demonstrated that amaranth albumin
proteins are capable to form disulfide bonds with gluten proteins [22]. The data do
not allow drawing out definitive conclusions about the modulatory effect of proteins
bioactivity. Otherwise, open a new way to further research in order to produce
innovative food formulations from which there could be expected more controlled
physiological effects. The literature search reveals continuous and intense research
efforts in the baking industry to incorporate gluten-free cereals and pseudocereals
[22, 63, 76].
2.3.4 Prebiotic Effects

Currently, the close association between the beneficial effects attributed to prebiotics
and prevention of gastrointestinal disorders is well accepted. Prebiotics are non-
digestible food ingredients that beneficially affect the host by stimulating the growth
and/or activity in specific intestinal bacteria. The latter, through the production of
short chain fatty acids, are able to modulate the intestinal inflammatory environment
[77]. Fermentation of prebiotics exert important metabolic consequences in liver-
related disorders contributes to lipid and cholesterol synthesis in the liver as well as
regulating the expression of different transcription factors.
Pseudocereals provide significantly higher amounts of dietary fiber than wheat
and related cereals [3]. Despite nutritional benefits, pseudocereals have been shown
favorable to the adaptability of yeast and most lactic acid bacteria of technological
interest [78, 79]. Preclinical studies demonstrated that enterobacteria and other
potential commensal harmful bacteria do not benefit from this prebiotic effect
of buckwheat [80]. In addition to metabolic effects, it has been reported the partic-
ipation of defined receptors (i.e., aryl hydrocarbon receptor) in the buckwheat-
mediated controlled of the proliferation of Bacteroides spp. [75]. This association
between the modulatory potential of buckwheat on microbiota composition and
receptors with an important role in cancer progression could open the way to
nutraceutical uses of defined components from pseudocereals.
2.3.5 Control of Glycemia

Typical Western diet is characterized by fast digestion and absorption of carbohy-
drates determining a high postprandial glucose and insulin responses, additionally
influencing the risk of liver and biliary tract cancers, although convincing evidence is
currently lacking [81]. Current data demonstrate higher glycemic values for gluten-
free breads as well as pasta than for their respective counterparts [82, 83]. To date,
scarce data support the positive effects of amaranth oil and seeds at controlling
insulin responses [84]. The effects of buckwheat on glycemic response(s) remain
controversial [85, 86]. Studies on the digestibility of pseudocereals starch predict a
poor glycemic value of those [25, 87]. In line with these studies, there have been
reported delayed fluxes of glucose release that are reflected in delayed AUC curves
of postprandial glucose in rats [88]. These effects were accompanied of an
upregulated hepatic expression of the PPARγ receptor that is a key regulator of
whole body glucose homeostasis and substrate distribution for energy expenditure.
Taking into consideration the influence of other nutrients than starch in the modu-
lation of glycemic values (i.e., protein, fiber and fat), pseudocereals constitute
promising “ingredients” to optimize food for populations at risk [45, 89]. Techno-
logical features of pseudocereals as well as their behavior during household pro-
cesses can also greatly impact its glycemic value.
2.3.6 Control of Cholesterolemia

Information from human trials supporting the cholesterol-lowering activity of
pseudocereals is scarce [90–92]. The main hypotheses to explain the potential
contribution of pseudocereals to the hypocholesterolemic effect are based on their
nutritional profile. Otherwise, distinct experimental approaches have been per-
formed to support the cholesterol-lowering activity of quinoa [92, 93], amaranth
[14, 94], and buckwheat [90, 91]. Some of those have also focused on the impact of
processing treatments (i.e., heat-expanded amaranth) [14] or to what extent can
alterations on lipid homeostasis [94] be controlled through pseudocereals intake.
Overall, the underlying mechanisms to explain the hypocholesterolemic effects of
pseudocereals remain unclear. For example, it has been proposed that buckwheat
cholesterol-lowering activity could be derived from its poor digestibility [95, 96] in
contrast to the commonly assumed high quality of its proteins. An interesting
hypothesis is also the assumption of metabolic effects beyond the changes in lipid
homeostasis derived from its nutritional profile [97]. Probably, at least in part, these
effects could occur through an increased energy expenditure promoting oxidative
metabolism. The existing uncertainties warrant further research in this sense.
3 Conclusion and Future Perspectives
Cereals have been associated to human diet from ancient times. Domestication of
cereals was facilitated by polyploidization events that led to genetic determinants,
which excluded potentially adaptive alleles. At present, an intense and continuous
social development, as well as commercial globalization, motivates the increase in
food production, where constant technological innovations are needed to respond to

the increasing demands of the consumer of high quality food. Thus, important
socioeconomic changes in most countries leading to a substantial change in dietary
habits favor the appearance of metainflammatory diseases (i.e., NAFLD, T2D,
obesity). Here, the strong social demand of gluten-free products increased world-
wide and even gluten-free foods started being consumed by many other groups of
population than those with immunological imbalances such as celiac patients.
Several distinct reasons are considered for this switch on “food choice” and con-
sumer’s behavior; nutritional quality and health benefits beyond this nutrition are
mostly considered. Pseudocereals represent a good option both from a nutritional
and technological point of view to improve the nutritional profile of food, while
reducing the intake of defined food ingredients associated to intestinal and immu-
nological disorders. Three distinct grains are majorly known as pseudocereals;
quinoa (Chenopodium quinoa), amaranth (Amaranthus cruentus), and buckwheat
(Fagopyrum esculentum and Fagopyrum tartaricum). These grains offer good
technofunctional options to use them as substitutes of wheat flour, thus producing
gluten-free foods. Moreover, ascription to the gluten-free diet is commonly associ-
ated to nutritional deficiencies. Here, the inclusion of pseudocereals improve the
nutritional profile and reduces the risk of deleterious effects on the nutritional status.
Pseudocereals consumption has been associated to a wide spectrum of beneficial
effects beyond its mere nutritional value in relation to immune regulation including
prevention of intestinal imbalances in gut microbiota composition and inflammatory
disorders. Many of these effects could derive from their potential to stimulate innate
branch of the immune system because of their particular composition on globulins,
but also to its prebiotic effect and potential capacity of other naturally occurring
biologically active compounds. Although the scarce data existing encourage the use
of pseudocereals as adjuvant in nutritional intervention strategies, a complete
description of the underlying molecular mechanisms and processes implied is still
unknown. This manuscript has been limited to those existing studies where key
aspects such as bioavailability of the different compounds as well as concentrations
used could result close to the effective physiological ones. The retrieval from
literature revision highlights the need to clarify these aspects in order to pave the
way for a full translational and transferable usage of pseudocereals into the clinical
practice and society to improve self-management strategies of diseases.
Acknowledgments JML thanks Spanish MINECO for his “Ramón y Cajal” contract. BF thanks
KNOW Consortium “Healthy Animal – Safe Food,” MS&HE Decision No. 05-1/KNOW2/2015.
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Part V
Food Carotenoids
Natural Food Pigments and Colorants
30
Delia B. Rodriguez-Amaya
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868
2 Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868
2.1 Chemical Structures, Properties, and Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 868
2.2 Stability and Alterations During Processing and Storage of Food . . . . . . . . . . . . . . . . . . . 871
3 Betacyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 875
4 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 878
5 Chlorophylls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
5.1 Structures, Properties, and Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 884
6 Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886
7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
Abstract
Extensive structure elucidation resulted in detailed information about anthocya-
nins, betacyanins, carotenoids, and chlorophylls, the major natural pigments
in plant-derived foods. Modifications of the basic skeleton form a broad diversity
of structures for anthocyanins and carotenoids. The chromophores responsible
for the pleasant colors and the factors affecting them have been delineated.
Identification of sources and determination of the composition in foods have
also been widely pursued. Stability and influencing factors, alterations during
D. B. Rodriguez-Amaya (*)
Faculty of Food Engineering, University of Campinas, Campinas, Sâo Paulo, Brazil
Universidade Federal da Fonteira Sul, Laranjeiras do Sul, Parana, Brazil

868 D. B. Rodriguez-Amaya
processing and storage of foods, and stabilization methods have been studied as
part of the effort to retain the natural color of foods and to substitute artificial food
dyes with natural colorants, this substitution being justified by concern about the
safety of artificial colorants and by the potential health benefits of the natural
colorants. Carotenoids have been the most investigated in terms of health effects,
involving epidemiological, in vitro, animal, and human intervention studies. A
wide range of biological activities have been attributed to anthocyanins, based
mainly on cell culture and animal studies; human clinical studies are lacking.
Investigations of the potential health benefits of betacyanin and chlorophyll are in
their initial stages.
Keywords
Natural pigments · Natural colorants · Anthocyanin · Betacyanin · Carotenoid ·
Chlorophyll · Bioactive compounds · Health benefits
1 Introduction
The natural color of foods of plant origin is due primarily to four groups of pigments:
the green chlorophylls, the yellow-orange-red carotenoids, the red-blue-purple
anthocyanins, and the red betacyanin. A few animal-derived foods are colored by
carotenoids. These pigments are also incorporated into food products by direct
addition or indirectly through animals’ feed.
Although earlier investigations were motivated by the color, recent studies have
been stimulated by potential health effects. These possible health benefits, along
with consumers’ concern about safety, have led to efforts to replace artificial food
dyes with natural colorants. This is not an easy task, however. Natural colorants are
usually less stable, more costly, and not as easily utilized as synthetic dyes, besides
having weaker tinctoral strength, interaction with food components, and limited
range of hues [1, 2].
2 Anthocyanins
2.1 Chemical Structures, Properties, and Occurrence
Anthocyanins are water-soluble pigments belonging to the family of compounds

called flavonoids. They are glycosides or acylglycosides of six commonly found
aglycone anthocyanidins: pelargonidin, cyanidin, delphinidin, peonidin, petunidin,
and malvidin (Fig. 1). Cyanidin is the most commonly occurring anthocyanidin in
nature. Anthocyanidins are flavylium (2-phenylbenzopyrylium) structures with
varying hydroxyl and methoxyl substituents. They consist of two aromatic rings
(rings A and B) linked by a three carbon heterocyclic ring (ring C) that contains
oxygen. Eight conjugated double bonds carrying a positive charge on the
30 Natural Food Pigments and Colorants 869
OH
OH
OH OH
3' OH
2'
1 4' O+ HO O+
8 +
O 2
HO
HO 7 5' OH
1'
3 6'
6 OH OH
5 4 OH
OH OH
OH
Delphinidin Pelargonidin
Cyanidin
OCH 3
OCH 3 OCH 3
OH
OH OH
HO O+
HO O
+ HO O+
OCH 3
OH
OH
OH OH
OH
OH OH
Malvidin
Peonidin Petunidin
Fig. 1 Six anthocyanins commonly found in foods
heterocyclic oxygen constitutes the chromophore responsible for the intense color of
anthocyanins. The color of anthocyanin-rich fruits and vegetables vary from vivid
red as in strawberry, purple as in eggplant, and dark blue as in blueberry.
Anthocyanins are known to vary in the number and position of hydroxyl groups;
the degree of methylation of these hydroxyl groups; the identity and number of sugar
moieties and the positions at which they are attached; and the extent of sugar
acylation and the identity of the acylating agent [3–5]. The most commonly encoun-
tered sugar is D-glucose, but anthocyanidins can also be conjugated to L-rhamnose,
D-xylose, D-galactose, arabinose, and fructose [6], as well as rutinose (6-O-α-L-
rhamnosyl-D-glucose), sophorose (2-O-β-D-glucosyl-D-glucose), gentiobiose (6-O-
β-D-glucosyl-D-glucose), sambubiose (2-O-β-D-xylosyl-D-glucose), xylosylrutinose,
and glycosylrutinose [3, 7]. Sugar is typically attached at C-3 on the C-ring (3-gly-
coside) or at both C-3 and C-5 on the A-ring (3,5-diglycoside). Glycosylation at C-7
on the A-ring and C-30 , C-50 , and C-40 on the B-ring (not at both C-30 and C-40 ) have
also been demonstrated.
The sugar moieties may be acylated with aromatic acids such as p-coumaric,
caffeic, sinapic, ferulic, gallic, and p-hydroxybenzoic acids and/or aliphatic acids
such as malonic, acetic, malic, succinic, or oxalic acids [8, 9]. The acyl substituents
are usually bound to the C-3 sugar, esterified to the 6-OH, or less frequently to the
4-OH of the sugars. Anthocyanins with complicated acylation patterns on different
sugar moieties had also been reported [10, 11].
In aqueous medium such as foods, anthocyanins undergo reversible structural
transformations with pH (Scheme 1), with concomitant change in color [1, 12, 13].
The red favylium cation predominates at pH below 2. At pH 3–6, rapid hydration of
the flavylium cation occurs at C-2 to form the colorless carbinol pseudobase. The
carbinol by ring-opening tautomerization gives rise to the (Z )-chalcone, which can
870
R1 R1 R1 R1
OH OH OH OH
OH OH
O HO
+
O O
O HO O HO
R2 R2 R2 R2
OR3 OR3 OR3 OR3

OH OH OH OH
Quinoidal base Flavylium cation Carbinol pseudobase (Z)-Chalcone

pH 6-7 pH < 2 pH 3-6 Pale yellow or
Colorless colorless
Blue Red
R1 and R2 = H, OH or OCH3, R3 = sugar
Scheme 1 Structural transformations of anthocyanins with pH

D. B. Rodriguez-Amaya
isomerize to the (E)-chalcone. At slightly acidic to neutral conditions, deprotonation

of the flavylium cation generates the blue quinoidal base.
Anthocyanins are found in fruits (especially berries), vegetables, nuts, grains,
roots, and flowers [14–18]. Major sources are purple grapes, cherry, plum, raspberry,
strawberry, blackberry, blueberry, black currant, cranberry, chokeberry, red cabbage,
and red wine [17–20].
The anthocyanin composition of foods is affected by several factors, such as
cultivar/variety [21–29], maturity [23, 24, 28], cultivation practices [27, 30], growing
area or season/climate [28, 31, 32], and storage conditions [33]. Amarowicz et al. [34]
reviewed extensively the influence of postharvest processing and storage on the
contents of flavonoids (including anthocyanins). They concluded that for most of the
subclasses in question, the effect of storage and processing on the polyphenol content is
negligible in comparison with the differences between different varieties of plants.
2.2 Stability and Alterations During Processing and Storage of

Food
Various factors affect anthocyanin color and stability, such as the chemical structure
and concentration of the anthocyanin, temperature, pH, light, oxygen, presence of
enzymes, proteins, metallic ions, other flavonoids and phenolics, ascorbic acid, and
sugar [5, 6, 35–40].
Generally, hydroxylation induces a bathochromic shift and reduces stability,
whereas methylation has the opposite effects [35, 41]. Glycosylation of the antho-
cyanin, especially in C-3, increases stability and solubility in water. The superior
color stability of red raspberry products compared to that of processed strawberry
and blackberry has been attributed to glycosylation with the disaccharide sophorose
[42]. Acylation of sugar moieties also improves stability. Diacylation in the red
radish anthocyanin as compared to the monoacylated anthocyanins in red-fleshed
potato might be responsible for its greater stability [9]. Diacylated anthocyanins
may be stabilized by a sandwich type stacking caused by hydrophobic interactions
between the planar aromatic residues of the acyl groups and the positively charged
pyrylium nucleus, thus diminishing the formation of the pseudobase [43]. In mono-
acylated anthocyanins, only one side of the pyrylium ring can be protected against
the attack of water [44].
Utilization of anthocyanins as food colorants and functional ingredients has been
limited by their low stability and interaction with other compounds in the food
matrix. Grape-skin extract has been used as a colorant for a long time. Research has
been directed toward the search for better sources and enhancement of extraction
efficiency and stability. Examples of possible sources of anthocyanins as colorants
are red radish [9, 45–48], red cabbage [23, 36, 49], black carrots [50, 51], red [48, 52],
and purple sweet potatoes [29, 52–54].
Because anthocyanins with complex patterns of glycosylation and acylation exhibit
remarkable stability to pH changes, heat treatment, and light exposure [55, 56],
attributed to copigmentation, self-association, and metal complexing [10, 44, 57],
stabilization efforts have been directed along this line. In nature, anthocyanins are
protected by these stabilizing mechanisms [43, 58, 59].
Copigmentation can occur through two mechanisms: (a) intramolecular interac-
tion, which is based on the stacking of the hydrophobic acyl moiety covalently
bound to sugar and the flavylium nucleus [56, 60]; and (b) intermolecular interac-
tions in which anthocyanins interact via van der Waals interactions (vertical π-π
stacking) between the planar polarizable nuclei of the anthocyanin with colorless
copigment (e.g., phenolics) [61, 62]. The anthocyanin-copigment complexes adopt a
sandwich-type stacking that stabilizes the favylium cation chromophore and par-
tially protect it from the nucleophilic attack of water, thereby preventing color loss
[10, 58]. This molecular association usually produces an increase in absorbance
(hyperchromic effect) and a shift to longer wavelength of the visible absorption
maximum (bathocromic effect) [43, 65]. A recent review revisited copigmentation,
providing a comprehensive description of the nature of binding (the dispersion and
electrostatic components of π-π stacking, the hydrophobic effect, and possible
hydrogen-bonding between pigment and copigment), and of spectral modifications
occurring in copigmentation complexes [7].
Many factors influence the magnitude of copigmentation, including the structures
and concentrations of the anthocyanin and copigment, their molar ratio, the pH,
temperature, and ionic strength [40, 61, 63, 64]. The effects of these different factors
were demonstrated in commercial anthocyanin extracts [57]. At a given pH, color
stability was mainly dependent on the structures of anthocyanins and of colorless
phenolic compounds. Colorants rich in acylated anthocyanins (purple carrot,
red radish, and red cabbage) exhibited great stability due to intramolecular
copigmentation. Protection of the red chromophore was greater for diacylated
anthocyanins in red radish and red cabbage. For colorants without acylated antho-
cyanins (grape-marc, elderberry, black currant, and chokeberry), intermolecular
copigmentation played a key role; colorants rich in flavonols and with the highest
copigment/pigment ratio showed remarkable stability.
Anthocyanins with ortho-dihydroxy system in the B-ring form complexes with
certain metal cations, such as AL, Fe, Sn, and Cu [65–67]. Glycosides of cyanidin,
delphinidin, and petunidin can form such complexes, but those of malvidin,
pelargonidin, and peonidin cannot.
Ascorbic acid accelerates anthocyanin degradation [68, 69]. Initially thought to
be due to a direct interaction between the two molecules, it is now believed that it is
hydrogen peroxide formed from the oxidation of ascorbic acid that attacks the C-2
position of the anthocyanin, provoking the cleavage of the pyrylium ring and
producing colorless esters and coumarin derivatives [37].
Monomeric anthocyanin combines with bisulfite at the pH of most foods and
beverages to form a colorless sulfonic acid addition adduct. Addition of the sulfonate
adduct was shown to take place at the C4 position [70], interrupting the conjugated
double bond system at the center of the molecule. Polymeric anthocyanins will not
undergo this reaction since the 4-position is unavailable, being covalently linked to
another phenolic compound [71].
Thermal processing reduces the anthocyanin content of foods [e.g., 26, 27, 29, 38,
72–78]. Monoacylated anthocyanins showed higher resistance against heat treatment
(steaming, pressure cooking, microwaving, and frying) than di- and nonacylated
anthocyanins [29]. In black currant, cyanidin-3-rutinoside was the most stable to
heat treatment at 95 C; cyanidin and delphinidin rutinosides were the most stable
during storage for 12 months at 8 C [79]. Cyanidin-3-rutinoside was also best
preserved in processing purees and low-sugar jams prepared from strawberry culti-
vars [27]. Pelargonidin-3-glucoside had the most losses.
Suggested pathways for anthocyanin alterations during processing involve poly-
merization (browning) or cleavage (loss of color) [37]. In alkaline or acid medium,
or in the presence of the enzyme β-glucosidase, anthocyanin is hydrolyzed, releasing
the anthocyanidin, which can be transformed first into the carbinol base and subse-
quently into α-diketone (Scheme 2). The latter can polymerize, forming brown
products, or fragment into an aldehyde and a derivative of hydroxybenzoic acid,
both of which are colorless. On the other hand, anthocyanin can also be converted to
carbinol and later into chalcone, and finally cleaved into a coumarin derivative and a
compound corresponding to the B ring. Coumarin 3,5-diglycoside is a common
product of the degradation of anthocyanins (3,5-diglycosides). The degradation
depends on the anthocyanin present and the temperature.
In heat-treated (over 6 h at 95 C, pH 3.5) elderberry and strawberry pigment
isolates, acylated anthocyanins initially underwent hydrolysis, splitting the acyl-
glycoside moieties from the flavylium backbone, forming anthocyanidin (aglycone)
[80, 81]. Pentoses were more readily split off than hexoses. Opening of the
pyrylium ring then followed. Finally, phenolic acids (4-hydroxybenzoic acid from
pelargonidin and protocatechuic acid from cyanidin) and phloroglucinaldehyde
(from both cyanidin and pelargonidin) were formed as terminal degradation prod-
ucts, residues of the B- and A-ring.
Stabilization methods for anthocyanins include the addition of copigment com-
pounds, such as polymers, phenolic compounds, and metals; exclusion of O2 during
processing and storage; encapsulation techniques [39, 82]. In model beverages, the
addition of polyphenols delayed color fading, the most notable improvement being
observed with green tea extract [83]. In blackberry juice, the addition of sugars,
especially trehalose, improved anthocyanin stability during thermal processing [77].
Encapsulation has been considered of great potential for protecting natural
pigments [84], such as anthocyanins [85]. Microencapsulation/encapsulation of
anthocyanins has been investigated to develop natural colorants with improved
stability, solubility, dispersibility, and bioavailability [51, 86–89]. In black soybean
anthocyanin, a combination of copigmentation and nanoencapsulation can be an
effective technique for improving the color and antioxidant stability of anthocyanin.
Anthocyanins and their interactions have a decisive role in the color intensity and
the stability of wine. Copigmentation appears to account for between 30% and 50%
of the color in young wines [58]. Evolution of the red wine color during aging is a
complex process that is attributed to copigmentation and to the progressive conver-
sion of the original athocyanins into new, more stable pigments [90, 91].
R1
OH
HO O+
R2
O-Glu
OH
Anthocyanin
pH> 7 or
acid or
b-glucosidase Glu H 2O
R1
OH
R1 OH
HO O
OH R2
HO O+
R2 O-Glu
OH
OH
Carbinol-glucoside
OH
Anthocyanidin
O2
R1
OH
OH
H2O HO O
R2
R1
OH O-Glu
OH
HO O OH
R2 Chalcone
OH
OH
Carbinol
R1
pH 3 - 10 HO O O OH
R1
OH
+
O-Glu R2
OH
HO O OH
R2
Coumarin-glucoside
O
R1
OH
HO OH
a-Diketone OH
+ CH2CHO
HOOC R2
OH
Brown
polymers Hydroxybenzoic acid Aldehyde
derivatives
Scheme 2 Structural alterations of anthocyanins during processing and storage of foods based on
[37] and [41]
3 Betacyanins
Betalains are water-soluble nitrogen-containing pigments, consisting of the red to

red-violet betacyanins and yellow-orange betaxanthins. They are immonium conju-
gates of betalamic acid with cyclo-dopa[cyclo-3-(3,4-dihydroxyphenylalanine)] and
amino compounds, respectively. The bathochromic shift of 50–70 nm of betacyanins
compared to betaxanthins is due to the aromatic ring of cyclo-dopa. Glycosylation of
betanidin results in a hypsochromic shift of the resulting betacyanin with glucose
attached at C-6 being less effective than that linked to C-5 [92]. Esterification with
aliphatic acyl moieties had little impact on the maximum absorption of betacyanins
[93, 94].
Khan and Giridhar [95] presented a list of betalain-accumulating plants reported
so far, highlighting pigment occurrence and accumulation pattern, and reviewed
betalain biosynthesis. Stintzing and Carle [41] focused on the technological aspects
and human health effects.
For a long time, beetroot was considered the sole source of betacyanin for use as
food colorant, betanin (betanidin 5-O-β-glucoside) being the most abundant pig-
ment. Recent years have seen an increased search for other sources, such as Ullucus
tuberosus, one of the most widely grown and economically important root crops in
the Andean region of South America [96]; Basella rubra, commonly known
as Malabar spinach, a leafy vegetable that accumulates pigments in its fruits [97];
cactus pear (Opuntia ficus-indica and O. stricta) [98–102]; red-purple pitaya
Hylocereus polyrhizus [93, 94, 99, 100, 103–106]; and Amaranthus species
[92, 107–110].

Food
Betalain stability is increased by high pigment content, high degree of glucosylation,

and acylation, low aw, low pH, antioxidants, chelating agents, low temperature,
darkness, and nitrogen atmosphere [104]. Degrading enzymes (peroxidase, polyphe-
nol oxidase, glucosidase), low degree of glucosylation and acylation, high aw, metal
cations, pH <3 or >7, high temperature, light, O2, and H2O2 lowers stability.
Glycosylation increased the half-life of betanidin and isobetanidin by about 17 times
in O2-saturated solutions, a behavior attributed to the lower oxidation-reduction
potential of betanidin as compared to betanin [111]. Betacyanin stability may also
be heightened by substitution with aromatic acids, the 6-O-substitution being more
effective than 5-O-substitution, due to intramolecular stacking, since the U-shape
folding of the molecule may protect the aldimine bond from hydrolytic attack [112].
Betalains have been reported to be stable over a broad pH range, from 3 to 7; the
pH optimum for betanin is between 4 and 6. Elevated temperature shifted this
optimum towards 6 [113]. In the presence of oxygen, betanin was most stable
between pH 5.5 and 5.8; under anaerobic conditions, lower pH values (4.0–5.0) were
more favorable [114]. Betanin stability decreased linearly with increasing oxygen
concentration [115]. Light and oxygen were observed to have additive effects, the
betanin degradation being 15.6 and 14.6%, individually, rising to 28.6% with the
simultaneous presence of both [116]. Since betanin undergoes water-dependent
hydrolysis, aw is a crucial factor for betanin susceptibility to aldimine bond cleavage
[107–109, 117]. Improved betanin stability was found at lower aw, the most effective
being below 0.63 [118]. In a stability study of encapsulated beetroot pigments, betanin
degradation was greatest at aw = 0.64 [119].Greater betanin stability was explained by
decreasing mobility of the reactants at lower aw and by dilution effects at higher aw.
Metal cations (Fe2+, Fe3+, Sn2+, Al3+, Cr3+, Cu2+) have been shown to accelerate
betanin degradation [120–122]. EDTA can prevent metal-catalyzed betanin degra-
dation by pigment stabilization and complex formation with metal ions [120].
Considerable decomposition may be catalyzed by betalain degrading enzymes.
Several polyphenoloxidases were isolated from red beet [123, 124]. A betalain
oxidase in red beet was reported to catalyze betanin degradation to cyclo-Dopa-5-
O-β-glucoside, betalamic acid, and 2-hydroxy-2-hydro-betalamic acid [125].
Betalain degradation on thermal processing has been reported in many papers [e.
g., 103–105, 113, 115, 121, 126, 127]. The color shift to orange of red beet juice on
thermal treatment was explained by the formation of yellow and orange-red degra-
dation products [127].
Betacyanin undergoes isomerization, deglosylation, hydrolysis, decarboxylation,
and dehydrogenation, with or without concomitant chromatic changes [117].
Betacyanins are generally accompanied by their respective isobetacyanins, the
ratio depending on the food source. However, isomerization can be induced by
both acidic [128–130] and alkaline conditions [128]. It has also been observed
during thermal treatment of red beet juice [127, 131], the betanin/isobetanin ratio
decreasing from 25:1 to 9:1 and 2.5:1 in raw, blanched, and sterilized red beet juice,
respectively [131]. On the other hand, thermal treatment of purple pitaya juice
elevated the betanin/isobetanin ratio [104]. Betanin and isobetanin exhibit the
same chromatic properties.
Under strongly acidic conditions [128], at high temperature [132], or in the
presence of β-glucosidase, the glucose moiety of betanin may be cleaved. This
deglycosylation results in a bathochromic shift of about 4 nm [117] and an increased
susceptibility towards oxidation [41].
At pH above 6 or during thermal processing, betanin undergoes cleavage to
the bright yellow betalamic acid and the colorless cyclo-Dopa-5-O-β-glucoside
[129]. This was found to be more pronounced during heat treatment in purified
solution than in red beet and purple pitaya juice [133]. Acylation with aromatic or
aliphatic acids may protect the aldimine bond from cleavage [112, 133].
Recondensation of betalamic acid and cyclo-Dopa derivatives occurs after short-
term heating [115, 134]. This regeneration is partial [131, 134] and is greater at pH 6
in the absence of oxygen [114]. No recondensation was observed at pH 7. Ascorbic,
isoascorbic, metaphosphoric, and gluconic acids improved the regeneration of red
beet juice pigments after heating [135].
Glu O HO
H H
+ 2 + 2
N HO N COOH
HO COOH
Isobetanin HOOC H
Red 17 17
15 N HOOC 15 N
H COOH COOH
H H
+ Betanidin
H strong Glu Violet-Red
+
H
ou
O
Glu O
H Glu O H C
+ 2 H 2O
N COOH H
+
HO
+
-H 2 O HO N COOH H
N COOH
H HOOC
Betanin H Cyclo-dopa-5-O-glucoside H
17
Red 15 N
HOOC COOH Betalamic Acid
H CO 2 Bright Yellow
CO 2
Glu O
H
+ 2
CO 2 HO
N
COOH
Glu O
Glu O H
+ 2
+ HO N
N 2 COOH
HO 17
15 N COOH
H
H
17
H 15 N 15-Decarboxybetanin
17 HOOC
HOOC 15 N
COOH
Red
H
H
17-Decarboxybetanin
Orange -Red
2-Decarboxybetanin
Red
Scheme 3 Structural alterations of betanin during processing and storage of foods based on [127]
Betanin may be decarboxylated at the C-2, C-15, and/or C-17 positions,

the carboxyl groups differing in their susceptibility toward decarboxylation [133].
Decarboxylation at either C-2 or C-15 do not alter the betanidin chromophore,
thereby maintaining the chromatic characteristics of the original betacyanin.
On the other hand, 17-decarboxy betanin exhibit an orange hue [117].
Monocarboxylated betanin, phyllocactin (malonyl-betanin, and hylocerenin
(30 -hydroxy-30 -methyl-glutaryl-betanin) were shown to be considerably more stable
toward degradation than nondecarboxylated betacyanins [114, 136]. Isobetanin
undergoes decarboxylations similar to those of betanin (not shown in Scheme 3).
Yellow neobetanin (14,15,-dehydrobetanin) was shown to be a natural constituent
of red beet [137, 138] and prickly pear [92, 139]. However, neobetanin formation
upon thermal treatment of red beet juice under aerobic conditions has been con-
firmed [127]. Moreover, dehydrogenation of phyllocactin and hylocerenin on
thermal treatment of purple pitaya juice resulted in the formation of the

corresponding yellow neo-derivatives [103]. Thus, dehydrogenation had been held
responsible for the noticeable color (hypsochromic) shift during heat treatment of red
beet juice and purple pitaya juice [103, 104, 117, 127].
On prolonged thermal treatment, a diversity of betacyanin degradation products
may ensue by multiple decarboxylation or by combined decarboxylation and dehy-
drogenation of both betanin and neobetanin [117]. Mixtures of mono-, di-, and
tridecarboxylated betacyanins, together with their corresponding neobetacyanins,
were identified in purified extracts of red beet and purple pitaya [106, 140]. Main
products were 17-decarboxy-betacyanin, 17-decarboxy-isobetanin, 2-decarboxy-
betanin, 2,17-bidecarboxybetanin, and 2,17-decarboxyisobetanin, and 14,15-
dehydrogenated-neobetanin. Heating of betanin, phyllocactin, and hylocerenin
resulted in decarboxylated neoderivatives together with the corresponding
decarboxylated betacyanins [133]. Thus, neobetanin is also decarboxylated in a
manner similar to that of betanin and isobetanin (not shown in Scheme 3).
Generation of neobetanin, neophyllocactin, and neohylocerenin was observed dur-
ing heat treatment of red beet and purple pitaya juice [103, 127].
4 Carotenoids
The lipophilic carotenoids are generally C40 tetraterpenes/tetraterpenoids formed

from eight C5 isoprenoid units joined head to tail, except at the center where a tail-
to-tail linkage reverses the order. The most distinctive structural feature is a centrally
located, conjugated double bond system. The basic linear and symmetrical skeleton
is modified in many ways, including cyclization, hydrogenation, dehydrogenation,
introduction of oxygen containing groups, migration of the double bonds,
rearrangement, chain shortening or extension, or combinations thereof, resulting in
a wide array of structures.
Carotenoids may be acyclic (e.g., lycopene, ζ-carotene) or may have a six-
membered ring at one (e.g., γ-carotene, δ-carotene) or both ends (e.g., β-carotene,
α-carotene) of the molecule, except capsanthin and capsorubin which have five-
membered rings. Hydrocarbon carotenoids (e.g., β-carotene, lycopene) are known as
carotenes, and the oxygenated derivatives are called xanthophylls. Common oxy-
gen-containing substituents are hydroxyl (as in β-cryptoxanthin), keto (as in cantha-
xanthin), epoxy (as in violaxanthin), and aldehyde (as in β-citraurin) groups.
Plants are able to synthesize carotenoids de novo. Thus, along with the principal
carotenoids, low levels of their biosynthetic precursors and derivatives are found in
plant-derived foods, making the carotenoid composition variable and usually com-
plex [141]. The carotenoids are located in subcellular organelles (plastids), mainly
associated with proteins in the chloroplasts and deposited in crystalline form or as
oily droplets in chromoplasts [142].
Carotenoids are not as widely distributed in foods of animal origin and the
composition is simpler. Unable to carry out carotenogenesis, animals are limited to
absorbing dietary carotenoids, which are accumulated unchanged or slightly altered
to form carotenoids typical of animal species.
Hydroxycarotenoids in ripe fruits are mostly esterified with fatty acids [143–147].
In a few fruits, particularly those that remain green when ripe (e.g., kiwi) [148],
limited or no esterification of carotenols occur. In green leaves [149], carotenols are
unesterified; those of corn [150, 151] are mostly unesterified. Lutein, the principal
carotenoid, occurs free or esterified in one (monoester) or both hydroxyl groups
(diester) in nasturtium [152] and marigold [153] flowers, with the esters pre-
dominating. Esterification occurs progressively during maturation and appears to
be important physiologically. Acylation increases the lipophilic character of the
xanthophylls, facilitating their accumulation in the chromoplasts [148].
Moreover, in red and hot chili peppers, mono- and diester carotenoids showed
greater processing stability than the nonesterified counterparts [154].
Astaxanthin is the main carotenoid of some fish, such as salmon and trout, as well
as crustaceans. It may be found free, esterified in one or both hydroxyl groups with
fatty acids, or as a complex with proteins (carotenoproteins) or lipoproteins
(carotenolipoproteins) [155]. Crustacean astaxanthin is a mixture of the three ester
forms.
Carotenoids in which the carbon skeleton has been shortened by removal of
fragments from one or both ends of the usual C40 structure are called apocarotenoids.
Natural examples are bixin, the major pigment of annatto, and crocetin, the main
coloring component of saffron. As with epoxy carotenoids, apocarotenoids are
initial products of the oxidative degradation of carotenoids. Thus, traces of
these compounds are often found in foods. For example, β-Apo-80 -carotenal,
β-Apo-100 -carotenal, and β-citraurin are common minor carotenoids of citrus fruits.
The conjugated double bond system comprises the light-absorbing chromophore
that confers the carotenoid’s attractive color and is mainly responsible for their
special properties and many functions [141]. As the light yellow ζ-carotene, a
carotenoid should have at least seven conjugated double bonds in order to have
perceptible color, phytoene (three conjugated double bonds) and phytofluene (five
conjugated double bonds) are colorless, whereas lycopene (11 conjugated double
bonds in an acyclic structure) is red. The monocyclic γ-carotene and the bicyclic
β-carotene, although having the same number of conjugated double bonds as
lycopene, are red-orange and yellow-orange, respectively. This is because cycliza-
tion takes the π electrons of the ring double bond out of plane with those of the chain
due to steric hindrance between the ring methyl group at C-5 and the hydrogen at C-8
of the polyene chain. Hydroxyl substituents do not affect the chromophore; thus,
both α-carotene and its dihydroxy derivative lutein are pale yellow. Similarly, the
monohydroxy and dihydroxy derivatives of β-carotene, β-cryptoxanthin and zea-
xanthin, have the same color as β-carotene. Capsanthin with a conjugated double
bond system consisting of nine double bonds in the polyene chain, one double bond
in the β-ring, and the carbonyl group double bond, and capsorubin with its nine
conjugated double bonds in the polyene chain extended by the double bonds of two
carbonyl groups, give the intense color of red pepper. Astaxanthin, which has nine
conjugated double bond in the polyene chain extended by two double bonds in β-
rings and two carbonyl group double bonds, is responsible for the vivid red color of
cooked shrimp, lobster, and crabs. In the raw crustaceans, astaxanthin is complexed
with protein, transforming the color to blue, black, or grey. Heating denatures the
protein and the red color of free astaxanthin ensues. Astaxanthin also gives the
reddish color of salmon and trout flesh.
In nature, carotenoids exist primarily in the all-E configuration. An exception
is bixin, which occurs naturally in the Z-form. Theoretically, each carbon-carbon
double bond in the polyene chain of carotenoids may exhibit E-Z isomerization.
However, some double bonds are prevented from undergoing this isomerization
because the Z-configuration is sterically hindered [156, 157]. The Z-isomers of
β-carotene [158–160] and zeaxanthin [160–163] commonly found in foods are the
9-Z-, 13-Z-, and the 15-Z-isomers. Carbon-carbon double bonds located in the cyclic
part of the carotenoid structure, as the C-5,6 double bond in β-carotene, are also
sterically hindered and are not isomerized. However, this double bond in the acyclic
lycopene is unhindered and 5-Z-lycopene is found in tomato and tomato products,
along with the 9-Z-, 13-Z-, and the 15-Z-isomers [164–167]. Being unsymmetrical,
all-E-α-carotene [158], β-cryptoxanthin [158], and all-E-lutein [160–163, 168] give
rise to 130 -Z- and 90 -Z-isomers in addition to 13-Z-, 9-Z-, and 15-Z-isomers in foods.
The carotenoids most commonly encountered in foods are β-carotene, α-carotene,
β-cryptoxanthin, lycopene, lutein, and zeaxanthin (Fig. 2) [169–174]. Rich sources
of β-carotene are some palm fruits, some squash cultivars, green leafy and non-leafy
vegetables, carrot, orange-fleshed sweet potato, cantaloupe, mango, and apricot.
β-Carotene is sometimes accompanied by α-carotene, as in carrot, some varieties
of squashes, pumpkins, and palm fruits. β-Cryptoxanthin is the main carotenoid of
orange-fleshed fruits, such as papaya, tangerine, orange, loquat, persimmon, and tree
tomato. Lycopene-rich foods are tomato and tomato products, pitanga, pink-fleshed
guava, red-fleshed papaya, and watermelon. The richest sources of lycopene, how-
ever, are the Asian gac (Momordica cochinchinensis) fruit [175, 176] and the
Spanish sarsaparilla [147]. Corn and corn products and some varieties of squash
are the major dietary sources of zeaxanthin/lutein. Leafy and other green vegetables
are the main sources of lutein.
The different factors affecting the carotenoid composition of the fresh produce
have been extensively investigated, such as cultivar/variety, maturity, climate or
season, geographic site of production, agronomic practices, part of the plant utilized,
harvesting, and postharvest handling [141, 148, 173, 174, 177–179].

Food
Carotenoid stability is affected by the nature of the carotenoid (carotene or xantho-

phyll, E- or Z-isomer, esterified or unesterified) and the food (fruit, root, leaf, juice,
puree), disruption (peeled, sliced, shredded) of the food matrix, oxygen, light,
a-Carotene
18' 4'
19 20 3'
16 17 5'
7 9 11 13 15 14' 12' 10' 8'
6 1'
2 6' 2'
1
8 9 10 12 14 15' 13' 11' 9' ' 7' '
17' 16'
3 5 20' 19'
4 18 b-Carotene
HO
b-Cryptoxanthin
OH
HO
Lutein
OH
HO
Zeaxanthin
Lycopene
Fig. 2 Carotenoids commonly found in foods
processing method and condition (especially temperature and duration), and storage
condition and duration, water content/activity, atmosphere, oxidizing enzymes,
antioxidants, metal catalysts and prooxidants, and free radical initiators and inhib-
itors [174, 180, 181, 182, 183, 184, 185].
Considering the individual effects along with the interplay of many influencing
factors, results of the numerous studies on the effects of home and industrial
processing may sometimes appear conflicting. Moreover, in spite of the great
progress in carotenoid analytical capability, existence of some errors in the analysis
and in the calculation of retention or loss cannot be ruled out. Isomerization and
oxidation of carotenoids during analysis and/or during storage of samples may also
be attributed erroneously to the processing and storage of foods.
Carotenoid degradation is known to increase with the destruction of the food
cellular structure, greater surface area or porosity, duration and severity of pro-
cessing conditions, duration and inadequate conditions of storage, permeability of
packaging material to O2, and exposure to light. Processing and storage effects on
food carotenoids had been reviewed [179–187], with tomato, carrot, and pepper as
the most investigated foods.
In paprika and chili powder, mono- and diesters showed greater processing
stability than their nonesterified counterparts [154]. This was probably due to the
more lipophilic nature of the esterified carotenoids, resulting in their better integra-
tion into membrane structures, thereby protecting them from thermal degradation.
Capsanthin, capsorubin, zeaxanthin, and β-cryptoxanthin mono- and diesters
exhibited similar stabilities, whereas the susceptibility to degradation of the non-
esterified carotenoids varied considerably.
A review of earlier studies [180] drew some conclusions, which continue to be
supported by recent studies [174]. Carotenoids differ in their susceptibility to
degradation. Moreover, their stability differs in different foods even when the
same processing and storage conditions are used. The main cause of carotenoid
loss during processing and storage of foods is enzymatic or nonenzymatic oxidation.
Enzymatic oxidation of carotenoids can occur to a greater extent than thermal
decomposition in many foods. Whatever the processing method chosen, retention
of carotenoids decreases with longer processing time, higher processing tempera-
tures, and cutting or maceration of the food. The heat treatment in blanching may
provoke some losses of carotenoids, but the inactivation of oxidative enzymes
will prevent further and greater losses during processing and storage. Because the
carotenoids are more concentrated in the peel than in the pulp, peeling and juicing
result in substantial losses of carotenoids, often surpassing those of heat treatment.
Exclusion of oxygen (through vacuum or hot filling, oxygen-impermeable packag-
ing, or inert atmosphere), protection from light, and storage at low temperatures all
protect carotenoids from decomposition.
Alteration or loss of carotenoids during processing and storage of foods occurs
through physical removal (e.g., peeling), geometric isomerization, and enzymatic or
nonenzymatic oxidation [174, 181].
Isomerization of all-E-carotenoids to the Z-isomers carotenoids is promoted by
acids, heat, and light. The release of organic acids during slicing, pulping, or juicing
of fruits can be sufficient to provoke this isomerization, but it occurs to a greater
extent during cooking or thermal processing, as shown for various carotenoids in
many foods [158, 162, 186, 188–195]. During typical cooking of tomatoes, β-car-
otene and lutein isomerized to a greater extent than δ-carotene, γ-carotene, and
ALL-E-CAROTENOIDS Z-CAROTENOIDS
heat, acid, light
Fragmentation Fragmentation
of polyene of polyene
chain chain
enzyme, O2 heat, light, O2
heat, light, metals,
metals, prooxidant
prooxidant volatile volatile
compounds compounds
ALL-E-EPOXY CAROTENOIDS Z -EPOXY CAROTENOIDS

ALL-E-APOCAROTENOIDS Z -APOCAROTENOIDS
ALL-E-HYDROXY CAROTENOIDS Z -HYDROXY CAROTENOIDS
LOW MASS VOLATILE COMPOUNDS LOW MASS VOLATILE COMPOUNDS
Scheme 4 General scheme for the oxidative degradation of carotenoids reproduced from [174]
lycopene [196]. Individual carotenoids and their isomeric forms also behaved
differently during production and storage of canned tomato juice [197]. Notably,
tetra-Z-lycopene was drastically reduced and isomerized to other geometric forms,
including all-E-lycopene.
The technological, analytical, and nutritional implications of the occurrence
of carotenoid Z-isomers in food were reviewed by Schieber and Carle [198].
Pronounced E-Z isomerization leads to a decrease in color intensity and the ability
to quench singlet oxygen [199, 200]. It also results in loss of provitamin A activity
and alteration of bioavailability and metabolism.
Enzyme-catalyzed oxidation takes place prior to heat treatment, during peeling,
slicing, and pulping. It can also occur in minimally processed food and in
unblanched frozen food during thawing [141, 174].
Nonenzymatic oxidation (also called autoxidation) is accompanied by isomeri-
zation, and both the Z- and E-isomers are oxidized [201, 202]. Oxidation initially
involves epoxidation, cleavage to apocarotenals, and hydroxylation [201, 203, 204].
Subsequent fragmentations result in a series of compounds of low molecular masses,
similar to those produced in fatty acid oxidation. Cleavages at different sites of the
polyene chain can also directly produce short volatile fragments [174]. An overall
carotenoid degradation scheme is shown in Scheme 4.
Epoxidation of β-carotene commences with oxygen attack in the terminal double
bond of the conjugated double bond system on one side of the molecule, and then on
the other side, forming β-carotene-5,6-epoxide and β-carotene-5,6,50 ,60 -diepoxide,
respectively. Rearrangement of the 5,6- to the 5,8-epoxide yields β-carotene-
5,8-epoxide and β-carotene-5,8,50 ,80 -diepoxide [201, 203–207].
Epoxidation of lycopene occurs both at the terminal conjugated double bonds and
at the isolated double bonds with the formation of lycopene-1,2-epoxide and lyco-
pene-1,2,10 , 20 -diepoxide, along with lycopene-5,6-epoxide and lycopene-
5,6,50 ,60 -diepoxide [202, 208, 209]. Combinations of these epoxides and cyclization
result in a greater number of products and a more complicated epoxidation scheme.
Introduction of the 5,6-epoxide moiety and transformation of this group to the
5,8-furanoid is a common reaction in foods. A good example is the conversion of
violaxanthin to auroxanthin, as observed in bottled mango juice [210], mango juice
and canned mango slices [211], and orange xanthophylls [212–214].
Cleavage of β-carotene results in β-apo-carotenals (β-apo-15-carotenal, β-apo-
140 -carotenal, β-apo-120 -carotenal, β-apo-100 -carotenal, and β-apo-80 -carotenal). A
similar scheme takes place with lycopene, forming apo-15-lycopenal, apo-140 -
lycopenal, apo-120 -lycopenal, apo-100 -lycopenal, apo-80 -lycopenal, and apo-60 -
lycopenal [202, 215].
Both Z- and epoxy-β-carotenes were detected in corn oil containing β-carotene,
oxidized in the Rancimat at 110 C from 1 to 14 h [216]. Likewise, some Z- and
epoxycarotenoids were found in cashew apple juice heated at 60 C and 90 C [217].
Volatile compounds are generated from carotenoids by direct cleavage of the
carotenoid polyene chain, sequential cleavage, and transformation of the initial
volatile [174]. These are mostly aldehydes, ketones, alcohols, hydrocarbons, furan,
and pyran [218–221]. Now devoid of color, they contribute to the desirable flavor of
foods and beverages, as in wine and tea, but also to off-flavor, as in dehydrated
carrot.
A prominent change during ripening of fruits is the enhanced biosynthesis of the
vividly colored carotenoids and their oxidative cleavage to volatile compounds that
contribute to the typical aroma/flavor of ripe fruits. There is similarity between the
volatile compounds resulting from autoxidation and the enzymatic oxidation of
carotenoids during ripening [174].
The utilization of carotenoids as colorant additives and functional ingredients in
foods and beverages can be problematic because of their insolubility in water,
instability, and low bioavailability. The first two problems have been addressed by
the formulation of water-dispersable market products, as colloidal suspensions,
emulsions, or dispersions in suitable colloids. In recent years, attention has centered
on encapsulation and nanoencapsulation [174].
5 Chlorophylls
5.1 Structures, Properties, and Occurrence
Chlorophyll a and b are the typical green pigments of higher plants, occurring in an
approximate ratio of 3:1 in lettuce leaves [222] and in five commonly consumed
Mediterranean leafy vegetables [223], but with a ratio of 1:2 to 1:4 in different
varieties of the tree tomato fruit [224]. Chlorophylls are porphyrins, which are
macrocyclic tetrapyrrole pigments in which the pyrrole rings are joined by methyne
bridges and the double bond system forms a closed, conjugated loop [37].
A centrally located magnesium atom is coordinated with the nitrogen of the four
pyrroles. Chlorophyll a has a methyl group and chlorophyll b has a formyl group at
C-3. Both have a vinyl and an ethyl group at the C-2 and C-4 position, respectively; a
carbomethoxy group at the C-10 position of an isocyclic ring; and a phytol group
esterified to propionate at the C-7 position. Phytol is a 20-carbon monounsaturated
isoprenoid alcohol. Chloropyll a appears blue-green and chlorophyll b yellow-green
[225]. The chlorophyll molecule has a hydrophilic part, the macrocycle, and a
hydrophobic segment, the phytol. The closed circuit of conjugated double bonds is
the chromophore that allows them to absorb light.

Food
The green color of vegetables and fruits, due to the presence of chlorophyll, is
affected by aging, enzymes, weak acids, oxygen, heat, and light. Degradation of
chlorophyll occurs during ripening of fruits, senescence of green vegetables, and
thermal processing of foods [226]. Epimerization is the first alteration observed
when chlorphyll is exposed to heat [37, 227, 228]. Mild heating is sufficient to
induce the inversion of the C-10 carbomethoxy group located in the isocyclic ring,
forming isomers designated as chlorophylls a0 and b0 .
The chlorophyll degradation route is shown in Scheme 5. It has long been known
that the olive brown color of cooked and canned vegetables is due to the formation of
pheophytin, the most common alteration of chlorophyll reported in thermally treated
green vegetables, such as spinach [229], broccoli [230], pepper [194], squash, green
beans, peas, leek, broccoli, and spinach [231]. Industrial processing resulted in
55–75% loss of chlorophyll a and 50–89% of chlorophyll b, and an increase in
pheophytin a and b in both broccoli florets and stems [230]. Disrupting and breaking
the cell walls, heat induces the release of acids, decreasing the pH. Under acidic
condition, the magnesium atom is replaced by hydrogen to form pheophytin.
Mg2+ CO2CH3
Chlorophyll Pheophytin Pyropheophytin
acid/heat heat
Enzyme
Phytol
Mg2+ CO2CH3
Chlorophyllide Pheophorbide Pyropheophorbide
acid/heat
Scheme 5 Alterations of chlorophyll during processing and storage of foods

The green chlorophyllide is formed by the removal of phytol, catalyzed by the

naturally occurring enzyme chlorophyllase. Prolonged heating causes elimination
of the carbomethoxy group at C-10, giving rise to pyropheophytins, as observed in
heat processed green vegetables [232].
Chlorophyll b was reported to be thermally more stable than chlorophyll a
[37, 233, 234], the higher thermal stability of the former being attributed to the
electron-withdrawing effect of its C-3 formyl group [235]. However, in a study on
the effect of microwave and conventional cooking methods on chlorophyll pigments
of six green vegetables, chlorophyll a was found more heat resistant compared with
chlorophyll b, except in peas [231].
A similar early stage degradation occurs in degreening senescent leaves, consisting
of the transformation of chlorophyll to chlorophyllide, pheophorbide, and pyropheo-
phorbide, catalyzed respectively by chlorophyllase, Mg-dechelatase, and pheo-
phorbide a oxygenase [226, 236, 237]. Pheophorbide a undergoes oxygenolytic
opening of the porphyrin macrocycle, catalyzed by pheophorbide a oxygenase;
subsequent reactions produce flourescent chlorophyll catabolites. The latter undergo
catabolism to nonflourescent chlorophyll catabolites. This general catabolic pathway
in senescent leaves was found to be also active in olive fruits during maturation [238].
Efforts to retain the green color in processed foods include: acid neutralization,
high temperature short-time processing, enzymatic conversion of chlorophyll to
chlorophyllide, and commercial application of metallo complex [37]. Pheophytin
and pyropheophytin will complex with copper and zinc ions to form complexes with
more attractive green color and are more stable to light. While pheophytin and
pyropheophytin were found in conventionally processed green beans, in Veri-
green canned beans, the green color was maintained due to the formation of the
more stable zinc complexes, Zn-pheophytin a and Zn-pyropheophytin b [239]. The
Veri-green process is a patented procedure in which blanching of green vegetables is
undertaken in the presence of zinc salts [240].
6 Health Benefits
Of the natural pigments, the most studied in terms of human health are the caroten-
oids. Their best established function is the provitamin A activity, but they have been
credited with other health-promoting effects, such as immunoenhancement and
reduction of the risk of developing chronic degenerative diseases, such as cancer,
cardiovascular diseases, cataract, and macular degeneration [241–245].
Carotenoids’ action against diseases has been widely attributed to their antioxi-
dant activity [246–248]. However, nonantioxidant mechanisms have been increas-
ingly reported for these bioactive compounds such as retinoid-dependent signaling,
modulation of carcinogen metabolism, regulation of cell growth, inhibition of cell
proliferation, enhancement of cell differentiation, stimulation of intercellular gap
junction communication, gene regulation, modulation of DNA repair mechanisms,
induction of detoxifying enzymes, hormonal and immune system modulation, and
filtering of blue light [241, 242, 244, 249, 250].
Lycopene’s possible role in human health has drawn considerable attention

[251–256], especially in relation to prostate cancer [257–260]. Lycopene has also
been associated with reduced risk for lung [261], pancreatic [262], colorectal [263],
and digestive tract cancer [264].
Lutein and zeaxanthin selectively accumulate in the macula of the human retina
[265, 266], and most epidemiological studies have shown that dietary intake or
plasma or retina levels of lutein and/or zeaxanthin are associated with reduced risk of
age-related macular degeneration, the major cause of irreversible blindness in the
elderly [e.g., 265, 267–272]. These two carotenoids have also been consistently
linked to the reduction of the risk for cataract [267, 270, 273, 274]. A meta-analysis
of six studies showed, however, that dietary lutein and zeaxanthin were significantly
related with reduced risk of late macular degeneration, but not with decreased risk of
early macular degeneration [275]. Supplementation with lutein could improve visual
function in patients suffering from macular degeneration and cataract [276, 277].
For the protective role against macular degeneration and cataract, lutein and zeaxan-
thin may act in two ways: (1) as filters of damaging blue light, and (2) as antioxidants
quenching excited triplet-state sensitizers or singlet oxygen and scavenging harmful
reactive oxygen species like lipid peroxides or the superoxide radical anion [278].
Dietary intake or serum levels of lutein and/or zeaxanthin have also been
inversely associated with cardiovascular diseases [279–281]. Based on their survey
of coronary mortality in 16 countries, Connor et al. [282] concluded that a diet low in
foods containing folate and lutein/zeaxanthin might be the major contributing factor
to increased coronary risk observed in the countries of Central and Eastern Europe.
As enumerated in review articles, a wide range of biological activities have been
attributed to anthocyanins, such as antioxidant, antiallergic, anti-inflammatory, anti-
viral, antiproliferative, antimicrobial, antimutagenic, antitumor activities; microcir-
culation improvement; and peripheral capillary fragility prevention [4, 13, 20, 283,
284]. Anthocyanins are associated with low prevalence or alleviation of some
diseases/disorders such as cancer, cardiovascular diseases, diabetes, obesity, and
cognitive decline.
Antidiabetic properties include lowering of blood glucose levels by protecting
β-cells, improving insulin resistance, increasing insulin secretion, improving liver
function, inhibiting carbohydrate hydrolyzing enzymes, antioxidant capacity, and
regulation of adipocyte function [283, 285].
Anticancer activity has been attributed to the additive effect of multiple mecha-
nisms, such as antimutagenic activity, anti-inflammatory activity, antiproliferative
effect, inhibition of DNA damage, inhibition of carcinogen activation, induction of
phase II enzymes for detoxification, cell cycle arrest, inhibition of COX-2 enzymes,
induction of apoptosis and antiangiogensis [13, 283, 286, 287].
Prevention of cardiovascular diseases encompasses increasing serum antioxidant
capacity, strong inhibition of lipid peroxidation, active oxygen radical scavenging,
capillary permeability, vasorelaxation, reduced plasmatic total cholesterol and hepatic
triglyceride levels, inhibition of atherosclerotic plaque progression, improving endo-
thelial dysfunction, inhibiting oxidized LDL formation [288, 289].Neuroprotection
and vision improvement have also been cited for anthocyanins [283, 284].
The numerous reported biological activities and health-promoting effects of

anthocyanins have been based mostly on in vitro cell studies and animal studies.
Properly designed human clinical trials need to be carried out to provide definitive
evidence.
Potential health-promoting activities (antioxidant, anti-inflammatory, anticancer,
antilipidemic, antimicrobial activities) have also been attributed to betalains
[290–292], but investigations are very much at the initial stage. Studies on the
possible health effects of chlorophyll (anticancer, antimutagenic, and anti-
proliferative activities; inhibition of heme-induced cytotoxic and hyperproliferative
effects) are even more limited [293–296].
7 Concluding Remarks
The impressive work that has been accomplished on the chemical and technological
aspects of natural pigments and colorants of foods, particularly anthocyanins,
betacyanins, carotenoids, and chlorophylls, has provided a wealth of information
on a wide range of important topics, including structures, chemical properties,
stability, alterations during processing and storage, and stabilization methods. The
potential health-promoting effects of carotenoids have been investigated for some
time, but because of some inconsistent or inconclusive results, more human clinical
studies are needed. The wide range of biological activities indicated by cell culture
and animal model research for anthocyanins require confirmation by more human
studies. Currently, at an initial stage, investigation of the health benefits of
betacyanin and chlorophyll should be continued.
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Lycopene: Metabolism and Functional
Aspects 31
Soma Srivastava
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 904
2 Biosynthesis of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905
3 Absorption and Metabolism Aspect of Lycopene in the Human Body . . . . . . . . . . . . . . . . . . . . 908
4 Lycopene and Its Role in Detoxification Pathway in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911
5 Mechanism Responsible for Health Benefits of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912
6 Stability and Functional Aspects of Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913
7 The Linkage of Health and Food Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 915
8 Regulatory Aspects of Functional Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917
Abstract
Lycopene is the most abundant carotenoid found in human serum and has been
recognized as the most effective antioxidant among all the carotenoids. Lycopene
has 11 conjugated double bonds in its structure. However, lycopene occurs
naturally in the all trans form in the dietary sources, found in as many as 18
different isometric forms in human serum and prostate cells mostly in cis form.
However, the absolute concentrations of individual carotenoids within specific
lipoprotein classes have not been reported; relative distribution of β-carotene,
α-carotene, and lycopene among the very low-density lipoprotein (VLDL), low-
density lipoprotein (LDL), and high-density lipoprotein (HDL) was similar, with
58–73% in LDL, 17–26% in HDL, and 10–16% in VLDL when separated by
conventional sequential flotation ultracentrifugation and quantified by high-
performance liquid chromatography. Lycopene has also been found helpful in
S. Srivastava (*)
Division of Agricultural Engineering and Renewable Energy,
Central Arid Zone Research Institute, Jodhpur, India

904 S. Srivastava
elimination of xenobiotics through stool or urine. Lycopene alters hormone and

growth factor signalling including IFG-1 (31.5% decrease in serum IFG-1 levels)
which is associated with cell proliferation. However its stability is a critical factor
for its functional aspects. Physical and chemical factors like elevated temperature,
exposure to oxygen and light, metallic ions (e.g., Cu2+ and Fe3+), extreme in pH,
and active surfaces affect its stability.
Keywords
Lycopene · Metabolism · Functional · Antioxidant · Carotenoids
Abbreviations
BV Biological value
CDP-ME Methylerythritol cytidyl diphosphate
CDP-MEP 4-Diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate
CTP Cytidine 50 -triphosphate
DMAPP Dimethylallyl pyrophosphate
DXR/IspC DXP reductoisomerase
HMBPP 4-Hydroxy-3-methyl-butenyl 1-diphosphate
IFG-1 Growth factor-1
IPP Isopentenyl pyrophosphate
IspD CDP-ME synthetase
LOOHs Hydroperoxides
MEcPP 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate
MEP 2C-methyl-D-erythritol 4-phosphate
MVA Mevalonic acid
PUFAs Polyunsaturated fatty acids
VLDL Very low-density lipoprotein
1 Introduction
Fruits and vegetables play a significant role in human nutrition, especially as

sources of vitamins, minerals, and dietary fiber. Tomatoes are important for both,
its large consumption and richness in health-related food components. It is one of
the most versatile vegetable crops, ranking second around the world after potato [1].
Lycopene, the carotenoid pigment responsible for the red color, is the most distinc-
tive compound present in tomatoes and has been recognized as the most effective
antioxidant among the carotenoids. The unique quality about the composition of
tomatoes and tomato products with respect to other fruits and vegetables is their high
content of lycopene, the acyclic carotenoid containing 11 conjugated double bonds.
There is a small amount of lycopene in few other fruits such as watermelon, pink
guava, pink grapefruit, strawberry, and papaya, but tomato products are the major
source of lycopene in human diet. Varietal effect is also prominent, and lycopene
31 Lycopene: Metabolism and Functional Aspects 905
content depends upon the variety, climatic conditions, and harvesting stage of
tomato.
In addition to lycopene, violaxanthin, neoxanthin, lutein, zeaxanthin,
α-cryptoxanthin, β-cryptoxanthin, α-carotene, β-carotene, γ-carotene, neurosporene,
phytoene, phytofluene, cyclolycopene, b-carotene 5, and 6-epoxide are other carot-
enoids commonly cited in tomato and tomato-derived products [2]. Lycopene is the
most abundant carotenoid found in human serum and, therefore, most important
in terms of net antioxidant activity [3]. However, lycopene occurs in all trans form
in dietary sources; it is always present in cis form in human tissues. The reason for
particular interest in lycopene is its pharmacological effect and potential use in
prevention of various metabolic diseases. Therefore, it is now regarded as a
neutraceutical or functional food ingredient.
2 Biosynthesis of Lycopene
Lycopene belongs to the carotenoid family which are the red, yellow, and orange
pigments found in dietary sources principally in fruits and vegetables. It was first
isolated by Millardet in 1876 who called it “solanorubine” [4]. It was again isolated
by Schunck in 1903 in pure form and named as “lycopene” [4]. It has an aliphatic
hydrocarbon chain with 13 double bonds in its structure, and the molecular formula
is C40H56 (Fig. 1). Lycopene has 11 conjugated double bonds in its structure, which
makes possible to assume 2048 geometrical configurations theoretically. However
naturally only 72 cis isomers of lycopene are found and are structurally favorable
ingredients. Scientists are conducting clinical trials to know the exact pharmacoki-
netics of lycopene in the human body.
Carotenoid synthesis in plants occurs by various biosynthetic precursors naturally
found in plants. It is basically de novo synthesis of carotenoids in plants which is
affected by the presence of these biosynthetic precursrs. Maturation or ripening in
fruits and fruit vegetables is generally accompanied by enhanced “carotenogenesis,”
the carotenoids increasing markedly both in number and quantity. Exposure to
sunlight and high temperature enhance carotenoid biosynthesis, so the tropical
foods are generally colored by carotenoids contrary to fruits of colder regions that
are mostly colored due to anthocyanins [5, 6].
Isopentenyl pyrophosphate (IPP), an isoprenoid, is the biological precursor
for all carotenoids including lycopene. Isopentenyl pyrophosphate is synthe-
sized from acetyl-coenzyme A by a well-established pathway via mevalonic
acid (MVA) (Fig. 2). Isomerized form of IPP which is known as dimethylallyl
pyrophosphate (DMAPP) acts as a starter molecule for chain elongation. When
the DMAPP is produced after isomerization of IPP, it again condenses with a
molecule of IPP to form geranyl pyrophosphate (C10) which further condenses
with a molecule of IPP to form farnesyl pyrophosphate (C15) and finally pro-
duces geranylgeranyl pyrophosphate (C20) by similar elongation. The presence
of enzymes which influence the chain elongation is not clear, but the mevalonic
kinase and 5-phosphokinase which are present in the chloroplast fragments are
906 S. Srivastava
Fig. 1 Common lycopene isomers
believed to be the core enzymes which affect this chain elongation process [7].
Phytoene which is the first (C40) compound is believed to be a carotenoid
precursor. Phytoene is stepwise dehydrogenated to phytofluene, ζ-carotene, and
neurosporene (Fig. 3). Cyclization begins at neurosporene level, and it leads to
the formation of various cyclic carotenes including lycopene (Fig. 4).
During postharvest transport or storage, carotenoid biosynthesis may
continue, raising the carotenoid content, provided that the fruit, vegetable,
CoASH CoASH
2CH3COSCoA CH3COCH2COSCoA+CH3COSCoA CH3C(OH)CH2COSCoA
CH2COOH
2NADPH, 2H+
2NADP+
2+
CH3C(OH)CH2CH2O P P Mn2+ CH3C(OH)CH2CH2O P Mn CH3C(OH)CH2CH2OH
CH2COOH CH2COOH CH2COOH

ADP ATP ADP ATP
ATP
ADP CH3 O P Pi CO2 CH3
C CH2CH2O P P CCH2CH2O P P
CH2 C O– CH2
Fig. 2 Conversion of acetyl-CoA into IPP
or root is kept intact, preserving the enzyme system responsible for

“carotenogenesis” [6].
For decades, the MVA pathway was thought to be the only pathway for the
biosynthesis of IPP and DMAPP. However, the 2C-methyl-D-erythritol 4-phosphate
(MEP) pathway was discovered in the 1990s [8–10]. This pathway is initiated with a
thiamin diphosphate-dependent condensation between D-glyceraldehyde 3-phosphate
and pyruvate to produce DXP which is then reductively isomerized to MEP by DXP
reductoisomerase (DXR/IspC). Subsequent coupling between MEP and cytidine
50 -triphosphate (CTP) is catalyzed by CDP-ME synthetase (IspD) and produces
methylerythritol cytidyl diphosphate (CDP-ME). An ATP-dependent enzyme (IspE)
phosphorylates the C2 hydroxyl group of methylerythritol cytidyl diphosphate, and
the resulting 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP) is
cyclized by IspF to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP). IspG
catalyzes the ring opening of the cyclic pyrophosphate and the C3-reductive dehydra-
tion of MEcPP to 4-hydroxy-3-methyl-butenyl 1-diphosphate (HMBPP). The final
step of the MEP pathway is catalyzed by IspH and converts (HMBPP) to both IPP and
DMAPP. Thus, unlike the MVA pathway, IPP/DMAPP isomerase (IDI) is not essential
in many MEP pathway-utilizing organisms [8].
The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway, in charge of the essen-
tial biosynthesis of isoprenoids, represents a promising and selective target for
developing new drugs against tuberculosis. To date, only fosmidomycin, a molecule
that targets the second enzyme of the MEP pathway, has reached clinical trials, but
recent advances elucidating the structure and kinetics of the MEP enzymes are likely
to change this scenario [11, 12].
908 S. Srivastava
Fig. 3 Stepwise dehydrogenation of phytoene to lycopene
3 Absorption and Metabolism Aspect of Lycopene in the

Human Body
As it has been discussed earlier that, however, lycopene occurs naturally in the all
trans form in the dietary sources, it is mostly found in cis form in the human tissues.
This supports the hypothesis that it is readily absorbed in cis form and present in the
same configuration in human tissues. Lycopene was found in as many as 18 different
Fig. 4 Conversion of neurosporene to α-carotene and β-carotene
isometric forms in human serum and prostate cells. According to the study of
Boileau et al. [13] when ferrets were given oral dose of only 9% cis isomers, the
cis-trans isomer ratios showed that cis isomers are more readily taken up by micelles
and making them more readily absorbed. However, the diet was containing only 9%
cis isomers, mucosa (58.8%), lymph (77.4%), and blood (52%), and tissues
contained significantly more cis-lycopene isomers than the stomach (6%) or intes-
tinal content (17.5%). Studies by Boileau et al. [13] also suggested that mucosal cells
contain 41% more cis-lycopene. In-vitro the mucosal cells were having more of cis-
lycopene, in vitro trials were conducted to understand the mechanism of lycopene
isomer uptake by bile acid micelles. However the complete mechanism of preferen-
tial absorption of cis isomers of lycopene is not clearly understood it was hypoth-
esized that the introduction of one or more double bonds into a lycopene molecule
reduces its length and thus fits well into micelles with greater ease. Alternatively, it
has also been suggested that linear all trans isomers may more readily aggregate
within the intestine and form crystals greatly reducing their uptake by micelles [14].
The hypothesis was also supported by the study of [15] when male F344 rats were
fed lycopene-containing diet for 8 weeks; they also achieved similar lycopene
concentration in tissues.
910 S. Srivastava
Intestinal lumen Brush Border uptake Intestinal mucosal cell Mesenteric lymph
Free lycopene
Fatty acids, monoglycerides
Bile salts
Iycopene
Chylomicrons
Micellarized lycopene
isomers Chylomicrons
Fig. 5 Uptake of lycopene through mucosal cells
All the carotenoids appear to be absorbed by duodenal mucosal cells by passive

diffusion, similar to that of cholesterol and triglyceride lipolysis (Fig. 5). Physical
matrix in which they are ingested is the critical and important factor which affects
their bioavailability and their dissolution in bulk lipids. Studies by Hernell et al.
[16] indicated that the ultimate structure produced during lipid digestion is a
discoidal mixed lipid micelle composed largely of bile salts, free fatty acids,
monoglycerides, and phospholipids with a diameter of 80 Å. The carotenoids are
included in micelles according to their structure and micellar lipid composition.
Carotenoids are taken up by the intestinal mucosal cells through the bile acid
micelles. Bile acid micelle formation is influenced by the fat intake, and hence
lycopene or some other carotenoid absorption is increased when it is taken with fat
or a meal containing fat [17].
Lycopene in the mixed lipid micelles is then taken up by the duodenal brush
border enzymes through passive diffusion. Different carotenoids and fat-soluble
vitamins like vitamin E compete with each other during absorption. Canthaxanthin
and lycopene reduced the 0–24-h plasma β-carotene response when given concur-
rently compared to administration of β-carotene alone [18]. Lycopene exits the
mucosal cell in chylomicrons, which are secreted via the mesenteric lymph system
into the blood. The distribution of carotenoids among the various lipoproteins
classes therefore appears to be determined by the physical characteristics of the
individual carotenoid and the lipid composition of the lipoproteins. However, the
absolute concentrations of individual carotenoids within specific lipoprotein clas-
ses have not been reported; relative distribution of β-carotene, α-carotene, and
lycopene among the very low-density lipoprotein (VLDL), low-density lipoprotein
(LDL), and high-density lipoprotein (HDL) was similar, with 58–73% in LDL,
17–26% in HDL, and 10–16% in VLDL when separated by conventional
sequential flotation ultracentrifugation and quantified by high-performance liquid

chromatography [19].
4 Lycopene and Its Role in Detoxification Pathway in

Humans
Cytochrome P-450 enzyme in animals constitutes one of the primary defense

systems against natural toxic chemicals from plants which are the major sources of
dietary toxins. The induction of cytochrome P-450 enzyme prevents acute toxic
effects from foreign chemicals but also results in oxidant by-products that damage
DNA. Cytochrome P-450 enzyme helps in detoxification via two different pathways
called phase I and phase II detoxification pathways. The study showed lycopene
significantly induced phase I enzymes in a dose-dependent manner and doubled
hepatic quinone reductase (QR), a phase II enzyme. Lycopene has also been found
helpful in elimination of xenobiotics through stool or urine. Xenobiotics are phar-
macologically, endocrinologically, and toxicologically active substances that must
be metabolized and degraded to such compounds so that it can be eliminated through
human excreta. Xenobiotics are also metabolized by cytochrome P-450 enzyme
phase I and phase II pathways.
Lipid oxidation is a normal biological process by which we obtain energy from
fat. Deleterious lipid oxidation occurring in the body is called peroxidation of
polyunsaturated fatty acids (PUFAs) and produces hydroperoxides (LOOHs).
Uncontrolled oxidation of lipids in biological membranes is a major contributor
in several diseases such as heart disease, cancer, and neurodegeneration. The
elevated level of LOOHs was observed during instances of cellular injury and
found correlated to the disruption of cellular membranes, inactivation of enzymes,
and damage to DNA and protein molecule. Initiation and propagation of lipid
peroxidation was studied in isolated microsomes of the liver. The reaction was
catalyzed by iron and microsomal NADPH-cytochrome P-450 reductase enzyme.
This enzyme is reported to produce superoxide anion formed by addition of an
extra electron onto the diatomic oxygen molecule. Aust and Svingen [20]
suggested that lipid peroxidation in microsomes of the liver occurs in two stages.
In initiation stage cytochrome P-450 reductase catalyzes reduction of ADP-Fe + 3
which is subsequently reacting with oxygen molecule to form a ADP-perferyl
radical. The perferyl radical is then responsible for initiating lipid peroxidation and
formation of lipid peroxides.
Oxidative DNA damage could be a major risk factor for the development of
tumors, so that dietary antioxidants are able to decrease such damage in vivo which
would be expected to have cancer prevention effects. Hence, antioxidant substances
when present in foods at low concentrations compared with those of oxidizable
substrate markedly reduce, delay, or prevent the oxidation of the substrate [21]. Due
to the safety concerns over synthetic compounds, research has been focused on novel
antioxidant components present in foods.
912 S. Srivastava
5 Mechanism Responsible for Health Benefits of Lycopene
There are five mechanisms that researchers proposed which may account for the
beneficial effects of tomato phytochemicals and their metabolite. Firstly, lycopene is
the strongest antioxidant compared to other commonly consumed carotenoids.
Decreased DNA damage has been reported in white blood cells after 15 days of
supplementation of tomato and tomato juice. It accounts for about 50% of caroten-
oids in human serum. Among the common dietary carotenoids, lycopene has the
highest singlet oxygen quenching capacity in vitro (Table 1). Another outstanding
feature is its high concentration in the testes, adrenal gland, and prostate. Lycopene
uptake varied with individuals, but peak serum concentrations were always reached
between 24 and 48 h. The carotenoid was eliminated from serum with a half-life of
2–3 d. The increase in peak serum concentrations was dose-dependent but not linear
with the dose.
Secondly, lycopene alters the biotransformation of xenobiotics; cooked tomatoes
and lycopene alter hormone and growth factor signaling in prostate cells. This
includes alterations in insulin-like growth factor-1 (IFG-1) activity. IFG-1 stimulates
cellular proliferation and decreases apoptosis, which is a mechanism by which
normal cell death happens. Eating cooked tomatoes was associated with a 31.5%
decrease in serum IFG-1 levels in a case-controlled study of 112 men. Beneficial
alterations of IFG-1 concentrations and its ability to stimulate cell division have also
been found in rats and healthy men. An in vitro study showed lycopene and tomato
polyphenols including quercetin, kaempferol, and rutin, to interfere with IGF-1
signaling, thus preventing the growth factor from stimulating cell proliferation.
In a number of cancer cell line including breast cancer cells and endometrial and
prostate cancer cells, lycopene halted cellular replication in vitro. Lastly, lycopene
and its metabolites may help fight some cancers by increasing connexin 43 levels.
Connexin 43 is a molecule involved in cell-to-cell communication, which is impor-
tant in the regulation of uncontrolled, rapid cell growth. In a metastatic prostate
cancer cell line, lycopene did not increase connexin 43; however, it did in another
prostate cancer cell line, a breast cancer cell line, and oral cancer cells. The inhibition
Table 1 Comparison of Rate constant for quenching of singlet oxygen

antioxidant capacities of Carotenoid Kq 109 (mol1 s1)
carotenoids
Lycopene 31
γ-Carotene 25
β-Carotene 19
α-Carotene 14
Lutein 8
Astaxanthin 24
Bixin 14
Canthaxanthin 21
Zeaxanthin 10
Source: Data from Di Masio et al. [22] and Miller et al. [23]
of connexin 43 in these cell lines was associated with an inhibition of cell growth,
suggesting that upregulation of connexin 43 may be important to the anticancer
action of lycopene. Since a synergistic effect appears to exist between tomato
phytochemicals, recommending the consumption of supplements made from
whole tomatoes and/or the consumption of two to four or more servings per week
of tomato products may reduce the incidence of prostate cancer and health-care costs
in our aging population.
In contrast to other carotenoids, its serum values are not regularly reduced by
smoking or alcohol consumption but by increasing age. Remarkable inverse relation-
ships between lycopene intake or serum values and risk have been observed in
particular for cancers of the prostate, pancreas, and to a certain extent of the stomach.
In some of the studies, lycopene was the only carotenoid associated with risk
reduction. Its role in cancer risk reduction still needs to be clarified. Patients with
HIV infection, inflammatory diseases, and hyperlipidemia with and without lipid-
lowering treatment may have depleted lycopene serum concentrations [24].
6 Stability and Functional Aspects of Lycopene
Stability is a critical aspect of lycopene functionality. As a highly conjugated

polyene, lycopene can undergo two types of changes: isomerization and oxidation.
Physical and chemical factors known to contribute to the degradation of other
carotenoids are also reported to affect in case of lycopene. These include elevated
temperature, exposure to oxygen and light, metallic ions (e.g., Cu2+ and Fe3+),
extreme in pH, and active surfaces [25, 26].
Cis and trans isomers of lycopene have distinct bioactivity and biostability.
In general cis isomers are more soluble in oil and hydrocarbon solvents than
their all trans counterparts. They are less prone to crystallization than trans isomers
due to their kinked structures. They are less intense in color which affect consumer’s
perception of food quality. All trans configurations predominate in fresh tomatoes
and gradually isomerize to cis configurations upon processing and storage.
Isomerization of lycopene has been shown to take place both in food systems and
in model systems. Trans-to-cis isomerization typically occurs during isomerization.
The storage of processed foods favors reversion from cis to trans because the cis
isomers are in a relatively unstable state compared to the trans isomer, which is
relatively a stable ground state. Mostly the 5-cis, 9-cis, and 15-cis isomers are found
in human serum when assessed using nuclear magnetic resonance (NMR) spectros-
copy. With its acyclic structure, a large array of conjugated double bonds and
important hydrophobicity lycopene exhibits a range of unique and distinct biological
properties. Of these properties its antioxidant potential is of particular interest. The
system of conjugated double bonds allows lycopene molecule to quench the singlet
oxygen and also other free radicals. In an in vitro study by Boileau et al. [13], it was
found an effective singlet oxygen quencher. Under appropriate conditions, it has
been found effective as an antioxidant at lower concentrations not only against O2
but also against lipid peroxidation and a highly destructive hydroxyl radical HO.
914 S. Srivastava
The antioxidant properties of lycopene most likely contribute to limit the risk of
some diseases [3].
Various factors such as heat, processing conditions, heat, oxygen, light, dehydra-
tion, and storage conditions affect the lycopene stability and cis-to-trans isomeriza-
tion of lycopene isomers. Time-temperature combination in heat processing of
lycopene is crucial and affects its isomerization and degradation. Besides time-
temperature combination reaction medium, physical matrix and environmental con-
ditions also affect the stability of lycopene. Basically two types of changes occur,
isomerization and degradation, due to the parameters described above. As is has
been discussed in earlier sections that majority of lycopene is present naturally in the
all-trans form in fruits and vegetables. However, the absorption and bioavailability
of the cis-lycopene is better than the trans isomers. During the heat processing, trans
isomers gradually change into the cis form. It has been observed that all trans-
lycopene isomerizes into mono- or poly-cis form due to changes in seven of the
conjugated double bonds initially during the thermal processing followed by degra-
dation of lycopene content. It has been reported by Hakette et al. [27] that degrada-
tion of lycopene started at a temperature as low as 25 C. At this temperature
degradation of lycopene occurs mainly due to oxidation, and isomerization occurs
at a very low rate. Isomerization of lycopene from trans to cis form occurs on a faster
rate at a temperature above 75 C [27, 28]. According to the study of Lee and Chen
[29], no significant change occurs in the lycopene content during the first 12 h of
heating at 50 C. However, at 100 C heating for 9 h, isomerization of lycopene from
all trans to mono-cis form followed by degradation of mono-cis to di-cis and poly-
cis form of lycopene occurs. Thus it can be concluded that the isomerization of
mono-cis-lycopene to di-cis-lycopene is the main phenomenon during heating in
initial phase followed by conversion to poly-cis form and subsequent degradation.
At the temperature above 100 C, lycopene was found highly unstable, and severe
deduction was noted in the lycopene content at higher temperature above 100 C.
No lycopene was detected after 10 min at heating over 100 C.
The exposure of light also induced similar kind of changes in the all trans-
lycopene. According to the study of Lee and Chen [29] and Shi et al. [30, 31], all
trans-lycopene first changes into 9-cis, 13-cis, and 15-cis isomers, and then degra-
dation of lycopene occurs. It has been also found during the study that cis isomers are
less stable than all trans isomers under light exposure. Lee and Chen [29] studied the
stability of lycopene standard at 25 C for 6 days at illumination intensity
(2000–3000 lx). All trans-lycopene was found to decrease with an increase in
illumination time, and total loss of lycopene was measured as 94% after 144 h of
illumination exposure.
Lycopene stabilization was found three times higher in the presence of oxygen
than under inert conditions. Vacuum and N2 packaging of tomato powder were
found highly effective to reduce the oxidation changes of lycopene. According to the
study of Sharma and Le Maguer [32], vacuum pack and dark storage combination
showed the lowest lycopene loss. Auto-oxidation of lycopene is irreversible and
leads to fragmentation of molecule. During the storage cis-trans reversion of lyco-
pene is also reported. During storage cis-trans re-isomerization and auto-oxidation
of lycopene are the main reasons of loss in lycopene content. Auto-oxidation

produce acetone methylheptenone, levulinic aldehyde and glycoxal also which is
responsible for loss in color and typical hay like odor [33].
7 The Linkage of Health and Food Choice
Health is one reason consumers mention when they are asked about factors that
influence their food choices correspondingly; nutrition experts emphasize that the
degree of healthy eating needs to be judged on the level of diet. Whereas no single
food product can be categorized as health promoting or not, this is true that from a
nutrition point of view, information about fat, vitamin, and other nutraceutical
contents of food tend to be categorized, for example, all fruits and vegetables are
rich sources of vitamin C and carotenoids or other pigment contents which are of
health-promoting value. Functional food is regarded somewhere between medicine
and conventional food, but there is no common shared definition of functional food.
Japan has its own legislation of “food for specified health uses,” called FOSHU, in
which functional foods are clearly regarded as food products that are eaten as part of
an ordinary diet. In Europe, the definition suggested by an EU-funded concerted
action project has widely been used. According to this definition, functional foods
are “satisfactorily demonstrated to affect beneficially one or more target functions in
the body, beyond adequate nutritional effects in a way that is relevant to either an
improved state of health and well-being and/or reduction of risk of diseases” [34].
Traditional nutritional education has emphasized the role of a health-promoting
diet. Following nutritional guidelines will result in reduced risk to develop obesity
and other chronic lifestyle-related diseases, such as cardiovascular disease, diabetes,
and cancer. The message on the benefit of a healthy diet may be hard to convey to the
consumer, as the end result is unsure and achieving the possible reward takes several
years or even decades. Functional foods offer a new kind of positive health benefit to
the people. Instead of avoiding certain kind of foods, these food products promise
positive physiological effect on bodily functions or even a reduction on risk level of
diseases through eating a single product. Functional food effects are easier to
understand for the consumer since they contain results that can be instrumentally
measured, such as lowering the level of cholesterol in the blood, decreasing the
blood pressure, or increasing the density of bone mass [35, 36].
The beneficial health effect of functional foods is due to the presence of a myriad
of bioactives that render their effects via a number of mechanisms. The diseases
of concern include coronary heart disease, certain types of cancer, type 2 diabetes,
brain health and mental disorders, immune response, inflammation, obesity, and
arthritis, in association with oxidative stress and metabolic syndrome. The sub-
stances that may influence such diseases often originate from plant sources and
sometimes animal sources and microorganisms. Examples include carotenoids
such as alpha- and beta-carotene; lutein; astaxanthin; lycopene; fucoxanthin; dietary
fiber; beta-glucan; soluble fiber; long-chain omega-3 fatty acids; phenolics such as
phenolic acid; phenylpropanoids; catechins; anthocyanidins; flavones; flavanones;
916 S. Srivastava
proanthocyanidins; lignans; sterols and stanols; pre-, pro-, and syn-biotics; and soy
isoflavones. Some proteins and biologically active peptides also come under the
category of functional foods.
8 Regulatory Aspects of Functional Foods
The success of a functional food depends to a large extent on the regulatory

framework in which it is allowed to be marketed. From country to country, the
method of regulating the labeling and composition of functional foods varies
enormously. The most important issue that must be considered related to functional
foods is “the nature of the ingredient.” What specific health benefit does it actually
confer on the consumer? A whole host of the products in the market profess to be
functional, ranging from vitamin- and mineral-enriched products to the products
containing added fiber ingredients and pre- and probiotics. Is there really a distinc-
tion between vitamin added for fortification and a vitamin added for its perceived
functional qualities?
Health claims of a product describe the relationship of a diet to the specific
disease, and only 11 health claims are approved by the FDA. An example is healthful
diet with adequate folate may reduce a women’s risk of having a child with brain or
spinal cord defect. Health claims traditionally have been approved based on the
concept of “significant scientific agreement” which the FDA has recently defined in
a guidance document as an agreement among qualified scientific experts that a
substance-disease relationship exists based on a sound body of scientific evidence.
The level of scientific evidence must be strong enough that it would unlikely to be
reversed by the further study [37].
Nutritional claims describe the level of a nutrient or dietary substances in a
product using terms such as free, high, and low, or they compare the level of a
nutrient in a food to that of another food, using terms such as more reduced, etc. An
accurate quantitative statement (e.g., 200 mg of sodium) that does not characterize
the nutrient level may be used to describe any amount of a nutrient present [37].
Structural and functional claims are statements of health-promoting or nutritional
benefit allowed on dietary supplement labels. They are not allowed to mention
disease conditions; they must describe the support or maintenance of the normal
functioning of the body. “Cranberry supports the health of urinary tract” is an
example of a model structure or functional claim.
9 Conclusion
Lycopene not only has the pharmacological and nutritional effect in humans and
animals but also has promising health benefit too. However its potential health
benefits have been known since the late 1950s; recently clinical and functional
aspects of lycopene and its metabolic fate have been stimulated their potential use
in the prevention of the chronic diseases. There has been a growing interest in the
role of lycopene in a variety of ailments in humans including some cancers and heart
diseases. Recent studies have shown that consumption of lycopene-rich foods
reduces the risk of such diseases. Lycopene is also a more potent antioxidant than
all other carotenoids found in human serum. However, it has no pro-vitamin A
activity, but it efficiently quenches the singlet oxygen and prevents degeneration of
proteins and DNA. Heat treatment has shown improved bioavailability of lycopene
after cooking, which means it is more easily absorbable by the body in the heat-
processed tomato products. Heat processing affects trans-cis isomerization of
tomato products. The loosely bound lycopene in tomato products is released during
heat processing, while the degree of isomerization is directly correlated with tem-
perature and duration of heat processing. Recent scientific and epidemiological data
has proven the anticancer properties of lycopene. The US National Research Council
of the Academy of Sciences, World Cancer Research Fund International, American
Cancer Research Institute, and WHO have strong documentary proofs and made
similar recommendations for possibility of reducing cancer risks. Considering the
possible health benefits of lycopene and functionality, it has a brighter scope for the
functional food domain and food processing industry.
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Sweet Potato: Bioactive Compounds and
Health Benefits 32
Remya Mohanraj
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920
2 Health Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921
2.1 Cardioprotective Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 923
2.2 Regulation of Blood Sugar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 923
2.3 Immune Booster and Anti-inflammatory Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 923
2.4 Benefits for the Gastrointestinal System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
2.5 Benefits for Respiratory System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
2.6 Cancer Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
3 Bioactive Compounds from Sweet Potato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
3.1 Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
3.2 Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
3.3 4-Ipomeanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931
Abstract
Sweet potato, a delicious root vegetable, possesses high nutritional value. It is
reported to exhibit anticancer, antidiabetic, and anti-inflammatory activities and
to be a natural alternative to estrogen therapy. Sweet potatoes are a rich source
of phytochemical compounds, and plant-derived compounds always have been
an important source of several clinically useful biomolecules. This chapter aims
to focus on the health benefits and phytochemical composition of sweet potato
with special emphasis on 4-ipomeanol. 4-Ipomeanol, produced by infected sweet
potatoes, is a potential anticancer agent. Earlier studies revealed that bioactivation
of 4-ipomeanol to a cytotoxic metabolite occurred particularly in tissues that are
abundant in specific P450 mixed function oxidase enzymes. Based on the above
R. Mohanraj (*)
Houston Community College, Houston, TX, USA

920 R. Mohanraj
rationale, 4-ipomeanol was the first agent to undergo clinical development as an

anticancer agent especially against lung cancer. 4-Ipomeanol as a potential
prodrug for P450-directed gene therapy of liver and brain cancers has also been
investigated. Recent findings suggest that 18F-labelled 4-ipomeanol could be used
in imaging tumors and monitoring enzyme/prodrug interactions.
Keywords
Sweet potato · 4-Ipomeanol · Anti-cancer activity · Phytochemical composition
1 Introduction
Sweet potato (Ipomoea batatas (L.) Lam, Convolvulaceae) (Fig. 1) is a root vege-
table rich in starch and sweet to taste [1, 2]. It ranks as the seventh important staple
crop in the world and in developing countries, it is ranked as the fifth following rice,
wheat, maize, and cassava [3]. The skin may be red, purple, brown, or white in color.
The flesh color ranges from white, yellow, and orange to purple [4].
Sweet potatoes are native to South America. Columbus introduced it to Europe
from where it eventually spread to other parts of the world [5]. From time imme-
morial, this root tuber that possesses an abundance of pharmacologically active
ingredients occupied a significant position in human nutrition and animal feeding.
Several reports point out to the use of sweet potatoes in traditional medicine. The
leaves are used by Akan tribes of Ghana to treat type 2 diabetes [6]; in Brazil, they
are used in the treatment of inflammatory and/or infectious oral diseases [7]. In
regions of Kagawa, Japan, sweet potatoes are used to treat anemia, hypertension, and
diabetes [8]. The stems are used for treatment of prostatitis [9]. In addition to the
above, sweet potatoes are reported to be alterative, aphrodisiac, astringent,
Fig. 1 Purple-skinned sweet

potatoes
32 Sweet Potato: Bioactive Compounds and Health Benefits 921
bactericide, demulcent, fungicide, laxative, and tonic. It is a folk medicine for

asthma, bugbites, burns, catarrh, ciguatera, convalescence, diarrhea, dyslactea,
fever, nausea, renosis, splenosis, stomach distress, tumors, and whitlows [10]. In
Mexico, the leaves are used for the treatment of inflammatory tumors, and their
decoction is used in baths and gargles for tumors of the mouth and throat [11].
Reed [12] described the plant as a tuberous-rooted perennial that is usually grown
as an annual. Slender, prostrate stems form a running vine up to 4 m long and
produce milky juice. Lateral stem branches that arise from the short stem are usually
not branched. Leaves are ovate-cordate and borne on long petioles, palmately
veined, angular or lobed, green or purplish depending on the variety. Fowers white
or pale violet, axillary, funnel-shaped, borne singly or in cymes on short peduncles.
Pods are round with 1–4 flattened, hard-coated, angular seeds per pod [12].
Keeping the above background in mind, this chapter aims at providing an
insight into the health benefits and recent advances in phytochemical composition
of sweet potato tubers with special emphasis on 4-ipomeanol, an anticancer
agent.
2 Health Benefits
Sweet potato is a staple food in many parts of the world and is a drought-tolerant crop
that holds promise in improving food and nutrition security [13]. Sweet potatoes
are especially significant because of their abundant nutraceutical components.
This tuberous root is a rich source of carbohydrates, dietary fiber, vitamin A (as
β-carotene), vitamin B6, vitamin C, manganese, copper, potassium, and iron [14].
Red sweet potatoes have been reported to have 12.98% of glucose and therefore gain
recognition as a good energy source. Besides this, sweet potatoes contain 2.78%
crude fiber as an added advantage for the consumers. In essence, sweet potatoes are
not only a good food diversification but also a food alternative that bestow food
security and promote health [15].
Sweet potatoes offer a near-balanced diet for the human body in that they possess
significant amounts of carbohydrates in comparison with other starchy foods such as
rice, maize, and sorghum porridge [16]. They encompass a broad range of macro-
and micronutrients, generous amount of vitamin C, reasonable amounts of vitamin B
complex (vitamins B1, B2, B5, and B6) and folic acid, as well as an adequate
amount of vitamin E [17, 18].
A colossal advantage is that the sweet potato crop could be made accessible
throughout the year in tropical and subtropical areas where warm conditions abound.
This offers a leverage in case drought poses a challenge for staple crops such as
cereals. The competence of sweet potatoes in being drought tolerant after establish-
ment raises its yield potential higher than that of other staple crops [13].
The US Food and Drug Administration summates the nutritional facts of sweet
potatoes as follows [19]:
922 R. Mohanraj
Serving size (gram weight/ounce weight) 1 medium (130 g/4.6 oz)

Calories 100
Calories from fat 0
Total fat 0
Sodium 70 mg
3% DV
Potassium 440 mg
13% DV
Total carbohydrate 23 g
8% DV
Dietary fiber 4g
16% DV
Sugars 7g
Protein 2g
Vitamin A 120% DV
Vitamin C 30% DV
Calcium 4% DV
Iron 4% DV
Percent Daily Values (% DV) are based on a 2,000 calorie diet
In the class of different varieties of sweet potatoes, orange-fleshed sweet potato is

reported to possess high amounts of β-carotene, a precursor for vitamin A. It has
been recorded that the amount of β-carotene is directly proportional to the intensity
of orange color of the sweet potato flesh [17, 20, 21].
Leighton [21] tested for β-carotene in different South African orange-fleshed
sweet potato varieties and concluded that β-carotene concentration varied with
depth of color.
Alam et al. [22] analyzed the nutritional composition of nine varieties of orange-
fleshed sweet potatoes. They also analyzed the total carotenoids and total polyphenol
content. Each variety showed a significant variation in the nutritional composition.
The quantification of total carotenoids and total polyphenol content was done using
spectrophotometry, and the proximate composition was done using AOAC. The
results obtained indicated that the total polyphenol content varied from 94.63 to
136.05 mg gallic acid equivalent/100 g fresh weight. The obtained results also
indicated that total carotenoids content ranged from 0.38 to 7.24 mg/100 g fresh
weight. Dark orange-colored flesh varieties had a higher content of total carotenoids
as compared to their light-colored counterparts. These findings suggest that the
sweet potato varieties studied are rich in proteins and carbohydrates, low in fat,
and high in polyphenol and carotenoids. Therefore sweet potatoes could serve as an
excellent source of dietary antioxidants which could potentially prevent free radical
damage and also prevent vitamin A malnutrition [22].
Sanoussi et al. [23] assessed the mineral composition of ten selected cultivars of
sweet potatoes (01 cream, 02 white, 03 yellow, and 04 orange flesh-colored) using
standard spectrophotometry, in order to establish a scientific basis for efficient
valorization and sustainable utilization of these crops in Benin. The mineral com-
position of the tubers on dry weight basis ranged from 0.53 to 0.73 mg/100 g for
iron; 0.23 to 0.27 mg/100 g for zinc; 23.04 to 29.97 mg/100 g for calcium; 21.30 to
25.40 mg/100 g for magnesium; 42.00 to 46.33 mg/100 g for phosphorus; 308.67 to
328.67 mg/100 g for potassium; and 29.00 to 34.00 mg/100 g for sodium. The
mineral salt recorded in highest amount in all the samples was potassium, which
could contribute on an average up to 19.78% and 15.83% of the recommended
dietary allowance of children and adults, respectively [23].
2.1 Cardioprotective Effects
Studies from Harvard University School of Public Health reveal that sweet potatoes
are a tremendous source of B6 vitamins, which promote breaking down of homo-
cysteine. It is noteworthy that homocysteine contributes to the hardening of blood
vessels and arteries. As an exceptional source of potassium that lowers blood
pressure and maintains fluid balance, sweet potatoes play a significant role in
improving heart health (American Heart Association) [19].
Phytochemical screening of aqueous tuber extract of Ipomoea batatas conducted
by Shafe et al. [24] showed the presence of tannin, saponin, flavonoid, terpenoid,
alkaloid, anthraquinones, reducing sugars, and cardiac glycosides. The administra-
tion of tuber extracts lead to a decrease in the activities of serum creatine and lactate
dehydrogenase. The results obtained suggest a potential cardioprotective effect of
the aqueous tuber extracts of sweet potato [24].
2.2 Regulation of Blood Sugar
According to sources from North Carolina State University, sweet potatoes help
regulate the levels of blood glucose. Linus Pauling Institute at Oregon State Uni-
versity reported that sweet potatoes are an excellent source of manganese which
helps the body metabolize carbohydrates and thus maintain healthy blood sugar
levels [19].
2.3 Immune Booster and Anti-inflammatory Properties
Mercy Margaret et al. [25] prepared the aqueous extract of I. batatas and evaluated
the in vitro anti-inflammatory activity of the extracts by membrane stabilizing
method. Phytochemical analyses of the extracts revealed the presence of phenols,
flavonoids, tannins, anthraquinones, and reducing sugars. The results indicated that
the anti-inflammatory potential of the extracts could be attributed to the presence of
phenols and flavonoids in the extracts [25]. Vitamins A and E present in sweet
924 R. Mohanraj
potatoes also support a healthy immune system and are powerful disease-fighting
antioxidants [19].
2.4 Benefits for the Gastrointestinal System
The high fiber content of sweet potatoes helps in retaining water. Also, magnesium,
which is present in sweet potatoes, aids in digestion. They are soothing for the
stomach and intestines. B-complex vitamins, vitamin C, beta-carotene, potassium,
and calcium present in sweet potatoes are beneficial in curing stomach ulcers.
Additionally, the roughage in sweet potatoes alleviates constipation and the resultant
acid formation, which diminishes the chance of ulcers [26].
2.5 Benefits for Respiratory System
Sweet potatoes are effective in providing relief from asthma by clearing congestion
of the nose, bronchi, and lungs. This property could be attributed to its typical aroma.
In addition to harboring vitamin C, iron, and other nutrients, sweet potatoes are
capable of warming up the body which help to cure bronchitis [26].
2.6 Cancer Prevention
Many scientific investigations have revealed that eating sweet potatoes have the
potential to decrease the risk of breast, colorectal, gallbladder, and kidney cancer. In
a 10-year study for evaluating the risk factors for kidney cancer death, 47,997 males
and 66,520 females aged 40 years and older were included. Researchers concluded
that eating sweet potatoes and potatoes regularly was associated with a decreased
risk of kidney cancer [27]. The various health benefits of sweet potatoes are
summarized in Fig. 2.
3 Bioactive Compounds from Sweet Potato
Sweet potatoes harbor an immense amount of bioactive compounds which contrib-

ute to the health benefits conferred by them. Earlier research studies have pointed out
that bioactive compounds are different in orange-fleshed sweet potato and white-
fleshed sweet potato. Phytochemical screening conducted by Shekar et al. [28]
revealed high percentage of carbohydrate, reducing sugar, and phenolics in white-
fleshed sweet potato and increased levels of total protein, flavonoids, anthocyanins,
and carotenoids in orange-fleshed sweet potato. In orange-fleshed sweet potato, it
was also observed that the rate of starch and cellulose degradation was lesser during
storage, which pointed out to a strict regulation of gene(s) involved in starch
anti diabetic
sweet potatoes
immune booster
Protects GI and
respiratory systems
cardio protective
anti cancer
Fig. 2 Health Benefits of Sweet Potatoes
degradation. These researchers also conducted comparative proteomics which

displayed a cultivar-dependent expression of proteins along with evolutionarily
conserved proteins [28].
Phenolic compounds and carotenoids have been reported to be present in sweet
potatoes. Phenolic compounds, such as phenolic acids and anthocyanins, are quite
predominant in the purple-flesh variety. In orange fleshed varieties, α-carotene,
β-carotene, and β-5 cryptoxanthin are predominant. Sweet potato is an abundant
source of dietary fiber, minerals, vitamins, beta-carotene, phenolic acids, and antho-
cyanins. These bioactive compounds confer distinctive flesh colors to sweet potatoes
such as cream, yellow, orange, and purple [29].
The tubers of sweet potato are rich in polyphenols such as anthocyanins and
phenolic acids. They also possess vitamins A, B, and C [30]. Sucharitha et al. [30]
reported that the root of Ipomoea batatas contains phytoconstituents like flavonoids,
carbohydrates, and tannins.
Phytochemical components such as alkaloids, saponin, tannins, steroids, antho-
cyanins, flavonoids, and anthraquinones were extracted by Anbuselvi and
Muthumani [14] using different solvents like ethyl acetate, methanol, chloroform,
and acetone.
Park et al. [31] determined the phytochemical diversity, including carotenoids,
flavonoids, anthocyanins, and phenolic acids, in sweet potatoes of varying flesh
colors (white, orange, and purple). They also made an attempt to identify the
hydrophilic primary metabolites from the different varieties. As a result of their
study, they reported that in orange-fleshed varieties, carotenoid content was consid-
erably higher among which β-carotene was the most plentiful. Only purple-fleshed
sweet potatoes showed the presence of anthocyanins. The levels of phenolic acids
and flavonoids were higher in purple-fleshed varieties as compared to the other two
varieties [31].
926 R. Mohanraj
Oluyori et al. [32] conducted phytochemical screening of the peels of sweet

potatoes and reported the presence of tannins/phenolic compounds, terpenoids,
reducing sugar, cardiac glycosides, alkaloids, and lipids.
Rosas-Ramírez and Pereda-Miranda [33] carried out the purification of the
chloroform-soluble resin glycosides from the roots of yellow-skinned sweet pota-
toes. This was done using preparative-scale HPLC, and six oligosaccharides, batatin
VII, and batatinosides VII–IX, with novel structures were collected, along
with previously known resin glycosides pescaprein I and batatinoside IV. Each
structure was characterized using high-field NMR spectroscopy and FAB mass
spectrometry [33].
Studies carried out by Kang et al. [34] indicated that sweet potato extracts inhibited
excessive production of pro-inflammatory mediators such as NO, iNOS, COX-2, and
TNF-α and thereby attenuated neuroinflammatory responses in LPS-activated BV-2
microglia. The results suggested that the anti-neuroinflammatory potential of sweet
potato extracts may be related to its strong antioxidant properties and its regulatory
actions on pro-inflammatory cytokine such as TNF-α. These results suggest the sweet
potato extracts might be developed as a promising candidate for the treatment of
neuroinflammation-mediated neurological disorders [34].
3.1 Anthocyanins
It has been reported that the purple-fleshed sweet potato anthocyanins protects
against acetaminophen induced hepatotoxicity in mice [35].
Cuevas Montilla et al. [36] compared different Japanese purple sweet
potato cultivars and reported a remarkable variation of anthocyanin profile. Ten
major pigments with non-, mono-, or diacylated structures of 3-O-(2-O-
-D-glucopyranosyl- -D-glucopyranoside)-5-O- -D-glucosides of cyanidin and
peonidin were characterized by ESI-MSn and NMR analyses.
Aldi et al. [37] reported that purple sweet potato peel has high levels of antho-
cyanins, which are powerful antioxidants. They conducted a study aimed at deter-
mining immunostimulatory effect of purple sweet potato peel. They observed
parameters like the activity and capacity of peritoneal macrophages, the total number
of leukocytes, and the percentage of leukocytes and spleen weights relative. The
results indicated that the ethanol extract of purple sweet potato peel posses
immunostimulatory effect that increased the activity and capacity of peritoneal
macrophage cells, the total number of leukocytes, and number of neutrophil
segments [37].
3.2 Phenolics
Jung et al. [38] used chromatography to isolate different polyphenolic

compounds possessing potent antioxidant activities, from methanolic and
hydromethanolic extracts of sweet potato tuber flour. The isolated compounds
Fig. 3 Bioactive components

of sweet potatoes Tannins
Anthocyanins Flavonoids
Bioactive
compounds
in sweet
potatoes
Phenolics Carotenoids
were 4-O- caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, and 3,5-di-O-

caffeoylquinic acid [38].
Pochapski et al. [39] carried out phytochemical screening that showed positive
results for triterpenes/steroids, alkaloids, anthraquinones, coumarins, flavonoids,
saponins, tannins, and phenolic acids. Total contents of alkaloids, anthraquinones,
and phenolic compounds in 100 g of the dry sample were reported to be 345.65,
328.44, and 662.02 mg, respectively [39].
Hesam et al. [40] reported that sweet potato contains endogenous amylases along
with the high starch content. The two predominant amylases are α- and β-amylases.
Since sweet potato is a promising source of β-amylase, they focused their investi-
gation on the properties of β-amylase from white-flesh sweet potato grown in Iran as
a potential source for industrial applications [40]. The various bioactive components
of sweet potatoes are given in Fig. 3.
4-Ipomeanol a furanoterpenoid produced from infected sweet potatoes has been
proven to possess anticancer properties. The following is a detailed description of
this wonderful yet less known compound from sweet potatoes.
3.3 4-Ipomeanol
4-Ipomeanol is a stress metabolite produced in response to general damage to sweet

potatoes [41]. Clark et al. observed that organisms like Plenodomus destruens,
Diaporthe batatatis, Diplodia tubericola, Fusarium solani, and Ceratocystis
fimbriata induced accumulation of relatively high concentration of 4-ipomeanol
(5–236 μg/g) in sweet potato [42]. However, it has been recently reported that
4-ipomeanol can also be produced in vivo in the root tubers of I. batatas without
specifically being infected [43, 44].
3.3.1 Physical and Chemical Properties

The various physical and chemical properties of 4-ipomeanol is given in Table 1
[42, 45]. The structure of 4-ipomeanol is shown in Fig. 4 [46].
928 R. Mohanraj
Table 1 Various physical and chemical properties of 4-ipomeanol [42, 45]

S. No. Properties Data
1 Molecular formula C9H12O3
2 Molecular weight 168.18978 g/mol
3 H-bond donor 1
4 H-bond acceptor 3
5 Rotatable bond count 4
6 Topological polar surface area 50.4
7 Description Colorless to yellowish color oil in state
8 Specific optical rotation þ7.86 deg at 25 deg C/D
9 Flash point >150 C
10 Maximum absorption 211 nm
11 Rf value 0.65 [methanol/benzene (1:10)]
12 GC-MS NIST number 131689
Fig. 4 Structure of
4-ipomeanol [46]
3.3.2 In Vitro Production

A procedure for isolation of 4-ipomeanol from infected sweet potatoes was
described by Boyd and coworkers [47]. 4-Ipomeanol was also synthesized chemi-
cally from diethyl 3,4-furandicarboxylate in a five-step process [48]. Krauss et al.
[49, 50] used another approach for the chemical synthesis of 4-ipomeanol in
four steps starting from commercially available furan-3-carbaldehyde. They also
synthesized analogues of 4-ipomeanol from furan-3-carbaldehyde and furan-2-
carbaldehyde [49, 50]. A protocol for in vitro bioproduction of 4-ipomeanol from
the rhizogenic callus of I. batatas was reported for the first time by Remya and Subha
[43, 44].
3.3.3 Covalent Binding Property

The cytochrome P450 system present in animal bronchial Clara cells activates
4-ipomeanol. Localized cytotoxicity is elicited by the resulting metabolites by
their binding to specific macromolecules [51]. Possibility of an influence by differ-
ences in species and strain in the covalent binding of 4-ipomeanol was studied by
Dutcher and Boyd [52]. Investigation of three strains of rat, Hartley guinea pigs,
albino New Zealand rabbits, golden Syrian hamsters, and six strains of mouse
divulged that lungs were the main target for 4-ipomeanol covalent binding and
toxicity. In addition to pulmonary damage, liver and kidney necrosis was reported
in the hamster and the mouse strains. Principal site of damage was found to be the
lung in rats, guinea pig, and rabbits. In all the six mouse strains studied, covalent
binding of 4-ipomeanol was approximately twice as high in kidney as in lung; there
was some lung damage and remarkable renal toxicity [52]. Though birds (Japanese
quail, chickens), devoid of Clara cells in their respiratory tracts, do not exhibit
pulmonary toxicity after 4-ipomeanol, they develop severe hepatic injury [53].
Following [14C] 4-ipomeanol administration to rats, Boyd et al. demonstrated
that radioactivity was concentrated in the lungs, and 90% was covalently bound [54].
Outcome of another study by Boyd showed that electron-dense granules which later
became necrotic were found specifically localized over Clara cells following the
administration of [14C] 4-ipomeanol to rats, mice, and hamsters. On the contrary, it
was observed that the adjacent ciliated bronchiolar cells and other major pulmonary
parenchymal cells were neither radiolabeled nor necrotic. When animals were
pretreated with piperonyl butoxide, an inhibitor of cytochrome P450, there was a
considerable reduction in covalently bound radioactivity, and there was no necrosis
in the Clara cells [55]. Covalently bound 4-ipomeanol was most heavily concen-
trated over the apical cap of Clara cells where the cytochrome P450 enzymes are
concentrated [56, 57]. Studies implied that formation of a reactive metabolite was
essential for covalent binding and that it was localized in lung proteins. It was also
observed that pretreatment with diethylmaleate helped increase covalent binding of
the reactive metabolite of 4-ipomeanol in both the lung and liver and reduced the
LD50 value. The “vital macromolecules” to which the reactive metabolite of
4-ipomeanol is covalently bound are tissue proteins and not nucleic acids. This
was revealed by studies that showed that hot trichloroacetic acid or perchloric acid
dislodged nucleic acids and the insoluble material left behind was mainly protein
[56, 58, 59].
3.3.4 Therapeutic Potential

4-Ipomeanol has been proven to have exhibited both in vitro and in vivo antitumor
activity against non-small cell lung cancer (NSCLC), and therefore was suggested as
an antitumor agent specific to lung cancer [60, 61]. The clinical development
of 4-ipomeanol as a lung cancer-specific agent was based on its organ-specific
toxicity in preclinical studies in mammals [62]. In animals like rabbits, rats, guinea
pigs, female mice, and dogs, preferential activation of 4-ipomeanol occurs in the
lung Clara cells. To a lesser extent the compound is also activated by type II
pneumocytes abundant in cytochrome P450 isoenzymes [55]. It could be observed
that toxicity is predominantly pulmonary, due to the binding of active intermediate to
nucleophilic macromolecules, leading to bronchiolar epithelial necrosis preferen-
tially involving Clara cells [53, 55, 63]. In preclinical toxicity studies, 4-ipomeanol
has produced dose-dependent pulmonary toxicity [63]. The primary sites of 4-
ipomeanol binding in mammals are lungs and proximal renal cortical tubules [51,
64]. In male rats administered with radiolabeled 4-ipomeanol, radioactivity was
highest in lung tissue followed successively by intestines, liver, and kidney [54].
Rowinsky et al. [65] observed cytotoxic effects in the human lung, liver, and
kidney, presumably because all these organs contain the isoforms of cytochrome
P450 that could metabolically activate 4-ipomeanol. These results suggested that
930 R. Mohanraj
clinical evaluation of 4-ipomeanol in humans should be extended to include liver

cancers, and renal cancers, in addition to lung cancers [65]. Phase I study on
4-ipomeanol in patients with non-small cell lung cancer (NSCLC) was performed
by Kasturi et al. [66].
It was noticed that 4-ipomeanol may be preferentially activated in the human liver
rather than the lung, and these results formed the basis for evaluating the clinical
activity and for evaluating the toxicity of 4-ipomeanol in patients with advanced
hepatocellular carcinoma [67].
Studies on the human CYP4B1 isozyme revealed that its metabolic activity
toward 4-ipomeanol is <1% of the rate of rabbit CYP4B1 [68]. Expression of rabbit
CYP4B1 in rat and human glioma cell lines made these cells highly susceptible to
4-ipomeanol [69]. Mohr et al. demonstrated that the CYP4B1/4-ipomeanol prodrug-
activating system is effective in inducing cell death of hepatocellular carcinoma cells
at low 4-ipomeanol concentrations. It was proposed that this system might be useful
for augmentation of standard chemotherapy or gene therapy [70].
Constanze et al. demonstrated that a proline residue at position 427 in human
CYP4B1 is important for 4-ipomeanol bioactivation. They developed a novel human
suicide gene system that could be used for adoptive cellular therapies by modifying
the human CYP4B1 enzyme for efficient activation of 4-ipomeanol [71].
Cytotoxicity induction by 4-ipomeanol (4-IM) in combination with ionizing
radiation in cells transfected with a fusion protein of rabbit cytochrome CYP4B1
under influence of EGR1 (radiation inducible promoter) was investigated by Hsu et
al. [72]. The results indicated that EGR1-CYP4B1/4-IM system is a feasible radia-
tion-gene therapy system that may allow effective control of cytotoxicity by thera-
peutic radiation fields.
Cytotoxic properties of cytochrome P450 4B1 (CYP4B1)-activated 4-ipomeanol
for prodrug-activated gene therapy was reported by Jang et al. [73].
Roellecke et al. recently conducted a study that was aimed at developing a
clinically relevant self-inactivating lentiviral vector for systematic co-expression of
CYP4B1 as an ER-located protein. The study lead to the development of a novel
human suicide gene systems that is based on human CYP4B1 and 4-ipomeanol.
4-Ipomeanol was found to induce apoptosis in primary T cells co-expressing mutant
CYP4B1 and the divergently located MACS selection and chimeric antigen receptor
genes [74].
Recent studies on 4-ipomeanol have revealed its potential to be used as an
imaging agent as well for gene prodrug activation therapy. (18)F-Labeled
4-ipomeanol could be used to image tumors and monitor enzyme-activating anti-
cancer prodrugs [75].
4 Conclusion
Sweet potatoes are rich source of nutrients and bioactive components. They have
been proven to exhibit anticancer, antidiabetic, cardioprotective, antimicrobial,
immune boosting, and hepatoprotective properties. Among the various secondary
metabolites from sweet potatoes, 4-ipomeanol holds promise as a potential antican-

cer agent and offers prospects for further research. Further research in the direction
of utilizing 4-ipomeanol to its maximum potential in the treatment of different types
of cancers through effective bioactivation, enhancement of metabolic stability,
structure modification, inclusion of biomarkers, evaluation of its utility in imaging
of specific types of tumors, and other techniques is essential.
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Anticancer Properties of Lycopene
33
Kazim Sahin, Cemal Orhan, Nurhan Sahin, and Omer Kucuk
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
2 Lycopene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938
3 Molecular Targets/Mechanisms of Action of Lycopene in Cancer Prevention . . . . . . . . . . . . 938
4 Lycopene in Cancer Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
4.1 Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
4.2 Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 944
4.3 Gastric Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
4.4 Liver Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
4.5 Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 951
4.6 Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 951
4.7 Skin Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952
4.8 Head and Neck Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 953
4.9 Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954
4.10 Renal Cell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
4.11 Ovarian Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 958
Abstract
Lycopene is an acyclic isomer of beta-carotene, found in red-colored fruits and
vegetables, including tomatoes, and their processed products, watermelon,
papaya, guava, carrots, red grapefruit, and sweet potatoes. It is synthesized by
plants or autotrophic bacteria but not by animals. This work provides an up-to-date
K. Sahin (*) · C. Orhan · N. Sahin

Department of Animal Nutrition, Faculty of Veterinary Science, Firat University, Elazig, Turkey
e-mail: [email protected]; corhan@firat.edu.tr; nsahin@firat.edu.tr
O. Kucuk
Winship Cancer Institute of Emory University, Atlanta, GA, USA
# Crown 2019 935

936 K. Sahin et al.
overview of mechanisms linking lycopene in the human diet and cancer, consid-
ering epidemiological, clinical studies, and experimental data. Dietary lycopene
supplementation may reduce the risk of cancers of many organs such as prostate
and at the same time retard the growth of tumors. The main protection properties of
lycopene against cancer include antioxidant, anti-inflammatory, anti-inhibitory of
cancer cell proliferation, anti-apoptotic, increased gap-junctional communication,
interferences in insulin-like growth factor 1 receptor signaling pathways, and cell
cycle progression and, the ability to improve the metabolic profile. In this context,
lycopene has been shown to exert a protective effect in humans or animals with
cancers including prostate, breast, gastric, colon, pancreatic, renal, and several
other cancers in many studies, although the obtained results are sometimes
inconsistent, which warrants further studies focusing on its bioactivity. In this
chapter, lycopene supplementation in cancer prevention is reviewed and possible
mechanisms of action are discussed in detail.
Keywords
Cancer · Nutrition · Prevention · Lycopene · Molecular mechanism
Abbreviations
4-NQO 4-Nitroquinoline-1-oxide
5-LOX 5-Lipoxygenase
ABCA1 ATP-binding cassette transporter 1
ACF Aberrant crypt foci
AOM Azoxymethane
ARE Antioxidant response element
BRCA Breast cancer
CAT Catalase
CDK Cyclin-dependent kinases
CI Confidence interval
COX-2 Cyclooxygenase-2
Cx43 Connexin 43
DEN Diethylnitrosamine
DMBA 7,12-Dimethyl-benz[a]anthracene
DMH 1,2-Dimethylhydrazine
ERK1 Extracellular signal-regulated kinase 1
GJC Gap-junctional intercellular communication
GSK-3β Glycogen synthase kinase-3β
HCC Hepatocellular carcinoma
HepG2 Human hepatocellular liver carcinoma cell line
HFD High-fat diet
IGF-1 Insulin-like growth factor
IGFBP-3 Insulin like growth factor binding protein 3
iNOS Inducible nitric oxide synthase
Keap-1 Kelch-like ECH-associated protein 1-
33 Anticancer Properties of Lycopene 937
LXRα Liver X receptor alpha

MCF-7 Human breast adenocarcinoma cell line
MMP-2 Matrix metalloproteinase 2
MNU N-methyl-N-nitrosourea
NADPH Nicotinamide adenine dinucleotide phosphate
NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells
NNK 4-(N-methyl-N-nitrosamino)-1-(3-pyridal)-1-butanone
Nrf2 Nuclear factor-E2-related factor 2
ORs Odds ratios
PCB Polychlorinated biphenyls
PCNA Proliferating cellular nuclear antigen
p-mTOR Phosphorylated mammalian target of rapamycin
PPARγ Peroxisome proliferator-activated receptor gamma
PSA Prostate-specific antigen
RCC Renal cell carcinoma
ROS Reduction oxidative stress
TNF-α Tumor necrosis factor-alpha
1 Introduction
There is a relationship between diet-related factors, especially nutritional factors

in foods and the risk of cancer [1–3]. Consumption of many bioactive substances
naturally found in foods, especially antioxidant compounds such as lycopene, is
related to a lower risk of cancer [4]. Lycopene, a natural pigment synthesized by
plants and microorganisms, is mostly found in tomato products, watermelon,
papaya, guava, and pink grapefruit [5]. Lycopene prevents the growth of tumors
and inhibits tumorigenesis through a variety of mechanisms, such as cell cycle
arrest and/or apoptosis stimulation, modulation of growth factor signaling, modula-
tion of redox status, alterations in cell growth-related enzymes, improvement of
gap junctional communication, and inhibition of inflammation [5]. Moreover, in
studying the relation of lycopene to cancer prevention, we have found that lycopene
improved the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1
expression and reduced NF-κB/cyclooxygenase-2, preventing the inflammatory
cascade and stimulating antioxidant signaling. Lycopene treatment also reduced
carcinogenesis associated rises in the phosphorylated mammalian target of
rapamycin (p-mTOR), phosphorylated p70 ribosomal protein S6 kinase 1 (S6 K1),
phosphorylated 4E-binding protein 1 (p-4E-BP1), and protein kinase B [6]. In this
chapter, we review the latest data from in vitro, animal, and human studies to
evaluate the protective effects of lycopene in cancer prevention and its mechanisms
of action.
938 K. Sahin et al.
Table 1 Dietary source Source μg/g wet weight

of lycopene [7–9]
Raw tomato 8.8–42
Tomato juice 86–100
Tomato sauce 63–131
Tomato ketchup 124
Pink grapefruit 3.6–34
Pink guava 54
Watermelon 23–72
Papaya 20–53
Rosehip puree 7.8
Apricot <0.1
2 Lycopene
Lycopene, found in tomatoes, red fruits, and vegetables, including carrots, water-
melons, apricot, papayas, guava, and strawberries, and discovered in 1999 by
Nguyen and Schwartz, is a tetra-terpene from the carotenoid family [7]. Processed
tomato products such as tomato sauce, tomato paste, and ketchup are more concen-
trated lycopene sources than unprocessed tomatoes. The lycopene-rich sources are
shown in Table 1 [7–9].
Lycopene’s chemical name is 2,6,10,14,19,23,27,31-octamethyl
2,6,8,10,12,14,16,18,20,22,24,26,30-dotriacontatridecaene. Its formula and molecular
weight are C40H56 and 536.85 [8, 9]. Physicochemical properties of lycopene are
shown in Table 2. Its structural formula is shown in Fig. 1.
The lycopene consists of eight isoprene units comprising 40 carbon and 56 hydro-
gen atoms [10]. Lycopene is an unsaturated hydrocarbon with 11 conjugates and
2 unconjugated double bonds, which are highly reactive with oxygen and free
radicals [11, 12]. Lycopene in natural plants presents mainly in trans configuration.
Conversely, induced via chemical reactions, light, and thermal energy, it can also
form cis-trans isomers including 15-, 13-, 11-, 9-, 7-, 5-cis isomers [13]. A study
presented that the 5-cis isomer of lycopene is the greatest stable one followed by the
all-trans, 9-cis, 13-cis, 15-cis, 7-cis, and 11-cis isomers [14]. Heat processes applied
to tomato and tomato products increase isomerization to lycopene cis form and
increase bioavailability [13–15].
3 Molecular Targets/Mechanisms of Action of Lycopene

in Cancer Prevention
Various studies have shown that lycopene has a protective influence against oxida-
tive stress, an important factor in cancer formation [9]. This may be because
lycopene is the most effective singlet oxygen quencher among carotenoids
Table 2 Physicochemical properties of lycopene [9].

Molecule Lycopene
Structure
IUPAC name (6E,8E,10Z,12Z,14E,16E,18E,20Z,22Z,24E,26E)-2,6,10,14,19,23,27,31-

Octamethyldotriaconta-2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene
Formula C40H56
Molecular 536.87264
weight (g/mol)
Solubility Ethanol and methanol, soluble in chloroform, hexane, benzene, carbon
disulfide, acetone, petroleum ether and oil, insoluble in water
Melting point 172–175
( C)
Stability Sensitive to high temperature, acids, catalyst, light, oxygen, metal ions. Store at
70 C. Combustible. Incompatible with strong oxidizing agents
[16]. Moreover, it was reported that lycopene is an antioxidant 10 times more potent
than alpha-tocopherol and twice as potent as beta-carotene [17]. Consumption of
tomato or tomato products reduces oxidative DNA damage [18], oxidative stress
sensitivity in lymphocytes [19], and lipid peroxidation [20]. Lycopene intake also
increases the production of superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase (GSHPx) enzymes [21, 22]. In an animal model, animals
reared under the stress conditions had lower lycopene concentration in sera and
tissue samples, SOD, CAT, and higher MDA levels than unstressed birds. Variations
were observed in serum and tissue lycopene and MDA concentrations and antiox-
idant enzymes as dietary lycopene level increased [21]. Lycopene may also scavenge
peroxynitrite, resulting in oxidized lycopene products [23].
Lycopene also induces phase II detoxification enzymes to protect cells from ROS
[24, 25]. It activates the transcription system of the antioxidant response elements/
electrophile response elements (ARE) transcription system by disturbing cytosolic
connections between the main ARE-activating Nrf2 and the inhibitor Keap1 [24]. In
another study, increased lycopene in the diet inhibited Keap1 expression in muscle
and improved Nrf2 expression, which was augmented by 150% and reduced by
40%, in response to stress [21].
Besides antioxidant effects, other important mechanisms that explain the anti-
carcinogenic effect of lycopene are: (i) upregulation of gap-junctional gene connexin
43 (Cx43), (ii) the inhibition of cancer cell proliferation and stimulation of differ-
entiation by regulating the expression of cell cycle regulatory proteins, (iii) regula-
tion of the IGF-1/IGFBP-3, (iv) inhibition of 5-lipoxygenase (5-LOX), (v) regulation
940 K. Sahin et al.
Fig. 1 Structure formula of lycopene
of carcinogenic metabolizing enzymes, (vi) modulation of immunity, (vii) regulation

of carcinogen-metabolizing enzymes, and (viii) decrease of oxidative stress by
reducing ROS-producing enzymes (Figs. 2 and 3).
Improved Cx43 expression and rises in gap-junctional intercellular communica-
tion (GJC) have been observed to arise after human cells were treated in culture with
various carotenoids including lycopene [26]. This effect is intensely associated with
the capacity of these carotenoids to suppress neoplastic transformation in model cell
culture [26, 27], which is an effect shared by retinoid [28]. Diminished expression of
connexins, considered as tumor suppressor genes, has been demonstrated in human
tumors compared to normal tissue [29–31]. Stahl et al. [32] reported that lycopene
Fig. 2 Mechanism of lycopene in the prevention of chronic diseases
Fig. 3 Molecular targets of lycopene

942 K. Sahin et al.
improved GJC fetal skin fibroblasts after 1 and 3 days of treatment. In addition,
Cx-43 expression was basically upregulated by lycopene administration in KB-1
human oral tumor cells, and to a much-reduced extent with β-carotene, thus pre-
venting proliferation and increasing GJC [33, 34]. On the other hand, lycopene acts
at multiple points to inhibit VEGF-mediated angiogenesis and suppresses Akt
activation by preventing phosphorylation of the molecule [35]. Lycopene also
inhibits the NF-κB pathway by inhibiting nuclear translocation and NF-κB DNA
binding [36]. Lycopene also acts to inhibit invasion and metastasis via reducing
plasma levels and activity of MMP-2 and -9 [37].
Lycopene treatment inhibits tumor formation by affecting various cellular pro-
cesses such as cell cycle progression and signal transduction pathways [38]. Nahum
et al. [39] reported that reduction of cyclin D1 expression by lycopene supplemen-
tation and consequent inhibition of cell cycle progression in the G0/G1 phase are
significant mechanisms for the decrease of mitogenic effect of IGF-1. Lycopene
supplementation significantly reduced serum IGF-1 levels in humans with colon
cancer [40]. One study reported that accumulation of lycopene and vitamin E in the
tumor and macroscopic estimation of tumors by magnetic resonance imaging pre-
sented a significant increase in necrotic area with lycopene plus vitamin E in a
prostate cancer model [41].
Lycopene acts via its anti-inflammatory effects to prevent cancer. For example,
lycopene inhibits the mRNA and protein expression of the proinflammatory cytokine
IL-8 through the NF-κB inactivation. Moreover, lycopene administration was
revealed to result in reduced p38MAPK ERK1/2 and JNK phosphorylation and
increased PPARγ expression, resulting in improved PTEN activity and the inactiva-
tion of AKT [42]. Lycopene treatment has been shown to induce NF-κB inactivation
by inhibiting the phosphorylation of IKKα and IKBα. It was also reported that
lycopene inhibits TNFα, COX-2, iNOS, and IL-6 secretion [37, 43] (Fig. 3).
Lycopene treatment may suppress levels of nonphosphorylated β-catenin protein
and Akt activation, and increase the phosphorylation of β-catenin, which were linked
with decreased cyclin D1 expression [35]. Therefore, lycopene treatment inhibits
Wnt/β-catenin pathways through the linking along the Akt/GSK3β/β-catenin
[44]. The 5-LOX signaling pathway is another potential pathway for lycopene
anticancer activity. In one study, it was estimated that the acyclic tomato carotene
lycopene and its natural dihydroxy analog lycophyll is bound by the high affinity in
the superficial cleft at the interface of the beta-barrel and the catalytic domain of
5-LOX [45].
4 Lycopene in Cancer Prevention
4.1 Breast Cancer
In the etiology of breast cancer, which is most common in women, environmental

factors, nutrition, and exercise play an important role. Studies have reported that
carotenoids including lycopene reduce the risk of breast cancer perhaps due to their
capability to scavenge DNA damaging free radicals, inhibit cell proliferation, induce
apoptosis, and suppress angiogenesis [46, 47]. For example, in an in vitro study,
treatment with a lycopene-rich extract from red guava (LEG) influenced the viability
of human breast cancer cells (MCF-7) but not murine fibroblast cells (NIH-3T3).
Additionally, LEG showed low cytotoxicity against BALB/c peritoneal macro-
phages and no hemolytic activity. LEG triggered a decrease in the cell proliferation
and induced cell cycle arrest, DNA fragmentation, alterations in the mitochondrial
membrane potential, and morphologic variations linked to granularity and size in
MCF-7 cells; nevertheless, it failed to cause any noteworthy damage to the cell
membrane or display necrosis or apoptosis [48]. In another cell study, mammary
tumor cell lines were cycle-arrested at G1/S phase after treatment with 10 μm
lycopene for 48 h by increasing breast cancer-1 (BRCA1) and breast cancer-2
(BRCA2) expression in estrogen-receptor (ER) positive cell lines (MCF-7 and
HBL-100) and decreasing them in ER-negative cell lines (MDA-MB-231) [49]. In
addition, expression of bcl-2 was reduced by 88% and cell cycle progress was
blocked at G(2)/M phase after treatment with lycopene in MCF-7 cell line
[50]. Peng et al. [51] also reported that lycopene caused MCF-7 cell contractility
and breakage, suggesting that its aggressiveness has decreased in a dose- and time-
dependent manner. Additionally, lycopene treatment caused a reduction in cell
proliferation and a rise in apoptosis, and it could also upregulate p53 and Bax
expression in the cells. Furthermore, lycopene could also increase the expression
of p53 and Bax mRNAs in MCF-7 cells [51]. Nahum et al. [52] reported that
lycopene treatment inhibits cell cycle progression by inhibiting the level of cyclin
D and retention of p27 in cyclin E-cdk2, and inhibiting CDK activities. Assar et al.
[36] found that lycopene inhibits breast cancer cell growth at physiologically
applicable concentrations of 1.25 μM. This also resulted in a 30–40% decrease in
the inhibitor of IκB phosphorylation in cells. In addition, inhibition was detected at
the same rate as lycopene for NF-κB activity [36].
In animal studies, it was shown that lycopene inhibits the occurrence and growth
of the chemically induced breast cancer [53–55]. In a study, our research group has
presented data showing inhibition of mammary cancer incidence, tumor weight and
tumor volume by lycopene (70, 48 and 18%), genistein (60, 61, and 35%) and their
combination (40, 67 and 65%) were observed in rats, respectively [54]. Combination
of lycopene and genistein treatment was more effective in preventing DMBA-
induced mammary tumors and regulating the apoptosis-associated protein expres-
sion than the treatment by each agent alone [54].
Numerous case-control trials have found associations between the lycopene
and mammary cancer in humans. Nevertheless, the results remain inconsistent.
Eliassen et al. [56] reported that significant preventive effects with breast cancer
were detected for α-carotene, β-carotene, lutein+zeaxanthin, lycopene, and total
carotenoids. When the data obtained from case control studies, lycopene decreased
breast cancer by 29.0% [46, 57]. A case-control study with 508 breast cancer cases
and 508 controls nested in the same cohort was performed and their plasma lycopene
levels were not changed. In another nested case control study, 1452 breast cancer
cases and 5239 women showed age-adjusted relative risks for breast cancer
944 K. Sahin et al.
increased by lycopene intake 1.0, 1.15, 0.93, 0.97, and 1.01 [58]. In a study
comparing lycopene levels in mammary adipose tissue, lycopene was found to be
inversely associated with breast cancer risk when adjusted for age, smoking status,
and menopausal status [59, 60].
4.2 Lung Cancer
Lung cancer, one of the most common cancers in the world, will be the main cause of
cancer deaths in 2020. Studies show that lung cancer patients have lower retinol,
α-tocopherol, β-carotene, lycopene, β-cryptoxanthin, selenium, and zinc concentra-
tions [61]. Epidemiological studies have shown that the lung cancer incidence is
inversely correlated with fruit and vegetable intake [62, 63]. Tomato and tomato
products have also been associated with a lower risk of lung cancer (Table 3)
[14]. Lycopene, a bioactive compound of tomato, has been postulated to prevent
lung tumorigenesis by cell cycle arrest and/or apoptosis induction, modulation of
growth factor signaling, modulation of redox status, cellular changes, alterations in
cell growth-related enzymes, and increase in GJC [64].
In an in vitro study, apo-100 -lycopenoic acid has been reported to inhibit the
normal human bronchial epithelial cells (NHBE), immortalized normal bronchial
epithelial cells (BEAS-2B), and nonsmall cell lung cancer cells (A549) by reducing
cyclin E levels, inhibiting cell cycle progression from G (1) to the S phase, and
improving cell cycle regulators p21 and p27 proteins [65]. In the same study,
pulmonary tumor growth was reduced from a mean of 16 tumors per mouse to
an average of 10, 7, and 5 tumors, in a dose-dependent manner, in groups
supplemented with apo-100 -lycopenoic acid at 0, 40 and 120 mg /kg [65]. In
addition, apo-100 -lycopenoic acid treatment results in nuclear transcription factor
Nrf2 accumulation in BEAS-2B human bronchial epithelial cells [66]. In these cells,
Nrf2 activation involves the induction of phase II detoxifying/antioxidant enzymes
including HO-1, NAD(P)H: quinone oxidoreductase1, glutathione S-transferases,
and glutamate-cysteine ligases.
Clinical trials have shown that high intake of lycopene reduces lung cancer risk
by approximately 20–30% [67, 68]. In a study in which human plasma and isolated
LDL and lycopene solutions were treated with cigarette smoke, depletion of all (E)-
lycopenin was higher in plasma than 5 (Z)-lycopenin or beta-carotene. However,
LDL was found to be more sensitive to both all (E)- and 5 (Z)-lycopenin than to beta-
carotene [69]. Shareck et al. [70] reported that the ORs linked with upper versus
lower tertiles of intake were 0.66 for β-carotene, 0.70 for α-carotene, 0.65 for
β-cryptoxantin, 0.75 for lycopene, and 0.74 for C vitamins. In a study conducted
in Spanish women (130 cases, 206 control), high lycopene consumption was found
to be inversely correlated with lung cancer when smoking status, E and C vitamins,
and total flavonoid consumption and other carotenoids were adjusted [71]. In another
case-control study, a significant inverse relationship between serum lycopene levels
and lung cancer mortality after adjusting for cigarettes and serum levels of other
33
Table 3 Summary of the effect of lycopene in different types of cancer

Cancer In vitro/in vivo studies Lycopene formulation Mechanistical effects References
Breast cancer Human breast Synthetic 0, 2, 4, 8, and 16 mM 200 mL Reduced cell proliferation and increased Peng et al.
carcinoma cell line culture medium apoptosis,upregulated the expression of [51]
MCF-7 p53 and Bax mRNAs in MCF-7 cells
Breast cancer Female Wistar rats Synthetic 20 mg/kg Inhibited of tumor growth and expression Sahin et al.
of apoptosis associated proteins [54]
Breast cancer female Wistar rats Synthetic 15 mg/kg BW Decreased tumor proliferation and Jain et al.
increased survival rate of treated animals [184]
Breast cancer MCF-7 breast cancer 0, 2, 4, 6, 8, and 10 μM of lycopene Modulated cell cycle proteins such as beta Uppala et al.
cells tubulin, CK8/18, CK19, and heat-shock [185]
proteins in MCF-7 breast cancer cells

Breast cancer Female rats Synthetic 50 mg/kg of diet MDA#, NO#, SOD", CAT", GPx" Al-Malki
et al. [186]
Lung cancer Mouse model 10, 40, and 120 mg/kg diet of Inhibited the growth of normal human Lian et al.
apo-100 -lycopenoic acid bronchial epithelial (NHBE) cells [65]
Lung cancer BEAS-2B human 10 mM, Apo-100 -lycopenoic acid, Induced both the nuclear accumulation of Lian and
bronchial epithelial apo-100 -lycopenol and Nrf2 protein and the induction of phase II Wang [66]
cells apo-100 -lycopenal detoxifying/antioxidant enzymes,
Colon cancer Human colon Synthetic 0, 0.1, 0.5, 1, and 2 μM Inhibited the phosphorylation of Akt, Lin et al. [44]
cancerHT-29 cells glycogen synthase kinase-3β (GSK-3β),
and ERK 1/2 proteins in human colon
cancer HT-29 cells
Gastric cancer Male Wistar rats Synthetic 50, 100, and 150 mg/kg BW Increased blood IL-2, IL-4, IL-10, TNF-α Luo et al.
levels and reduced IL-6 level and enhance [86]
blood IgA, IgG, and IgM levels in gastric
cancer rats
Decreased MDA and increased blood and
gastric antioxidant parameters (SOD, CAT
and GSH-Px)
945
(continued)
946
Table 3 (continued)
Liver (hepato SK-Hep-1 cell line Synthetic 10 μM Decreased the activities and protein Yang et al.
carcinoma) expression of MMP-2 and -9; increased [187]
the protein expression of nm23-H1 and the
tissue inhibitor of MMP (TIMP)-1 and -2;
suppressed protein expression of Rho
small GTPases; inhibited focal adhesion
kinase-mediated signaling pathway, such
as ERK/p38 and PI3K-Akt axis
Pancreas Rat pancreatic acinar Synthetic 2, 10, 20,100 nmol/L Inhibited the decrease in Ku70 in whole- Seo et al.
AR42J cells cell extracts and nuclear extracts, induced [188]
by G/GO in AR42J cells
Renal cancer Male Sprague-Dawley Synthetic 10 mg/kg Improved against renal damage caused by Oguz et al.
rats Mtx, presumably via antioxidant and anti- [189]
inflammatory activities
Prostate cancer Human Natural (derived from tomato, red Decreased the risk of prostate cancer Giovannucci
watermelon, pink grapefruit, papaya, et al. [190]
guava, rose hip canned)
Prostate cancer Human Synthetic 15 mg (twice daily) Reduced IGF-1 level; increased IGFBP-3 Kucuk et al.
level; decreased tumor growth [161]
Prostate cancer TRAMP:Bco2 mice Tomato powder,384 and 462 mg Lowered incidence of prostate cancer Tan et al.
lycopenekg diet [191]
Prostate cancer Human study Natural, 30 mg of lycopene daily Lowered PSA level in patients with Paur et al.
nonmetastatic prostate cancer [163]
Prostate ccancer LNCaP cells Synthetic 2.5–10 μM Involved the activation of the Yang et al.
PPARγ-LXRα-ABCA1 pathway, leading [192]
to reduced cellular total cholesterol levels
Prostate cancer LNCaP cancer cells Synthetic 10 μM Inhibited DU145 cell proliferation via Yang et al.
PPARγ-LXRα-ABCA1 pathway [151]
K. Sahin et al.
33
Prostate cancer DU145 prostate cancer Lycopene and apo-120 -lycopenal Unaltered the levels of the gap junction Ford et al.
cells protein, connexin 43, decreased cell [156]
apoptosis rates
Ovarian cancer NOD/SCID mice 2.0 ve 5.0 μM lycopene Decreased the expression of the ovarian Holzapfel
cancer biomarker, CA125 et al. [177]
Ovarian cancer Laying hens 200 or 400 mg of lycopene Reduced the expression of NF-κB while Sahin et al.
increasing the expression of nuclear factor [176]
erythroid 2 and its major target protein,
heme oxygenase 1
Photocarcinogenesis Human dermal Synthetic, 0.5–1.0 μM Suppressed the increase of collagenase Offord et al.
(exposed to UV-A fibroblasts GT-F metalloproteinase 1 (MMP-1) mRNA [193]
radiation) expression
Skin cancer Human foreskin Chiang et al.
Synthetic, 10 μM Inhibited PDGF-BB induced fibroblasts

fibroblasts and migration, attenuated PDGF-BB induced [194]
metastatic melanoma phosphorylation, and reduced PDGF-BB
induced signaling.
Skin cancer Albino rats Lycopene extract from tomatoes As lycopene is involved in the synthesis of Butnariu and
vehiculated in nanoemulsions prostaglandins and phospholipids Giuchici
components of cell membrane [195]
Skin cancer NMRImice (ears) (~ 0.05%) lycopene extract Reduced inflammatory infiltrate in higher Ascenso et al
extension, which supports the beneficial [196]
potential of lycopene.
Skin cancer ICR mice Synthetic 10–40 mg/kg BW Reduced the tumor incidence and volume, Shen et al.
inhibited the formation of ROS and MDA, [134]
protected against the loss of GSH, affected
the activities of several oxidant enzymes
in skin, translocated higher levels of
NF-E2-related factor 2
(continued)
947
948
Table 3 (continued)
Skin cancer SENCAR mice Synthetic Decreased the epidermal thickness, Kowalczyk
reduced the number of mice with mutation et al. [197]
in 61 codon of Ha-ras (anti-initiation
effect)
Skin cancer SKH-1 hairless and Red tomato powder Lowered tumor numbers, metabolomic Cooperstone
immunocompetent analyses elucidated compounds derived et al. [133]
mice from tomato glycoalkaloids (including
tomatidine and hydroxylated-tomatidine)
as significantly different metabolites in
skin after tomato exposure
Skin cancer SKH-1 hairless mice Synthetic Attenuated UVB-induced cell hyper- Chen et al.
proliferation and promoted apoptosis, [132]
accompanied by decreased cyclin-
dependent kinase 2 (CDK2) and CDK4
complex in both human keratinocytes
K. Sahin et al.
carotenoids were observed [72]. On the other hand, there was no significant rela-
tionship between lycopene and lung cancer in VITAL study [73].
4.3 Gastric Cancer
Gastric cancer, the fourth most common cancer in the world, is the second leading
cause of cancer death. Regular and high intake of fruits and vegetables, and the
bioactive substances contained therein such as lycopene, can decrease the gastric
cancer risk [74]. These bioactive compounds including lycopene have been observed
to increase in the serum and tissues of those who consume them, and high serum
levels of lycopene and other carotenoids decrease risk of gastric cancer [75]. In
another study, it was reported that lycopene inhibits gastric tumorigenesis by
upregulating GSH-dependent hepatic detoxification systems including GSH, GPx,
GST, and GR [76]. The combined use of S-allylcysteine, found in garlic, and
lycopene decreased gastric cancer via induction of apoptosis by regulating Bcl-2,
Bax, and caspases [77]. In an in vitro study, lycopene exhibited antiproliferative
action in HGC-27 cell lines by enhancing LC3-I, p-ERK expression. In addition,
lycopene treatment significantly decreased tumor weight in gastric cancer nude mice
model [78]. In clinical trials, lycopene supplementation did not affect the risk of
gastric cardiac cancer but reduced the risk of gastric noncardiac cancer by <33%
[79]. In a cohort study, the overall relative risk of gastric cancer for fruits and
vegetables were 0.82 and 0.88 [80]. In another case-control study, it was reported
that consumption of tomato and tomato products had strong preventive effects on
stomach cancer development and its incidence [81]. Moreover, serum lycopene
concentrations were lower in patients with H. pylori [82]. In a case-control study
with 723 gastric cancer patients and 2.879 matched controls in Italy, a significant
trend was observed in the reduction of cancer risk due to raw tomato consumption
after adjusting for age, gender, education, calorie consumption, alcohol intake, and
smoking [83]. In a study involving 191 cases and 570 age-matched controls, the
odds ratio for gastric cancer in the highest versus lowest quartile of prediagnostic
lycopene was found to be 0.55 [84]. However, Zhou et al. [85] reported that
only β-carotene and α-carotene consumption reduced gastric cancer risk. Luo and
Wu [86] showed that lycopene treatment to gastric cancer-induced rats mainly
up-regulated the redox status and immunity with reduction the risk of gastric
carcinoma.
4.4 Liver Cancer
Liver cancer is the fifth most common cancer type in the world. [87]. The most
important risk factors for this cancer are hepatitis B and hepatitis C viruses, chronic
liver diseases, alcoholism and long-term aflatoxin exposure and other chronic
infections [88, 89]. Phytochemicals are powerful potential therapeutic agents for a
950 K. Sahin et al.
multitude of chronic diseases such as cancer, diabetes, and neurodegenerative

diseases. It has been reported that supplemental phytochemicals including lycopene
and resveratrol are beneficial in the liver cancer treatment [87]. In addition, there is a
noteworthy contrary link between serum lycopene, retinol, and retinol-binding
protein 4 (RBP4) levels with fibrosis phase in the liver. Serum β-carotene and
lycopene are also closely related with their relevant liver levels [90]. In addition,
lycopene treatment inhibited metastasis both in vitro and in nude mice in hepatoma
SK-Hep-1 cells [91, 92], while the Nrf2-ARE system in HepG2 cells was active
[93]. In an experimental study, it was reported that lycopene decreased the number of
liver tumor-bearing mice by 49.7% and decreased the average number of tumors per
mouse by 88% [94]. Hepatocytes treated with lycopene and beta-carotene are
protected from the effects of carcinogenic aflatoxin at both cellular and molecular
levels [95]. Conversely, in a study conducted with Long-Evans Cinnamon (LEC)
rats, it was stated that lycopene treatment did not diminish spontaneous liver cancer
for 70 weeks. In another study, it was found that NADPH oxidase 4 (NOX4)
expression and intracellular ROS levels in SK-Hep-1 cells inhibited by 64.3%
with lycopene treatment at the highest level in 2.5 μM [96]. In a study, we reported
that lycopene supplementation improved the liver CAT, SOD, GSHPx and decreased
the NF-κB/COX-2. In addition, lycopene treatment decreased the rises in p-mTOR,
p-S6 K1, p-4EBP-1, and protein kinase B [6]. Lycopene treatment is effective
against the preneoplastic foci in the liver [97]. Lycopene treatment prevents liver
tumor formation in rats spontaneously developing liver tumors and in rats with
induced tumor formation with DEN and high fat diet [98]. Cheng et al. [99] showed
that treatment of apo-100 -lycopenoic acid in a dose dependent is effective in pre-
venting migration and invasion, suppression of angiogenesis and of liver cells by
inhibition of MM-2 expression and activation.
Lycopene supplementation for 26 weeks inhibited the tobacco carcinogen
4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced expressions
of α7 nicotinic acetylcholine receptor in the lung and NF-κB and CYP2E1 in the
liver and reduced the NNK-induced mortality and pathological lesions in both the
lungs and livers [100]. Moreover, lycopene treatment for 24 weeks reduced liver
NK-κB p65 and STAT3, IL6, and inflammatory foci in mice. Conversely, lycopene
treatment in BCO2-KO were related with decreased hepatic endoplasmic reticulum
stress-mediated unfolded protein response through reducing ER(UPR)-mediated
protein kinase RNA-activated like kinase-eukaryotic initiation factor 2α activation,
and inositol-requiring 1α-X-box-binding protein 1 signaling. Lycopene in BCO2-
KO mice inhibited Met mRNA, β-catenin, and mTOR complex 1 activation related
with augmented liver microRNA (miR)-199a/b and miR214 levels [101]. In another
study, it was reported that lycopene decreased levels of ALT, AST, ALP, LDH in
mice during initial stages of N-nitrosodiethylamine (NDEA)-induced liver cancer.
Lycopene treatment of NDEA mice also resulted in decrease of AFP, HIF-1α,
VEGF, CD31, MMP-2, and MMP-9 expression in comparison with NDEA group
alone [102].
4.5 Pancreatic Cancer
Pancreatic cancer is the eighth most common cause of cancer death in Europe and
the fourth in the United States [103]. Chronic pancreatitis is thought to be related
with high intake of alcohol, tobacco smoke exposure and obesity, consumption of
animal protein and fat and antioxidant deficiencies [104]. Fruits and vegetables may
have a role in the inhibition of pancreatic cancer because they comprise potentially
protective substances such as carotenoids, tocopherols, and other phytochemicals
[103, 105]. These bioactive complexes play a protective role contrary to free radical
damage to DNA, improve immune properties, and inhibit IGF by binding to IGF
receptors [103, 106]. In a case-control study, there was a substantial contrary
relationship between lycopene consumption and pancreatic cancer after adjustment
for age, body mass index, smoking, calorie intake, and education [107]. Huang et al.
[108] reported an inverse link between lycopene uptake and risk of pancreatic cancer
in Caucasians; this association was found to be insignificant in the mixed population.
Another meta-analysis study showed that alpha-carotene and lycopene could reduce
the risk of prostate cancer [109]. In another case-control study (44 matched controls
and 22 pancreatic cancers), it was reported that lycopene levels were lower in cases
than in controls, after adjusting for smoking, education, and serum levels of other
carotenoids [110]. However, Jeurnink et al. [103] reported that contrary relations
with pancreatic cancer risk were detected for serum β-carotene, zeaxanthin, and
α-tocopherol [103].
4.6 Colorectal Cancer
There is an inverse relationship between the lycopene intake or tomato products and
colorectal cancer [111, 112]. For example, in in vitro study, it was reported an
important decrease in the number of viable cells in human colon adenocarcinoma
cells (HT-29) and human colon carcinoma cells (T-84) after 48 h of treatment by
lycopene. In the same study, it was also demonstrated that lycopene induced cell
cycle arrest followed by decreased cell viability in the common of cell lines after
96 h as compared to controls. Moreover, lycopene has been shown to prevent cell
proliferation in HT-29 cells with IC50 value of 10 μM. Lycopene also inhibited Akt
activation and β-catenin levels in human colon cancer cells [113]. Lycopene
inhibited leptin-mediated cell invasion and MMP-2 expression in HT-29 cells
[44]. In addition, lycopene plays an important role in leptin-mediated MMP-7
expression and cell invasion of MAPK /ERK and PI3K/Akt signaling pathways.
Lycopene could effectively inhibit the phosphorylation of Akt, glycogen synthase
kinase-3 (GSK-3p), and ERK1/2 proteins. Huang et al. [114] tested the lycopene and
gold nanoparticles (AN) effects on the inhibition of the HT-29 cells. They reported
that their combination (AN 10 ppm plus lycopene 12 μM) and nanoemulsion
(AN 0.16 ppm plus lycopene 0.4 μM) caused in a 5- and 15-fold rise in early
952 K. Sahin et al.
apoptotic cells of HT-29 compared with the control treatment. Also, the nano-
emulsion decreased the caspases 8, 3, and 9, PARP-1, and Bcl-2, while Bax
expression was improved. A fivefold decrease in the migration ability of HT-29
cells was detected for this nanoemulsion when compared to control, with the
invasion-related markers being reversed through the upregulation of the epithelial
marker E-cadherin and downregulation of Akt, NFκB, MMP-2, and MMP-9 [114].
In an animal study, we presented that 5% of the dietary tomato powder decreased
the aberrant crypt foci (ACF) ratio and also decreased adenocarcinoma growth and
azoxymethane (AOM)-induced colorectal cancer formation in rats. Moreover,
tomato powder supplementation shows chemopreventive action by modulation
Nrf2/HO-1 pathway in colon tissue while preventing COX-2 levels and inducing
apoptosis via the NF-κB pathway [115]. In an in vivo study, lycopene has been
shown to be correlated with anticancer effects by suppression of COX-2, PGE2, and
phosphorylated ERK1/2 proteins [116]. However, postinitiation treatment with
lycopene did not reduce the development of ACF in male B6C3F1 mice [117]. In
contrast, lycopene treatment throughout the postinitiation stage decreased ACF
growth but not colon tumors in female Fischer 344/NSIc rats [118].
A meta-analysis showed that an inverse relationship was detected between
lycopene intake and the colon cancer [112]. In a clinical study, the serum lycopene
level was detected to be lower in the patients with colorectal cancer than in the
control subjects [119]. However, a study stated that no important relationship
was determined between colorectal cancer risk and lycopene consumption in a
cohort study, middle-aged men smokers [120]. Case-control studies have
reported a 60% decline in the risk of colorectal cancer linked with higher intake of
tomatoes [121–123].
4.7 Skin Cancer
UV light penetrates different skin layers and can cause skin damage by affecting
specific biological structures at each level. These effects depend on penetration
depth, absorption, wavelength, and structural properties affecting reflection and
light scattering, molecular patterns, and pigmentation [124]. Carotenoids including
lycopene can be used to reduce the adverse effects of UV light that cause damage to
the skin. A linear relationship between lycopene consumption and a prevention of
UV-induced erythema formation and a reduced risk of nonmelanoma skin cancer
were shown [9, 125–127]. In addition, plasma lycopene levels were associated with
lycopene levels in skin and a positive effect was found between the β-carotene in the
serum and the β-carotene in the skin. However, plasma-skin associations were lower
and not significant for lutein, zeaxanthin, and β-cryptoxanthin. More skin lycopene
is destroyed compared to β-carotene when skin is exposed to UV light [128]. Con-
sumption of products rich in lycopene and synthetic lycopene for 10–12 weeks
decreased the sensitivity toward UV-induced erythema in volunteers [129,
130]. Andreassi [131] reported that lycopene intake could protect skin against the
effects of UV-B radiation, particularly when linked with vitamins C and E. Another
study reported that lycopene did not affect the apoptotic status and necrotic and
viable cells in unirradiated cells. Nevertheless, irradiated cells were subjected to a
rise with lycopene in both dead and viable subpopulations compared to unexposed
irradiated cells [127] by resulting in overexpression of Bax gene.
Chen et al. [132] demonstrated that lycopene pretreatment reduced
UVB-induced cell hyperproliferation with apoptosis and reduced cyclin-dependent
kinase 2 (CDK2) and CDK4 complex both in human keratinocytes and SKH-1
hairless mice. While FOXO3a is phosphorylated in response to UVB irradiation
and is involved in cytoplasm, lycopene treatment rescues this sensitization. Gene
ablation of FOXO3a showed a lycopene-induced reduction in cell hyperproliferation,
CDK2 and CDK4 complexes, a critical role for FOXO3a in the lycopene-stimulated
antiproliferative effect of keratinocytes during UVB irradiation. Furthermore, loss of
AKT induced more enhanced lycopene-induced FOXO3a dephosphorylation, while
the loss of mTORC2 by transfection with RICTOR siRNA induced levels of AKT
phosphorylation comparable to those obtained with lycopene. Conversely, over-
expression of AKT or mTORC2 decreased the lycopene effects on AKT phosphor-
ylation as well as on the expression of FOXO3a, suggesting that lycopene is due to
negative modulation of mTORC2/AKT signaling [132]. In an animal study of mice
exposed to 2240 J/m2 UV-B irradiation and fed with red tomato powder diet for
35 weeks, the number of tumors was found to be significantly lower in mice
consuming red tomato diet [133].
Shen et al. [134] reported that pretreatment with lycopene for oxidative stress and
skin cancer induced by DMBA/12-O-tetradecanoylphosphoryl-13-acetate (TPA) in
female ICR mice significantly delayed tumor growth and decreased the tumor
incidence and volume. In addition, lycopene prevented the production of ROS and
MDA, thereby inhibiting the loss of glutathione and activating antioxidant enzymes
in the skin of mice. Furthermore, animals treated with lycopene showed higher levels
of Nrf2 into the nucleus compared with control animals [134]. On the other hand,
there are also studies reporting that lycopene has no effect on skin photo-
carcinogenesis [135, 136]. The reason for these conflicting reports could be the
lycopene levels, the UVB radiation dosage, the different experimental conditions
and models [132].
4.8 Head and Neck Cancer
Numerous reports have suggested that lycopene and tomato products may have
useful properties in the head and neck cancer therapy [13, 33, 137, 138]. For
instance, Lodi et al. [139] reported that lycopene significantly reduced multiple
forms of HNSCC including laryngeal, oral, and pharyngeal cancer. Lycopene and
β-carotene (5–40 μM) dose- and time-dependently reduced the viability of the
EC109 human esophageal squamous carcinoma cells. Lycopene and β-carotene
treatments upregulated the expression of PPARγ and p21WAF1/CIP1 and down-
regulated the expression of cyclin D1 and COX-2. These modulatory effects of the
carotenoid treatments were suppressed by GW9662, an irreversible PPARγ
954 K. Sahin et al.
antagonist, suggesting that the inhibition of EC109 cell viability by lycopene and
β-carotene involves PPARγ signaling pathways and the modulation of p21WAF1/
CIP1, cyclin D1, and COX-2 expression [140]. Mayne et al. [138] found that only
serum lycopene levels were significantly inversely related with total mortality and
mortality in nonsmoking patients. In a clinical trial (754 oral cancer cases and 1775
control), lycopene intake was not significantly linked with oral cancer risk [141]. In
another study, high tomato consumption reported that OR was 0.49 for oral cancer
[142]. In addition, lycopene inhibited the proliferation of human oral cancer cell line
KB-1 by enhanced the expression of the gap junctional protein connexin 43 [33].
In an experimental study, an inverse relationship was detected between lycopene
at 2.5 mg/ kg lycopene dose and squamous cell carcinomas with buccal cuts in
hamsters. In a study on hamsters, consumption of lycopene 2.5 mg/kg resulted in an
inverse relationship between buccal pouch squamous cell carcinomas
[143]. El-Rouby [144] found that lycopene treatment at a dose of 2.5 mg/kg BW
once a day with intragastric intubation decreased the incidence of laryngeal cancer
and increased E-cadherin and β-catenin expression in the lycopene-treated group
compared to the control group. Another study showed no carcinomas in lycopene or
mixed carotenoid groups [99]. In a study of 228 cases and 491 hospital-based
controls, tomato consumption was linked with a decrease in the risk of 0.30, while
tomato sauce-rich foods had a protecting effect of 0.57 [145]. The nutrient group
consisting of raw and tomato-rich foods presented a strong inverse association with
head and neck cancer. Lycopene was also related with a reduced risk of 0.22.
4.9 Prostate Cancer
Prostate cancer is the second most common cancer in men in the world and the fifth
most common cancer death in men [146]. There are several ways in which lycopene
treatment can protect against prostate cancer, including the prevention of DNA
damage by scavenging free radicals [147], modulating gene expression related to
prostate cancer [148] and slowing of cancer cell growth [149, 150], inhibiting
prostate cancer cell proliferation via the PPARγ-LXRα-ABCA1 pathway, [151],
and progression of prostate cancer is hampered by apoptosis induction and angio-
genesis suppression [149, 152]. The lycopene intake was significantly linked with
decreased PCa risk, and dose-response studies shows a significant linear effect for
the dietary lycopene and PCa risk, thus PCa reduced by 1% for every 2 mg of
lycopene intake. In addition, for each 10 μg dL-1 circulating lycopene in the linear
and nonlinear models, the PCa risk decreased by 3.5% to 3.6% [153]. A substantial
inverse association between PC and serum lycopene levels has been found between
the highest and lowest quintiles of intake [154–156]. It has been reported that
upregulation of miR-let-7f-1 targeted AKT2 and AKT2 in PC3 cells may improve
the properties caused by miR-let-7f-1 [157]. Van Hoang et al. [150] reported that the
risk of PC reduced by lycopene consumption, tomatoes, tomato products, and
carrots. There was no statistically significant correlation for the consumption of
α-carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, and other carotenoids.
In a case-control study [158, 159], it was reported that in patients with high-
concentration lycopene, the risk of aggressive prostate cancers was reduced. In
particular, a significant relationship has been established between high plasma
lycopene concentration and a strong reduction in aggressive prostate cancers. Sim-
ilarly, a meta-analysis found an inverse relationship between lycopene consumption
or serum lycopene concentrations and the risk of prostate cancer, with a stronger
relationship, detected for the circulating lycopene compared to the highest to the
lowest level [109]. However, Morgia et al. [160] reported that Se and lycopene were
not related with higher prostate cancer risk.
In a randomized two-arm studies, plasma prostate-specific antigen (PSA) con-
centration were stated to be reduced by 18% in the intervention group and by 14% in
the control group. Eleven of fifteen patients had no extra prostatic tissue involvement
with surgical limitations and/or cancer in the intervention group, compared with 2 of
the 11 patients in the control group. In the lycopene treatment group, 12 of 15 patients
had tumors measured 4 cc or less, 5 of 11 in the control group [161]. In the same
study, Cx43 in the malignant part of the prostate gland was higher in the group using
lycopene than in the control group. Phase II randomized clinical trial of 15 mg of
lycopene intake twice a day for 3 days before radical prostatectomy found a
reduction in IGF-I concentration, but no significant change in Bax and Bcl-2
[162]. Another study reported that the median PSA of the tomato group was lower
in postoperative-moderate-risk patients than in the control group [163]. Patients with
the highest rise in serum lycopene, Se, and C20: 5 n-3 fatty acid had a 1% reduction
in median PSA values compared to the lowest increase in lycopene, Se and C20:
5 n-3 fatty acids. Additionally, PSA was reduced in patients with the highest increase
in lycopene group. It has also been shown that neither prediagnosis nor post-
diagnosis dietary lycopene was associated with PC-specific mortality [164]. Con-
sumption of either low or high dose of lycopene (4 and 16 mg/kg) and a single dose
of β-carotene (16 mg/kg) was reported to strongly inhibit tumor growth and
decreased prostate tumor volume and tumor weight in nude mice.
Lycopene and β-carotene at high dose levels significantly decreased PCNA
expression in tumor tissues and the plasma IGF-binding protein-3 levels. Soares
et al. [166] found a significant reduction in primary prostate cancer cell viability
upon with lycopene treatment obtained tomato-based food products. Moreover,
lycopene, containing tomato extract and tomato sauce, showed a 50-fold increase
in the proportion of apoptotic cells by upregulation of TP53 and Bax expression and
also downregulation of Bcl-2 compared to the control group [166].
4.10 Renal Cell Carcinoma
It is expected that 63.340 new cases of renal cell carcinoma (RCC) (42.680 in men
and 22.660 in women), the most common type of renal cancer in adults, will occur in
2018, and about 14.970 people (10.010 men and 4.960 women) will die from renal
cancer. Renal carcinoma is among the 10 most common cancers in both men and
women. In general, the risk of developing kidney cancer in men and women is about
956 K. Sahin et al.
1 in 48 and 1 in 83 [167]. Many studies suggest that micronutrients may be used as

dietary supplements, including α-carotene, β-carotene, lutein, zeaxanthin, lycopene,
vitamin A, vitamin C, and selenium. These agents may inhibit oxidative DNA
damage, mutagenesis, and tumor growth [168–170]. Nevertheless, some studies
have reported that there is no significant relationship between renal cancer and
consumption of antioxidant substances [171], but others have provided evidence
that some agents may have a protective effect [169].
Ho et al. (2015) indicated that lycopene intake was inversely linked with the
risk of renal cancer; when compared with the lowest quintile of lycopene intake,
the highest quartile was related with a 39% lower renal cancer risk. The authors
described the potential anticancer effects of lycopene as follows: (i) In contrast to
provitamin A carotenoid (α-carotene, β-carotene, and β-cryptoxanthin), it is
recommended that lycopene, along with other carotenoids without provitamin A
activity (lutein and zeaxanthin), has a different enzymatic affinity. For this reason,
absorption and metabolism of lycopene cannot be controlled by vitamin A con-
centration through intestinal-specific homeobox transcription factors [169, 172].
(ii) Lycopene, one of the most hydrophobic carotenoids, has the ability to affect a
variety of transcriptional pathways at the molecular level, unlike other carotenoids.
It has also been shown that lycopene has the greatest singlet oxygen quenching
activity of any carotenoid [12]. These properties make lycopene a potential
anticancer agent. In a study done in the predisposed TSC2 mutant Eker rat
model, the incidence of tumor changed from 94% in control rats to 65% in the
lycopene-treated rats, but the change was not significant. However, the mean
number of tumors was significantly reduced in rats treated with lycopene com-
pared to the controls rats. In the same study, it was also reported that in the
lycopene-treated rats, number of tumors reduced and the numbers tended to
reduction linearly as lycopene level increased from 0 to 200. Control rats fed
only basal diet had a greater length of tumors than rats fed lycopene supplement
groups. Furthermore, length of tumor reduced and tumor length inclined to reduc-
tion linearly as dietary lycopene level increased from 0 to 200 mg/kg. All tumors
presented strong staining with antibodies against mTOR, phospho-S6, and EGFR
[173]. However, a clinical trial revealed that RCC risk was inversely associated
with β-carotene, α-carotene, β-cryptoxanthin, vitamin A, vitamin C, but not with
lycopene or vitamin E [174]. A case-control study also suggested a significant
opposite relationship between lycopene and renal in the nonsmoking group, but
not in the smoking group [175].
4.11 Ovarian Cancer
Ovarian cancer is the seventh most frequently diagnosed cancer in women and the
sixth leading cause of death from cancer among women. Recently, we found
significantly decreased tumor incidence and serum MDA levels and increased
serum lycopene levels in the hens fed a lycopene-enriched diet compared to control
groups. NF-kB and STAT-3 expression were reduced and Nrf-2, PIAS-3, and HO-1
were improved in the ovarian tissues of lycopene-fed animals [176]. Holzapfel et al.
[177] reported that lycopene administration decreased the metastatic load of ovarian
cancer-bearing mice, while treatment with lycopene reduced the tumor burden.
Lycopene administration also improved the antitumorigenic effects of paclitaxel
and carboplatin. Tumor and metastatic tissues for Ki67 revealed that lycopene
reduced the number of proliferating cancer cells. They also reported that lycopene
reduced the expression of CA125. These effects were complemented by regulation
of expression of ITGA5, ITGB1, MMP9, FAK, ILK, and EMT markers, reduced
expression of integrin α5.
In a human study, Li and Xu [178] established an insignificant contrary link
between lycopene intake and ovarian cancer risk, and subgroup analysis stratified by
study design, location, histological type of ovarian cancer, and length of dietary
recall presented no significant results. In addition, a case-control study conducted by
Cramer et al. [179] indicated a significant inverse relationship between consumption
of lycopene and tomato sauce and risk of ovarian cancer mainly in premenopausal
women. In another study, it was found that fruit- and vegetable-rich nutrition was
related with a reduction in the risk of ovarian cancer, and among the most convincing
were tomatoes [180]. Helzlsouer et al. [181] found no correlation between serum
lycopene levels and ovarian cancer risk. Nevertheless, Jeong et al. [182] have found
an inverse link between serum lycopene levels and ovarian cancer risk. rGO-Ag, a
new biomolecule, lycopene, and tricostatin A have been shown to inhibit the
viability of the human ovarian cancer cell (SKOV3) in a dose-dependent
manner [183].
5 Conclusion
In conclusion, lycopene consumption and higher levels of lycopene in the plasma

have been associated with a lower risk of cancer in clinical and experimental
studies. Lycopene may lower cancer risk by decreasing oxidative stress through
modulation of ROS-producing enzymes including COX-2 and 5-LOX and inducing
antioxidant/detoxifying phase II enzymes. These phase II enzymes are also modu-
lated by the Nrf2-ARE system. The NFκB and Nrf2/HO-1 signaling pathway is
suggested to be an important primary target for chemoprevention by lycopene.
Lycopene can inhibit the proliferation and induce differentiation of cancer cells by
modulating the expression of cell cycle regulatory proteins, modulating the IGF- 1/
IGFBP-3 system and other mechanisms including the prevention of oxidative DNA
damage and modulation of the immune function, as well as the inactivation of
growth factors. There is a need for more detailed experimental studies and larger
clinical trials investigating the effect of lycopene on cancer prevention and integra-
tive cancer treatment.
Acknowledgments The study was supported in part by Turkish Academy of Sciences (KS).
958 K. Sahin et al.
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Part VI
Food Polyphenols
Bound Phenolics in Foods
34
Liliana Santos-Zea, Javier Villela-Castrejón, and
Janet A. Gutiérrez-Uribe
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974
2 Bound Phenolics in Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 976
2.1 Conjugates with Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 976
2.2 Conjugates with Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 976
3 Bound Phenolic Extraction and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
3.1 Traditional Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
3.2 Emerging Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977
4 Bioactive Potential of Bound Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978
4.1 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978
4.2 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
4.3 Carbohydrate and Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984
4.4 Interactions with Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
Abstract
Bound phenolic compounds are widely distributed among several plants, espe-
cially cereals. In most of the cases, covalent bonds are formed with polysac-
charides, proteins, or lipids. But additionally, hydrophobic interactions
L. Santos-Zea · J. Villela-Castrejón
Tecnologico de Monterrey, School of Engineering and Science, Centro de Biotecnología-FEMSA,
Monterrey, Mexico
J. A. Gutiérrez-Uribe (*)
Tecnologico de Monterrey, School of Engineering and Science, Department of Bioengineering and
Science, Campus Puebla, Puebla, Mexico

974 L. Santos-Zea et al.
may affect their release from the food matrix. Many studies have reported their
bioactivity after their release from foods, in most of the cases involving acid or
basic hydrolysis and further extraction with organic solvents. Besides their
antioxidant activity, bound phenolics have important effects on the inhibition
of cancer cell growth, key enzymes involved in the metabolism of carbohy-
drates, as well as in the regulation of inflammatory processes. Food processing
and gastrointestinal digestion affect the bound phenolic bioavailability and in
consequence the potential benefits to human health. Recent studies have dem-
onstrated that microbiota composition in the gastrointestinal tract affect the
release of bound phenolics and their metabolism. Therefore future studies will
help us to understand the complex interactions between bound phenolics and
gastrointestinal microbiota and produce natural controlled released bioactive
phenolic compounds.
Keywords
Bound phenolics · Antioxidant · Prebiotic · Extraction
1 Introduction
Phenolic compounds (PCs) are plant secondary metabolites necessary for physi-
ology and cellular metabolism. Among the main functions in plants are the
protection against insects and animals, structure, growth, and reproduction [1].
PCs come from the phenylpropanoid pathway, and the two main precursors are the
amino acid phenylalanine from the shikimate pathway and malonyl-CoA from the
acetate pathway [2]. The phenolic acids possess one or more aromatic rings
hydroxyl-substituted. Currently, there are 8000 known phenolic structures, which
are classified according to the carbon chain (Fig. 1) [3]. Furthermore, the location
within the plant and the chemical structure makes possible to categorize phenolic
compounds as soluble and insoluble. Soluble phenolics are composed of free
simple phenols, glycosides (with single or multiple sugar moieties), and low
molecular weight phenolics not bound to the other plant cell major components.
Bound phenolic (BP) group is conformed by condensed tannins, phenolic acids,
and other compounds of low molecular weight [4]. Particularly in cereals, the
insoluble fraction account for the major part of phenolic acids [5].
Phenolic compounds have gained a lot of attention, since these molecules have
proved their antioxidant, antimutagenic, antiviral, and antibacterial activities
[6–10]. Epidemiological studies have linked the consumption of foods
and beverages high in phenolic acids with a reduced risk of development of
several diseases [3]. Since bound phenolics are poorly absorbed in the small
intestine, their fermentation products generated by the microorganisms in the
large intestine generate positive effects on microbiota and inhibit the growth of
pathogens.
34
Bound Phenolics in Foods
Fig. 1 Diversity of phenolic compound structure

975
2 Bound Phenolics in Nature
Bound phenolic compounds are widely distributed among several plants, such as cereal
grains (>62% of the total phenolic are bound), whereas fruits and vegetables have most
of their phytochemicals in the free or soluble form (<24% of the total phenolic are
bound) [11]. Phenolics, proteins, and polysaccharides are separated in different com-
partments in the cell, normally they do not interact until an external stimuli triggers the
intracellular contact and then several subsequent reactions can occur and force the
interaction (adsorption, oxidation, solubilization, and migration) [12].
PCs are linked to the cell wall structure macromolecules, such as arabinoxylans,
pectin, cellulose, lignin, or proteins through ester, ether, and C-C bonds [13]. PCs
can interact through hydrogen bonding, covalent bonding, hydrophobic interactions,
and ionic bonding with other macromolecules [14]. Some phenolic acids are more
abundant in the bound fraction, such as in the case of whole grain oats, where ferulic
and p-coumaric acids were found 10- to 100-fold higher in concentration with
respect to the free phenolic fraction. However, the profile was less diverse in the
bound fraction than in the free phenolics [15].
In the past 20 years, there has been an important increment in research in the field
of phenolic interactions with other macromolecules, but there is still limited knowl-
edge about the changes that occur during plant development, postharvest treatments,
or interactions produced during food processing and that may affect the release of
free phenolics.
2.1 Conjugates with Polysaccharides
Phenolic compounds, mainly phenylpropanoids and simple phenolic acids, can be

bound to carbohydrates. Hydrogen bonding and hydrophobic interactions are the
driving mechanisms for this type of complexes [14]. The non-covalent complexation
between cyclodextrin and phenolic acids is one of the most studied interactions. This
complexation is driven by the size of cyclodextrin and its polarity [12].
2.2 Conjugates with Proteins
The molecular weight, affinity of the phenolic acid for water, structural flexibility,
and the hydroxyl groups are the main characteristics that dictate the binding of
phenolic acids with proteins [16]. In addition, this type of reaction is favored under
neutral and basic pH conditions. One of the most studied interactions is the complex
with the bovine serum albumin (BSA) [17]. Irreversible interactions involve the
oxidation of phenolic compounds and the formation of o-quinones or o-semi-
quinones or the cleavage of proanthocyanidin interflavanic bonds.
Hydrolysable tannins are conformed by simple phenolic acids (gallotannins)
or hexahydroxydiphenic acids (ellagitannins) esterified to polyols (most commonly
glucose). Condensed tannins or proanthocyanidins are polymers and oligomers of
34 Bound Phenolics in Foods 977
flavan-3-ol unit that are distinguished by their degree of polymerization and type of
linkage [12]. Tannins can interact with proteins due to hydrophobic interactions between
the aromatic ring of the tannin and the hydrophobic region of the protein and the
hydrogen bonds. The tannin-protein complexes depend on the size, length, pH, and
flexibility of tannins. These molecules are able to precipitate proteins from an aqueous
solution. Interaction with PCs can increase the thermal stability of proteins [14, 18, 19].
Protein aggregation is affected by the average size of the condensed tannins rather than
the hydroxylation pattern. In special, the procyanidins and cis-flavan-3-ol units contrib-
uted most to the tannin interactions on the BSA surface [20].
3 Bound Phenolic Extraction and Analysis
For the precise identification and quantification of phenolic compounds, they need to
be correctly extracted. Several extraction processes underestimate the total phenolic
compounds due to recovery inefficiency [21].
3.1 Traditional Methods
The most common method is the solid-liquid extraction; it involves the extraction of
fresh or freeze-dried materials. But a critical step to extract bound phenolic com-
pounds is their release from the insoluble food matrix. Acid or alkaline hydrolysis
has been extensively used for this purpose. Alkaline hydrolysis has been proved to
be more effective but time, temperature, and base concentration need to be optimized
for each crop.
Once the compounds have been released from the matrix and the pH adjusted,
extraction with solvents is required to recover bound phenolics. The most used solvents
are water, methanol, ethanol, acetone, or several mixes. The parameters in this process
are extraction time, temperature, solvent type, solvent to sample ratio, and number of
repeated extractions. These parameters must be optimized in order to obtain a substantial
recovery of PCs. The main disadvantages of solid-liquid extraction include the need to
remove contaminants or other unwanted compounds from the extract, the use of
hazardous organic reagents, and the time required for extraction [21].
Traditional food processing releases bound phenolics. Boiling is still the most
common cooking method, and it has a negative impact on the total phenolics content
of food since bound phenolics are released. After their release, they may form new
irreversible covalent bonds with proteins due to the hydrothermal energy [22].
3.2 Emerging Technologies
Several physical methods have been recently used to release bound phenolics from
the matrix. They may be used in combination with alkaline hydrolysis. Microwaves,
ultrasound, far infrared radiation, pulsed electric field, and pressurized liquids are
examples of emerging technologies for the release of bound phenolics [11]. All these
methods increase the extraction yield and reduce the hydrolysis time significantly.
4 Bioactive Potential of Bound Phenolics
Over the years, diverse biological activities from dietary and medicinal plants have
been attributed to phenolic compounds, in both free and bound forms. Antioxidant
capacity has been the most widely studied effect of phenolics, but effects, such as
antiproliferative and apoptotic effect on cancer cells, inhibition of carbohydrate and
lipid metabolism enzymes, and anti-inflammatory and vasodilator effects, among
others, have been recognized (Table 1). It is important to point out that in these
studies, bound phenolics are initially subjected to hydrolysis, as documented in the
previous section, to release the phenolic moiety from the carbohydrate or protein
moiety and therefore determine their biological effects.
With respect to in vivo potential, bound phenolics are essentially considered non-
bioavailable, as they reach the colon still covalently linked. In general, it has been
determined that only about 5–10% of insoluble bound phenolics are absorbed in the
small intestine and the majority reaches the colon intact [13]. These compounds can
be released by pH conditions in the stomach, processed by ß-glycosidase in the
intestinal border brush cells, transported as glycosides by sugar transporters, or
enzymatically released by fermentation of the colonic microbiota [11, 13]. Addi-
tionally, during and after absorption, phenolics undergo conjugation by intestinal
and liver enzymes, such as methylation, sulfation, glucuronidation, increasing
hydrophilicity, and therefore excretion [23].
4.1 Antioxidant
Antioxidant capacity of bound phenolics, particularly in the case of grains, has been
well studied [24]. In most cases only the in vitro or chemical capacity as antioxidants
has been analyzed by diverse methods, such as total oxygen scavenging capacity
(TOSC), ferric reducing antioxidant power (FRAP), or oxygen radical antioxidant
capacity (ORAC) (Table 1). To determine the impact that phenolic compounds can
have in a real biological system, models such as LDL cholesterol oxidation, super-
coiled DNA fragmentation, and cellular antioxidant activity assays have become
more widely used in recent years [46].
4.1.1 Antioxidant Capacity by Chemical Methods

With respect to chemical antioxidant capacity of bound phenolics, Adom and Liu
(2002) determined that in the case of whole grains, such as corn, wheat, and oats, this
fraction corresponds to over 60% of the total antioxidant capacity evaluated by the
TOSC assay, and this activity was highly correlated to total phenolics and ferulic
acid content in the grains [24]. In the case of dehulled highland barley, antioxidant
capacity evaluated by ORAC showed that around 50–60% of total antioxidant
Table 1 Potential bioactivity of bound phenolic compounds

Source Phenolics Bioactivity Dose/concentration Reference
Corn, wheat, Ferulic acid and Chemical antioxidant 50–150 μmol vit C [24]
oats, rice other phenolic capacity evaluated by eq/g grain
acids total oxygen scavenging
capacity (TOSC)
Dehulled Catechin, phenolic Cellular antioxidant 11.3–19.2 μmol [25]
highland acids capacity on HepG2 cells QE/mL
barley
Model system Quercetin TEAC antioxidant 2.0–4.6 mM as [26]
of quercetin- capacity of BSA-bound BSA-quercetin,
BSA quercetin was increased 3.0–4.0 as peptide-
hydrolyzing the protein quercetin
Rice soluble Ferulic acid bound Antioxidant capacity 1.14–1.24 mg [27]
fiber to arabinoxylans evaluated by inhibition
of ß-carotene oxidation
(IC50)
White, red, Phenolic acids Chemical antioxidant 20–100 mM [28]
and tannin bound to capacity evaluated by Trolox equivalents
sorghum bran, arabinoxylans ORAC (TE)/g
corn fiber
Cinnamon, Phenolic acids, Chemical antioxidant 2.6–34.6 mmol [29]
oregano, and cinnamaldehyde, capacity evaluated by FeSO4/100 g and
clove coumarin, eugenol FRAP and DPPH 2.3–17.7 mmol TE/
methods 100 g
Ginger Cinnamic and Inhibition of lipid 5.2 μg GAE/gliver, [30]
p-coumaric acids peroxidation (IC50), 2–4 μg GAE
inhibition of DNA
fragmentation
Blackberry, Phenolic acids, Chemical antioxidant 5–68 μmol TE/g, [6]
black catechins, capacity, LDL 0.5 mg/mL,
raspberry, and flavonoids, and cholesterol oxidation 0.10 mg/mL
blueberry meal anthocyanins inhibition (48–60% at
seeds 22 h), supercoiled DNA
strand breakage
inhibition (80–98%)
Chlorogenic Model system Binding of chlorogenic 1–10 μM [31]
acid-BSA acid to BSA inhibits chlorogenic acid
LDL oxidation in a þ4% BSA
dose-response way
Blueberries Phenolic acids, Cellular antioxidant 13.5–63.5 μmol [32]
flavonoids, and capacity on HepG2 cells QE/100 g
anthocyanins
Litchi pulp Phenolic acids, Cellular antioxidant 7.4 1.5 μmol [33]
()-epicathechin, activity on HepG2 cells, QE/100 g,
4-methylcatechol, ORAC antioxidant 286.1 1.5 μmol
rutin, quercetin capacity TE/100 g
Cancer treatment or prevention
(continued)
Table 1 (continued)
Maize lime- Feruloyl Induction of phase II 1.28–3.07 μg/mL [34]
cooking putrescines, enzyme quinone
wastewater ferulic acid reductase
(nejayote)
Dehulled Catechin, phenolic Antiproliferative activity 66.4–159.4 mg/mL [25]
highland acids on hepatic cancer
barley HepG2 cells (IC50)
Cactus Isorhamnetin Apoptotic activity on 4.9–34.8 μg/mL [35]
cladodes glycosides colon cancer HT-29
(Opuntia ficus- (IC50) cells, mediated by
indica) caspase 3/7
Tea catechins Epigallocatechin Antiproliferative activity 10–20 μg/mL [36]
encapsulated gallate bound to on colon cancer HT-29
in milk casein micelles cells (IC50)
Foxtail millet Phenolics Apoptotic activity on 0.28–1.43 mg/mL. [37]
(Setaria colon cancer HCT-116 0.57–0.88 mg/mL,
italica) bran cells, mediated by ROS 0.5–0.7 mg/mL
pathway, and inhibition
of NF-κB pathway
Carbohydrate and lipid metabolism
Citrus fruits Phenolic acids, Inhibition of Not reported [38]
hesperidin α-glucosidase
(49.3–80.7%)
Foxtail and p-coumaric, Inhibition of α-amylase 38.3–54.3 FAE μg/ [39]
little millet caffeic and ferulic (IC50) and α-glucosidase mL, 31.3–35.5
acids, quercetin (IC50) FAE μg/mL
Whole grain Phenolic acids Modulation of maltose 34–85 GAE μg/mL [15]
oats and hydrolysis (49–61%) and 8.5–17 GAE
products Inhibition of intestinal μg/mL
glucose transport
(31–67%)
Shaddock Not reported Inhibition of α-amylase 80–320 μg/mL [40]
(Citrus (12.9–64-2%) and
maxima) peels α-glucosidase
(45.9–95.9%)
Soybeans Not reported Inhibition of α-amylase 320.5 and [41]
(IC50) and α-glucosidase 458.7 μg/mL
(IC50)
Black quinoa Phenolic acids and Mild inhibition of 37.6–55.6 mg/mL [42]
seeds flavonoids α-glucosidase (IC50) and and
pancreatic lipase (IC50) 9.0–10.5 mg/mL
Grapefruit peel Resveratrol, HMG-CoA reductase 115.3 μg/mL [43]
caffeic acid, (IC50) inhibition
ellagic acid, and
other phenolic
acids
Inflammation and cardiovascular health
[44]
(continued)
Table 1 (continued)
Cactus Isorhamnetin In vivo decrease of cell 5 mg/kg (rat
cladodes glycosides infiltration (51.8%); animal model)
(Opuntia ficus- inhibition of: NO•
indica) (77.2–81.4%), COX-2
(76.3–77.7%), TNF-α
(85.2%), and IL-6
(53.0%)
Antihypertensive and antiulcerative
Grapefruit peel Resveratrol, Angiotensin converting 137.4 μg/L [43]
caffeic acid, enzyme (ACE)
ellagic acid, and inhibition (IC50)
other phenolic
acids
Raw and Not identified ACE inhibition (40%) 8 mg FAE/mL. [45]
cooked by pigmented rice and 4 mg FAE/mL
pigmented and (20%) by nonpigmented
nonpigmented rice extracts
rice
Ginger Cinnamic and p- Parietal cell proton 1.5 μg/mL, 38 μg/ [30]
coumaric acids pump inhibition (IC50), mL
Helicobacter pylori
growth inhibition (MIC)
capacity corresponded to the bound phenolic fraction [25]. The proportion of

bound phenolics with respect to total content may vary among different cultivars
or varieties of the same species. In different colored bran rice, it was seen that
bound phenolic DPPH radical scavenging capacity represented from 7% to 41% of
the total antioxidant capacity of rice, while the ORAC activity varied from 26% to
75% of the total capacity coming from the bound fraction [48]. These results
indicated that, in the case of whole grains, evaluating the effect of only free
phenolics would underestimate the total antioxidant potential of these particular
matrices.
Feruloyl arabinoxylans form an interesting group of bound phenolic compounds
with antioxidant potential. The antioxidant capacity of arabinoxylans obtained from
rice was found to be a synergy between the phenolic and carbohydrate moieties.
Lower IC50 values were reported (1.14–1.24 mg) with respect to the predicted
amount (54.8–56.9 mg), calculated as the equivalent concentration of ferulic acid
bound to the fiber [27]. The authors of this work concluded that ferulic acid is also
able to exert antioxidant capacity without needing to be released. Similar results
were observed for arabinoxylans extracted from sorghum and maize bran, in which
antioxidant capacity in the extracts was attributed to ferulic acid and other bound
phenolics to this cell wall component [28]. Since arabinoxylans can be used as
ingredients in gluten-free bread formulation, antioxidant capacity of the bound
phenolics to the carbohydrates can give added value to such food products [49].
With respect to other non-grain sources, the contribution of bound phenolics to

antioxidant capacity is generally lower than 50%, depending on the source. In the
case of spices such as cinnamon, oregano, and clove, bound phenolics represented
between 3% and 17% of the total antioxidant capacity evaluated by the FRAP
method and 3–28% by the DPPH method [29]. In the case of litchi pulp, variation
in total antioxidant capacity and the proportion represented by bound phenolics was
variable among different cultivars, from 10% to 49% of the antioxidant capacity
measured by FRAP assay, and EC50 values in DPPH method demonstrated that
bound phenolics were three to ten times less effective than free phenolics [50].
Similar results were obtained for litchi pulp when using the ORAC method, where
bound antioxidant capacity was reported as 286.1 μmol TE/100 g, while free ORAC
values reached up to 3406.8 μmol TE/100 g, representing 7.7% of the total antiox-
idant capacity [33]. Therefore, in general terms, contribution of bound phenolics to
antioxidant capacity of herbs and fruits is lower when compared to cereals and other
grains. However, in fruit seeds, a higher amount of antioxidants can be found in the
bound fraction. For example, 35–70% of total antioxidant capacity from blackberry,
black raspberry, and blueberry seed meals were accounted after hydrolyzing bound
phenolics [6].
Binding of PCs to proteins, such as bovine serum albumin (BSA), can affect
their antioxidant potential. Such was the case with a quercetin-BSA complex, which
had a lower Trolox equivalent antioxidant capacity (TEAC) (2.0–4.6 mM), at
concentrations from 7.9 to 17.5 μg of quercetin/mg of protein, with respect to the
same amounts of free quercetin (4.4–6.0 mM). Furthermore, a higher antioxidant
capacity (3.0–4.0 mM) of the peptide containing 12.2 μg of quercetin/mg of protein
obtained after trypsin hydrolysis with respect to the same amount of free quercetin
(5.0 mM) [26].
4.1.2 Antioxidant Capacity in Biological Systems

So far, the antioxidant capacity has been discussed in terms of chemical assays;
however, the real protective potential of a compound requires evaluation in more
complex systems. Antioxidant activity of ginger (Zingiber officinale) extracts was
analyzed by two methods to determine its protective effect on biomolecules. Results
showed that cinnamic acid and p-coumaric-rich extract inhibited lipid peroxidation
(48–60%) at 5.2 μg gallic acid equivalent/g rat liver homogenate (μg GAE/gliver) and
protected against DNA oxidative fragmentation (80–98% inhibition) when adding
2–4 GAE μg [30]. Blackberry, black raspberry, and blueberry seed meal extracts at
0.5 mg/mL inhibited LDL cholesterol oxidation from 46% to 60% after 22 h
incubation, and DNA strand breakage was inhibited at levels up to 98% with
addition of 0.10 mg/mL of extract [6]. Cellular antioxidant activity is a measurement
that takes into account the internalization of the compounds into the cell, therefore,
making a more realistic approach to the actual antioxidant activity of a given
compound or extract in a living system [51]. This assay has been used to evaluate
the antioxidant potential of sources, such as blueberry extracts, where CAA values
were found in the range of 13.5–63.5 μmol quercetin equivalents (QE) per 100 g
fresh weight of berries, from which 8% to 20% of the total phenolic content
corresponded to the bound fraction [32]. In the case of litchi pulp, bound phenolic
fraction exerted a lower antioxidant capacity (7.4 1.5 μmol QE/100 g) than its
corresponding free fraction, which reached up to 56.7 μmol QE/100 g, where bound
fraction represented 11% of the CAA [33].
As described with respect to chemical antioxidant evaluation, PCs bound to
proteins have also exhibited activity in other models. One example is the inhibition
of LDL cholesterol oxidation when incubated in the presence of 4% BSA solutions
with different doses of chlorogenic acid. It was shown that up to 80% delay in
oxidation initiation occurred when 10 μM chlorogenic acid was used in the system,
after removing the non-bound phenolic acid from the solution [31]. This study shows
promising activity for these complexes, particularly when considering that protein-
binding can confer protection during the digestion process but allowing release and
increase of bioavailability, as well as stability during storage [16, 52].
In general, these studies have been conducted after hydrolyzing the bound
phenolic fraction from the matrix, by any of the methods discussed in Sects. 3.1
and 3.2. This approach allows us to determine whether bound phenolics should be
taken into account when evaluating the antioxidant capacity of a given food source.
However, when considering bioavailability, bound phenolics are usually not released
and absorbed during gastrointestinal digestion [23]. Food processing such as fer-
mentation, malting, extrusion, or cooking allows the release of this fraction, increas-
ing its uptake and making it available to target organs [11].
Therefore, most of the classical assays underestimate the true antioxidant capacity
of foods since most of the compounds responsible of this activity are bound to
insoluble matrices. Most of the research on the total antioxidant capacity of foods has
ignored the interactions that may occur due to the coexistence of multiple antioxi-
dants. In fact, the multiple extraction procedures used as a preliminary step for
classical antioxidant determination hinder the generation of a standardized database.
“QUENCHER” (Quick, Easy, New, CHEap, Reproducible) is a simple and direct
assay for antioxidant capacity measurement that is extraction-independent and may
enable interlaboratory comparison [47]. With this method, the functional groups of
the soluble and insoluble bound antioxidants in solid samples make contact with free
radicals via the liquid-liquid type of reaction and solid-liquid type of reaction,
respectively [47].
4.2 Cancer
Antioxidant mechanisms are proposed as one of the primary ways in which PCs exert
their bioactive potential [3]. However, there is evidence of additional anticancer
mechanisms for preventive and prophylactic effect of PCs against cancer (Table 1).
One of the ways in which BPs can contribute to chemoprevention of cancer is
inducing phase II enzymes, such as NADPH-dependent quinone reductase (QR),
which work in vivo to inactivate free radicals and electrophiles [53]. Acosta-Estrada
et al. (2015) determined that a bound phenolic extract obtained from maize lime-
cooking wastewater solids was able to induce QR activity at a concentration of
76 μg/mL and presented a chemopreventive index of 4.58 (cytotoxicity requires a

4.58-fold higher concentration). In this study, QR induction was attributed to
coumaroyl-feruloyl and di-feruloyl putrescines that were able to induce QR at
1.28 μg/mL and 3.07 mg/mL, respectively [34].
Antiproliferative effect of BPs released from different sources has been assayed
on diverse cell lines. Catechins and phenolic acids from dehulled highland barley
showed antiproliferative potential on hepatic cancer HepG2 cells (66.4 μ IC50
159.4 mg/mL), observing variation among four barley varieties [25]. In this study, a
lower bioactivity occurred with bound phenolics with respect to free phenolics.
Glycosylated flavonoids released by alkaline hydrolysis from Opuntia ficus-indica
cactus pads and subsequently purified presented a high antiproliferative effect.
Alkaline crude extracts were effective on HT-29 (IC50=4.9–9.1 μg/mL) and Caco-
2 (IC50=8.2–16.7 μg/mL) colon cancer cell lines. Purified isorhamnetin diglycosides
presented IC50 values from 8.6 to 15.2 μg/mL on HT-29 and 25.8–36.7 μg/mL Caco-
2 [35]. The authors determined that glycosylation pattern of the isorhamnetin
derivatives had an important impact, since diglycosides were more effective than
triglycosides [35].
Haratifar et al. (2014) demonstrated that PCs capacity to bind to protein could
allow encapsulation of compounds, such as epigallocatechin gallate, in casein
micelles, while preserving antiproliferative activity on HT-29 cells (16 IC50
20 μg/mL) [36]. This PC-protein complex could be an alternative for delivery of PCs
as a food ingredient or dietary supplement, such as fortified breads, dairy products,
and processed meats [16, 52].
Besides the evaluation of the antiproliferative effect, some mechanisms related to
the anticancer effect of BPs have been explored. In the case of Opuntia ficus-indica
alkaline extracts, an increase in caspase 3/7 was observed by flow cytometry in HT-
29 (33%) and Caco-2 (28–43%) cells treated with the extracts when compared to
non-treated cells. Moreover, apoptosis was observed in 32–53% of HT-29 cells
treated with isorhamnetin diglycosides and 59% when crude extract was used [35].
On the other hand, bound phenols from foxtail millet induced apoptosis by gener-
ation of reactive oxygen species (ROS) in HCT-116 colon cancer cells at concen-
trations from 0.57 to 0.88 mg/mL. Additionally, apoptotic effect was shown by a
decrease in Bcl-2 protein and increase in BAX protein, along with inhibition of the
NF-κB pathway [37]. Foxtail millet bran contains BPs such as catechin, myricetin,
daidzein, luteolin, quercetin, apigenin, naringenin, and kaempferol, likely account-
able for the bioactive effect [39].
4.3 Carbohydrate and Lipid Metabolism
Phenolic compounds, such as phenolic acids and flavonoids, are known inhib-
itors of starch-hydrolyzing enzymes α-amylase and α-glucosidase, capable of
delaying carbohydrate digestion [54, 55] or even absorption by glucose trans-
porters [15]. Bound phenolics also have potential to interfere with the activity of
the fat-digesting enzymes known as lipases, due to PCs affinity with proteins,
diminishing the rate of fat absorption [56].
The BP fraction of several citrus fruits showed α-glucosidase inhibitory activity
[38]. The main compounds found in these extracts were ferulic acid, p-coumaric
acid, vanillic acid, and the flavonoid hesperidin. It can be noted that the authors
reported in the same study that these extracts activated α-amylase and angiotensin
converting enzyme (ACE) instead of inhibiting them [38]. Other authors report a
strong inhibitory potential, as observed with BP extracts from foxtail
(IC50=35.21–35.26 μg ferulic acid equivalents per mL) (μg FAE/mL) and little
millet (IC50=38.27–46.9 μg FAE/mL); in both cases the extracts were obtained
from hull and bran [39]. Pradeep and Sreerama (2017) determined that p-coumaric,
caffeic, and ferulic acids were the major component of the extracts. Additionally,
α-glucosidase inhibition was observed for BPs obtained from whole grain oats, with
a 49.6–61.3% decrease in maltose hydrolysis at 34–85 μg gallic acid equivalents per
gram (GAE μg/g). In this case, ferulic acid represented from 52% to 89.3% of bound
phenolics from whole grain oats [15]. The effect of black quinoa seed BPs was
weaker, with IC50 in the range between 37.6 and 55.6 mg/mL, and the main
components of the extract were phenolic acids and flavonoids [42].
With respect to α-amylase, inhibition by PCs has also been reported. Foxtail and
little millet showed IC50 values of 41.7 and 38.3 FAE μg/mL, respectively, in the hull
fraction and 54.3 and 46.9 FAE μg/mL in the bran fraction [39]. Bound phenolics
from the citrus shaddock (Citrus maxima) fruit peel were able to inhibit α-amylase
activity up to 64.2% in a dose-dependent manner, at concentrations from 80 to
320 μg/mL [40]. Soybean BP extracts were found to inhibit 50% of α-amylase
activity at 320.5 μg/mL [41]. In general, phenolics are stronger inhibitors of
α-glucosidase than of α-amylase, such as in the case of BPs extracted from little
millet and shaddock peels, where significant differences were observed [39]. This
difference in BPs effect on both enzymes could be important when considering their
use as a therapeutic agent for hyperglycemia, since inhibiting α-glucosidase preferen-
tially can prevent side effects from excess inhibition of α-amylase [40, 41]. However, it
should be taken into account that in some cases α-amylase was activated by treatment
with BPs, as happened with whole grain oat phenolics and citrus fruits [15, 38].
4.4 Interactions with Microbiota
Bound phenolics help to maintain a “healthy” microbiota. For example, cocoa

bound flavanols increased the number of Lactobacilli and Bifidobacteria [57].
Many bound phenolics resist enzymatic digestion (oral, gastric, and small and
large intestine phases) and exert their antioxidant activity in the large intestine
[58]. But more importantly, their release is slow and continuous [59]. The slow
release of bound phenolic compounds could be related to the complexity of the
matrix and the diversity of microorganisms required to breakdown the chemical
interactions [60].
5 Conclusions
For many years, bound phenolics were considered as poor contributors to improve
human health due to their strong interactions with the food matrix and low bioavail-
ability. Particularly in cereals, bound phenolics are major contributors to total phenolic
content and antioxidant activity. Most of the studies on bound phenolic compounds
involve their extraction after acid or base hydrolysis, and therefore a lot about their
chemical structure is known. But when gastrointestinal microbiota is taken into consid-
eration as an important element in the digestibility of dietary fiber, the release of bound
phenolics is more complex as well as their potential bioactivity. Microbiota heteroge-
neity is important to have a vast enzymatic tool to release bound phenolics continuously
and slowly to maintain a constant dose of circulating bioactive compounds.
Acknowledgments We appreciate the support from NutriOmics Chair from Tecnológico de

Monterrey.
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Tea, Coffee and Health Benefits
35
Sumio Hayakawa, Yumiko Oishi, Hiroki Tanabe, Mamoru Isemura,
and Yasuo Suzuki
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
2 Effects on Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
2.1 Effects of Tea on Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996
2.2 Effects of Coffee on Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003
2.3 Simultaneous Evaluation of the Anticancer Effects of Tea and Coffee . . . . . . . . . . . . . 1009
3 Effects on Metabolic Syndrome and Related Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
3.1 Effects of Green Tea on Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011
3.2 Effects of Green Tea on Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1014
3.3 Effects of Green Tea on Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1016
3.4 Effects of Green Tea on Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018
3.5 Effects of Coffee on Metabolic Syndrome and Related Disorders . . . . . . . . . . . . . . . . . . 1020
3.6 Effects of Coffee on Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
3.7 Effects of Coffee on Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025
3.8 Effects of Coffee on Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026
3.9 Comparison of the Effects of Tea and Coffee in Simultaneous Studies on
Metabolic Syndrome and Related Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1030
S. Hayakawa (*) · Y. Oishi

Department of Cellular and Molecular Medicine, Medical Research Institute, Tokyo Medical and
Dental University, Bunkyo-ku, Tokyo, Japan
H. Tanabe
Department of Nutritional Sciences, Faculty of Health and Welfare Science, Nayoro City
University, Nayoro-City, Hokkaido, Japan
M. Isemura
Tea Science Research Center, University of Shizuoka, Suruga-ku, Shizuoka, Japan
Y. Suzuki
Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa,
Hyogo, Japan

992 S. Hayakawa et al.
4 Effects on Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1030

4.1 Effects of Green Tea on Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1030
4.2 Effects of Coffee on Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035
Abstract
A number of epidemiological studies and clinical trials have reported the bene-
ficial effects of both green tea and coffee on human health, including anticancer,
anti-obesity, antidiabetic, antihypertensive, and hepatoprotective effects. Further-
more, these findings in humans are supported by cell-based and animal experi-
ments. These effects have been attributed to epigallocatechin gallate (EGCG) in
green tea and chlorogenic acid (CGA) in coffee, which have been proposed to
function via various mechanisms of action, the most important of which appears
to implicate reactive oxygen species (ROS). Both EGCG and CGA can exert
conflicting dual actions as an antioxidant and a prooxidant. Their antioxidative
action can scavenge ROS, leading to downregulation of nuclear factor-κB to
produce various favorable effects such as anti-inflammatory effects and cancer
cell apoptosis. The prooxidant actions, however, can promote the generation of
ROS leading to the activation of 5’AMP-dependent protein kinase, which mod-
ulates various enzymes and factors with beneficial roles. At present, it remains
unclear how EGCG and CGA can be directed to act as either a prooxidant or an
antioxidant, although their cellular concentrations, the presence of metal cations
such as Cu+ and Fe++, and the redox state of the cells appear to be important
factors. Notably, several human studies did not report the beneficial health effects
of green tea and coffee. The inconsistent results may have been caused by various
confounding factors including smoking, intestinal microbiota, and genetic fac-
tors. This chapter examines the current information on these properties of green
tea and coffee with the aim of improving the understanding of a way to enjoy
healthy longevity.
Keywords
Green tea · Coffee · Polyphenol · Catechin · EGCG · Chlorogenic acid · Human
health · ROS · NF-κB
Abbreviations
ACC Acetyl-CoA carboxylase
ACE Angiotensin-converting enzyme
ACF Aberrant crypt foci
ALT Alanine aminotransferase
AMPK 50 -AMP-activated protein kinase
ANI α-Naphthylisothiocyanate
AOM Azoxymethane
AST Aspartate aminotransferase
35 Tea, Coffee and Health Benefits 993
BBN N-Butyl-N-(4-hydroxybutyl)-nitrosamine
BMI Body mass index
C/EBP CCAAT/enhancer-binding protein
CGA Chlorogenic acid
CLL Chronic lymphocytic leukemia
COX Cyclooxygenase
CPP Coffee polyphenols
DBP Diastolic blood pressure
EC ()-Epicatechin
EGCG ()-Epigallocatechin-3-gallate
ERK Extracellular signal-regulated kinase
FASN Fatty acid synthase
G6Pase Glucose-6-phosphatase
GCE Green coffee extract
GLUT Glucose transporter
GST Glutathione S-transferase
GTC Green tea catechin
GTE Green tea extract
GTP Green tea polyphenol
HbA1c Hemoglobin A1c
HBV Hepatitis B virus
HCV Hepatitis C virus
HFD High-fat diet
HNF Hepatocyte nuclear factor
HO Heme oxygenase
HR Hazard ratio
HuR Human antigen R
IFN Interferon
IGF Insulin-like growth factor
IL Interleukin
IRS Insulin receptor substrate
LPL Lipoprotein lipase
LXR Liver X receptor
MAPK Mitogen-activated protein kinase
MetS Metabolic syndrome
MMP Matrix metalloproteinase
mTOR Mechanistic target of rapamycin kinase
NAFLD Nonalcoholic fatty liver disease
NF-κB Nuclear factor-kappa B
NO Nitric oxide
NOS Nitric oxide synthase
Nrf2 Nuclear factor, erythroid 2 like 2

OR Odds ratio
PCa Prostate cancer
PEPCK Phosphoenolpyruvate carboxykinase
PKC Protein kinase C
PPAR Peroxisome proliferator-activated receptor
PPE Polyphenon E
QHD Qushi Huayu Decoction
RR Relative risk
RXR Retinoid X receptor
SBP Systolic blood pressure
SHR Spontaneously hypertensive rats
SREBP Sterol-responsive element-binding protein
STAT Signal transducer and activator of transcription
STZ Streptozotocin
T2DM Type 2 diabetes mellitus
TNF Tumor necrosis factor
Treg Regulatory T
VEGF Vascular endothelial growth factor
1 Introduction
Green tea is produced by the processing of tea leaves from the plant Camellia
sinensis (Theaceae) and is popularly consumed worldwide, particularly in Japan
and China. Green tea has been shown to have beneficial effects on human health
such as anticancer, anti-obesity, antidiabetic, anti-cardiovascular, anti-infectious, and
hepatoprotective effects [1–6]. Most of these biological effects are thought to be
ascribable to polyphenol catechins, specifically ()-epigallocatechin-3-gallate
(EGCG) (Fig. 1), which is the major catechin. A single 200 mL cup of typically
brewed green tea supplies 240–320 mg of catechins, of which EGCG accounts for
60%–65%, together with much lower quantities of other polyphenols including
quercetin, myricetin, and kaempferol [7]. Green tea is a rich source of caffeine
(Fig. 1), which has strong physiological effects on bodily systems such as the central
nervous, respiratory, cardiovascular, urinary, and gastrointestinal systems [8].
Black tea is also produced from C. sinensis through enzymic processing (some-
times called fermentation) by intrinsic enzymes and microorganisms during which
catechins are polymerized to yield catechin derivatives such as theaflavin (Fig. 1)
and theasinensins [9]. It has been shown to have physiological effects similar to
those of green tea, albeit with a lesser efficacy than green tea in most cases.
Coffee is also consumed worldwide and, like green tea, exerts various health-
related effects. It contains about 2,000 chemicals, including caffeine, and the major
polyphenols are chlorogenic acid (CGA) or 5-caffeoylquinic acid (Fig. 1) and its
derivatives, which amount to about 3 g per 100 g of roasted coffee powder [8].
Fig. 1 Chemical structures of compounds pertinent to this chapter
A single serving of coffee provides 20–675 mg of CGAs [10]. It should be noted that
a recent analysis using ultrahigh-performance liquid chromatography showed that
green tea leaves collected from public markets in Brazil contained 1.1 g of CGAs per
100 g of dried leaves [11].
In this review, we discuss recent evidence that supports the beneficial effects of
tea and coffee consumption in relation to the mechanistic aspects of catechins and
CGAs by focusing on selected diseases in which we have studied the action of green
tea. Caffeine, a highly bioactive constituent contained in both tea and coffee, has
been comprehensively reviewed by Temple et al. [12] and is discussed only briefly
here. For the sake of readability, 95% confidence interval values and statistical
p-values, which an original datum contains in statistical evaluation, are not presented
here unless otherwise described.
2 Effects on Cancer
2.1 Effects of Tea on Cancer
2.1.1 Human Epidemiological Studies

A number of human epidemiological studies have shown that green tea exerts
beneficial effects against various cancers [1–6]. A review article by Yang et al.
reported an inverse association between green/black tea consumption and cancer risk
for various types of cancer including bladder, breast, colon, gastric, kidney, lung,
ovarian, pancreatic, and prostate cancers in 51 of 127 case-control studies and 19 of
90 cohort studies which were carried out from 1965 to 2008 [1]. Yuan and co-
workers reviewed the results of 13 studies in which green tea consumption was
associated with a significant risk reduction for breast, colorectal, gastric, esophagus,
liver, lung, oral cavity, and prostate cancers in 9 studies [13, 14].
Recent studies also demonstrated the beneficial effects of tea, with some exam-
ples as follows. The European Prospective Investigation into Cancer and Nutrition
study, involving 486,799 men/women for a median follow-up of 11 years, found that
increased tea intake was associated with a 59% reduction in the risk of developing
hepatocellular carcinoma (HCC) [15]. The analysis of 87 datasets from 57 studies,
which included a total of 49,812 subjects, showed that high tea consumption was
associated with a reduced risk of oral cancer with a risk ratio (RR) of 0.72, although
it had no significant effect on the risk of bladder, breast, colon, gastric, liver, lung,
rectal, ovarian, pancreatic, and prostate cancers or gliomas. In a subgroup analysis of
individuals in Western countries, the consumption of tea was associated with a
reduced risk of bladder cancer, although the consumption of black tea was associated
with an increased risk of breast cancer [16].
The results of a hospital-based, case-control study including 160 cases and 320
controls in China showed that the regular consumption of larger amounts of green tea
(35 g/week) was associated with a lower risk of stomach cancer, with odds ratios
(ORs) of 0.72 and 0.53, respectively. Among regular tea drinkers, lower temperature
and longer interval between tea being poured and drunk also reduced the risk,
suggesting that green tea was inversely associated with risk of stomach cancer [17].
In a study to determine the prostate cancer (PCa) risk associated with green tea
and EGCG intake among Hong Kong Chinese men, data from 32 cases and 50
controls showed that habitual green tea drinking had an adjusted OR of 0.60. An
inverse association was also found between intake of EGCG and PCa risk [18].
Similarly, data from the Japan Public Health Center-Based Prospective Study
showed that green tea intake may decrease the risk of advanced PCa [19].
A case-control study in Vietnamese men showed that after adjustment for
confounding factors, increased tea consumption was associated with a reduced risk
of PCa [20]. The adjusted ORs were 0.52 and 0.30 for participants drinking
100–500 mL/day and >500 mL/day, respectively, relative to those drinking
<100 mL/day. Significant inverse dose-response relationships were also observed
for years of drinking and number of cups consumed daily, showing that habitual tea
consumption was associated with a reduced risk of PCa. A meta-analysis of seven
epidemiological studies and three randomized controlled clinical trials indicated that
green tea consumption reduced the incidence of PCa with a linear dose-response
effect and significantly reduced the risk of PCa risk at more than 7 cups/day [21].
A meta-analysis of eight studies comprising 18 independent reports on biliary
tract cancer showed that tea intake reduced the risk of cancer by about 34%
compared with a no-intake group. This inverse relationship was statistically signif-
icant in women but not in men [22]. Chen et al. conducted a meta-analysis to
evaluate relationships between tea intake and the risk of biliary tract cancer in 29
qualified studies. The summary OR of developing colorectal cancer for the highest
versus the lowest tea consumption was 0.93. A stratified analysis revealed that tea,
especially green tea, had a protective effect in female and rectal cancer patients. The
dose-response analysis showed that there was a significant inverse association
between an increment of 1 cup/day of tea consumption and colorectal cancer risk
(OR, 0.68) in women [23].
A meta-analysis performed in April 2016 in a total of 18 (11 case-control and
7 cohort) studies, comprising data for 701,857 female subjects including 8,683
ovarian cancer cases, showed that tea consumption had a significant protective effect
against ovarian cancer (relative risk [RR], 0.86). The relationship was
confirmed after adjusting for family history of cancer (RR, 0.85), menopause status
(RR, 0.85), education (RR, 0.82), body mass index (BMI) (RR, 0.85), and smoking
(RR, 0.83) [24].
2.1.2 Clinical Studies

One of the most significant studies may be that of an Italian research group which
showed that green tea catechins (GTCs) were safe and highly effective for the
treatment of premalignant lesions prior to the development of PCa. Only 1 tumor
was detected among a group of 30 men with precancerous lesions who received daily
oral administration of 600 mg GTCs, as compared with 9 detected tumors among 30
placebo-treated male patients after 1 year [25]. A later systematic review of 15
studies with 11 reports on the effect of green tea consumption on PCa prevention and
4 reports on the effect of green tea on treatment revealed that green tea appeared to be
an effective chemopreventive agent for PCa, particularly in patients with high-grade
prostate intraepithelial neoplasia, although evidence of efficacy in the treatment of
PCa is currently lacking [26].
To investigate whether erythrocyte oxidative stress was associated with PCa and
whether daily consumption of green tea improved the oxidative phenotype, Lassed et
al. performed a study on 70 Algerian PCa patients and 120 age-matched healthy
subjects. The results at baseline showed reduced glutathione levels and catalase
activity and a high level of malondialdehyde in erythrocytes from PCa patients. The
consumption of 2–3 cups of green tea per day for 6 months significantly increased
glutathione concentration and catalase activity and decreased malondialdehyde
concentration. Green tea also significantly decreased oxidative stress in these
patients, indicating that regular consumption of green tea for a long period may
prevent the development of PCa or at least delay its progression [27].
In a clinical trial in ten patients with stage 0 chronic lymphocytic leukemia (CLL)
and ten healthy subjects administered oral green tea extract (GTE) therapy for
6 months, eight out of ten patients showed a reduction in lymphocytosis and absolute
number of circulating regulatory T (Treg) cells. Only one nonresponding patient had
disease progression at 5 months after the end of GTE administration and chemo-
therapy. These findings suggest that green tea can control lymphocytosis and prevent
disease progression [28].
In a clinical trial in 124 subjects who were recruited and randomly assigned to
low-dose GTCs (500 mg), high-dose GTCs (1,000 mg), or placebo for 3 months,
urinary fumonisin B1, a carcinogen, was significantly decreased after 1 month in the
high-dose group compared with the placebo group, with reduction rates of 18.95% in
the low-dose group and 33.62% in the high-dose group. After a 3-month interven-
tion, urinary levels of fumonisin B1 were reduced to 40.18% in the low-dose group
and 52.6% in the high-dose group compared with both the placebo group and
baseline levels. These findings suggest that supplementation with GTCs may repre-
sent a useful chemopreventive strategy for reducing co-exposure to aflatoxin B1 and
fumonisin B1 [29].
In a randomized placebo-controlled trial, 99 women received either Polyphenon
E (PPE), a green tea polyphenol formulation primarily consisting of EGCG, or
placebo once a day for 4 months. A complete response, defined as negative for
high-risk human papilloma virus and normal histopathology, was noted in 17.1%
and 14.6% of women in the PPE and placebo arms, respectively, showing a
preferable effect of PPE [30].
In a phase II pharmacodynamic prevention trial of PPE, patients with bladder
tumors were randomized to receive PPE containing either 800 or 1,200 mg of EGCG
or placebo for 14–28 days prior to transurethral resection of the bladder tumor or
cystectomy. EGCG levels in plasma and urine increased significantly, and the
expression of proliferating cell nuclear antigen and clusterin was downregulated in
the bladder tissues. Despite the limitations of this pilot study, the authors pointed out
that the observed pharmacodynamics and desirable biological activity warranted
further clinical studies of PPE in bladder cancer prevention [31].
In a randomized clinical trial to evaluate GTE for the prevention of metachronous
colorectal polyps, 143 patients who underwent the endoscopic removal of colorectal
adenomas were divided into a supplementation group (0.9 g GTE/day for 12 months)
and a control group without supplementation. Follow-up colonoscopy after conducted
12 months found that the incidence of metachronous adenomas was 42.3% in the
control group and 23.6% in the GTE group. The number of relapsed adenomas also
decreased in the GTE group compared with that in the control group [32].
Although several studies have demonstrated the anticancer effects of green tea, as
described above, conflicting results have also been reported [1, 4]. For example, Je
and Park identified five eligible cohort studies comprising 231,870 female partici-
pants and 1,831 cases of endometrial cancer [33]. The pooled RR of the three studies
conducted in the United States, in which black tea was consumed by most people,
was 1.00. These findings do not support an association between tea consumption and
endometrial carcinogenesis risk. Furthermore, a meta-analysis of 25 case-control
studies (15,643 patients and 30,795 controls) and 7 prospective cohort studies (1,807
cases and 443,076 participants) showed that tea consumption was not significantly
associated with bladder cancer risk [34].
In a double-blind randomized controlled trial, subjects with primary multifocal
high-grade prostatic intraepithelial neoplasia and/or atypical small acinar prolifera-
tion received 35 mg lycopene, 55 μg selenium, and 600 mg GTCs, or placebo, per
day for 6 months. The results indicated that the administration of high doses of
lycopene, GTCs, and selenium in men was associated with a higher incidence of
PCa, suggesting that the use of these supplements should be avoided [35].
In a comprehensive review article, Yang and Wang concluded that the results of
human studies on GTCs, mostly from small randomized clinical trials, have been
inconsistent [6]. The authors highlighted the following examples. An earlier ran-
domized clinical trial on oral cancer prevention in China showed that a mixed tea
product (3 g/day) caused a significant decrease in cancer growth, but a later phase II
randomized clinical trial in the United States showed that GTE (500, 750, or
1,000 mg/m2, twice daily) for 12 weeks resulted in only potentially beneficial
effects, which were not significant in reducing oral premalignant lesions. Further-
more, in spite of seemingly promising results reported in the Italian intervention
study mentioned previously, a subsequent trial in Florida with a similar design
showed that catechin supplementation for 6–12 months did not cause a reduction
in the number of PCa cases compared with placebo [6].
Thus, further studies are needed to determine the chemopreventive effects of
green tea, but its potentially beneficial effects are supported by Yang and Wang in a
phase 2 trial in patients with early CLL where oral doses of PPE (2,000 mg twice/
day) caused a sustained decline in absolute lymphocyte count and/or lymphadenop-
athy in the majority of patients [6]. Furthermore, in a study to assess salivary
antioxidant alterations in smokers, participants who consumed 2 cups of green tea
per day (2 g of green tea dissolved in 150 mL hot water per cup) had increased levels
of salivary antioxidants, suggesting that green tea may reduce the rate of oral cancer,
given the likely association between oxidative stress and oral cancer [36].
This optimistic expectation may also be supported by a highly encouraging case
report of an EGCG-based ointment (PPE/sinecatechins), approved by the US Food and
Drug Administration, which was successfully used to treat anogenital warts. Rob et al.
found that after application of the ointment for 10 weeks in an 11-year-old child, the
warts disappeared completely without recurrence during a 12-week follow-up [37].
2.1.3 Laboratory Studies and Mechanism of Action

A large number of animal and cell-based experiments have indicated the anticancer
effects of green tea [1–6, 38, 39]. For black tea and oolong tea, however, fewer
studies are available. Hibasami et al. reported for the first time that catechins,
including EGCG, induced apoptosis or programmed cell death in cancer cells [40].
Our research group also observed similar apoptosis-inducing actions of EGCG and
has proposed that the binding of EGCG to the cell surface Fas protein is involved in
its anticancer activity [41]. Tachibana et al. found that the 67 kDa laminin receptor
on the cell surface is an EGCG receptor and mediates various types of EGCG
activity, including its anticancer activity [42]. The role of the protein-binding
capability of EGCG in its mechanism of action has been reviewed elsewhere by
Yang et al. as well as our research group [1, 2].
The number of animal and cell-based experiments showing anticancer effects of
EGCG and GTCs continues to increase, as described next. C3H/He mice (8 weeks
old; n = 46) were treated with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine
(BBN) solution for 14–24 weeks. Mice in the BBN þ GTP group (n = 47) were
additionally treated with 0.5% GTP solution over the same period. Cytoplasmic
human antigen R (HuR) expression in cancer cells increased at 14 and 24 weeks in
the BBN group compared with that in the control group and was associated with
increased invasion of tumor cells in muscle. However, these effects were not
observed in the BBN þ GTP group. GTP was independently associated with
cyclooxygenase (COX)-2 and heme oxygenase (HO)-1 expression, while cytoplas-
mic HuR expression was associated with COX-2 and vascular endothelial growth
factor (VEGF)-A levels. Expression of COX-2 and HO-1 was associated with cell
proliferation while that of VEGF-A and HO-1 was associated with angiogenesis.
Therefore, GTP potentially suppresses tumor cell proliferation and angiogenesis
both directly and indirectly via HuR-related pathways in bladder cancer [43].
When the cytotoxic effects of GTPs were examined in human cell lines (MCF-7,
A549, Hela, PC3, and HepG2 cells), GTPs were found to inhibit cell growth,
particularly in MCF-7 cells. Mechanistic studies showed that the main modes of
cell death induced by GTCs were cell cycle arrest at G1/M and G2/M and apoptosis.
GTP also caused a reduction in mitochondrial membrane potential, increased the
generation of reactive oxygen species (ROS), induced DNA fragmentation, and
activated caspase 3 and caspase 9 [44].
The cancer-preventive activity of PPE was demonstrated in an animal model of
colorectal cancer induced by azoxymethane (AOM). Dietary PPE increased the
plasma and colonic levels of tea polyphenols and decreased tumor multiplicity and
size. It also decreased β-catenin nuclear expression, induced apoptosis, and
increased the expression of retinoid X receptor (RXR)-α, β, and γ in adenocarci-
nomas. These results demonstrate the inhibitory effects of orally administered PPE
on colon carcinogenesis [45].
Posadino et al. investigated the impact of PPE on PCa cells. PPE treatment at 30
and 100 μg/mL significantly decreased cell viability and proliferation. PPE-induced
cell death was associated with mitochondrial dysfunction and the downregulation of
Akt activation. Cell exposure to the ROS scavenger N-acetylcysteine prevented
PPE-induced ROS increase, Akt activation impairment, and cell death, indicating the
causative role of ROS. In cells over-expressing Akt, PPE failed to cause ROS
increase, Akt activation impairment, and cell death. Thus, PPE induced apoptotic
cell death through a prooxidant rather than an antioxidant mechanism [46].
When estrogen receptor α-positive breast cancer T47D cells were treated with
0–80 μM EGCG, cell viability was decreased, and EGCG at 80 μM increased the
gene expression of PTEN, caspase 3, and caspase 9 but decreased that of Akt.
Furthermore, EGCG increased the Bax/Bcl-2 ratio of gene and protein expression
and decreased the gene expression of hTERT. These findings suggest that EGCG
may be a useful adjuvant therapeutic agent for the treatment of breast cancer [47].
Chen et al. found that EGCG inhibited the spheroid formation of colorectal cancer
cells as well as the expression of colorectal cancer stem cell markers, suppressed cell
proliferation, and induced apoptosis. EGCG downregulated the activation of the
Wnt/β-catenin pathway, supporting its potential as an anticancer agent targeting
colorectal cancer stem cells through the suppression of this pathway [48]. Aberrant
expression of β-catenin is associated with the progression of various cancers,
including head and neck cancer. Shin et al. found that EGCG induced apoptosis in
KB and FaDu cells via the suppression of β-catenin signaling and promotion of
ubiquitin-mediated 26S proteasomal degradation. These effects of EGCG were
confirmed in a syngeneic mouse model [49].
Harati et al. found that EGCG suppressed the proliferation and viability of
liposarcoma, synovial sarcoma, and fibrosarcoma cells [50]. Cornwall et al. showed
that EGCG at concentrations ranging from 25 to 100 μg/mL induced apoptosis in
CLL B-cells but did not affect healthy control B-cells. They also showed that,
in contrast to healthy controls, T-cells from CLL patients underwent apoptosis in
the presence of EGCG. Thus, EGCG differentially induces apoptosis in CLL B- and
T-cells but not in healthy B- and T-cells [51].
In an attempt to identify its anticancer activities against cholangiocarcinoma cells,
Kwak et al. found that EGCG inhibited the growth of HuCC-T1 cells but not of
human embryonic kidney 293 T cells, indicating that EGCG induced apoptosis in
cancer cells without adverse effects in normal cells. EGCG inhibited the expression
of mutant p53 and induced apoptotic molecular signals such as Bax/Bcl-2, caspases,
and cytochrome c. EGCG also inhibited the activity of matrix metalloproteinase
(MMP)-2/9, invasion, and migration. In an animal tumor xenograft model using
HuCC-T1 cells, EGCG inhibited tumor growth and suppressed carcinogenic molec-
ular signals such as Notch1, MMP-2/9, and proliferating cell nuclear antigen [52].
Similarly, Luo et al. showed that treatment of bladder cancer SW780 cells with
EGCG resulted in the significant inhibition of cell proliferation by induction of
apoptosis, without obvious toxicity to normal bladder epithelium SV-HUC-1 cells.
EGCG also inhibited SW780 cell migration and invasion at 25–100 μM. EGCG
induced apoptosis in SW780 cells by the activation of caspases 8, 9, and 3; Bax;
Bcl-2; and PARP. Animal studies demonstrated that EGCG decreased tumor volume
and weight in mice bearing SW780 tumors and downregulated the expression of
nuclear factor-kappa B (NF-κB) and MMP-9 at both the protein and mRNA level in
tumor and SW780 cells [53]. As exemplified by this finding and the studies described
above, EGCG exerts stronger apoptosis-inducing effects on cancerous cells than on

normal cells. Our research group has also provided evidence that differentiated HL-60
cells are notably less susceptible to apoptosis than undifferentiated cells [54].
Interestingly, Ward et al. reported that different diets may have different effects on
the action of GTE. They hypothesized that GTE would have different effects on
colon carcinogenesis, body composition, and lipid metabolism in mice fed a basal
diet formulated to promote health and growth (AIN93G) compared with total
Western diet, which emulates the typical American diet. Mice were fed either
AIN93G or the total Western diet for 18 weeks with or without GTE. The quantity
of a precancerous marker, aberrant crypt foci (ACF), was nearly three times greater
in AOM-treated mice fed the total Western diet than in those fed AIN93G. The
consumption of GTE suppressed ACF development only in mice fed the total
Western diet. Similarly, supplementation with GTE suppressed weight gain and
fasted glucose level only in mice fed the total Western diet, while GTE suppressed
fat mass gain in mice fed either diet, suggesting that diet is an important factor for the
efficacy of GTCs [55].
In their review of animal and cell experiments, Yang and Wang extensively
discussed the molecular mechanisms by which GTCs exert anticancer actions. For
example, in tumorigenesis of the small intestine in ApcMin/þ mice, EGCG action
was associated with increased levels of E-cadherin on the plasma membrane and
decreased levels of nuclear β-catenin, c-Myc, phospho-Akt, and phospho-ERK1/2 in
tumors [6]. In a model of AOM-induced precancerous lesions, the inhibitory activity
of PPE was associated with decreased levels of nuclear β-catenin and cyclin D1 and
increased levels of RXR-α. In male C57BL/KsJ-db/db mice, the inhibition of AOM-
induced ACF formation by EGCG was associated with the suppression of insulin-
like growth factor 1 (IGF1) signaling. EGCG also increased the levels of IGF1
receptor (IGF1R), phospho-IGF1R, phospho-GSK3, and β-catenin in colonic
mucosa. Yang and Wang further described that oral administration of 0.5% PPE or
0.044% caffeine in drinking water to tumor-bearing A/J mice inhibited the progres-
sion of lung adenomas to adenocarcinomas by enhancing apoptosis and decreasing
the levels of c-Jun and phospho-ERK1/2 in adenocarcinomas.
In a study which examined the cytotoxicity of green, black, and purple tea
infusions, green tea inhibited breast cancer 4TI cell proliferation to the greatest
extent with an IC50: 13.12 μg/mL. Results also revealed the differential expression
of apoptosis-related genes. Caspases 8, 9, 3, and 6 and 8AP2, Aifm1, Aifm2, and
Apopt1 genes were significantly upregulated, indicating the process of apoptosis was
initiated and executed [56].
In a PCa model, the antitumor action of GTC was associated with the modulation
of IGF1 and IGFBP3 levels with reduced levels of phosphatidylinositol 3-kinase
(PI3K), phospho-Akt, and phospho-ERK1/2. Furthermore, GTC significantly
decreased the levels of angiogenic and metastatic markers such as VEGF-A,
MMP-2, and MMP-9 [6].
Tumor metastases are responsible for approximately 90% of all cancer-related
deaths [57]. In these events, cancer cells released from the tumor invade surrounding
tissues, enter the blood vessels, and extravasate to spread to new organs through
several steps including attachment to and subsequent degradation of the endothelial

basement membranes [2]. In 1992, Taniguchi et al. reported that GTCs rich in EGCG
inhibited the metastasis of melanoma B16-F10 and BL6 cells in both experimental
and spontaneous metastasis systems [58]. Our research group has also observed
similar effects of green tea in an animal model of metastasis [59].
In 2001, GTEs were demonstrated to inhibit metastasis in transgenic adenocar-
cinoma of the mouse prostate (TRAMP) mice, and a later study showed that PPE
was an effective chemopreventive agent in preventing metastasis of PCa in this
mouse model [60, 61]. A likely molecular basis for the inhibition of metastasis by
green tea and GTCs is their ability to inhibit the enzymatic activity and gene
expression of MMPs [2]. The results of our research group suggest that EGCG
inhibits MMP activity by directly binding to MMPs [62].
Theaflavins have been shown to inhibit the growth and metastasis of HCC in an
orthotopic model and a lung metastasis model. Theaflavins induced apoptosis by
activating the caspase pathway, suppressing the phosphorylation of constitutive and
inducible signal transducer and activator of transcription 3 (STAT3), and down-
regulating downstream proteins regulated by STAT3, including antiapoptotic pro-
teins (Bcl-2 and survivin) and invasion-related proteins MMP-2 and MMP-9 [63].
Angiogenesis is required for many physiological processes, including embryo-
genesis and postnatal growth, but pathological angiogenesis is a hallmark of several
diseases including cancer and subsequent metastasis. Rashidi et al. reviewed the
ability of green tea constituents to suppress angiogenesis signaling, summarized
the mechanism by which EGCG might act on the VEGF family of proteins,
and highlighted the microRNAs affected by green tea that are involved in anti-
angiogenesis [64].
2.2 Effects of Coffee on Cancer
2.2.1 Epidemiological Studies

Some early epidemiological studies of the health effects of coffee showed that coffee
consumption was associated with an increase in cancer risk [65]. For example, Yu et
al. reported that daily coffee consumption was a risk factor for renal cell carcinoma
in women [66]. However, several later studies have demonstrated the beneficial
effects of coffee on cancer. A comprehensive review by Wierzejska showed that
coffee consumption reduced cancer risk in 1/4 studies on bladder cancer, 10/17
studies on breast cancer, 6/9 studies on colorectal cancer, 5/5 studies on liver cancer,
2/4 studies on pancreatic cancer, and 3/4 studies on PCa, while 3/3 studies on lung
cancer showed an increased risk [65]. Since then, a growing number of studies have
sought to further examine these effects, as reviewed here.
In a study of 64,603 participants with a follow-up period of up to 15 years for
breast cancer, consumption of 4 cups/day of boiled coffee was associated with a
reduced risk of cancer (hazard ratio (HR): 0.52) as compared with <1 cup/day,
although no association was found for all cancer sites combined or for prostate and
colorectal cancers. An increased risk of premenopausal breast cancer and a reduced
risk of postmenopausal breast cancer were found for both total coffee (HR, 1.69 for
premenopausal breast cancer; HR, 0.60 for postmenopausal breast cancer) and
filtered coffee (HR, 1.76 for premenopausal breast cancer; HR, 0.52 for postmeno-
pausal breast cancer). Boiled coffee was associated with an increased risk of
respiratory tract cancer (HR, 1.81), a finding limited to men. The main results for
less common cancer types included reduced risk for renal cell cancer with total
coffee (HR, 0.30) and increased risk for pancreatic cancer for boiled coffee (HR,
2.51). These findings demonstrate that coffee has beneficial effects in certain types of
cancer and that the effect may be dependent on the brewing method [67].
A population-based prospective cohort study of >215,000 men and women in
Hawaii and California with an 18-year follow-up period showed that high levels of
coffee consumption were associated with reduced risk of incident HCC and chronic
liver disease mortality. Compared with non-coffee drinkers, those who drank 2–3
cups/day had a 38% reduction in risk for HCC (RR, 0.62), while those who drank 4
cups/day had a 41% reduction in HCC risk (RR, 0.59). Compared with non-coffee
drinkers, participants who consumed 2–3 cups of coffee/day had a 46% reduction
in risk of death from chronic liver disease (RR, 0.54), and those who drank 4 cups/
day had a 71% reduction (RR, 0.29). These findings suggested that coffee
consumption reduces the risk of HCC and chronic liver disease in multiethnic US
populations [68].
A study of 738 middle-aged Japanese patients with adenoma and 697 controls
showed that high coffee consumption was associated with a reduced risk of ade-
noma. A multivariate-adjusted OR for the highest versus lowest quartile of coffee
intake was 0.67, suggesting a protective effect of coffee drinking on colon adenoma,
a precursor of colon cancer [69].
Results from a Danish case-control study from 1995 through 1999 showed that
both coffee (OR, 0.90 per cup/day) and total caffeine consumption from coffee and
tea combined (OR, 0.93 per 100 mg/day) decreased the risk of ovarian cancer [70].
The results of the European Prospective Investigation into Cancer and Nutrition
study in a total of 335,060 women from 1992 to 2000 indicated that higher
caffeinated coffee intake may be associated with a lower risk of postmenopausal
breast cancer, with a null association in the case of decaffeinated coffee [71].
A prospective study of breast cancer in the Swedish Women’s Lifestyle and
Health study of 42,099 female participants suggested that coffee consumption and
caffeine intake were inversely associated with the overall risk of breast cancer and of
estrogen receptor-positive/prolactin-negative breast cancer [72].
In a case-control study, during and 6 months after adjuvant chemotherapy, 953
patients with stage III colon cancer prospectively answered questionnaires which
included the dietary intake of caffeinated coffee, decaffeinated coffee, and non-
herbal tea. The results showed that patients who consumed 4 cups/day of total
coffee had an adjusted HR of 0.58 for colon cancer recurrence or mortality, com-
pared with nondrinkers. The daily consumption of 4 cups of caffeinated coffee
resulted in reduced cancer recurrence or mortality risk (HR, 0.48), while decaffein-
ated coffee was not associated with cancer outcome [73].
In a prospective cohort study in 307 patients over 4 years, the risk of colorectal
tumor recurrence was significantly lower (OR, 0.21) in patients who consumed >3
cups of coffee/day compared with those who did not consume coffee. In a sub-
analysis of tumor location, OR of colorectal tumor recurrence in the proximal colon
showed a tendency toward reduction as coffee consumption increased; however,
increased coffee consumption significantly increased colorectal tumor recurrence in
the distal colon [74].
A meta-analysis with a total of 1,534,039 participants from 13 published studies
showed that the RR of total coffee consumption and endometrial cancer was 0.80. A
stronger inverse association between coffee intake and cancer incidence was found
in patients who had never received hormone therapy (RR, 0.60) and subjects with a
BMI 25 kg/m2 (RR, 0.57). The overall RR for caffeinated and decaffeinated coffee
was 0.66 and 0.77, respectively. Endometrial cancer risk decreased by 5% for every
1 cup of daily coffee intake, 7% for every 1 cup of daily caffeinated coffee intake,
4% for every 1 cup of daily decaffeinated coffee intake, and 4% for every 100 mg of
daily caffeine intake. These findings suggest that coffee and caffeine may reduce the
incidence of endometrial cancer and that these effects may be modified by BMI and
history of hormone therapy [75].
A systematic review and meta-analysis of nine observational studies with a total
of 927,173 study participants showed that the pooled RR for melanoma among
regular coffee drinkers was 0.75 compared with controls. The pooled RR for
melanoma among decaffeinated coffee drinkers was, however, not statistically
significant [76].
A systematic review and meta-analysis of prospective cohort studies including 12
studies on HCC (3,414 cases) and 6 studies on chronic liver disease (1,463 cases)
found that the summary RRs for HCC were 0.66 for regular, 0.78 for low, and 0.50
for high coffee consumption, respectively. The summary RRs for chronic liver
disease were 0.62 for regular, 0.72 for low, and 0.35 for high consumption and
0.74 for an increment of 1 cup/day. These findings indicate an inverse relation
between coffee consumption and the risk of HCC and chronic liver disease [77].
In a meta-analysis of observational studies published until February 2016, the
intake of caffeinated coffee was inversely associated with nonmelanoma skin cancer
risk (summary RR, 0.82 for those in the highest versus lowest category of intake), as
was the intake of caffeine (summary RR, 0.86). In a subgroup analysis, these
associations were limited to the basal cell cancer histotype. There was no association
between decaffeinated coffee intake and summary RR, suggesting that caffeine may
contribute to the risk reduction [78].
A meta-analysis of 9 cohort and 13 case-control studies involving 7,631 cases and
1,019,693 controls reported a summary RR for gastric cancer of 0.94 for the highest
category of coffee consumption compared with the lowest category and 0.93 for
coffee drinkers compared with nondrinkers. The pooled RRs for the population
consuming <1 cup/day, 1–2 cups/day, and 3–4 cups/day compared with that of
nondrinkers were 0.95, 0.92, and 0.88, respectively, indicating that an increase in
coffee consumption was associated with a decreased risk of gastric cancer [79].
Another meta-analysis of the cohort and case-control studies reported a summary

RR for nonmelanoma skin cancer of 0.96 for 1 cup of coffee, 0.92 for 1–2 cups, 0.89
for 2–3 cups, and 0.81 for >3 cups/day, respectively. The results suggest that
caffeinated coffee might have dose-dependent chemopreventive effects against
basal cell carcinoma [80].
The results of a study of 18 cohorts, involving 2,272,642 participants and 2,905
cases, and of 8 case-control studies, involving 1,825 cases and 4,652 controls,
showed that increased consumption of caffeinated coffee and, to a lesser extent,
decaffeinated coffee was associated with reduced risk of HCC, including in patients
with preexisting liver disease. An extra intake of 2 cups/day of coffee was associated
with a 35% reduction in the risk of HCC [81].
However, several studies have failed to demonstrate the beneficial effects of
coffee, as exemplified by the following studies. A cohort study with 560,356
participants in the UK Million Women Study found no significant association
between endometrial cancer risk and consumption of coffee [82]. In a systematic
meta-analysis of 2,803 cases and 503,234 controls in ten independent studies, Chen
et al. found that coffee consumption was significantly associated with the increased
risk of laryngeal carcinoma (RR:1.47) [83].
A meta-analysis of 9 prospective cohort studies involving 1,250,825 participants
and 3,027 gastric cancer cases demonstrated that coffee consumption was not
associated with overall gastric cancer risk and that it may even be a risk factor for
gastric cardia cancer [84].
A meta-analysis of 17 studies (5 cohort and 12 case-control studies) involving
12,276 cases and 102,516 controls showed that coffee intake was associated with an
increased risk of lung cancer. Particularly over the past 5 years, studies have
consistently indicated that lung cancer risk is significantly increased by 47% in the
population with the highest category intake of coffee compared to that with the
lowest category intake [85].
In a large population-based case-control study in Italy, no association was
observed between regular coffee consumption and any type of leukemia [86].
A meta-analysis of 12 case-control studies, comprising a total of 3,649 cases and
5,705 controls, showed that high maternal coffee consumption was associated with
increased risk of acute lymphoblastic leukemia (OR, 1.43) and acute myeloid
leukemia (OR, 2.52) in children. The finding indicates a detrimental association
between maternal coffee consumption and childhood leukemia risk [87].
A multicentric case-control study on 690 bladder cancer cases and 665 hospital
controls conducted in Italy between 2003 and 2014 showed that decaffeinated
coffee, tea, cola, and energy drinks were not related to bladder cancer risk [88].
A meta-analysis of 13 prospective cohort studies with 20 independent reports
involving 3,368 patients with gastric cancer and 1,372,811 participants during a
follow-up period ranging from 4.3 to 8 years did not support the hypothesis that
coffee consumption was associated with the reduced risk of gastric cancer and even
indicated an increased risk of gastric cancer for participants in the United States [89].
The results of a population-based prospective cohort study in Japan on 89,555
people aged 45–74 years showed no clear association between coffee consumption
and biliary tract, gallbladder, or extrahepatic bile duct cancer [90]. Furthermore,
based on the meta-analysis of five cohort studies and nine case-control studies, Akter
et al. concluded that the evidence was insufficient to support that coffee drinking
increased or decreased the risk of colorectal cancer [91].
These conflicting results may have been caused by several confounding factors,
including the methods of quantifying coffee consumption, coffee temperature,
cigarette smoking, alcohol consumption, and differences in genetic and environ-
mental factors such as race, sex, age, intestinal microbiota, and lifestyle as in the case
of tea consumption [1, 2, 92].

A total of 31 men and 33 women were randomly assigned to two groups with two
intervention periods of 2 weeks separated by a washout period of 8 weeks, and they
consumed 1,000 mL of cafetière (French press) coffee daily or no coffee [93]. The
results showed that unfiltered coffee significantly increased the glutathione content
in the colorectal mucosa by 8% and in plasma by 15%. Unfiltered coffee did not
influence the colorectal mucosal proliferation rate, but appeared to cause an increase
in detoxification capacity and antimutagenic properties in the colorectal mucosa by
increasing the glutathione concentration. Thus, the findings suggest a possible
reduction of colon cancer risk by coffee consumption.
A controlled intervention trial with a crossover design in which 38 participants
consumed 800 mL coffee or water daily over 5 days demonstrated that the propor-
tion of DNA migration attributable to the formation of oxidized purines was
decreased by 12.3% after coffee intake. However, other biochemical parameters
including the total antioxidant levels in plasma, glutathione concentrations in blood,
and superoxide dismutase and glutathione peroxidase activity in lymphocytes were
not markedly altered. These results indicate that coffee consumption prevents the
endogenous formation of oxidative DNA damage in humans [94].
A clinical trial in which ten participants consumed 1 L of unfiltered coffee/day
over 5 days showed a weak induction of glutathione S-transferase (GST) and a
threefold increase in the induction of placental-type GST in blood, whereas the level
of GST-α was not altered [95]. Although serum cholesterol levels were increased
without statistical significance, other clinical parameters (creatinine, alanine amino-
transferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase),
which are markers for organ damage, were not altered. In a similar trial with seven
participants who consumed 1 L coffee/day over 3 days, a significant threefold
induction of placental-type GST was observed. The effects were identical between
filtered and unfiltered coffee, indicating that caffeine concentration was not respon-
sible for this result. In a further trial, the consumption of unfiltered coffee (1 L/day
for 5 days) resulted in a 45% reduction effect. These findings show that coffee
induces placental-type GST, which may confer protection against chemical carcino-
genesis [95].
However, a placebo-controlled intervention trial on 160 healthy human subjects
who consumed 3 or 5 cups of coffee per day for 8 weeks showed that blood pressure,
oxidation of DNA and lipids, blood glucose level, insulin, cholesterol, triglycerides,
and inflammatory markers were unchanged, although a slight elevation of serum

creatinine level and a significant elevation of serum γ-glutamyl transaminase level
were observed in the 5 cups/day group. These findings indicated that there was no
detectable effect, either beneficial or harmful, of coffee consumption on human
health [96]. Given the conflicting results of these clinical studies, further studies
are warranted to understand the relationship between coffee and cancer.

Numerous cell-based and animal experiments have demonstrated the anticancer
effects of CGAs, the most common polyphenols found in coffee.
A cell-based experiment found that CGA inhibited the viability of colon cancer
HCT116 and HT29 cells. CGA induced ROS production and cell cycle arrest at the S
phase and suppressed the activation of ERK. These events may lead to a reduction in
viability of cancer cells and suggest that CGA is a potential treatment for colorectal
cancer. Deka et al. found that CGA killed MDAMB-231 and MCF-7 breast cancer
cells with an IC50 of about 76 and 53 μg/mL, respectively. CGA bound to protein
kinase C (PKC) with a dissociation constant of about 29 μM and caused the
translocation of PKC from the cytosol to the plasma membrane, leading to cell
cycle arrest at the G1 phase. CGA induced apoptosis through a mitochondrial
pathway which involves a reduction in mitochondrial potential and the release of
cytochrome c into the cytosol [97].
Salomone et al. reviewed the effects of coffee and its components in experimental
models of liver cancer. Coffee exerted beneficial effects, including a reduction in
preneoplastic lesions in an aflatoxin-induced liver cancer model, a reduction in
tumor growth and metastasis in hepatoma-bearing rats, and a reduction in the
incidence of liver tumors in an aminopyrine-sodium nitrite-induced cancer model.
Regarding the mechanism of action of coffee and CGA, the authors highlighted the
antioxidant effects associated with nuclear factor, erythroid 2 like 2 (Nrf2) signaling,
the activation of which leads to (1) the induction of enzymes involved in xenobiotic
detoxification processes and cellular antioxidant defenses; (2) the induction of gene
expression of hepatic and intestinal NAD(P)H/quinone oxidoreductase 1, GST class
α1, intestinal uridine-50 -diphosphoglucose-glucuronosyl transferase 1A6, and the
glutamate cysteine ligase catalytic subunit; (3) the induction of the transcription of
several UDP-glucuronosyltransferases in hepatoma cells; and (4) an increase in
hepatic superoxide dismutase, catalase, and glutathione peroxidase activity [98].
When G422 glioma cells were injected subcutaneously into the right flank of ICR
mice in a xenograft model experiment and the mice were intraperitoneally adminis-
tered either CGA or vehicle daily for 2 weeks, CGA inhibited glioblastoma growth.
Moreover, CGA increased the population of CD11c-positive M1 macrophages and
decreased the distribution of CD206-positive M2 macrophages in tumor tissue via
the promotion of STAT1 activation and inhibition of STAT6 activation, respectively,
suggesting its therapeutic potential for the reduction of glioma growth [99].
CGA was also shown to have therapeutic effects in breast cancer, brain tumors,
lung cancer, colon cancer, and chronic myelogenous leukemia. Suggested mecha-
nisms of action of CGA include (1) the induction of GSK-3 β and APC genes; (2) the
inhibition of the β-catenin gene; (3) the inhibition of activator protein-1, NF-κB, and
MAPKs; and (4) the induction of phase 2 detoxifying enzyme activity [100].
The bark of Odina wodier is one of the Indian tribes for treating inflammatory
disorders, and its major constituent is CGA. Ojha et al. demonstrated that both the
methanol extract of bark and CGA exerted significant anti-inflammatory activity,
inhibiting the expression of tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6,
and IL-12. The expression of murine TLR4, NF-κBp65, MyD88, iNOS, and COX-2
molecules was also reduced in CGA-treated groups. These findings suggest that
CGA can inhibit inflammation by downregulating the TLR4/MyD88/NF-κB signal-
ing pathway. Through these activities, CGA may be beneficial for the prevention of
chronic inflammatory diseases including cardiovascular diseases, diabetes, and
cancer [101].
In contrast, Choi et al. found that neither caffeine, caffeic acid, nor CGA showed
any cytotoxicity against colon adenocarcinoma HT-29 cells, while a coffee
diterpene, kahweol, did [102].
Cancer metastasis may be prevented by coffee polyphenols. Weng and Yen
reviewed several studies to show anti-invasive and anti-metastasis activities of
dietary phenolic compounds including CGA and caffeic acid in various cancer
cells such as hepatoma Hep3B and SKHep1 cells, glioma U-87 cells, prostate cancer
PC3 cells, fibrosarcoma HT1080 cells, and colon adenocarcinoma CT26 cells [103].
The authors concluded that the daily consumption of natural dietary components that
are rich in phenolics could be beneficial for the prevention of cancer metastasis.
2.3 Simultaneous Evaluation of the Anticancer Effects of Tea and

Coffee
Several epidemiological studies have simultaneously evaluated the anticancer effects

of tea and coffee. These results are briefly summarized in Table 1. In the European
Prospective Investigation into Cancer and Nutrition study in 486,799 subjects, the
results from a median follow-up of 11 years showed that increased coffee and tea
intake was associated with lower HCC risk. Coffee and tea consumers in the highest
quintile had a lower HCC risk by 72% and 59%, respectively, compared with the
lowest quintile [15].
The results of a Danish case-control study indicated that both coffee and total
caffeine consumption from coffee and tea combined decreased the risk of ovarian
cancer, while no relationship was observed between tea consumption and ovarian
cancer risk [70].
In a middle-aged Japanese population, Budhathoki et al. found that high coffee
consumption was associated with a reduced risk of colorectal adenoma, with a
multivariate-adjusted OR of 0.67 for the highest versus the lowest quartile of coffee
intake, indicating a protective effect of coffee drinking against colon adenoma, a
precursor of colon cancer. Green tea intake was not found to be associated with
colorectal adenoma risk [69].
Table 1 Examples of the simultaneous evaluation of the effects of tea and coffee on human cancer riska
Cancer type Tea Coffee Reference
Liver cancer ― # [106]
Hepatocellular carcinoma # # [15]
Colorectal adenoma ― # [69]
Colorectal tumors ― # [74]
Laryngeal carcinoma ― " [83]
Breast cancer ― # [71]
Breast cancer " # [72]
Ovarian cancer ― # [70]
Endometrial cancer ― ― [82]
Endometrial cancer ― # [104]
Bladder cancer ― ― [88]
Biliary tract cancer # ― [90]
Nonmelanoma skin cancer ― # [78]
Brain tumor ― # [105]
Leukemia # ― [86]
All cancers combined # ― [104]
a
Cancer risk is reduced (#), increased ("), or not affected/evaluated (―)
A systematic meta-analysis of 2,803 cases and 503,234 controls in ten indepen-

dent studies, including case-control and cohort studies, showed that tea drinking was
not associated with laryngeal carcinoma. However, coffee consumption was posi-
tively associated with laryngeal carcinoma (RR, 1.47) [83].
A cohort study with 560,356 participants in the UK Million Women Study found
no significant association between endometrial cancer risk and the consumption of
either tea or coffee [82].
The prospective study of breast cancer in the Swedish Women’s Lifestyle and Health
study among 42,099 female participants suggested that coffee consumption and caffeine
intake reduced the risk of both overall and estrogen receptor-positive/prolactin-negative
breast cancer, while tea consumption increased the risk [72].
A multicenter case-control study on 690 bladder cancer cases and 665 hospital
controls conducted in Italy between 2003 and 2014 showed that consumption of
decaffeinated coffee, tea, cola, and energy drinks was not related to bladder cancer
risk [88].
In a meta-analysis of observational studies reported up to February 2016, intake
of both caffeinated coffee and caffeine was inversely associated with nonmelanoma
skin cancer risk (summary RR, 0.82). In a subgroup analysis, these associations were
limited to the basal cell cancer histotype. There was no association between intake of
decaffeinated coffee and green tea and nonmelanoma skin cancer risk [78].
A meta-analysis of 12 case-control studies, comprising a total of 3,649 cases and
5,705 controls, showed that high maternal coffee consumption was associated with
increased risk of acute lymphoblastic leukemia (OR, 1.43) and acute myeloid leukemia
(OR, 2.52). On the contrary, low-to-moderate tea consumption was inversely associated
with overall leukemia (OR, 0.85), although the trend was not significant. These findings
indicate the detrimental association between maternal coffee consumption and child-
hood leukemia risk. In contrast, an inverse association was found with tea, implying that
other micronutrients contained in this beverage could potentially counterbalance the
deleterious effects of caffeine [87].
The results of a population-based prospective cohort study in Japan on 89,555 people
aged 45–74 years showed no clear association between coffee consumption and biliary
tract, gallbladder, or extrahepatic bile duct cancer. However, the findings suggested that
high green tea consumption might lower the risk of biliary tract cancer [90].
In a large population-based case-control study in Italy, no association was
observed between regular coffee consumption and any type of leukemia. A small
protective effect of tea intake was found among myeloid malignancies, which was
more evident among acute myeloid leukemia (OR, 0.68) [86].
In a study of 97,334 eligible individuals, 10,399 developed cancers including 145
head and neck, 99 esophageal, 136 stomach, 1137 lung, 1703 breast, 257 endometrial,
162 ovarian, 3037 prostate, 318 kidney, 398 bladder, 103 glioma, and 106 thyroid
cancers. Coffee intake was not associated with the risk of all cancers combined, whereas
tea drinking was associated with an overall decreased risk of cancer (RR, 0.95 for 1 cup-
increment/day versus <1 cup/day). For endometrial cancer, a decreased risk was
observed for coffee intake (RR, 0.69) of 2 cups/day [104].
A Japanese cohort study with 106,324 subjects (50,438 men and 55,886 women)
found a significant inverse association between coffee consumption and brain tumor
risk in both total subjects (3 cups/day; HR: 0.47) and in women (3 cups/day;
HR: 0.24) [105]. No association was observed between green tea and brain tumor
risk. A prospective cohort study with 18,815 subjects aged 40–69 years showed that
coffee consumption may reduce the risk of liver cancer regardless of hepatitis C virus
(HCV) or hepatitis B virus (HBV) infection status, whereas green tea may not [106].
Thus, there are conflicting results related to the effects of both tea and coffee in a
variety of human cancers. These differences may have arisen from several
confounding factors, including the method of quantifying tea consumption, tea
temperature, cigarette smoking, alcohol consumption, and differences in genetic
and environmental factors such as race, sex, age, and lifestyle [1, 2, 6, 92]. In
addition, caffeine consumption is an important factor to be adjusted for. Intestinal
microbiota and genetic polymorphisms may also have influenced the effects of
coffee in these studies [107]. The differences in results between human and animal
experiments may have been due to different doses of tea and coffee [3].
3 Effects on Metabolic Syndrome and Related Disorders
3.1 Effects of Green Tea on Metabolic Syndrome

Metabolic syndrome (MetS) is diagnosed based on variables related to the five
components of obesity, blood triglycerides, high-density lipoprotein (HDL) choles-
terol, systemic hypertension, and fasting glucose. Any agent that exerts beneficial
effects on these components may potentially prevent MetS [108]. Several human
studies have suggested that tea is one such agent.
Grosso et al. conducted a cross-sectional survey among 1,889 inhabitants in
Sicily, southern Italy, and found that tea consumption was inversely associated
with MetS (OR, 0.51) after adjusting for all covariates. Although no direct associ-
ation between caffeine intake and MetS or its components was observed, tea and
coffee were significantly related to reduced OR of MetS. Similarly, results from a
cross-sectional population-based survey including 8,821 adults of the Polish arm of
the Health, Alcohol and Psychosocial Factors in Eastern Europe cohort study
showed that a high consumption of tea was inversely related to MetS, and the
analysis stratified by gender revealed a significant association for men but not for
women. After adjusting for potential confounding factors, tea consumption was
inversely associated with MetS (OR, 0.79) [109].
In a comprehensive review, Yang et al. provided several examples of reports,
including two epidemiological studies to demonstrate the mitigating effects of tea on
MetS [7]. One example is the study by Vernarelli and Lambert in 6,472 US adults.
Hot tea consumption was inversely associated with obesity, mean waist circumfer-
ence, and BMI and also increased HDL cholesterol and reduced blood triglyceride
levels in women [7, 92]. It should be noted that these associations were not observed
with iced tea consumption.
In contrast, some studies did not show a beneficial effect of tea on MetS [7, 92].
For example, a cross-sectional study by Tsubono and Tsugane found no association
between green tea intake and serum lipid levels [110]. An epidemiological study on
1,902 Japanese men and women showed no correlation between green tea intake and
MetS, since green tea consumption did not influence blood pressure, abdominal
circumference, fasting plasma glucose, or lipid levels [111]. The results of a cross-
sectional study that enrolled 554 adults in Tokushima, Japan, showed that green tea
consumption was not associated with the prevalence of MetS. Thus, epidemiological
studies have provided conflicting results, which may have resulted from various
factors as discussed above. Further studies are therefore required to determine
whether there is an association between tea consumption and MetS [112].

Legeay et al. reviewed six human intervention studies and found that EGCG was
associated with decreased BMI (three cases), body weight (four cases), low-density
lipoprotein (LDL) cholesterol (five cases), blood pressure (three cases), triglycerides
(two cases), and blood glucose (two cases) [113]. In one of these studies, a random-
ized, double-blind trial in 115 women with central obesity, significant weight loss,
from 76.8 kg to 75.7 kg, was observed as well as decreased BMI and waist
circumference after 12 weeks of high-dose EGCG treatment, with a consistent
trend of reduced total cholesterol and decreased LDL plasma levels [114].
Amiot et al. conducted a systematic review of dietary polyphenols on subjects
with MetS and summarized the effects of green tea and pu-erh tea extracts in ten
studies [115]. These studies showed significant improvements in BMI (eight cases),
weight circumference (seven cases), blood pressure (one case), LDL cholesterol
(five cases), triglycerides (four cases), and blood glucose (two cases) in subjects with
MetS. One example showed that in older adults with MetS, the consumption of 3
cups of green tea per day for 60 days was effective in inducing weight loss and
reducing both BMI and waist circumference [116].

A number of experiments using animal models and cultured cells have demonstrated
the beneficial effects of tea consumption on MetS and its components and underlying
molecular mechanism. For example, a study to evaluate the effect of GTE on drug-
induced weight gain and metabolic abnormalities in rats found that GTE exerted
protective effects against obesity, partially due to its lowering effect on leptin [117].
In this study, GTE significantly decreased body weight gain and average food and
water intake, improved the lipid profile and fasting blood glucose levels, and
decreased hyperleptinemia and hypertension in this animal model.
A study using a rat model of benign prostatic hyperplasia accompanied with MetS
induced by a high-fat diet (HFD) combined with testosterone injection demonstrated
that orally administered EGCG decreased the levels of glucose, total cholesterol,
triglycerides, insulin-like growth factors, and inflammatory cytokines, normalized
the activities of antioxidant enzymes, and increased the prostatic expression of
insulin-like growth factor-binding protein-3 and peroxisome proliferator-activated
receptors (PPARs) [118].
Yang et al. have described two major mechanisms of action for tea: one is the
action of tea constituents in the gastrointestinal tract in decreasing the digestion and
absorption of macronutrients or by altering the gut microbiota, and the other is that
produced by tea constituents following systemic absorption, namely, the inhibition
of anabolism and stimulation of catabolism in liver, muscle, adipose, and other
tissues [7]. GTCs may decrease the digestion and absorption of nutrients through
the inhibition of pancreatic lipase, phospholipases, and lipid transporters to reduce
body weight gain. Furthermore, green tea consumption can increase the proportion
of favorable intestinal bacteria such as Bifidobacterium species [7].
Yang et al. have proposed the “AMPK hypothesis,” in which the activation of
5’AMP-activated protein kinase (AMPK) is the main mechanism by which EGCG
and other catechins influence energy metabolism to alleviate MetS [7]. AMPK
activated by phosphorylation can decrease the expression of enzymes involved in
gluconeogenesis, such as phosphoenolpyruvate carboxykinase (PEPCK); lipogene-
sis, such as fatty acid synthase (FASN); adipogenesis; and protein synthesis, such as
homolog of target of rapamycin (mTOR), and increase the expression of those
involved in lipolysis, such as acyl-CoA dehydrogenase (ACAD). Activated
AMPK can also modulate the expression of transcription factors such as HNF,
SREBP, PPAR-α, and PPAR-γ [7]. Through the activation of AMPK, GTCs can
activate the ERK1/2-PPAR-γ-adiponectin pathway, leading to a reduction in fat
deposits in HFD-fed rats [119].
Thus, the AMPK hypothesis can explain many of the beneficial actions of tea/
GTCs on MetS and other diseases (Fig. 2). However, it is not clear how GTCs
activate AMPK. One possible explanation is the GTC-mediated generation of ROS,
Fig. 2 EGCG and CGA as a prooxidant can modulate gene expressions via promoting ROS
production. Related genes to those described in the text are shown. AMPK may possibly suppress
NF-κB activity [224, 225], leading to modulation shown in Fig. 2.
which may activate AMPK (Fig. 2) [120]. On the other hand, the scavenging ROS
activity of GTCs is well established (Fig. 3) [4, 107]. The factor which directs GTCs
to act as either an antioxidant or a prooxidant agent thus remains to be determined.
3.2 Effects of Green Tea on Obesity

A limited number of epidemiological studies on obesity are available to date. Some
of these studies have indicated a beneficial effect of tea on obesity [107]. For
example, Vernarelli and Lambert found an inverse association between hot tea
consumption and obesity as a component of MetS, as described above [121].

Several clinical studies have demonstrated a beneficial effect of tea on obesity
[92, 115]. For example, in a meta-analysis of 11 studies which met the inclusion
criteria, Hursel et al. found that GTCs significantly decreased body weight and also
significantly maintained body weight after a period of weight loss [122].
A clinical trial in which 240 Japanese subjects with visceral fat-type obesity
ingested green tea containing 583 mg of catechins (catechin group) or 96 mg of
catechins (control) per day for 12 weeks found that decreases in body weight, BMI,
Fig. 3 EGCG and CGA as

an antioxidant can modulate
gene expressions via
elimination of ROS. Related
genes to those described in the
text are shown.
body fat ratio, body fat mass, waist circumference, hip circumference, visceral fat
area, and subcutaneous fat area were greater in the catechin group than in the control
group [123].
As mentioned previously, Yang et al. provided several examples of studies that
indicated the preventive effect of tea on MetS through the mitigation of obesity [7].
Similarly, a systematic review by Amiot et al. reported the beneficial effects
of green tea on BMI [115]. In a literature search, Ferreira et al. found that six out
of eight randomized intervention studies showed a beneficial effect of green tea
on obesity, such as a reduction in body weight and body fat, while two studies
did not [124].
Green tea may also be useful in the treatment of obesity, as suggested by
Suliburska et al. [125]. In their randomized, double-blind, placebo-controlled
study, 46 obese patients were randomly assigned to receive either 379 mg of green
tea extract or placebo daily for 3 months. They found that 3 months of GTE
supplementation decreased BMI, waist circumference, and the levels of total cho-
lesterol, LDL cholesterol, and triglycerides.
By contrast, a randomized controlled trial showed that two kinds of Japanese
brand green tea did not affect body weight, although they did show an LDL
cholesterol-lowering effect [126].
3.2.3 Laboratory Studies

The molecular mechanisms for the anti-obesity effects of green tea have been
presented in many studies, as described in above section. A comprehensive article
published by Huang et al. in 2014 reviewed the anti-obesity effects of green tea in
human and laboratory studies and summarized the mechanisms of action of GTC.
These included (1) interference with energy absorption and metabolism to inhibit the
proliferation of preadipocytes and induce apoptosis in the preadipocyte and matured
adipocyte; (2) inhibition of preadipocyte differentiation and the adipogenesis of
maturing adipocytes; (3) inhibition of the activity of gastrointestinal digestive

enzymes, luminal emulsification, and micellar solubilization of lipids; (4) interfer-
ence with the uptake and intracellular processing of lipids and secretion of chylo-
microns in enterocytes; (5) enhancement of fecal excretion; (6) downregulation of
hepatic gene expression of lipogenic enzymes and related transcription factors; (7)
upregulation of hepatic mRNA levels of β-oxidation genes; (8) stimulation of fatty
acid oxidation and glucose uptake in skeletal muscle; (9) stimulation of the gene
expression of lipolysis and fatty acid oxidation-related genes in adipose tissue; and
(10) suppression of glucose intake and gene expression of lipogenesis-related genes
in adipose tissue [127]. Many of these actions may be explained by the “AMPK
hypothesis” of Yang et al. [7, 107] (see Fig. 2).
A recent cell-based experiment also provided one possible mechanism. In adipo-
cytes differentiated from C3H10T1/2 cells and immortalized preadipocytes in vitro,
EGCG reduced the triglyceride content. While EGCG did not affect protein kinase A
signaling or brown adipocyte marker expression in adipocytes, it did increase
autophagy and reduce mitochondrial membrane potential and intracellular ATP
levels. Although mTOR signaling was not upregulated by EGCG treatment,
AMPK phosphorylation was induced and lipophagy was activated. These results
indicated that EGCG upregulated autophagic lipolysis in adipocytes, supporting the
therapeutic potential of EGCG as a caloric restriction mimetic to prevent obesity and
obesity-related metabolic diseases [128]. AMPK activation by EGCG was also
demonstrated in the brown adipose tissue of diet-induced obese mice [129].
For black tea, detailed information is available in the review article by Pan et al. [130].
3.3 Effects of Green Tea on Hypertension

As compared with clinical studies, less information is available for epidemiolog-
ical cohort studies on the antihypertensive effect of tea/green tea. In a cross-
sectional epidemiological study of Singaporean Chinese residents aged 40 years,
consumption of green tea at least 150 mL per week was associated inversely with
hypertension risk (OR, 0.63). Drinking combination of green tea and British tea
was associated with higher reduction in the risk of hypertension (OR, 0.58),
suggesting that consumption of tea lowers the risk of hypertension [131]. By
contrast, a study of the population-based prospective cohort that recruited
63,257 Chinese aged 45–74 years and residing in Singapore found that daily
drinkers of green tea/black tea had slight increase in the hypertension risk, but
these risk estimates were attenuated and became nonsignificant after adjustment
for caffeine [132].

Several studies have demonstrated the beneficial effects of tea on blood pressure. For
example, a meta-analysis of 10 studies (834 participants) published from 1946 to
September 27, 2013, showed statistically significant reductions in systolic blood
pressure (SBP, mean differences 2.36 mmHg) and diastolic blood pressure (DBP,
mean differences 1.77 mmHg) with tea consumption in individuals within
the prehypertensive and hypertensive blood pressure ranges [133]. Similarly, a
meta-analysis of 14 randomized controlled trials in 971 participants found that
green tea or GTE supplementation caused a small but significant reduction in
blood pressure [134].
A crossover, randomized, double-blind, placebo-controlled clinical trial in 20
middle-aged women found that GTE supplementation for 4 weeks resulted in a
significant decrease in SBP compared with placebo, but not in DBP [135].
In contrast to reports describing the favorable effect of green tea on hypertension,
several studies did not show such an effect. For example, Suliburska et al. found that
daily supplementation of 379 mg of GTE for 3 months had no effect on blood
pressure in a randomized, double-blind, placebo-controlled study in 46 obese
patients, although the supplementation decreased BMI, waist circumference, and
the levels of total cholesterol, LDL cholesterol, and triglycerides [125].
3.3.3 Laboratory Studies

Several laboratory studies have shown the favorable effects of green tea on hyper-
tension and cardiovascular disease (CVD). For example, Yi et al. found that EGCG
delayed the progression of hypertension in spontaneously hypertensive rats (SHR).
SHR have higher mean arterial pressure, plasma pro-inflammatory cytokines, and
circulating norepinephrine levels compared with normotensive control rats and also
manifest increased NF-κB activity and higher levels of the subunit of NAD(P)H
oxidase, ROS, and pro-inflammatory cytokines and lower levels of IL-10 in the
hypothalamic paraventricular nucleus. The bilateral hypothalamic paraventricular
nucleus infusion of EGCG (20 μg/hour) for 4 weeks improved these parameters in
SHR, and the involvement of ROS and NF-κB activity in the mechanism of action of
EGCG was suggested [136] (see Fig. 3).
In another experiment in rats fed a high-NaCl diet, supplementation with GTE
was shown to reduce blood pressure, concentration of TNF-α, and antioxidant status
compared with a control group that did not receive GTE [137].
Kluknavsky et al. found that subchronic ()-epicatechin (EC) (Fig. 1) signifi-
cantly prevented the development of hypertension, increased the total antioxidant
capacity of blood, and decreased blood nitrotyrosine concentration in young SHR. In
the aorta, EC significantly increased nitric oxide (NO) synthase (NOS) activity and
elevated NO-dependent vasorelaxation [138]. These findings suggest that a pathway
involving ROS and NF-κB is associated with the antihypertensive activity of EGCG.
In addition, the direct action of EGCG on angiotensin I-converting enzyme
(ACE) may also mediate its antihypertensive effect. Takagaki and Nanjo demon-
strated that EGCG and its metabolites produced by intestinal bacteria showed
inhibitory activity against ACE and that a single oral intake of metabolites decreased
SBP in SHR [139]. A molecular docking mechanism may explain the potential of
EGCG as a new class of ACE inhibitors. Further chemical modification via fragment
modification guided by structure and ligand-based computational methodologies
may lead to the discovery of better inhibitors as clinical candidates [140].
3.4 Effects of Green Tea on Diabetes

Many epidemiological studies have reported the antidiabetic effects of tea and
GTE [92]. For example, Panagiotakos et al. reported that long-term tea intake reduced
levels of fasting blood glucose and was associated with a lower prevalence of diabetes.
In a cohort of 937 older adults living on Mediterranean islands, the consumption of 1–2
cups/day of green tea and/or black tea was associated with 70% lower odds of
developing type 2 diabetes mellitus (T2DM), irrespective of age, sex, body mass,
smoking, physical activity status, dietary habits, and other clinical characteristics
[141]. In a literature review of the antidiabetic effects of tea, Fu et al. identified six,
ten, and one epidemiological studies published from 2006 to 2016 showing the bene-
ficial effects of green tea, black tea, and oolong tea, respectively [142].
However, several epidemiological studies failed to demonstrate antidiabetic
effects [92, 142]. For example, Pham et al. found a rather positive association
between green tea consumption and insulin resistance in 1,440 participants aged
18–69 years [143]. Thus, further studies are necessary to confirm the antidiabetic
effects of tea in human subjects.

A number of intervention studies have reported the antidiabetic effect of green tea
[92]. For example, a randomized controlled trial conducted in 66 Japanese T2DM
patients found that daily ingestion of GTE containing 544 mg catechins for 2 months
caused significant reductions in hemoglobin A1c (HbA1c) levels and DBP [144].
Similarly, a 2-month intervention study in 60 patients with mild hyperglycemia
showed that the daily ingestion of GTE decreased the HbA1c level, although other
biomarkers were unaffected [145].
In a randomized, double-blind, placebo-controlled trial performed in 92 Taiwan-
ese subjects, the ingestion of 500 mg GTE three times a day for 16 weeks amelio-
rated the expression levels of an insulin resistance marker and the secretion of
glucagon-like peptide-1 in T2DM patients [146]. In a double-blind randomized
intervention study in nondiabetic overweight or obese male subjects in the United
Kingdom, Brown et al. found that twice-daily ingestion of 400 mg EGCG for
8 weeks resulted in reduced DBP, although no significant effects on glucose toler-
ance, insulin sensitivity, or insulin secretion were observed [147].
In a randomized, double-blind study in 42 diabetic subjects with a urinary
albumin-creatinine ratio >30 mg/g, patients were randomly assigned to two groups
to receive either GTP containing 800 mg of EGCG (17 patients with T2DM and 4
with type 1 diabetes) or placebo (21 patients with T2DM) for 12 weeks. The results
indicated that GTP reduced the urinary albumin-creatinine ratio by 41%, while the
placebo group had a 2% increase, suggesting that GTP may reduce the risk of
diabetic nephropathy [148].
In contrast, several intervention studies found no beneficial effects of green
tea on diabetes. For example, a double-blind, placebo-controlled, randomized
multiple-dose (0, 350, or 750 mg catechins and theaflavins for 3 months) study
conducted in the United States showed no effect on the level of HbA1c in

patients with a medical history of diabetes of more than 6 months [149]. A crossover
randomized controlled trial in southern Sweden showed that no glucose or insulin-
lowering effects were demonstrated by the consumption of 300 mL of green tea
or water [150].
Furthermore, a meta-analysis of randomized controlled trials found that the
consumption of green tea did not decrease the levels of fasting plasma glucose,
fasting serum insulin, hemoglobin HbA1c, or the insulin resistance index in
populations at risk of T2DM [151]. Thus, clinical trials in humans on the effects
of green tea in diabetes have demonstrated conflicting results. The discrepancy may
be explained as discussed in the previous section.

Multiple studies using cultured cells and laboratory animals have demonstrated the
antidiabetic effects of green tea and GTCs [92, 152].The underlined mechanisms
include (1) inhibition of α-amylase and α-glucosidase activity, (2) inhibition of
glucose absorption in the small intestine, (3) protection of pancreatic β-cells, (4)
improvement of insulin sensitivity in peripheral organs, and (5) inhibition of glucose
production from noncarbohydrates such as amino acids in the liver, known as
gluconeogenesis.
Inhibition of α-amylase and α-glucosidase results in reduced glucose production
leading to the prevention and suppression of diabetes by impeding the rise in blood
sugar levels [92, 152]. Similarly, the inhibition of glucose absorption in the small
intestine suppresses the increase in blood sugar levels [92, 152]. Improvements in
insulin sensitivity would result in the rapid suppression of blood glucose levels by
promoting glucose uptake by peripheral tissues. Green tea ingredients such as EGCG
have exhibited insulin-like activity in terms of increased glucose uptake [153].
EGCG may also protect insulin-secreting pancreatic β-cells from injury since
EGCG protected IL-1β and interferon (IFN)-γ-mediated cytotoxicity in an
insulinoma cell line, presumably through the inhibition of NF-κB activation [154]
(see Fig. 3).
Several studies have reported inhibitory effects of EGCG on gluconeogenesis.
EGCG exhibited insulin-like activity by suppressing the gene expression of
gluconeogenic enzymes, glucose-6-phosphatase (G6Pase), and phosphoenolpyr-
uvate carboxykinase [155]. One of the proposed mechanisms is the suppression of
transcription factor, hepatocyte nuclear factor-4α (HNF4α) expression by EGCG,
leading to a decrease in expression of these gluconeogenic enzymes and resulting in
diminished glucose production [2, 92]. The suppressive action of EGCG on HNF4α
expression may be explained by its prooxidative activity to generate ROS, leading to
the activation of AMPK which is known to inhibit HNF4α expression [2, 4] (see
Fig. 2). Collins et al. postulated that ROS was involved in the activation of AMPK
by EGCG and the suppression of hepatic gluconeogenesis [120].
In addition, several other mechanisms have been proposed for the antidiabetic
activity of tea. These include (1) improvement of endothelial dysfunction, (2)
modulation of cytokine expression, and (3) amelioration of insulin resistance,
as reviewed by Fu et al. [142]. These authors pointed out the involvement of GTCs in
the modulation of gene expression under its antidiabetic effects [142]. GTE may
increase the mRNA levels of glucose transporter family proteins. EGCG can also
attenuate the formation of advanced glycation end products; activate Nrf2, which
regulates the expression of antioxidant proteins protecting against oxidative damage;
and inhibit the expression of the AGE receptor. Green tea decreases the expression of
receptor activator of NF-κB and pro-inflammatory cytokine TNF-α in a rat model of
diabetes and increases anti-inflammatory cytokine IL-10. EGCG may also suppress
the expression of genes related to inflammation such as IL-1β, TNF-α, IL-6, CD11s,
CD18, and MCP-1 in adipose tissues in another animal model [142] (see Fig. 3).
3.5 Effects of Coffee on Metabolic Syndrome and Related

Disorders

Hino et al. conducted a population-based health check in 1999 and found that all
components of MetS (blood pressure, waist circumference, fasting plasma glucose,
and lipid profiles) except for HDL cholesterol were inversely related to the con-
sumption of coffee but not of green tea after adjusting for confounding factors [111].
Using the PubMed, Embase, Scopus, and Science Direct databases, Yesil et al.
found that four out of six human studies showed an inverse association
between coffee consumption and the risk of MetS, although two studies
showed no such association in a cohort consisting of young persons with a low
prevalence of MetS [156].
In addition, the following studies reported beneficial effects of coffee. From May
2009 to December 2010, a cross-sectional survey was conducted on 1,889 inhabi-
tants living in Sicily, southern Italy. As a result, coffee consumption (OR, 0.43) was
found to be associated inversely with MetS after adjusting for all covariates. No
association was observed between caffeine intake and MetS. Triglycerides were
inversely associated with the consumption of espresso coffee. Thus, coffee con-
sumption was demonstrated to have a reduced OR for MetS [108].
Results from a cross-sectional population-based survey including 8,821 adults
showed that high coffee drinkers had lower BMI, waist circumference, SBP and
DBP, and triglycerides and higher HDL cholesterol than those drinking < 1 cup/day.
After adjusting for potential confounding factors, higher coffee consumption was
demonstrated to be inversely associated with MetS (OR, 0.75) [109].
A Mendelian randomization study with 93,179 individuals from two large general
population cohorts found that in a cross-sectional analysis, there was a lower risk of
MetS with higher coffee intake. Compared with individuals with no coffee intake,
ORs for MetS were 0.91, 0.89, 0.88, 0.83, 0.84, and 0.89 for 0.1–1, 1.1–2, 2.1–3,
3.1–4, 4.1–5, and >5 cups/day, respectively [157].
Shang et al. conducted a meta-analysis of 11 studies published between January
1999 and May 2015 with a total of 159,805 participants to determine the association
between coffee intake and MetS risk. The aggregated result for the highest versus
lowest category of coffee consumption was 0.872, suggesting that coffee consump-
tion was associated with a reduced risk of MetS [158]. A cross-sectional study of a
random sample of 5,146 participants aged 20 years found that moderate drinkers
had 17% lower odds of developing MetS compared with nondrinkers. Tea consump-
tion was related to some components, but not to MetS in general [159].
Although these findings appear to show beneficial effects of coffee on MetS,
caution should be taken as coffee, particularly instant coffee mix, may have a
potentially harmful effect arising from the excessive intake of sugar and powdered
creamer [160].

Several clinical studies on the effect of coffee and CGAs have been reported. Patti et
al. evaluated the impact of a natural supplement, Kepar, which contains several plant
extracts such as curcuma longa, silymarin, guggul, CGAs, and inulin, in 78 patients
with MetS. Although Kepar exerted beneficial effects on body weight, BMI, and
waist circumference, fasting glucose and total cholesterol levels, further studies are
required to verify whether CGAs indeed contribute to these effects [161].
Santana-Gálvez et al. reviewed several clinical trials to evaluate the effects of
CGA on the prevention and treatment of obesity and hypertension, which are the
component disorders of MetS. These studies are discussed in the Sects. 3.6.2 and
3.7.2 [162].

Many studies have evaluated the effect of CGA on MetS or associated disorders,
including obesity, dyslipidemia, diabetes, and hypertension. Santana-Galvez et al.
reviewed the literature and found the beneficial effects of CGA on MetS and its
components [162].
Similarly, Yesil and Yilmaz identified three animal studies on the effects of coffee
on MetS and five on the risk of fatty liver infiltration. All showed a protective effect
of coffee against the development of MetS and nonalcoholic fatty liver disease
(NAFLD) [156].
In a study investigating the effects of Colombian coffee extract in an animal
model of MetS, rats were fed a corn starch-rich diet, whereas two other groups were
given a high-carbohydrate, HFD with 25% fructose in drinking water for 16 weeks.
The high-carbohydrate, HFD group showed the symptoms of MetS leading to
cardiovascular remodeling and NAFLD. Colombian coffee extract supplementation
attenuated the impaired glucose tolerance, hypertension, cardiovascular remodeling,
and NAFLD without affecting abdominal obesity and dyslipidemia. This study
suggests that Colombian coffee extract can attenuate diet-induced changes in the
structure and function of the heart and the liver without changing abdominal fat
deposition [163].
Tsumura-Suzuki obese diabetic mice are a newly developed MetS model which
spontaneously exhibits obesity, diabetes, hyperlipidemia, and nonalcoholic
steatohepatitis with liver nodules. When animals were divided into two coffee intake
groups (with and without caffeine), coffee intake did not affect obesity and
hyperlipidemia. However, coffee intake caused various degrees of improvement in

pancreatic β-cell damage and steatohepatitis with liver carcinogenesis. Most of the
effects were likely caused by a synergistic effect of caffeine with other components
such as polyphenols, and the anti-fibrotic effects of coffee appeared to be attributable
to the polyphenols rather than the caffeine [164].
Ma et al. conducted two sets of experiments. In one experiment, 6-week-old
C57BL/6 mice were fed regular chow or HFD for 15 weeks with twice-weekly
intraperitoneal injection of CGA (100 mg/kg) or vehicle. In another experiment,
obese mice (average weight of 50 g) received intraperitoneal injection of CGA
(100 mg/kg, twice a week) or vehicle for 6 weeks. CGA significantly inhibited the
development of diet-induced obesity but did not affect body weight in the obese
mice. CGA treatment also curbed HFD-induced hepatic steatosis and insulin resis-
tance; suppressed the hepatic expression of PPAR-γ, Cd36, Fabp4, and Mgat1 genes;
and attenuated inflammation in the liver and white adipose tissue accompanied by a
decrease in mRNA levels of macrophage marker genes including F4/80, Cd68,
Cd11b, Cd11c, and TNF-α, Mcp-1, and Ccr-2 encoding inflammatory proteins.
These results suggest that CGA is a potent compound for preventing diet-induced
obesity and obesity-related MetS [165].
Santana-Galvez et al. proposed a possible mechanism of action for CGA,
whereby it may act as an antioxidant to reduce ROS, which stimulate inflammation
leading to fat accumulation, weight gain, and insulin resistance (see Fig. 3). The
antioxidative activity of CGA may also stimulate NO production, leading to an
improvement in endothelial function and blood pressure. CGA may also improve
lipid metabolism by downregulating transcriptional factors such as LXRα and PPAR
and the gene expression of enzymes such as FASN, acetyl-CoA carboxylase (ACC),
and HMGCR and by upregulating PPAR-α, adiponectin, and AMPK phosphoryla-
tion [162] (see Fig. 2).
Although these studies support a beneficial effect of CGA in MetS, others failed to
show such effects. For example, in a controlled dietary intervention of over 12 weeks in
which male C57BL/6 mice were divided into three groups: (i) normal diet, (ii) HFD, and
(iii) HFD plus CGA (1 g/kg of diet), Mubarak et al. found that CGA supplementation in
mice fed HFD did not reduce body weight compared with mice fed HFD alone. CGA
caused increased insulin resistance compared with mice fed HFD and led to reduced
phosphorylation of AMPK and ACC-β, a downstream target of AMPK in the liver. The
livers of mice fed an HFD supplemented with CGA had a higher lipid content and more
steatosis relative to mice fed an HFD, indicating impaired fatty acid oxidation. This
study suggests that CGA supplementation in HFD does not protect against the charac-
teristics of MetS in diet-induced obese mice [166].
3.6 Effects of Coffee on Obesity

Few studies have been published on the effect of coffee on obesity. The result of
cross-sectional analyses in a Mendelian randomization study showed that higher
coffee intake of up to 4 cups/day was associated with a lower risk of obesity.

Compared with individuals with no coffee intake, ORs were 0.82, 0.86, 0.86, 0.83,
0.95, and 1.02 for 0.1–1, 1.1–2, 2.1–3, 3.1–4, 4.1–5, and >5 cups/day, respectively
[157]. A study on 137 patients with NAFLD and 108 controls found that coffee
consumption was inversely associated with insulin resistance and obesity [167].

Several clinical studies have reported the effects of coffee or CGAs on obesity. In a
meta-analysis of randomized clinical trials, Onakpoya et al. found a significant
reduction (2.47 kg) in body weight in a green coffee extract-treated group com-
pared with placebo. The magnitude of the effect was moderate, and there was
significant heterogeneity among the studies. The authors summarized that the results
from these trials were promising but that the studies were of poor methodological
quality. More rigorous trials are thus required to assess the usefulness of green coffee
extract as a weight-loss tool [168].
In a randomized controlled trial, overweight men with a mild-to-moderate eleva-
tion of fasting plasma glucose were randomly allocated to a 16-week intervention of
the consumption of 5 cups of caffeinated (n = 17) or decaffeinated (n = 15) instant
coffee per day or no coffee (n = 13). The results indicated that waist circumference
decreased by 1.5 cm in the caffeinated coffee group, increased by 1.3 cm in the
decaffeinated coffee group and decreased by 0.6 cm in the non-coffee group. Body
weight at 16 weeks showed a similar pattern; the corresponding changes from
baseline were 1.1 kg, 0.5 kg, and 0.6 kg, respectively. The authors proposed
that the decreases in the caffeinated coffee group were attributable to caffeine, which
increases thermogenesis and fat oxidation [169].
In a review article, Santana-Gálvez et al. listed two clinical studies showing the
effect of CGA on obesity [162]. One study was a randomized, double-blind,
12-week study in 30 overweight people. The result indicated that the average loss
in body weight among subjects who consumed CGA-enriched coffee was 5.4 kg,
while that in the normal instant coffee groups was 1.7 kg, suggesting a beneficial
effect of CGA on body weight reduction [170]. Another study was a placebo-
controlled, double-blind, crossover intervention study in 18 healthy male subjects
in which those who consumed 185 mL of a test beverage with or without CGAs
(329 mg) per day for 4 weeks showed no effects on body weight, BMI, or body fat,
although a significantly higher postprandial energy expenditure was observed in the
CGA group compared with the control group [171]. Thus, further studies are
required to determine the effects of coffee and CGAs on obesity.

A number of laboratory studies have provided evidence for the anti-obesity effect of
coffee and its mechanism of action. Hsu et al. found that the addition of coffee
phenols to culture medium decreased the cell growth of 3T3-L1 preadipocytes. The
IC50 values of CGA, gallic acid, o-coumaric acid, and m-coumaric acid on the
preadipocytes were 72.3, 43.3, 48.2, and 49.2 μM, respectively. A relationship
analysis indicated that there was a linear correlation between the influence of
phenolic acids on cell growth and their antioxidant activity. The treatment of
preadipocytes with CGA, o-coumaric acid, and m-coumaric acid caused cell cycle
arrest in the G1 phase. These results suggest that coffee phenols have anti-obesity
effects [172].
When C57BL/6 J mice were fed a control diet, HFD, or HFD supplemented with
0.5–1.0% coffee polyphenols (CPP) for 2–15 weeks, CPP supplementation reduced
body weight gain, abdominal and liver fat accumulation, and infiltration of macro-
phages into adipose tissues. The mRNA levels of sterol regulatory element-binding
protein (SREBP)-1c, ACC-1 and -2, stearoyl-CoA desaturase-1, and pyruvate dehy-
drogenase kinase-4 in the liver were also significantly lower in CPP-fed mice than in
HFD and control mice (see Fig. 2). Similarly, CPP suppressed the expression of
these molecules in Hepa 1-6 cells, concomitant with an increase in microRNA-122.
Structure-activity relationship studies of nine quinic acid derivatives isolated from
CPP suggested that CGA and di-caffeoylquinic acids were active substances in the
beneficial effects of CPP in these cells. Furthermore, CPP and 5-CQA decreased the
nuclear active form of SREBP-1, ACC activity, and cellular malonyl-CoA levels.
These findings indicate that CPP enhances energy metabolism and reduces lipogen-
esis by downregulating SREBP-1c and related molecules, leading to the suppression
of body fat accumulation [173].
When a commercially available supplement composed of cocoa, coffee, green
tea, and garcinia which contains 196 mg/g of total polyphenols and 4.0 mg/g of
EGCG was given to high-energy diet (HED)-induced obese rats, a reduction was
observed in the levels of free fatty acids, triglycerides, total cholesterol, LDL-C, and
LDL-C/HDL-C, AST, ALT, and ketone bodies in serum as well as hepatic triglyc-
erides and total cholesterol content, while the levels of HDL-C in serum and lipase
activity in fat tissues increased compared with the HED group. These results suggest
that the supplement stimulated lipid metabolism in HED-induced obese rats through
fat mobilization from adipose tissue [174].
When hyperlipidemia was induced in Wistar rats using HFD, the animals given
CGA complex from green coffee bean, CGA7 (50, 100, and 150 mg/kg body
weight), showed decreased triglycerides and free fatty acid levels in plasma and
the liver compared with the control group. CGA7 administration led to the activation
of AMPK and a subsequent increase in the levels of carnitine palmitoyltransferase 1
and a decrease in ACC acetyl-CoA carboxylase (ACC) activity (see Fig. 2). These
results suggest that CGA7 complex may be suitable as an active ingredient in
nutrition for obesity management [175].
Maki et al. found that coffee intake significantly suppressed HFD-induced metabolic
changes such as increased body weight and the accumulation of adipose tissue and the
upregulation of glucose, free fatty acid, total cholesterol, and insulin levels in the blood.
In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed
delayed cell cycle entry into the G2/M phase, termed as mitotic clonal expansion. Coffee
extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBP-β)
by preventing its phosphorylation by ERK and suppressed adipogenesis-related events
such as mitotic clonal expansion and C/EBP-β activation through the downregulation of
insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly
decreased by treatment with coffee extract, due to proteasomal degradation. These

results showed an anti-adipogenic function for coffee intake and identified IRS1 as a
novel target for coffee extract in adipogenesis [176].
Although these results indicate the favorable effects of coffee and/or its extract in
obesity, several other studies do not support these findings. For example, Cheong et
al. carried out a study in which C57BL6 mice were randomly divided into the
following experimental groups: (i) normal diet, (ii) HFD, or (iii) HFD supplemented
with 0.5% w/w GCE rich in CGA. The results showed that groups (ii) and (iii)
displayed MetS symptoms more profoundly than group (i) and that GCE did not
attenuate HFD-induced obesity, glucose intolerance, insulin resistance, or systemic
oxidative stress [177].
3.7 Effects of Coffee on Hypertension

A meta-analysis of 7 cohorts including 205,349 individuals showed a 9% significant
decreased risk of hypertension per 7 cups of coffee per day in a nonlinear analysis,
while in a linear dose-response association, there was a 1% decreased risk of
hypertension for each additional cup of coffee per day. The analysis also suggested
smoking as an important confounder [178].
In contrast, a large prospective study during 112,935 person-years of follow-up in
5,566 cases of incident hypertension showed that neither caffeinated coffee nor
caffeine intake was associated with mean SBP or DBP but that decaffeinated coffee
intake was associated with a small but clinically irrelevant decrease in mean DBP.
Decaffeinated coffee intake was not associated with mean SBP. Thus, no anti-
hypertension effects were demonstrated, but caffeinated coffee, decaffeinated coffee,
and caffeine appeared not to be risk factors for hypertension in postmenopausal
women [179].
A cross-sectional study conducted in 2012 among 1,164 individuals aged
63 years showed that among the 715 hypertensive participants, those consuming
3 cups of coffee per day showed higher 24-hour SBP and DBP than non-coffee
drinkers. Compared with non-coffee drinkers, ORs for uncontrolled BP among
those consuming 1, 2, and 3 cups of coffee/day were 1.95, 1.41, and 2.55,
respectively [180].

Santana-Gálvez et al. reviewed human studies on the effects of CGA on blood
pressure and found that CGA or coffee extracts reduced SBP and DBP in three out
of four human intervention studies [162]. In a similar approach, Tajik et al. found
that five out of six human intervention studies showed favorable effects on blood
pressure [181]. One example is the report by Revuelta-Iniesta and Al-Dujaili, who
conducted a randomized pilot crossover study in healthy subjects. The results
indicated that green coffee consumption reduced SBP, arterial elasticity, BMI, and
abdominal fat [182].

Several studies have demonstrated the beneficial effects of CGA on blood pressure
and suggested the underlying molecular mechanisms. Suzuki et al. demonstrated that
a single ingestion of CGA (30–600 mg/kg) reduced blood pressure in SHR, an effect
that was blocked by the administration of an NOS inhibitor, N(G)-nitro-L-arginine
methyl ester. When SHR were fed diets containing 0.5% CGA for 8 weeks (approx-
imately 300 mg/kg per day), the development of hypertension was inhibited. The
authors proposed, as the underlying mechanism, that dietary CGA reduces oxidative
stress and improves NO bioavailability by inhibiting the excessive production of
ROS in the vasculature, leading to the attenuation of endothelial dysfunction,
vascular hypertrophy, and hypertension in this animal model [183].
Zhao et al. summarized that CGA may exert an antihypertension effect through
(1) inhibition of NAD(P)H oxidase expression and activity, leading to reduction in
free radical production; (2) direct free radical scavenging; (3) stimulation of NO
production by the endothelial-dependent pathway; and (4) inhibition of ACE in the
plasma and possibly also in the organs and tissues. The anti-inflammatory effects of
CGA may also contribute to the effect [184].
3.8 Effects of Coffee on Diabetes

Ding et al. conducted a systematic review and meta-analysis of 28 prospective
studies with 1,109,272 participants and 45,335 cases of T2DM for coffee consump-
tion and disease risk. The results showed that compared with no or rare coffee
consumption, RR for diabetes was 0.92, 0.85, 0.79, 0.75, 0.71, and 0.67 for 1–6
cups/day, respectively. Thus, the decreases in RRs for every 1 cup/day were 9% for
caffeinated coffee consumption and 6% for decaffeinated coffee consumption,
indicating that the consumption of both types of coffee is inversely associated with
the risk of T2DM [185].
Based on three large cohort studies of men and women in the United States,
Bhupathiraju et al. found that coffee consumption was associated with a lower risk of
T2DM. During 1,663,319 person-years of follow-up, participants who increased
their coffee consumption by >1 cup/day over a 4-year period had an 11% lower risk
of T2DM in the subsequent 4 years compared with those who made no changes in
consumption. Participants who decreased their coffee intake by >1 cup/day had a
17% higher risk for T2DM [186].
In another study in 90,317 US adults, where 8,718 deaths occurred during
805,644 person-years of follow-up from 1998 through 2009, coffee drinkers had a
lower HR for several diseases, including diabetes, compared with nondrinkers [187].
Nordestgaard et al. conducted a Mendelian randomization study among 93,179
individuals from two large general population cohorts. The results indicated that
higher coffee intake was associated with a lower risk of obesity, MetS, and T2DM.
However, per-allele meta-analyzed ORs for T2DM were in the range of 0.98–1.01,
indicating that there was no genetic evidence to support corresponding causal

relationships [157].
In 2001–2002, a random sample of 1,514 men and 1,528 women was selected to
participate in a study in the Athens metropolitan area in Greece. This 10-year follow-
up study showed that individuals who consumed 250 mL of coffee had 54% lower
odds of developing diabetes compared with abstainers, indicating the significance of
long-term habitual coffee drinking in the onset of diabetes. The inverse association
between habitual coffee drinking and diabetes was suggested to be mediated by
serum amyloid-A levels [188].
A cross-sectional study in a large Brazilian cohort of 12,586 middle-aged and
older individuals provided evidence of a protective effect of coffee on the risk of
adult-onset diabetes. The results showed 23% and 26% lower odds of diabetes
among those consuming coffee 2–3 and >3 times per day, respectively, compared
with those reporting no or almost no consumption of coffee. An inverse association
was also found for 2-hour post-load glucose but not for fasting glucose concentra-
tions, suggesting the action of coffee on postprandial glucose homeostasis [189].
A population-based cohort study over a follow-up period of 4 years was
conducted to examine the association between habitual coffee intake and the risk
of T2DM and to determine whether this association varied by genetic polymor-
phisms related to T2DM. The results on 4,077 Korean adults aged 40–69 years with
a normal glucose level at baseline showed an inverse association between coffee
intake and the combined risk of T2DM and prediabetes. This inverse association was
found among individuals with the GT/TT of IGF2BP2 rs4402960, GG/GC of
CDKAL1 rs7754840, or CC of KCNJ11 rs5215 polymorphisms, which are known
to be related to T2DM in East Asians [190].
The European Prospective Investigation into Cancer and Nutrition-Potsdam study
involving 27,548 middle-aged participants found that coffee consumption was
inversely associated with diacyl-phosphatidylcholine and liver injury markers in
both sexes and positively associated with certain acyl-alkyl-phosphatidylcholines
in women. Coffee consumption showed an inverse relationship with C-reactive
protein in women and with triglycerides and phenylalanine in men. These findings
may partly explain the inverse association between long-term coffee consumption
and T2DM risk [191].
A population-based cohort study on first-trimester coffee and tea intake and risk
of gestational diabetes among 71,239 nondiabetic women with singleton pregnan-
cies showed that moderate first-trimester coffee and tea intake was not associated
with the risk of gestational diabetes and possibly may have a protective effect [192].
A recent review article based on review papers from in vivo, ex vivo, and in vitro
experimental studies in animals and human tissues as well as epidemiological studies
reported a reduced risk of developing T2DM in regular coffee drinkers of 3–4 cups a
day. The effects were proposed as attributable to CGAs and caffeine [193].
In a cross-sectional epidemiological study aimed to investigate the mechanism of
the association of coffee with liver injury, caffeinated coffee showed a significant
inverse association with ALT, AST, and NAFLD liver fat score but not with fetuin-
A, another liver injury marker. However, there was no significant association
between decaffeinated coffee intake and markers of liver injury. These results
indicate a beneficial impact of caffeinated coffee on liver morphology and/or
function and suggest that this relationship may mediate the inverse association of
coffee with risk of T2DM [194].
By reviewing the literature, Chrysant concluded that coffee consumption has
either neutral or beneficial effects on blood pressure, CVD, heart failure, cardiac
arrhythmias, and diabetes, and that the established concept that coffee consumption
is a risk factor for hypertension, heart disease, or diabetes is no longer warranted,
although some caution should be exercised in vulnerable populations [195].

Santana-Gálvez et al. reviewed clinical trials to evaluate the effects of CGA on
MetS-related diseases and found that five studies showed beneficial effects on
diabetes [162]. One such study was a randomized crossover trial for the effects of
12 g decaffeinated coffee, 1 g CGA, and placebo (1 g mannitol) on glucose and
insulin concentrations during a 2-hour oral glucose tolerance test in 15 overweight
men. The results indicated that CGA ingestion significantly reduced blood glucose
and insulin concentrations [196].

Evidence has been accumulating to support the beneficial effects of CGA on diabetes
in cell-based and animal experiments. A comprehensive review by Meng et al.
indicated that CGA stimulated glucose uptake in murine adipocytes and L6 muscle
cells and inhibited G6Pase in hepatic cells [197]. It also reported that more than ten
animal experiments found favorable effects of CGA such as improvement in blood
glucose levels, glucose tolerance, and insulin resistance and inhibition of α-gluco-
sidase, together with AMPK activation and the upregulation of hepatic PPAR-α
[197] (see Fig. 2).
Similarly, a recent review of five animal studies found that CGA had beneficial
effects in diabetes through a reduction in the levels of blood glucose and HbA1c,
prevention of the development of sugar cataracts, acceleration of wound healing, and
inhibition of hepatic G6Pase levels [162].
In an experiment in which female db/db mice were administered 80 mg/kg/day
CGA by lavage for 12 weeks, the percentage of body fat and the fasting plasma
HbA1c level decreased compared with that in the control group; transforming
growth factor-β1 protein expression and aldose reductase activity in the kidney
also decreased, while the adiponectin level in visceral adipose increased. CGA
significantly upregulated phospho-AMPK in the liver and skeletal muscle and
downregulated G6Pase in the liver, while upregulating GLUT4 (see Fig. 2). Thus,
CGA lowered the levels of fasting plasma glucose and HbA1c during late diabetes
and improved kidney fibrosis through the modulation of adiponectin receptor
signaling pathways in db/db mice [198].
Several other investigations also demonstrated the beneficial effects of CGA

in diabetes. For example, in a study aimed to investigate whether short-term
treatment with plant polyphenols, including CGA, could improve endothelial
dysfunction related to diabetes, streptozotocin-induced diabetic mice received
CGA (0.03 mmol/kg/day) by injection for 5 days. This treatment improved the
NO components of relaxation without the normalization of acetylcholine-stimu-
lated NO production. In addition, CGA treatment suppressed the acetylcholine-
stimulated level of thromboxane B2 in aortas from streptozotocin-induced dia-
betic mice. Thus, the treatment caused basal NO production and a prompt
improvement in endothelial function in diabetic mice, which may involve the
normalization of thromboxane B2 levels but not NO production under acetyl-
choline stimulation [199].
In an animal experiment to evaluate the effects of CGA on diabetic auditory
pathway impairment, CGA was shown to prevent the progression of auditory
pathway dysfunction caused by diabetes. The study also found that CGA may aid
in the recovery from outer hair cell and otic hair cell damage. Thus, CGA appears to
have beneficial effects in the management of diabetic sensorineural auditory
dysfunction [200].
There is a possibility that CGA can protect kidney function against oxidative
stress in diabetic nephropathy. Ye et al. showed that CGA decreased the levels of
blood glucose, blood urea nitrogen, and serum creatinine in a rat model of diabetic
nephropathy. CGA increased the activity of superoxide dismutase, glutathione
peroxidase, and catalase and decreased the level of lipid peroxidation. Immunohis-
tochemical analysis showed that CGA downregulated COX-2 protein expression in
renal tissues. In addition, CGA blocked the expression of activating transcription
factor-6 and C/EBP homology protein as well as the phosphorylation of eukaryotic
initiation factor 2α and double-stranded RNA-activated protein kinase-like endo-
plasmic reticulum kinase. These findings suggest that CGA attenuates oxidative
stress in diabetic nephropathy, leading to modulation of the endoplasmic reticulum
signaling pathway [201].
In alloxan-induced diabetic rats, oral administration of aqueous infusions of
Artemisia herba-alba Asso and Ajuga iva Schreber, used in folk medicine, was
shown to reduce blood glucose levels. Since A. herba-alba infusion contains CGA as
the main compound, CGA may contribute to this effect [202].
On the other hand, some studies did not show the beneficial effects of CGA in
diabetes. For example, in a study using 3T3-L1 cells, coffee was shown to reduce
the accumulation of lipids during adipocytic differentiation of these cells, which
may explain the antidiabetic action of coffee. Coffee also inhibited expression of
PPAR-γ, a transcription factor which upregulates the differentiation of adipocytes.
However, CGA showed no effect on PPAR-γ gene expression, suggesting that
CGA does not contribute to the antidiabetic activity of coffee [203].
In a study aimed to determine whether bioactive substances in coffee increase
insulin secretion from β-cells and improve insulin sensitivity in skeletal muscle cells,
cafestol and caffeic acid but not CGA were found to increase insulin secretion, both
acutely and chronically [204].
3.9 Comparison of the Effects of Tea and Coffee in Simultaneous

Studies on Metabolic Syndrome and Related Disorders
A cross-sectional epidemiological study among Singaporean Chinese residents aged

40 years reported that drinking at least 150 mL green tea per week was associated
with lower hypertension risk (OR, 0.63). Drinking a combination of green tea and
British tea was associated with a higher reduction in the risk of hypertension
(OR, 0.58). In contrast, consumption of coffee (OR, 1.44–1.46) was found to be a
potential risk factor for hypertension [131].
A population-based prospective cohort study among 63,257 Singapore Chinese
aged 45–74 years found that, compared to those who drank 1 cup of coffee/day, the
HRs were 0.87 for <1 cup/week drinkers and 0.93 for 3 cups/day drinkers.
Compared to <1 cup/week drinkers, daily drinkers of black or green tea had a
slight nonsignificant increase in risk, after adjustment for caffeine. Compared with
the lowest caffeine intake (<50 mg/day) group, the highest caffeine intake
(300 mg/day) group had a 16% increase in risk [132].
In a study of the effects of tea and coffee consumption in cardiovascular diseases
and risk factors such as hypertension, hyperglycemia, and hyperlipidemia, Di
Lorenzo et al. concluded that data from the clinical literature showed that tea
consumption reduced some risk factors, especially in overweight people and obese
subjects. For coffee, the results were controversial and did not allow conclusions to
be drawn [205].
4 Effects on Liver Disease
4.1 Effects of Green Tea on Liver Disease

Several human studies have indicated that GTC has beneficial effects in liver
diseases such as hepatitis, hepatoma, and liver fibrosis [92]. In a meta-analysis of
studies published from 1995 to 2014, Yin et al. found that there was a significant
reduction in the risk of liver disease with GTC intake. RRs were 0.74, 0.65, 0.57,
0.56, and 0.49 for hepatocellular carcinoma, liver steatosis, hepatitis, liver cirrhosis,
and chronic liver disease, respectively [206].
A retrospective cross-sectional study on 1,018 patients with NAFLD, HCV, and
HBV infection found that patients who drank 2 cups of coffee per day had a lower
liver stiffness, which is indicative of less fibrosis and inflammation, but that tea
consumption had no effect [207].

Only a few studies have examined the effects of green tea in liver disease. In a
clinical experiment on nine cases of intractable chronic hepatitis C with a high viral
load of >850 kIU/mL, Sameshima et al. found that combination therapy of 6 g of
green tea powder/day and interferon/ribavirin showed 3.5-fold efficacy compared
with interferon/ribavirin therapy alone [208]. This result and that of another clinical
trial in which a single 400 mg oral dose of EGCG was safe and well tolerated by all
11 patients with hepatitis C and detectable viremia strongly encourage further
studies [209].

Several cell-based and animal experiments demonstrated the hepatoprotective
effects of EGCG [5]. For example, our research group showed that consumption
of an EGCG-rich green tea beverage reduced liver damage in rats with galactos-
amine-induced hepatitis [210]. Administration of the beverage restored the plasma
levels of inflammatory factors TNF-α and IL-1β and their hepatic expression
upregulated by galactosamine. Similarly, in concanavalin A-induced hepatitis
mice, oral administration of EGCG (10 or 30 mg/kg) twice daily for 10 days prior
to concanavalin A injection was associated with decreased immunoreaction and
pathological damage by reducing inflammatory factors, such as TNF-α, IL-6, IFN-γ,
and IL-1β (see Fig. 3). EGCG also exhibited antiapoptotic and anti-autophagic
effects by inhibiting the expression of proapoptotic protein BNIP3 through the
IL-6/JAKs/STAT3 pathway [211].
Steinmann et al. reviewed the antiviral and antibacterial activities of EGCG
against human pathogens including HBV, HCV, human immunodeficiency virus,
and influenza A virus. In most of the studies, EGCG exhibited antiviral properties
within physiological concentrations in vitro [212]. The actions of EGCG were
suggested as being mediated through (1) the inhibition of viral entry by interference
with binding to target cells, (2) the inhibition of integrase and reverse transcriptase,
(3) the destruction of virions by binding to CD4 and interference with gp120
binding, (4) the inactivation of virus particles, (5) the inhibition of intracellular
virus growth and viral protease, (6) the alteration of physical integrity of virus
particles, and (7) the suppression of viral replication via modulation of cellular
redox milieu [212]. One of the studies reviewed by the authors showed that HCV
infection was suppressed by EGCG in Huh7.5.1 cells infected with the JFH1-GFP
chimeric virus when monitored for HCV RNA and protein expression levels. The
inhibitory mechanism was proposed to involve the suppressive effects of EGCG on
both the HCV entry and RNA replication steps. In addition, 50 and 25 μM EGCG
was shown to eliminate HCV from cell cultures after two and five passages,
respectively [213].
Xu et al. demonstrated that EGCG inhibited transcription of the HBV promoter in
HEK293 cells co-transfected with expression plasmids of farnesoid X-activated
receptor-α and RXR-α, suggesting that EGCG, as an antagonist of farnesoid
X-activated receptor-α in liver cells, has the potential to be employed as an effective
anti-HBV agent [214].
4.1.4 Hepatotoxicity
Green tea is generally considered to be safe for human health. However, a consid-
erable number of reports have described hepatotoxicity related to GTE. For example,
Navarro et al. found that since 2006, there have been more than 50 reports of
clinically apparent acute liver injury with jaundice attributed to GTE [215]. The
illness was generally self-limiting, but fatal instances have been reported in up to
10% of cases, typically among those who presented with acute hepatocellular injury
and jaundice. These authors pointed out that in most reports of GTE hepatotoxicity,
the human dose of EGCG (generally less than 12 mg/kg daily) did not appear to be
excessive or in the range that might have direct toxicity (estimated for humans to be
30–90 mg/kg), suggesting that liver injury associated with GTE is an idiosyncratic
reaction, typical of conventional drug-induced liver injury. Naturally, the excessive
consumption of catechin supplements should be avoided.
A recent investigation by Wang et al. suggested that melatonin may be useful for
preventing the potential adverse effects of EGCG in its application for MetS
alleviation and body weight reduction [216]. Future studies may explore effective
ways to prevent or reduce the possible adverse effects of tea constituents.
4.2 Effects of Coffee on Liver Disease

Coffee intake may exert beneficial effects in the liver. In population studies among
persons with an unknown diagnosis of liver disease, greater coffee intake was
associated with lower risk of cirrhosis, chronic liver disease, and HCC [217]. In a
large prospective study of patients with chronic hepatitis C and advanced liver
disease who had failed to achieve a sustained virological response with peginterferon
plus ribavirin treatment, Freedman et al. found an inverse association between coffee
intake and liver disease progression. Drinkers of 3 cups of coffee/day had a 53%
lower risk of liver disease progression than non-coffee drinkers. In contrast, no
association was observed for the consumption of black or green tea [217].
Yesil et al. identified two cross-sectional studies and three case-control studies
investigating the association between coffee consumption and the risk of NAFLD.
All of these studies suggest a reduced risk of NAFLD associated with coffee
drinking [156].

Wadhawan and Anand reviewed the literature for an association between coffee and
liver disease and provided clinical evidence of the benefit of coffee consumption in
hepatitis B and C, as well as in NAFLD [218]. Coffee consumption was associated
with an improvement in liver enzymes such as ALT and AST, especially in individ-
uals at risk for liver disease. Coffee intake of 2 cups/day in patients with pre-
existing liver disease was shown to be associated with lower incidence of fibrosis
and cirrhosis, lower HCC rates, and decreased mortality [218]. Similarly,
Wijarnpreecha et al. found a significantly decreased risk of NAFLD among coffee
drinkers and significantly decreased risk of liver fibrosis among patients with
NAFLD who drank coffee on a regular basis [219].
A study to examine whether or not coffee consumption was associated with lower
serum aminotransferases in the general Korean population and in those at high risk
for hepatic disease showed that the proportions of individuals with elevated AST
were 32.5%, 33.1%, and 26.7% in subjects who drank coffee <1, 1, and 2 times/
day, respectively. The ORs for elevated ALT and AST were significantly
lower in subjects who drank 2 cups of coffee/day than in those who drank
<1 cup/day. Thus, higher coffee consumption may be associated with lower
risk of liver disease [220].

Twenty-five male 129/Sv mice were administered CGA, and an ɑ-naphthyli-
sothiocyanate (ANI) challenge was performed at 75 mg/kg on day 4 after treatment.
CGA almost totally attenuated the resulting drug-induced liver damage and chole-
stasis compared with the untreated group. A dose of 50 mg/kg of CGA significantly
prevented drug-induced changes in serum levels of ALT, alkaline phosphatases, total
bile acid, direct bilirubin, indirect bilirubin (5.3-, 6.3-, 18.8-, 158-, 41.4-fold,
respectively), and AST (4.6-fold). Expression of the altered bile acid metabolism
and transport-related genes was normalized by treatment with CGA. The expression
of IL-6, TNF-α, and suppressor of cytokine signaling 3 was found to be significantly
decreased (1.2-fold, 11.0-fold, and 4.4-fold, respectively) in the CGA/ANI group.
Western blotting revealed that CGA inhibited the activation and expression of signal
transducer and activator of transcription 3 and NF-κB. These data suggest that CGA
inhibits both ANI-induced intrahepatic cholestasis and liver injury by down-
regulating STAT3 and NF-κB signaling [221] (see Fig. 3).
Feng et al. examined the impact of the Chinese herbal medicine Qushi Huayu
Decoction (QHD) and its active components (geniposide and CGA) on the NAFLD
liver transcriptome and gut microbiota using NAFLD rats. Increased expression of
genes required for glutathione production and decreased expression of genes
required for lipid synthesis were observed in NAFLD livers treated with QHD and
CGA. CGA treatment decreased serum lipopolysaccharide, which could be
explained by reduced mucosal damage in the colon of CGA-treated rats. In addition,
the results suggested an increased abundance of Treg cell-inducing bacteria, which
stimulated Treg cell activity in the CGA-treated colon, which in turn downregulated
inflammatory signals, improved gut barrier function, and consequently reduced
hepatic exposure to microbial products. These findings suggested that QHD simul-
taneously enhanced the hepatic antioxidative mechanism, decreased hepatic lipid
synthesis, and promoted Treg cell-inducing microbiota in the gut [222].
Watanebe et al. found that coffee intake did not affect obesity or hyperlipidemia in
TSOD mice, but that it did cause various degrees of improvement in pancreatic β-cell
damage and steatohepatitis with liver carcinogenesis. Most of the effects appeared to
be caused by a synergistic effect between caffeine and other components such as
polyphenols. The anti-fibrotic effects of coffee were likely attributable to the poly-
phenols rather than to caffeine [164].
Arauz et al. examined the hepatoprotective properties of coffee and caffeine in a
model of chronic bile duct ligation in Wistar rats. Western blot assays showed
decreased protein expression levels of transforming growth factor-β1, connective
tissue growth factor, α-smooth muscle actin, and collagen 1 in the coffee- and
caffeine-treated ligation groups compared with untreated rats. Similarly, coffee

decreased the mRNA levels of these proteins. The results indicated that coffee
prevented bile duct ligation-induced liver cirrhosis by attenuating the oxidant pro-
cesses, blocking hepatic stellate cell activation, and downregulating the main pro-
fibrotic molecules involved in extracellular matrix deposition [223].
The beneficial effects of coffee in liver disease have also been supported by a
recent review by Salomone et al. [98]. The authors described that in experimental
models of fibrosis, caffeine inhibited hepatic stellate cell activation by blocking
adenosine receptors and may favorably impact angiogenesis and hepatic hemody-
namics, while CGA suppressed liver fibrogenesis and carcinogenesis by reducing
oxidative stress and counteracting steatogenesis through the modulation of glucose
and lipid homeostasis in the liver.
In contrast, Aoyagi et al. reported that CGA did not contribute to the coffee’s
suppressive effect on PPAR-γ gene expression. They observed that coffee reduced
the accumulation of lipids during the adipocytic differentiation of 3T3-L1 cells and
that 5% coffee decreased the accumulation of lipids by about 50% compared with
control. Coffee inhibited the expression of PPAR-γ, a transcription factor that
controls the differentiation of adipocytes, and reduced the expression of other
differentiation marker genes aP2, adiponectin, C/EBP-α, GLUT-4, and lipoprotein
lipase (LPL) during adipocyte differentiation (see Fig. 2). Major bioactive constitu-
ents of coffee extracts such as CGA, caffeine, trigonelline, and caffeic acid showed
no effect on PPAR-γ gene expression. The inhibitory activity of coffee was found to
be produced by the roasting of coffee beans [203].
5 Conclusions
Multiple human studies have shown that both green tea and coffee exert beneficial
effects in human diseases, and most animal and cell-based experiments support these
outcomes. Clearly, GTCs and coffee CGAs are implicated in these activities. Several
mechanisms of action have been proposed, among which the involvement of ROS
appears to be the most prominent. Two conflicting actions have been proposed:
GTCs and CGAs can either scavenge ROS or promote its generation. The scaveng-
ing of ROS results in the inhibition of NF-κB activity, leading to the modulation of
cytokines and apoptosis-related factors to yield various favorable outcomes such as
anti-inflammatory effects and the induction of apoptosis in cancer cells (Fig. 3)
[4, 5]. It is also possible that the generation of ROS results in the activation of
AMPK, leading to the modulation of various enzymes and factors with roles in
health promotion (Fig. 2) [4, 107]. In addition, AMPK activation has been suggested
to downregulate NF-κB (Fig. 2), leading to modulation shown in Fig. 3 [224, 225].
At present, no explanation is available for these dual actions of GTCs and CGAs,
although some conditions may direct them to act as either prooxidant or antioxidant
agents. These conditions include their concentrations, the presence of cations such as
Cu+ and Fe++, cellular concentrations of antioxidants and oxidoreductive enzymes, and
the cellular redox state [4, 107]. These issues remain to be resolved in future studies.
Furthermore, conflicting results in human studies have also been reported. The
reasons for these inconsistencies include incomplete adjustment for confounding
factors and lack of necessary questions in questionnaires, such as the temperature of
tea or coffee, intestinal microbiota, and genetic factors. Nevertheless, we may expect
the overall favorable effects of green tea and coffee consumption to promote healthy
longevity in view of the growing of evidence presented in this chapter.
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Theobroma cacao and Theobroma
grandiflorum: Bioactive Compounds and 36
Associated Health Benefits
Maria Inés Genovese and Helena Rudge de Moraes Barros
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
2 Chemical Composition of Cocoa and Cupuassu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1051
3 Bioactive Compounds of Cocoa and Cupuassu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
4 Cocoa Polyphenolic Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055
5 Cupuassu Polyphenolic Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055
6 Effect of Processing on Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1056
7 Health Benefits Associated to Cocoa and Cupuassu Ingestion . . . . . . . . . . . . . . . . . . . . . . . . . . 1057
8 Effects of Cocoa and Cupuassu on Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1057
9 Effects of Cocoa and Cupuassu on Glucose Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063
10 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
Abstract
The genus Theobroma comprises about 20 species, among them cocoa
(Theobroma cacao L.), with the highest economic importance, and cupuassu
(T. grandiflorum), of growing interest. Chemical compositions of cocoa and
cupuassu unprocessed fresh seeds, pulps, and products (chocolate and cupulate)
are presented, and the effects of processing in profile and quantity of the bioactive
compounds, namely polyphenols and methylxanthines, are discussed. Dietary
consumption of cocoa and dark chocolate has been associated to beneficial effects
on health, mainly related to polyphenols and their antioxidant and anti-inflam-
matory activities affecting important signaling pathways and also modulating
intestinal microbiota. Vasodilation and cardioprotective effects of cocoa poly-
phenols are related to release of nitric oxide (NO) through activation of
M. I. Genovese (*) · H. R. d. M. Barros

Department of Food Science and Experimental Nutrition, University of São Paulo, São Paulo,
Brazil

1050 M. I. Genovese and H. R. d. M. Barros
endothelial NO synthase. Significant improvement of insulin resistance and flow-

mediated dilatation (FMD) and reductions in diastolic blood pressure and mean
arterial pressure were reported. In particular, the effects of cocoa and cupuassu
polyphenols on obesity and glucose metabolism are reviewed.
Keywords
Cocoa · Cupuassu · Polyphenols · Obesity · Glucose metabolism
Abbreviations
AA Arachidonic acid
ALT Alanine aminotransferase
AMPK AMP-activated protein kinase
AST Aspartate aminotransferase
BMI Body mass index
DW Dry weight
FA Fatty acid
FFA Free fatty acids
FMD Flow-mediated dilatation
FW Fresh weight
GK Glucokinase
GLP Glucagon-like peptide
GLUT Glucose transporter
GPx Glutathione peroxidase
GR Glutathione reductase
GS Glycogen synthase
GSK3 Glycogen synthase kinase 3
HDLc High-density lipoprotein
HF High fat
HO-1 Heme oxygenase-1
HOMA-B Homeostatic model assessment of cell function
IRS Insulin receptor substrate
JNK Jun N-terminal kinase
LDLc Low-density lipoprotein
MDA Malondialdehyde
MES-WAT Mesenteric white adipose tissue
NF-κB Nuclear factor kappa B
NO Nitric oxide
Nrf2 Nuclear factor erythroid 2-related factor
PEPCK Phosphoenolpyruvate carboxykinase
T2D Type 2 diabetes
Tb/Cf ratio Theobromine/caffeine ratio
TBARS Thiobarbituric acid reactive substances
36 Theobroma cacao and Theobroma grandiflorum: Bioactive. . . 1051
TG Triacylglycerol
TLR4 Toll-like receptor 4
UCP Uncoupling protein
VLDLc Very low-density lipoprotein
1 Introduction
The genus Theobroma comprises about 20 species, and several of them produce
edible seeds, notably cacao and cupuaçu. The highest economic importance is of the
cultivars (varieties) “Criollo,” “Forastero” and “Trinitario,” a hybrid of Criollo and
Forastero, of the species Theobroma cacao L., which are used for chocolate pro-
duction [1]. On the other side, the species T. grandiflorum (cupuassu or cupuaçu) is
getting more relevance for the South American market where the fruit pulp is highly
appreciated and seeds are used for the manufacture of chocolate-like products.
Cupuassu pulp can be consumed as juices, mousses, drinks, ice-creams, jellies and
candies, although not usually consumed directly due to its strong acidity. The seeds
contain high amounts of fat and may be used in food products and in a variety of
cosmetics. The seeds can also be processed in a similar way as cocoa to yield a
chocolate-like product called “cupulate ®,” firstly described and patented by the
Brazilian Research Institute Embrapa (Empresa Brasileira de Pesquisa
Agropecuária) [2].
Processing of raw cocoa involves several stages and starts with a fermentation
step, crucial for the development of the desirable flavor of cocoa products. After
drying/roasting and separation of shells, the grinding of beans results in cocoa nibs,
which are then milled to a liquid paste known as cocoa liquor. Pressing of cocoa
liquor removes cocoa butter and leaves a solid cake which is pulverized into cocoa
powder. Chocolate is produced adding more cocoa butter and other ingredients such
as sugar, milk, and emulsifying agents to cocoa liquor, in different proportions
depending on the type of chocolate desired. All these processing steps result in
changes in composition and quantity of the bioactive compounds present in cocoa,
mainly a sharp decrease in concentration after fermentation and drying [3].
2 Chemical Composition of Cocoa and Cupuassu
Cocoa butter is the major nutrient in cocoa beans. Differences in chemical compo-
sition of cocoa beans are due to different geographic origin, genetic variability and
post-harvesting processing. Cocoa beans from five regions of Cameroon, the fourth
largest producer of cocoa in the world, variety Trinitario, showed fat contents
ranging from 24.6% to 43.1%, moisture from 3.1% to 4.2%, carbohydrates from
24.4% to 43.4%, and proteins from 11.7% to 13.4% [4]. Ecuadorian unroasted cocoa
beans showed higher moisture (5.95%), fibre (19.5%) and total fat (43.4%) content
than Ghanaian beans, lower carbohydrates (33.8%), and similar contents of protein
(12.8%) and ash (~4%) [5].
Table 1 Chemical compositions of cocoa and cupuassu fresh seeds and products (%)
Sample Moisture Ashes Lipids Proteins Carbohydrates
Cupuassu
Fresh seeds 53 1a 1.46 0.06a 22 1a 4.3 0.1a 16 1a
Fermented and 8.6 0.1b 2.76 0.01b 45 3b 10.0 0.4b 31 1b
dried seeds
Fermented and 3.2 0.1c 3.02 0.02c 48 2b 10.0 0.2b 31 2b
roasted seeds
Liquor 2.64 0.03d 3.0 0.2c 54 2c 10.5 0.3b 27 1c
Cupulate 1.12 0.01e 1.78 0.05d 37 2d 7.0 0.2c 52 3d
Cocoa
Fresh seeds 46 2f 2.09 0.05e 20 1a 9.1 0.2b 19 1a
Chocolate 1.5 0.1g 1.55 0.04a 35 2d 7.8 0.6c 53 4d
Values in the same column with different letters are statistically different ( p < 0.05). Data from
Pugliese [7]
Fresh seeds of cupuassu, from three different harvest seasons, presented ca 53%
moisture, 1.5% ashes, 22% lipids, 3.8–4.2% proteins, and 14–18% carbohydrates.
Fresh pulps presented higher moisture (90%), as expected, and lower ashes (0.5%),
lipids (1%), proteins (0.7%) and carbohydrates contents (5–6%) [6]. For comparison,
cocoa fresh pulp from the same origin (Bahia, Brazil) presented 85% moisture,
0.43% ashes, 0.22% lipids, 0.77% proteins, and 12% carbohydrates [7].
Chemical compositions of cocoa and cupuassu unprocessed fresh seeds and
products prepared by CEPLAC (Comissão Executiva do Plano da Lavoura
Cacaueira, Cocoa Processing Unit, Fazenda Riachuelo, Bahia, Brazil) are presented
in Table 1. Although with similar lipid content, protein content of cocoa fresh seeds
was more than double that of cupuassu fresh seeds. Cupulate and chocolate, on the
other side, presented a similar composition.
Similar to what happens to cocoa, processing results in alteration of the chemical
composition of cupuassu seeds. Fermentation and sun-drying reduce drastically
moisture content of seeds, leading to a proportional increase of the other constitu-
ents. After the elimination of shells from roasted seeds, there is a reduction in
carbohydrate content (fiber) and an increase in lipid content of the resulting nibs,
which are then ground to produce the liquor (Table 1). Finally, cupuassu liquor
(52%) is mixed with cupuassu butter (8%), milk powder (6%), lecithin (0.5%) and
sugar (33.5%), resulting in an increase in carbohydrates content in the final product,
cupulate [7].
Quantitatively C16:0 (hexadecanoic acid or palmitic acid) (>25%), C18:0 (octa-
decanoic acid or stearic acid) (>33%) and C18:1 (octadecenoic acid or oleic acid)
(>34%) are the most important fatty acids in unroasted cocoa beans of both origins
Ecuadorian and Ghanaian [5]. Among the fatty acids detected in cocoa beans from
five different regions of Cameroon, stearic acid was the most abundant (37.6–39.5%)
followed by oleic acid (30.4–33.2%) and palmitic acid (24.8–28.2%), and saturated
fatty acids were most abundant (63.9–66.8%), followed by monounsaturated
(30.4–33.2%) and finally by polyunsaturated fatty acids (2.7–3.2%) [4].
Table 2 Fatty acid composition of cupuassu fresh seeds and cupuassu liquor (% total lipids)
Fresh seeds Liquor
Formula Fatty acid % %
16:0 Palmitic 7.54 0.08a 6.86 0.05b
17:0 Margaric 0.19 0.01a 0.18 0.01a
18:0 Stearic 32.6 0.1a 31.4 0.2b
18:1 (n-9) Oleic 41.31 0.06a 43.5 0.1b
18:1 (n-7) Vaccenic 0.47 0.02a 0.46 0.01a
18:2 (n-6) Linoleic 4.9 0.1a 5.04 0.01a
18:3 (n-3) α-linolenic 0.20 0.01a 0.13 0.00b
20:0 Eicosanoic 10.51 0.04a 10.23 0.09b
20:1 (n-9) Eicosenoic 0.34 0.01a 0.37 0.01b
22:0 Docosanoic 1.72 0.01a 1.70 0.05a
24:0 Tetracosanoic 0.19 0.00a 0.17 0.01a
Total Saturated 52.81 0.07a 50.5 0.4b
Monounsaturated 42.12 0.04a 44.4 0.1b
Polyunsaturated 5.07 0.11a 5.17 0.01a
Values in the same row with different letters are statistically different ( p < 0.05). Data from
Pugliese [7]
Fatty acid composition of cupuassu fresh seeds and liquor is shown in Table 2.
Although the fatty acid composition is very similar to cocoa, as it can be seen,
cupuassu fat stands out for the prevalence of oleic acid (18:1, 41%), followed by
stearic acid (18:0, 33%), and much lower amounts of palmitic acid (16:0, 7.5%) than
cocoa fat (~26%). Cupuassu fat also presents ten times more arachidic acid (20:0)
than cocoa fat [8]. The fatty acid composition of cupuassu fat provides softness and
enables its use in chocolates, replacing cocoa butter [9]. According to Lucas [10], in
spite of the higher melting point of cupuassu butter (33.9 C) compared to cocoa
butter (31 C), the high content of monounsaturated fatty acids, mainly oleic acid, of
cupuassu fat turns it softer than cocoa fat.
Bezerra et al. [11] also found that the primary fatty acids in cupuassu fat are oleic
(41.6%) and stearic (31.6%) acids, and that the fat has low atherogenicity and
thrombogenicity indexes, melting point of 38.1 C, and can be combined in blends
with suitable physicochemical properties for many applications.
Among by-products, cupuassu peel flour stands out for presenting an elevated
dietary fiber content (~80%), mainly insoluble fiber (~78%), higher than that of cocoa
peel (~64%, 52% insoluble fiber), and for being able to be incorporated in whole-
bread at 6% level without affecting color, aroma, texture and flavor attributes [12].
3 Bioactive Compounds of Cocoa and Cupuassu
Cocoa is rich in polyphenols and methylxanthines, and the higher the amount of cocoa
solids, the higher the content of methylxanthines and polyphenols in cocoa products
such as cocoa liquor, cocoa powder, dark, milk, and powdered chocolate [13].
Besides, processed cocoa beans and cocoa-based products may present other sub-
stances and chemical compounds of microbial and fungal origin which can be also
related to human health benefits. Bacteria and fungi which drive spontaneous
postharvest fermentation of cocoa beans immediately after bean harvesting produce
various primary and secondary metabolites with antifungal, antineoplastic, anti-
infective and cholesterol-lowering activities [14].
The physiological effects of methylxanthines are mainly mediated by adenosine
receptors blockage and include central nervous system stimulation, diuresis, cardio-
vascular and metabolic effects, bronchial relaxation and increased secretion of
gastric acids [15]. The main methylxanthines of cocoa are theobromine (0.8–2%
on a dry weight basis, DW) and caffeine (about 0.2%) and concentrations vary
depending on bean type, plant species, and degree of fermentation [16]. As expected,
concentrations of theobromine and caffeine decrease in cocoa products, following
the order cocoa > baking chocolate > dark chocolate > milk chocolate > cocoa
butter, and theophylline is normally below limit of detection. The benefits of
chocolate on mood seem to be mainly exerted by caffeine whereas theobromine
may be related to other effects [15].
Methylxanthine contents of fresh cocoa beans from different cocoa varieties
grown in Peru were in the range of 1.09–1.45%, for theobromine, and 0.20–0.41%
DW, for caffeine, decreasing during fermentation and drying. Liquor from these
samples presented theobromine contents from 0.84 to 1.14, and caffeine contents
from 0.08 to .165% DW [17]. The average contents of total methylxanthines
decreased from 15.05 mg/g dry matter in non-fermented to 12.94 mg/g dry matter
in fermented cocoa samples from Nicaragua [18]. Although methylxanthines do not
undergo chemical degradation during fermentation, about 30% of alkaloids are lost
due to diffusion out of the bean cotyledons [19].
The theobromine/caffeine (Tb/Cf) ratio is considered an important parameter for
cocoa, and lower ratios (˂3) are typical of Criollo cocoa (less bitter), as it presents
lower concentration of theobromine and higher levels of caffeine relative to other
genetic groups, Trinitario (˂ 9 Tb/Cf >3) and Forastero (Tb/Cf >9) cocoas [16–18].
Caffeic acid was reported to be present in a concentration range of 1.1–2.1 mg/g
dried extract, theobromine in a concentration range of 4.7–11.6 mg/g dried extract
and ()-epicatechin in a concentration range of 1.1–142.9 mg/g dried extract in
cocoa beans from five different regions of Cameroon [4].
Cupuassu presents a high amount of phenolics, especially on seeds, and of
ascorbic acid on pulp. However, a much lower content of theobromine (1 mg/g)
and caffeine (0.5 mg/g) was reported for ground seeds (both raw and roasted) of
cupuassu in comparison to cacao seeds (33–36 mg/g theobromine, 3.3–5.6 mg/g
caffeine) [20]. Fresh pulp of cupuassu showed a high content of ascorbic acid (ca
102 mg/100 g DW sample, or 17 mg/100 g FW) but commercial frozen pulps seem
to suffer a drastic loss during processing, presenting a ten times lower ascorbic acid
content (9–13 mg/100 g DW), probably as a result of oxidative reactions during pulp
separation and thermal processing [6].
Cocoa by-products are also important sources of bioactive compounds. Cocoa
husks are part of the cocoa bean and are separated from the cotyledons after the
roasting process. They have a high content of total dietary fibre, mainly insoluble
fibre, and represent a secondary source of theobromine, caffeine, and phenolics
(these last in the range 2.6–4.1 mg/g DW) [21].
4 Cocoa Polyphenolic Composition
Polyphenols in cocoa beans can be classified into three main groups: flavan-3-ols or
catechins (37%), anthocyanins (4%) and proanthocyanidins (58%), and the main
catechin is ()-epicatechin with up to 35% of polyphenol content [3]. Polyphenols
represent about 10% of the whole bean’s dry weight, with epicatechin concentrations
among freshly harvested beans ranging from 21.9 to 43.3 mg/g of dry defatted
sample [1].
Besides these major compounds (catechin, epicatechin and their dimers pro-
cyanidin B1 and procyanidin B2), other polyphenols have been reported in cocoa
beans or cocoa products: procyanidin B3 = catechin-(4α ! 8)-catechin, procyanidin
B4 = catechin-(4α ! 8)-epicatechin, procyanidin B5 = epicatechin-(4 β ! 6)-
epicatechin, procyanidin C1 = epicatechin-(4β ! 8)-epicatechin-(4β ! 8)-
epicatechin, procyanidin D = epicatechin-(4 β ! 8)-epicatechin-(4 β ! 8)-
epicatechin-(4 β ! 8)-epicatechin, higher oligo- and polymers, mostly homologues
of epicatechin with 2–18 monomeric units, as well the flavanols quercetin, quercetin-
3-O-glucoside (isoquercitin), quercetin-3-O-galactoside (hyperoside), quercetin-3-
O-arabinoside, the flavones apigenin, apigenin-8-C-glucoside (vitexin), apigenin-6-
C-glucoside (isovitexin), luteolin and luteolin-7-O-glucoside, dihydroquercetin,
dihydroxykaempferol, kaempferolrutinoside, naringenin, naringenin-glucoside,
myricetin-glucoside, and also caffeic acid, chlorogenic acid, coumaric acid, ferulic
acid, phenylacetic acid, phloretic acid, protocatechuic acid, syringic acid, vanillic
acid, clovamide and dideoxyclovamide [1].
5 Cupuassu Polyphenolic Composition
Nine known flavonoids were found in cupuassu seeds: (þ)-catechin, ()-

epicatechin, isoscutellarein 8-O-β-D-glucuronide, hypolaetin 8-O-β glucuronide,
quercetin 3-O-β-D-glucuronide, quercetin 3-O-β-D-glucuronide 600 -methyl ester,
quercetin, kaempferol, and isoscutellarein 8-O-β-D-glucuronide 600 -methyl ester.
Two new sulphated flavonoid glycosides, theograndins I and II, were also described
[22].
Pugliese et al. [6] reported that the main flavonoids both in seeds and pulp of
cupuassu were the glycosyl flavones, mainly the glucuronide and glucuronide-
sulfates: Hypolaetin-8-O-β-D-glucuronide, Hypolaetin-8-O-β-D-glucuronide-300 -O-
sulfate (Theograndin II); Isoscutellarein-8-O-β-D-glucuronide; Hypolaetin-30 -
methyl ether-8-O-β-D-glucuronide; Isoscutellarein-8-O-β-D-glucuronide-300 -O-sul-
fate (Theograndin I); Hypolaetin-30 -methyl ether 8-O-β-D-glucuronide- 300 -O-sul-
fate, with a total flavone concentration above 31 mg/g of unfermented cupuassu
seeds. Similar flavonoid profiles were observed for fresh and commercial frozen
pulps, but commercial frozen pulps presented lower contents than did fresh pulps, on
average 0.5 and 2.2 mg/g, respectively.
Cupuassu did not show any anthocyanidins, which are common in cocoa seeds.
The total proanthocyanidin content of unfermented cupuassu seeds was 23 mg/g.
Cupuassu pulp has a phenolic content similar to that found in other tropical fruits,
like passion fruit [23].
6 Effect of Processing on Polyphenols
Polyphenols in the cocoa beans are stored in the cotyledons. Processing of cocoa
seeds for chocolate manufacture is responsible for a significant decrease in polyphe-
nol content, mainly during fermentation and drying. Polyphenols diffusion from
storage cells results in exposition to other bean constituents and oxidation reactions
to produce insoluble high molecular weight tannins, both nonenzymatic and cata-
lyzed by polyphenol-oxidase. Fermentation of cocoa beans is crucial for the devel-
opment of precursors for chocolate flavour. Anthocyanins that represent ca. 4% of
the total polyphenol content are hydrolyzed during the fermentation process of cocoa
bean. During drying, additional loss of polyphenol occurs, mainly due to non-
enzymatic browning reactions. After fermentation and drying, the nibs obtained
are roasted and ground, resulting in the cocoa liquor. Removal of part of the cocoa
butter from the liquor results in cocoa powder, and these products can be further
alkalinized (Dutching), a process that also leads to significant losses of cocoa poly-
phenols [3, 24, 25].
A total of 11 different cocoa powder products available in the Spanish market
presented a mean content of total flavonoids equivalent to 2.65 mg/g, with a high
prevalence of ()-epicatechin. The alkalinization process of cocoa powder resulted in
60% loss of the flavonoid content. Among flavanols, ()-epicatechin presented a larger
decline (67%) than (þ)-catechin (38%), probably because of its epimerization into ()-
catechin, a less bioavailable form of catechin. Among flavonols, quercetin suffered the
highest loss (86%), whereas quercetin-3-glucuronide, quercetin-3-arabinoside, and iso-
quercitrin showed a similar decrease (58, 62, and 61%, respectively). Di-, tri-, and
tetrameric procyanidins also declined, 69% for dimer B2, 67% for trimer C1, and 31%
for tetramer D [26]. Todorovic et al. [27] also reported a 1.8 times lower polyphenol
content in alkalized cocoa samples than in natural ones with a decrease in epicatechin/
catechin ratio (2.21 in natural and 1.45 in alkalized cocoa powders).
Processing also affects the bioavailability of cocoa polyphenols. The comparison
of an unfermented, nonroasted, and blanch-treated cocoa powder with a conven-
tional one showed four times more procyanidins, and eight times more epicatechin
and procyanidin B2. A crossover intervention with healthy volunteers consuming
cocoa milk drinks prepared with these powders showed that the content of
epicatechin glucuronide, the main metabolite detected in plasma, was five-fold
higher upon consumption of the low-processed as compared with the conventionally
processed cocoa powder [28].
7 Health Benefits Associated to Cocoa and Cupuassu

Ingestion
Dietary consumption of cocoa and dark chocolate has been associated to beneficial
effects on health, mainly related to polyphenols and their antioxidant and anti-
inflammatory activities affecting some important signaling pathways, such as Toll-
like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) /signal transducer and
activator of transcription. Vasodilation and cardioprotective effects of cocoa poly-
phenols are related to inducing release of nitric oxide (NO) through activation of
endothelial NO synthase. The effects of chocolate, cocoa, and flavan-3-ols on major
cardiovascular risk factors were systematically reviewed by Hooper et al. [29] who
observed significant improvement of insulin resistance and flow-mediated dilatation
(FMD) and reductions in diastolic blood pressure and mean arterial pressure. Also,
cocoa polyphenols also modulate intestinal microbiota, leading to the growth of
bacteria that trigger a tolerogenic anti-inflammatory pathway in the host [30].
However, commercial cocoa and chocolate products present a high variability in
polyphenols content [26], and as a consequence, it is very difficult to generalize any
associated health benefits of their consumption and/or deriving from cocoa poly-
phenols. Below we examine specifically the reported effects of cocoa and cupuassu
polyphenols on obesity and glucose metabolism.
8 Effects of Cocoa and Cupuassu on Obesity
Although the scientific research on the beneficial properties of cocoa or dark

chocolate has increased, clinical studies determining their effects on weight regula-
tion have rarely been conducted. Many studies were related to cardiovascular
diseases and body fat is only assessed as part of routine measurement of the research
[31–33]. So far, there are no studies with humans evaluating the effects of
Theobroma grandiflorum (cupuassu) consumption on weight control.
Interventional and observational studies that relate cocoa consumption with
antiobesity effects have presented many controversial results until now. Some
studies found an inverse correlation between body mass index (BMI) and regular
cocoa consumption at small portions [31, 34, 35, 36]. However, other studies did not
detect significant changes in weight following the regular consumption of dark
chocolate [37–41], even when offered along with a restrictive diet [42]. In contrast,
Desch et al. [43] found a slight increase in body weight of patients with hypertension
that consumed 25 g of dark chocolate daily for 3 months. Greemberg et al. [44]
found that there was an increase in body weight with chocolate consumption by
healthy individuals in a dose-response manner, concluding that the hypothesis that
chocolate intake was associated with lower body weight did not apply to participants
without preexisting illness.
Therefore, this led to more focused animal studies on the effects of cocoa on
weight control. Some studies have been conducted to examine whether cocoa may
reduce weight gain, hyperlipidemia and inflammation and, thus, improve weight
control and other outcomes related to obesity [33, 45–49]. Some animal studies have
also reported the effects of cupuassu on weight control [50, 51]. These studies are
summarized in Table 3 and potential mechanisms by which cocoa could exert
antiobesity effects are discussed below.
Almost all studies listed in Table 3 demonstrated reduced weight gain in high fat
mice induced obesity by cocoa ingestion, while cupuassu demonstrated no changes
or only attenuated weight gain. However, supplementation with both fruits promoted
improvements in lipid-related outcomes as dyslipidemia and hepatic steatosis, glu-
cose-related outcomes as hyperinsulinemia and hyperglycemia, inflammation and
oxidative stress. Different outcomes may be explained by the wide range of doses
used, as well as the variety of intervention methods (gavage, in diet), different types
of products utilized (powder, extract, liquor, pure compounds) and the role of
intervention (preventive or treatment).
Among treatments for obesity, one of the most promising strategies to decrease
energy intake without altering central mechanisms is the development of nutrients
digestion inhibitors [52]. Thus, interactions of polyphenols with digestive enzymes
altering their activity may represent a significant caloric restriction and contribute to
improve the energy metabolism regulation [53]. Cocoa extracts and mainly pro-
cyanidins with higher degree of polymerization have demonstrated inhibition of
pancreatic lipase and secreted phospholipase A2 activities in a dose-dependent
manner in vitro [54]. In this sense, a lower weight gain and adipogenesis observed
in mice fed a high-fat diet supplemented with cocoa was suggested to be in part due
to cocoa’s inhibitory effect on pancreatic lipase activity [46, 47, 55]. Cocoa supple-
mentation resulted in reduced fat absorption from the gut as indicated by the
presence of a high fecal fat content in mice fed a high fat diet [47]. Cupuassu
phenolic extract also showed inhibition of pancreatic lipase in vitro and increased
lipid fecal excretion in mice fed high-fat high-sucrose diet, followed by reduction of
liver triacylglycerol contents and cholesterol levels in plasma [50]. However, cocoa
flavanols and cupuassu polyphenols have not been evaluated for inhibition of lipid
digestion and absorption in humans yet.
In addition to the reduction of fat absorption, several studies have shown that
cocoa decreases diet induced lipogenesis and hyperlipidemia by enhancing fat
oxidation and inhibiting the syntheses of fatty acids in liver and adipose tissue of
mice fed a high-fat diet [49, 56, 57]. Ali et al. [56] demonstrated that cocoa poly-
phenols treatment down-regulated the expression of PPAR-γ, SREBP-1c, Acaca and
Mcat genes in white adipose tissue and Fasn and Scd1 genes in the liver, the key
enzymes of fatty acid synthesis. Conversely, the expression of genes involved in
fatty acid oxidation PPAR-α, CPT1, and Prkaa1 was significantly higher in liver and
in adipose tissue of mice treated with cocoa [56, 58]. In accordance with these
results, Matsui et al. [48] found that cocoa ingestion decreased the expression of
genes for fatty acid synthesis in the liver, however without activating the expression
of genes for fatty acid oxidation in this tissue. In adipose tissue, cocoa ingestion
decreased the expression of genes for fatty acid transport molecules and their
transcription factor PPAR-γ with concurrent activation of thermogenesis. In contrast,
other study [33] showed that cocoa and their polyphenolic derivatives were
Table 3 Animal studies related to the effects of cocoa and cupuassu polyphenols on obesitya
36
Duration of
Vehicle Dose intervention Model Animals Biomarkers affected References
Crude polyphenolic 600 mg/kg 4 weeks Obesity induced Sprague # body weight gain, liver and MESWAT [58]
extract by gastric gavage bw/day by high-fat diet Dawley rats weight
(treatment) # liver lipid contents
# FFA and insulin serum levels
#lipogenesis related genes in liver
"β-oxidation related genes in liver
Crude polyphenolic 600 mg/kg 4 weeks Obesity induced Sprague # body weight gain, WAT and liver [56]
extract by gastric gavage bw/day by high-fat diet Dawley rats weight
(treatment) #lipogenesis related genes in liver and
visceral fats
# TG, TC, FFA and insulin serum levels
"β-hydroxybutirate serum levels
# liver lipid contents
#lipogenesis related genes in liver and
visceral fats
"β-oxidation related genes in liver and
visceral fats
Cupuassu phenolic extract 2.25 and 8 weeks Obesity induced Male Attenuated body weight gain [50]
by oral gavage 4.5 mg EC/kg by high-fat high C57BL/6 J # liver weight
Theobroma cacao and Theobroma grandiflorum: Bioactive. . .
bw/day sucrose diet mice "fecal TG content

(prevention) # ALT, AST, LDL and VLDL serum
levels
# liver peroxidation and TG contents
Improved fasting glucose and glucose
tolerance
Cocoa flavanol extract or a 25 mg/kg 12 weeks Obesity induced Male Attenuated body weight gain and [46]
flavanol fraction enriched bw/day by high-fat diet C57BL/6 J adipose tissue gain
with procyanidins in diet (prevention) mice #insulin serum levels
#endotoxin plasmatic levels
1059
(continued)
1060
Table 3 (continued)
Duration of
Vehicle Dose intervention Model Animals Biomarkers affected References
Cocoa powder in diet 10% of diet 4 weeks Obesity induced Swiss male # body weight gain [57]
by high-fat diet mice # liver lipid contents
(prevention) # oxidative stress
Cocoa powder in diet 8% (w/w) of 18 weeks Obesity-related Male No body changes [47]
diet Inflammation C57BL/6 J # inflammatory genes expression in
induced by mice adipose tissue
high-fat diet # AA contents, eicosanoid-generating
(prevention) enzymes and NF-κB in WAT
# inflammation related genes in WAT
# endotoxin plasmatic levels
"GLP-2 plasmatic levels
Cocoa powder in diet 8% (w/w) of 10 weeks Obesity-related Male # body weight gain and WAT weight [59]
diet Inflammation C57BL/6 J "fecal lipid content
induced by mice " adiponectin plasmatic levels
high-fat diet # insulin, ALT and pro-inflammatory
(treatment) mediators plasmatic levels
# lipid contents in liver
# inflammation related genes in WAT
Cocoa powder in diet 12.5% (w/w) 3 weeks Obesity induced Male Wistar # body weight gain and MES-WAT [48]
of diet by high-fat diet rats weight
(prevention) #TG serum levels
"TC and HDL serum levels
#gene expression of the FA synthesis in
the liver
#gene expression of the FA synthesis and
transport and "thermogenesis in the
adipose tissue
M. I. Genovese and H. R. d. M. Barros
36
Cocoa polyphenol extract 40 and 5 weeks Obesity induced Male # body weight gain, liver and epididymal [55]
dissolved in sodium 200 mg/kg by high-fat diet C57BL/6 N weight
carboxytmethylcellulose of bw (prevention) Mice # food intake
#TG plasmatic levels
Cupuassu and cocoa liquor 2.1 and 4 weeks Obesity induced Male Wistar "antioxidant capacity in plasma and liver [51]
phenolic-rich extracts by 7.2 g/kg bw by high-fat diet rats # alt, AST
oral gavage (treatment) # glucose intolerance and insulin
resistance (only cupuassu)
Cocoa powder, cocoa 1 g/ kg, 8 weeks Obesity induced Male Wistar #body weight gain, attenuated fat mass [33]
extract and its flavanols 100 mg/Kg by high-fat high rats accumulation
(Epicatechin, Catechin and and 10 mg/kg sucrose diet # food intake
Procyanidin B2) bw/day (prevention) #TG, TC and LDLc serum levels
By gastric gavage # fasting glucose and insulin serum
levels
# adipocytokine levels in retroperitoneal
adipose
" genes involved in FA uptake and β-
oxidation
Cacao liquor procyanidin 0.5 or 2% 13 weeks Obesity induced Male # body weight gain [49]
extract in diet (w/w) of diet by high-fat diet C57BL/6 # hyperglycemia and hyperinsulinemia
(prevention) mice # TC and leptin plasmatic levels
" adiponectin plasmatic levels
Theobroma cacao and Theobroma grandiflorum: Bioactive. . .
" translocation of GLUT-4, UCP

expression and AMPK phosphorylation
a
The arrow indicates an increase (") or decrease (#) in the levels or activity of the different parameters analyzed. AA Arachidonic acid, ALT alanine
aminotransferase, AMPK AMP-activated protein kinase, AST aspartate aminotransferase, FA fatty acid, FFA free fatty acids, GLP Glucagon-like peptide,
GLUT glucose transporter, HDLc high-density lipoprotein, LDLc low-density lipoprotein, MES-WAT mesenteric white adipose tissue, NF-κB factor nuclear
kappa B, TC total cholesterol, TG triacylglycerol, UCP uncoupling protein, VLDLc very low-density lipoprotein
1061
associated with the upregulation of the expression of genes involved in fatty acid
uptake (PPAR-γ and FAT/CD36) suggesting an improvement in adipose tissue
function with a better capacity to store nutritional overload and reduce ectopic
lipid deposition in other tissues and lipoproteins levels. However, fat oxidation in
adipose tissue was also enhanced by the upregulation and activation of PPARα,
PGC1α and SIRT1. In addition, some studies also related the antiobesity effect of
cocoa with the increase of the gene and protein expressions of the UCPs in the
adipose tissues and skeletal muscle, which are involved in the reduction of
weight, lipid contents in tissues and lipoprotein levels [33, 48, 49]. Although
the intake of cupuassu polyphenols did not demonstrate an effective reduction in
weight gain, improvements related to lipotoxicity could be observed. Supple-
mentation with cupuassu phenolic extract significantly reduced the plasma
concentrations of VLDL and LDL cholesterol, and the accumulation of triglyc-
erides in the liver [50, 51]. This suggests that the presence of flavones, which
are not found in cocoa, in combination with flavanols, may also help to alleviate
the metabolic changes triggered by a diet rich in fats and sugars. However,
further studies are needed to elucidate the mechanisms associated with these
effects.
Evermore, obesity chronic inflammation seems to be a key mediator of other
pathologies including insulin resistances and fatty liver disease. Cocoa has been
found to ameliorate obesity related inflammation, insulin resistance, and fatty liver
disease in HF-fed mice, possibly by reducing pro-inflammatory gene expression and
adipokines in white adipose tissue and modulating eicosanoid metabolism, gut
barrier function and metabolic endotoxemia [46, 47, 59]. Cocoa supplementation
also significantly increased plasmatic adiponectin levels in mice compared to HF-fed
controls, which may contribute to the decreased triglyceride accumulation in liver
and protect from inflammation and fibrosis [33, 49, 59].
In fact, adiponectin has been associated with maintaining glucose homeostasis
through activation of AMPK. Cocoa has the potential to prevent insulin resistance
and obesity by activating the adiponectin–AMPK pathway. Interestingly, cocoa also
reduced insulin plasmatic level without having effect on fasting glycaemia in high-
fat fed induced obesity studies. However, cupuassu improved fasting glycaemia and
glucose tolerance, which was associated with reduced lipotoxicity and oxidative
stress [50, 51]. The effects of Theobroma on glucose metabolism will be discussed in
the next section.
Until now, these antiobesity effects of cocoa can be explained by the activation of
AMPK and PPARα signaling, molecules responsible for suppressing lipogenesis
(fatty acid synthesis) and promoting lipolysis (fatty acid oxidation). These effects
also appear to be due in part to the modulation of dietary fat absorption, modulation
of gut microbiota and the down-regulation of NF-κB target gene expression, reduc-
ing inflammation. However, other genes and/or transcription factors may play a role
in beneficial effects. Although research to date has not specifically addressed the
mechanisms that Theobroma can act on obesity, there is reason to believe that
consumption of cocoa and cupuassu may induce beneficial metabolic changes
through the effects described above.
9 Effects of Cocoa and Cupuassu on Glucose Metabolism
As antidiabetic, cocoa polyphenols have been found to attenuate postprandial

glycemic responses while improving acute insulin secretion and insulin sensitivity
in animal models and in a limited number of human studies [46, 60–64]. In fact,
cocoa ingestion has been shown to protect β-cells by reducing oxidative stress and
decreasing levels of blood glucose, limiting the risk factors for diabetes and other
chronic diseases [63, 65, 66].
Currently, animal studies continue to evaluate the physiological effects of
Theobroma’s intake on different diabetic models. These studies are summarized in
Table 4 and potential mechanisms by which cocoa could exert antidiabetic effects are
discussed below.
The possible mechanisms of Theobroma’s action include inhibition of carbohy-
drate digestion and glucose absorption in the intestine, stimulation of insulin secre-
tion from the pancreatic β-cells, modulation of glucose release from the liver,
activation of insulin receptors and glucose uptake in the insulin-sensitive tissues,
and modulation of intracellular signaling pathways and gene expression. These
positive effects on glucose homeostasis are also supported by epidemiological
evidence on polyphenol-rich diets [67–69].
Experimental data from in vitro studies have suggested that cocoa and its main
phenolic compounds can act as potential antidiabetic agents by inhibition of carbo-
hydrate digestion [54, 70] and glucose absorption in the intestinal lumen [71, 72].
Cocoa extract demonstrated an in vitro inhibition of α-amylase activity in a dose-
dependent manner, which was enhanced by the degree of polymerization of cocoa
procyanidins [54, 70]. Cupuassu extract rich in flavonoids also demonstrated a potent
inhibition in vitro of α-amylase activity when compared to other Brazilian fruit
extracts [73]. However, these results were not related to procyanidins contents because
the extracts were obtained through a solid phase extraction in a polyamide SC6
column, which eliminates procyanidins, sugars, vitamins and minerals. In addition,
in this study, flavanol contents of other fruit extracts seemed to be related to inhibitory
activity of α-amylase. Interestedly, in contrast with other studies [6, 50, 51], flavanol
contents were not detected in cupuassu in this study, what suggests that flavones may
also exert effects on α-amylase activity. In another study [50], cupuassu phenolic
extract also inhibited α-glucosidase activity in a dose-dependent manner and was
more effective than acarbose in inhibiting α-glucosidase in vitro.
Chronic hyperglycemia and hyperlipidemia exposure increases intracellular reac-
tive oxygen species (ROS) generation, disturbs mitochondrial function and can exert
deleterious effects on β-cell function impairing insulin secretion [69, 74, 75]. Cocoa
polyphenols have been suggested to protect pancreatic β-cells from oxidative stress
and improve insulin secretion, thus alleviating hallmarks of T2D [63, 65, 66].
Studies in vitro demonstrated that cocoa polyphenols could protect INS-1 cells
against oxidative stress and modulate insulin secretion [65, 66]. Supporting these
findings, a study using diabetic fatty rats showed that cacao rich diet ingestion
increased β-cell mass and function by preventing beta cell apoptosis and improving
antioxidant defenses in pancreas [66]. In accordance with these data, ob/ob rats fed
Table 4 Animal studies related to the effects of dietary cocoa and cupuassu polyphenols on
glucose metabolisma
Vehicule Dose Duration Model Animals Outcomes References
Cocoa powder Cocoa 9 weeks Type 2 Male # body weight [60]
rich diet diabetes Zucker gain and liver
(10%) (pre-diabetic diabetic weight
stage) fatty rats # glucose and
(mutation insulin
in the plasmatic
leptin levels
receptor) Improved
glucose
tolerance and
insulin
resistance
Liver:
# p-(Ser)-IRS-
1, p-GS and
PEPCK
" p-GSK3,
GK and
GLUT-2
# p-JNK and
p-p38
rich diet diabetes Zucker gain
(10%) (pre-diabetic diabetic # glucose and
stage) fatty rats insulin
plasmatic
levels
" insulin
sensitivity
Liver:
# ROS,
carbonyl
groups, HO-1,
p-Nrf2, p65-
NFĸB
" sod
rich diet diabetes Zucker gain
(10%) (pre-diabetic diabetic # glucose and
stage) fatty rats insulin
plasmatic
levels
# HOMA-B,
# HOMA-IR
Pancreas:
"cell mass,
" Bcl-xL,
" GPx, " GR,,
# carbonyl
groups, # Bax,
#caspase-3
activity,
# TBARS
(continued)
Table 4 (continued)
Vehicule Dose Duration Model Animals Outcomes References
Cocoa extract by 600 mg/ 4 weeks High-fat diet Male # FFA, 8- [76]
oral gavage kg bw in STZ injection Sprague- isoprostane
diet Dawley plasmatic
rats levels
Cupuassu and 3.6 and 40 days Streptozotocin Male #TG plasmatic [78]
cocoa liquors 7.2 g/kg (STZ)- Wistar levels
bw induced rats Plasma HDLc
diabetic rats Plasma
antioxidant
capacity
# lipid
peroxidation
in plasma and
tissues
Cacao liquor 0.5%, 3 weeks Female, #blood [64]
Proanthocyanidin 1.0% db/db glucose in a
(w/w) in Mice dose
diet (obese, dependent
diabetic) manner
Cacao liquor 0, 05 Acute Male ICR # glucose and [77]
Procyanidin or 1% and and insulin
(w/w) 7 days C57BL/6 plasmatic
50 or mice levels
250 mg/ " GLUT4
kg bw translocation
in skeletal
muscle
a
The arrow indicates an increase (") or decrease (#) in the levels or activity of the different
parameters analyzed. FFA free fatty acids, GK glucokinase, GLUT glucose transporter, GS glycogen
synthase, GSK3 glycogen synthase kinase 3, GPx glutathione peroxidase, GR glutathione reductase,
HDLc high-density lipoprotein, HO-1 heme oxygenase-1, HOMA-B homeostatic model assessment
of cell function, IRS insulin receptor substrate, JNK Jun N-terminal kinase, NFkB nuclear factor-
kappa B, Nrf2 nuclear factor erythroid 2-related factor, PEPCK phosphoenolpyruvate
carboxykinase, ROS reactive oxygen species, SOD superoxide dismutase, TBARS thiobarbituric
acid reactive substances, TG triacylglycerol
cocoa extracts demonstrated decreased oxidative stress and enhanced glucose-stim-

ulated insulin secretion, thereby enhancing functional β-cell mass [76].
In addition, cocoa might improve insulin secretion by inhibiting lipid accumula-
tion in the cells. Considering this hypothesis, the supplementation of cupuassu
polyphenols in mice fed a diet high fat high sucrose diet reestablished the phosphor-
ylation of AKT and therefore insulin signaling in liver. These results were associated
with a reduction of triacylglycerol and oxidative stress in liver, as evidenced by the
reduction of MDA production and plasma transaminase activities [50]. However,
further studies are needed to identify the exact mechanisms by which these effects
are induced.
Liver is a key organ in the maintenance of blood glucose levels in tight cooper-
ation with peripheral tissues. In this sense, Cordero-Herrera et al. [60] demonstrated
that cocoa ameliorates hyperglycemia through its ability to preserve the hepatic
functionality by preserving the levels of GLUT-2 and glycogen content and to
modulate gluconeogenic and glycolytic enzymes, as it decreases hepatic PEPCK and

enhances hepatic GK. Although enzymes involved in hepatic production have not
been evaluated in the cupuassu study, the increase of AKT phosphorylation suggests
inhibition of hepatic glucose production through the improved insulin signaling in
this tissue and reduced glucose plasmatic levels [50].
Cocoa polyphenols may also exert important antidiabetic effects by improving
glucose uptake in muscles and adipocytes, through the action of GLUT4 [77]. Since
cupuassu administration did not demonstrate a significant reduction in adipose tissue
and body weight in mice fed a high fat high sucrose diet, the decrease in glycolipo-
toxicity demonstrated could be explained by an increase in GLUT4 activity in
adipose tissue. However, this hypothesis was not evaluated for cupuassu yet.
10 Conclusions
Considering the evidences presented, cocoa may be useful in ameliorating insulin

resistance, body weight gain and lipid alterations, thus reducing metabolic damage
associated to diabetes and obesity. Numerous studies on the effect of cocoa on endo-
thelial function also point to a possible effect on insulin sensitivity. However, additional
studies to confirm these effects in humans are needed, mainly establishing a dose-
response relationship between cocoa polyphenols and their overall effect. Cupuassu,
another Theobroma, but little studied, also deserves attention as a potential polyphenolic
source to act against metabolic alterations caused by diabetes and obesity.
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Olive Oil and Health Effects
37
Álvaro Hernáez, Julieta Valussi, Alejandra Pérez-Vega,
Olga Castañer, and Montserrat Fitó
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
2 Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073
3 Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074
4 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075
4.1 Type-II Diabetes and Impaired Glucose Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075
5 Cardiovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
5.1 Arterial Hypertension and Endothelial Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
5.2 Haemostasis and Platelet Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
5.3 Lipid Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
5.4 Lipid Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
5.5 HDL Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1082
5.6 Inflammatory Processes and Systemic Oxidative Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
5.7 Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084
5.8 Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085
6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1086
Abstract
Beneficial health effects of olive oil and its phenolics are presented in light of the
Mediterranean diet (MD), which is characterized by (1) a high intake of cereals,
vegetables including leafy greens, legumes, nuts, and fruit; (2) a moderate intake
Olga Castañer and Montserrat Fitó have contributed equally to this work.
Á. Hernáez · J. Valussi · A. Pérez-Vega · O. Castañer · M. Fitó (*)
Cardiovascular Risk and Nutrition Research Group (CARIN), Hospital del Mar Medical Research
Institute (IMIM), Barcelona, Spain
CIBER of the Physiopathology of Obesity and Nutrition (CIBEROBN), Institut Hospital del Mar
d’Investigacions Mediques (IMIM), Barcelona, Spain
e-mail: mfi[email protected]

1072 Á. Hernáez et al.
of poultry, fish, eggs, milk and dairy products, as well as a regular but moderate
ethanol consumption (generally in the form of wine during meals); and (3) a low
intake of red and processed meat and industrial confectionary. Olive oil (OO) is
the main fat used in all preparations of the MD. The health benefits of consuming
OO have been known since antiquity and were traditionally attributed to its high
MUFA content, mainly oleic acid. A large number of epidemiological and
laboratory studies suggest beneficial and protective effects of OO in reduced
risk of suffering cardiovascular disease (CVD) and cerebrovascular diseases,
diabetes mellitus, metabolic syndrome, certain cancers, and neurodegenerative
diseases. Potential effects of OO on various diseases-related parameters are
discussed in relation to OO and its phenolics.
Keywords
Olive oil · Mediterranean diet · Phenolic compounds · Virgin olive oil · PUFA ·
MUFA
Abbreviations
CRP C-reactive protein
EFSA European Food Safety Agency
GcMAF Gc protein-derived macrophage activating factor
ICAM-1 Intercellular adhesion molecule-1
IL Interleukin
MD Mediterranean diet
MUFA Monounsaturated fatty acid
OO Olive oil
OOPC Olive oil phenolic compound
PUFA Polyunsaturated fatty acid
SFA Saturated fatty acid
VCAM-1 Vascular cell adhesion molecule-1
VOO Virgin olive oil
1 Introduction
In a number of studies, the Mediterranean diet (MD) has been connected with
longevity and a reduced risk of morbidity and mortality. Life-style factors, such as
regular physical activity, a healthy diet, and the existing social cohesion in southern
European countries have been recognized as candidate protective elements which
may explain the Mediterranean paradox. The term MD was coined in the 1960s by
Ancel Keys within the framework of the Seven Countries Study. This epidemiolog-
ical study with more than 12,000 individuals reported that Italian and Greek
populations had lower mortality rates, and a reduced incidence of cancer and
cardiovascular disease (CVD), compared to those from other European countries,
37 Olive Oil and Health Effects 1073
America, and Asia [1]. Such findings led to an exponential increase from 1999 of
original articles regarding the MD [2]. High adherence to this diet pattern has been
associated with a reduction in the risk of suffering CVD and cerebrovascular
diseases, diabetes mellitus, metabolic syndrome, certain cancers, and neurodegen-
erative diseases [3]. The link between adherence to the MD and a reduction in total
mortality has also been confirmed [4, 5].
In general, the MD is characterized by (1) a high intake of cereals, vegetables
including leafy greens, legumes, nuts, and fruit; (2) a moderate intake of poultry,
fish, eggs, milk and dairy products, as well as a regular but moderate ethanol
consumption (generally in the form of wine during meals); and (3) a low intake of
red and processed meat and industrial confectionary [4–7]. Along with some other
traits of the Mediterranean diet, the use of olive oil (OO), as the main source of fat
(especially dressings), is common in southern European countries. A relatively high
fat consumption (up to 40% of total energy intake), mostly from monounsaturated
fatty acids (MUFAs) (up to 20% of total energy consumption) is characteristic of this
diet [8]. In the Mediterranean area, it is estimated that subjects consume between 25
and 50 mL of OO per day (raw and that used for cooking). Nevertheless, its
consumption varies around the Mediterranean countries.
The health benefits of consuming OO have been known since antiquity and were
traditionally attributed to its high MUFA content, mainly oleic acid. In this regard, a
recent meta-analysis of 32 cohort studies with the aim of studying MUFA (of both
plant and animal origin), oleic acid, MUFA/saturated fatty acid (SFA) ratio, and OO
intake, indicated that, when comparing the upper to the lower tertile of consumption,
OO, but not MUFA, was associated with a reduced risk of all-cause mortality, CVD
events, and stroke [9]. Thus, the extra constituents of OO may also have a protective
potential [10–12].
2 Cancer
Cancer is a multifactorial disease in which several aberrant processes are involved

(deregulation of cell cycle, abnormal expression of pro-oncogenes, deregulated
angiogenesis, excessive oxidative stress, chronic inflammatory responses, etc.).
Several lifestyle factors such as smoking, unhealthy eating, and sedentary habit
can increase the predisposition to develop cancer. Thirty to 40% of all cancers
could be prevented by appropriate diets, physical activity practice, and maintenance
of appropriate body weight [13]. Traditionally, a high consumption of fruit and
vegetables has been inversely related to chronic degenerative diseases such as cancer
[13, 14]. In the EPIC study, fiber intake from cereals, fruit, and vegetables showed a
protective effect on colorectal cancer, and fruit consumption was also associated
with lower rates of lung cancer [15]. Among the Mediterranean countries, meta-
analyses of ecological and cohort studies found that cancer morbidity and mortality
are lower than in other ones [16]. A high adherence to the MD, in which OO is the
main source of fat, has been associated with reduced mortality for all type of cancers
in prospective studies [5]. Around 25% of colorectal cancer incidence, 15% of breast
cancer, and 10% of prostate, pancreas, and endometrial cancer could be prevented by
a TMD in the Western countries [17]. Recently, a 4.8-year adherence to a traditional
MD (supplemented with virgin olive oil (VOO) or nuts) was found to protect against
breast cancer development in an elderly women [18]. In contrast, in Mediterranean
cohorts within the framework of the EPIC Study, OO consumption was not linked to
lower breast cancer risk in postmenopausal women. Nevertheless, an inverse asso-
ciation between OO intake and the levels of estrogens and progesterone receptor-
negative tumors was suggested [19]. Also within the EPIC Study, the adherence to
an MD was associated with a decrease in the incidence of gastric adenocarcinoma
[20]. In this respect, the antimicrobial activity conferred by OO against Helicobacter
pylori (a microorganism related to gastric ulcers and subsequent carcinomas) could
play a role in such an observation [21]. Finally, in Caucasian populations in the
Mediterranean basin, prostate cancer incidence is lower than that in Caucasian males
from other areas. The richness of MUFAs versus SFAs in the MD pattern has been
postulated as a possible explanation [22].
With reference to OO, an inverse association between its intake and the appear-
ance of different types of cancers has been described mainly from case-control
studies [23]. In this respect, a meta-analysis of case-control studies assessing the
effects of OO and MUFA intake was performed (N = 19, 13,800 cancer patients and
23,340 controls). Subjects in the group with the highest OO consumption presented
lower odds of suffering any type of cancer (logOR = 0.41; CI: [ 0.53, 0.29]).
Considering different cancer origins, the meta-analysis showed that high OO intake
is linked to a lesser probability of having cancer of the digestive tract
(logOR = 0.36; CI: [ 0.50, 0.21]) and breast cancer (logOR = 0.45; CI:
[ 0.78, 0.12]) [24].
In a clinical trial, patients with advanced cancer received a dietary intervention
rich in oleic acid and Gc protein-derived macrophage activating factor (GcMAF)
which can inhibit cancer cell proliferation. The diet, which was low in carbohy-
drates, rich in proteins, fermented milk products (which contains naturally-produced
GcMAF), Vitamin D3, and omega-3 fatty acids, enhanced the immune system, and a
25% reduction in tumor volume was observed [25].
3 Neurodegenerative Diseases
Chronic inflammatory status, together with oxidative stress, affects neuron function-
ality. Oxidation and inflammation processes promote the deposition of a number of
proteins inside neurons, consequently, the correct function of the mitochondria
becomes impaired. When these processes are perpetuated over a long period of
time, the development of several neurodegenerative diseases can occur. In particular,
Alzheimer’s disease, which is one of the greatest challenges of any national health
system, given the population aging. As there is no curative treatment to date,
preventive strategies based on a healthy aging are being promoted. A number of
epidemiological studies suggests that several nutrients (such as antioxidants, E and
B-vitamins, and polyunsaturated fatty acids) [26], and foods (such as fish,
vegetables, fruit, and wine) [27] may decline cognitive impairment, including
Alzheimer’s disease [28]. As oxidative stress can play a role in the development of
neurodegenerative diseases [29], the intake of phenolic compounds (PCs), with a
considerable spectra of bioactivities in vitro and in vivo, has been proposed for their
management [30].
Given the association between cardiovascular risk factors and neurodegenerative
ones, the cardiovascular health benefits of the MD can also confer protection against
neurodegenerative disorders [31]. While diets rich in saturated fats and simple
carbohydrates are linked to neurodegenerative diseases [32], the MD is able to
enhance cognitive function. An association between MD adherence and delay in
cognitive decline was observed in elderly subjects in a prospective cohort study in
France [6]. Moreover, in elderly individuals at high cardiovascular risk following the
MD, better cognitive efficiency [28] and performance were reported [33]. The
majority of these studies have been performed in the Mediterranean areas; never-
theless, other projects have been conducted in non-Mediterranean countries which
suggest that the healthy benefits of an MD can be transferred to other populations. In
this respect, greater adherence to the MD pattern was linked to a reduced risk of
Alzheimer’s disease with approximately 2000 subjects in New York [7].
The reduced vascular comorbidities observed with an MD, together with its
richness in compounds with antioxidant and anti-inflammatory effects, can play a
role in central nervous system benefits [34–36]. In this regard, VOO, one of the key
foods of the MD pattern, has been linked to improved cognitive function due to its
relevant bioactive compounds [28]. Hydroxytyrosol, oleuropein, and especially
oleuropein aglycon have been shown to inhibit Tau aggregation in vitro [37].
4 Diabetes Mellitus
4.1 Type-II Diabetes and Impaired Glucose Tolerance
Impaired glucose tolerance is defined as high blood glucose levels after eating,
whereas impaired fasting glucose is defined as high blood glucose after a period of
fasting. People with impaired glucose tolerance are at high risk of developing DM2.
Unsurprisingly, impaired glucose tolerance shares many characteristics with DM2
and is associated with obesity, advancing age, and the inability of the body to use the
insulin it produces. Not everyone with impaired glucose tolerance goes on to
develop DM2 [38]. DM2 is a chronic disease that occurs either when the pancreas
does not produce enough insulin or when the body cannot effectively use the insulin
it produces. Hyperglycemia is a common effect of uncontrolled diabetes and over
time leads to serious damage to many of the body's systems, especially the nerves
and blood vessels [39]. Some 382 million people worldwide, or 8.3% of adults, are
estimated to have diabetes. According to International Diabetes Federation, the
causes are still unclear but people with overweight, poor diet, lack of physical
activity, and family story of diabetes are at high risk of DM2 [38].
Several studies have found a link between MD and glucose metabolism. The
ATTICA study observed that adherence to MD was related to better homoeostasis
factors related to fasting glucose (fasting plasma glucose, insulin levels and the
insulin resistance index: HOMA) in normoglycemic people [40]. Moreover, PRE-
DIMED study found that a long-term intervention with a high-quality dietary pattern
based on traditional MD and rich in EVOO could reduce the incidence of DM2 in
older people at high cardiovascular risk. This beneficial effect was mainly due to the
overall composition of the dietary pattern and not to calorie restriction, increased
physical activity, or weight loss [41]. A couple of meta-analysis [42, 43] also found a
significant association between adherence to dietary patterns and decreased risk of
DM2. One of the previous meta-analysis specifically concluded that adherence to a
MD is associated with a decreased risk of becoming diabetic at a reasonable
magnitude (19%) [43]. Other systematic review of eight meta-analyses and five
randomized controlled trials studied the effect of MD on the treatment of DM2 and
prediabetic states. This review indicated that in DM2 patients, the adherence to MD
was associated with lower glycated hemoglobin (HbA1c) levels and improved
cardiovascular risk factors, as compared with control diets, mainly lower fat diets
[44]. Thus, it is suggested that the Mediterranean diet is not only suitable for
prevention but it is also an appropriate dietary pattern for the management of DM2.
Another meta-analysis of prospective cohort studies to assess the association
between different diets and prevention of DM2 found that although the diets
associated with prevention of DM2 may vary in their composition, nonetheless
they shared several common components, including whole grains, fruit, vegetables,
nuts, legumes, protein sources such as white meat and seafood, little or moderate
alcohol, and reduced intake of red and processed meats and sugar-sweetened bev-
erages, and noteworthy healthy table oils (i.e., olive oil) [45]. MD covers all of the
above being one of its emblematic characteristics is the use of olive oil.
One of olive oil’s characteristics is its high content in monounsaturated fatty acids
(MUFA). Evidence suggests that diets enriched in MUFAS from OO have a positive
effect in glycemic control. A study examining intakes of MUFA through the
increased consumption of olive oil found an association with lower fasting plasma
glucose concentration [46, 47]. Another cohort study of women found a similar
result after a 22 years follow-up. The published results showed that higher olive oil
intake was associated with modestly lower risk of DM2 and that hypothetically
substituting other types of fats and salad dressings (stick margarine, butter, and
mayonnaise) with olive oil was inversely associated with the onset of DM2 [48]. In
another cross-sectional study in Spain (Pizarra study), it was found that insulin
resistance was significantly lower in people who consumed OO than in those who
consumed sunflower oil or a mixture [167].
Furthermore, a randomized trial with two groups of DM2 patients, following a
MD using OO or a control low-fat diet presented that the MD with OO can improve
endothelial dysfunction, inflammation, and oxidative in DM2 patients compared to a
control diet [49]. The PREDIMED trial also provides strong evidence that long-term
adherence to a MD supplemented with EVOO, which is high in MUFAS and
bioactive polyphenols, results in a substantial reduction in the risk for DM2 among
older people with high cardiovascular risk [41].
Oleic acid (cis C18:1 n-9), the predominant MUFA on OO, has been related to a
lower insulin resistance as well. A study addressing [50] the relationship between
changes in membrane fatty acid composition and glucose transport as an index of
insulin sensitivity found a reduction in insulin resistance when a polyunsaturated
(linoleic acid; C18:2 n-6) rich diet was changed to an oleic acid (C18:1 n-9) rich diet.
This improvement was related to the change in membrane fluidity, as an oleic acid-
rich membrane would be less fluid to its linoleic acid-rich equivalent [46]. Similar
results were found in another study [51] where changes in membrane fatty acids and
signaling proteins induced by VOO consumption in elderly persons with DM2
compared to a control group were analyzed. The long-term consumption of VOO
induced changes in the fatty acid content of erythrocyte membranes from elderly
DM2 participants. These changes were due to an increase amount of oleic acid in the
membranes and in consequence biochemical changes in the amount of signaling
proteins (G proteins and protein kinase C). The above could explain the mechanisms
of glycemic homeostasis after consumption of olive oil.
In addition to MUFA, extra virgin olive oil contains also other bioactive compo-
nents, some of them are called the phenolic compounds such as oleuropein and
hydroxytyrosol, flavonoids, specially flavones, and lignans. Apart from PRE-
DIMED, other clinical trials have evaluated the effect of EVOO rich in polyphenols
on glycemic biomarkers [52]. Daily consumption of polyphenol-rich EVOO (25 mL/
day, 577 mg of phenolic compounds/kg) for 8 weeks significantly reduced fasting
plasma glucose and HbA1c, as well as other circulating inflammatory adipokines
(visfatin), in overweight patients with DM2 [53], in a crossover randomized trial.
Mediterranean-type dietary patterns are known to improve several parameters of
the postprandial state with high atherogenic potential, such as the glycemic load and
the secretion and clearance of chylomicrons [54]. The consumption of OOPCs has
been able to decrease oxidative stress (as observed in several biomarkers related to
oxidative modifications of plasma lipids, proteins, and DNA) in healthy volunteers
after meals [10] and in long-term interventions [11]. Concretely, a single dose of
25 mL of OO does not promote postprandial oxidative stress whereas doses equal or
superior to 40 mL do [10, 55]. In addition, phenolic compounds from OO have been
able to modulate postprandial oxidative stress in healthy volunteers [10]. The
glycemic load of a meal can depend on the bioavailability of carbohydrates and
food preparation. In this regard, in a study with 12 women with obesity and insulin
resistance, food fried in VOO improved both insulin and C peptide responses after
a meal [56]. VOO may additionally contribute, within the context of a traditional
MD, to the long-term improvement of the glycemic load and the dietary glycemic
index [57].
In conclusion, there is already widespread experience of the benefits of olive oil in
glucose metabolism, both in a medium- and long-term period and in the postprandial
phase, in the context of dietary patterns, of olive oil by itself, and the bioactive
compounds of olive oil.
5 Cardiovascular Diseases
The development of an atherosclerotic plaque in the endothelium of blood vessels is

common to CVDs. The plaque is formed by the accumulation of cholesterol inside
the macrophages and other cells, such as the smooth muscle ones, located in the
intima media. This generates an inflammatory chronic response that eventually can
trigger acute thrombotic vascular disease, including myocardial infarction, stroke,
and sudden cardiac death [58]. Atherosclerotic plaques are linked to the coexistence
of several processes related to inflammation, lipid oxidation, dysfunction of the
vascular endothelium, exacerbated activation of immune cells, and migration of
vascular smooth muscle cells, among others [59].
Since Ancel Keys presented the MD as a health protecting one [1], there have
been many studies supporting this link [5, 7, 16, 60]. The most impressive
benefits of this diet, however, have been related to reductions in cardiovascular
morbidity and mortality [61]. In general, countries from southern Europe present
the lowest values of accumulated incidence and mortality rate of coronary heart
disease [62, 63]. The paradox of the Mediterranean countries with a low inci-
dence rate of cardiovascular disease [64–66], in spite of a marked prevalence of
classical cardiovascular risk factors [67, 68], is attributed in part to a high degree
of adherence to the MD. Nevertheless, most studies were observational so that
any causal inference was hindered by residual confounding among other biases.
Thus, large-scale randomized trials using dietary patterns, and assessing clinical
end-points, are needed to provide a high level of scientific evidence. In this
regard, very few randomized trials have been performed. On one hand, the
Lyon Diet Heart Study, a secondary prevention trial, showed a large reduction
in rates of coronary heart disease events with a modified MD enriched with alpha-
linolenic acid [69]. On the other hand, the PREDIMED study, a multicenter,
randomization, intervention trial with the MD, reported the protection of the
traditional Mediterranean Diet (TMD) on incident CVD in high cardiovascular
risk individuals, in primary prevention [70]. In addition, the PREDIMED Study
has also provided evidence of the efficacy of the TMD on primary prevention for
stroke [70], atrial fibrillation [71], type-2 diabetes (DM2) [72], and peripheral
vascular disease [73].
Nowadays, the relevance of overall high-quality diet patterns, rather than focus-
ing on single nutrients and foods, has highlighted the need to address the complexity
of dietary exposures. Nevertheless, it is well-known that OO plays a pivotal role in
this diet pattern. Data from EPIC cohorts showed an inverse relationship between
OO consumption and coronary heart disease mortality and incidence [74–76]. Also,
results from the Three-City Study reported an inverse relationship between OO
consumption and stroke risk in women [77]. Finally, results of the PREDIMED
Study showed that consumption of OO, specifically the extra-virgin variety, within
the framework of the Mediterranean diet, reduces the risk of CVD and mortality in
elderly high cardiovascular risk individuals [78].
It is increasingly accepted that chronic degenerative diseases, such as CVD,
cancer, and neurodegenerative ones, share common risk factors. Besides the classical
cardiovascular risk factors (diabetes, hypertension, lipid profile, tobacco, and obe-
sity), perpetuated molecular dysregulation with respect to oxidation, low-grade
inflammation, LDL atherogenicity, HDL function, and endothelial dysfunction,
among others, may be behind their onset.
5.1 Arterial Hypertension and Endothelial Dysfunction
OO has also been linked to an improvement of endothelial dysfunction and blood

pressure state. In a meta-analysis of randomized controlled assays (N = 12, with a
duration of 6 months or longer) high- versus low-MUFA intake (low MUFA intake
was considered a MUFA consumption below 12% of total daily energy consump-
tion) was compared, and MUFA-rich diets decreased systolic (mean effect:
2.26 mmHg; CI: [ 4.28, 0.25]; p = 0.03) and diastolic blood pressure (mean
effect: 1.15 mmHg; CI: [ 1.96, 0.34]; p = 0.005]. In addition, an improvement
of the fat mass 1.94 kg, CI: [ 3.72, 0.17], p = 0.03] was additionally reported
[79]. MUFA intake also has effects on doses of antihypertensive drugs prescribed to
patients. A MUFA-rich diet (17.2% of total daily energy intake by MUFAs, 3.8% by
PUFAs) was able to decrease blood pressure relative to a polyunsaturated fatty acid
(PUFA)-rich one (10.5% of total daily energy intake, 10.5% by PUFAs) and,
moreover, decreased the daily dosage of hypertensive agents [80]. A decrease of
diastolic blood pressure in hypertensive women was observed after an MD inter-
vention, enriched with VOO or nuts, within the context of the PREDIMED Study
[81]. Nitric oxide and endothelin-1 levels, together with endothelin-1 receptor gene
expression, could play a role in blood pressure improvement [82].
Regarding OOPCs, in a meta-analysis with 13 studies concerning the effects of
high phenolic OO oil on cardiovascular risk factors, medium effects for lowering
systolic blood pressure (n = 69; mean effect: 0.52; CI: [ 0.77, 0.27]; p<0.01)
were described; however, no effects for improving diastolic blood pressure were
reported (mean effect: 0.20; CI: [ 1.01, 0.62]; p = 0.64) [83].
Endothelial function improved in hypercholesterolemic patients at postprandial
state with phenol-rich OOs and OOPC-enriched functional OO intake [84, 85], after
a 4-month intervention of a daily intake of VOO in patients with incipient athero-
sclerosis [86], and after 2 months in women with high-normal blood pressure or
stage-1 essential hypertension [87].
Endothelial homeostasis can be disturbed by oxidative stress and chronic inflam-
matory processes and finally lead to endothelial dysfunction [58]. An increase in
nitric oxide metabolites was observed after a phenol-rich OO postprandial intake
versus a low-phenolic OO [84]. Also, a decrease in systolic blood pressure after the
intake of VOO was described, with a decrease in lipid oxidation markers,
in hypertensive stable patients [88]. Finally, in two randomized crossover studies,
a 50 mL intake of phenol-rich OO decreased the postprandial leukotriene B4 (a pro-
inflammatory eicosanoid) and thromboxane B2 (a vasoconstrictor eicosanoid)
levels in comparison to refined OO in healthy [169] and mildly dyslipidemic
subjects [89].
5.2 Haemostasis and Platelet Aggregation
Olive oil has been shown to improve the production of coagulation factors and bio-
markers linked to platelet aggregation, thus improving the thrombogenetic profile [90].
With regard to monounsaturated fats, a MUFA-rich diet such as the MD decreases
postprandial levels of coagulation factor VIIc [91]. In addition, oleic acid (75% of
OO fatty acids) attenuates the prothrombotic state in the postprandial phase [91–93].
Regarding OOPCs, the consumption of phenol-rich OOs improved the postpran-
dial prothrombotic state (activated coagulation factor VII, tissue factor, tissue plas-
minogen activator, plasminogen activator inhibitor type-1, and fibrinogen) in a
number of randomized controlled trials in both healthy [94] and hypercholesterol-
emic subjects [90]. In long-term interventions, a decrease in plasma fibrinogen in
women with high baseline fibrinogen concentrations has been reported after OO
consumption, in a randomized crossover trial [95].
5.3 Lipid Profile
Improved dietary fat quality, achieved through a replacement of SFA by unsaturated

fat, enhances the lipid profile by reducing low density lipoprotein (LDL) cholesterol
and increasing high-density lipoprotein (HDL) cholesterol [96]. Olive oil intake
assures an increase of MUFA, without a significant rise of SFA, and guarantees an
appropriate intake of (PUFA). As a consequence, the MUFA/SFA ratio is much
higher in areas where an MD is followed [97]. An updated review suggested a small,
but potentially relevant, reduction in cardiovascular risk with low saturated fat intake
[98]. The replacement of energy from saturated fat with polyunsaturated fat provides
more healthy benefits than with carbohydrate. Regarding the substitution of SFAs for
MUFAs, although the benefits are more modest, some positive effects on lipid profile
have been observed. The reduction of dietary saturated fat, and partial replacement
by unsaturated fats, has been proposed to achieve maximum health benefits although
the ideal type of unsaturated fat is still unclear [98].
Schwingshackl et al. published a meta-analysis including 32 studies with over-
weight and obese patients. On one hand, decreases in total cholesterol (weighted
mean difference 0.12 mmol/L, 95% CI: [ 0.28 to 0.03]; P = 0.01) and LDL
cholesterol ( 0.08 mmol/L, 95% CI: [ 0.12 to 0.04]; P<0.0001) were more
marked after low-fat diets. On the other hand, a rise in HDL cholesterol
(0.06 mmol/L, 95% CI: [0.03, 0.09]; P<0.0001) and a reduction in triglyceride
( 0.095 mmol/L, 95% CI: [ 0.15, 0.04]; P = 0.001) were more pronounced after
the high-fat diet groups [99].
Regarding OO, in a recent meta-analysis about the effects of PC-rich OO on
cardiovascular risk factors, no significant effects in improving lipid profile were
observed (total cholesterol N = 400; mean effect: 0.05; CI: [ 0.16, 0.05];
p = 0.33); HDL cholesterol (N = 400; mean effect: 0.03; CI: [ 0.14, 0.08];
p = 0.62); LDL cholesterol (N = 400; mean effect: 0.03; CI: [ 0.15, 0.09];
p = 0.61); and triglycerides (N = 360; mean effect: 0.02; CI: [ 0.22, 0.25];
p = 0.90). Nevertheless, the small number of studies (N = 8) is a limitation [83].

Specifically, the beneficial effects of OO polyphenols on blood HDL cholesterol
concentrations were evaluated by the European Food Safety Authority (EFSA) and
they concluded that evidence was insufficient to establish a cause-effect relationship
[100]. A recent review indicated that the intake of PC-rich OO induced no significant
increases in HDL cholesterol levels [83], while several high-quality randomized
controlled trials have pointed to a dose-dependent increment in HDL cholesterol
after the consumption of OOPCs [11, 12, 101].
5.4 Lipid Oxidation
In November 2004, the USA Federal Drug Administration [102] permitted a claim
on OO labels regarding “the benefits on the risk of coronary heart disease of taking 2
tablespoons (23 grams) of OO daily, due to monounsaturated fat.” However, if the
effect of OO is only attributed to its MUFA content, any type of MUFA-rich food
(such as rapeseed oil, canola oil, or MUFA-enriched fats) would provide the same
beneficial effects for health. The minor components of OO (1–2% of the total
content) are classified in two groups: (1) the unsaponifiable: squalene and other
triterpenes, sterols, tocopherol, carotenoids, and pigments and (2) the soluble frac-
tion which includes the PCs [168]. In this respect, the EFSA released a health claim
concerning the protection of OOPC (5 mg/day) against LDL oxidation [103]. Based
on well-designed intervention trials, a cause-effect relationship was established by
the EFSA between the consumption of VOO and the protection of LDL particles
from oxidative damage [103].
Not only changes in LDL cholesterol are involved in cardiovascular risk, oxida-
tion modifications of the LDL particle may play a major role in atherosclerosis [58,
104, 105]. Several OO components contribute to decreasing LDL oxidizability. On
the one hand, MUFAs are less prone to becoming oxidized when compared to other
unsaturated fats [106]. On the other hand, OO antioxidants (vitamin E, carotenoids,
and OOPCs) can bind to the LDL particle and protect it from oxidative
modifications.
A recent meta-analysis (N = 13) reported that oxidized LDL levels decrease
significantly after the consumption of high-phenolic OO (N = 300; mean effect:
0.25; CI: [ 0.50, 0.00]; p = 0.05) [83]. In the EUROLIVE Study, 200 volunteers
were given 25 mL/day of raw OO with high (366 mg/kg), medium (164 mg/kg), and
low (3 mg/kg) phenolic content in a randomized, crossover, and controlled trial [11].
Covas et al. reported a decrease of the in vivo lipid oxidative damage (concretely in
oxidized LDL, uninduced conjugated dienes, and hydroxy fatty acids) in a linear
manner with the phenolic content of the OO administered [11]. As potential mech-
anisms to explain this benefit, we could consider an increase in the content of
vitamin E and OOPC in LDL that may counteract locally oxidative modifications
[107, 108]. In addition, the reductive effect of the OOPC on oxidized LDL could be
due to the generation of antibodies [109]. Vázquez-Velasco et al. observed, in
healthy volunteers, a decrease in the concentration of oxidized LDL when
hydroxytyrosol-enriched sunflower oil (45–50 mg/d) was administered during 3

weeks [110]. In 40 males suffering from stable coronary heart disease, VOO was
able to decrease the oxidized LDL in plasma compared with another olive oil with a
smaller amount of PC in a randomized crossover trial [88]. Finally, LDL
oxidizability has also been evaluated in vitro, and it decreased after the consumption
of MUFAs [111, 112] and phenol-rich OOs [11, 101, 113, 114].
Finally, Fitó et al. reported an improvement of circulating oxidized LDL after a 3-
month MD intervention in high cardiovascular risk patients [115].
5.5 HDL Functionality
Although low levels of HDL cholesterol are considered an independent cardiovascular

risk factor [116], it has been recently observed that presenting high HDL cholesterol
levels does not always lead to a decrease in cardiovascular risk [117–119]. An increase
in HDL cholesterol is one of the goals of clinical management of cardiovascular diseases
[117]. In this regard, recent studies have shown that the functionality of HDL can be of
greater relevance than its amount [120]. Decreased values of the most relevant HDL
function have been reported to be related to a high incidence of subclinical atheroscle-
rosis [121] and coronary events [122].
The functionality of the HDL particle involves the promotion of cholesterol efflux
from macrophages and peripheral cells, which constitutes part of the so-called
“reverse cholesterol transport” [123]. Furthermore, HDLs play a crucial role in
inhibiting the oxidation of plasma lipids (mainly, the ones in LDLs) and also present
anti-inflammatory and vasoprotective capacities [124]. Oxidative modifications of
the HDL can additionally affect the lipid and/or protein of the lipoprotein and alter its
physiological properties [120, 124–126] and, in this regard, antioxidants linked to
the particle could be able, direct or indirectly, to counteract such oxidation. An
increment in HDL fluidity, enhanced HDL composition, and better HDL size
distribution could also mediate improvements in HDL function [127–129].
Regarding OO, the consumption of a MUFA-rich diet improved the cholesterol
efflux capacity of HDLs in a linear trial [130]. This functional improvement could be
due to a lower degree of oxidative modifications of the lipoprotein after increasing its
MUFA content [131]. Regarding OOPCs, Hernáez et al. reported for the first time an
increase in cholesterol efflux after the daily intake of 25 mL of VOO (366 mg/kg) in
healthy volunteers within the framework of the EUROLIVE Study [132]. In parallel,
biological metabolites of OOPC bound to the HDLs (hydroxytyrosol sulfate, and
homovanillic acid sulfate, and glucuronate) were determined. The improvement of
HDL fluidity and a triglyceride-poor core can also result in a more functional HDL
particle [132]. In this respect, functional VOO supplemented with olive and thyme
phenols versus a VOO intervention produced an increase in the lecithin-cholesterol acyl-
transferase concentration which esterifies free cholesterol and mediates its migration into
the particle core [133]. The consumption of OOPCs has also been shown to be able to
increase the levels of the main HDL antioxidant enzyme, paraoxonase-1, as well as
HDL anti-inflammatory ability, in a noncontrolled trial [134]. Besides the direct
antioxidant effect of OOPCs on HDL particles, these compounds have also been
observed to be able to improve the gene expression related to HDL function [135].
Finally, a TMD, especially when supplemented with real-life doses VOO, was
able to improve HDL functionality [136]. Concretely, a 1-year intervention with an
MD increased cholesterol efflux capacity and, in particular, the VOO-rich MD
enhanced nitric oxide synthesis by endothelial cells promoted by HDLs, decreased
cholesteryl ester transfer protein activity, and increased HDL ability to esterify
cholesterol and paraoxonase-1 arylesterase activity [136].
5.6 Inflammatory Processes and Systemic Oxidative Status
Oxidation and inflammation are intertwined processes which, when sustained for a
long period, may induce the onset of a number of chronic degenerative diseases,
such as CVD, diabetes, neurodegenerative diseases, and cancer. The increased
circulating concentrations of pro-inflammatory analytes (tumor necrosis factor-α,
monocyte chemotactic protein-1, soluble vascular cell adhesion molecule 1, and
soluble intercellular adhesion molecule-1) perpetuate the inflammatory response in
the subendothelial space and establish endothelial dysfunction.
Traditionally, the health benefits of the MD have been attributed to its richness in
antioxidants. Among the approximately 230 chemical compounds of OO, the main
antioxidants are carotenes and phenolic compounds, including lipophilic and hydro-
philic phenols [137]. Current evidence points to oxidative damage as a promoter of
pathophysiological processes in oxidative stress-related diseases such as coronary
heart disease, cancer, neurodegenerative pathologies, and, in addition, aging
[138–140]. Although phenolic compounds are good antioxidants in vitro, their
in vivo effects can be indirectly mediated through the activation of several
nutrigenomic pathways and not only by their intrinsic antioxidant activity
[141, 170]. Benefits of olive oil on lipid oxidation have been extensively explained
in the Sect. 5.4 apart.
Several benefits on the levels of pro-inflammatory biomarkers have been proven
in human trials with OO or MUFA-rich diets. A daily intake of real-life doses of OO
has been shown to produce a decrease of the C-reactive protein (mean effect:
0.64 mg/L; CI: [ 0.96, 0.31]; p<0.0001) and IL-6 (mean effect: 0.29; CI:
[ 0.70, 0.02]; p<0.04) in a recent systematic review [43]. Nevertheless, the
heterogeneity of design among studies makes further research necessary. With
regard to other MUFA-rich diets, a randomized controlled trial with 28 hyper-
triglyceridemic and 14 healthy males who followed a diet rich in refined OO
(high-oleic acid diet) or a diet rich in high-palmitic sunflower oil, revealed a
postprandial decrease of the soluble adhesion molecules (VCAM-1, ICAM-1) after
the high-oleic intervention [142]. And in another randomized controlled trial, a
2-month MUFA-rich diet decreased the expression by peripheral blood mononuclear
cells of ICAM-1 in healthy males [143]. In contrast, a randomized crossover trial in
healthy subjects regarding the effect of three Malaysian diets: palm olein, coconut
oil, and OO (the fat source providing approximately 20% of total energy intake in
each case), the postprandial and 2-week fasting circulating concentrations of a

number of inflammatory biomarkers (tumor necrosis factor-α, IL-1β, IL-6, IL-8,
CRP, and interferon-γ) were not affected [144].
The consumption of OO, which provides oleic acid as the main fatty acid, also
yields a moderate intake of PUFA (mainly omega-6 linoleic acid and omega-3
alpha-linolenic acid) without a noticeable increase in the intake of SFA. Omega-6
fatty acids, in which PUFAs are predominant (especially in the Western diets), are
precursors of pro-inflammatory eicosanoids (2-prostaglandins, tromboxanes, and
4-leukotrienes) [145]. In this regard, omega-3 fatty acids in OO may play a role in
the inhibition of the inflammatory process boosted by omega-6 fatty acids [146].
It has been reported that a low incidence of coronary heart disease is related to a
diet rich in omega-3 fatty acids [147].
The OOPCs have also shown effects on pro-inflammatory biomarkers. Surpris-
ingly, Beauchamp et al. described oleocanthal anti-inflammatory properties that were
similar to those of ibuprofen (cyclooxygenase-2 inhibition and nuclear factor kappa
beta counteract) [148]. Moreover, the intake of phenol-enriched OO (50 mL/day)
decreased C-reactive protein (CRP) levels at postprandial state relative to refined
olive or corn oils in healthy individuals, in a randomized controlled trial. In this
regard, a regular intake of OO at long term can contribute to controlling the
postprandial inflammatory burden [149]. Also, a combined consumption of white
wine (2–3 glasses/daily) and extra VOO (ad libitum) for a 2-week period decreased
the levels of CRP and IL-6 in both patients with chronic kidney disease and healthy
subjects [150]. Finally, a raw real-life daily dose of 50 mL of VOO for 3 weeks
decreased the IL-6 and CRP concentrations (versus refined OO) in a randomized
controlled trial with stable coronary heart disease patients [151].
Within the context of a traditional MD, supplementation with VOO or nuts
decreased the systemic levels of CRP, IL-6, sVCAM1, and sICAM1 in high cardio-
vascular risk volunteers within the framework of the PREDIMED Study [81]. In
addition, the traditional MD (supplemented with VOO or nuts) was able after 3
months of intervention to decrease the expression of CD49d (an adhesion molecule)
and CD40 (a pro-inflammatory ligand) in monocytes isolated from the blood of high
cardiovascular risk volunteers [152].
The MD participants had lower plasma concentrations of atherosclerosis-related
inflammatory markers (IL-6, IL-8, MCP-1, and MIP-1β) after 3 and 5 years of an
MD intervention versus a low-fat diet control group. In addition, IL-1β, IL-5, IL-7,
IL-12p70, IL-18, TNF-α, IFN-γ, GCSF, GMCSF, and ENA78 decreased especially
after an MD supplemented with VOO [153]. Finally, the reduction in CD49d and
CD40 expressions in T lymphocytes and monocytes at 3 years were greater in the
MD group than in the low-fat diet control one [154].
5.7 Immune System
Regarding MUFAs, they have been reported to modulate a number of biological

pathways of immune competent cells [155]. In this regard, in a randomized
controlled trial with healthy males, there was a significant decrease in the expression
of intercellular adhesion molecule 1 by peripheral blood mononuclear cells after
MUFA-rich 2 month-diet. Nevertheless, natural killer cell activity and the prolifer-
ation of mitogen-stimulated leukocytes were not affected [143].
The consumption of a functional VOO showed an increase in the proportion of
IgA-coated bacteria, which indicates a local stimulation of the intestinal mucosal
immunity [156]. Based on the effects of OO intake on the immune system, it has
been suggested that OO consumption may have benefits on rheumatoid arthritis
[155]. In contrast, other authors have published that OO-rich diets do not alter host
resistance to infection [157, 158]. Given that data are scarce, more studies are
required to establish the possible immunostimulation of OO in vivo.
5.8 Microbiome
Gut microbiota, a complex and dynamic ecosystem, is an emergent factor of a

number of diseases including obesity and type-II diabetes [159]. PCs can selectively
stimulate growth bacteria, such as Lactobacillus [160], which can play a role in
lowering cholesterol levels [161]. Intestinal lactobacilli produce bile-salt hydrolase,
which deconjugates bile acids, prevents their reabsorption, and therefore promotes
their fecal excretion [162]. This can be one of the mechanisms involved in the
decrease of systemic cholesterol after PC consumption [163]. PCs may also be
related to the growth of other bacterial populations (such as Bifidobacterium)
which might be linked to a lesser development of the atherosclerotic plaque [164].
Since most PCs are not fully absorbed into the upper gastrointestinal tract and
reach the large intestine, they can be metabolized by gut microflora [165]. A 3-week
intake of a phenol-enriched VOO (with OO and thyme PCs) increased populations of
Bifidobacteria and the levels of microbial metabolites of antioxidant PCs, such as
protocatechuic acid and hydroxytyrosol, in hypercholesterolemic individuals [166].
6 Conclusion
In summary, current evidence indicates the potential benefits of olive oil, within the
framework of a healthy diet such as the Mediterranean one, on the prevention of
chronic degenerative diseases including cardiovascular and degenerative ones. To
date, the majority of these effects have been demonstrated from the perspectives of
atherosclerosis and cardiovascular risk research. Nevertheless, there is increasing
evidence that such beneficial properties also display a protection towards other
diseases including neurodegenerative ones and cancer.
The daily diet represents the consumption of a mixture of foods in which the
type of cooking plays an indisputable role in the availability and properties of
nutrients. Furthermore, synergism among nutrients and foods, and their cumula-
tive effects, must also be taken into consideration. With respect to the
Mediterranean diet, olive oil as the main source of fat is determinant with respect
to the properties attributed to this dietary pattern.
Olive oil intake facilitates more vegetable consumption and thus benefits on health
can be maximized. Nutrient-specific biases and type of olive oil should be considered in
order to disentangle the benefits of MUFA and/or other minor components. Further well-
designed, large-scale cohort studies in Mediterranean and non-Mediterranean areas are
needed to reaffirm the therapeutic properties of olive oil and establish in which subjects
and under which conditions benefits can be more easily achieved.
Acknowledgments This work was supported by: Agència de Gestió d’Ajuts Universitaris i de
Recerca (2014-SGR-240), Instituto de Salud Carlos III (CB06/03/0028, JR14/00008, JR17/00022,
CD17/00122, and CES12/025), and the Spanish Ministry of Education, Culture and Sport (FPU12/
01318). The CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) is an initiative of
the Instituto de Salud Carlos III.
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Anthocyanins: Nutrition and Health
38
Iva Fernandes, Cláudia Marques, Ana Évora, Ana Faria,
Conceição Calhau, Nuno Mateus, and Victor de Freitas
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
1.1 Chemistry, Occurrence, and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1098
1.2 Anthocyanin Food Bioaccessibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1103
2 Interactions of Anthocyanins with Biomolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104
2.1 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104
2.2 Biomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1106
2.3 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1106
3 Bioavailability and Metabolism of Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107
3.1 Oral Cavity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107
3.2 Stomach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1108
3.3 Intestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1111
4 Obesity/Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1111
5 Gut Microbiota Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1113
6 Gut-Brain Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
7 Skin Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117
7.1 Skin Aging and UV Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1118
7.2 Anthocyanins and Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1122
8 Functional Foods and Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1126
I. Fernandes · A. Évora · N. Mateus · V. de Freitas (*)

REQUIMTE/LAQV, Department of Chemistry and Biochemistry, Faculty of Sciences, University
e-mail: [email protected]; anasfi[email protected]; [email protected]; [email protected]
C. Marques
Nutrição e Metabolismo, NOVA Medical School, Faculdade de Ciências Médicas, Universidade
Nova de Lisboa, Lisboa, Portugal
ProNutri - Clinical Nutrition and Disease Programming, CINTESIS - Center for Research in Health
Technologies and Information Systems, Porto, Portugal
Faculty of Medicine, University of Porto, Porto, Portugal

1098 I. Fernandes et al.
Abstract
After consumption of anthocyanin-rich foods, there is a long journey before these
bioactives may exert a health-promoting property. They must pass through the
oral cavity, the gastrointestinal tract, undergo metabolism events, pass cellular
barriers, and eventually trigger a biological event.
Hence, before looking at the health effects of anthocyanins, some topics
related to their food bioaccessibility, interaction with biomolecules (proteins,
biomembranes, and DNA), and bioavailability/metabolism will be described as
possible interferers to their bioactive effects.
There are several reports on the health preventing properties of anthocyanins
on several diseases like cardiovascular diseases, some types of cancers, diseases,
diabetes, allergies and osteoporosis. In this chapter some of the less revisited ones
giving emphasis to obesity/metabolic syndrome, microbiota modulation, neuro-
degenerative diseases and skin health will be reviewed.
Keywords
Anthocyanins · Bioactives · Disease prevention · Food
1 Introduction
1.1 Chemistry, Occurrence, and Stability
Anthocyanins constitute the largest group of water-soluble pigments widespread in

the plant kingdom arising from plant secondary metabolism, being responsible for
the colors displayed by many flowers, fruits, and leaves of angiosperms (Fig. 1).
Chemically, anthocyanins are flavonoids, hence based on a C15 skeleton with a
chromane ring bearing a second aromatic ring B in position 2 (C6–C3–C6). In
nature, anthocyanidins are usually glycosylated in one or more positions of the
basic flavanic nucleus by different sugars (Fig. 2). According to the literature,
A. Faria
REQUIMTE/LAQV, Department of Chemistry and Biochemistry, Faculty of Sciences, University
C. Calhau
38 Anthocyanins: Nutrition and Health 1099
1) Food Bioacessibility 2) Oral processing

Cytoplasm
Vacuole
CellWall Nucleus
Pecticpolysaccharides Complexes with salivary proteins
Oral cavity Modulation by Oral microbiota
ß-glycosidase
Insoluble polysaccharides complexes
Phase II metabolites activity
Catabolites
3) Gastric absorption 4) Physiologycal interactions
Stomach
Lipase Trypsin Pepsin α-glucosidase

MKN-28 cell line
Elastase Collagenase Tyrosinase
Anthocyanin Inhibition of digestive/ECM enzymes
• Protective antioxidant effect

• Antiproliferative effect
• Modulation by diet components

Polyphenols, proteins, pH, carbohydrates, alcohol and monosaccharides
• Gold Nanoparticles
Insertion in biomembranes
Transient silencing
5) Phase II metabolism and intestinal absorption 6) Microbiota modulation – Brein axis
STANDARDS
Phase II
metabolization
Cólon
Liver
Human intestinal Catabolites

absorption
Small
Intestine • Neuroprotetive effects
Caco-2 cell line
7) Obesity/ metabolic syndrome 8) Skin health
Anthocyanin
HaCat Cell line

HFF-1 Cell line
Normalweight (NW) Overweight/obese (OW) Extracellular
Matrix (ECM)
• Wound-healing effect
• UV protection
• Modulation of glucose homeostasis
and obesity biomarkers
Fig. 1 Schematic representation of anthocyanin long journey after consumption and some of their
biological targets
Fig. 2 Basic structure of

anthocyanidin pigments
(flavylium form) in which R
could be H, OH, or OCH3.
The nomenclature for
numbering carbons is
indicated in the structure
Table 1 Anthocyanidins Name Position of substitution

found in nature [3]
Substituted with a hydroxyl group
Apigeninidin 5,7,40
Aurantinidin 3,5,6,7,40
Cyanidin 3,5,7,30 ,40
Delphinidin 3,5,7,30 ,40 ,50
6-Hydroxycyanidin 3,5,6,7,30 ,40
Luteolinidin 5,7,30 ,40
Pelargonidin 3,5,7,40
Triacetidin 5,7,30 ,40 ,50
Substituted with a methyl ether group
Apensinidin 5,30 ,50
Europenidin 5,30
Hirsutidin 7,30 ,50
Malvidin 3,50
5-Methylcyanidin 5
Poenidin 30
Petunidin 30
Pulchellidin 5
Rosinidin 7,30
there are 17 anthocyanidins with differences in the number and position of hydroxyl
groups and/or methyl ether groups, but six of them are the most common
anthocyanidin in the plant kingdom (Table 1, in bold). From these 17 structures,
the ones with at least one sugar molecule give origin to anthocyanin compounds. The
range of anthocyanin is greatly increased by the sugar diversity and all the possible
structural positions of glycosylation (glucose, rhamnose, xylose, galactose, arabi-
nose, and fructose). Additionally, many acylated anthocyanins show in their
structures ester bonds between sugars and organic acids such as coumaric, caffeic,
ferulic, p-hydroxybenzoic, synaptic, malonic, acetic, succinic, oxalic, and malic.
While acylated anthocyanins in general have increased stability due to co-pig-
mentation effects, this effect may also decrease the absorption of these compounds
during digestion [1]. In fact, in human studies have found that non-acylated antho-
cyanins are more bioavailable than acylated anthocyanins [2].
The degree of complexity of those structures in aqueous solution increase because
they are in different equilibrium forms depending on the pH [4].
In very acidic aqueous solution (pH < 1), these pigments are present as red
flavylium cations. The increase of pH leads to a reduction of the intensity of the
color due to a decrease of the concentration of the flavylium cation that is converted
into its colorless hemiketal form through nucleophilic attack of water. The hemiketal
further undergoes a tautomerization reaction to give the pale yellow cis-chalcone
(Cc), which isomerizes to trans-chalcone (Ct). At low acidic, neutral, and basic pH
values, deprotonation of the flavylium cation also occurs, giving rise to the violet/
blue quinoidal forms (Fig. 3) [4]. Indeed, anthocyanins GI absorption is highly affect
by this pH dependence (Fig. 3).
Anthocyanins are responsible for many attractive colors, from scarlet to blue, of
flowers, fruits, leaves, and storage organs. Anthocyanins display several functions in
plants: antioxidant, photoprotection, defense mechanism, as well as other reproduc-
tive functions such as pollination and seed dispersal. The type of anthocyanins in
plants is so variable that some plants present only one type of anthocyanin, whereas
others have mixtures. In the same trend, some fruits are a source of one anthocyanin:
cyanidin in apple, cherry, fig, and peach and delphinidin in eggplant and pomegran-
ate; some fruits have two main anthocyanins such as cherry sweet and cranberry
(cyanidin and peonidin), while others have several anthocyanins (grape, myrtillus).
In general, the anthocyanin concentration in most of the fruits and vegetables goes
from 0.1 up to 1% dry weight [3].
Other anthocyanin derivatives may be found in processed foodstuff and in
fermented and unfermented fruit juices including pyranoanthocyanins and polymeric
anthocyanins (Fig. 4) derived from these common anthocyanins by the reaction with
a reaction partner that may include hydroxycinnamic acids, vinylphenols,
vinylflavanols, pyruvic acid, acetaldehyde, acetone, or the genuine pigment [4].
Since anthocyanins are the most important coloring compounds between flavo-
noids, they provide characteristic colors to fruits, vegetables, and food beverages
having a huge impact on this key quality parameter that influences consumer
acceptance.
From a nutritional perspective, anthocyanins are consumed not only in fresh
fruits and vegetables but also in many foodstuffs that are thermally processed prior
to consumption, and this process can greatly influence anthocyanin content in the
final product. Heating at lower temperatures (~40 C) for up to 3 days may not
be detrimental, but heating at higher temperatures (60–125 C) for more than 8 h
results in considerable loss of anthocyanins [6]. Bleaching by the effect of heat
occurs because the anthocyanin equilibrium is moved toward the uncolored forms,
as shown in Fig. 3. The flavonoid structure opens to form a chalcone, which is
1102
Food
pH 1-5 pH 5-7.4 pH 7.4-8
Oral cavity
Stomach
Small
Intesne
Cólon
Fig. 3 Anthocyanin equilibrium forms according to the GI tract pH. (Adapted from [5])
I. Fernandes et al.
Fig. 4 Pyranoanthocyanins and polymeric anthocyanins identified in processed foodstuffs and in

fermented and unfermented fruit juices
further degraded to brown products. In slightly alkaline solutions (pH 8 to 10),

highly colored ionized bases are formed [7].
This thermal degradation can have a dramatic impact on color and may also affect
nutritional properties. Still, the thermal degradation of anthocyanins could be accom-
panied by the formation of products which may also possess antioxidant properties.
Therefore, it is not clear whether the formation of these components results in an
overall reduction in antioxidant activity, besides affecting other food features [8].
Besides, anthocyanin stability is not merely dependent on the final processing tem-
perature but is in turn influenced by the intrinsic properties of the product and the process
such as pH, storage temperature, chemical structure and concentration of anthocyanins,
light, oxygen, the presence of enzymes, proteins, metallic ions, and carbohydrates.
1.2 Anthocyanin Food Bioaccessibility
The structural diversity and physical-chemical properties of anthocyanins greatly

interferes with their absorption and digestion and also with their final excretion
during metabolic processes [1]. Before the endogenous factors such as pH, the
presence of food, digestive enzymes, bile acids, microbiota, and the motility and
permeability of the gastrointestinal (GI) tract may exert their actions, food
bioaccessibility is one major impairment of anthocyanins absorption.
In order for anthocyanins in fruits and vegetables to become available for absorp-
tion within the human GI tract, these compounds must be released from the vacuole of
plant cells [9]. When cells are ruptured during processing or oral mastication, antho-
cyanin and plant cell wall component (cellulose and pectin) complexes come into
contact for the first time with other in vivo components [9, 10]. In processed foodstuffs,
interactions between different anthocyanins and the food matrix (carbohydrates, pro-
teins, fibers, other polyphenols) are likely to affect their bioaccessibility (i.e., the
amount of anthocyanins released from the food matrix prior to absorption) of antho-
cyanins. This could potentially have an impact on the nutritional content and func-
tional potential of diets [11]. Besides the initial mechanic and enzymatic action in the
oral cavity (further discussed), foodstuffs are ingested as small pieces, so these types of
interaction may be kept until the gastric environment.
In a recent work, it was shown that the flavylium cation (at pH 1) and hemiketal
forms (at pH 3–5) of two anthocyanins (cyanidin-3-glucoside and delphinidin-3-
glucoside) interact with ionic carbohydrates (pectin) [12]. This is indicative that this
type of complexes may be stable at the gastric environment and prevent absorption
and metabolization. In addition, the more polar anthocyanins revealed a stronger
binding to pectin [13].
Anthocyanin uptake in the small intestine appears to be very likely considering
the increase in their plasmatic levels [14], but their release from complexes may not
be easily achieved, and their maintenance in the intestine/gut in that form may
resemble a prebiotic effect.
The co-ingestion of anthocyanins with other meal components is also detrimental
for the absorption of anthocyanins [15, 16]. Normally, to reduce the biological
samples complexity, animal and human studies are performed with purified antho-
cyanins or extracts dissolved in water or even red fruit consumed after overnight
fasting, thereby not taking into account possible interactions of anthocyanins with
other dietary constituents. These are highly reactive pigments, and their possible
reaction with other polyphenols and interaction with carbohydrates or proteins may
greatly alter their pharmacokinetics.
Likewise, Xiao and coworkers concluded that milk did not influence the oral relative
bioavailability of pelargonidin anthocyanins under meal conditions; however, the oral
relative bioavailability of pelargonidin anthocyanins was reduced by 50% by milk
under meal conditions ( p < 0.05) [15]. The simultaneous intake of a food source
reduces anthocyanins absorption and urinary excretion [16]. However, the additional
food matrix does not appear to have an effect on anthocyanin metabolism [16].
2 Interactions of Anthocyanins with Biomolecules
2.1 Proteins
During oral consumption, a variety of molecular interactions can occur, namely,

interaction with food proteins resulting from the mastication processes or with
salivary proteins, and later in the digestive process, the released anthocyanins may
exert their effect in the modulation of the activity of digestive enzymes [17].
Only some local effects in the oral epithelium may be considered. Anthocyanins
are not usually directly linked to astringency sensation, although they may contribute
to the bitterness of some fruits and drinks [18]. The bitter taste perceived after the
ingestion of some foodstuffs, for example, red or port wine, may be attributed to the
activation of some bitter receptors [19], or anthocyanins could form complexes with
saliva proteins [18].
Moreover, anthocyanins may display inhibitory effects on carbohydrate digestion
and glucose absorption. Suppression of the activity of pancreatic lipase, α-glucosi-
dase, and α-amylase is positively associated with the reduction of fat and sugar
absorption from the gastrointestinal tract [20]. This ability may be explored as part of
a strategy to reduce postprandial hyperglycemia by optimizing the functionality of
foods with anthocyanin extracts that would strengthen efforts to reduce the risk of
T2D.
Inhibition of pancreatic α-amylase and α-glucosidase activity was correlated with
anthocyanin ingestion resulting in a reduction of blood glucose levels after starch-
rich meals [20, 21]. A range of berry polyphenols (e.g., flavonols, anthocyanidins,
ellagitannins, and proanthocyanidins) can inhibit protease activities at levels which
could affect protein digestion in the gastrointestinal tract [21, 22].
In addition, anthocyanidins with vicinal hydroxyl groups in ring B (cyanidin and
delphinidin) were found to inhibit glycogen phosphorylase activity being able to
affect glucose/glycogen homeostasis [23].
This in vitro data is supported by several human trials reporting the regular
consumption of berries in association with a reduction in the risk of T2D. Growing
evidence from randomized controlled trials suggests that berry extracts, purees, and
nectars acutely inhibit postprandial glycemia and insulinemia following oral carbo-
hydrate loads [24]. Consumption of black currant extract in amounts roughly
equivalent to 100 g black currants reduced postprandial glycemia, insulinemia, and
incretin secretion, which suggests that inclusion of black currant polyphenols in
foods may provide cardio-metabolic health benefits [24, 25].
Berry extracts and anthocyanins inhibit the activities of pancreatic α-amylase and
α-glucosidase in the gut lumen and interact with intestinal sugar transporters,
sodium-dependent glucose transporter 1, and GLUT2, hence reducing the rate of
glucose uptake into the circulation. This topic is further explored in the bioavail-
ability section.
In a fundamental work, the affinity of Cy3glc for human serum albumin (HSA)
was studied showing to be stronger at pH 7, thereby underlining its potential in the
transport and distribution of anthocyanins in the organism [26]. Hydroxyl substitu-
ents and glycosylation of anthocyanins decreased the affinity for binding to HSA at
lower pH (especially pH 4) but increased the strength of binding at pH 7.4. Con-
versely, methylation of a hydroxyl group enhanced the binding at acidic pH, while
this substitution reduced the strength of binding at pH 7.4. According to these
findings, binding of anthocyanins to HSA is influenced not only by their structure
but also by the pH of the medium [27].
Some questions still remain as to how far such interactions may improve the
stability of anthocyanins and how far such interactions may influence their
bioavailability.
2.2 Biomembranes
In a recent work of Trouillas and collaborators, the capacity of various anthocyanin

derivatives to insert lipid bilayer membrane was evaluated [28]. Malvidin-3-O-
glucoside was studied in its different charge forms (flavylium cation, neutral and
anionic quinonoidal bases) as well as its deglycosylated, hydrated, and conjugated
derivatives. Most of the derivatives were theoretically predicted to insert rather deep
in the membrane, i.e., embedded in between lipid chains, therefore being prone to
scavenge both the initiation and propagation stages of lipid peroxidation.
Besides this protective antioxidant effect, in a recent in vitro study, the effect of
anthocyanins (pelargonidin-3-O-glucoside chloride and cyanidin-3-O-galactoside
chloride) on erythrocytes morphology showed that their incorporation into the
outer region of the erythrocyte membrane affects its shape and lipid packing order,
which is reflected in the increasing number of echinocytes (abnormal red blood cell
membrane) [29].
This involvement of anthocyanins in cellular morphologic alterations through
membrane interactions may be associated with the antiproliferative effect of antho-
cyanins present in several foodstuffs [30]. Similar to what was observed by other
authors, anthocyanidins were shown to interact with the deeper regions of the lipid
bilayers in a structure-dependent manner and to decrease their fluidity. Greater
membrane interacting potency was associated with a 3-hydroxyl group, 30 ,40 -
dihydroxyl groups of ring B, and 5,7-dihydroxyl groups of ring a, and
anthocyanidins meet these structural requirements.
The amphiphilic properties of flavonoids allow them to interact not only
hydrophobically with phospholipid acyl chains but also electrostatically with phos-
pholipid polar heads. In addition to hydrophobic interaction and hydrogen bonding,
steric configuration also participates in the interaction between anthocyanins and
membranes. The presence of polyhydroxyl groups, the heterocyclic ring C (pyran or
pyrone), and structural hydrophobicity are important for flavonoids to interact with
biomimetic membranes. The lower membrane interactivities are attributable to the
more hydrophobic anthocyanins, like delphinidin.
2.3 DNA
Nucleic acids such as DNA and RNA have also been proved to act as highly reactive
ligands for anthocyanins. The interaction of anthocyanins with calf thymus DNA
(ctDNA) was reported [31]. The anthocyanin-DNA complexes might be a possible
defense mechanism against oxidative damage to DNA and might have normal
physiological functions in vivo. This interaction is thought to occur between the
positively charged flavylium cation and the PO2 group; the ctDNA complex led to
phosphate charge neutralization and helix stabilization [31]. In vivo, this type of
interaction is not likely to occur since the nuclear pH is around 7.4, and at this value,
the positively charged fraction of the anthocyanin equilibrium forms is almost
absent.
On the other hand, the DNA triplex stabilization property of seven natural
anthocyanins (five monoglucosides and two diglucosides) has been determined by
triplex denaturation experiments [32]. It was assumed that the difference between the
diglucosides and monoglucosides could be due to the supplementary sugar moiety at
position 5 for the diglucosylated compounds, which would make anthocyanins too
crowded and close together to allow their interactions with the triplex. Furthermore,
the most active compounds among the monoglucoside series were the only antho-
cyanins that bear a catechol B-ring in their structure, which could be important for
such an interaction.
This ability of anthocyanins may be a possible mechanism for the anti-
proliferative against cancer cell lines described for this class of pigments.
3 Bioavailability and Metabolism of Anthocyanins
Considering the wide distribution of anthocyanins in the diet, it is reasonable to

assume that humans are very likely to consume these compounds in large amounts.
In a survey with Italian subjects, anthocyanin daily intake was in the range
25–215 mg/person, depending on gender and age, and this intake is largely enough
to induce pharmacological effects [3]. Nonetheless, among all polyphenol classes,
anthocyanins are the ones considered to have the lowest bioavailability. However,
recent reports suggest an underevaluation of the real bioavailability of these
compounds.
The early appearance of anthocyanins in stated blood and urine after consumption
of anthocyanin-rich foods is not compatible with an exclusive absorption at the
intestinal level, as initially accepted [33]. Furthermore, when considering the metab-
olites of anthocyanins resulting from the action of gastrointestinal enzymes and flora,
their bioavailability increases substantially [33].
3.1 Oral Cavity
The oral cavity is the first environment for the absorption and digestion of anthocy-
anins in humans. In chokeberry juice, among the several anthocyanins present in the
original juice, Cy3glc was the one preferentially accumulated in epithelium cells
[17]. This result suggests that anthocyanin structure affects oral cell uptake and
therefore the potential anthocyanin type and concentration available for further
absorption. Due to the short residence time of food in this compartment and the
transient passage of drinks, its contribution to the oral uptake of anthocyanins is still
in discussion, although their possible adhesion to the surface of the oral cavity cells
should be explored, together with the effect of chronic exposure (dairy intake). This
adhesion could be prompted by interaction of anthocyanins with membrane proteins.
Besides the physiological conditions (pH and temperature) impact, the dental
plaque and saliva oral microorganisms may contribute to anthocyanin degradation
during oral intake [17, 34]. Some reports point for the oral degradation of
anthocyanins after 5–60 min of residence in in vitro or ex vivo simulations or even in

human intervention studies [17, 34]. These may be attributed to enzymatic action of
oral microbiota, high oral temperatures, and pH and to binding of anthocyanin to
salivary proteins [18, 35]. In fact, in the oral cavity (pH ~6.8), the chalcone form of
cyanidin 3-O-glucoside may account for as much as 30% of the original amount of
standard cyanidin 3-O-glucoside incubated with ex vivo saliva [35].
Besides pH interconversion, deglycosylation of anthocyanins due to oral native or
microbiota-related glycosidases is one of the main products identified in the oral
cavity [35, 36]. In this latter study, Cy3glc microbiota metabolite, protocatechuic
acid, was also detected in saliva. The enzymatic machinery for phase II metabolism
and enteric recycling of anthocyanin was identified in the oral cavity of humans [36].
Furthermore, saliva samples (collected at 5, 60, 120, and 240 min) contain
glucuronidated anthocyanin conjugates and glucuronidated metabolites, consistent
with intracellular uptake and phase II conversion of anthocyanins [36]. Besides, as
subtracts of phase II enzymes, anthocyanins seem also to contribute to their activa-
tion [36].
3.2 Stomach
Based on the short residence time of anthocyanins in the oral cavity, their bulk
portion should reach the stomach in their ingested native form.
Within the stomach and due to its quite acidic pH, anthocyanins are in their most
stable medium. Besides being the main breakdown site in the digestion system of the
human body for other food ingredients, in the case of anthocyanins, it may also have
an important role in their absorption and metabolism. In fact, previous studies
indicated that significant amounts of anthocyanins could be absorbed quickly and
efficiently in the stomach in their native forms [37, 38]. In vitro studies using the
human MKN-28 and NCI-N87 as gastric barrier models also indicated that the
stomach could be an important site for anthocyanin absorption [39, 40].
By using in vitro cell barrier models, the gastric absorption mechanism was
described, and the influence of different parameters was determined. The transport
efficiency of anthocyanins is dependent on incubation time, pH, and anthocyanin
polarity and size and independent of the presence of ethanol [39, 41].
The presence of D-(+)-glucose was found to decrease anthocyanins gastric uptake
suggesting the involvement of glucose transporters [39]. In addition, gastric cells
pretreated with anthocyanins were found to increase anthocyanin transport, possibly
by inducing the expression of glucose transporters.
Using gene silencing technology in the same in vitro gastric model, it was
possible to attribute to glucose transporters the partial implication on the transport
of anthocyanins independent on the position of the glucose residue in the anthocy-
anin moiety. The fact that these compounds can reach an equilibrium in different
structural forms at a specific pH value (Fig. 3) suggests that anthocyanin transport in
vivo is dependent on the molar fraction of each equilibrium form. This highlights for
the fact that this particular class of polyphenols can be absorbed in different forms by
different transporters that contribute to the net transport efficiency determined. This
ability of anthocyanins to share the transport mechanism with glucose may give to
these compounds the capacity to modulate glucose absorption, which could be
important for several pathologies.
In addition, anthocyanins can be absorbed in fermented drinks like red wine in the
form of derivative pigments like carboxy-pyranoanthocyanins (type A vitisins) and
methyl-pyranoanthocyanins [42]. The gastric absorption of pyranoanthocyanin
derivatives is slightly reduced in comparison with the parental anthocyanins possibly
due to some steric interferences resulting from the higher size of these molecules.
The slightly higher gastric absorption value observed for oxovitisin among other
pyranoanthocyanins may be related to the absence of charge on this molecule [42].
The importance of pH on the anthocyanin transport efficiency that may be related not
only with the main anthocyanin forms present but also with the influence on the
possible transporters involved in their transport was already reported [39]. Besides
the proven gastric absorption of these pigments, also the intestinal compartment may
contribute to the circulatory appearance of anthocyanin derivative pigments [43].
Few in vivo studies have already proven the rapid detection of anthocyanin deriv-
atives in rat plasma after oral administration of malvidin-3-glucoside-pyruvic acid
adduct [44].
Considering studies with human volunteers, additional information may be
obtained from the pharmacokinetic parameters and from the chromatographic pro-
files of the biological samples. Moreover, besides extracts or purified anthocyanins,
different foods are consumed as anthocyanin sources, which will give a more
realistic scenario of the matrix effect, especially in the crossover studies.
Previous studies evaluating the bioavailability of red wine anthocyanins have
only looked for the main native anthocyanin (Mv3glc) in plasma and urine,
underestimating the total anthocyanin content in biological samples [45, 46].
Increases in plasma of malvidin-3-O-glucoside (Mv3glc) concentrations were not
significantly different after the consumption of either red wine or dealcoholized red
wine [45]. In the work of Garcia-Alonso et al. where volunteers consumed 180 mg of
a grape anthocyanin extract in a sugar-sweetened yogurt, the main pigment detected
in plasma was native Mv3glc followed by peonidin-3-O-glucoside (Pn3glc), but the
authors were already able to identify some anthocyanin metabolites in plasma
including glucuronyl conjugates of malvidin and peonidin [47]. Similarly, after
anthocyanin-rich grape juice consumption, the most abundant native anthocyanins
found in plasma and urine were malvidin and peonidin and glucuronidated metab-
olites [48].
In another work, several anthocyanin conjugates (methylated, glucuronidated,
and sulfated) were detected both in plasma and urine after ingestion of blackberry
purees, with or without ethanol, by two groups of volunteers with different body
mass indexes [14]. Altogether these works highlight for the possible implication of
the gastric epithelium on the metabolization of these pigments. In fact, previous in
vitro works had already detected methylated anthocyanins after incubation with total
protein extract of gastric cells [33]. The majority of these compounds may be
produced in the liver and kidneys since the plasma concentration of total
anthocyanin conjugates continues to increase after 60 min [14]. Moreover, this study
indicated for the first time that ethanol enhances cyanidin-3-O-glucoside (Cy3glc)
metabolism potentiating its conversion into methylated and glucuronidated deriva-
tives (Me-Cy-Glucr and 30 -Me-Cy3glc). This effect was more pronounced in over-
weight and obese individuals. These results should prompt the attention of the
scientific community to the fact that the kinetics of these compounds is influenced
by ethanol and body mass index.
Recently, a crossover trial with healthy volunteers was performed using table and
Port red wine in order to compare the pharmacokinetics of anthocyanins in these two
red wines [49]. In this study, red wine anthocyanins were detected in their intact
forms in both plasma and urine samples, but MvGlucr and PnGlucr were the two
main derivatives detected after both red wines consumption. For the first time, and
supported by the synthesis of Mv3Glucr, the main pathway followed by Mv3glc
after absorption was described and involve the anthocyanidin conjugation with
glucuronic acid after glucose removal.
According to these and other works performed with radiolabel anthocyanins [50],
this class of pigments seem to be more bioavailable than previously reported.
Nevertheless, the high impact of phase II conjugates detected in human plasma
and urine, anthocyanin catabolites, seem also to be produced in huge amounts. The
most important catabolites corresponded to products of anthocyanin degradation
(i.e., benzoic, phenylacetic, and phenylpropanoic acids, phenolic aldehydes, and
hippuric acid) and their corresponding phase II conjugates, which were found at 60-
and 45-fold higher concentrations than their parent compounds in urine and plasma,
respectively [50] (Fig. 5).
All in all, this data suggests that anthocyanins would be as bioavailable as other
flavonoid subclasses, such as flavan-3-ols and flavones, which have relative bio-
availabilities between 2.5% and 18.5% [51].
Fig. 5 Schematic representation of anthocyanins phase II metabolites and catabolites

3.3 Intestinal Tract
During the absorption process, anthocyanins can efficiently cross the intestinal wall
[38]. To describe the intestinal transport mechanism, in vitro studies with Caco-2 cell
line as an intestinal barrier model have been done. This cell line has an enterocyte
phenotype after differentiation, and it expresses hexose transporters, ATP-binding
cassette (ABC) gene family, and H+-dependent monocarboxylic acid transporter in a
similar way as small intestinal cells [52–55].
The pretreatment of caco-2 cells with red grape anthocyanin extract seems to
increase (glucose transporter 2) GLUT2 expression, and this effect was positively
correlated with anthocyanin and glucose transport [55]. In addition, cyanidin 3-O-
glucoside absorption is dependent not only on the activity of GLUT2 but also on the
activity of SGLT1 (sodium-dependent glucose transporter 1) although no implica-
tion of P-gp and MRP2 was observed [53]. Anthocyanins and anthocyanidins have
moderate to high affinities for the human efflux transporter BCRP and moderate to
low affinities for MDR1, so they may be actively transported out of intestinal tissues
and endothelia, limiting their bioavailability [56]. Anthocyanidins may alter phar-
macokinetics of drugs that act as BCRP and MDR1 substrates [56].
By using a phenolic berry extract containing 60% of anthocyanins, an opposite
effect was observed as GLUT2 and SGLT1 mRNA expression was reduced by acute
or longer pretreatments [52]. This contradictory results may once more highlight for
the importance of matrix effect. Since the standards for metabolites detected in in
vivo samples are not commercially available, no evidence for the transport mecha-
nism has ever been described.
Besides the obvious implication of the small intestine in the absorption of
anthocyanins, the huge impact of colon was also recently described. Using ileostomy
subjects, a high proportion of anthocyanins from blueberry (up to 85%, depending
on the attached sugar moiety) were found in the ileostomy bags unchanged; this
amount would therefore reach the colon under physiological conditions and be
subjected to microbial degradation [57]. Comparison of the bioavailability of antho-
cyanins in healthy subjects versus ileostomists revealed substantially higher amounts
of anthocyanins and degradants in the plasma/urine of subjects with an intact gut
[58]. The results suggested that the colon is an important site for absorption of
bioactive components such as anthocyanins and their degradation products.
4 Obesity/Metabolic Syndrome
Epidemiological studies have established an association between unhealthy dietary

behaviors and chronic diseases such as cardiovascular diseases (CVD) and type 2
diabetes, which are leading causes of death in high-income countries. In this context,
the interest on functional food ingredients that might confer health benefits to the
populations has increased.
Several reviews have been published concerning the effects of anthocyanins on
obesity and metabolic syndrome, a set of interrelated risks factors for cardiovascular
disease and diabetes [59–62]. However, the number of robust and well-designed
clinical trials about the effects of anthocyanins on obesity and metabolic syndrome is
still limited to sustain health claims. Without solid scientific evidence, governmental
authorities do not allow health claims on foods.
A search at the http://ClinicalTrials.gov database, a service of the US National
Institutes of Health, for “anthocyanins” retrieved a total of 87 studies (last update
February 9, 2018). From those addressing some of the metabolic syndrome features,
three important randomized, double-blind placebo-controlled studies are
highlighted.
One study was conducted on hypercholesterolemic participants, assigned either to
the anthocyanin group (n = 75; 31 males and 44 females) or to the placebo group
(n = 75; 32 males and 43 females), for 12 weeks. The anthocyanin capsules used
(80 mg anthocyanins per capsule, 4 per day) provided a total daily intake of 320 mg
anthocyanins. Compared with the control group, the authors observed significant
increases in HDL cholesterol and a significant reduction in the LDL cholesterol
concentrations [63].
In the second study conducted on a total of 58 diabetic patients, 160 mg of
anthocyanins (80 mg anthocyanins per capsule) or placebo were provided twice
daily (n = 29 in each group). Here, the authors found that anthocyanin supplemen-
tation significantly decreased serum LDL cholesterol (by 7.9%; p < 0.05) and
triglycerides (by 23.0%; p < 0.01) and increased HDL cholesterol (by 19.4%;
p < 0.05), compared with placebo, after the 24-week intervention period [64].
Anthocyanin supplementation exerted beneficial metabolic effects in subjects with
type 2 diabetes by improving dyslipidemia, enhancing antioxidant capacity, and
preventing insulin resistance [64].
Lastly, in the most recent study, a total of 160 eligible participants with either
prediabetes or early untreated diabetes were recruited and randomly assigned to two
intervention arms to receive either purified anthocyanins (320 mg/day) or placebo
(n = 80 in each group). Compared with placebo, purified anthocyanins moderately
reduced HbA1c (0.14%; p < 0.05) and LDL cholesterol (0.2 mmol/L; p < 0.05)
after the 12-week intervention period [65].
These trials provided solid evidence on the effects of anthocyanins in the treat-
ment of hypercholesteremia and diabetes by using commercialized capsules of
purified anthocyanins extracted from bilberries and black currants (Medox®). The
effects observed in these studies can be attributed exclusively to anthocyanins rather
than other compounds (e.g., fibers) often present in anthocyanin-rich foods (e.g.,
berries).
Several mechanisms may explain the effects of anthocyanins on these metabolic
syndrome features. Anthocyanins are antioxidant and anti-inflammatory, but only a
few studies have considered their modulatory effects on the gut microbiota compo-
sition [66]. The effects of anthocyanins on gut microbiota are discussed in the next
sections; nevertheless, it is important to bear in mind that the gut microbiota might
compromise itself the effects of anthocyanins on obesity and metabolic syndrome.
The gut microbiota plays an important role in the bioavailability of anthocyanins.
Anthocyanins are absorbed mainly in the intestine. However, those that are not
absorbed in the upper part of the gastrointestinal tract, in the colon might be
metabolized by the existing bacteria, originating low molecular weight metabolites
[67]. Given the enterohepatic recirculation, these compounds may prevail in the
human body for several days [67]. This reveals the importance of the gut microbiota
in anthocyanins metabolism. Since the gut microbiota differs between obese and
lean individuals, this factor might be taken into account when evaluating the effects
of anthocyanins in obese individuals [68]. In fact, two studies have shown that there
are differences between normal weight and obese individuals regarding anthocya-
nins’ metabolism [14, 69]. The disrupted gut microbiota as well as the generalized
metabolic dysfunction unveiled by obese individuals may be behind the variability
observed between these two groups. Obesity may comprise the metabolism of these
compounds which deserves special consideration since obese individuals might be
the ones who would benefit the most from anthocyanins intervention. It is important
to note that anthocyanins are extensively metabolized in the organism (either by host
or bacterial enzymes), and therefore, their metabolites are probably responsible for
the health benefits of anthocyanins rather than the parent compounds that naturally
exist in plants. Thus, future clinical trials should address this issue to achieve the
optimal dose of anthocyanins according to the body mass index of the individuals.
5 Gut Microbiota Modulation
The human body harbors a collection of trillions of microorganisms. Bacteria,

archaea, virus, fungi, and other eukaryotes live inside different organs establishing
a symbiotic relationship with the host [70–72]. The microbes that collectively
inhabit the gut – the gut microbiota – constitute the largest and most diverse
community in the body [73]. Most of them are bacteria belonging to the Firmicutes,
Bacteroidetes, Actinobacteria, Proteobacteria, or Verrucomicrobia phylum [74].
Supported by the technological advances, the scientific community have been
exploring the dynamics of such complex ecosystem. The role these microorganisms
play in our health is still a mystery, but emerging evidence suggest that gut
microbiota dysbiosis is implicated in a number of diseases ranging from localized
gastrointestinal disorders to autoimmune, hepatic, respiratory, cardiovascular, onco-
logic, metabolic, neurologic, and psychiatric diseases [75].
The expansion of pathobionts (commensal microorganisms that can cause pathol-
ogy under uncontrolled proliferation), the loss of beneficial bacteria, and the loss of
diversity (microbial richness) are common characteristics of a dysbiotic state [76,
77]. These features have detrimental consequences for the host (may initiate obesity
and metabolic diseases as well as neurologic disorders) and can be caused by
environmental factors such as dietary modifications that encompass high contents
of fat.
Diet is one of the most important factors that can modulate the gut microbiome
and rapidly induce alterations in its composition [23]. High-fat and high-sucrose
diets (HF diets) cause profound alterations in the gut microbial ecology, reducing
bacterial diversity [24, 25]. In addition to HF diets, antibiotics, environmental
pollutants [28, 29], and food additives such as dietary emulsifiers [30–32] and
noncaloric artificial sweeteners [33] have been shown to disrupt the gut microbiota
(causing dysbiosis) in a similar way to what is observed in HF diet-induced obesity
models. On the other hand, modulation of the gut microbiota composition by pre-
biotics might be a dietary strategy to prevent or counteract gut microbiota dysbiosis.
To fulfill the criteria of a previous prebiotic definition, the most studied prebiotics
are nondigestible carbohydrates [78]. However, the definition of prebiotics has been
modified to “a substrate that is selectively utilized by host microorganisms confer-
ring a health benefit” [79]. Other substances, besides carbohydrates, might fit
the updated definition if convincing evidence demonstrates their health benefits.
Compounds such as polyphenols, namely, anthocyanins, may be considered pre-
biotics, according to this new definition.
Anthocyanins turn out to be particularly relevant since their bioavailability is
considered to be low. Therefore, after consumption of anthocyanin-rich foods, it is
expected that high amounts of anthocyanins reach the intestine to modulate bacterial
growth at the same time they are metabolized by the existing bacteria.
Table 2 summarizes the studies evaluating the effects of anthocyanins on gut
microbiota composition. There are substantial differences in the results since the
type and the amount of anthocyanins used vary widely among the studies. Further-
more, the methodology used to characterize the gut microbiota composition is
focused only on specific microbial groups which may not be sufficient to have a
complete picture of the gut microbiota modifications that anthocyanins might bring
about. For this reason, the gut microbiota composition of rats fed either a standard or
a HF diet and supplemented with an anthocyanin-rich blackberry extract (BE) was
recently evaluated, using next-generation sequencing (NGS) technology, the gold
standard method. The dose chosen was 25 mg/kg body weight/day, easily achievable
with 100 g of blackberries for a 60 kg man [14, 88]. Interestingly, BE was able to
counteract some of the features of HF diet-induced dysbiosis by decreasing
Ruminococcus and increasing Sporobacter, and, in the context of a standard diet,
BE increased bacteria belonging to Pseudoflavonifractor genus [89].
6 Gut-Brain Axis
The gut-brain axis encompasses the strong bidirectional communication between the
gut microbiota and the central nervous system (CNS) [90]. Multiple mechanisms
may be involved in this bilateral communication: e.g., the gut microbiota has the
capacity to produce many neurotransmitters and neuromodulators such as gamma-
Aminobutyric acid (GABA), noradrenaline, serotonin, and acetylcholine [91, 92]
and has the ability to control host tryptophan metabolism, regulating the fraction of
tryptophan available for serotonin synthesis and, on the other hand, the production of
neuroactive and neurotoxic metabolites along the kynurenine pathway [93–95]. As a
result, modulation of the gut microbiota might be a tractable strategy to the devel-
opment of novel therapeutics for complex CNS disorders.
Table 2 Studies reporting the effects of anthocyanins or anthocyanin-rich food sources on gut
microbiota
Changes in gut
Study design Dose/duration Methods microbiota composition Ref.
Fecal batch Red wine extract FISH = Bacteria (EUB mix) [80]
culture (0.6 mg/ml) = Bifidobacterium spp.
fermentation • 5, 10, 24, 30 and 48 h (Bif 164)
(n = 3) = Lactobacillus/
Enterococcus spp.
(lab 158)
= Bacteroides spp.
(bac 303)
= C. histolyticum
(chis 150)
Fecal batch Malvidin-3-glucoside FISH " Bifidobacterium spp. [81]
culture (200 mg/l) (Bif 164)
fermentation • 5, 10, 24 h " Lactobacillus/
(n = 3) Enterococcus spp.
(lab 158)
= Atopobium spp.
(Ato 291)
= Bacteroides spp.
(bac 303)
# C. histolyticum
(chis 150)
" Eubacterium
rectale Clostridium
coccoides (Erec 482)
" Total bacteria
(EUB338)
Human Red wine (272 ml/day) and Real-time " Enterococcus [82]
crossover trial de-alcoholized red wine qPCR " Bifidobacterium
(n = 10) (272 ml/day) " Eggerthella lenta
• 4 weeks " Blautia coccoides –
Eubacterium rectale
groups
Male BALB/cJ Bilberry powder T-RFLP = bacterial diversity [83]
mice. I/R- (1.62 g/mouse) (Shannon -wiener
induced model • 10 days diversity index)
(n = 8) Bacterial populations
are influenced by dietary
supplementation of
bilberry
21-day-old Jussara (Euterpe edulis Real-time " Lactobacillus spp. [84]
offspring of Mart.) freeze-dried powder qPCR
Wistar rats supplement (5 g/kg control
(n = 6–9) diet)
• Pregnancy and lactation
Fecal Red cabbage and Selective " Lactobacillus spp. [85]
suspension anthocyanin-rich extract growth " Bifidobacterium spp.
(pool of 14 digests media " Clostridium spp.
• 48 h " Bacteroides spp.
(continued)
Table 2 (continued)
Changes in gut
Study design Dose/duration Methods microbiota composition Ref.
healthy " Enterococcus spp.
volunteers) " Enterobacteriaceae
" Total anaerobic
bacteria
Anaerobic Purple sweet potato FISH " Bifidobacterium spp. [86]
fermentation anthocyanins (Bif 164)
broth • 6, 12, 24 h " Lactobacillus/
(n = 8) Enterococcus spp.
(lab 158)
# Bacteroides spp.
(bac 303)
# C. histolyticum
(chis 150)
= Total bacteria
Cross-sectional Strawberries Real-time # Lactobacillus [87]
study (13.67 29.47 mg qPCR
(n = 124) anthocyanins/day)
Obese male Blueberry and black currant Gut low- " Bacteroidetes [66]
C57BL/6 J anthocyanins (400 μg/g HF density " Actinobacteria
Mice diet) array
(n = 8) • 12 weeks (GULDA)
FISH, fluorescence in situ hybridization; HF, high-fat; I/R, ischemic/reperfusion; qPCR, quantita-
tive polymerase chain reaction
Anthocyanins have emerged as anti-inflammatory agents and are promising

candidates for the prevention of neuroinflammation, a common hallmark of obe-
sity-associated neuropsychiatry disorders and neurodegenerative diseases [60, 61,
96, 97]. The mechanisms underlying these effects might be related to the interaction
of anthocyanins with neuron and microglia biology. Recent studies suggest that
anthocyanins may protect neurons against neuroinflammatory injury by stimulating
the production of proteins involved in synaptic plasticity [96, 98].
Anthocyanins and their metabolites are able to cross the blood-brain barrier [99].
Nonetheless, to be neuroprotective, anthocyanins do not necessarily need to reach
the brain. By changing the gut microbiota and acting on the gut-brain axis,
anthocyanins may exert a biological activity even without being absorbed. Alter-
ations in gut microbial composition afforded by anthocyanins were shown to result
in changes in the levels of tryptophan and kynurenic acid which has been implicated
in CNS inflammation, excitation, and behavior (Fig. 6) [89]. Besides, the trypto-
phan/kynurenine ratio has been implicated in neuropsychiatry conditions such as
depression and anxiety [95]. Therefore, by decreasing this ratio, anthocyanins
might constitute a new promising strategy for the treatment of neuropshychiatry
disorders.
Fig. 6 Mechanisms behind the neuroprotective effects of anthocyanins, in the context of HF diet-
induced obesity. Anthocyanins act directly in the brain increasing the expression of fractalkine, a
chemokine extremely important in the crosstalk between neurons and microglia during synaptic
plasticity [96]. On the other hand, anthocyanins modulate the gut microbiota composition, increas-
ing the bacterial genus Sporobacter and alter host tryptophan metabolism. The amount of trypto-
phan available is decreased by anthocyanins to undergo the kynurenine pathway and originate
kynurenic acid, a metabolite whose neuroprotective actions were recently identified [100, 101].
Through these routes, anthocyanins counteract the HF diet-induced neuroinflammation and may
attenuate the neurological complications of obesity as well as neurodegenerative diseases. Antho-
cyanins might constitute, therefore, a new class of psychobiotics
7 Skin Health
Consumers have grown eager for natural solutions toward daily problems, paying
more and more attention to natural sources of botanical products as bioactives in the
products they consume. The cosmetic and skin health industry is no exception, and
companies start to worry more about this issue, creating organic lines of products
and natural solutions to treat a variety of problems and diseases of the skin. At the
same time, general concern from the population is rising toward the visible changes
in skin structure and functionality associated with chronological aging that are
aggravated by sun exposure. Consumers look up for innovation that englobes natural
compounds when treating skin aging manifestations, as wrinkles and hyper-
pigmentation. On the other hand, while one out of three diagnosed cancers is a
skin type cancer (nonmelanoma and melanoma), as revealed by the World Health
Organization, WHO, there are not many products with natural bioactives to prevent
these diseases.
Although cosmetic products with natural compounds and extracts usually claim a
large variety of effects and benefits related to skin aging attenuation, truth is that
there is a lack of scientific evidence to support those claims in terms of the role,
stability, and absorption of such actives in cosmetic formulations. Despite this,
fundamental knowledge regarding botanical compound bioactivity, particularly
polyphenols and, most recently, anthocyanins, toward skin conditions is rising,
and several works are now published about their role in skin aging, cancer, and
other skin diseases. This section reviews the scientific literature existing on the
potential of anthocyanins as skin actives to be used in preventing skin aging and
skin diseases.
7.1 Skin Aging and UV Protection
The sun emits a wide spectrum of electromagnetic waves of which UV is the most
cytotoxic, namely, UVB (295–315 nm) and UVA (325–400 nm), responsible for
many skin diseases [102]. UV damage happens via two different mechanisms: the
production of reactive oxygen species through the interaction of UVA with endog-
enous photosensitizers, which leads to the formation of oxidized products, such as
lipid hydroperoxides and protein carbonyls, or the absorption of UVB by the
heterocyclic bases of the DNA, causing damage to the genetic material. Hence,
even if UV radiation possesses some health benefits, such as the stimulation of
cholecalciferol (vitamin D) production, it is also responsible for some photosensitive
diseases such as sunburn, immunosuppression, photocarcinogenesis, and premature
skin aging (also called photoaging) [103].
Photoaging affects all layers of the skin, particularly epidermis and dermis [104].
The epidermis is the most external layer of the skin, and it is manly composed of
keratinocytes. The dermis, located bellow the epidermis, is rich in connective tissue
(collagen and elastic fibers), and it is the tissue responsible for the strength, exten-
sibility, and elasticity of the skin. In physical and anatomical terms, skin aging is
manifested by the appearance of skin wrinkles, dryness, thinning of the skin, loss of
subcutaneous fat, and uneven pigmentation [105].
These visible changes of the skin depend on many molecular alterations which
happen under UV irradiation. One of the key phenomena is the overexpression of
metalloproteinases (MMPs), which are a family of zinc-dependent endopeptidases
capable of degrading proteins of the ECM (the extracellular matrix), i.e., collagen
and elastin, which are important processes happening during skin regeneration and
cell migration. When overexpressed, matrix maintenance is impaired, which leads to
the loss of skin elasticity and resilience, leading to the appearance of wrinkles. At the
same time, UV radiation triggers the increase in redox-sensitive transcription factors
such as the nuclear factor-kappa B (NF-kB) and activator protein-1 (AP-1). On the
other hand, as skin ages, cellular regeneration slows, making the recovery from
mechanical injuries harder. This has led to an increasing research for compounds
capable of blocking UV radiation or stopping its consequences, by a number of
strategies, namely, by suppressing the oxidative damage, inhibiting MMP over-
expression, modulating NF-kB/AP-1 pathways, and acting as wound-healing agents.
The harmful effects of UV radiation on the skin are usually fought using sun-
screens. These are topically applied and can absorb or reflect UV radiation, pre-
venting the damage to cellular material and diminishing the risk of skin diseases.
There is a growing interest from the consumers’ part toward the implementation of
compounds from natural sources in general consumables, which is leading the
markets toward natural alternatives. Botanical supplements with antioxidant activity
have been proposed as sunscreen actives, usually in combination with well com-
monly used compounds [106]. Polyphenols [107–109] and particularly anthocya-
nins [110–112] have been described as potential agents in skin aging prevention,
firstly because they absorb strongly in the visible and UV spectrums, with maximum
absorbances in the ranges of 500–550 and 280–320 nm. This characteristic varies
depending on their specific aglycone, sugar conjugation, and acylation patterns. An
in vitro study with a cosmetic formulation containing anthocyanins from purple
sweet potato (TNG73) investigated the potential of their absorbance patterns for
dermatological purposes. Researchers found that, at a concentration of 0.61 mg/
100 g of cream, anthocyanins could act as solar protectors, once they were shown to
absorb 46% of the incident UV radiation [113], hence preventing damages to the
dermal tissue.
The effects of anthocyanins in preventing skin damage have been studied using a
different set of cellular and animal models, exploring which molecular pathways
these compounds could be modulating (Fig. 7). Anthocyanins from blueberry could
reduce UVA-stimulated ROS formation and lipid peroxidation in keratinocyte cells
[114], and an extract from bog blueberry rich in cy3glc, petunidin-3-glucoside
(Pt3glc), malvidin-3-glucoside (Mv3glc), and delphinidin-3-glucoside (Dp3glc)
also prevented UVB-induced overexpression of MMPs and reduced the inflamma-
tory response, modulating NF-kB- and MAPK-dependent pathways [115]. Another
report showed that Cy3glc inhibited UV-induced translocation of the transcription
factors NF-kB and AP-1, as well as the overexpression of IL-8, caspase-3 activation,
and DNA fragmentation in human keratinocytes [116]. Blackberry anthocyanin-
enriched fractions could reduce UV-mediated oxidative injury toward skin
keratinocytes, by reducing reactive oxygen species and superoxide ion [117]. Skin
photoprotection by anthocyanins has also been studied by Pornpimpa
Phetpornpaisan et al., 2013, reporting that a black rice bran extract with high content
of cyanidin-3-glucoside has a high antioxidative effect. This extract was able to
inhibit MMP-2 and MMP-9 and possessed wound-healing effects by the
Oxidative Stress (ROS)

UV radiation Inflammation
MMPs overexpression
Activation of NF-kB/AP-1
Keratinocyte Epidermis
Langerhans Cell
Melanocyte
Fibroblast
Anthocyanins
Collagen fibers
Antioxidants
Elastic fibers Dermis
Anti-inflammatory
Anti-proliferative
Fig. 7 Schematic representation of the skin layers and the main skin damages resulting from UV
irradiation and potential actions of anthocyanins in pharmaceutical and cosmetic formulations
enhancement of collagen production and fibroblast proliferation [118]. A study using

an anthocyanin-rich red orange extract showed that these compounds could reduce
the translocation of NF-kB factor and AP-1 upon irradiation of cultured
keratinocytes with UV light, as well as to reduce inflammation [119]. Green tea
polyphenols were shown to be capable of preventing UV-induced immunosuppres-
sion [120], and cyanidin-3-glucopyranoside could prevent UVA-induced damage to
keratinocytes [121]. At the same time, malvidin and cyanidin derivatives from açai
fruit were able to counteract UVA-induced oxidative stress in fibroblasts, keeping the
glutathione and lipid peroxidation levels close to the normal cellular values [122].
According to Tsoyi et al. (2008), anthocyanins from black soybean coats offer
protection against UV radiation, not only to cultured keratinocytes but also in vivo to
hairless mice skin, by reduction of cyclooxygenase-2 (COX-2) and prostaglandin E2
(PGE(2)) through NF-kB-dependent pathways and by the prevention of the apopto-
tic cell death, inhibiting caspase-3 activation and reducing the levels of proapoptotic
Bax protein [112]. In a different study, cyanidin-3-glucoside, the main anthocyanin
in black soybean coats, was reported to inhibit the ROS/COX-2 pathway in HaCat
cells (human normal keratinocytes cell line), scavenging reactive oxygen species by
decreasing COX-2 expression [123].
Delphinidin, the most abundant anthocyanidin found in pomegranate fruit extract,
has also shown a protective effect against oxidative stress and apoptosis in HaCat
cells and in mouse skin in an in vivo experiment [124]. In another in vitro study, this
anthocyanidin was capable of restoring the elastic modulus of irradiated
keratinocytes, regenerating the mechanical properties of these cells [125], and of
reducing the UV-induced MMP-1 expression in human dermal fibroblasts [126].
Using a reconstituted human skin – EpiD5erm™ FT-200, Afaq et al., 2009,
explored the bioactives of pomegranate anthocyanin-enriched extracts. The extracts
were applied 1 h prior to a 12-h UVB irradiation period (60 mJ/cm2), and it was
noted that they could prevent UVB-induced damage to the dermal tissue [110].
These anthocyanins were able to significantly inhibit protein oxidation and to reduce
the presence of cyclobutane pyrimidine dimers and 8-dihydro-20 -deoxyguanoside.
On the other hand, anthocyanins showed some bioactive properties against the
extracellular matrix of the skin, decreasing the UVB-induced overexpression of
various MMPs, such as collagenase (MMP-1), gelatinase (MMP-2 and MMP-9),
and elastase (MMP-12) [110].
Another group investigated the effect of encapsulated anthocyanins from H.
sabdariffa on melanogenesis process using A375 melanocytes. It was found that
these flavonoids, besides their potent antioxidant activity, could inhibit tyrosinase
activity and suppress its expression alongside with the microphthalmia-associated
transcription factor (MITF), involved in melanin production [127].
Another important feature of an aged skin is the slowing down of the cell cycle
and the difficulty in regenerating injured skin. This leads to the investigation of
compounds that, on one hand, may induce the production of collagen by fibroblasts
on the dermis and, on the other hand, may accelerate recovery from injury. A few
studies have tried to understand the role of anthocyanins in helping skin recover
from wounding. Cell migration assays using HaCat and fibroblast cells were
performed with black soybean anthocyanins (Cy3glc, Dp3glc and Pt3glc) in which
the positive effect of anthocyanins in cellular migration was observed. At the same
time, this study explored the production of VEGF (angiogenic factor) which was
increased in both cell lines in the presence of anthocyanins. The authors also found
that anthocyanins prevented TNF-α-mediated inflammatory responses, including
ROS accumulation, NF-kB activation, and monocyte adhesion in endothelial cells
[128].
Another model has been employed to study wound healing in a more reproduc-
ible way than with traditional assays such as the scratch assay, using a biophysical
system – the electrical cell-substrate impedance sensing (ECIS). This system corre-
lates impedance readings with biological behaviors of cells in culture. In one study, a
model was developed to study the wound healing of skin cell monolayers such as
keratinocytes (HaCat cell line) and fibroblasts [129]. Wound was electrically trig-
gered with a higher voltage, and cells on top of the working electrode died. The
healing was followed up by impedance readings, as the cells migrated and prolifer-
ated on top of the electrode, recovering from the damage. The time of recovery was
analyzed in the absence or presence of red wine and blackberry anthocyanins,
particularly mv3glc and cy3glc. It was shown that anthocyanins could reduce the
time of recovery by more than half [130]. Although there are not many studies
exploring the effects of anthocyanins in this phenomenon, the few existing suggest
that anthocyanins may accelerate wound healing, promoting regeneration of the
skin. This requires further studies with different anthocyanins and in a more reliable
model of skin.
Even though these in vitro studies suggest the potential roles of anthocyanins in
fighting photoaging, there is a huge gap in terms of dermatological preclinical and
clinical studies performed with formulations containing anthocyanins. A study by
Leonel E. Rojo et al. (2013) showed that anthocyanins could be bound to protein-
rich matrices and be stabilized without losing their pharmacological properties up to
50 weeks [131]. Anthocyanin-rich protein matrices were tested toward their inhibi-
tion ability of MMP-9, and it was observed that cranberry-rich protein matrix could
abolish enzyme activity, followed by maqui berry and blueberry anthocyanins,
inhibiting 85 and 80% of the enzyme activity, respectively. Another group recently
performed an assay with a strawberry-based cosmetic formulation to test its efficacy
against UV-induced damage to dermal fibroblasts [132]. They proposed the use of a
strawberry/reduced CoQ10-based formulations as sunscreens, because it was
observed that the conjugation of strawberry extract and CoQ10 could reduce ROS
and pro-inflammatory markers, as well as to improve the mitochondrial function and
the expression of antioxidant enzymes. Even though the class of polyphenols present
in the extract was not identified, this kind of studies opens doors to the investigation
of new ways to stabilize anthocyanins in formulations and to study their effect in
vitro and in vivo in respect to aging conditions.
7.2 Anthocyanins and Skin Diseases
Anthocyanins have a variety of physiological functions, such as antioxidant activity

and playing a protecting role in some chronic and age-related diseases that include
some skin diseases and conditions.
A study performed using a three-dimensional reconstituted human skin model
investigated the role of delphinidin in psoriasis. Psoriasis is an autoimmune, chronic,
and inflammatory disease that affects 2–3% of the population [133]. The histologic
marks reveal marked epidermal hyperproliferation and thickening, and the clinical
features include skin inflammation with demarcated erythematous papules and scaly
dermatological plaques [133]. Delphinidin has been reported to suppress prolifera-
tion and inflammation as well as to induce epidermal differentiation markers; thus
this anthocyanidin is said to possess pro-differentiation effects [134]. In addition to
this, delphinidin significantly decreased the thickness of the viable normal and
hyperplastic epidermis, consolidating the differentiating and cornified layers of the
skin equivalent [134]. Another study identified delphinidin as a novel potent dual
PI3K/mTOR inhibitor, which possesses antipsoriatic activity in vitro in cell-free and
cell culture, as well as in vivo in preclinical IMQ-induced psoriasis-like disease in
Balb/c mice [135]. Another research group explored the effects of the topical
application of delphinidin to flaky skin of mice and noted that it reduced the
pathological markers of psoriasis lesions, by inducing the differentiation and reduc-
ing the proliferation and inflammation [136]. On the other hand, delphinidin also
modulated the expression of tight junction proteins and increased the expression of
protein JunB [136]. When using a three-dimensional reconstituted human skin
model of psoriasis, researchers found that this anthocyanidin could significantly
decrease the thickness of the viable normal and hyperplastic epidermis and consol-
idate the differentiating and cornified layers of the skin equivalent. This differenti-
ation is correlated with an increase in expression of both caspase-14 and profilaggrin.
These set of studies indicate that delphinidin could be an important agent in

ameliorating psoriasis lesions and suggest that preclinical and clinical studies should
be performed to understand the efficacy of this anthocyanidin on the management of
psoriasis. Heather R. Austin et al. (2014) investigated the potential role of cyanidin
as an agent in psoriasis therapy by exploring its effect on the expression of late
cornified envelope (LCE) genes as their deletion is a risk factor for psoriasis. In this
study they show that this cyanidin upregulates the five LCE genes in cultures of
differentiating primary human keratinocytes [137].
The effect of anthocyanins and other polyphenols from blueberry in atopic
dermatitis has also been studied. This is a chronic and relapsing inflammatory skin
disease with a prevalence of 10–20% and is more common among children [138].
Cyanidin-3-glucoside and quercetin fraction could inhibit inflammation of the lesion
skin, correcting the Th1/Th2 balance and reducing IL-17. Anthocyanin-rich but not
anthocyanidin-rich bilberry extract were shown to alleviate chronic pruritus of
experimental dermatitis, probably by inhibition of mast cell degranulation [139].
Skin cancer is one of the most common of cancers in pale skin individuals, and it
is directly related to sun exposure, namely, because of UV radiation. There are some
studies regarding the use of anthocyanins as chemopreventive agents, particularly
with pomegranate fruit extract (PFE), juice, and seed oil. Tests have been performed
in cell culture, reconstituted human skin models and animal models of skin cancer
and pomegranate was reported to exhibit immense potential for preventing UVB-
induced skin cancer. PFE contains several polyphenols (such as catechins, gallic and
ellagic acids) and anthocyanins. Pretreatment of human normal keratinocytes with
PFE resulted in a dose- and time-dependent inhibition of UVB-induced phosphor-
ylation of ERK1/2, JNK1/2, and p38 proteins and inhibition of the activation of
nuclear factor-KB (NF-kB) pathway [140]. PFE treatment also resulted in a signif-
icant inhibition of UVA-induced expression of Ki-67 and PCNA and led to an
enhanced expression of pro-apoptotic Bas and Bad with downregulation of anti-
apoptotic Bcl-xL protein [141]. The chemopreventive properties of PFE were further
evaluated in mice exposed to UVB radiation. Oral administration of PFE inhibited
UVB-induced epidermal hyperplasia, leukocyte infiltration, and protein oxidation
and also attenuate the activation of key inflammatory and cell proliferative pathways
such as NF-kB and MAPK [142]. Oral administration of PFE in drinking water also
reduced UVB-induced skin tumor incidence and tumor multiplicity in SKH-1
hairless mice [143]. In a study using delphinidin, it was also shown to inhibit
UVB-induced COX-2 expression in JB6 P+ cells (a skin mouse cell line) by
blocking the MAPKK4 and PI-3 K pathways and suppressing AP-1 and NF-kB
activities, indicating that delphinidin may suppress UV-mediated skin carcinogene-
sis [144]. Diaconeasa Z. et al. (2017) performed a study on the effect of anthocyanins
in melanoma inhibition. Different anthocyanin sources, namely, red grapes and
chokeberries, were applied to melanoma cells and were able to reduce proliferation,
to diminish mitochondrial membrane potential, and to induce oxidative damage,
without showing these negative effects on normal skin cells [145].
A clinical study was performed to evaluate the effects of anthocyanins toward a
skin condition. A formulation containing anthocyanins and glutathione was tested in
patients with erythema resulted from radiation therapy of breast cancer. This report
does not present statistical significance or information on doses and types of
anthocyanins used, but a trend was still observed toward improvement from the
erythema in the group with the tested formulation. The theory behind this formula-
tion is that while the breast is being irradiated, glutathione may clean up oxygen and
carbon radicals formed, and anthocyanins may then help on the recycling of the
glutathione so it can keep working.
Skin is the most superficial organ and the most exposed to environmental
aggressions, UV radiation being one of the most important aggressors and the
cause of skin photoaging and some skin diseases such as melanoma cancer.
Oxidative damage, inflammation, apoptotic cell death, and overexpression of
MMPs are some of the consequences of UV irradiation of the skin. In here it is
suggested by accumulating scientific evidence that anthocyanins may play a role
in delaying and fighting skin aging, as well as in protecting this organ against
cancerous lesions. Even though the evidence obtained from different in vitro and
in vivo studies shows the potential of these polyphenols, this might be insufficient to
guarantee that the skin-protective effects are solely from anthocyanins, because in
most studies, extracts are used and there is always the possibility that other com-
pounds may be present. Furthermore, there is a gap in technology for the develop-
ment of chemically stable and clinically effective anthocyanin-rich formulations for
dermatological applications that must be addressed in the future.
8 Functional Foods and Cosmeceuticals
The concept of functional foods appeared first in Japan in the 1980s and mentioned
foods that were developed specifically to promote health or to reduce the risk of
disease. In Europe, the European Commission’s Concerted Action on Functional
Food Science, coordinated by the International Life Science Institute, defined
functional foods as “a food that, together with the basic nutritional impact, has
beneficial effects on one or more functions of the human organism thus either
improving the general and physical conditions or/and decreasing the risk of the
evolution of diseases” [146]. The amount of intake and form of the functional food
should be as it is normally expected for dietary purposes. Consequently, it could not
be in the form of pill or capsule just as normal food [147]. Therefore, they are usually
considered to be those foods which are intended to be consumed as part of the
normal diet and that contain bioactive components with potential health benefits
such as vitamins, fatty acids, or dietary fiber or foods that include phytochemicals
and other antioxidants or probiotics. These types of foods are being integrated into
the corporate mainstream and being increasingly accepted by the public.
Anthocyanins are widely distributed in the human diet, and their chemical and
biological functions have been extensively studied. These natural pigments are
broadly used in the food industry as natural colorants, and their use is not only
valuable for improving the overall appearance but also because anthocyanins have
beneficial properties to our health. Anthocyanin extracts have been demonstrated to
play a role in several diseases and conditions, displaying antioxidative and radical-
scavenging properties [148, 149], acting as chemopreventive agents [150], but also
playing a role in diabetes [151–154]. Because of their coloring and biological
properties, anthocyanins are now being considered as potential functional food
ingredients and dietary supplements. This section will discuss the possibility of
using anthocyanins in functional foods.
Anthocyanins are widely spread in fruits and vegetables, as well as in red wines
which results in high ingestion and a daily intake ranging from several milligrams to
hundreds of milligrams per person [155]. These flavonoids are now extensively
studied and commonly used as food colorants especially in acidic foods that favor
their chemistry, by stabilizing the flavylium cation form. Some vegetable sources
like black carrot, red-fleshed and purple sweet potato, as well as red cabbage have
been shown to provide high amount of acylated anthocyanins [156–158] that are
more stable to pH than non-acylated [159]. An example of anthocyanins used as
colorants is red rice, which was approved by the Chinese Ministry of Health as a
modern food additive to increase the color and delicacy of meat, fish, and soybean
products [160]. Besides this, red rice is also used as a functional food to treat
abdominal pain and cardiovascular disease [161]. Even so and to this day, there
are not many works performed on the manufacture of products that include antho-
cyanins as functional ingredients, but as the consumers interest on the incorporation
of bioactives into popular foods is growing, innovative work is rising. An example of
a product that can incorporate anthocyanins as functional ingredients is bread. This
can be used by the production of anthocyanin-enriched flours as explored by
Donatella Ficco et al. in 2017 [162]. They performed the micronization followed
by air classification to select anthocyanin-rich fractions of durum and soft wheats
and noted that the pigmented durum wheat showed the greatest acceptability in terms
of aroma and taste of the bread obtained.
Anthocyanins have a variety of biological properties and may be used as potential
functional foods against some metabolic syndromes such as obesity and type 2
diabetes [24]. Some anti-obesity strategies include the increase of physical activity
and the consumption of non-starch polysaccharides and fiber, accompanied by the
reduction of micronutrient-poor diets [163]. Another strategy that may be employed
to fight obesity is related to the use of inhibitors of some digestive enzymes, such as
α-glucosidase and α-amylase. Inhibitors may help retarding the increase of sugar in
the bloodstream after a meal, and anthocyanins and other polyphenols, such as
tannins, may be employed to this end [164]. On the other hand, cyanidin-3-glucoside
(cy3glc) has been shown to reduce blood glucose and to enhance insulin sensitivity
by downregulating retinol binding protein-4 expression in type 2 diabetic mice
[165]. In two other studies, the effect of anthocyanins in diabetes was explored,
and it was found that, even though black raspberry anthocyanins do not alter the
development of obesity in mice that were fed an obesogenic high-fat diet [166],
purified anthocyanins from blueberry as well as blueberry juice could prevent
dyslipidemia and obesity in the same animal model [167]. Another study revealed
that blueberry anthocyanins could reduce glucose production in hepatocytes and
body weight gain [168]. Another source of anthocyanins, maqui berry rich in
delphinidin-3-sambubioside-4-glucoside, also decreased the glucose production and

downregulated glucose-6-phosphatase in liver cells, which is a gluconeogenic
enzyme [169]. Edirisinghe et al. (2011) also found that the strawberry anthocyanin
pelargonidin-3-O-glucoside could reduce inflammation and increase insulin sensi-
tivity in overweight adults [170]. These studies suggest the potential of anthocyanins
to be used as functional foods to fight obesity and type 2 diabetes and to lead the way
to the conception of new products enriched in these flavonoids to be consumed as
supplement in patients with these conditions or as a fundamental tool to convert our
diet in a functional one, preventing food-related diseases. Because anthocyanins are
usually unstable outside their environment, work has been done toward the stabili-
zation of anthocyanins for their use in the food industry which may be of importance
when conceiving new functional foods [171–173]. Anthocyanin-loaded chitosan
nanoparticles, for example, were found to slow the breakdown of anthocyanins in
simulated gastrointestinal fluid and improved the stability of these compounds in a
beverage [172].
Anthocyanins are now known as bioactives with potential health benefits exten-
sively studied in several field such as at metabolic syndromes but also play a role in
cardiovascular disease and possess anti-inflammatory, chemopreventive, and anti-
oxidant activities, among others. The development of functional foods and supple-
ments incorporating these compounds is now being promoted as the information on
the biological properties of these flavonoids arises. Soon, it might be possible to
purchase enriched beverages to aid in the treatment of diabetes or purple bread to
incorporate in a more functional diet as well as other products usually consumed but
improved not only in its nutritional value but also in health promoting and functional
role in our diet.
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Wine Polyphenols and Health
39
Giovanna Giovinazzo, Maria A. Carluccio, and Francesco Grieco
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1136
2 Soluble Acids, Flavonols, and Stilbenes: Bioactive Compounds in Wine . . . . . . . . . . . . . . . . 1138
2.1 Phenolic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138
2.2 Flavonols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1139
2.3 Stilbenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1140
3 Technological Approaches to Enhance Polyphenol Content and Antioxidant Activity
in Wine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142
4 Wine Polyphenol Mechanism of Action Against Cardiovascular Diseases . . . . . . . . . . . . . . . 1145
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150
Abstract
The various polyphenol families present in wine are important for a number of
technological properties of wine such as clarity, hue, and palatal taste. Dietary
polyphenols are associated with a wide range of health benefits, protecting against
chronic diseases and promoting healthy aging. However, basic and clinical
science is showing that the reality is much more complex than this and that
several issues, notably daily intake, bioavailability, or in vivo antioxidant activity,
are yet to be resolved. The concentration of phenolic compounds in wine is
determined by viticulture and vinification practices, peculiar of different coun-
tries. Interesting are the effects of different yeast strains on the final concentration
of polyphenols in red wine. We here summarize the recent findings concerning
G. Giovinazzo (*) · F. Grieco

Institute of Sciences of Food Production (ISPA), National Research Council, Lecce, Italy
M. A. Carluccio
Institute of Clinical Physiology (IFC), National Research Council, Lecce, Italy

1136 G. Giovinazzo et al.
the effects of specific classes of polyphenol (soluble acids, flavonols, and stil-
benes) on human health and propose future directions for research to increase the
amount of these healthy compounds in wine.
Keywords
Wine · Soluble acids · Flavonols · Stilbenes · Yeast · Health
1 Introduction
Wine contains a large and diverse class of phenolic compounds known variously as
“polyphenols” or “biophenols.” These compounds contribute to the characteristic
colors and flavors of wine and act as natural wine preservatives that allows a long
aging process [1]. Polyphenols are extracted during crushing and fermentation when
the juice is in contact with the grape skins and seeds. The amount of phenolic
compounds in red wine is about six times higher than that in white wine because
red juice has longer contact time with the grape skins and seeds. The concentration of
polyphenolic compounds in red wine is approximately 1800–3000 mg/L [2].
There is emerging evidence that a functional diet can help in modulating the
immune system responses to the inflammation processes through a variety of
mechanisms based on the absorption and utilization by the human metabolism of
specific compounds.
Polyphenols are the principal compounds related to the wine consumption ben-
efits due to their antioxidant and free radical scavenging properties. This class of
compounds has been proven to exert important health effects, acting against cancer
pathologies [3] as well as reactive oxygen species (ROS) which are considered the
main cause of different cardiovascular and neurodegenerative diseases.
Grapevine (Vitis spp) is the most cultivated fruit crop in the world, with an area
dedicated to viticulture. The berries are harvested primarily for winemaking but also
to provide fresh table grapes, raisins, and other minor products. Phenolic composi-
tion of grape is genetically driven and greatly affected by environmental factors.
A major challenge for breeding of grapevine cultivars adapted to climate change and
with high potential for winemaking is to dissect the complex plant metabolic
response involved in adaptation mechanisms. Among plant products are the poly-
phenols, a large family of secondary metabolites, which are involved in plant
responses to biotic and abiotic stresses. The most represented polyphenols in plants
are the flavonoids, the cinnamic and benzoic acids, and the stilbenes. They derive
from the phenylpropanoid metabolism, but flavonoids are ubiquitous in plants,
whereas stilbenes are specific to certain plant families.
Other polyphenols, as the phytoalexins, are antimicrobial compounds synthesized
in response to pathogen or herbivore attack. However, other roles have been
described for stress-induced polyphenols, including the defense signaling of
responses and protection against ultraviolet (UV) light damage [4, 5].
Phenolic compounds are secondary metabolites synthesized [4] during normal
development of the berry grape in response to stress conditions.
39 Wine Polyphenols and Health 1137
A large-scale experiment involving cultivation of an association panel of a large

number (more than 200) V. vinifera cultivars designed to represent the genetic and
phenotypic variation encountered in cultivated grapevine and metabolomics analysis
targeted to a large number of polyphenolic compounds (polyphenomics) was
performed. Chemometrics analysis of the data showed large differences in polyphe-
nol composition related to genetic factors, environmental factors (i.e., water stress),
and genetic-environment interactions. Correlation networks shed light on the rela-
tionships between the different polyphenol metabolites and related biosynthetic
pathways. In addition, detailed polyphenomics analysis confirmed that polyphenol
reactions described in wine take place in the berries.
Finally, was reported a large-scale study demonstrating an influence of environ-
mental influence (water stress) on the different classes of polyphenols but also
cultivar differences in the types and extents of drought responses, with different
molecules affected either positively or negatively and different impacts on grape and
wine quality [6].
Grape phenolic compounds comprise several families, divided between
non-flavonoids (hydroxybenzoic acids, hydroxycinnamic acids, and stilbenes) and
flavonoids, based on the same C6-C3-C6 skeleton (flavonols, dihydroflavonols,
flavan-3-ols, and anthocyanins). Each family is represented by several compounds
differing by their hydroxylation level and by substitution of the hydroxy groups
(methylation, glycosylation, acylation). For example, anthocyanins, the red grape
pigments, are based on six aglycones, which can be mono- or di-glucosylated and
further acylated with acetic, p-coumaric, and caffeic acid, giving rise to a large
number of compounds [6]. Grape flavan-3-ols also show high diversity. They
include several monomers (catechin, epicatechin, gallocatechin, epigallocatechin,
and epicatechin 3-gallate) that are the constitutive units of oligomers and polymers
(proanthocyanidins or condensed tannins), with degrees of polymerization ranging
from 2 to over 100 in grape skin [6].
The flavonoid family includes the flavonols, such as myricetin, quercetin, and
kaempferol, which exist both as aglycones and sugar conjugates. The non-flavonoids
encompass hydroxybenzoic acids such as gallic acid, hydroxycinnamic acids,
including p-coumaric, caffeic, and caftaric acids, and the stilbenes, such as trans-
resveratrol and cis-resveratrol [5]. The synthesis of stilbenes in grape berry tissues
is activated in response to fungal attack, to berry injury, and to ultraviolet
irradiation [5].
The healthy physiological effects are especially associated to flavonoids and
stilbenes [7], namely, quercetin, (+)-catechin, gallic acid, and trans-resveratrol [8].
The stilbene trans-resveratrol has gained great attention, and a number of scientific
papers have appeared related to the moderate consumption of red wine for its ability
to inhibit platelet aggregation and LDL oxidation and its beneficial effects in health.
Since trans-resveratrol is postulated to be involved in the health benefits associated
with a moderate consumption of red wine, it is one of the most extensively studied
natural products.
The various polyphenol families present in wine [7, 9] are important for a number
of technological properties of wine [7]. The knowledge about their qualitative and
quantitative profile in grapes is very important to predict wine aging attitude and can
help to solve problems related to color stability, especially in the case of red wines
that are destined to long aging periods [10]. The wine aging also changes the
phenolic composition, as these compounds can suffer diverse transformations, like
oxidation processes, condensation and polymerization reactions, and extraction from
wood, usually associated to the changes in color and colloidal stability [11], flavor,
bitterness, and astringency [12, 13]. The polyphenolic fingerprint can be useful for
the classification of wines, since it can give us information about the variety, the
geographic and winery origin, and even the applied winemaking technology [14].
During winemaking, only a fraction of the grape flavonoids is selectively trans-
ferred to the wine and a final yield strongly depending on the management of the
contact of the liquid must, containing berry skin and seeds, with the solid parts of the
grape bunches and on the grape variety [10].
The data concerning the extractable phenolics of the grape cannot be simply
generalized to predict the wine composition, since a high variability in the extraction
yield from grape to wine is introduced by the technological factors governing the
winemaking process (such as temperature, duration and intensity of the liquid-solid
contact, final ethanol concentration).
Many conditions (i.e., genetic, agronomic, technological, storage, etc.) linked to
each other by complex and multifactorial phenomena affect both profiles and
concentrations of bioactive compounds, either in grape or in wine [15].
2 Soluble Acids, Flavonols, and Stilbenes: Bioactive

Compounds in Wine
Wine is a complex mixture of hundreds of molecules, some of them showing

important biological properties, while others are mainly associated with its organo-
leptic characteristics. In particular, we describe specific classes of polyphenols such
as phenolic acids (hydroxybenzoic and hydroxycinnamic acids), flavonols, and
stilbenes (Fig. 1).
2.1 Phenolic Acids
In wine, there are two groups of phenolic acids: hydroxybenzoic acids and
hydroxycinnamic acids [16]. Hydroxybenzoic acids, including gallic acid, pro-
tocatechuic acid, gentisic acid, p-hydroxybenzoic acid, vanillic acid, and syringic
acid, derive from benzoic acid. Chlorogenic acid, as the main constituent of the
hydroxycinnamic acid derivative group, increased with harvest time delay, and the
same occurred with sinapic acid. The converse was true of caffeic acid and ferulic
acid, which were also esterified with tartaric acid as the known compounds caftaric
acid and fertaric acid, respectively [16, 17]. Hydroxycinnamic acids have gained an
increasing interest in health because they are known to be potent antioxidants.
Fig. 1 Polyphenol biosynthesis pathway in grape tissues. CHS chalcone synthase, STS stilbene
synthase
These compounds have been described as chain-breaking antioxidants acting

through radical scavenging activity that is related to their hydrogen or electron
donating capacity and to the ability to delocalize/stabilize the resulting phenoxyl
radical within their structure. The free radical scavenger ability of antioxidants can
be predicted from standard one-electron potentials.
Phenolic acids represent important fraction of wine phenolics, but their biological
effects have been scarcely investigated. The interrelationship between antioxidative
capacity and vasodilatory activity, two potentially beneficial biological effects, of
phenolic acids from wine was examined. Antioxidative and vasodilatory effects
of phenolic acids relate to the number of hydroxyl groups in the phenyl ring, degree
of compactness and branching of molecules, and three-dimensional distributions of
atomic polarizability of the tested molecules [18]. Caffeic acid has been shown to
have neuroprotective effects against injury induced by 5-S-cysteinyl-dopamine,
against Aβ-induced neurotoxicity and by inhibiting peroxynitrite-induced neuronal
injury [19, 20]. Ferulic acid has been cited as an antidiabetic effect by lowering
blood glucose and by increasing plasma insulin [21].
2.2 Flavonols
Four aglycones belong to the flavonols class: myricetin, quercetin, kaempferol, and
isorhamnetin. The diversity in the flavonols structure is due to changes in the basic
skeleton introduced by enzymes such as glycosyl transferase, methyl transferase, and
rhamnosyl-transferase. In one plant species, dozens of different flavonoids may be
present, and many of these are conjugated to various types of sugars. Both the basic
structure and the level of glycosylation determine the biological function and
bioavailability of polyphenols in the human and animal intestines. Depending on
their structure, these molecules have diversified activities.
Flavonols are found throughout the plant foods. The best-known flavonols are
quercetin and kaempferol. Quercetin, kaempferol, myricetin, and isorhamnetin are
flavonols presents in grape skins and stems as several different glycosides. Quercetin
accumulates in grape skins to protect against damage from ultraviolet (UV) light.
There are high concentrations of quercetin in wine made from sun-exposed grapes.
Quercetin is readily extracted from grape skins during fermentation. Stems may
contribute additional flavonols in whole cluster fermentations.
Quercetin glycosides may be hydrolyzed in wine to form quercetin aglycone.
This process is similar to the hydrolysis that can occur with anthocyanins. Unlike
anthocyanins, flavonol aglycones are stable in wine and can be used to monitor
hydrolysis reactions. Quercetin may cause problems as a precipitate in bottled wines.
Flavonols can interact with anthocyanins, enhancing their red color in a process
known as co-pigmentation. This process may also help anthocyanin color stability.
Dietary flavonols inhibit LDL oxidation and so reduce the primary risk factor for
atherosclerosis and related diseases. The animal studies are supported by human
epidemiological studies, which show inverse correlations between the occurrence of
CVD, certain cancers, and age-related degenerative diseases and the consumption of
flavonol-rich diets [22–25]. Flavonols have been linked to protective effects against
several specific cancers, including leukemia and pancreatic, breast, cervical, pros-
tate, uterine, and urinary tract cancers. Subjects with regular flavonol intake have a
10–60% lower incidence of these types of cancer compared with subjects with low
flavonol intake.
This protective activity results from both the action of flavonols as stimulators of
antioxidant defenses and their direct inhibitory effects on cellular proliferation.
Quercetin consumption has been reported to be inversely associated with breast
cancer incidence [26].
2.3 Stilbenes
Stilbenes are a class of compounds with multiple pharmaceutically relevant proper-

ties. The stilbenes production in grape berry tissues is considered to be a part of the
general defense mechanism since they display strong antifungal and antimicrobial
activities [27].They are a group of plant phytoalexin polyphenols found in high
concentrations in grapes, berries, nuts, and teas. In plants, their main function is to
protect the plants against pathogens and fungi; therefore, their content is highly
variable and increases with stress exposure. UV radiation, heavy metal exposure, and
fungal infection may thus be utilized to enrich grapes with stilbenoids [28, 29].
Resveratrol (3,4_,5-trihydroxystilbene) may be found especially in red wine,
grapes, and berries, at a concentration ranging from 0.1 to 15 mg/L. Nowadays,
the primary source of resveratrol is the roots of perennial plant Polygonum
cuspidatum (Japanese knotweed) [29]. Due to the positive health effects attributed
to resveratrol through so-called French paradox, its biological activity has been
extensively studied. Most biological effects are assigned to trans-resveratrol, the
more stable of the two isoforms. However, also cis-resveratrol, which is formed from
trans-resveratrol upon UV light exposure, exhibits certain anti-inflammatory activity.
Unless otherwise stated, the activities mentioned here apply to trans-resveratrol.
Stilbenes are non-nitrogen polyphenols with acidic and amphiphilic character which
causes their enrichment in biomembranes, where many of their targets occur (COX,
5-LOX, protein kinase B) [30]. Structurally, stilbenoids possess two aromatic rings
connected by an ethylene or ethene bridge with a variety of substituents. Even
though the presence of double bond suggests that stilbenoids exist in cis- as well
as trans-form, the trans-form is more stable and the biologically relevant form. In
nature, stilbenoids are synthesized from phenylalanine through multiple enzymatic
reactions [31]. Stilbenoids are heterogeneously spread throughout the plant king-
dom. They are especially abundant in Gnetaceae, Pinaceae, Cyperaceae, Fabaceae,
Dipterocarpaceae, and Vitaceae families [32]. Resveratrol has a function of phyto-
alexin produced by specific plant species in response to biotic and abiotic challenges.
It is thought to be one of the principal agents in the health-promoting effects of red
wine [33]. Results of clinical studies show that the most important source of
resveratrol and piceid are wines (98.4%) and grape and grape juices (1.6%), whereas
peanuts, pistachios, and berries contribute to less than 0.01% [34]. Wine is the major
source of resveratrol and piceid to the diet, ranging from 95% and 98.7% for trans-
resveratrol and trans-piceid to 99.9% and 99.7% for cis-resveratrol and cis-piceid,
respectively. Other food items such as grapes contribute by amount of 3.8% of total
trans-resveratrol, whereas other contributors such as pistachios or berries provide
less than 1% of the dietary total amount of trans-resveratrol and trans-piceid.
Resveratrol possesses numerous important bioactivities, including anti-
inflammatory, antioxidant, anti-aggregatory functions, and modulation of lipoprotein
metabolism [35–37]. It has also been shown to detain chemo preventive properties
against certain forms of cancer and cardiovascular disorders and to have positive
effects on longevity [38–42].
Anticancer activity of this compound is mainly due to induction of apoptosis via
several pathways, as well as alteration of gene expressions, leading to a decrease in
tumor initiation, promotion, and progression [43]. Resveratrol blocks the growth of
lymphoma cells and increases their rate of cell death [44]. Resveratrol sensitizes
chemotherapy-resistant lymphoma cells to treatment with paclitaxel-based chemo-
therapy [45], also reduces the production of growth factors, such as vascular
endothelial growth factor and interleukin [33], and may reduce the ability of
lymphoma cells to spread to other organs [46]. Finally, it was demonstrated that
in vitro administration of resveratrol favorably altered gene expression in the
androgen axis and in cell cycle regulators, providing potential anticancer benefit
for prostate cancer [47]. Moreover, trans-resveratrol appears to protect against
diabetes [48] and neurodegenerative disorders [49], due to induction of sirtuin 1
genes [50]. Trans-resveratrol might also contribute to increasing the life span of
metazoans and mice by miming the effect of caloric restriction and thus decreasing
age-related signs [51, 52]. Experimental studies have shown that resveratrol exhibits
both an anti-inflammatory and cardioprotective potential by inhibiting the expres-

sion of inflammatory mediators and the monocyte adhesion to vascular endothelial
cells [53, 54]. Although resveratrol exhibits potent anticancer activities against
transformed cells, its effectiveness is limited by its poor bioavailability, and as
a dietary phytonutrients, it is most effective against tumors with which it comes
in direct contact, such as skin cancers and tumors of the gastrointestinal tract.
Furthermore, inhibition of sirtuin 1 by both pharmacological and genetic means
abolished protein de-acetylation and autophagy as stimulated by resveratrol, but not
by piceatannol, indicating that these compounds act through distinct molecular
pathways. In support of this notion, resveratrol and piceatannol synergized in
inducing autophagy as well as in promoting cytoplasmic protein de-acetylation [55].
3 Technological Approaches to Enhance Polyphenol Content

and Antioxidant Activity in Wine
The winemaking steps determine the phenolic content of red wines that able the
extraction of phenolic compounds from the grape berries. Numerous winemaking
procedures have been developed to enhance the extraction of phenolic molecules, by
preventing the several motives that affect the release of polyphenols from the berry
tissues [56, 57]. Several investigations have studied the effect of the fermentation
temperature during the winemaking process on the extraction of polyphenols, thus
demonstrating that their concentration increased when wines were produced at
higher fermentation temperatures [58, 59]. Moreover, it has recently shown the
efficacy of thermo-vinification to improve a number of factors, such as the antiox-
idant potential and the polyphenolic and resveratrol content, in Pinot noir, Prokupac,
Merlot, and Cabernet Sauvignon wines [60].
The impact of maceration time and of the utilization of enological additives
(enzymes, sulfur dioxide) on the polyphenol content has been studied [57]. Gambuti
and coworkers [61] have examined the effects of those factors during vinification of
Aglianico grape must. The authors indicated that the simultaneous addition, during
the pre-fermentation stage, of pectolytic enzymes and SO2 increased the release of
these molecules from grape tissues, thus resulting in a higher concentration of
polyphenols in the produced wine. Comparable evidences were recently obtained
by investigating experimental vinification of grape must from the Vranec cultivar
[62, 63]. Also in this case, both increased maceration time and SO2 addition
augmented the final concentration of total polyphenols, anthocyanins, flavonoids,
and flavan-3-ols in the final product, this effect action not dependent by the wine
aging.
The action of diverse winemaking approaches in determining polyphenolic
profile of red wines obtained from the Italian red cultivar “Negroamaro” has been
recently studied. Were compared three different pre-fermentative steps: the tradi-
tional (7 days of maceration at 25 C), the cryomaceration, (24 h at 0 C using dry
ice), and ultrasound (37 kHz, 150 W, 15 min at 30 C) [64]. The authors demon-
strated that the ultrasound action enhanced the release of all polyphenol classes,
whereas the cryomaceration only improved the anthocyanin content in the

produced wine.
This evidence has been recently supported by a recent study of Ferraretto and
Celotti [65]. They evaluated the effect of high-power ultrasound (20 kHz) on the
phenolic structure of red wines and demonstrated that this physical treatment
promote the polymerization of the phenolic compounds as the wine matures and
consequently speed up the aging course of wines.
Another investigation [66] has evaluated the consequence of different technolog-
ical approaches on the polyphenolic profile and the antioxidant activity of wine
produced from grape of the Italian cultivar Primitivo. The addition of tannins was
more efficient in enhancing the concentration of phenolic molecule when compared
in musts to the other considered technologies and the aging to get better wine
antioxidant activity. Furthermore, a recent report indicated that protracted post-
fermentation maceration up to 50 days increased the polyphenol concentration
and, consequently, the potential healthy effect of the obtained wine [67]. However,
a number of reports do not agree with this hypothesis.
However in contrast with the above findings, Mulero and coworkers [68] did not
detect variation in the concentrations of the different types of phenolic compounds in
three wines from Monastrell grapes, produced by separately adopting the protracted
maceration, must fermentation by adding enzymes, or the traditional procedure
above three technological approaches.
The pulsed electric field (PEF) technology represents a very promising approach
because of its ability to enhance the extraction of polyphenolic compounds through-
out the vinification process. The PEF pretreatments on Cabernet Sauvignon must
gave substantial differences in the produced wines, since the same PEF-treated must
showed an increase of 97% in the content of total flavonols of 32% in the content of
total phenolics and of 62%, in the color, when compared to the untreated control
[59]. The above findings were confirmed by similar investigations that analyzed the
vinification of Cabernet [69], Merlot [70], and Syrah [71] grape musts after the
application of the PEF step. Moreover, PEF treatment allowed the acceleration and
enhancement in the extraction of phenolic compounds throughout the maceration
step of winemaking process [72].
An interesting research have recently assessed the effects of a novel fermentation
technologies based on the “Ganimede” that is able to trap the carbon dioxide (CO2)
generated during the alcoholic fermentation on the phenolic contents of Cabernet
Sauvignon wines [73] indicating that this device was able to increase the concen-
tration of anthocyanins superior in the wines produced.
The mechanisms through which yeast influences the color and content of poly-
phenolic compounds of wine are currently being researched, but three modes of
interaction between the yeasts and the polyphenolic component have been already
identified. Some strains of yeast adsorb polyphenols on the cell wall. However,
although it has been shown that yeast is one of the factors able of inducing the
reduction of the polyphenol concentration in wines, it is unknown whether their
adsorption on the cell walls is the only mechanism responsible of this behavior. The
amount of biomass produced during the alcoholic fermentation is capable to adsorb
on the cell walls a significant amount of polyphenols. This capacity is likely to be

strain-specific, since different yeast strains have a different composition of the cell
wall. In fact, it could be hypothesized that specific yeast strains could perform a
“differentiated” adsorption of the diverse polyphenol classes. The second type of
interaction between yeast strains and the wine polyphenol is related to the microbial
β-glucosidase enzymatic activity. In fact, the greatest part of anthocyanins is found in
the wine as glucosidase derivatives (linked to a sugar); thus, in this state they are
much less sensitive to chemical or enzymatic oxidation; therefore the β-glucosidase
activity decreases color and stability, since it produces free anthocyanins in wine.
The third mechanism regards the strain-specific secretion throughout the alcoholic
fermentation, by some wine yeast, of polysaccharides capable to combine with the
polyphenols and to form with them stabile complexes.
It has been shown that different yeast metabolites, including pyruvic acid, can
react with the anthocyanins of the grapes giving rise to stable pigments during the
maturation and aging of red wines [74]. Taken together, the above considerations
highlight the pivotal role played by yeasts in modifying the polyphenolic profile
of wines.
Indeed, several investigations have highlighted the ability of different wine yeast
strains and of enological additives of microbial origin to improve the phenolic profile
of red wines. The addition of β-glucanases or other yeast-derived enzyme prepara-
tions as enological additives increased the antioxidant potential of sparkling wines
[75]. On the opposite, yeast lees have been demonstrated to lower the amounts of
polyphenolic compounds [76] and anthocyanins [74] in wines, because they formed
stabile complexes with the mannoproteins released by yeasts after their yeast.
Even though previous studies did not show any effect of yeast starters on the
polyphenolic profile of Pinot noir [77], Cabernet Franc, and Merlot [78] wines,
several recent reports indicated a correlation between the utilized yeast strain and the
antioxidant capacity of the produced wine.
Brandolini and coworkers [79] carried out an investigation by evaluating the
properties of wines produced by the separate inoculation of 19 strains of Saccharo-
myces cerevisiae in the same grape must. The antioxidant capacity and the polyphe-
nol profile detected in the different wines were extremely different, thus showing the
strain-specific yeast feature to differentially adsorb polyphenols during the vinifica-
tion process. Kostadinović and coworkers [62] showed analogous evidence on
Vranec and Merlot wines in Macedonia. The authors used different starter cultures
to carry out vinification tests, where the strains demonstrated that they were capable
to affect specifically the trans-resveratrol concentration and antioxidant activities in
the final wines.
The employment of different yeast starter strains allowed the production of Pinot
noir wines with a dissimilar polyphenolic content [80]. This study has analyzed how
yeast selection can modify phenolic content in Pinot noir wine. In fact, five different
yeast starters were tested in multiple vinifications, where the Saccharomyces
cerevisiae strain RC212 was able to raise conspicuously the concentrations of total
pigment, anthocyanin, and tannins. Recently, Carrascosa and collaborators [81] have
recently shown that different yeast strains were able to produce Albariño wines
denoted with a specific polyphenol composition. The above findings were further
confirmed by a recent report [82], where an unambiguous correlation between the
yeast starter utilized to promote the fermentation process and chemical profile of
wine was recognized, thus underlining the strain-specific yeast property to modify
the color and the polyphenolic composition of the final product.
Moreover, a recent study has produced the identification of yeast starter cultures
able to enhance the quality of the wine produced from the Italian red cultivar
“Gaglioppo” a cultivar with reduced synthesis of anthocyanins [83]. The obtained
evidences further evidenced the strain-specific capacity of wine yeast to modify the
final amounts of total anthocyanins, total polyphenols, and total tannins.
Recently, Giovinazzo and coworkers (manuscript in preparation) highlighted a
positive role of autochthonous yeast starter cultures for the enhancement of poly-
phenol content throughout the industrial production of Negroamaro wine. The
statistical assessment of the experiment showed that the use of autochthonous strains
increased the concentrations of several classes of polyphenols in the produced wines
when compared to wines produced from the Sama grape must with commercial
starter strain.
Taken together, the above-described scientific outcomes emphasize the relevance
of the development and the industrial application of innovative biotechnological
approaches in order to exalt the presence of healthy molecules in wine, thus
improving “functional parameters” with the consequential enhancement of the
final wine quality.
4 Wine Polyphenol Mechanism of Action Against

Cardiovascular Diseases
Wine polyphenols have garnered much attention, especially with regard to their
potential role in the protection against cardiovascular diseases. Indeed, red wine is
thought to be responsible for the “French paradox” [84], a term used to describe the
low incidence of cardiovascular disease in the French population despite their high
intake of saturated fats.
Many preclinical and some clinical studies have identified a number of mecha-
nisms and targets by which specific wine polyphenols could exert benefits against
cardiovascular diseases. Wine polyphenols, including flavonols and resveratrol,
have been shown to modulate the expression of inflammation-related genes involved
in the atherosclerotic process as well as in chronic degenerative diseases.
Flavonols (quercetin, kaempferol, and myricetin) significantly downregulate the
coordinated expression of the endothelial adhesion molecules, E-selectin, vascular
cell adhesion molecule (VCAM)-1, and intercellular adhesion molecule (ICAM)-1,
in human cultured endothelial cells activated by inflammatory triggers [85], thus
decreasing the adhesion and subsequent trans-endothelial migration of monocytes
into the intima of the vascular wall, i.e., processes that constitute the initial steps in
the development of atherosclerosis. Flavonols have also been reported to signifi-
cantly downregulate the expression of monocyte chemoattractant protein (MCP)-1
and macrophage colony-stimulating factor (M-CSF); both pro-inflammatory endo-

thelial proteins guide monocytes into the subendothelial space, during inflammatory
state [85]. The anti-inflammatory flavonol effects were mediated by the reduced
activation of NF-κB and AP-1, whose binding sites are present in the promoter
region of the pro-inflammatory genes including VCAM-1, ICAM-1, E-selectin, as
well as MCP-1 and M-CSF [86]. Furthermore, flavonols also affected the inflam-
matory response by downregulating the expression of inflammation-related genes,
like interleukin (IL)-6, IL-4, and tumor necrosis factor (TNF)-α. In human vascular
endothelial cells, our research group reported that quercetin reduced inflammatory
angiogenesis, a key pathogenic process contributing to atherosclerotic lesion forma-
tion, progression, and vulnerability, through inhibition of the pro-inflammatory
enzyme cyclooxygenase (COX)-2 and gelatinases, the matrix metalloproteinase
(MMP)-9 [87].
A limitation of the anti-inflammatory effect by flavonols in these in vitro studies is
the use of flavonols at supraphysiological concentrations and as aglycone forms. It
has been proven that after oral absorption, flavonols are rapidly converted to
circulating conjugates through glucuronidation, sulfidation, or methylation. This
accounts for the very low aglycone concentrations in human plasma (in the nano-
molar range).
Few studies have investigated the effects of flavonol metabolites on vascular cell
function. It has been shown that human quercetin plasma metabolites at physiolog-
ically significant concentrations were able to inhibit COX-2 expression in human
lymphocytes. Furthermore, Tribolo et al. showed that quercetin and its phase II
metabolites affected the expression of VCAM-1, ICAM-1, and MCP-1 in inflamed
endothelial cells [88]. However, at 10 μM, quercetin metabolites showed a reduced
ability to decrease the stimulated expression of these genes when compared with
quercetin. This suggested that the chemical transformation of quercetin during phase
II metabolism resulted in a reduction of bioactivity, at least with respect to regulation
of inflammatory gene expression. However, at a vascular level, quercetin glucuro-
nides can be freed of their sugar moiety by a deconjugation process performed by
β-glucuronidases, and the free aglycone is delivered to tissues, particularly under
inflammatory conditions [89]. In a vascular co-culture model represented by human
arterial smooth muscle cells and endothelial cells, quercetin and its phase II metab-
olites at physiologically relevant concentration significantly decreased the stimu-
lated expression of the vasoconstrictor endothelin-1 [90], involved in the endothelial
regulation of vascular tone.
Many results obtained in cell culture studies have been replicated in animal model
but not in human trials. However, several human studies have shown that quercetin
can reduce blood pressure in hypertensive patients [91], although the precise mech-
anisms have not been elucidated.
In addition to flavonols, the anti-inflammatory action of wine polyphenols is
exerted by resveratrol.
Resveratrol, like quercetin, has been reported to modulate the expression of
inflammation-related genes involved in the cellular processes that control adhesion
and migration of monocytes to vascular endothelium [92, 93]. We reported that
a
PWPE NWPE
GROUPS POLYPHENOLS
mg/mL mmol/L mg/mL mmol/L
p-Coumaric acid 0.04 ± 0.03 0.24 0.04 ± 0.02 0.27

HYDROXYCINNAMIC ACIDS Caffeic acid 1.26 ± 0.54 6.98 1.52 ± 0.81 8.41
Caftaric acid 8.23 ± 1.12 26.37 8.01 ± 0.98 25.64
Total Hydroxycinnamic Acids 9.53 9.57
Kaempferol 0.05 ± 0.02 0.18 0.08 ± 0.04 0.30
FLAVONOLS Quercetin 0.13 ± 0.05 0.42 0.14 ± 0.03 0.47
Myricetin 0.04 ± 0.01 0.12 0.09 ± 0.02 0.29
Total Flavonols 0.22 0.31
trans-Resveratrol 0.06 ± 0.02 0.24 0.02 ± 0.01 0.10
STILBENES
trans-Piceid 0.20 ± 0.05 0.51 0.09 ± 0.03 0.24
Total Stilbenes 0.26 0.11
TOTAL POLYPHENOLS 10.00 10.00
b
HYDROXYCINNAMIC ACIDS FLAVONOLS STILBENES
R1 OH
O
OH
R1
R2
HO O R1
R2
HO
OH HO
R1 R2 OH O R1 R2 R1
p-Coumaric acid (CMR) H OH Kaempferol (KMP) H H trans-Resveratrol (RSV) OH
Caffeic acid (CFF) OH OH Quercetin (QRC) OH H trans-Piceid (PCD) OGlucose
Caftaric acid (CFT) OH C4O6H5 Myricetin (MYR) OH OH
Fig. 2 Characterization of Primitivo wine polyphenol extract (PWPE) and Negramaro wine
polyphenol extract (NWPE) polyphenol content and chemical structure of polyphenols. (a) Poly-
phenol content PWPE and NWPE (10 μg/mL). (b) Chemical structures of polyphenol groups
identified in red wine extracts: hydroxycinnamic acids (CFF caffeic acid, CMR p-coumaric acid,
CFT caftaric acid), flavonols (KMP kaempferol, QRC quercetin, MYR myricetin), and stilbenes
(RSV trans-resveratrol, PCD trans-piceid)
resveratrol decreased monocytoid cell adhesion to human endothelial cells via

reduction of VCAM-1 gene expression and by suppressing VCAM-1 promoter
activity (see Figs. 2 and 3) [15]. In addition to VCAM-1, resveratrol also inhibited
ICAM-1 and E-selectin, as well as MCP-1 and M-CSF [85, 93]. In human endothe-
lial cells, monocytes, and smooth muscle cells, resveratrol strongly inhibited the
expression of MMPs [85, 93, 94], responsible for the degradation of extracellular
matrix, an essential event in atherosclerotic process, thus preventing plaque devel-
opment, progression, and vulnerability. Furthermore, in endothelial and mononu-
clear cells, resveratrol inhibited, in a dose-dependent manner, the stimulated
expression of tissue factor [96], a key regulator in the extrinsic pathway of blood
coagulation. These anti-inflammatory and anti-thrombotic effects of resveratrol were
at least in part mediated by lowered levels of intracellular ROS and the reduced
activation of redox-sensitive transcription factors, NF-κB and AP-1 [85, 93, 96]. Part
a LPS
b
LPS
120
U937 adhesion (% vs LPS)

#
100
* *
80
CONTROL 1 10 50 PWPE (µg/mL)
60 ** **
40 ** **
20
0
(µg/mL) - - 1 10 50 1 10 50
PWPE NWPE
LPS 1 10 50 NWPE (µg/mL)
c d e
E-Selectin expression (% vs LPS)

LPS LPS LPS
ICAM-1 expression (% vs LPS)
VCAM-1 expression (% vs LPS)
120 120 120

# # #
100 100 * 100
* * *
* ** *
80 80 * 80
** **
60 ** ** 60 60
** **
40 40 40
20 20 20
0 0 0
(µg/mL) - - 1 10 50 1 10 50 (µg/mL) - - 1 10 50 1 10 50 (µg/mL) - - 1 10 50 1 10 50
PWPE NWPE PWPE NWPE PWPE NWPE
Fig. 3 Inhibitory effects of PWPE and NWPE on the monocyte adhesion to endothelial monolayer
and on the expression of endothelial adhesion molecules. (a, b) HUVEC were pretreated with
Primitivo wine polyphenol extract (PWPE) and Negramaro wine polyphenol extract (NWPE)
(1–50 μg/mL) or vehicle (control) for 1 h and then stimulated with LPS 0.5 μg/mL for 16 h.
HUVEC were co-cultured with calcein AM-labeled U937 monocytes for 1 h. The number of
adherent U937 cells was monitored by fluorescence microscope (a) or measured by the fluorescence
plate reader (b). (c–e) Cell surface expression of ICAM-1 (c), VCAM-1 (d), and E-Selectin (e) was
analyzed by cell surface EIA. Each experiment was performed in triplicate. Data are expressed
as the percentage of LPS-induced expression (mean SD). #p <0.01 versus control; *p <0.05;
**p <0.01 versus lipopolysaccharide (LPS) alone
of the beneficial effects of resveratrol was also mediated by the upregulation of

endothelial nitric oxide synthase (eNOS), involved in the regulation of vascular
homeostasis. Most of the studies about the cardiovascular beneficial effect of
resveratrol were performed by using its aglycone form; however it has been shown
that piceid, a glycosidic form of resveratrol, also preserved the vascular anti-
inflammatory properties, although at a lesser extent [85, 94].
As a critical point of in vitro studies, the cardio-vasculo-protective effects of
resveratrol have been shown to occur at supraphysiological (10 μM) concentrations,
which cannot be achievable through dietary intake. However, some beneficial
properties of resveratrol have been also observed at dietary doses. In human endo-
thelial cells, resveratrol at physiological concentrations decreased the stimulated
expression of VCAM-1, ICAM-1, and MCP-1 [54], as well as the cytokines, IL-6
and CCXL2. A significant increase in eNOS expression in HUVEC has been
reported also at lower concentration of resveratrol (1 μM), following repeated
stimulation for 6 days [97]. Physiological concentrations (0.1–1 μM) of resveratrol

have also been reported to modulate the expression of genes involved in cell
proliferation, blood pressure regulation, oxidative stress response, and autophagy
in endothelial cells [98].
Another critical point for resveratrol efficacy is its low bioavailability. It is rapidly
absorbed, metabolized, and excreted; however, in spite of its low bioavailability,
evidence that beneficial activities occur in humans is beginning to emerge, and
this phenomenon has been described as the “resveratrol paradox.” This paradox
could thus be related to a possible action of resveratrol metabolites [99] and/or
to a synergistic effect of resveratrol with other polyphenols or micronutrients.
Accordingly, resveratrol as a blend of polyphenols from grape extracts exhibited a
greater inhibitory effects on the expression of inflammatory markers in vascular cell
than resveratrol alone [85, 95], suggesting the occurrence of a synergism among
resveratrol and other polyphenols.
Though resveratrol’s potential utility in preventive medicine has been demon-
strated using in vitro models, few clinical trials have also evaluated the effects of
resveratrol on clinically relevant biomarkers. In healthy individuals, Agarwal and
collaborators evaluated the effects of 1-month resveratrol treatment on endothelial
response and plasma biomarkers [100]. Exposing cultured human coronary artery
endothelial cells to plasma drawn post-resveratrol supplement resulted in signifi-
cantly lower mRNA expression of VCAM-1, ICAM-1, and IL-8 than plasma drawn
from the same subjects at baseline. This clinical trial highlighted for improved gene
expression in vascular endothelium by resveratrol. A triple-blind, randomized,
placebo-controlled, 1-year treatment with a resveratrol-containing grape supplement
on stable patients with coronary artery disease [101] showed dose-dependently an
increase of the anti-inflammatory serum adiponectin and a decrease of plasminogen
activator inhibitor-1. Moreover, the transcriptional profiling showed a down-
regulation of pro-inflammatory genes and a modulation of inflammatory transcrip-
tion factors, confirming previous in vitro findings [102].
Moreover, in peripheral blood mononuclear cells of type 2 diabetes and hyper-
tensive patients with coronary artery disease [102], the supplementation with
resveratrol-containing grape supplement significantly reduced the expression of
the pro-inflammatory cytokines CCL3, IL-1, and TNF-α and modulated
inflammatory-related microRNAs. These clinical studies support the conclusion of
beneficial anti-inflammatory and immunomodulatory effects of grape extract
enriched in resveratrol for secondary prevention of patients with coronary artery
diseases.
5 Conclusion
The precise nature of the role played by polyphenols in human health has been
largely highlighted in these last years. A better knowledge concerning the compo-
sition and dynamics of polyphenol profile in red grape will help vinedresser and
winemakers in producing grape-derived products and wines with high content of
phenolic antioxidants and considerable antioxidant activity, maintaining optimal

organoleptic properties and a significant link with the original terroir.
Even though, a better understanding is still requested about the several different
cellular mechanisms and complex pathways involved in wine polyphenol metabo-
lism, the present findings suggest that the contribution of antioxidant phenols
through a reasonable daily drinking of red wines may offer additional protection
against in vivo oxidation and other damages of human cell components.
Acknowledgments This research was partially supported by the Apulia Region in the framework
of the Projects NEWINE (Bando “Ricerca e sperimentazione in Agricoltura”; Project code
PRS_042), SOLBIOGRAPE (Bando “Ricerca e sperimentazione in Agricoltura”; Project code
PRS_053), and DOMINA APULIAE (POR Puglia FESR – FSE 2014-2020-Azione 1.6. –
InnoNetwork; Project code AGBGUK2).
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Natural Estrogenic Substances, Origins,
and Effects 40
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1158
2 The Natural Estrogenic Substances and Their Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159
2.1 Estrogenic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159
2.2 Isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162
2.3 Coumestans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162
2.4 Lignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
2.5 Resorcylic Acid Lactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
3 Human Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.1 Isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1175
3.2 Coumestans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
3.3 Lignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
3.4 Resorcylic Acid Lactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
4 Effects in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
4.1 Isoflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
5 Effects in Humans and Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184
5.1 Beneficial Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184
5.2 Deleterious Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1187
6 Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
6.1 Estrogen-Dependent Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
6.2 Other Side Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
Abstract
Some natural substances have been scientifically identified as estrogenic since the
late 1930s when they were found to be deleterious at high doses for cattle
C. Bennetau-Pelissero (*)
Department of Life Science and Health, University of Bordeaux, Bordeaux, France

reproduction. Several compounds belonging to different chemical families are

considered here: isoflavonoids, coumestans, lignans, and resorcylic acid lactones.
This list is not exhaustive. The vegetable sources of these compounds are
probably not all identified yet, but all the compounds presented here were
shown to act as endocrine disruptors, i.e., modifying the hormonal natural
balance, at dietary doses either in human, in cattle, or in other vertebrates.
Estrogenic compounds mimic estradiol activities and can interact nearly with
all biological functions in lower and higher vertebrates. Some mollusks are also
sensitive to estrogens. The effective dose is crucial to consider since as other
endocrine disruptors the natural substances may have opposite effects at low,
dietary, and pharmacological concentrations. Different cell pathways are trig-
gered by the natural estrogenic substances, including some that are not influenced
by estradiol itself, and this explains why their effect is not a monotonic
dose–response line. This questions the classical toxicological approach which
considers acute exposure (short and high concentrations) as the key of the toxicity
evaluation. The history of human exposure to isoflavones was recently casted on
doubt, reinforcing the need for careful study of these compounds’ occurrence and
effects on humans. It is clear now that the traditional soy food makings were able
to remove isoflavones from foodstuffs. This is no longer the case in modern
processing, and this means that the exposure to this estrogenic substances has
increased markedly in recent times. Estrogens in mammals can have both bene-
ficial and harmful effects which are evoked here.
Keywords
Natural estrogens · Food sources · Modern exposure · Bioavailability ·
Mechanism of actions · Breast cancer · Bone health · Thyroid · Reproductive
disruption
1 Introduction
Nowadays, as the concern on endocrine disruptors is back to front stage, scientists

realize that natural estrogenic compounds which were ignored for ages have to be
included in the panel of active and potentially deleterious compounds [1]. This is
mainly due to the recent discovery that humans were not traditionally exposed
to natural estrogenic compounds [1] and that this recent exposure is synchronized
with that to other anthropoid endocrine disruptors such as pesticides or wrapping
agents [2]. Humans were not traditionally exposed to natural estrogenic compounds
because they used to prepare all grain legumes, which are the major sources of
estrogenic substances in human diet, by prolonged soaking, cooking, or simmering
in water. The water was then discarded, and because natural estrogenic compounds
are mainly under a glycosidic form in plants, they leaked into the water during the
cooking steps and were then eliminated with water [3]. Reproductive problems were
recently reported in multigenerational toxicological studies in animal models [4] and
40 Natural Estrogenic Substances, Origins, and Effects 1159
in humans [5]. They occur at actual dietary exposure levels, although the acute
toxicity studies performed classically on short-term durations and with pharmaco-
logical concentrations do not show major adverse effects [6]. Since endocrine
disruptors were discovered, the toxicology studies dealing with reproductive issues
were moved to multigenerational exposure and to low doses. Following this guid-
ance, studies can show hormonal disruption of reproduction including epigenetic
effects visible even in generations not exposed per se but issued from exposed
genitors [7]. For the compounds studied here, the toxicity studies all converge in
claiming an estrogenic activity. The present chapter is therefore focusing on the
so-called phytoestrogens taken in their large definition since it included the myco-
toxins zearalenone (ZEN) and zearalenol (ZOL) produced on grains by fungi. They
also focus on natural estrogenic substances produced by plants or by the human gut
after plant-substance absorption, i.e., coumestans, isoflavones, isoflavans, and
enterolignans. Flavonoids can also have estrogenic effects, but at nutritional expo-
sure, their effects remain low enough to be neglected here. However, if tomorrow a
synergy with other anthropological endocrine disruptors taken at environmental
doses is demonstrated, they will also have to be considered. Sources and concentra-
tions, roles in plants, and effects in plant consumers are exposed together with the
questions remaining on their beneficial and deleterious effects in human and domes-
tic animal consumers.
2 The Natural Estrogenic Substances and Their Sources
2.1 Estrogenic Effects
The estrogenicity of a substance being anthropogenic or natural relies on two major

pillars: its efficiency on estrogen-dependent gene transcription which is approached
by its efficient dosage in in vivo and in vitro systems and its bioavailability at the
target cell. The estrogenic effects of natural substances are based on their chemical
structures which share some similarities with that of the 17β-estradiol (17β-E2)
molecule, which is the most potent estrogen in vertebrate animals. 17β-E2 is an
aromatized C18 steroid with hydroxyl groups at 3- and 17-β-position (Fig. 1a). In
humans, it is produced primarily by the cyclic ovaries and the placenta. It is also
produced by the brain, the adrenal cortex, and the adipose tissue of men and
postmenopausal women. It is also crucial for male tract development [8] and
adequate sperm production [9]. As seen in Fig. 1, 17β-E2 is a three-dimensional
molecule with its two hydroxyl groups at a distance of 10 angstroms (Å). 17β-E2
bears a phenolic ring called cycle A.
The hydroxyl groups form an angle allowing the best interaction with the specific
estradiol receptors (ERs) [10]. These receptors are proteins with specific quaternary
structures allowing estradiol binding via a ligand-binding domain (LBD) and a
subsequent activation of gene transcription via phosphorylation processes and acti-
vation of transcription factors. This transcription phenomenon can either follow the
canonical route or be induced via cell phosphorylation pathways. The canonical
Steroid
OH Coumestan OH
O
Cycle A
Estradiol = E2 HO O O Coumestrol
HO CAS: 50-28-2 CAS: 479-13-0
Isoflavones
OH O OH
OH O O OH
H3CO
O Genistein O HO O Glycitein
HO HO Daidzein
CAS: 446-72-0 CAS: 40957-83-3
CAS: 486-66-8
Isoflavan OH OCH3 O OCH3

OH O
HO O S-Equol O HO O Formononetin
HO Biochanin A
CAS: 531-95-3 CAS: 485-72-3
CAS: 491-80-5
Enterolignan
Resorcylic Acid Lactone
HO
OH O CH3 O
O Zearalenone O
CAS: 17924-92-4
HO Enterolactone
CAS: 77756-20-8 OH
O
Fig. 1 17β-Estradiol structure together with the structures of the main phytoestrogen families:
isoflavones, coumestans lignans, and resorcylic acid lactones
route involves a direct DNA receptor interaction, and it is known to rely on both ERα
and ERβ estradiol receptor subtypes. These two estradiol receptors derive from two
different genes: ESR1 located in humans on chromosome 6 and ESR2 located
on chromosome 14, respectively [11]. The two receptors derive from a common
ancestor but evolved differently, and nowadays they do not exhibit the same primary
structure, and their activation by various ligands can be rather different.
These receptors present a LBD with a hydrophobic pocket constituted by the E/F
domain (C-terminal domain) of the protein and a part of the A/B domain
(N-terminal domain) folded in its vicinity [11]. The E/F domain is constituted of
several helix structures, and the spatial position of the H12 helix depends on the
chemical structure of the ligand. Estradiol taken as reference is a full agonist of its
ERs. Other estrogens can exhibit selective modulating effects depending on the way
they fit into the ligand-binding pocket of the ERs. If their structure induces a
structural conformation of the H12 helix which does not allow the recruitment of
all transcription factors, then the estrogenic effect can be partial. Because the
transcription factors are not the same in all cell types, referring to their differenti-
ating history, then some compounds can induce the transcription of estrogen-
dependent genes in certain cell types only. This situation refers to the SERM
(selective estrogen receptor modulators) concept [12]. The 17β-E2 spatial structure
is so relevant for the ligand–receptor interaction that the natural 17-α-isomer of

estradiol binds only weakly to the ERs and exhibits little estrogenic–genomic
activity, considering its natural concentrations, in estrogen-responsive tissues
[13]. 17β-E2 physiological plasma concentrations in women usually vary from
10 to 20 pg/mL (i.e., from 36 to 72 pM) in the metestrous phase of the female
cycle to 500–600 pg/mL (i.e., 2 to 4 nM) in the estrous phase of the female cycle.
During pregnancy these 17β-E2 levels can reach values over 30 ng/mL (i.e., over
110 nM). In the meantime, 17β-E2 levels in men are below 50 pg/mL and below
40 pg/mL in postmenopausal women (i.e., below 200 pM in both cases). Estradiol
receptors coded by ESR1 and ESR2 are also present in cell membranes. This is due
to the palmitoylation of the ERs at the AA corresponding to the 451 DNA base [14]
since a mutation at this point prevents membrane location. The physiological roles
of the membrane forms of the ER subtypes are currently under investigation [15]. A
role of the membrane ERs as transmembrane channels for steroid hormones is
evoked by some authors [16].
Other estradiol receptors were found in cell endoplasmic reticulum and mito-
chondria membranes, the so-called GPER or GPR-30. This is a completely different
molecule belonging to the seven transmembrane G-protein-coupled receptors of the
rhodopsin receptor family. These receptors were shown to react with estradiol and
with several estrogens, including phytoestrogens [17, 18], and to induce cell signal-
ing pathways and growth factor receptor cross talk [19]. Finally and although these
last receptors do not transmit estrogenic effects, the orphan estrogen-related recep-
tors, i.e., ERRα, ERRβ, and ERRγ, were found to bind with different affinities
several categories of phytoestrogens [20]. In some cases, the ERRs are involved in
physiological processes known to be influenced by estrogens. The ERRs’ function is
currently under investigation, but they seem to be involved in the cell energy balance
and considering an integrative view in metabolic syndrome in humans.
Because of a partial similarity (Fig. 1), the affinities of isoflavones for the ERs
are lower than that of 17β-E2. They range from 1/1000 to 1/10,000 for ERα when
genistein (GEN) and kievitone are considered (with the following order of
magnitude decrease: GEN > daidzein (DAID) > glycitein > biochanin
A > formononetin > phaseolin > kievitone) [11, 21]. For ERβ the affinity of
isoflavones is known to be higher, 1/60 and 1/150 for GEN and DAID, respec-
tively. Other compounds like glyceolin were also found to exhibit estrogenic
activity. However, these activities are not fully characterized yet [22] even though
they are expressed at nutritional doses. Although the affinities of the natural
estrogens for the ERs can be low, natural estrogenic substances can be present at
mg level in edible plants, and some of them can reach plasma concentrations
10,000 to 100,000 times higher than those of 17β-E2. Namely, isoflavones which
present the highest bioavailability can reach micromolar concentrations in plasma,
whereas 17β-E2 is currently found at picomolar to nanomolar (pregnancy) concen-
trations in those same plasmas. Cell bioavailability specifically controlled by phase
III enzymes (ABC transporters) acting after phase I (CYP) and II (Glut and Sult)
enzymes can also play a role in the tissue-specific activities of the natural sub-
stances. See [11] for more details.
2.2 Isoflavones
The estrogenic isoflavonoids studied here include GEN, DAID, glycitein, and the
DAID metabolite S-equol produced by the gut flora and which is an isoflavan
(Fig. 1). Two other isoflavones are methylated precursors of estrogenic compounds,
the so-called formononetin and biochanin A (Fig. 1). All these compounds are
secondary metabolites from legumes and mainly present in Fabaceae. Some of
them contain high amounts of isoflavones with estrogenic properties (i.e., up to
several hundred mg/100 g of dry matter [23]). The most concentrated Leguminosae
are, namely, black beans (a variety of soybean) [21], soy (Glycine max) [24], kudzu
(Pueraria lobata or tuberosa) [25], alfalfa (Medicago sp.) [23], and clover (Trifolium
sp.) [26]. Estrogenic isoflavones are also found in a smaller quantity in pulses
traditionally edible in Western countries like beans, lentils, broad beans, and chick-
peas, but in these vegetable sources, the concentrations are about 500 times lower
than in the plants previously cited [27, 28] (Table 1). These compounds were also
found in more than 300 different plants, including many species of Leguminosae
like Baptisia, Cytisus, Dalbergia, Genista, Lupinus, Medicago, Phaseolus, Teline,
Trifolium, and Ulex and in several species of Rosaceae like Prunus [29, 30]. In
Western countries, these plants were traditionally used as sources of antifertility
agents [29, 30].
2.3 Coumestans
The main estrogenic substance in the coumestan family is coumestrol (COUM)

although it also exists as methylated substances (40 -O-methyl and 7-O-methyl
derivatives) which were found in alfalfa (Medicago sativa) [39]. When they reach
the liver, the methylated forms can be demethylated in COUM by phase I enzymes.
Coumestrol estrogenic potency is by far the highest in vitro especially through
ERα [40]. However, its bioavailability appears to be lower than that of isoflavones
in rat [41]. No data were found in humans since this compound is considered as
toxic, and therefore, pharmacokinetic studies have not been published yet in humans.
In addition, this compound is present in significant amounts in alfalfa especially after
fungi’s attacks (Pseudopeziza medicaginis) [42] or in clover sprouts [43].
However, if these pulses can be used for animal feeding, they are only anecdotally
used in humans, and in that case, it is essentially as dietary supplements. According
to [44], clover sprouts, raw, contain 14.079 mg COUM/100 g; kala chana, mature
seeds, raw, contain 6.130 mg COUM/100 g; and pinto beans, mature seeds, raw,
contain 1.805 mg COUM/100 g. In addition, alfalfa seeds, sprouted, raw, contain
1.596 mg COUM/100 g; mung beans, mature seeds, sprouted, raw, contain 0.932 mg
COUM/100 g; and split peas, mature seeds, raw, contain 0.812 mg/100 g. Still
according to [44], soymilk, original and with vanilla, unfortified, can contain
0.807 mg COUM/100 g, and soybeans, mature seeds, sprouted, raw, can contain
0.341 mg COUM/100 g. However, soybean does not seem to be a constant source of
COUM. It is likely that COUM synthesis depends on the plant culture conditions and
its contamination by fungi.
Table 1 Isoflavones and coumestrol in several edible plants
40
Isoflavones Coumestans
Biochanin A Daidzein Genistein Glycitein Coumestrol
Fruits and (CAS 491-80- (CAS Formononetin (CAS (CAS 40957- (CAS
vegetables 5) 486-66-8) (CAS 485-72-3) 446-72-0) 83-3) 479-13-0) Total Authors
μg/100 g of fresh weight μg/100 g
Alfalfa sprout 67 152 3,899 118 na 105 4,341 [31]
Alfalfa sprout nd nd 340 nd na 4,680 5,020 [32]
Alfalfa sprout nd nd 5,170 nd na 72,010 77,180 [32]
(freeze-dried)
Almond 25 <1 <1 1 <1 na 26 [33]
Apricot (dry) tr nd nd tr na nd 0 [31]
Asparagus (white) na nd na nd na na 0 [34]
Asparagus (white) nd 58 nd tr na tr 58 [31]
Asparagus (green) na nd na nd na na 0 [34]
Basilic na nd na nd na na 0 [34]
Beet na nd na nd na na 0 [34]
Natural Estrogenic Substances, Origins, and Effects
Black bean freeze- nd 77,440 nd 79,640 na nd 157,080 [32]

dried
Black bean seeds nd 69,850 nd 61,220 na nd 131,070 [32]
1, dry
Black bean seeds nd 26,950 nd 27,710 na nd 54,660 [32]
2, boiled
Black trumpet na nd na nd na na 0 [34]
mushrooms
Black-eyed bean 173 nd nd nd na nd 173 [32]
seeds, dry
Brazil nut 13 6 <1 85 <1 na 104 [33]
(continued)
1163
Table 1 (continued)
1164
Broad beans nd nd 210 129 na nd 339 [32]
(grilled)
Broccoli nd 44 nd nd na nd 44 [31]
Broccoli nd 5 nd 7 na na 12 [35]
Brocoli sprouts nd 44 nd nd na na 44 [27]
Brocoli sprouts nd 44 nd nd na nd 44 [31]
Brussel sprouts nd tr nd nd na nd 0 [31]
Brussel sprouts na nd na nd na na 0 [34]
Capers na nd na nd na na 0 [34]
Carrotts nd 2 nd 2 na nd 4 [31]
Carrotts 5 nd nd nd na na 5 [31]
Cashew nuts 7 nd 2 2 1 na 12 [33]
Celery na nd na nd na na 0 [34]
Ceps na nd na nd na na 0 [34]
Chanterelle na nd na nd na na 0 [34]
Chick pea 126 nd nd 640 na 6,130 6,896 [32]
Chick pea 2 40 140 60 na na 242 [35]
Chinese peas, 101 nd nd nd na nd 101 [32]
boiled
Chinese peas, 126 nd nd nd na nd 126 [32]
freese-dry
Clover sprout 440 nd 2,280 350 na 28,060 31,130 [32]
Clover sprout 751 71 4,020 71 na 98 5,011 [31]

40
Clover sprout 8,810 nd 561,140 6,940 na 561,140 1,138,030 [32]

freese-dried
Coconuts 6 nd nd nd 4 na 10 [33]
Coconuts powder na nd na nd na na 0 [34]
Codea nd 50 nd tr na na 50 [31]
Dog Rose Berry na nd na nd na na 0 [34]
Garbanzo bean 152 nd nd nd na nd 152 [32]
seeds, dry
Garbanzo bean 1.52 nd nd nd na na 1.52 [36]
seeds, dry
Garbanzo bean 1,394 nd 52 67 na na 1,513 [31]
seeds, dry
Garbanzo bean 2,822 33 258 82 na na 3,195 [35]
seeds, dry
Garlic tr nd nd tr na nd 0 [31]
Garlic nd nd 23 1 na na 24 [35]
Ginger na nd na nd na na 0 [34]
Grape na nd na nd na na 0 [34]
Grape nd tr nd tr na nd 0 [31]
Grapefruit tr 36 tr 27 na 50 113 [31]
Grapefruit na nd na nd na na 0 [34]
Great northern 60 nd nd nd na nd 60 [32]
bean seeds, dry
Green bean nd nd 211 nd na nd 211 [32]
freese-dried
Green bean fresh tr nd tr nd na nd 0 [32]
boiled
Green bean fresh nd nd 15 nd na nd 15 [32]
raw
1165
(continued)
1166
Table 1 (continued)
Haricots en grains 838 11 215 73 na na 1,137 [35]
Hazelnut 12 <1 <1 9 <1 na 21 [33]
Horse radish na nd na nd na na 0 [34]
Kiwi na nd na nd na na 0 [34]
Large lima beans nd nd 10 nd na nd 10 [32]
seeds, boiled
Leek na nd na nd na na 0 [34]
Letuce na nd na nd na na 0 [34]
Licorice nd 293 1,493 599 na nd 2,385 [31]
Mungo bean tr 387 26 424 na 2,000 2,837 [31]
sprout
Mungo bean nd 745 nd 1,902 na na 2,647 [37]
sprout
Mungo bean nd nd tr nd na na 0 [35]
sprout
Mungo bean nd nd tr nd nd na 0 [32]
sprout
Mungo bean nd tr nd nd na na 0 [32]
sprout freese-dry
Mungo Beans nd nd 61 nd na na 61 [32]
Olive na nd na nd na na 0 [34]
40
Orange (juices) tr tr tr tr na 53 53 [31]

Origan na nd na nd na na 0 [34]
Peach nd nd nd nd na nd 0 [31]
Peanuts (fresh) 8 <1 4 48 10 na 70 [33]
Pear na nd na nd na na 0 [34]
Pecan nut 30 nd 2 2 nd na 34 [33]
Pine nuts 27 <1 <1 4 <1 na 31 [33]
Pink Bean nd nd 105 nd na nd 105 [32]
Pinto bean 56 nd nd nd na 361 417 [32]
Plums nd nd nd nd na nd 0 [31]
Red Bean 196 nd nd 9 na na 205 [31]
Red Bean nd 10 nd 5 na na 15 [35]
Red Bean boiled 41 nd nd nd na nd 41 [32]
Red Bean dry 560 nd nd nd na na 560 [36]
Red Bean dry nd 20 nd 520 na na 540 [31]
Red Bean dry 4 42 nd 98 na na 144 [38]
Red Bean dry nd nd nd 310 na na 310 [32]

Red Bean freese- 132 nd nd nd na nd 132 [32]
dried
round split peas, nd nd nd nd na 811 811 [32]
dry
Rutabaga na nd na nd na na 0 [34]
Shitake na nd na nd na na 0 [34]
Small lima bean 37 nd 55 nd na nd 92 [32]
seeds (dry)
Small white bean nd nd 82 74 na nd 156 [32]
seeds, dry
Soft potatoes nd nd nd tr na nd 0 [31]
1167
(continued)
1168
Table 1 (continued)
Sunfolower seeds nd nd nd nd na nd 0 [31]
Turnip na nd na nd na na 0 [34]
Wallnuts 17 1 <1 11 <1 na 29 [33]
Yellow split peas, 86 nd nd nd na nd 86 [32]
dry
Yellow split peas, nd 726 nd nd na nd 726 [32]
dry
2.4 Lignans
Lignans are not estrogenic substances per se, but some compounds in the lignan
family (Fig. 2) can be transformed into enterolignans exhibiting low or partial
estrogenic effects [45]. As far as it is known, the classical precursors of the
estrogenic metabolites enterolactone (ENL) and enterodiol (END) are lariciresinol,
matairesinol, medioresinol, pinoresinol, secoisolariciresinol, sesamin, sesamolin,
and syringaresinol [46, 47]. Figure 2 gives their chemical formula.
However, in rats it was shown that lignin could be metabolized into enterolignans
via its hydrolysis into lignans [48]. The current sources of enterolignan precursors
are given in Table 2. However, this list is most likely not exhaustive.
Therefore, the exposure to enterolignan precursors is far to be precise, and in some
populations, data are missing. As a consequence, the exposure is more reliably
evaluated based on blood or urine measurements of enterolignans. This is particularly
true since the ability to form enterolignans varies largely between human subjects [11].
2.5 Resorcylic Acid Lactones
The estrogenic resorcylic acid lactones are the mycotoxins ZEN and ZOL types α
and β. All are produced by fungi of the Fusarium family developing on maturing
corn, wheat, barley, rye oats, soybeans, sorghum, peanuts, and other food and feed
Lignans (non estrogenic)
OCH3
OCH3 OCH3
Pinoresinol OH
Lariciresinol OH O
OH
CAS: 27003-73-2 Sesamin O O
CAS: 4263-88-1 O OH
CAS: 607-80-7 O OCH3
H3CO
H3CO H3CO O
O O O
O HO Medioresinol
HO HO O CAS: 40957-99-1
OCH3 Secoisolariciresinol
OH HO
CAS: 29388-59-8
O O
HO
O
CH2OH H3CO
OCH3 Sesamolin O
O
H3CO CH2OH CAS: 526-07-8 O
H3CO
O OCH3
Matairesinol
HO Syringaresinol O OH
OCH3 O O CAS: 580-72-3
OCH3 CAS: 1177-14-6 OH O
Enterolignans (estrogenic)
HO HO CH2OH
O
CH2OH
O
Enterolactone Enterodiol
OH OH CAS: 80226-00-2
CAS: 77756-20-8
Fig. 2 Chemical structure of enterolignans and of some of their known precursors

1170
Table 2 Lignan content in several edible fruits and vegetables

Matairesinol Lariciresinol Pinoresinol Syringaresinol Medioresinol
Fruits and Secoisolariciresinol (CAS (CAS (CAS (CAS 21453- (CAS
vegetables (CAS 148244-82-0) 580-72-3) 27003-73-2) 487-36-5) 69-0) 40957-99-1) Total Author
μg/100 g of wet weight μg/100 g
Alfalfa sprouts tr nd na na na na 0 [31]
Ananas 7 18 24 3 na na 52 [49]
Apple na 3 55 na na na 58 [49]
Apricot dry 328.3 tr na na na na 328.3 [31]
Artichoke 171.45 0 153.76 3,479.71 21.89 56.65 3,883.47 [46]
Asparagus 743 14 92 na na na 849 [49]
Asparagus 68 tr na na na na 68 [31]
Avocado 47 6 31 272 na na 356 [49]
Baby corn na na 25 16 na na 41 [49]
Baby corn 7 na 14 na na na 21 [49]
Bambou 38 na na na na na 38 [49]
sprouts
Banana nd 17 na 19 na na 36 [49]
Basilic 546 1.9 na na na na 547.9 [34]
Black trumpet 1.3 0.1 na na na na 1.4 [34]
mushrooms
Broad Bean 240.39 34.89 319.23 24.67 57.34 4 681.29 [46]
Broccoli tr nd na na na na 0 [31]
Brussel Sprouts 30 nd na na na na 30 [31]
Brussel Sprouts 21 0.6 na na na na 21.6 [34]
Capers 44.5 15.1 na na na na 59.6 [34]
40
Carob bean 1,266.51 108.41 1,770.74 294.99 12,965.7 336.08 16,742 [46]
Carrots 38 tr na na na na 38 [31]
Cauliflower 91 na 36 85 na na 212 [49]
Celery 12 na 16 43 na na 71 [49]
Celery 9.9 0 na na na na 9.9 [34]
Ceps 0.6 0.3 na na na na 1 [34]
Chanterelles 3.9 7.3 na na na na 11.2 [34]
Cherry tomato 17 na 43 11 na na 71 [49]
Cherry tomato 16 na 38 19 na na 73 [49]
Clover sprouts nd nd na na na na 0 [31]
Coconut 34 0 na na na na 34 [34]
(powder)
Cucumber 41 na 65 na na na 106 [49]
Dog Rose berry 78.8 2 na na na na 80.8 [34]
Eggplant 8 na 40 51 na na 99 [49]
Garlic 55 na 84 45 na na 184 [49]
Garlic 27 37 na na na na 64 [31]
Ginger na 16 na 15 na na 31 [49]
Ginger 21.3 0 na na na na 21.3 [34]
Grape tr 52 na na na na 52 [31]
μg/100 g de poids frais μg/100 g
Grape 2 1.3 na na na na 3.3 [34]
Grapefruit nd tr na na na na 0 [31]
Grapefruit 26.3 0 na na na na 26.3 [34]
Green 78.3 3.4 na na na na 81.7 [34]
Asparagus
(continued)
1171
1172
Table 2 (continued)
Green Pepper 5 na 73 6 na na 84 [49]
Grilled corn 14.46 0 15.33 0 331.55 3.01 364.35 [46]
Horse radish 14 0 na na na na 14 [34]
Horse radish 37 49 219 16 na na 321 [49]
Kiwi fruit 174.6 1.2 na na na na 175.8 [34]
Kiwi fruit 106 na 20 13 na na 139 [49]
Leak 11.8 0 na na na na 11.8 [34]
Lentils 1.38 245.17 233.24 86.93 196.63 17 781.1 [46]
Mung bean 82 1 32 33 na na 148 [49]
sprouts
Mung bean tr tr na na na na 0 [31]
sprouts
Nashi 7 na 21 na na na 28 [49]
Olive 55.9 2.7 na na na na 58.6 [34]
Orange Navelle 14 na 128 24 na na 166 [49]
Orange 56 na 193 51 na na 300 [49]
Valencia
Oranges (Juice) tr tr na na na na 0 [31]
Oregano (dry) 44.4 1 na na na na 45.4 [34]
Oriental Pears 0.28 0 6.9 179.99 23.43 9 220.21 [46]
Paprika 8 79 2 na na na 89 [49]
Pea 129 na 9 6 na na 144 [49]
Pea na na 59 50 na na 109 [49]
40
Pea 2.73 6.79 150.59 79.81 175.36 24.2 439.48 [46]

Pea 1 98.82 83.73 7.11 1.82 0 192.63 [46]
Pea 2.61 87.75 133.75 8 1.81 0 234.14 [46]
Peach 11 na 38 83 na na 132 [49]
Peach 28 nd na na na na 28 [31]
Pear 2 na 34 2 na na 38 [49]
Pear 9.9 0.7 na na na na 10.6 [34]
Plums 16 na 20 42 na na 78 [49]
Plums 76 tr na na na na 76 [31]
Pumpkin 20 na 29 na na na 49 [49]
Pumpkin 29 3 58 8 na na 98 [49]
Radish na na 29 43 na na 72 [49]
Radish 1 15 na 40 na na 56 [49]
Red bean nd nd na na na na 0 [31]
Red Pepper 9 na 73 1 na na 83 [49]
Rice Bread 33 4 47 44 na na 128 [49]
Rice Bread 7 1 18 16 na na 42 [49]

Rutabaga 3.7 0.6 na na na na 4.3 [34]
Salade (Letuce) 105.6 0.6 na na na na 106.2 [34]
Shitake 0.2 0 na na na na 0.2 [34]
Spanish melon 23 na 69 12 na na 104 [49]
Spinach 8 na 110 31 na na 149 [49]
Spinach 13 1 79 15 na na 108 [49]
Strawberry 51 na 33 21 na na 105 [49]
Strawberry 61.37 18.3 134 5.21 60.83 3.89 284.49 [46]
Sunflower seed 128 nd na na na na 128 [31]
(continued)
1173
1174
Table 2 (continued)
Sweet potato 65 2 79 16 na na 162 [49]
Sweet potato nd 41 na na na na 41 [31]
Tomato 3 na 11 12 na na 26 [49]
Turnip 6.3 0 na na na na 6.3 [34]
Watermelon 5 na 67 5 na na 77 [49]
White 28.7 0.6 na na na na 29.3 [34]
Asparagus
crops in the field and in grain during transportation or storage. Zearalenone and ZOL
(Fig. 1) are mainly produced by F. graminearum and F. semitectum [50]. Due to its
structural similarity to the naturally occurring estrogens, ZEN is an estrogenic
mycotoxin that induces obvious estrogenic effects in animals [51]. Zearalenone
and ZOL productions are favored by high-humidity and low-temperature conditions.
They can occur simultaneously with other mycotoxins such as deoxynivalenol
(DON) and less frequently with aflatoxins [52]. Zearalenone is stable in food
under regular cooking temperatures but can be partially eliminated under deep
heating [53]. In the human diet, the main sources are grain milling products, for
human consumption, e.g., breakfast cereals, breads, and rolls. Cooked pastas only
contain small amounts of ZEN, and meat and meat products are always below the
detection limits (Table 3).
3 Human Exposure
3.1 Isoflavones
Isoflavones nowadays are present in almost all domestic animal food except when an
alternative breeding practice is adopted, namely, if grass or other human residues are
used instead of the conventional diet. In most animal breeds, the protein input is
brought by pulses. The most nutritionally interesting are soy, alfalfa, or clover. All
three can contain natural estrogenic compounds at high concentrations as previously
mentioned. However, estrogenic isoflavones, although having a good bioavailability,
have a low distribution volume and are usually eliminated for the most part during
the 24 h coming after intake. Because animals are currently fasting for at least 12 h
before slaughtering, the amount of isoflavones remaining in their flesh is generally
low [55]. Meat or fish, even though they have been fed pulses during their life, are
not good isoflavone vectors in human diet.
In human, the exposure is mainly due to soy intake, although clover, alfalfa, and
kudzu extracts can be used in food supplements. Until recently, this exposure was
thought to be safe assuming that isoflavones had always been part and parcel of the
human diet in Asia. However, it was recently shown that all traditional Asian recipes,
which were elaborated sometimes over several centuries, included prolonged
cooking, simmering, or soaking in water. These steps empirically removed the
glycosylated isoflavones which are soluble in water but heat resistant. As an
example, for traditional tofu, a rough soy meal is simmered and soaked in water
for a total of 2–3 h before curdling. Then, water is squeezed out by pressing to make
the soy cheese. In the same way, kudzu, which contains high levels of isoflavones
with inverse proportions of GEN and DAID compared to soy, is directly powdered to
prepare medicines. It is also boiled in water to be eaten as we could do with potatoes.
The boiling step can last over 2 h and removes isoflavones. In 2004, looking at
isoflavone intakes in rural Chinese women who are more likely to have kept their
traditional tofu making, Liu and co-workers [56] found that 65% of them had an
isoflavone intake below 15 mg/day, the majority being below 5 mg/day. Nowadays,
Table 3 Occurrence of ZEN in unprocessed grains from EFSA [54]

>LOD Concentrations (μg/kg)
Food group Nb % LB/UB Mean P50 P75 P95 Max
Unprocessed grains 9,877 41 LB 33 0.0 15 160 2,969
UB 40 7.0 27 161 2,969
Wheat 5,318 38 LB 22 0.0 7.0 89 2,969
UB 27 5.0 20 90 2,969
Barley 1,071 37 LB 10 0.0 5.0 49 775
UB 13 5.0 10 50 775
Corn 2,460 56 LB 76 16 76 319 2,700
UB 87 40 78 319 2,700
Oats 596 23 LB 21 0.0 0.0 76 1,590
UB 23 1.5 5.0 98 1,590
Rice 43 7.0 LB 0.8 0.0 0.0 10 15
UB 5.5 5.0 5.0 10 15
Sorghum 55 53 LB 96 50 147 450 700
UB 116 50 147 450 700
N, number of samples; > LOD, indicates the percentage of results above the LOD or LOQ; LB,
lower-bound; UB, upper-bound; (lower-bound, values below the detection limit are extrapolated to
0; upper-bound, values below the detection limit are extrapolated to detection limit)
P50, 50th percentile; P75, 75th percentile; P95, 95th percentile
a
If N < 60, then the calculated P95 should be considered only as an indicative value [54]
in Western countries as in the developed countries of Asia, soy is essentially

produced using industrial techniques. In all cases, cooking steps were reduced in
time to reduce energy costs, and water is used either in small proportion (steaming)
or not used at all. As a consequence, the amounts of isoflavones can be very high.
Therefore, the amount of isoflavones in modern products can be high when a portion
is considered (Table 4). Soy is also incorporated in processed food as soy flakes
(Table 4) which can contain from 90 to 182 mg of isoflavones for 100 g [1]. There-
fore, isoflavones can be present in significant amounts in processed food containing
soy flakes (Table 4).
Why is there such amount of isoflavones in modern soy preparation? This is
because when the industrial processes were set up, in the 1930s, hardly anything was
known about isoflavones, and the processes were designed essentially to prevent
protein degradation. As an example, defatted soy flakes are treated with hexane
which removes fat and concentrates isoflavones in the protein matrix. The matrix is
then extruded. Extrusion destroys most of the anti-nutritional factors but not iso-
flavones. Generally speaking, in the industry, the cooking steps were shortened to
save energy and costs. When it was possible to assay isoflavones accurately, in the
early 1980s, the assays were performed in several popular areas essentially eating
industrial soy-based products produced in mass for city populations. The study by
Liu [56] is one of the very few dealing with exposure in remote Chinese areas still
consuming soy food traditionally cooked. It showed that the largest proportion of
rural women was exposed to low doses of isoflavones. Therefore, nowadays and
Table 4 Quantities of isoflavones measured in modern dishes based on soy or containing soy as an
ingredient
Intake for
Genistein Daidzein Total (μg/ Reasonable 1 portion
(μg/g) (μg/g) g) portion size (μg)
Foodstuffs based on soy juice
Tonyu 1 91.37 49.57 140.94 1 bowl 49,330
( 6.52) ( 3.47) (350 mL)
Tonyu 2 51.32 39.83 91.14 1 bowl 31,899
( 8.16) ( 4.39) (350 mL)
Yogurts 44.70 37.40 82.20 1 yogurts 8,220
( 3.17) ( 3.61)
Soft yogurt 125.00 129.19 254.31 1 yogurt 30,510
( 8.75) ( 9.12)
Vanilla soy cream 29.82 19.39 49.21 1 cup (100 g) 4,921
( 2.16) ( 1.36)
Caramel soy 40.00 17.90 57.89 1 cup (100 g) 5,789
cream ( 2.86) ( 0.7)
Soy juice 107.00 70.00 178.75 1 mug 58,987
chocolate taste ( 7.49) ( 4.92) (330 mL)
Vanilla dessert 159.50 63.20 224.10 1 cup (100 g) 22,410
( 11.16) ( 4.42)
Powdered 1,310.00 1,070.00 2,390.00 1 mug 49,790
soy “milk” ( 81.62) ( 64.96)
Herb cheese 368.39 357.23 725.62 50 g 36,280
made of soy ( 25.86) ( 22.99)
Cream substitute 70.08 63.16 134.95 20 mL 2,700
made of soy ( 5.11) ( 4.42)
Foodstuffs based on soy
protein
Sausages made of 82.21 40.64 122.85 2 sausages 11,060
soy (1) ( 5.76) ( 2.87) (90 g)
soy (2) ( 10.55) ( 5.53) (80 g)
soy (3) ( 10.42) ( 20.70) (90 g)
Soy biscuits 95.38 87.74 183.12 4 biscuits (80 g) 14,650
with figs ( 6.36) ( 6.16)
Buckwheat 228.50 154.00 382.50 1 pancake 38,250
pancakes with ( 15.86) ( 10.35) (100 g)
tofu
Soy pancakes 202.30 116.92 319.22 1 pancake 31,920
with tomatoes ( 14.16) ( 8.56) (100 g)
Soy pancakes 227.15 129.48 356.63 1 pancake 35,663
“provençale” ( 15.57) ( 11.02) (100 g)
Smoked tempeh 165.33 112.00 277.33 3 slices (50 g) 13,870
( 11.56) ( 7.84)
(continued)
Table 4 (continued)
Intake for
Instant powder 99.69 106.11 205.80 3 spoons 8,026
for drinks ( 6.96) ( 7.35)
Japanese soft tofu 117.87 70.92 188.79 100 g 18,879
( 10.29) ( 2.88)
Natural tofu 225.27 117.22 342.49 125 g 42,810
( 75.14) ( 7.43)
Japanese soft tofu 117.87 70.92 188.79 100 g 18,879
( 10.29) ( 2.88)
Traditional tofua 224.43 100.92 325.35 100 g 32,535
( 11.79) ( 12.48) ( 24.26)
Whey from 744.36 459.93 1,204.30 60 mL 72,258
traditional tofua ( 39.89) ( 60.17) ( 100.06)
Tofu 48.00 46.16 95.27 1 portion 9,530
( 3.26) ( 3.16) (100 g)
Breaded tofu (1) 150.33 71.22 221.55 1 portion 22,150
( 10.52) ( 5.57) (100 g)
Breaded tofu (2) 289.29 188.49 477.78 1 portion 47,778
( 6.04) ( 2.00) (100 g)
Tofu with garlic 216.74 138.25 354.99 1 portion (80 g) 28,400
( 9.60) ( 4.09)
Smoked tofu 273.54 178.54 452.08 1 portion 45,210
( 15.33) ( 5.50) (100 g)
Legumes mixed 98.81 62.90 161.71 1 dish (300 g) 48,510
with tonyu sauce ( 1.90) ( 6.35)
Vegan steak 332.44 222.65 555.09 1 steak (90 g) 49,960
tomato and ( 11.39) ( 3.29)
onions
Vegan 244.73 155.40 400.13 120 g 48,020
“Bolognese for ( 81.73) ( 8.25)
pasta”
Conventional soy 490.98 347.68 838.67 100 g 85,421
grain ( 6.62) ( 78.26)
Toasted soy grain 1,360.00 1117.42 2,477.42 20 g 49,550
(appetizers) ( 51.32) ( 119.03)
Slimming 223.59 135.44 359.03 1 pack (50 g) 16,510
dish (soup) ( 15.46) ( 9.24)
Slimming dish 185.03 98.36 283.39 1 pack (50 g) 13,030
(breakfast) ( 11.95) ( 7.56)
Slimming dish 287.17 193.64 480.81 1 pack (50 g) 22,110
(meal) ( 18.53) ( 13.22)
Soy lecithin 0.17 0.68 0.97 10 g 9.7
extract (1) ( 0.01) ( 0.04)
Soy lecithin 0.86 2.36 3.52 10 g 35.2
extract (2) ( 0.06) ( 0.16)
(continued)
Table 4 (continued)
Intake for
Foodstuffs with
hidden soy
Minced beef pie 4.66 1.53 6.20 1 portion 1,860
(Parmentier) ( 0.32) ( 0.11) (300 g)
Minced beef 73.92 49.34 12.22 1 steak 12,220
portions ( 5.11) ( 3.43)
Stuffed tomatoes 33.02 26.94 59.99 2 tomatoes 8,960
( 2.82) ( 1.96)
Stuffed cabbages 33.04 25.48 58.48 2 cabbages 9,040
( 2.55) ( 1.53)
Meatballs (1) 78.14 54.23 132.37 4 balls (125 g) 16,546
( 6.16) ( 3.56)
Meatballs (2) 82.55 59.60 142.15 4 balls (125 g) 17,768
( 6.68) ( 2.59)
Minced veal 55.96 35.32 91.28 1 steak (100 g) 9,128
(breaded) ( 3.86) ( 2.45)
Brownies 65.24 43.92 109.16 3 Pieces (90 g) 9,824
( 4.56) ( 3.15)
Figures are mean SD of three measures performed on three different microtitration plates
a
The traditional tofu is an industrial product
especially in developed Asian countries where soy is traditionally eaten but where
the processing has been industrialized, the isoflavone intakes have risen dramati-
cally, sometimes over the 45 mg/day which shown to have an effect on pre-
menopausal menstrual cycle [57]. According to recent studies, the mean human
exposure in Japan lies between 45 and 60 mg/day [58], in Korea the highest value
recorded was 33.6 mg/day for adults [59], and in China it tends to increase nowadays
[60] with 16.2 mg/day for adults and 27 mg/day for adolescents and a large
interindividual variation. In Western countries, the exposure to isoflavones also
rose recently although it is most likely underestimated. Usually, in Western coun-
tries, the isoflavone intake is based on the evaluation of pulse consumption and
essentially based on soy which is the major provider [61]. Therefore, many countries
consider the intake below 2 mg/day especially because they tend to calculate a mean
intake over the total population and taking only soy food intake into account. In the
USA, the exposure was estimated to be less than 2.7 mg/day in 2001 [31] and
confirmed later by subsequent studies [62, 63]. However, the amount of isoflavones
in modern soy-based food is higher than that found in the past in traditional
foodstuffs (Table 4). Hence, soy juices which are very popular in the West, together
with their side products (yogurts and creams), can be good vectors of isoflavones,
since in this case, isoflavones are concentrated at the juice filtration step [1]. Soy is
also prepared as flakes containing large amount of isoflavones. As already
Table 5 Isoflavone intakes by infants fed with soy-based infant formula in different countries
Countries Isoflavones mg/liter Daily isoflavone intake (mg/kg bw) Reference
USA 20.9–47 2.3 to 9.3 [68]
UK 18–46.7 1.7 to 4.4 [68, 69]
Australia 17.2–21.9 [68]
New Zealand 17.1–33 2.9 to 3.8 [68, 70]
Brazil 10–47.4 0.8–1.6 to 6.6 [68, 71, 72]
France 19.4–42.3 2.3 to 6.04 [73]
mentioned, flakes are incorporated into many recipes marketed as traditional but
made by the food industry. Soy is then incorporated as an ingredient for its nutri-
tional and technological properties or for economic reasons. Most studies have
underestimated this exposure, but it is so significant that in recent studies no urine
samples were found to be devoid of isoflavones in American men [5]. Isoflavones
were also detected in 88% of the urine of American pregnant women [64], in all
Israeli adults [65], and in all German adolescents [66] of a study cohort. Finally, it
must be pointed out here that the human populations most exposed nowadays are
infants or children on soy-based infant formula [67]. These formulas were advised to
children with lactose intolerance mainly in the 2000s. In the USA and according to
authors, 17–30% of infants received these formulas in the last past years. Table 5
shows data recorded in different countries by several authors.
In their paper [67], Badger and co-workers also showed that infant exposure to
soy isoflavones is a modern practice. They explained the exposure to estrogens of
infants fed with soy-based infant formula is dramatically different from that of Asian
children enjoying traditional Asian soy intake. Traditional soy consumption in Asia
was based on solid foodstuffs (tofu, natto, miso, tempeh), which were not suitable for
babies. Traditionally, these foodstuffs were prepared following recipes including
prolonged cooking, simmering, or soaking in water and most probably contained
much less isoflavones than the modern industrial products. Therefore, the isoflavone
exposure calculated in [67] was nearly nil before 12 months in Asian children but
rose progressively thereafter. By comparison, in Western babies fed with soy-based
infant formulas, the exposure rate is high as soon as soy formula intake begins,
which rises and continues to rise progressively until food diversification. Exposure
levels are currently 10–20 times higher than those inducing an estrogenic effect in
women, as shown on breast reactivity [74–76], menstrual cycle lengthening [57, 77],
and impaired sperm production [78–80]. The plausible consequences of such expo-
sures will be discussed later.
3.2 Coumestans
Coumestrol as a phytoalexin is essentially produced by pulses following fungal

attacks. Therefore, and because human foodstuffs are usually better controlled, this
substance is more likely to be present in animal food rather than in human diet.
Although, it is not excluded that it could be present in food supplements such as

those based on alfalfa. In England the study of Clarke and co-workers [81] failed to
detect any COUM in the samples analyzed. In France, in 2011, according to the
EAT2 study [82], the consumer exposure was considered to be at least 1,891 ng/day.
However, the authors tend to think that this exposure is underestimated since all the
contributors could not be analyzed in the EAT2 study. Recently a protein supplement
based on alfalfa was authorized by EFSA although the supplier was able to measure
milligrams of COUM in his extracts, i.e., 78 mg/kg [83]. The supplier also showed
an estrogenic effect in mice and showed a large variability of phytoestrogen content
from one batch to another. If such a supplement is to be used widely by the European
consumers as it is proposed, the mean exposure to COUM will probably rise
dramatically. However, in South Korea, it was reported in 2009 that the mean
level of exposure was 0.3 mg per capita per day due to a local specific foodstuff
consumption like soybean sprout and arrowroot [59].
3.3 Lignans
As already mentioned, lignans are not estrogenic in plants. The molecules present in
grains and in vegetables are eventual precursors of estrogenic enterolignans
depending on the composition and efficiency of the human gut flora. Therefore,
the enterolignan exposure is difficult to assess because not all precursor sources are
identified yet [49] and because not all consumers are able to produce them after
ingestion. According to Zamora-Ros and co-workers [84], in European populations,
lignans were the most abundant contributor of phytoestrogen intakes when the
global intake is low. This study reports a low lignan intake (1.02 mg/day) in
Mediterranean countries and even lower in Italy (0.67 mg/day). It also reported a
higher intake (1.26–1.60 mg/day) in non-Mediterranean countries. The study also
compared their data with those obtained in the Netherlands (1.24 mg/day), in
Sweden (0.50–2.81 mg/day), and Finland (1.22 mg/day). Still according to [84], in
other Western countries such as Mexico, the USA, and Canada, lignan intakes were
considered to be much lower (from 0.35 to 0.86 mg/day). However, when pharma-
cokinetic studies are performed in humans, the enterolignan levels recorded in the
blood of ENL producers are never null [85]. Moreover, in urine and in serum, of
citizen of Western countries, they are of the same range concentrations with iso-
flavones [86, 87]. As an example according to [86] in the USA (Albany), the urine
lignan concentrations range from 0.99 to 1230 ng/mL and were found in 94% of the
population. In parallel, still in the same study, isoflavone urine levels range from 1.33
to 1290 ng/mL. The detection rate reached 100% for DAID in women. In the
UK and according to [87], the enterolignans in urine sample ranged from 0 to
10,229 μg/mmol creatinine and isoflavones (DAID, GEN and equol) excreted in
urine ranged from 0 to 1,442.6 μg/mmol creatinine. This shows that the sources of
enterolignan precursors are far from being identified and that the exposure is only
approached and most probably underestimated. In addition, this lignan intake cannot
be readily assimilated to a phytoestrogen exposure because the gut metabolism

differs between consumers [88].
3.4 Resorcylic Acid Lactones
Because of a careful screening of plant matter, usually the exposure to the

mycoestrogens, ZEN, α-ZOL, and β-ZOL in the European populations are consid-
ered to be low. More precisely based on a bio-monitoring approach linking the scarce
pharmacokinetic data of ZEN in human and data obtained on urinary levels [89], it
appeared that zearalenone daily exposure is most probably below the tolerable daily
intake (TDI) of 0.25 μg/kg body weight in European study cohorts. However, some
individuals from Haiti and in African countries, where corn is a major food source,
may have an exposure that exceeds the TDI. Table 6 from [54] gives exposure
calculations in different categories of European consumers.
4 Effects in Plants
4.1 Isoflavones
Isoflavones in plants act as phytoalexin [21]. They are antimicrobial and may have a
role in plant protection. As an example, the antifungal activity of lupin isoflavones
was demonstrated [90]. In soybean cultivars, an increase in isoflavone concentra-
tions was shown to be a specific response to the attack of a saprophytic fungi Mucor
ramosissimus. In the soybean strains resistant to the fungus, isoflavones (including
glyceollins I, II, and III, glycinol, glyceocarpin, GEN, isoformononetin, and
N-acetyltyramine) were induced by the fungal attack, all compounds possessing
antifungal activity with the exception of GEN [91]. A contamination of a soy strain
with the fungus Diaporthe phaseolorum f. sp. Meridionalis induced the accumula-
tion of isoflavones (GEN, DAID), pterocarpans (glyceolins), and flavones (apigenin
and luteolin) via a nitric oxide synthase pathway [92]. To go on with interactions of
the plants with microorganisms, there is some evidence that the symbiotic relation-
ship between Rhizobium lupine and Lupinus albus stimulates an increase in the
production of prenylated isoflavones in the root nodules [93]. These prenylated
isoflavones possess in vivo activities against a number of other Rhizobium species.
Zhang and Smith [94] also showed that GEN plays a major role in the establishment
of the symbiosis between Bradyrhizobium japonicum, the arbuscular mycorrhyzas
such as Glomus mosseae, and their host, i.e., soy (Glycine max). Genistein is the soy
recognition molecule inducing the greatest plant-to-bacterium signal. In fact, the
binding of GEN to B. japonicum activates many of the bacteria nod genes.
There is solid proof that farming practices directly influence the levels of iso-
flavones in soy. As a matter of fact, irrigation was shown to enhance isoflavone
content in soybean by as much as 2.5-fold [95]. A deficit in nitrogen fertilizer
increases the estrogenic activity of a clover pasture [96]. On the other hand, a
Table 6 Daily exposure of European citizen to zearalenone as calculated by EFSA in ng/kg/b.w [54]
Age class Summary statistics of exposure to zearalenone (ng/kg/b.w. per day)
Minimum Median Maximum
LB UB LB UB LB UB
Mean dietary exposure in total population
Infantsa 3.3 87 6.4 87 9.4 88
Toddlers 9.3 51 13 83 23 100
Older children 5.7 29 11 44 22 75
Adolescents 3.6 17 6.1 26 12 42
Adults 2.4 14 4.3 18 7.2 29
Elderly 2.0 13 3.4 16 6.4 26
Very elderly 2.3 12 2.9 16 7.1 29
95th percentile exposure in total populationb
c c c c
Infants 33 217d
Toddlers 24 104 31 182 50 277
Older children 9.9 59 22 80 42 124
Adolescents 7.5 38 15 53 26 76
Adults 4.7 28 9.5 35 14 54
Elderly 3.5 25 7.5 31 12 42
Very elderly 7.0 26 7.7 35 13 47
b.w., body weight; LB, lower-bound; UB, upper-bound (lower-bound, values below the detection
limit are extrapolated to 0; upper-bound, values below the detection limit are extrapolated to
detection limit)
a
Estimates based on only two dietary surveys
b
The 95th percentile estimates obtained on dietary surveys/age classes with less than 60 observa-
tions may not be statistically robust [54], and therefore they should not be considered in the risk
characterization. Those estimates were not included in this table
c
Not calculated
d
Estimates are based on one dietary survey only
supplementation in the same fertilizer decreases the occurrence of isoflavones with

estrogenic activity in a clover pasture [96]. A study where soy cultivars (Glycine
max) were bred for 3 years demonstrated that levels of isoflavones were related
to both environmental and genetic characteristics and could be susceptible to
selection [97]. The genomic regions implicated in this process have been
identified [98]. Vyn and co-workers [99] showed that GEN, DAID, and glycitein
contents in soy are correlated with potassium in roots and leaves and with crop
management using potassium-rich fertilizers. Lindner in [23] gave an evolutionist
theory about the ability of Leguminosae to produce estrogenic isoflavones. Indeed, it
appears that isoflavonoid compounds are synthesized by the plants in response to
bacterial or fungal attacks or to water stress. Estrogenic isoflavones can then be
considered as protective compounds. In the case of overgrazing of a pasture by
mammalian predators, the production of great quantities of estrogenic compounds
which could impair predator reproduction would result in the reduction of the
predator pressure. Finally, because estrogenic isoflavones are also specific attractants
for symbiotic bacteria of the Rhizobium gender and of mycorrhizal fungi of the
Glomus gender, and because these bacteria can fix the atmospheric nitrogen, human
selection against the production of isoflavones in pulses would result in the loss of
one of their greatest interests in crop management.
5 Effects in Humans and Animals
5.1 Beneficial Effects
5.1.1 Beneficial Effects in Animals

Estrogens are used in cattle as anabolic agents. Therefore, some studies attempted to
figure out if a phytoestrogen supplementation can enhance cattle or poultry growth
and meat quality [100]. Beside a potential interest in antioxidant content of meat
from animal raised on estrogenic food [100], it was seen that a DAID supplemen-
tation in the diet increased marbling meat score in steers [101]. If the meat quality
was significantly improved considering several parameters, the carcass bone pro-
portion was also greater probably reflecting a positive effect of phytoestrogens on
osteoblast function. In another study [102] and at low doses, i.e., 300 and 400 mg/
day, DAID was also shown to enhance immune function in late-lactation cows under
heat stress. However, the DAID supplementation does not strictly mimic a legume
intake since in all the roughages based classically on pulses, DAID or its precursor
formononetin is present together with other compounds either estrogenic per se
(GEN) or precursors of these estrogenic isoflavones (biochanin A) playing in
interaction.
There is no positive effect reported for lignans, COUM, or ZEN on farmed
animals although ENL was found in milk of cows fed with flaxseed or forages
rich in ENL precursors [103].
5.1.2 Menopause in Women
Isoflavones
Menopausal symptoms encompass hot flushes, night sweats, vaginal dryness, mam-
mary density, and mood fluctuations. Menopause is not a disease. In Western
countries, menopausal symptoms were treated for more than 20 years using hormone
replacement therapy (HRT). However, several studies including the Women’s Health
Initiative (WHI) showed that deleterious effects of the US treatments on breast or
endometrial cancers and those on cardiovascular diseases may overcome their
benefits [104]. More than 543 epidemiological studies were performed to examine
if the consumption of soy and isoflavones with estrogenic activities may prevent hot
flushes. This effect can be suspected since it is known that hot flushes and night
sweats are controlled by the hypothalamus preoptic area implicated in the body core
temperature regulation. In addition, the neurones in this region have estradiol
membrane receptors involved in body temperature and energy homeostasis [105].
This area induces shiverings or sweatings when it records a body temperature over
a low and an upper threshold. The amplitude of the neutral zone situated between
these thresholds is under serotonin and noradrenergic control. 17β-E2 is known to
induce the local synthesis of these two neuromediators as well as their respective
receptors [106]. The recent meta-analysis of Chen and co-workers [107] showed a
reduction in hot flushes with isoflavone intake. The study is based on 15 randomized
clinical trials comparing phytoestrogens vs. placebo for which the mean age of the
subjects ranged from 48 to 60.1 years. The number of participants was at least 30 and
up to 252. The study duration ranged from 3 to 12 months. The meta-analysis of the
7 studies reporting the Kupperman index gathering 11 menopausal symptoms
showed no significant effects of phytoestrogens as compared to placebo. However,
the meta-analysis of ten studies showed a significant reducing effect of
phytoestrogens on hot flush frequency compared to placebo. The meta-analysis of
the five studies that planned to report side effects of phytoestrogens did not show
any significant difference between the two groups. Menopausal symptoms are
known to be influenced by psychological elements. Therefore, a placebo effect
exists. Nevertheless, in accordance with an estrogenic effect in women especially
when estradiol receptors are present, the phytoestrogen effect can be observed.
Coumestrol
In women some data exist linking the use of some herbal medicine, some of them
containing COUM with other substances with or without estrogenic activities [108].
This is the case for clover or alfalfa preparations. However, menopausal symptoms
are influenced by psychological factors [109], and some meta-analyses failed
to really correlate isoflavones and/or COUM with a decrease of menopausal
symptoms [110]. For COUM its level in the preparation is generally low even if it
can also be highly variable. In addition, little is known about its serum bioavailabil-
ity. Therefore, its effect is very difficult to truly associate to any observation.
Lignans
Only eight randomized, double-blind, placebo-controlled clinical trials were found
trying to prove the effectiveness of flaxseed on the management of menopausal
symptoms [111–116]. None of them, whatever the amount of flaxseed or secoisolar-
iciresinol diglucoside (SDG) tested, whatever the age and number of volunteers
involved, was able to show an effect over placebo treatment. This could be expected
at least for two reasons. First, enterolignan precursors are ubiquitously distributed in
human diet, and therefore the placebo group also receives estrogenic enterolignans
in rather variable amount. See Table 2 and the known sources of lignans. This, of
course, reduces the power of the interventional studies. Second, only END or ENL
can possibly exert estrogenic effect, which could explain hot flashes relief. However,
only a small portion of the population harbors the gut flora able to produce these
enterolignans in sufficient amount [117]. Therefore, the enterolignan effect should
only be seen if women able to produce enterolignans were compared to women
unable to produce them. This would require a selective procedure at the entrance of
the clinical studies and enterolignan monitoring in blood to account for the dietary
exposure through casual diet. None of the studies done until now did undergo such
protocol. Note that urine measurements may not be precise enough since an elimi-
nated compound is not an active one and that there may be interindividual variations
in the elimination pathways of enterolignans.
Zearalenone
Zearalenone and its metabolites α- and β-ZOL as mycotoxins have not been tested in
clinical intervention on menopausal symptom relief.
5.1.3 Bone Health in Women
Isoflavones
Two meta-analyses converge in saying that isoflavone from soy may be protective on
bone density in menopausal women but only at high doses, i.e., over 80 mg/day. The
first one [118] considered only supplementation by soy isoflavone extracts (not soy
protein or foods containing isoflavones) on bone mineral density (BMD) in meno-
pausal women. It was based only on randomized controlled trials published in
English, Japanese, or Chinese reporting the effects of soy isoflavone extracts on
lumbar spine or hip BMD in menopausal women. Only 11 studies in total were found
to match the given criteria. The meta-analysis included data from 1,240 menopausal
women. It revealed that daily ingestion of an average of 82 mg of soy isoflavones
(aglycone) for 6 to 12 months significantly increased spine BMD. Treatment dura-
tion, geographic origin, and basal BMD had a major influence on the effect observed.
No significant effects on femoral neck, hip total, and trochanter BMD were found,
while soy isoflavone extract supplements increased BMD feature that was restricted
to the lumbar spine in menopausal women. The second meta-analysis [119] included
randomized controlled trials (RCTs) examining the effect of a soy isoflavone sup-
plementation in women for at least 1-year duration. The main outcomes were BMD
changes from baseline at the lumbar spine, total hip, and femoral neck. Ten RCTs
gathering 896 women were found to be eligible according to the study criteria. A
mean dose of 87 mg of soy isoflavones for at least 1 year did not significantly affect
BMD changes. However, when doses were stratified, it was shown that only a large
dose over 80 mg/day of isoflavone tended to have a weak beneficial effect on spine
BMD. The BMD was not increased but preserved.
Coumestrol
No data seem to exist on the effect of COUM on menopausal women bone health.
Only one study tested the effect of this compound in rodents [120], showing a
preventive effect against bone loss in an ovariectomized rodent model. The estro-
genic activity of COUM in vivo on bone cells has also been shown on the prevention
of osteoclast differentiation [121] and the enhancement of osteoblast formation
[122]. These are classical estrogenic effects.
Lignans
The only study which was found dealing with the association of lignans and bone
health is likely to be insufficient to prove any effect [111]. It deals with an exposure
evaluated from a dietary questionnaire, and since median intake of total

phytoestrogens was estimated at 876 μg/day, the study most probably
underestimated the exposure. Phytoestrogen plasma assays should have been done
for this study to really help in determining the enterolignan effects. Nevertheless, the
authors said that enterolignans estimated from food intake irrespective of the gut
flora efficiency were positively associated with bone density in postmenopausal
women. However, this association became nonsignificant when dietary Ca2+ was
added to the model. In light of this finding, further data are needed before any
definitive conclusion was drawn out.
Zearalenone
Because of the toxicity of the molecule and of its metabolites α- and β-ZOL, no study
was performed and published in humans. As for COUM, some data can confirm the
estrogenic effects of ZEN and metabolites on bone cells in vitro [123] but also
in vivo in rodent models [124]. Again the main outcomes are characteristic of
estrogenic effects, with a prevention of bone loss [125], although in some studies
chromosome aberrations were also reported [126 and publications included].
5.2 Deleterious Effects
Looking at deleterious effects is a challenge since in several occasions it can be seen

that the experts do not agree on what is adverse and what is safe. Before the 2010s
hormonal changes were not always considered as adverse, and therefore two types of
noneffective levels were defined for phytoestrogens: the no observable adverse
effect level (NOAEL) and the no observable effect level (NOEL). Conventional
toxicology relies upon measures of exposure that induce pharmacological or phys-
iological adverse effects. Hence, the NOEL is the highest dose tested at which there
is no measurable effect, and the NOAEL is the highest dose tested at which there is
no measurable adverse effect. Then the definition of what is an adverse effect is
crucial to consider. Nowadays, the concept of endocrine disruption has emerged, and
all hormonal modifications especially those dealing with reproductive hormones can
be considered as adverse. However, such adverse effects are usually observed on a
long-term basis, and therefore multigenerational studies may sometimes be required
to see an effect. For example, if chronic classical toxic effects are considered
for GEN, the NOAEL on liver endpoint is 50 mg/kg/day. However, considering
hormonal endpoints and according to McClain and co-workers, the NOEL is
5 mg/kg/day [6]. If hormonal effects are seen as endocrine-disrupting effects, then
the NOAEL of GEN switches from 50 mg/kg/day to 5 mg/kg/day. This NOAEL is
sustained by the recent studies of the National Toxicology Program (NTP) in the
USA [4]. However, the NTP study also showed reduced birth weight and reduced
weight at weaning of pups at all doses tested including 0.3 mg/kg/day in females and
over generations. The authors concluded that if this effect is considered as a toxic
effect, then the NOAEL is below 1.2 mg/kg/day in rat. It also showed a reduced
anogenital distance reduction in pups of both sexes at various doses and in several
exposure conditions. Litter size was also impaired after several generation of expo-
sure for the highest dose tested. These effects sign a physiological impairment which
can have consequences on reproduction which is finely tuned by complex endocrine
balances. Therefore, effects which could have been neglected in the past come to the
front nowadays because science progresses. NOAEL and LOAEL (lowest observed
adverse effect level) can be used to establish mean tolerable daily intake (MTDI). A
TDI is an estimate of the amount of a substance that can be taken daily over a lifetime
without appreciable health risk. TDIs are calculated on the basis of laboratory
toxicity data to which uncertainty factors are applied. According to [127], reasonable
uncertainty factors should be applied. When a NOAEL in rat is available in chronic
study, this uncertainty should include a factor accounting for the difference of
sensitivities among humans and a factor accounting for the relative sensitivities of
animals compared to humans. In that case, the MTDI in human is 46 times lower
than the NOAEL in rat. When only LOAEL are available, the uncertainty factor
should also account for uncertainty in extrapolating from a low-risk level. Therefore,
the global uncertainty factor between LOAEL and MTDI increases to 184. Here, the
adverse effect can be considered to be a hormonal disruption. As TDIs are regarded
as representing a tolerable intake for a lifetime, short-term exposure to levels
exceeding the TDI is not a cause for concern, provided the individual’s intake
averaged over longer periods of time does not appreciably exceed the level set.
The uncertainty factors used to establish a TDI provide assurance on the relative
safety of the TDI. However, there should be concern if the TDI is substantially
exceeded for long time periods. In order to have a quick overview of what can be
said about the molecules retained here, we propose Table 7 which gathers NOAEL or
LOAEL obtained in rat. It also gives an overview of the estimated exposure and a
weight limit under which the exposure is due to overcome the MTDI. This table is
partly deduced from that of Hendrich [128].
These data have to be considered with caution, since here again, science evolves
and toxicological studies performed more than 10 years ago may not reach the
standard of quality actually required. However, if they have not been reproduced
recently, they should be considered as valid. The MTDI presented in this table are
compared to the mean daily exposure expressed in mg/day. The values given here
may be corrupted by many biases when they were estimated from food frequency
questionnaires. First these questionnaires may not exactly reflect all population
exposure, and second the large variability of the content in the phytoestrogen of
interest may not be taken into account. Some food sources can be completely omitted
as for isoflavones. Hence, as already mentioned, soy is nowadays ubiquitously
present in industrially processed food, and the isoflavone intake is so common that
they can be found in a large proportion of consumers’ urine samples (100% in the US
study by Mumford [5], 100% in the German study by Degen [66], 98% in the Israeli
study by Berman [65]). Note that for lignans, no data of exposure were found in
Asian consumers. This is due to the fact that many plants including plants used for
personal care in Asia are sources of the estrogenic-enterolignan precursors. All these
precursors are not known yet, and this has to be combined to the fact that not all the
consumers harbor the competent gut flora for ENL production. Therefore, when the
40
Table 7 Summary of NOAEL or LOAEL of natural estrogenic substances together with the consumer estimated exposures and the limit at which the mean
dietary tolerable intake can be overcome
NOAEL in Human Estimated daily
toxicological MTDI mean intake Inferior weight limit
Phytoestrogens studies References (mg/kg) (mg/day) Country References for MDTI overcome
Isoflavones 5 mg/kg [6] 0.11 2.7 USA (general pop) [31] 24.55
50 Japan (general pop) [58] 454.55
2.257 UK (general pop) [84] 20.52
1.12 France (women) [84] 10.18
0.1782 Spain (women) [84] 1.62
0.22 Greece (women) [84] 2.00
0.317 Italy (women) [84] 2.88
0.7675 Netherlands [84] 6.98
(women)
0.46 Denmark (women) [84] 4.18
0.893 Sweden (women) [84] 8.12
1.285 Norway (women) [84] 11.68

Coumestrol 4 mg/kg [129] 0.022a 0.01243 USA [130] 0.57
(LOAEL) Trace China [60] –
0.06259 Europe (men, mean) [84] 2.85
0.04348 Europe (women, [84] 1.98
mean)
Lignans 100 mg/kg [131] 0.540a 0.155 USA [132] 0.29
(LOAEL) nd Asia – nd
1.371 Europe (men, mean) [84] 2.54
1.207 Europe [84] 2.24
(women, mean)
(continued)
1189
1190
Table 7 (continued)
NOAEL in Human Estimated daily
toxicological MTDI mean intake Inferior weight limit
Phytoestrogens studies References (mg/kg) (mg/day) Country References for MDTI overcome
Zearalenone 0.2 mg/kg [133] 0.0001 0.000055 Europe mean (UB) [54] 0.01
0.000973 New Zealand (mean) [134] 0.19
0.000019 Canada (young men) [135] 0.19
0.000047 Canada (young [135] 0.19
children)
0.000098 Canada (adults) [135] 0.47
0.0017 USA [136] 17.00
0.00153 France (adults UB) [82] 15.30
0.001155 France (children [82] 11.55
UB)
The inferior weight limit for MDTI overcome is obtained dividing the estimated daily intake by MTDI
MTDI mean tolerable daily intake
a
extrapolated from the LOAEL; nd, not determined
effect of ENL is analyzed, the substance is assayed in the urine, but the correlation of
the urine dose to a dietary intake is far to be simple. Finally, when the dose is low, the
detection technique may not be reliable enough to get good results. Therefore,
looking at the exposure to lignans in Asia, it was impossible to find any published
data. The last column of the table gives a figure which materialized the inferior
weight limit for MTDI overcome. It is calculated by dividing the estimated daily
intake by the MTDI. As an example, for GEN in the USA, considering a mean daily
intake of 2.7 mg in the general population, it appears that all people, whose body
weight is lower than 24.55 kg, overcome the MTDI. Because this limit is considered
as safe, the concern about this situation can arise only from its chronicity and from
the degree of overtaking from the MTDI. The MTDI is a mean of the population
exposure, and some people can be exposed to much higher doses. The situation in
Japan could be considered as worrying since the calculation indicates that all people
under 454.55 kg overcome the MTDI, considering the daily intake estimation. In that
case, a fertility problem over generation should be investigated because it is seen in
experimentally exposed rats.
5.2.1 Reproduction in Animals
Isoflavones
Isoflavones were considered for many years as estrogenic endocrine disruptors and
studied as such by several authors [137–144]. These compounds can be present at
high doses in estrogenic pastures based on certain clover species or on alfalfa. In
sheep on these pastures, estrogenic isoflavones were shown to induce permanent
estrus, heavy vaginal secretion, uterus prolapse, early abortion, reduced prolificity in
reproductive ewes, and mammary fluid production in nulliparous ewes and castrated
males. Castrated rams also exhibited abnormalities in their reproductive tracts,
including enlargement of the prostate, the bulbourethral gland, and the membrane
of the vasa differentia. In ewes affected by clover diseases, clover isoflavones were
shown to delay LH secretion via a GnRH interaction [142]. This LH delay in
secretion, which led to progesterone secretion impairment, was considered to be
responsible for early abortion. More recently, a renewed interest emerged among
scientist for the soy and isoflavone effects on reproduction of domestic animals. This
is because it was recently figured out that isoflavone deleterious effects on cattle
could have been missed out because soy was progressively incorporated to cattle
food while genetic selection was undertook to improve production features including
reproduction. Therefore, the antagonistic effects of both processes could have partly
masked each other. As an example, it was shown that soy isoflavones can prevent in
cows as in rodent the response of the corpus luteum to GnRH [145]. This result
obtained, in vivo, is confirmed by data recently obtained ex vivo on Prim Holstein
cows [146]. In addition, clover isoflavones were shown to block the early proges-
terone synthesis in Holstein heifers fed with a Trifolium alexandrinum-rich roughage
after insemination [147]. This disruption was thought to be responsible for lesser
(P = 0.054) conception rate and the greater (P = 0.062) percentage of heifers
returning to estrus when compared to the control silage-fed heifers. As demonstrated
for COUM, GEN and DAID are able to increase the secretion of oxytocin by cow
corpus luteum [148]. This can induce early abortion in these females and reduce
global fertility and hence financial incomes to farmers relying either on veal or milk
production. Recently, it was shown that isoflavones intake through Egyptian clover
(Trifolium alexandrinum) as estrogenic roughage exerted a deleterious effect on
fertility. Comparing the estrogenic roughage to the control diet based on maize
silage, the % of Holstein heifers returning to estrus was 7.70 (1/13) under the control
diet, and it was 38.46 (5/13) when cows ate clover roughage. Still in cows, it was
shown that soy phytoestrogens increase prostaglandin secretion in cattle during
estrous cycle and early pregnancy [149]. The number of cows involved in the trial
was low. However, it appeared that soy-derived phytoestrogens and their metabolites
disrupt reproductive efficiency and uterus function by increasing the ratio of pros-
taglandin F2-alpha (PGF2-α) to prostaglandin E2 (PGE2). Therefore, phytoestrogens
led to high, nonphysiological production of luteolytic PGF2-α in cattle during the
estrous cycle and early pregnancy. The consequence of this increased PGF2-α
production is a higher failure rate of pregnancy. In addition, in vitro but at physio-
logical doses, GEN, DAID, and equol were shown to inhibit bovine adrenal 3-beta-
hydroxysteroid dehydrogenase directly involved into the synthesis of progesterone
[150]. This inhibition seems to be possible in vivo considering the active doses of
isoflavones in vivo. On top of the LH disruption, this direct effect may be a way by
which isoflavones reduce progesterone secretion in early pregnancy. This can cause
early abortion.
The phenomenon is generally enough to have led to endocrine disruption also
observed in lower vertebrates of economic importance like birds or fish. Although the
isoflavone effect in hens and laying hens does not seem to be deleterious, some long-
term effects were recorded in ducks [151]. Noteworthy, ducklings were significantly
smaller at hatching when exposed maternally to DAID supplementation. The differ-
ence in size compared to control animals disappeared at 4 weeks of age. It was
accompanied by changes in the secretion of metabolic hormones and expression of
growth-related genes. Although the negative effect of maternal DAID on embryonic
growth could be eliminated 4 weeks after hatching, the long-term effect of maternal
DAID on reproductive function was noted. Namely, it was an obvious downregulation
of hypothalamic GnRH mRNA expression observed in ducklings maternally exposed
to DAID. Fish in fish farms fed with a soy-containing diet can also exhibit altered
reproductive performances or at least endocrine-disrupting features [152–154]. These
effects were recently confirmed in goldfish considered as a model for other cyprinids
[155] and also in other species of commercial interest like catfish or sea bass [156,
157]. Both estrogenic and thyroid functions involved in smoltification were shown to
be impaired by soy phytoestrogens in the Atlantic salmon [158].
Coumestrol
Coumestrol was shown to exhibit deleterious effects on grazing animal reproduction
[159]. Coumestrol was also involved in the clover disease affecting ewe reproduc-
tion in New Zealand in the late 1940s, and these endocrine disruptions still attract
scientific interest [160]. According to Adams [161], cows are more sensitive to
COUM than to isoflavones most probably due to differences in metabolism when
compared to ewes. As an example, COUM was shown to increase the production of
oxytocin by cows decreasing the ratio of progesterone to oxytocin. This endocrine
modification induces a higher failure rate to pregnancy. Still in the same study,
COUM was shown to decrease PGE2 secretion. These endocrine-disrupting effects
can increase the risk of early abortion in this species [162] and therefore can affect
the % of females returning to estrus as well as the calving to calving interval. In
mare, COUM exerts endocrine-disrupting effects on chronic exposure leading to
ovulation impairment [163]. The authors conclude to a potential infertility syndrome
due to estrogenic silage (clover and alfalfa) in mares. The first reports of the
deleterious effects of COUM in rat were produced in 1970 [164]. Then the Whitten’s
group published several coherent data showing the estrogenic effects of dietary
COUM on the rat puberty and cycle [165], on uterus development [166], on the
embryo implantation [167], and on the pituitary and hypothalamus hormone disrup-
tions [167]. They also pointed out the remaining effects after the end of exposure
[168] as well as sexual behavior impairments [169]. Other studies showed repro-
ductive adverse effects on males’ semen production which could be explained by a
precocious estrogenic effect affecting the pituitary hormones and steroid secretion
[170]. According to Hendrich [128], there is no NOAEL for COUM, and the
LOAEL is 4 mg/kg/b.w./day.
Lignans
There is few data on the role of estrogenic enterolignans in domestic animals.
These lignans can be present in significant amount especially when linseeds are
used to modify the meat quality toward a greater proportion of omega-3 polyun-
saturated fatty acid (PUFAs). According to [171], ENL concentration is increased
in follicular fluid from cows fed with flaxseed-rich diet, although the serum levels
are not affected. These increased enterolignan concentrations are correlated with
increased concentrations of estradiol locally addressing the question of an effect of
ENL on follicular steroid biosynthesis. Still in cattle, [172] showed that ENL
deriving from a flaxseed-enriched diet or given alone in parallel of specific
mixtures of PUFAs can decrease PGE2 and PGF2α concentrations in endometrial,
stromal, and epithelial cells. This reduction was associated with lower mRNA
abundance of the PG synthase genes in stromal cells. An omega-3-enriched PUFA
mixture increased the effect of ENL compared to ENL alone and PUFA mixture
rich in omega-3 alone. The authors concluded that considering the known
luteolytic properties of PGF2α, a reduction in endometrial PGF2α secretion
would favor the establishment and maintenance of pregnancy. Enterolignans
were found in the early 1980s in human and cattle semen. In both cases, their
concentrations were shown to be 2.5 times higher than the corresponding plasmas
[173]. In rat they were found to alter pregnancy outcome and reproductive
development [131].
Resorcylic Acid Lactones

Zearalenone and ZOL are known as endocrine-disrupting agents from fungi since the
early 1960s [51]. In cattle, ZEN was shown to lower the conception rate of the
heifers [174]. In addition, ZEN contaminating sugar beet pellets was considered to
be responsible for a reduced embryo transfer efficiency from a dairy farm experienc-
ing low success rates of embryo transfer [175]. More recently, thanks to better
detection techniques, it was shown that low ZEN contamination (below the allowed
limit in Japan) was able to modify anti-Mullerian hormone levels in cow showing an
effect in the ovarian antral-follicle populations in cows [176]. In swine the delete-
rious effects of ZEN on reproduction have been studied scientifically as early as in
the 1970s [177]. It was later shown that concentrations of 25, 50, or 100 ppm of 95%
purified ZEN fed to groups of healthy, multiparous sows during pre-estrus or
throughout the gestation period (or both) produced multiple reproductive deficien-
cies [178]. These disorders included infertility, constant estrus, pseudopregnancy,
diminished fertility, reduced litter size, offspring’s small size, offspring
malformations, juvenile hyperestrogenism, and probably fetal resorption. Sows’
reproductive organs exhibited lesions, and their uterus, uterine duct, and cervix
showed marked epithelial changes, i.e., squamous metaplasia. Similar features
were found in the vagina and mammary glands. Transgenerational effects were
also documented in sows [179], and ZEN was shown to reduce the quantity of
healthy follicles, which probably lead to premature oocyte depletion in adulthood. In
rat toxicity studies were undertook using ZEN (in corn oil) at doses of 0, 1, 2, 4, or
8 mg/kg/b.w. in order to determine the NOAEL for in utero development [180]. The
fetal and pups’ body weights were decreased in a dose–response manner in both
sexes. Fetal development was delayed in all treated groups and linked to maternal
toxicity. Zearalenone delayed skeletal ossification at 4 and 8 mg/kg. Fetal anogenital
distance was increased in all treated groups, and fetal viability was decreased at
8 mg/kg. The weight of several maternal organs was modified at 4 and 8 mg/kg
doses. Gonadotrophins were only marginally affected. Prolactin was significantly
increased at 8 mg/kg. Estradiol dose dependently decreases at 2, 4, and 8 mg/kg.
Therefore, ZEN was maternally toxic and fetotoxic but not teratogenic. Based on the
dose-related maternal and fetal toxicity in all treated groups, the NOAEL for
reproductive and teratogenic effects was less than 1 mg/kg.
5.2.2 Reproduction in Human
Isoflavones
Fertility: Unfortunately, as regards the human fertility, no clear-cut data are currently
available about the effect of the modern exposure to estrogenic isoflavones. In 2014,
an epidemiologic study performed on Adventist populations [181] sorted into six
groups of isoflavone consumers showed that in young women (26 years old on
average), the highest exposure of >50 mg/day was associated with a significant
decrease in the number of birth. The same isoflavone intake was also correlated to an
increased risk in women over 50 years of never being pregnant. Fertility also
depends on the ability of men to produce efficient gametes. Noteworthy, sperm
production is a long process, starting from spermatogonial recruitment, continuing

by spermatogenesis in the testis, and ending by the capacitating steps into rete testis
and epididymis. In men the first step in the testis lasts for about 74 days [182]. Sperm
presence in the rete testis and epididymis can vary from 2 days up to 18 days. It is
only after sperm has undergone all steps that it can be ejaculated for fertilization and
studied for its ability to fecundate. Therefore, the whole process from production to
ejaculation can be considered as lasting about 3 months [182]. Consequently, when-
ever the effects of any endocrine-disrupting compound need to be studied on sperm
production in men, exposure to those compounds should exceed 3 months to avoid
any effect remaining undetected. As the capacitating process in the epididymis
requires adequate thermal conditions, environmental conditions can exert bias on
the final sperm quality. It is well-known that sperm production is influenced by both
androgens and estrogens and that environmental endocrine disruptors (androgens,
antiandrogens, estrogens, antiestrogens) can disturb sperm production in quality and
in quantity [183]. As certain interventional studies on sperm production and quality
like those of [184] or [185] were, unfortunately, performed over a short duration (less
than 50–60 days), those studies cannot be retained when arguing about the effects of
isoflavones on full spermatozoa production. Interindividual variation in sperm
production and release, over the study period, is also a confounding factor that is
not always taken into account and which can explain unexpected results [186]. In
addition, the exposing dose should be in the dietary range. The data given in [1]
indicate that, if a deleterious effect should be confirmed regarding sperm production
and fertility, this should be addressed at a demographic level with data from
industrial soy-eating countries and traditional soy-eating countries being treated
separately. Noteworthy, the two countries that are most exposed to isoflavones
because they have been eating industrial soy since the 1960s are South Korea and
Japan. IndexMundi in 2017 indicates that the total fertility rate being the average
number of children that would be born to a woman over her lifetime is 1.41 for Japan
and 1.25 for Korea. These figures are lower than those recorded in all other
developed countries with the exception of China territories. In France the total
fertility rate is 2.07, and it is 1.87, 1.44, and 1.89 in the USA, Germany, and the
UK, respectively. Noteworthy and still from the same sources, the contraceptive
prevalence rate for women between 15 and 49 in Japan is only 33% when it is 80% in
South Korea, 88% in France, 74.1% in the USA, 66% in Germany, and 84% in the
UK. In addition, in both Asian countries consuming industrial soy, the demography
regressed since 1983, i.e., one generation after soy-processing industrialization. This
is also concomitant with the massive introduction of plant protection products in
crop production all over the world. Moreover, in all studies that did show a
correlation between decreased sperm quality and isoflavone intake, it was never
possible to definitively exclude the interacting effects of other steroidal estrogens
(in the case of obese men [78]) or of other endocrine disruptors like plant protection
products [5, 79, 80]. In addition in [5], Mumford showed a positive interaction of
sperm abnormalities with BMI. This indicates that the deleterious action of iso-
flavones on sperm production is probably influenced by other endocrine disruptors,
present or not in the environment, and by endogenous estrogens.
Plausible consequences of early exposure: From a physiological point of view, a

significant exposure to estrogens of babies under 6 months of age comes with its
inevitable load of consequences [187]. During that period, differentiating processes
involving estrogens and androgens are still at work, and steroid receptors are present
in the different target tissues. These steroids, which were shown to potentiate
isoflavone actions, are well-known to be disturbed in animals [4, 188] or in vitro
[189] by isoflavones. The study from Strom et al. [190] showed that early isoflavone
exposure via soy-based infant formula led to an endocrine impact with ulterior
consequence on menstrual cycle impairment. It should be noted that cycle impair-
ments were noticed in the rats of the NTP study [4] that examined the effects of GEN
on the reproductive physiology of rats. However, the subjects enrolled in [190] were
too young to allow a fertility impact of early isoflavone exposure to be shown.
Noticeably, the sperm quality and production of men exposed to early soy were not
assessed either. The number of stillborns in the group of women fed with soy (3 out
of 74 births) was not significantly higher than the number of stillborns in the cow
milk-fed group (0 out of 149 births), because only a small number of women had
given birth at the time of the study. In fact Gilchrist and co-workers [191] discovered
that 4-month-old boys fed with soy-based infant formula (S-BIF) from 3 weeks of
age had a lower testis volume than that of their counterparts fed with breast milk
( p < 0.03). Their testes volume was lower but not significantly than that of boys fed
with cow’s milk. This can be explained by a decrease of LH and testosterone
neonatal production due to isoflavone intake. This is sustained by the study of
Sharpe et al. on paired twin marmosets fed with either breast milk or soy-based
infant formula prepared for human infants [192]. The soy-fed baby marmosets had
lower testosterone plasma levels than their twin brother fed with cow’s milk formula.
In marmosets the exposure was lower than in boys exclusively fed with S-BIF, and
no differences were seen in testes histology when the monkeys reached adulthood
[193]. Adgent and co-workers reported a masculinization of the play and toy
preferences in girls fed with S-BIF from 3 weeks to 6 months of age [194]. The
observation was done at 42 months of age and tended to vanish at 57 months
apparently due to social pressure. This observation can be linked to the masculini-
zation effects of estrogens on the sexually dimorphic area of the hypothalamus
during neonatal exposure (see [11] for further details). In addition and still according
to the same team, a subtle acceleration of menarche could be provoked by an early
exposure to estrogenic isoflavones via S-BIF [195]. These results although not fully
convincing could also be linked to data published by Kim and co-workers [196]
associating high serum concentrations of DAID (P = 0.0202), GEN (P = 0.0021),
and total isoflavones (P = 0.0009) with central precocious puberty in young South
Korean girls. As already mentioned, South Korea is in 2017 the country with the
world’s lowest total fertility rate [197]. More recently, it was shown that S-BIF can
epigenetically modify the promoter of the PRR5L exon 1 gene [198]. The role of this
gene is not yet fully understood, but it seems to regulate cell migration via an
interaction with mTORC2 and PKC-delta. The study essentially points out that
early exposure to isoflavones can have noticeable consequences later in life. The
reason why reproductive deleterious effects linked to early isoflavones intake were
not yet observed at population level is essentially because the appropriate studies
have not so far been performed [68]. A longitudinal study, which could be the best
approach, is unlikely to show significant effects, if it does not involve a huge number
of subjects, especially during childhood reproductive dormancy [199]. In [190]
Strom et al. were focused on too early a stage, before both adult men and women
really want to have children, and the study was unable, therefore, to help in
determining whether early soy feeding could induce a fertility problem. One conse-
quence is that, even if no alarming effects have been reported so far, it is not because
they do not exist but rather because they may be expressed long after the initial
causal exposure. Accordingly, the effect of this initial exposure may be masked by
other psychological, social, economic, or even chemical disrupting factors, inducing
large interindividual variability. If a reproductive effect really exists, it should be
seen in those populations which are largely exposed to isoflavones through industrial
modern soy food and not in those consuming traditional soy. Because it is now
recognized that there can be interactions with other anthropogenic endocrine
disruptors [2] like bisphenol A or glyphosate [200], the effects of isoflavone expo-
sure can be potentiated by other environmental chemicals. Careful monitoring,
which would allow these effects to be separated, should be undertaken in order to
discover what is really happening. The expected effects could induce lower sperm
production in men and an increased incidence of abnormal menstrual cycle, of early
or late abortion, and of larger uterine fibroids in women exposed during their infancy
[190, 201]. According to [198], a higher risk of uterine cancer may be observed. If
the percentage of infants fed with soy-based infant formula in a given population was
monitored for many years, then it could be possible to check whether that percentage
is maintained or increased in male adults consulting for fertility problems.
Physiological effects of actual exposure: Nutritional doses of modern soy iso-
flavones (45 mg/day) have estrogenic effects in women, which can lead to a modest
lengthening of the menstrual cycle [57, 77]. Noteworthy, when the diet is fully
monitored to avoid the presence of hidden isoflavones, the menstrual cycle of Amer-
ican premenopausal women can be significantly lengthened by 2 days [57]. This
lengthening is due to a retardation of the LH surge at mid-cycle. The same conse-
quence is also observed in ewes eating clover rich in isoflavones [142]. This phenom-
enon was also observed in Japanese women. In that case, the mean menstrual cycle
length is 30 days as opposed to 28 days in the West [77], with the mean modern
isoflavone intake in Japan and Korea currently being evaluated at between 45 and
60 mg [58]. In this context, Nagata and co-workers [77] showed that supplementing
the current Japanese diet with 50 mg of extra isoflavones from soy juice can lead to a
menstrual cycle length of 32 days. Although this lengthening may possibly be
protective against estrogen-dependent cancers, it can also reduce, however, the oppor-
tunity of ovulation in fertile women. This may well contribute to reducing the
reproductive efficiency of a given population. In addition, as shown in domestic
animals, it can be hypothesized that modern dietary intake of isoflavones can impair
embryo implantation and induce early abortion. In women so far, only luteal phase
deficiency was associated with isoflavone intake among healthy eumenorrheic
women. It is a modest correlation with an adjusted odds ratio (aOR) of 1.38 (95%
CI: 0.99, 1.92), P = 0.06, because the subpopulation studied was small. Because luteal
phase deficiency can lead to miscarriages and impaired fertility, it may be responsible
for a progressive demographic regression at the population level. Moreover, according
to [202], high soy intake for long durations can have even more significant effects. In
this study, the authors reported that high soy intake was responsible for abnormal
bleeding in women under norethisterone (contraceptive pills). This means that their
large soy intake was able to transform a contraceptive treatment based on progesterone
into an estro-progesterone form of contraception. A similar case involving a 32-year-
old woman who drank a liter of soy juice, 3 times a week, after her basketball training,
was also observed by our team. Her cycle impairment regressed when soy intake was
stopped. In [202], the authors also alerted against uterine fibroids and repeated
endometriosis features in the three cases they followed. They reported that all the
problems regressed when soy intake was stopped.
Vaginal and endometrial health: Both the vaginal mucosa and endometrium
develop under estradiol stimulation and therefore bear all the known estradiol
receptors: ERα, ERβ, GPER, as well as ERRs [203–205]. The estrogenic effects of
isoflavones were first shown on the uterotrophic test and even before on the uterus
maturation of the New Zealand ewes on clover pasture. Therefore, negating an effect
of estrogenic isoflavones on the endometrium makes no sense. However, in animals,
isoflavones effects were reported either in reproductive females or in female freshly
ovariectomized. In all cases, this means that estradiol receptors were available to
mediate an estrogenic effect of isoflavones. In humans, most of the studies were
performed in menopausal women. Most often their distance to menopause was
largely variable, and the availability of ERs in the vagina and endometrium, although
known to decrease after the arrest of ovarian function [205, 206], was not checked.
Therefore, no study showed a significant effect on the endometrium especially in
Western postmenopausal women [207], and very few studies were able to show an
effect on the vagina in postmenopausal women. Among them is that of Lima and
co-workers [208] which showed a significant estrogenic effect of a vaginal gel
containing isoflavones on vaginal dryness, on dyspareunia, and on maturing index
of vaginal cells. The last endpoint evolves similarly in the isoflavone group and in
the conjugated equine estrogen group [208]. Interestingly, a study performed in
Japanese students by Watanabe and co-workers [209] dealt with the supplementation
on normal Japanese diet with either 20 or 40 mg isoflavones. Considering the mean
urinary levels at baseline, the basal isoflavone exposure was most probably close to
25 mg/day. In their study, Watanabe found that extra isoflavone lengthen the
menstrual cycle by 2 days and lengthen menstruations and bleedings in a dose-
dependent manner. They observed that not all students react equally to the treatment.
On the blood samples collected from three volunteers, they did not show any
significant modification of steroid or gonadotrophin hormones. This suggests a direct
effect of isoflavones on the endometrial mucosa. To conclude, isoflavone effects on
the vagina and uterus of premenopausal women must not be rejected although they
require additional work for evidence. In postmenopausal women, a vaginal effect of
isoflavones may be achieved, while an endometrial effect has never been clearly
shown despite a large number on trials.
Coumestrol
Although there is a great deal of studies reporting the effect of COUM on rodent
genital tract, there is little data in women. Only in epidemiology studies COUM is
sometimes investigated. Each time the level of intake is usually low (a few μg per
day) and can be omitted in front of other phytoestrogens considering its poor
availability and its large distribution in human fluid. As an example, in the study
by Xia [80], showing a deleterious effect of phytoestrogens in Chinese men pre-
senting idiopathic infertility, the 19th percentile exhibiting the largest impairment
presented 619.36, 408.86, and 504.90 μg/g of excreted DAID, equol, and GEN,
respectively, while they excreted 4.04 μg/g of COUM. The total excreted isoflavones
was then 1533.12 μg/g. This is more than 350 times higher than the dose of COUM.
Lignans
Because not all humans can produce the estrogenic metabolites of dietary lignans,
only studies based on either blood or urine measurements can be taken into account
while considering the effects of enterolignans on reproduction. Hence, urinary levels
reflect an elimination process that reduces the blood bioavailability. Therefore,
whenever possible plasma or serum levels should be preferred. Considering these
restrictions, the study by Tang and co-workers [210] indicated that when sorted in
four quartiles (i.e., 16.54, 85.53, 325.91, 891.7 μg/g of urine) in pregnant women at
delivery, birth outcomes were significantly modified. The higher the enterolignans’
urinary levels, the lower the gestation length. There is also a tendency toward lower
birth weight with END. The decrease in gestation length can be explained by a
precocious induction of oxytocin receptor production increasing the action of oxy-
tocin at the time of labor delivery. This has been shown for estrogens, not yet for
enterolignans [211]. The smaller birth weight can be linked to a vasoconstriction of
placental vessels due to estrogens which are not harvested by the alpha-fetoprotein
because their affinity for this protein is lower than that for the estradiol receptor
[212]. In addition, it was also shown in American men that urinary END and ENL
were associated with subtle deterioration of spermatozoa quality parameters in a
dose–response manner [5]. However, it should be kept in mind that lignans are
biomarkers associated with grain, vegetable, and fruit consumption. All these items
can be contaminated by plant protection products, and this effect may reflect at least
a synergistic effect between several endocrine disruptors. Contrarily to isoflavones,
enterolignans are associated with better reproductive incomes in humans [213]. Like-
wise, Mumford and co-workers showed an association of urinary ENL and shorter
time to pregnancy. This may be due to the specific effect of enterolignans on the
ERα [45].
Zearalenone
There is little convincing information about the effects of ZEN in humans because
such an effect could only be recorded on long-lasting exposure if an effect has to be
correlated with a serum or urine level. Nevertheless, ZEN was pointed out in the
occurrence of precocious puberty, in Italy [214] and Puerto Rico [215], although
levels were not actually assessed in any biological fluid. In Hungary [216], an
increased incidence of early thelarche was reported in patients with serum ZEN
levels of 18.9–103 μg/mL. In 2010 it was published that in Viareggio (Tuscany), the
incidence of precocious puberty was 22 to 29 times higher than in the neighboring
areas [217]. Among 63 cases, 6 of them had high serum ZEN (933.7 200.3 pg/mL)
and α-ZOL (106.5 1.9 pg/mL). But only 5 of 36 patients with early thelarche
presented detectable levels of mycoestrogens. In 2003, ZEN was associated with
“endemic enlargement disease” in China [218]. All grains analyzed were contami-
nated by mold, and 34% exhibited Fusarium species and COUM. More recently it
was found that mycoestrogens were detectable in a large proportion of the urine of
the 163 girls, aged 9 and 10 years, participating in the Jersey Girl Study and enrolled
in the survey by Bandera et al. [219]. The subjects presenting the highest values were
also being significantly of shorter stature and less likely to have reached the onset of
breast development. This may sign an estrogenic effect on the pituitary gland with
resulting anti-FSH effect. Such effects were reported in rodents [220] and more
recently in pigs [221].
6 Questions
6.1 Estrogen-Dependent Cancers
6.1.1 Isoflavones
There has been a long-lasting controversy about the positive or negative effect of
isoflavones on breast, endometrial, or prostate cancers all considered being more or
less estrogen-dependent. Their incidence is also lower in Asia.
Endometrium
As far as endometrial cancers are concerned, in 2015, the EFSA opinion [222]
indicated that no definitive opinion could be given on any deleterious effects of
isoflavones on uterine cancer. However, the study only concerns healthy peri- and
postmenopausal women, because it was based on the scientific literature in which
volunteers are recruited following specific inclusion criteria, and such criteria do not,
therefore, exactly reflect the global population. No case studies were performed to
see an effect on endometrial cancer, and therefore neither the quality of the popula-
tion nor its number was designed to demonstrate an effect. According to [222], there
are very few studies triggering specifically perimenopausal women, and the number
of studies was insufficient to conclude on this specific population. No conclusion
could be rigorously drawn out on perimenopausal women because they cannot be
assimilated to postmenopausal women. Noteworthy, their production of endogenous
estrogens was still active, and estradiol receptors were still present at the target
tissues. On the opposite, there is a progressive decrease in endometrium estradiol-
receptor bioavailability in postmenopausal women [206]. Therefore, a lack of action
of isoflavones in the uterus of these women is expected, because the estrogenic
effects of isoflavones require an interaction with the estradiol receptors. On top of
other things, this tissue bears both alpha and beta subtypes of ERs [206], and
isoflavones affinity is greater for the beta subtype [223, 224]. As the activation of the
ER-beta was shown to negate the proliferative action of the ERα [225], via its AF-1
function [226], normal soy intake is not likely to affect the rate of endometrial cancer
of healthy postmenopausal women.
Prostate
For prostate cancer, there is also a controversy. If the incidence of prostate cancer is
lower in Asian population, the postmortem tumor detection indicates that the
frequency of prostate cancer is equivalent between Asia and the West [227]. To
add to the controversy, Nakamura and co-workers [228] recently developed a
patient-derived prostate cancer xenograft model. They grafted clinical prostatectomy
samples into nude athymic mice fed with 0 or 2 or 10 mg/kg genistein. For these
doses, they showed dose-dependent increases in lymph nodes and secondary organ
metastases (liver, lungs). They also showed the aggregation of invasive malignant
cells in the secondary organs of the genistein-treated groups and not in the control.
Their data suggest that the effect of genistein may depend on the kind of prostate
tumor considered. A recent meta-analysis [229], performed on 21 case-control and
2 cohort studies, found that GEN and DAID may, in some cases, be associated with
decreased risk of prostate cancer. However, when studies were sorted out according
to the way isoflavone intakes were assessed, it appeared that the risk was not reduced
when isoflavones were assayed in the serum or urine, although it was reduced when
GEN and DAID exposure was assessed via food questionnaires. Because this last
method is less accurate than the assay in biological fluid, the validity of these results
remains questionable.
Breast
Although things are still unclear for endometrial or prostate cancers, there are now
enough data to conclude that both positive and negative effects can occur consider-
ing isoflavone effects on estrogen-dependent breast cancer. Sufficient data now exist
to consider that isoflavone through soy intake may be preventive against the
occurrence of breast cancer [230–232] playing its role during the initial phase of
cancer development [1]. However, there are also many data indicating that estro-
genic isoflavones exert a proliferative effect on breast estrogen-dependent cancer cell
lines. These data now exist in vitro [233], in vivo in animals [234], and in
women [235].
The mechanism by which cancer prevention occurs is not yet clear and may be
related to different mechanisms including early cell differentiation [236, 237], onco-
suppressor expression [238], or pro-oncogene depletion [239]. Unfortunately, not all
the protective mechanisms were so far recorded in vivo, using plausible dietary
doses. Nevertheless, this preventive effect of isoflavones is in accordance with the
demonstrations made by Lamartiniere on rats exposed to dimethylbenz(a)anthracene
(DMBA) in which the preventive effect of GEN appears during the initial phase
[240]. This protective effect is also sustained by the study of the National Toxicology
Program [241] examining the carcinogenic effect of GEN in rats. Hence, they found
that females from the first generation exposed to 5 and 100 ppm of GEN from
conception exhibited lower incidences of combined mammary adenoma and adeno-

carcinoma than the control or than the 500 ppm group. The differences were not
statistically significant, but they may support the idea that a low exposure from
conception would protect against mammary tumor incidence. Of course, reducing
the occurrence of cancer cells via inhibiting their occurrence by any pathway during
the initial step would result in a decreased incidence of cancer at the population level.
However, most established breast cancers rely on the activation of the alpha
estradiol receptor subtype cross talking with the IGF receptor [242, 243]. This
means that 17β-E2 and other estrogens act as growth factors of the tumors [40,
244, 245]. At least three studies showed an estrogenic effect of soy isoflavones at
modern dietary doses (45–60 mg/day) on the mammary gland of premenopausal
women [74–76]. The three studies were undertaken for short durations and showed a
positive impact of the isoflavone dietary intake on estrogenic biomarkers (pS2,
nipple aspirate apolipoprotein D, progesterone receptor). In premenopausal
women, an interaction of isoflavones with endogenous 17β-E2 could not be
excluded. Therefore, this effect cannot be extrapolated to postmenopausal women.
This does not, however, negate the estrogenic effect of 45–60 mg of isoflavones per
day in premenopausal women. Such active doses in premenopausal women are
sustained by other physiological data concerning the menstrual cycle length dura-
tion, as explained before. Unfortunately, when they can exert their estrogenic effect,
isoflavones increase cell proliferation speed on already established estrogen-depen-
dent cancer cells [233]. This was also shown in vivo in implanted nude mice
[234]. This is also sustained by the study of Shike et al. [235] in women with a
declared breast cancer. Examining the proliferative effect of soy isoflavones in
women diagnosed with breast cancer is both very difficult and ethically disputable,
which would explain why only one study [235] has been published so far. The study
is not fully conclusive because of a short treatment duration (between 7 and 30 days)
and a low compliance with the treatment. Therefore, the biomarkers followed
probably reflect a large interindividual response to a large interindividual variation
in exposure. Therefore, in [235], the authors relied on isoflavone plasma levels to
sort the treated subjects and then went on to perform genomic analysis on prolifer-
ation markers. The results they obtained in vivo with plasma forms (i.e., conjugated
forms) of isoflavones are consistent with the in vitro data obtained by many other
authors, who showed an estrogenic effect of isoflavones on breast cancer cells (i.e.,
proliferative effect) when used in the μM range (0.2 to 5) [246]. Shike’s study
indicates that both in vivo and in humans the circulating forms of isoflavones can,
as already shown, have estrogenic effects [74–76]. Hence, at modern dietary doses,
they can act as growth factors for estrogen-dependent tumors. Although the studies
in nude mice implanted with human breast cancer cells cannot help in determining an
active dose, the NTP study [241], which showed a significant increase in adenocar-
cinomas in the mammary and pituitary gland of rats exposed to 500 ppm GEN from
conception to sampling, is particularly important. This classical toxicology study
was designed to define the mean tolerable daily intake in humans. In that case, the
LOAEL is 100 ppm (5 mg/kg/day for rats), and the NOAEL is 5 ppm in the diet
(1.2 mg/kg/day for rats).
Finally, some breast cancer cells can be triple negative for ER, PR, and Her2/neu.
They are considered to be resistant to hormone therapy based on antiestrogens or on
anti-aromatase and therefore to have particularly negative prognosis [247]. It was
shown in some occasions that, in the absence of ER, breast cancer cells such as
MDA-MB-231 did not proliferate under isoflavone treatments [248]. This triple-
negative cell line only bears trace amounts of GPER [249]. However, some triple-
negative cancer cell lines such as HCC1806 were shown to have a large proportion
of GPER in their membranes [249]. Because of that although they were first
considered as estrogen independent, they can proliferate under estrogen stimulation
[250]. GPER can mediate rapid 17β-E2-induced non-genomic signaling pathways. In
addition, by the effect of transactivation of epidermal growth factor (EGF) receptors,
GPER induces mobilization of intracellular calcium (Ca2+) stores and activation of
mitogen-activated protein kinase and PI3K signaling pathways [247]. Furthermore,
when present in triple-negative cancer cells, GPER seems to be with poor clinical
outcome. It has been shown to upregulate cyclins and BCL-2 genes favoring cell
proliferation and survival. Therefore, GPER possibly plays an important role in the
carcinogenesis process. The GPER-induced proliferation can also be blocked in vivo
by inactivating GPER [251] with siRNA, using GPER antagonist like G-15 [252] or
even better estriol [251], opening a new way for breast cancer therapy. In Egypt,
where breast cancers represent 17.5% of all malignant tumors, it was observed that
these mammary cancers were more aggressive than that encountered in the West
[247]. In this country, it was shown by immunostaining that 65% of archival
formalin-fixed paraffin-embedded cases of invasive ductal carcinoma were GPER
positive. GPER as a membrane receptor of the rhodopsin-like family is activated by
low doses of many xenoestrogens including bisphenol A (BPA) [253]. As mentioned
earlier, GEN and DAID are ligands of GPER with affinity 2 to 3 times higher than
that of BPA. The IC50 for GEN is 133 nM, which is only ten times higher than that of
estradiol [253], and GPER pathway activation is achieved using 0.2 μM. This dose
can be obtained in human blood with dietary soy intake. Equally, DAID is able to
induce GPER pathways at doses as low as 0.1 μM [254]. Therefore, the effects of
phytoestrogens on breast cancer should take into account the GPER pathway as
another target for proliferation induction. Lastly, data on the effect of isoflavones on
breast cancer cell lines through the estrogen-related receptors (ERR) α, β, or γ are
presently too scattered to conclude on a clinical effect. When tested, the doses of
isoflavones required for the ERR pathway activation do not fit with a dietary
isoflavone intake [255].
6.1.2 Coumestrol
Because this substance is only anecdotally present in human food at least in Western
developed countries, its effects on estrogen-dependent cancers are only scarcely
examined. There is no interventional study that is strictly controlled, and there are
a few observational studies mentioning COUM as a parameter which have been
taken into account because it was above the assay detection limit in biological
samples. In 2006, Hedelin and co-workers showed that COUM together with a
precise Snp present in the promoter of the ERβ subtype was correlated with a strong
significance to a decreased risk of prostate cancer [256]. However, this correlation

should be taken with caution since the COUM urine levels measured are close to the
detection limit.
6.1.3 Lignans
Two major problems linked to the exposure evaluation of the estrogenic
enterolignans can explain the paucity of clear data gathered on enterolignans and
estrogen-dependent diseases. As already mentioned, not all sources of lignans are yet
characterized in food [257], and secondly not all human being can efficiently
transform lignan precursors into estrogenic enterolignans [258].
Prostate
It is in the late 1980s that lignans were correlated to reduced risk of prostate cancer
[259]. Despite this early hope, in 2010, Saarinen and co-workers made a point on
population and intervention studies dealing with enterolignans and prostate cancer
and found no clear correlations at the population level [260]. In population studies
even when ENL was followed in plasma and/or urine, a clear association with
prostate cancer incidence, progression, or risk could be pointed out. However,
three studies dealing with the administration of flaxseed in men with a diagnosed
prostate cancer did show a decreased serum total PSA and proliferation rate of
benign epithelium and a significant decrease in total testosterone and free androgen
indices. Among men with Gleason score below 6, they reported decreased tumor
proliferation index and increased tumor apoptotic scores when comparing the
flaxseed group to historic controls. Finally, they also mentioned a significantly
reduced tumor proliferation rates with flaxseed supplemented diets. Their conclusion
was to say that enterolignans may have a protective effect on prostate cancer but that
the normal dietary intake is currently insufficient to induce such protective effect.
More recently the study by Azrad and co-workers [261] came to confirm that
prostate cancer preoperating treatment with flaxseed supplement was associated
with the decrease of several tumor biomarkers. Namely, there was a significant
decrease in NFκB, Ki67, and VEGF. The authors conclude that flaxseed supplemen-
tation inhibits cancer cell growth and possibly reduces tumor angiogenesis in
patients with prostate cancer.
Breast
Enterolignans have a greater affinity for the ERα receptor; however, ENL which is
currently the most concentrated in human plasma was shown to better recruit the
AF-2 transactivation factors and therefore to reduce the proliferative effects of
compounds activating gene transcription through the AF-1 domain [45]. This
in vitro mechanism was further confirmed in vivo on nude mice transplanted with
MFC-7 cells [262]. In this study, ENL and END were tested alone and together with
GEN. They were able to prevent the proliferation induced by GEN. Foods
containing the precursors of enterolignans were also tested in the nude mice model
[263]. The study showed that secoisolariciresinol diglucoside (SDG), the most
classical precursor of enterolignans in the scientific literature, was less efficient than
sesamin in protecting against breast tumor proliferation. Sesamin was converted
in vivo by the nude mice in unknown compound proportion that was able to increase
significantly apoptosis of the cells. If the mice metabolism is involved in this effect,
which is most plausible, then the result cannot be translated directly to human.
Nevertheless, in some cases, studies showed that a higher intake of lignans was
associated with lower risk of developing cancer. In several occasions, the receptor
status of cancers was mentioned [264, 265]. The discrepancy between the studies can
be explained by at least two factors: (1) not all women can produce enterolignans
from their lignan precursors, and if no plasma or urine measurements are available,
this induces confusion; (2) not all the sources of lignans are yet characterized, and
this reduces the power of the analyses. To illustrate the first point, the study by Buck
and co-workers [265] showed a better reduction in the breast cancer risk when data
were collected based on an ENL measurement rather than on a lignan dietary intake.
When the lignan intake was taken globally, the ER-negative tumors seemed to be
more susceptible to a protective effect. Therefore, and because lignan intake was not
systematically associated with estrogenic enterolignan production, the action may
not be an estrogenic or antiestrogenic effect. Regarding an effect of enterolignans
through the GPER pathway, there are too few such data to hypothesize any effect.
However, several epidemiological studies gathered in the meta-analysis [266]
showed overall that a decreased risk of death by breast cancer could be associated
with serum ENL. This study performed on 2,182 patients divided the population into
4 quartiles based on their serum ENL levels. In each case, the highest quartile
corresponding to serum levels over 70 nM was associated with lower risks. The
endpoints which were monitored were all-cause mortality, breast cancer-specific
mortality, distant disease-free survival, and risk of recurrence [266]. In this study
and thanks to a large number of subjects, it was possible to see that enterolignans
could have a positive effect per se, distinctive of a healthy lifestyle pattern. Finally,
as for isoflavone, a preventive effect at the initial phase of breast cancer progression
is plausible since it was shown that ENL at a dose of 10 mg/kg/day was able to
decrease the occurrence and size of chemo-induced mammary tumors using DMBA
in ovariectomized rat [267]. Because DMBA is able to transform a healthy cell in
cancer cells, this effect of ENL triggers the initial phase of mammary cancer
progression.
6.2 Other Side Effects
6.2.1 Thyroid
Links were made between soy consumption and alterations of fragile thyroid func-
tion. In the 1960s when the first soy-based infant formulas were commercialized,
hypothyroidism goiters were observed [268] in infants and led to iodine supplemen-
tation in these formulas [269]. However, nowadays hypothyroid infants can still
develop a goiter under soy-based infant formula, and their thyroid function is still
difficult to manage under these soy-based formulas [270–272]. Generally speaking,
until now, whenever isoflavone effects were reported on hypothyroidism, it was

always in persons with preexisting thyroid impairments [271, 273–275]. In parallel
to these data is the case of a woman who was treated with Levothyrox for hypothy-
roidism. It showed that the intake of soy-based food supplements interacts with the
drug absorption [274]. As a result, the doses required to balance her thyroid function
had to be increased when soy-based food supplement was taken simultaneously with
Levothyrox treatment. Consequently, in Europe, these medications are generally
advised avoiding soy consumption. The mechanism of action is not fully character-
ized, but GEN was found to bind to thyroid hormone receptors [276] and to compete
with triiodothyronine (T3) on its receptor at concentrations of 1 μM which are dietary
relevant. In addition, in the presence of iodide ions, genistein and daidzein blocked
TPO-catalyzed tyrosine iodination by acting as alternate substrates in vivo [277]. This
mechanism would explain the greater need of iodine for correct thyroid function in
the presence of soy. Many other studies, however, showed no adverse effects from
soy or isoflavones on thyroid function of healthy subjects [278–282]. In addition, in
Japan, isoflavones are consumed at the highest rate on earth; until recently [283] no
adverse effects were consistently reported. The exception [283] is the case of a
72-year-old woman with preexisting lymphocytic thyroiditis who was admitted to
the hospital with acute hypothyroidism. It was due to the consumption of a health
drink containing soy. Symptoms progressively disappeared after soy-based health
drink arrest. However, the traditional Japanese diet is based on a large variety of
seafoods which are rich in iodine. They may usually compensate for the effect of soy
and/or isoflavones on an alteration of the thyroid function. Finally, for many years, it
was unclear if any soy component or specifically isoflavones were responsible for the
hypothyroid effect of soy. The study by Sathyapalan and co-workers [284] finally
answered the question. When isoflavones were removed or added to soy, and fed to
volunteers with preexisting mild hypothyroidism, the isoflavone supplementation
clearly aggravated the hypothyroid symptoms. Taking all these data together, it
seems that the adverse effects of soy isoflavones may occur only in persons with
previous hypothyroidism. In that case, a balance in the thyroid function is more
difficult to achieve medically.
6.2.2 Autoimmune Diseases

Autoimmune diseases occur when the immune system attacks and destroys the
organs and tissues of its own host. Several autoimmune diseases occur with a higher
frequency in women than in men [285]. Although links with several factors
expressed by the X chromosomes have been related to the occurrence of autoim-
mune diseases [286], the role played by female sex steroids is currently under
investigation. Hence, the incidence of autoimmune diseases is generally higher in
premenopausal women than in children (girls) and decreases after menopause [287].
At the cell level, 17β-E2 was shown to induce the expression of many different
factors playing a role in the activation of autoantibodies. As an example, 17β-E2 at
physiological doses increases lymphocyte B and plasma dendritic cell differentia-
tions in vitro [288, 289]. This process is inhibited by the pure antiestrogens ICI
182 780 or by tamoxifen. Plasma dendritic cell activation and IFNα production are
enhance in vitro and in vivo by 17β-E2 and reduced when the ERα receptor is
reduced or blocked [289]. Autoimmune processes seem to be reduced during
pregnancy, while progesterone is present in the body at high concentrations [290].
Testosterone also seems to exert a preventive effect [291]. For systemic lupus
erythematosus (SLE), the sex ratio distortion is one of the highest with currently
nine women and one man being hit by the disease in France [292]. In Japan, the ratio
seems to be more in favor to men with eight women and two men being sick [287]. In
addition, the disease evolves by flares which occurrence is still difficult to prevent
because of unknown causes. Among other causes, environmental factors have been
suggested to be involved [293, 294]. Dietary phytoestrogens, because of their
estrogenic potencies and their mostly unknown sources of exposure in modern
diet, are good candidates for further studies. In a transgenic mouse model of SLE,
the MRL/Mp-lpr/lpr mouse GEN and DAID at nutritional doses were shown to
aggravate the SLE-induced nephropathy and to induce earlier mortality when com-
pared to a diet devoid of soy [295]. Noteworthy, at pharmacological doses, iso-
flavones seem to have the opposite effect [296]. Fort and co-workers [297] examined
the incidence of autoimmune diseases in children fed with soy-based infant formula
compared to their counterpart fed with other formulas in their infancy. They found
out that the frequency of feedings with soy-based milk formulas in early life was
significantly higher in children with autoimmune thyroid diseases (prevalence 31%)
as compared with their siblings (prevalence 12% p < 0.01) and healthy nonrelated
control children (prevalence 13%, p < 0.02). More recently working in rodent, Tran
and co-workers [298] showed that GEN, DAID, or glycitein and a soy extract
suppressed iodine uptake and stimulated the production of autoimmunogen in rat
thyrocytes in vitro. However, cells were from rat, and the efficient doses were higher
than 1 μM. Therefore, the transposition to the clinical situation should be taken with
caution. Finally, Portman and co-workers [299] recently showed an association
between soy and the incidence of Kawasaki disease (KD) in children. A significant
increased KD risk was observed in children for total isoflavone intake (OR, 2.33; CI
95%, 1.37–3.96) and for genistein intake (OR, 2.46; CI 95%, 1.46–4.16), when
comparing high-soy consumers vs. non-consumers. KD risk was also significantly
increased in Asian-American children with the highest isoflavone consumption.
Hence, total isoflavones were highly significantly correlated to an increased risk
(OR, 7.29; CI 95%, 1.73–30.75), and this was also the case for genistein (OR, 8.33;
CI 95%, 1.92–36.24) when compared to white children. The authors concluded that
childhood dietary isoflavone consumption, but not maternal isoflavone intake during
pregnancy and nursing, strongly relates to KD risk in an ethnically diverse US
population. To conclude, the association of autoimmune disease with estrogens is
clear, and progesterone and testosterone are most probably protective.
Phytoestrogens being estrogenic, antiandrogenic [300], and able to reduce proges-
terone levels at dietary doses [301] can be good environmental candidate to solve the
reason of unexplained autoimmune disease flares. However, additional studies are
needed to ascertain this effect and therefore to go toward disease prevention.
7 Conclusion
Natural potent estrogens are ubiquitous in our environment. Resorcylic acid lactones
and COUM, because of their strong known effects on rodent reproductive physiol-
ogy, have been adequately monitored so far. Hence, the human exposure, except in
specific circumstances, is not a cause of concern. Lignans, although leading to the
eventual production of estrogenic compounds by the gut bacteria, are far less
studied, and the exposure to the weak estrogen enterolignans is far from being
correctly assessed. A large interindividual response is expected from the exposure
to dietary lignans. However, as far as we can approach them with our current tools,
these compounds act as SERMs, and their effects appear to be more beneficial than
adverse. Their effects on reproduction appear to be positive, and they were shown to
reduce the proliferation on estrogen-dependent breast cancer cell lines. Their expo-
sure being associated with grain, fruit, and vegetable intakes does not seem to
suppress the benefit of a diet rich in these foodstuffs. Inducing more concerns is
the case of isoflavones. Their estrogenic effects in vitro range between ENL on one
hand and COUM and ZEN on the other hand. Their bioavailability is the highest of
all compounds listed here. Many effects, them being beneficial or having adverse
effects, have been reported so far for these compounds. However, based on the
assumption that they had always been part and parcel of the human diet, they were,
until now, essentially considered as inactive. This discrepancy vanishes if it is
admitted that the isoflavone exposure is modern and essentially due to changes in
the cooking process of soy. Then the compounds can be considered for what they
are, i.e., estrogenic compounds, possibly exerting endocrine-disrupting effects in
synergy with other anthropoid compounds recently liberated in the environment. The
actual data tend to indicate that their most pronounced deleterious effect is observed
on reproduction. Their deleterious effects on already established estrogen-dependent
cancers seem to occur at higher doses based on toxicology studies. These effects are
counterbalanced by a preventive effect probably due to a favorable interaction at the
initial step of breast cancer progression. More mechanistic data are required to
ascertain this mechanism. Therefore, although more research is still required to
take the best part of these compounds, the question of their endocrine-disrupting
effect should be taken into consideration. Because the history shows that isoflavone
can be reduced easily in food, this should be done for both human and domestic
animals to reduce the exposure to endocrine disruptors and improve farmers’
incomes.
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Jaboticaba: Chemistry and Bioactivity
41
Natália Crialeison Balbo Vall Ribeiro, Andressa Mara Baseggio, and
Vicki Schlegel
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226
2 Distribution and Botanical Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1227
3 Nutritional Composition of Jaboticaba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1228
4 Health Benefits of Jaboticaba or Its Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230
4.1 Antioxidative Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230
4.2 Immune System Health and Anti-inflammatory Properties . . . . . . . . . . . . . . . . . . . . . . . . . 1234
4.3 Cancer Prevention and Mitigation Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1236
4.4 Atherosclerosis Prevention and Mitigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1238
4.5 Gastrointestinal Health: Gut Microbiota Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
4.6 Metabolic Syndrome: Obesity and Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1242
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244
Abstract
The jaboticaba is a fruit native to Brazil that grows in the wild throughout the
country but is also cultivated on a low-scale basis by small farmers. Research is
currently being reported that jaboticaba is a rich source of bioactive compounds,
particularly the phenolic compounds. For example, high levels of the anthocya-
nins, cyanidin-3-O-glucoside and delphinidin-3-O-glucoside, and ellagitannins/
ellagic acid are the predominant phenols present in jaboticaba and reside primar-
N. C. B. V. Ribeiro · A. M. Baseggio
Department of Food Science, Faculty of Food Engineering, University of Campinas,
Campinas, Brazil
V. Schlegel (*)
Department of Food Science and Technology, University of Nebraska Lincoln, Lincoln, NE, USA
# This is a U.S. government work and not under copyright protection in the U.S.; 1225
foreign copyright protection may apply 2019
1226 N. C. B. V. Ribeiro et al.
ily in the peel and seeds of the fruit. These substances have been linked to
multiple health benefits, including the prevention and/or mitigation of oxidation,
inflammation, atherosclerosis risk factors, cancer, and conditions involved with
metabolic syndrome. The fruit, or substances therein, has also been shown to
enhance the immune system and gut microbiome. Therefore, the objective of this
manuscript is to review the health-promoting properties exerted by jaboticaba or
the compounds that reside in the fruit demonstrated throughout the literature, with
an emphasis on the phenols.
Keywords
Jaboticaba · Bioactive agents · Phenols · Flavonoids · Anthocyanins · Ellagic
acid · Gallic acid · Health benefits · Dietary bioactivity · Ellagitannins
ATP Adenine triphosphate (ATP)
CD40 Cluster of differentiation 40 protein
GI Gastrointestinal
H2O2 Hydrogen peroxide
hBD- Human beta defensin 2
HIV Human immunodeficiency virus
iNOS Inducible nitric oxide synthase
LOX-1 Lectin-like oxidized low-density lipoprotein receptor
MetS Metabolic syndrome
MMP Matrix metalloproteinase
NO Nitric oxide
RNS Reactive nitrogen species
SLP1 Secretory leukocyte protease inhibitor
w/w Weight/weight
1 Introduction
The jaboticaba (Plinia sp.) is an edible tropical fruit native to Brazil that grows
wildly throughout the country with the Minas Gerais and São Paulo states reporting
the highest yields. This tiny black berry belongs to the family, Myrtaceae, which is
41 Jaboticaba: Chemistry and Bioactivity 1227
the same family as the guava tree. The fruit has a thin, smooth skin and a moisty
white, translucent squash-like center that is sweet and slightly acidic [1]. Chemical
characterization of the jaboticaba, particularly the peel, has revealed the presence of
multiple health-promoting compounds, including phenolic compounds (e.g., flavo-
noids and phenolic acids) and insoluble and soluble fiber (e.g., pectin) [2]. More
specifically, ellagic acid and its derivatives are important phenolic molecules present
in jaboticaba due to their powerful antioxidative properties [3]. Other flavonoid
compounds predominant in the jaboticaba peel are the anthocyanins, cyanidin-3-O-
glucoside and delphinidin-3-O-glucoside. Anthocyanins are a class of flavonoid
pigments that are also powerful antioxidants but have other multiple cellular stresses
and disease-preventing properties [4–6]. Finally, pectin is a heteropolysaccharide
composed of galacturonic acid, arabinose, galactose, and rhamnose, which contrib-
utes to its high capacity for forming gel, thereby aiding in the digestive health.
A decrease in macronutrient absorption, specially fats and sugars, potentially leading
to glycaemia, or improving the plasma lipid profile, is yet another benefit of pectin
intake [7]. As a climacteric fruit, the maturation process continues postharvest,
resulting in the activation of many metabolic pathways that degrade or modify
many of the previously cited beneficial compounds with the anthocyanins being
the most affected [2, 8].
2 Distribution and Botanical Characterization
The jaboticaba is cultivated by small farmers on a low-scale basis but is ubiqui-

tously distributed throughout Brazil, especially in the Atlantic Forest biome [2, 9].
The jaboticaba tree has novel characteristics that include white blossoms located on
both the trunk and branches that develop into the berry [10] (Fig. 1). In addition, the
fruit matures rapidly (between 40 and 60 days); this aspect is related to the scientific
name of the first jaboticaba species discovered in Brazil (Plinia cauliflora). The
jaboticaba has a spherical shape that is 2.0–3.5 cm in diameter and contains one to
four seeds. The pulp is mainly white and sweet, while the peel is dark purple when
mature [10, 11] (Fig. 1). The color of the jaboticaba peel is due to the presence of
high levels of anthocyanin pigments, primarily cyanidin 3-O-glucoside and
delphinidin-3-O-glucoside. Two other jaboticaba species are also indigenous to
the Brazilian territory, i.e., Plinia cauliflora (“Jabuticaba assú,” in popular lan-
guage) and Plinia jaboticaba (“Jabuticaba sabará,” in popular language). Differ-
ences between species are evident in the tree characteristics. The Plinia cauliflora is
a small tree (3–6 m in height) with long leaves (2–6 cm in length), while the Plinia
jaboticaba is a taller tree (6–9 m in height) with smaller leaves (2–4 cm in length)
[11]. The Plinia jaboticaba is the most widely cultivated and recognized species
that grows mainly in the states of Minas Gerais and São Paulo [2]. Jaboticaba
postharvest shelf life is relatively short due to its high moisture levels along with a
fragile skin requiring mild industrial processes. Jaboticaba perishability also affects
the functional features of the berry.
Fig. 1 Jaboticaba tree

showing berries on trunk and
branches [10]
3 Nutritional Composition of Jaboticaba
As stated previously, several studies have reported that the jaboticaba is a rich source
of chemically diverse nutrients. For example, carbohydrates are the most abundant
macronutrient followed by proteins and lipids [9, 12–17] (Table 1). High concen-
trations of fructose are located in the pulp [13], while higher fiber levels (soluble and
insoluble) are present in the peel [14]. A wide variety of minerals are also present
in jaboticaba with the predominant being potassium, magnesium, and phosphorus
[13, 15] (Table 1). Moreover, whole fresh jaboticaba contain high levels of vitamin C
[16] (Table 1). It must be emphasized that non-nutrients (as bioactive compounds) of
the jaboticaba are currently receiving intensive interest, especially those that reside
in the peel. These compounds are the secondary metabolites produced by the plant
that provide protection against external hazards, such as disease, seed/insert pres-
sure, and various types of bacterial/yeast infections. Similarly, these compounds
have shown multiple human health benefits [12, 18–21]. Table 2 provides a sum-
mary of seminal research conducted on the health-promoting properties, while these
and other studies are described in more details throughout the document on the fruit
41
Table 1 Different species of jaboticaba: chemical characterization

Macronutrients (g/100 g) Micronutrients (mg/100 g)
Species Origin CHOa PTNb LIPc Dietary fiber Specific minerals Specific vitamins Ref.
Whole fruit
Plinia cauliflora São Paulo 78.2 1.02 0.01 0.55 0.01 K = 1320 – [13]
Mg = 120
Plinia jaboticaba São Paulo 76.5 0.94 0.02 0.40 0.10 K = 1000 – [13]
Jaboticaba: Chemistry and Bioactivity
Mg = 100
Plinia jaboticaba Minas Gerais 90.1 0.01 5.0 0.10 1.8 0.00 38.2 0.06 K = 700.7 81.20 Ascorbic acid = 8.6 0.20 [9]
Mg = 72.3 7.60
Plinia cauliflora Santa Catarina 77.64 0.78 0.00 0.53 0.17 9.26 0.03 – – [17]
Plinia cauliflora – – – – – Ascorbic acid=238 2.2 [16]
Jaboticaba peel
Plinia jaboticaba São Paulo 62.75 7.31 0.17 2.71 0.17 28.79 0.37 – – [12]
Plinia jaboticaba São Paulo 72.06 0.25 6.85 0.05 2.94 0.02 33.86 1.38 – – [14]
a
CHO = Total carbohydrate content
b
PIN = Total protein content
c
LIP = Total lipid content
1229
Table 2 Bioactive compounds identified in jaboticaba

Fruit Main bioactive compounds and
Species part concentration Health effects Ref.
Plinia Peel Cyanidin-3-O-glucoside In vitro: High antioxidant [18]
jaboticaba (1541 45.51 mg/100 g) and capacity, antiproliferative
Delphinidin-3-O-glucoside activity against tumor cells
(634.75 1.83 mg/100 g)
Plinia Peel Gallic acid 4.03 mg/100 g Sprague-Dawley rats fed [19]
jaboticaba with high-fat diet: # lipid
peroxidation in the brain
Ellagic acid 348.08 mg/100 g " antioxidant defenses in the
Quercetin 4.82 mg/100 g liver
Plinia Peel Gallic acid 177.76 2.26 μg/g Wistar rats fed with high-fat [12]
jaboticaba Ellagic acid 1581.61 135.50 μg/g diet " antioxidant defenses in
Cyanidin-3-O-glucoside liver and plasma
34,242 594.70 μg/g
Plinia Peel Cyanidin-3-O-glucoside Swiss mice fed with high-fat [20]
jaboticaba 1113.38 19.37 mg/100 g diet: Improvement of
Ellagic acid cognitive function and
710.10 33.77 mg/100 g prevention of insulin
resistance and fat weight gain
Plinia Peel Cyanidin-3-O-glucoside In vitro: " total antioxidant [21]
jaboticaba 2866 40.1 mg/100 g capacity (especially
ellagitannins)
Delphinidin-3-O-glucoside In vivo: " serum antioxidant
356.3 1.0 mg/100 g capacity in humans
Ellagic acid 142.8 11.7 mg/100 g
and/or its components. Moreover, Fig. 2 shows the structure of the main phenolic
components present in the jaboticaba. However, readers are referred to [10] for a
comprehensive illustration of the multiple structures of the phenol compounds with
only the primary phenols present in jaboticaba presented in Table 3.
4 Health Benefits of Jaboticaba or Its Components
4.1 Antioxidative Properties
The term “oxidative stress” is defined as imbalance in metabolism that leads to

the accumulation of reactive oxygen species and/or reactive nitrogen species (ROS/
RNS) in the cells [21]. Excessive levels of free radicals are able to overwhelm the
endogenous antioxidant capacity, resulting in oxidation damage to structural, trans-
port, and regulatory molecules, such as proteins, DNA, and lipids [21]. If left
unchecked, oxidative stress can lead to the onset of chronic degenerative diseases
A. Anthocyanins: Basic structure.

Delphinidin-3-O-glucoside: R3’=OH, R4’=OH, R5’=OH, R3=O-Glucoside, R5=OH, R6=H, R7=OH, R8=H
Cyanidin-3-glucoside: R3’=OH, R4’=OH, R5’=H, R3=O-Glucoside, R5=OH, R6=H, R7=OH, R8=H
B. Gallic acid
C1. Ellagic acid C2. Valoneic acid dilactone C3. Ellagic acid-pentose
(Basic Structure) Conjugate of ellagic acid Conjugate of ellagic acid
Fig. 2 Common small phenols present in jaboticaba
that include coronary heart disease and cancer, as reviewed in [18]. Dietary antiox-
idants have been shown to protect or mitigate oxidative stress by scavenging the free
molecules and/or binding reactive metal ions [22, 23]. However, the phenolic
antioxidants are more likely to aid in the activation or upregulation of antioxidative
enzymes, such as superoxide dismutase or catalase, or inhibit or downregulate
oxidative enzymes, such as xanthine oxidase or inducible nitric oxide synthase
(iNOS) [24–27].
Several studies have demonstrated the potent antioxidant properties of the jabo-
ticaba. For example, the antioxidant capacity of extracts obtained from seven
Brazilian fruits was evaluated using the 1,1-diphenyl-2-picrylhydrazyl radical test
and the β-carotene-linoleic acid-coupled oxidation assay [28]. While all the fruits
exhibited high antioxidative capacity, jaboticaba produced the highest activity for
both tests. From a potential human health perspective, Leite et al. [29] analyzed
the antioxidant status of rats fed with a high-fat-inducing obesity diet but
supplemented with freeze-dried jaboticaba peel (at 1, 2, and 4% (w/w)). Compared
to the animals fed with the high-fat diet only, the supplemented diets resulted in
lower serum saturated fatty acids, higher plasma antioxidant defenses, decreased
Table 3 Other phenols reported in jaboticaba

Phenola Classification
Hexahydroxydiphenoyl group Ellagitannin
Hexahydroxydiphenoyl-galloyl-glucose Ellagitannin
Casuariin Ellagitannin
Casuarinin Ellagitannin
Tellimagrandin I Ellagitannin
Tellimagrandin II Ellagitannin
Pedunculagin Ellagitannin
Casuaricitin Ellagitannin
Iso-oenothein Ellagitannin
Oenothein C Ellagitannin
Pedunculagin Ellagitannin
Casuaricitin Ellagitannin
2-O-(3,4-Dihydroxybenzoyl)-2,4,6-trihydroxyphenyl acetic acid Depside
jaboticaba
Syringin Phenolic acid aglycone
Syringin-O-glucoside Phenolic acid glucoside
O-Coumaric acid Phenolic acid aglycone
Protocatechuic acid Phenolic acid aglycone
Methyl protocatechuic acid Methylated phenolic acid
Peonidin Anthocyanin methoxy
derivative
Peonidin-3-O-glycoside Anthocyanin glucoside
Isoquercitrin Flavonoid glucoside
Quercimeritrin Flavonoid conjugate
Myricetin Flavonoid aglycone
Quercetin Flavonoid aglycone
Rutin Flavonoid glucoside
a
Information obtained from [11] but tabulated instead of illustrated
lipid peroxidation of the liver and brain, and higher antioxidative status of the
kidneys, depending on peel dosage. Moreover, Calloni et al. [30] reported that
extracts from the jaboticaba peel were able to reduce H2O2-induced mitochondrial
oxidative stress in human lung fibroblast cells by mitigating the decrease in the
activity of the mitochondrial complex 1 protein and ATP levels. The researchers
proposed that this berry may provide protection to the mitochondria when exposed
to dysfunctional conditions. Martins de Sá [31] showed that fermented jaboticaba
beverages (red, rose, and white) exerted a significant vasorelaxant effect on isolated
arteries, which was inversely associated with the antioxidative capacity of a given
beverage. The red drink produced the highest antioxidative activity and the lowest
vasorelaxant response followed by the rose and then the white fermented beverages.
While other studies have also shown jaboticaba as a rich source of antioxidative
agents, the above-discussed reports provide a representation of the related
experiments currently being conducted [18, 32–34] with the majority attributing the
phenols as the responsible components in protecting against oxidation, especially the
anthocyanins.
The basic structure of flavonoids consists of two phenolic rings (Fig. 1) with a
center heterocyclic ring bonded to either hydrogen, water, hydroxyl groups, carbo-
hydrates, or organic acids at carbon sites on the rings. The various type of hydroxyl
binding to the rings and position of the rings is specific to a particular flavonoid
class [35]. Individual anthocyanins are distinguished from other flavonoids by the
number and position of hydroxyl and methoxy groups on its basic structure and also
by the type and the number of bound sugars or acids (Fig. 1) [5]. Importantly,
anthocyanins are the only flavonoid able to exist as a cation or anion [8], resulting in
color changes from deep purple to red depending on the pH. Among the multiple in
vitro and in vivo studies that have been conducted to understand the antioxidative
benefits of anthocyanins [36, 37], the following studies are discussed to provide
support, in part, to the hypothesis that anthocyanins are the dominant antioxidative
protective agents in jaboticaba. Using rats subjected to hepatic ischemia-reperfusion
to produce oxidative stress after consumption of an anthocyanin supplemented diet,
Tsuda et al. [38] determined that the consumption cyanidin-3-glucoside and its
aglycone form produced an antioxidant response similar to that of α-tocopherol,
specifically in preserving the liposomes, liver, and erythrocyte membranes. Ramirez-
Tortosa et al. [39] also utilized a rat model, but the animals presented with a
vitamin E deficiency making the animal susceptible to oxidative stress. Yet, when
treated with an anthocyanin extract supplementation, the plasma antioxidative
capacity increased relative to the control. As a result, the damage caused by the
vitamin E deficiency, lipid peroxidation, and DNA damage was reduced. Based on
the French Paradox, this study was conducted on the anthocyanins present in red
wine. Importantly, the researchers determined that dietary anthocyanins were able to
protect against the risks associated with oxidative stress.
Ellagic acid may or may only slightly contribute to the antioxidant protective
properties of jabocatica. While this molecule is able to scavenge free radicals [40]
and other characteristics in terms of protection against lipid peroxidation and DNA
damage and increasing the activities of the antioxidative enzymes that include super
oxide dismutase, catalase, and glutathione peroxidase in the presence of an oxidant
using cell culture models [41, 42], its antioxidant protection in human health is
sporadic, especially compared to other phenols. Meyer et al. [43] showed that five
different phenols were able to inhibit copper-catalyzed low-density lipoprotein
(LDL). The flavan-3-ol, catechin, exhibited the highest antioxidant protection
followed by the anthocyanin, cyanidin, which was similar to caffeic acid, followed
by quercetin and finally ellagic acid. The differences were attributed to the structural
dissimilarities of the compounds. Interestingly, when the phenols were combined, an
additive response was obtained with the notable exception of ellagic acid, which
exerted a substantial antagonistic response. Yet, ellagic acid was able to ameliorate
the effect against negative effects of cisplatin, a cytotoxic agent drug used in
chemotherapy, using a rat model administered with this drug through gavage
delivery. Turk et al. [44] showed that rats supplemented with ellagic acid were
presented with decreased plasma, sperm, and testicular T-bar levels (a secondary
oxidation indicator). The ellagic acid supplement also activated the activity of
catalase and glutathione peroxidase.
4.2 Immune System Health and Anti-inflammatory Properties
The immune system is a complex network composed of a variety of molecules, cells,

tissues, and organs that act to defend their host against multiple invading infectious
agents or mechanistic dysregulations [45]. When the immune system is exposed to
these external/internal stresses, the inflammation response is initiated, which is the
body’s way of dealing with localized tissue damage and infection, and thus is critical
to our survival. As such, maintaining a robust immune system is essential to human
health. According to recent studies, the diet plays an important role in either
adversely or positively regulating the immune system. For example, researchers
determined that chronic, or silent, inflammation stimulates a self-perpetuating immu-
nological responses that are linked to nutrient overload intake, which, in turn, can
lead to multiple health risks [46–48]. On the other hand, certain foods or their
components can prevent the onset of harmful cellular stressors or diseases that
enhance and strengthen the immune system by acting upon or preventing chronic
inflammation.
Chan et al. [49] completed a study using monocyte-induced dendritic cells treated
with gallic acid (a phenolic acid ubiquitous to many foods, including jaboticaba)
followed by a cocktail of pro-inflammatory cytokines. The results showed that
various markers involved in the maturation of dendritic cells were suppressed
by gallic acid, such as the stimulatory proteins, CD40/CD83 and CD80/CD86
responsible for activating antigen-presenting cells and T cells via co-stimulation,
respectively [49].
Moreover, consuming ellagic acid-rich fruits and vegetables may aid in
maintaining oral homeostasis as this phenolic acid can also enhance the integrity
of epithelial oral cells, as reviewed in more detail by Dale [50]. Ellagic acid consists
of two resonant phenolic rings, each bound to para-hydroxyl groups and two
heterocyclic [51] (Fig. 2). This compound has been shown to be particularly potent
in protecting the immune system from a variety of onslaughts. A study completed
by Fakhry et al. [52] used oral epithelial cells to determine that ellagic acid increased
the expression of beta-defensin 2 and antileukoproteinase. Beta-defensin 2 consists
of an antimicrobial factor produced by neutrophils that attaches gram-negative
microorganisms [53]. Alternatively, antileukoproteinase protects epithelial cells
from serine proteases, which if left unchecked, degrade peptide bonds of proteins,
thereby contributing to damages to mucosal cells. Such a response to ellagic acid
contributes to a healthy immune response as strong mucosal cells promote physical
and chemical barriers against pathogens. The physical barrier consists of cell-to-cell
attachments, while the chemical barrier is constructed by antimicrobial peptides,
cytokines, and chemokines [53]. Another important factor related to the health of
mucosal cells as mediated by ellagic acid includes the prevention of HIV transmis-
sion. The human β-defensin peptide, Hbd2, and the secretory leukocyte protease
inhibitor, SLPI, have been recognized as mediators that not only enable the mucosal
to defend against microbial infections but also inhibit adenoviral infections, such as
HIV, as demonstrated by QuiÃones-Mateu et al. [54]. Yet another study completed
by Promsong et al. [55] used ellagic acid-treated primary human gingival epithelial
cells and showed that this bioactive agent (in concentrations of only μmoles) was
able to increase the expression of Hbd2 and SLPI by 3.7- and 2.5-fold relative to the
control (untreated cells). In addition, Modi et al. [56] showed that the ellagic acid
inhibits HIV-1 integrase enzyme, which is responsible for integrating the virus DNA
to the genetic content of the host DNA. As approximately 90% of HIV transmission
occurs via mucosal cells, ellagic acid-rich foods, such as jaboticaba, may be yet
another important preventative measure for contracting HIV by acting on the cited
potential targets.
Additionally, anthocyanins have been linked to immune health, particularly
via their anti-inflammatory properties. By enlisting female subjects (18–76 year,
n ~ 1250) Jennings et al. [57] determined their intake of total flavonoids as well as
individual subclasses, including the anthocyanins by using a frequency question-
naire. In terms of chronic inflammatory, the high-sensitivity C-reactive protein
marker was monitored and showed that higher anthocyanin intake was associated
with lower levels of this marker. In fact, no other associate with total or other
flavonoid subclasses occurred with this inflammation marker. In yet another study
using human subjects (31–315), Hassellund et al. [58] monitored the effects
of anthocyanins on inflammation risk factors in prehypertensive male subjects
(35–51 year, n = 27). The men were divided into two groups with one provided
an anthocyanin supplement (640 mg) and the other a placebo for 4 weeks followed
by a 4-week washout. No beneficial responses were detected in multiple inflamma-
tory cytokines monitored, but endothelial dysfunction improved with the anthocy-
anin supplementation. It must be noted that the sample size was small in this study
and trial duration short. Yet, Mena et al. [59] provided a review on the potential of
anthocyanins, a powerful anti-inflammatory agent.
However, foods are not purified compounds acting on single targets but rather
complex mixtures that may affect many biochemical pathways. Nonetheless, studies
remain limited on the benefits of the whole jaboticaba fruit or the peel with following
exception to our knowledge. Batista et al. [19] used Swiss mice to determine the
effects of a high-fat diet supplemented with only 4% of jaboticaba peel on multiple
markers for development of tauopathies, including hippocampal inflammatory
markers, tumor necrosis factor-α, and interferon-γ. Firstly, the added peel supple-
ment did not adversely affect either inflammatory markers as the animals fed with the
high-fat supplemented diets presented with significantly similar values as those on
the normal diet. Yet, compared to the mice fed with the high-fat diet only, these
markers were 100% lower for the animals fed with the jaboticaba peel supplemented
diet. Along with the anthocyanins and ellagic acids, the ellagitannins may also have
exerted a positive effect to mitigate inflammation exhibited the jaboticaba as

supported by the following review [60].
4.3 Cancer Prevention and Mitigation Properties
Cancer is leading cause of death in many western countries, including the United
States, second only to heart disease [61]. This disease is caused by uncontrolled cell
proliferation leading to the formation of tumors. Tumors are complex structures that
are composed of cells that have altered regulatory processes allowing a given tumor
to grow in most types of tissues and/or organs. Proliferating cells (or neoplastic cells)
are capable of shutting down the senescence pathway of a normal cell securing their
perennial life [62, 63]. Furthermore, cancer cells release chemical signals that
promote angiogenesis around the tumor structure, thereby supplying oxygen,
blood, and other nutrients needed for growth. These remarkable cancer features are
the primary reason this disease leads to high mortality rates [62, 63]. Additionally,
proliferating cells reach a development point where they are able to combine
signaling with taxis capacity and, as a consequence, metastasize to other tissues
different from their original point of origin, which is yet another lethal characteristic
associated with cancer [63, 64].
An estimated one-third of the cancer deaths are related to lifestyle that include
diet factors [65]. Yet, diets rich in various natural systems, such as tomatoes, carrots,
and berries, have exerted impressive effects in mitigating cancer [64], with jaboti-
caba providing potent anticancer properties among the berries [66]. A recent study
conducted by Wang et al. [33] tested different sections of the fruit to identify the
fractions that exerted most effective anticarcinogenic response. In order to accom-
plish this aim, human oral carcinoma cells obtained from the apical layer of the
epithelial tissue were treated with extracts (aqueous or ethanoic) recovered from the
seed, stem, and peel of the jaboticaba. The results showed that the water-extracted
seed fraction provided the most potent effects in lowering cellular proliferation
compared to the control by acting as inhibitors of apoptosis. Leite-Legatti et al.
[18] reported that polar and nonpolar peel extracts of jaboticaba were able to provide
effective antiproliferative properties against leukemia cells (K-562) and prostate
cancer cells (PC-3), respectively, of the 11 different cancer cell types tested. It is
evident from these two isolated studies that more research is needed to determine the
anticancer potency of the whole fruit or its individual parts in vivo as well as to
characterize the anticancer mechanism.
Nonetheless, several studies have reported on the anticancer mode of action
exerted by the primary bioactive agents in jaboticaba credited (ellagic acid, gallic
acid, anthocyanins, and ellagitannins) as shown by the following seminal reports.
Firstly, ellagic acid has been shown to initiate a cascade of events in normal human
lymphocytes that resulted in declining rates of DNA synthesis during the cell cycle.
Also, the acid induced cell apoptosis by generating DNA molecule fragmentation,
thereby preventing the neoplastic cells from proliferating [51]. Losso et al. [67]
evaluated the antiproliferative activities of ellagic acid against eight different cell
lines with doses ranging from 10 to 100 mol/L. The most susceptible cells to the
antiproliferative activity of ellagic acid were those derived from the colon (Caco-2),
but the breast and prostate lines were also strongly impacted. As evidenced by
microscopic examination of the cell gross morphology, ellagic acid prevented
proliferation via induction of apoptosis. Moreover, lower ATP levels were produced,
which is essential for cancer cell viability, indicating that ellagic acid exerts both a
selective cytotoxicity and an anti-proliferation effect. Finally, ellagic acid was able to
prevent fetal bovine stimulation of cell migration at the highest concentration used,
suggesting that this compound may also mitigate metastasis. Edderkaoui et al. [68]
determined that ellagic acid induced apoptosis in pancreatic cancer lines, which,
in turn, prevented proliferation by approximately 20-fold at a concentration of
50 mmol/L. These results were associated with the depolarization of the mitochon-
dria, release of cytochrome C, and activation of downstream caspase, which were
attributed to the ellagic acid dose-dependent decrease of the binding activity of the
transcription factor, nuclear factor kappa β. For more information about the ellagic
acid effects in response to cancer and potential mechanisms, refer to Stoner and
Muktar [69], Maas et al. [70], and Landete [60].
Secondly, as the largest phenolic compound present in jaboticaba, ellagitannins
do not absorb into the bloodstream but rather are physiologically hydrolyzed to
ellagic acid in the large intestine by the microbiota (refer to Sect. 4.5), which, in turn,
is metabolized to smaller metabolites, such as the urolithins [71]. Urolithins A and C
in turn have shown to inhibit HT-29 (colon cancer cells) cell proliferation via B0/G1
and G2/M arrest followed by the induction of apoptosis. Urolithins are also advan-
tageous to cancer prevention as they are able to remain in the colon through
enterohepatic circulation [72]. As an outcome to its metabolism into multiple but
potent health-promoting metabolites, ellagitannins have been shown to have multi-
targeted effects [73]. For example, Seeram et al. [74] showed that 25 miRNA,
including the let family members, MiR-370, MiR-373, and miR526b, were likely
targets of preventing proliferation and differentiation of Hep-G2 liver cancer cells.
Indeed, other studies have been completed to show the chemopreventive properties
of ellagitannins against prostate, colon, breast, oral/gastric, liver, cervical, lung, and
skin cancer as reviewed by [72, 73].
Thirdly, current research has shown that gallic acid has exceptionally potent
anticarcinogenic properties due to its ability to act on multiple targets. For example,
gallic acid is able to prevent cancer by targeting angiogenesis, which is accom-
plished by concurrently inhibiting its initiation and by preventing additional neo-
vessel growth after angiogenesis has already commenced [68–75]. More specifically,
Lu et al. [75] determined that gallic acid was able to reduce glioma cell-mediated
angiogenesis, in addition to inhibiting cell viability, proliferation, and invasion
with U87 and U251 glioma cells as the cancer models. Using nude mice that
were subcutaneously injected with the prostate cell lines 22Rv1 and DU145, Kaur
et al. [76] reported that gallic acid consumption reduced the microvessel density in
tumor xenografts as compared to the control. This study also showed that gallic acid
decreased cell viability in a dose-dependent manner in both cell lines primarily by
apoptosis induction. A recent study completed by Ho et al. [78] showed that the
exposure of squamous carcinoma cells to gallic acid resulted in inhibiting the

activation of two matrix metalloproteinase (MMP) enzymes, MMP-2 and MMP-9,
which are responsible for modulating cytokines and growth factors involved in
angiogenesis as well as the invasion and migration of cancer cells [79]. As such
the gallic acid may execute an important function against the tumor development.
For more information on the anticancer effects of gallic acid, the reader is directed to
excellent reviews presented by Verma et al. [77] and Locatelli et al. [80].
Lastly, the anthocyanins protect against cancer development due, in part, to their
ability to bind directly to cell cycle regulator proteins, such as p53, p21, and cyclins
[81, 82] and thus preventing cell proliferation. Additionally, these bioactive agents
are able to increase the membrane potential of the mitochondrial membrane l and
induce the accumulation of ROS resulting in cell apoptosis [83]. Other studies have
shown that anthocyanins are able to downregulate the expression of MMP, which, in
turn, decreases the ability of the tumor cell to degrade the extracellular matrix and
invade new tissues [84, 85]. Anthocyanins have been shown to target the regulation
of various cytokines involved in angiogenesis [86]. (Wang et al. [87] provides an in-
depth review of the anticancer mode of action by anthocyanins.)
4.4 Atherosclerosis Prevention and Mitigation
Atherosclerosis is a condition caused by accumulating lipids and fibrous compounds

in major arteries [88, 89], resulting in narrowing the arteries and thereby reducing
blood flow to vital organs. This cardiovascular state is responsible for approxi-
mately 50% of reported fatal heart diseases, including cardiovascular disease [90].
Atherosclerosis is diagnosed by established biomarkers, including total cholesterol
(TC) that is highly associated with the ratio of low-density lipoprotein (LDL) to
high-density lipoprotein (HDL) levels. The higher the ratio, the more serious the
condition as LDL is easily oxidized, which is a significant contributing factor to
atherosclerosis [88]. Oxidized LDL molecules enhance the production of reactive
oxygen species (ROS), decrease the formation of NO (nitric oxide, an endogenous
antioxidant), and stimulate the endothelium to express adhesion molecules and
chemokines, which in turn recruit monocytes and T cells to the arterial wall [91,
92] ultimately forming foam cells and perpetuating inflammation. Effective treat-
ments must be able to decrease LDL cholesterol (LDL-C) and/or raise HDL choles-
terol (HDL-C) in the bloodstream.
Natural alternatives to the commonly used treatments, mainly statin drugs, have
been of intense interest for decades due, in part, to the side effects and cost of these
drugs [93]. Because of the presence of several potent bioactive agents, jaboticaba is
now being studied as a potential food or source of agents to protect against
atherosclerosis, although research again remains limited on the fruit itself with the
following exceptions. Batista et al. [94] evaluated the effects of obese rats fed with a
high-fat diet (lard) supplemented with only 1, 2, and 4% dried jaboticaba peel on the
lipid profiles present in serum, liver, and fecal material. Although hepatic and serum
lipid contents were not impacted by the peel, fecal triglyceride excretion increased,
particularly for the animals fed with the 1 and 2% supplements. Most importantly,
evaluation of lipid peroxidation of the liver showed that the 2% supplement was able
to lower peroxide values by 28.35% compared to the positive control fed with the
high-fat diet only. In contrast, Alezandro et al. [95] showed that total plasma
cholesterol levels and triglycerides were reduced by 32% and 50%, respectively, in
streptozotocin-induced diabetic rats fed with 1.0 and 2.0 g dry weight/kg of body
weight of jaboticaba extracts. The difference in serum levels between the two studies
may be due to the induction stressors used to elevate the lipids, with the dietary
inducer being more resistant. Another report by Batista et al. [19], who again fed rats
lard as the atherosclerosis inducer and freeze-dried jaboticaba supplements (2% and
4%), showed that that peel reduced serum saturated lipids, another risk factor for
atherosclerosis [96].
Relative to specific bioactive agents present in jaboticaba, ellagic acid may be a
primary candidate responsible for protecting against cardiac damage caused by
atherosclerosis. Several studies have shown that this molecule acts directly on
LDL oxidation responses by inhibiting the proliferation of aortic smooth muscle
cells and decreasing the levels of induced ROS generation that, in turn, activate
adhesion molecules [97–99]. To prevent ROS induction, ellagic acid downregulates
the expression of the lectin-like oxidized low-density lipoprotein receptor 1
(LOX-1), which is also responsible for the expression of pro-inflammatory mole-
cules, such as cytokines, and chemokines in the endothelial cells. Moreover, this
phenolic compound suppresses the expression of iNOS, which catalyzes a pathway
that produces nitric oxide (NO). Under normal conditions, NO regulates arterial wall
homeostasis. However, when the production of NO is upregulated by the oxidized
LDL, as in the case of the iNOS pathway, the NO molecules bind with tyrosine
residues that are essential for the vascular tone, thereby remediating further compli-
cations to the endothelial dysfunction [100–102]. Ellagitannins most likely act in the
same manner due to their hydrolysis to ellagic acid and the urolithins. For more
information on vascular health in response to ellagitannins and its metabolites, refer
to the [103].
In addition, Xia et al. [104] determined that anthocyanins exert a unique effect in
mitigating the progression of endothelial dysfunction, namely, by reducing the risk
for catastrophic ruptures by reducing or stabilizing plaque. In this study, animals
prone to the formation of weakened plaques (apolipoprotein E-deficient mice) were
treated with diets rich in anthocyanins. At the end of the 20-week feeding study,
plaques decreased by 18% and were firmer in texture, and necrotic core was not
enlarged compared to the control group. Although the mechanisms involved in this
phenomenon are not clear, the authors proposed that the anthocyanins augmented
the antioxidant levels inside the plaque leading to increased stability. Moreover, the
action of the anthocyanins can be related to the lipid serum level improvement as
the anthocyanin-fed animals presented with reduced plasma cholesterol, LDL, and
triglyceride levels. As in most of the studies related to bioactive compounds, there
remains a critical lack of information regarding the pathways involved in protecting
against atherosclerosis or related risk factors. Nonetheless, it is clear from several

reviews [60, 105–107] that flavonoids and other types of phenolic molecule are able
to prevent or ameliorate the atherosclerosis status.
Although not a phenolic compound, which this review has been the focus, pectin
is particularly high in jabocatica and must be recognized as such, particularly on its
effects on risks for atherosclerosis. Multiple studies that date back to the early 1960s
have shown the advantageous impact of pectin consumption, which are reviewed in
[108, 109]. The most commonly reported response is that pectin facilitates the
excretion of cholesterol and by inhibiting bile acid absorption [110].
4.5 Gastrointestinal Health: Gut Microbiota Modulation
The symbiotic relationship between human health and the gastrointestinal (GI)
microbiome is garnering intense research with diet playing a major role
[111–114]. Although studies pertaining to gut microbiota health and jaboticaba
have not been reported to our knowledge, related research has been conducted on
the prevalent bioactive agents of this berry. Firstly, the ellagitannins and ellagic acid
are partially absorbed by the intestinal tract as their bioavailability is low. However,
the remaining unabsorbed compounds remain intact until each reaches the large
intestine where it is metabolized by the bacterial population, resulting in smaller
polyphenolic metabolites (Table 4) [115–118]. These metabolites have been
reported to exhibit both anti-inflammatory and antiproliferative properties [118,
119], among other health-promoting properties [60]. In addition, ellagic acid has
been shown to modulate the gut microbiome to a healthy state. For example, Selma
et al. [120] compared the production of urolithin A, a metabolite of ellagic acid, and
other GI metabolized components of human subjects fed an ellagic acid-rich extract
of pomegranate extract. After analyzing the composition of their fecal material
subsequent to consumption of the supplement, the researchers determined that only
certain test individuals presented with elevated production of ellagic acid metabo-
lites while levels of these agents were low in other subjects. Those subjects that
produced high levels urolithin A also had elevated Clostridium coccoides
populations. This difference in metabolite production and the simultaneous alter-
ation in the microbiome composition suggest a strong relationship between the two
factors [120]. Another study completed by Li et al. [121] analyzed the relationship
between the production of urolithin isomers and the gut microbiota composition in
response to duration of consumption of an ellagic-rich pomegranate extract. In the
1st week, urolithins were present in the stool of only 30% of the individuals. By the
4th week, 50% of the individuals who did not produce urolithin excreted significant
amounts of ellagic acid. This difference provides additional evidence that ellagic
acid is likely playing an important role in the alteration of the gut microbiota
composition but is subject specific. The same study reported a change in the
Firmicutes/Bacteroidetes ratio present in the fecal material of the test subjects
after extraction administration. Those who were able to produce urolithin A
Table 4 Microbial metabolism of ellagitannins, ellagic acid, and anthocyanins

Ellagitannins Ellagic acid Anthocyanins
Ellagic acid Nasutin A m-Hydroxyphenyl propionic acid
– Isonasutin Isoferulic acid
– Urolithin M-5 3,4-Dihydroxyphenylacetic acid
– Urolithin M-6 Ferulic acid
– Urolithin M-7 Catechol
– Urolithin C 3,4-Dihydroxphenylpropionic acid
– Urolithin D m-Hydroxyphenylacetic acid
– Urolithin A Dihydroferulic acid
– Urolithin B Phloroglucinaldehyde
– Isourolithin A Vanillic acid
– Isourolithin B p-Hydroxybenzoic acid
– – Protocatechuic acid
– – Gallic acid
– – Tyrosol
– – Resorcinol
– – Pyrogallol
– – Caffeic acid
– – Homovanillic acid
– – Syringic acid
– – p-Coumaric acid
experienced a decrease in the Firmicutes population with a concomitant increase in

bacteria from the genus Prevotella, which belongs to the phylum Bacteroidetes. The
ratio between those two phyla has been shown to be closely related to lean and
obese phenotypes with higher levels of Bacteroides and lower levels of formicates
associated to lower body weight [122, 123]. This fact is useful in the development
of functional foods, such as jaboticaba, as means to target obesity through gut
microbiota modulation.
In the case of anthocyanins, multiple studies have been reported based on their
ability to modulate the gut microbiota, as reviewed by Faria et al. [116]. Again, the
majority of anthocyanin levels consumed are not absorbed by the upper GI tract,
thereby reaching the lower intestinal microbiota. Similar to ellagitannins and ellagic
acid, anthocyanins are biotransformed into smaller but multiple metabolites, which
are then absorbed through the large intestine (Table 4). Once absorbed, these
compounds have been shown to exert different biological properties compared to
their parent counterparts [124]. Using an in vitro fecal fermentation approach,
Hidalgo et al. [125] further reported that anthocyanins were able to modulate the
gut microbiota. More specifically, malvidin-3-glucoside enhanced the growth of the
putatively beneficial bacteria, Bifidobacterium spp. and Lactobacillus spp., but did
not affect Bactericides spp. growth. However, the two predominant anthocyanins
present in jaboticaba, delphinidin 3-glucoside and cyanidin 3-glucoside [18], were
not studied in isolation in terms of their ability to modulate the gut microbiome.
Rather, they were combined with malvidin-3-glucoside, peonidin-3-glucoside, and

petunidin-3-glucoside to monitor the combined response on the gut microbiota. The
results showed even higher levels Bifidobacterium spp. and Lactobacillus spp. with
latter growth even higher than the positive control, which used the proven prebiotic
fructooligosaccharide [126]. The authors attributed these results to a synergistic
effect exerted by the anthocyanins in combination. As such, it is reasonable to
propose that the anthocyanins present in jaboticaba peel are capable of modulating
the gut microbiota to a healthy community.
Pectin in jaboticaba may be able to modulate the gut microbiota to a health
state. Jiang et al. [127] showed that apple-derived pectin supplement into a high-
fat-inducing obesity diet prevented a decrease in the Bacteroidetes population and
an increase in the Firmicutes phylum. Parkar et al. [128] investigated the effects of
six different pectins obtained from kiwifruit on gut health in terms of influence on
bacterial adhesion on intestinal epithelial cells (Caco-2 cells). The pectin which
originated from the kiwifruit was more effective, enhancing the adhesion of
Lactobacillus rhamnosus while decreasing the adhesion to Salmonella
typhimurium. Still the adhesion of Bifidobacterium bifidum significantly increased
in the presence of inulin and citrus pectin. Indeed, studies are needed on pectin
present in jaboticaba to determine its effect on the microbiome as another study
completed by Nazzaro et al. [129] further confirmed that pectin source affects gut
microbe modulation.
4.6 Metabolic Syndrome: Obesity and Diabetes
Metabolic syndrome (MetS) is a cluster of conditions that involve risks for cardio-
vascular diseases and type 2 diabetes and is prevalent with obese state [130, 131].
Specific markers of MetS include excessive central adiposity, higher serum glucose
levels, increased serum triglycerides and LDL-C, lower HDL-C, and high blood
pressure [132–135]. (As the latter conditions have already been discussed previ-
ously, with the exception of central adiposity and serum glucose levels, this section
will mainly focus only on health risks associated with obesity and diabetes.)
According to the World Health Organization [133], obesity is defined an excessive
accumulation of fat tissue that can cause various negative effects on the human body,
such as chronic oxidative and pro-inflammatory states [134, 135]. Diabetes is
defined as the dysregulation of glucose absorption and breakdown pathway leading
to glucose resistance and further ketosis, tissue damage, and many other metabolic
failures [135]. As this MetS is becoming a worldwide epidemic [136], researchers
are now focusing their efforts on discovering bioactive compounds that could
prevent or decrease the damages caused by these alarming clinical markers.
The relationship between cellular metabolic responses and the antioxidants pre-
sent in the peel of the jaboticaba fruit is a main focus of several research studies. For
example, in a recent study conducted by Lenquiste et al. [137], freeze-dried jaboti-
caba peel was administered at 1%, 2%, and 4% (w/w) of the diet as a supplement to
rats fed with a high-fat diet added to an AIN-93 M diet. Consumption of the high-fat
diet þ jaboticaba supplementation resulted in lower weight gain compared to the
high-fat diet. Moreover, the antioxidant profile of the liver improved for rats fed with
the high-fat þ supplement diet, while those on the high-fat diet presented with
significantly higher levels of lipid peroxidation. Meanwhile, serum insulin levels
reduced by 47%, 57%, and 52% with respect to the animals fed with the 1%, 2%, and
4% freeze-dried jaboticaba peel supplemented diet. A previous study using freeze-
dried jaboticaba peel supplement fed to a high-fat-induced obese rat model showed
that incidence of type 2 diabetes was directly associated with plasma cholesterol
ester levels and total short-chain fatty acids [19]. These factors are closely related to
inflammation and insulin resistance [135]. In another study, Batista et al. [94]
reported that high fat obesity induced animals fed three different concentrations of
freeze-dried jaboticaba extracts (1%, 2%, and 4%) resulted in increased triglyceride
output, decreased hyperinsulinemia, decreased hepatic inflammation, and improved
insulin sensitivity. However, only the 4% diet exerted these benefits. The authors
proposed that results were associated with a decrease in plasma short-chain fatty
acids, which has been linked to a downregulation in the expression of lipogenic
genes [83].
The anthocyanins present in jaboticaba may, in part, play a role in mitigating
the risk factors of MetS by inhibiting the release of inflammatory adipokines
secreted by the hypertrophic adipose tissue that contribute to insulin resistance as
evidenced by Matsukawa et al. [138]; albeit only the anthocyanin, cyaniding-3-
glucoside, was used. Still, this effect was attributed to the anthocyanin interacting
with proteins involved in insulin signaling that induce a state of insulin resistance.
Garcia-Diaz et al. [139] showed that anthocyanin-rich fractions obtained from
blackberry and blueberry beverages were able to reduce intracellular fat accumu-
lation when applied during the differentiation of 3T3-L1 adipocytes followed by
the inhibition of lipolysis of the mature cells. Moreover, the blends partially
restored the insulin-induced glucose uptake by the adipocytes. This effect was
attributed to the interaction of the anthocyanins with proteins involved in insulin
signaling that induce a state of insulin resistance. Using a mice model, Prior et al.
[140] fed three groups increasing levels of fat that contributed to 10%, 45%, or
60% of their daily calories. The diets were supplemented with whole blueberries,
strawberries, or purified anthocyanin extracts recovered from each of the fruits.
The whole fruit, blueberry or strawberry, did not prevent the weight gain that
accompanied the groups fed with the 45% and 60% high-fat caloric diets. Yet,
consumption of the purified anthocyanins obtained in either fruits resulted in
reduced obesity.
The above-discussed documents represent only a cross section of the numerous
studies that have been reported on health benefits of anthocyanins as well as tannic,
ellagic, and gallic acid on preventing or remediating MetS and/or its risk factors/
conditions. Therefore, for more information on the possible mechanisms of action of
these phytochemicals on MetS responses, the reader is referred to [141–143] for
excellent reviews on this topic.
5 Conclusions
The jaboticaba fruit is an important crop indigenous to Brazil due, in part, to the
numerous nutrients and phytochemicals that protect against multiple diseases cur-
rently afflicting western cultures. Despite its increasing importance, jaboticaba
consumption is low in other parts of the world. This review provides information
on jaboticaba, with an emphasis on their phenolic composition and links to human
health benefits. Although our understanding of the latter health attributes is increas-
ing, critical gaps in knowledge remain on the role that jaboticaba has on protecting
against multiple disease risks or states. As jaboticaba contains multiple nutrients and
other phytochemicals, they most likely exert these effects as synergists or additives
within the complex berry system, but again research remains limited on the whole
fruit with more data available on the peel. Such studies are particularly important
considering that theses berries are consumed primarily as a whole product, with the
market class and production location most likely affecting their compositional pro-
files and therefore its potential health effects.
However, the presented studies show the potential of the jaboticaba fruit as a
highly effective functional food capable of providing multiple health benefits. Foods
that have been branded as a functional food (or disease preventing/remediating
foods), such as cranberries and grapes, have expanded approximately tenfold in
the last decade, growing at three to four times the rate of conventional foods due to
the increasing preference for natural interventions [144]. Based on this encouraging
growth, expanding jaboticaba into the functional food category is expected to
increase overall consumption and demand, which in turn can support and increase
prices. By increasing the demand on the global scale for this currently
underconsumed fruit, it is expected that a positive economic impact would affect
all segments of the production and distribution chain.
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Pomegranate Bioactive Molecules and
Health Benefits 42
Saeed Akhtar, Tariq Ismail, and Anam Layla
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254
2 Biochemical Composition of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1255
3 Health Benefits of Pomegranate and Its Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1257
3.1 Pomegranate Promotes Cardiac Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1257
3.2 Pomegranate and Its Role in Cancer Chemoprevention . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260
3.3 Antidiabetic Properties of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262
3.4 Hepatoprotective Role of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
3.5 Pomegranate and Brain Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263
3.6 Anti-Inflammatory and Bone Health Promotion Properties . . . . . . . . . . . . . . . . . . . . . . . . 1264
3.7 Microbial Pathogenesis Inhibitory Role of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
3.8 Anthelmintic Activities of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266
3.9 Gut Health Promotion Properties of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266
3.10 Role of Pomegranate in Modulating Renal Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
3.11 Pomegranate Perspective Potential in Improving Fertility . . . . . . . . . . . . . . . . . . . . . . . . . 1267
3.12 Wound Healing Properties of Pomegranate Accessions . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268
3.13 Cosmo Care Properties of Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268
3.14 Pomegranate Role in Maintaining Oral Hygiene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1269
4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1269
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1270
Abstract
Fruits contribute an abundant supply of antioxidants to human diet and act as the
first line of defense against the risks of chronic diseases occurrence. Pomegranate
is one among the highly explored and appreciated fruits on account of its
promising health-promoting and disease-preventing properties. Pomegranate
fruit and its key components including rind, seed, and membranous network
S. Akhtar (*) · T. Ismail · A. Layla

Institute of Food Science and Nutrition, Faculty of Agricultural Sciences and Technology,
Bahauddin Zakariya University, Multan, Pakistan

1254 S. Akhtar et al.
have been evidently reported as carriers of a wide range of bioactive compounds

including ellagitannins, hydroxycinnamic acids, hydroxybenzoic acids, flavons,
flavonol-3-ols, anthocyanidins, anthocyanins, and conjugated and nonconjugated
fatty acids, phytosterols, vitamins, and minerals. Traditional aspects of pome-
granate exploitation as remedy against infections and gastrointestinal ailments
have generated a basis for the modern-age research. Findings of the research
carried out in the last two decades manifest fruit, flower, seeds, and peel of
pomegranate as natural strategy to treat microbiological and parasitic pathogen-
esis and to act as a chemopreventive and therapeutic approach against inflamma-
tory and infectious chronic ailments. Forthcoming sections of this chapter review
fundamental biochemical composition of pomegranate and its anatomical frac-
tions and provide recent updates on pomegranate perspective applications against
the risks of various forms of cancers, cardiovascular diseases, diabetes, acute and
chronic liver injury, renal disorders, impaired gut health, neurodegenerative
disorders, microbiological pathogenesis, and parasitic infestation.
Keywords
Pomegranate · Ellagitannins · Inflammation · Cancer · Neurodegeneration
1 Introduction
Pomegranate (Punica granatum L; “seeded apple”) was lauded in the Torah, Bible,
and Al-Quran as a sacred fruit deliberating good luck, wealth, and power of fertility
[1]. The mystical fruit traditionally implicated as folk or ethnic medicine to cure
various health disorders [2]. The fruit-bearing plant is a predominant shrub of family
Punicaceae that typically grows up to 12–16 feet and has several spiny branches with
glossy lance-shaped leaves. The grenade-shaped fruiting body encloses clusters of
delicious arils separated by pale white spongy mesocarp, with deep red leathery skin
[1]. Since ancient times, the fruit is native and under cultivation from the northern
Himalayas to Iran, Afghanistan, India, Pakistan, and China. The pomegranate
cultivation was stretched from west of Persia (old name of Iran) over the entire
Mediterranean region to Turkey and dried regions of American Southwest, Mexico,
California, and Arizona [3].
The pomegranate plant, fruit, and their anatomical segments including flowers,
fruit rind, leaves, bark, seeds, and roots encompasses a variety of biomolecules
including phenolics, hydrolysable tannins, anthocyanins, flavonoids, and a wide
range of essential micronutrients. The unique biochemical profile of the fruiting
body and its major fractions attribute strong antioxidative, anti-inflammatory, apo-
ptotic, and antimutagenic properties to curtail chronic maladies [4, 5]. Prophylactic
properties of pomegranate are of broad spectrum and are anticipated to mitigate
microbiological and parasitic pathogenesis [6, 7], gastric damage [8], cardiovascular
disease [9], type 2 diabetes [10], several types of cancers [3], renal illnesses [11],
liver complications [12], infertility [13], osteoarthritis [14], oral and dental health
[15], and skin melanoma [16].
42 Pomegranate Bioactive Molecules and Health Benefits 1255
The consumption trend of fruit and associated structures is diversely alike fresh or
dried arils, fresh or fermented juice, powdered extracts, rind powder tablets, capsules
and soft gels, and extracts-based ointments and decoctions [5, 17, 18].
Pomegranate fruit waste including peel and seeds provides substantial amount of
phytonutrients that can be exploited for their potent health benefits. The mechanistic
properties of pomegranate fruit and peel extracts such as antioxidant, antimicrobial,
flavoring, and colorant may also be implicated as a natural additive in the food
industry for quality enhancement and food preservation [4, 19]. In addition, pome-
granate seed oils and rind extracts deliberate photoprotective effect and can be
utilized in cosmo care products [20]. The peel powders also have biosorption
capacity for heavy metal from water and are capable of acting as phyto-remediating
agent to ensure cost-effective waste water treatment and decontamination of chro-
mium, nickel, and zinc contamination [21, 22].
A plethora of research has been conducted on health-promoting and food features
of pomegranate and its anatomical fractions as in the last two decades. This chapter
comprehensively covers the recent updates from research carried out to explore
pomegranate fruit and its waste fractions’ capability in delivering preventive and
therapeutic aspects against certain human acute and chronic ailments of critical
concern (Figs. 1, 2, and 3).
2 Biochemical Composition of Pomegranate
Globally, higher acceptability of pomegranate as a miraculous fruit is linked to its

remarkable health benefits. Predominately, a unique biochemical profile ornamented
with more than 124 phytochemicals is the differential factor responsible for a broad
range of antioxidant, anti-inflammatory, and antimutagenic properties of
Fig. 1 Pomegranate flower

Fig. 2 Pomegranate ripened

fruit
Fig. 3 Pomegranate edible

fleshy sacs
pomegranate. Pomegranate fruit and its different anatomical fractions like flower,
peel, juicy sacs, and seeds are referred as reservoirs of high molecular weight
hydrolysable tannins, i.e., ellagitannins, alongside a wide range of anthocyanins,
hydroxybenzoic acids, hydroxycinnamic acids, minerals, essential lipids, and com-
plex polysaccharides [23–26]. Approximately 50% of the fruit weight is attributed to
the rind portion that constitutes 30% of the fruit anthocyanidins contents.
Pomegranate peel, the waste fraction of the fruit, has been reported to carry 10%
w/w crude polysaccharides that could further be exploited as a functional ingredient
in attributing gelling and prebiotic properties to food formulations. A recent study
from Tunisia reported 6.8–10.1% pectin contents in peel portion of four Tunisian
pomegranate cultivars [27, 28]. Pomegranate seeds constitute 15–25% of the edible
juicy arils and are considerably good sources of phytosterols, omega-5 fatty acids
including punicic acids, tocopherols, and isomers of conjugated alpha-linolenic acid
[29–31].
Variability in concentration of pomegranate bioactive compounds including
phytochemicals of human health significance and their biological properties varies
with the cultivar, anatomical part of the fruit, maturity period, type of extraction, and
mode of phenolics extraction. Low-cost and efficient recovery techniques of pome-

granate phytochemicals, polyphenols, and seed oil have been introduced in the
recent past including pulsed ultrasound-assisted extraction, green ultrasound-
assisted extraction, ultrasound-assisted enzymatic extraction, enzyme-assisted
supercritical fluid extraction, microwave-assisted extraction, microwave-treated
supercritical CO2, high-voltage electrical discharge, and hydroalcoholic extraction
system [32–42].
Selection of a cost-effective, safer, and intelligent biomolecules extraction tech-
nique for pomegranate and its various fractions ensures higher rate of phenolics
recovery with better ability of extracts/fractionated compounds in dispensing various
biological promoting properties of human health concern (Table 1).
3 Health Benefits of Pomegranate and Its Fractions
3.1 Pomegranate Promotes Cardiac Health
Global estimates predict death toll due to cardiac complication to be 40% by the year
2020 [44]. Several epidemiological studies correlate inverse relation between adher-
ence to relatively high intake of plant polyphenols and incidence of cardiovascular
diseases (CVD). Experimental evidence suggested polyphenol-rich pomegranate
fruits consumption to present modulatory effect in improving cardiac muscles
tone, mitigating oxidative stress-mediated arterial hardening, and attenuating ath-
erosclerosis. Healthy heart features are attributed predominately by the pomegranate
ellagitannins especially punicalagin isomers α and β. Pomegranate hydrolyzable
ellagitannins after being converted to ellagic acid are further metabolized into certain
types of urolithins by intestinal microbiota. Studies on pomegranate consumption
and association with improved cardiac health correlate urolithins as one among the
known ellagitannins metabolites that stand responsible for attenuating various car-
diac complications [9, 45]. Arterial lipid accumulation is a hallmark of atheroscle-
rosis and is encouraged by low-density lipoprotein (LDL) cholesterol oxidation. A
double-blind pilot scale study conducted in dyslipidemic obese patients receiving
500 mg pomegranate rind extract daily for a period of 8 weeks demonstrated a
significant reduction in systolic blood pressure and improved lipid profile via
reduced triglycerides and LDL levels and increased HDL level proclaiming pome-
granates a potent antiatherogenic agent [46]. In a study conducted by Razani et al.
[47], 100 hospitalized patients diagnosed with myocardial ischemia and unstable
angina were randomly assigned to consume 220 ml pomegranate juice in addition to
medical treatment; pomegranate diet therapy presented cardioprotective effect by
significant reduction in serum level of malondialdehyde and troponin, intensity of
angina pectoris, and attenuating reperfusion injury [47].
Pomegranate fruit and peel extracts have also been reported to exert an amelio-
rative role in vascular inflammation, oxidative stress, and elevated cardiac enzymes
markers. Pomegranate extracts supplements (625 mg carrying 200 mg punicalagin)
delivered to hyperlipidemic animal models have been found to prevent coronary
Table 1 Major biochemical constituents of pomegranate leaf, flower, fruit, peel, seeds, bark and
root
Chemical Anatomical
Compound class distribution Structures
H
Punicalagin Ellagitannins Peel, leaf, bark, root H

O
O
H
O
O
H
O
H
O
H
O O
H O
O O
H
O
H
O
O
H
H
O
H
O
O O
H O O H
H
H O
O H O
O
O H
O
O
H
O
O
H O H
Punicalin Ellagitannins Peel, leaf, bark, root o

o
o o o
o
o
o o o
o o o
o o
o
o o
o o
Pedunculagin Ellagitannins Peel H

O
H O
O
O
O
H
H
O O
H
O
H
H O
O O O O
H
O
O O
H
H
O O
H
H O O O O H
H H
Corilagin Ellagitannins Peel, leaf O

O O
O
O O
O O
O O
Casuarinin Ellagitannins Peel O

O O
O O O
O
O O
O
O
O O
O
O O
O
O
O
O
O
O O
O
HO OH HO OH
Granatin A Ellagitannins Peel HO OH
O O
HO O
O O
H
HO OH O
HO
HO OH
O
Granatin B Ellagitannins Peel HO

OH
OH
OH O
O OH
O O
OH
O OH
O
O
O OH
O
O
HO OH
OH
HO OH OH
Tellimagrandin I Ellagitannins Peel O

O O
O O
O
O
O
O
O
O
O
O
O O
O
O
O
Pelargonidin Anthocyanins Juice, peel O

H
O
H
O O
+
O
H
(continued)
Table 1 (continued)
Chemical Anatomical
H
Delphinidin Anthocyanins Juice, peel O

H
O
H O
O O
+
O
H
O
H
Cyanidin Anthocyanins Juice, peel O

H
O
H
O O
+
H
O
O
H
Catechin Flavanoids Juice, peel O

H
O
H
O O
H
O
O
H
Epicatechin Flavanoids Juice, peel O

H
O
H
O O
O
H
OH
Epigallocatechin 3- Flavanoids Juice, peel H

OH
gallate HO O
OH
O
H
OH OH
O
OH
OH
Quercetin Flavanoids Juice, peel O O

H
O
H O
O O
H
O
O
H
Kaempferol Flavanoids Peel O O

H
O
H
O O
O
H
Luteolin Flavanoids Peel O O
H
O O
H
O
O
H
Apigenin Flavanoids Leaf O O
H
O O
H
O
Juice, peel, flower

O
Gallic acid Organic acid O

O
O
O
O O
O O
O
O
O
O
Ellagic acid Organic acid Juice, peel, seed O
O
O
Punicic acid Organic acid Seed H

H
H
O
H H
H
O
Chlorogenic acid Organic acid Juice, peel H O O

H
O
H H
O H
O O
H
O
O H
H
(continued)
Table 1 (continued)
Chemical Anatomical
Quinic acid Organic acid Juice, peel O
H
H O O
H H
O O
O
H
Glucose Simple sugars Juice
Fructose Simple sugars Juice
Sucrose Simple sugars Juice
Source: Lansky and Newman [3]; Ambigaipalan et al. [43]
endothelial dysfunction. The study further delineates inhibition of vascular inflam-

mation and stress-mediated coronary DNA damage and stimulating Akt/endothelial
nitric oxide-synthase pathway to improve vasodilatory values as the fundamental
mechanism of pomegranate extracts in preventing coronary endothelial dysfunction
[9]. Oxidative stress-mediated myocardial infarction results in coronary muscle
atrophy and necrosis. Pomegranate fruit extracts have been found to present an
ameliorative effect in isoproterenol-toxicated myocardial infarction by attenuating
oxidative stress and level of cardiac enzymes markers, i.e., CK-MB and troponin
[48]. Clinical findings are therefore suggestive of pomegranate fruit juice, seeds, rind
extracts, and other derived products as potent source of antioxidants and nutrients
offering cardioprotective and therapeutic properties.
3.2 Pomegranate and Its Role in Cancer Chemoprevention
Cancer is one of the leading causes of death in global perspective, and it has been
reported that new cancer cases will increase by 70% at the end of 2030. Healthier
eating practices and dietary preferences toward more fruits and vegetable consump-
tion have been reported to reduce risks of cancer insurgencies and associated
mortalities [49, 50]. Pomegranate ranks among the fruits that carry highest concen-
tration of cancer chemopreventive and therapeutic biomolecules including
ellagitannins, flavonoids, and anthocyanins. Pomegranate polyphenols dispensed

either in the form of crude, purified fruit extracts or as fruit juice furnish anticancer
activities by inducing apoptosis, cell cycle arrest, antiangiogenesis, and anti-
mutagenesis activities [49].
Pomegranate anticancerous properties alike other chemopreventive agents have
been reported to be associated with antioxidant activities of biomolecules. Pome-
granate juice prospectively contains 2 g/L ellagitannins of which punicalagin
accounts 1500–1900 mg/L [51]. It’s among the reasons that pomegranate juice
adds three times higher antioxidant capacity in terms of inhibition of the production
of DNA oxidation products, reactive nitrogen species, lipid peroxidation, and in
scavenging reactive oxygen species [51–54].
Clinical findings are indicative of pomegranate biomolecules metabolites accu-
mulation in prostate tissues of prostate cancer patients administrated with 250 ml
pomegranate juice consecutively for a period of 4 weeks [55]. However, statistically
nonsignificant impact of pomegranate juice administration on cancer development
and progression markers was indicated probably due to shorter treatment duration.
Another 18-month study wherein patients were provided with 1 or 3 g pomegranate
extracts was found to extend prostate-specific antigen doubling time from
12–19 months and 12–17.5 months, respectively for low-dose and high-dose treated
groups [56]. Inflammation – one among the cancer initiation factors activates cancer
cells and induces DNA damages alongside epigenetic changes. Pomegranate as
juice, fruit extracts, and individually fractionated biomolecules exerts significant
impact on expression of inflammatory cell signaling protein in cancer cells [57]. A
study carried out in human colon cancer cell line indicated pomegranate juice,
pomegranate tannins, and punicalagin to significantly reduce expression of cyclo-
oxygenase – 2 (COX-2) expression. COX-2 proteins stand responsible for produc-
tion of prostanoids that induce inflammation [58]. Ellagic acid – another metabolite
of pomegranate ellagitannins has been reported to inhibit intestinal inflammation by
downregulating certain inflammation-mediating compounds such as COX-2 and
iNOS and blocking cell signaling pathways including NF-kB, p38 MAPK, IL6,
and STAT3 in tissues of the colon [59].
A study carried out by Mehta and Lansky [60] highlighted fermented pome-
granate juice carrying 10 μg/ml polyphenols to exert chemopreventive properties
by 90% suppression of mammary cancerous lesion formation [60]. Pomegranate
extracts possessing up to 200 μg/ml bioactive phenolic compounds have been
reported to reduce breast cancer cells viability and block mammary cancer cell
cycle progression [61]. Pomegranate as fruit and peel extracts exhibits skin
protection properties against UVA- and UVB-mediated reactions. Pretreatment
with pomegranate fruit extracts protects skin fibroblast from UV-mediated cell
death. A significant inhibition in production of UV-induced reactive oxygen
species and increment in intracellular antioxidant levels however can only be
achieved with too higher pomegranate polyphenols concentration, i.e., 500 to
10,000 mg/L [62, 63].
Albeit, a plethora of research have been undertaken to explore chemopreventive
and therapeutic properties of pomegranate against various types of cancers including
breast, colon, liver, bladder, skin, lung, and cerebral cancer, yet the bioavailability of
pomegranate bioactive compounds including ellagitannins, flavonoids, and antho-
cyanins in the target cells need to be determined. Furthermore, maximum tolerable
doses of combined extracts and individual compounds capable of delivering che-
mopreventive and therapeutic role and exact underlying anticancer mechanism are
yet to be explored.
3.3 Antidiabetic Properties of Pomegranate
Traditionally pomegranate fruit and associated structures alike flowers and leaves
were recommended in conventional therapy of diabetes [64]. Since last decade,
scientific work has consolidated effects of fruit in curing type 2 diabetes and its
complications. Oxidative stress-mediated damage of pancreatic β-cell is one of the
proposed mechanism involved in the onset of type 2 diabetes. Pomegranate being
the healing fruit owing to its free radical scavenging properties has the ability to
safeguard pancreatic β-cells from injury through neutralizing the effect of free
radicals [65, 66]. In human trials, fruit juice and derived products presented a
diabetes-preventive role that may be related with enhanced activity of antioxidative
enzymes such as paraoxonase 1 (PON1), reduction of lipid peroxidation, and
inhibition of inflammation mediator transcription factor NF-kB activation [67,
68]. Oral supplementation of pomegranate peel powder (200 mg/kg B.W) for
20 days substantially improved serum level of ALT and AST, glutathione and
SOD contents, hepatic glucose-6 phosphate dehydrogenase activity, and
regenerated pancreatic β-cells in streptozotocin-induced diabetic animal model
[69, 70]. In vivo and in vitro application of fruit extracts and their individual
components like punicalagin, punicalin, ellagic acid, and gallic acid exhibited
anti-glycation properties and reduced levels of glycation products alike serum
AGEs, hemoglobin A1c, and glycoalbumin [71]. Ellagic acid and its glycosides
have a profound effect on insulin-sensitizing activity through increasing gene
expression of PPAR-γ and GLUT4 that activate insulin signaling pathways for
the glucose uptake in L6 myotubes [72]. Phenolic-rich pomegranate peel and
flower extracts have been reported to offer α-glucosidase inhibitory activity with
IC50 value of 5.56 μg/ml and 29.77 μg/ml, respectively. The studies further suggest
ellagitannins to bind the active site of α-glucosidase surface rendering it inactive
and delaying carbohydrate digestion that ultimately facilitate in lower glucose
absorption [10, 73]. The fruit also exhibits ameliorative effect in treating diabetic
nephropathy via improving blood glucose level, kidney hypertrophy index,
strengthening glomeruli tubules, increasing basement membrane thickness, and
reducing renal disease markers [74, 75]. On account of abovementioned explor-
atory features, pomegranate, fruit, and peel-derived extracts and fractionated com-
pounds may be suggested as functional food and nutraceutical ingredient in
diabetes care.
3.4 Hepatoprotective Role of Pomegranate
The liver is principally the metabolic powerhouse of the human body that stands
responsible for the metabolization of nutrients and detoxification. Oxidative stress is
among the mechanisms involved in liver pathogenesis and progression of chronic
liver disease. Pomegranate fruit on account of its polyphenols including
hydrolysable tannins, anthocyanins, flavonoids, and compounds of alike structure
prevent progression of liver illnesses. Oral feeding of pomegranate (800 mg/kg B.W)
for 2 weeks have shown to protect sepsis-mediated acute liver injury. The study
further demonstrated free radical scavenging properties and anti-inflammatory activ-
ities of the fruit phenolics as the fundamental protection mechanism of pomegranate
phenolics to protect and allay the risks of acute liver injury. Pomegranate phenolics
consumed in the form of juice or extracts activate SOD and glutathione activity and
reduce serum malondialdehyde contents that further help in mediating TLR4 and
NF-kB inflammatory pathways [76].
Administration of pomegranate peel extracts rich in ellagitannins to animal
models with clinical signs of steatosis (fatty liver) showed considerable protective
effect on hepatic morphology, inhibited lipogenesis in cytoplasm of hepatocytes, and
improved liver enzymes [12]. Ellagic acid, one of the key pomegranate phenolic
attenuates alcohol-induced hepatotoxicity by reducing expressions of pro-fibrogenic
and pro-inflammatory cytokines like interleukins, TNF-α, and TGF-β that are
reported to increase in alcohol-induced liver fibrosis and inflammation [77, 78].
Pomegranate seed extracts alike peel extracts have been recorded to offer remarkable
protective effect against xenobiotics (e.g., CCl4)-mediated liver fibrosis. The mech-
anism might be correlated with secondary metabolites of ellagitannins like ellagic
acid, urolithins, increased free radical scavenging activity, and inhibition of TGF-β1
level and collagen synthesis [77, 79].
3.5 Pomegranate and Brain Health
Brain aging is generally recognized by irreversible loss in gross motor and sensory
neurons and associated physiological functions including cognition. Alzheimer’s
disease is a suboptimal neurodegenerative disorder symptomized with dementia and
cognitive impairment. Senile plaque formation through accretion of amyloid β
peptides and generation of neurofibrillary tangles are the hallmark lesions associated
with Alzheimer’s disease. Research findings have shown redox impairment to be
involved in the amyloid β-associated neurotoxicity [80, 81]. Fruits like pomegranate
bearing substantial free radical quenching properties could serve as a natural
neuroprotective agent. A study conducted in transgenic amyloid precursor protein
(APP) mutated mice models-associated diet consumption enriched with 4% pome-
granate juice to generate significant impact on boosting cognitive performance of the
animal models [82]. The study further witnessed pomegranate juice consumption to
anticipate improved memory, learning ability, and motor coordination and reduced
anxiety level in treated mice model as compared to standard diet-fed mice. In another
study wherein pomegranate peel extracts in combination with Gmelina arborea bark
extracts were found to present gamma secretase modulatory and acetylcholinesterase
inhibitory effect in reducing pathogenesis of Alzheimer’s diseases, it concluded that
the extracts derived from both plants offer neuroprotective effect in cognitive
dysfunction [83]. Human trials further endorsed consumption of pomegranate
juice (8 ounce/day) for a duration of 4 weeks to boost memory and brain function-
ality via strengthening brain antioxidants pool [84].
3.6 Anti-Inflammatory and Bone Health Promotion Properties
Inflammation is an undefined immune system hyperactivity that progressively trig-

gers transcription factors, i.e., TNF-α and NF-kB, that elevate level of pro-inflam-
matory cytokines like prostaglandin E2 (PGE-2) and interleukin (IL-1, IL-6, IL-8)
and overexcite production of reactive oxygen and nitrogen species [85, 86]. A recent
study reported that punicalagin A & B, ellagic acid and gallic acid possess anti-
inflammatory effect by exerting potent inhibitory effect on lipopolysaccharides
stimulated macrophages that downregulate COX-2 protein release from
RAW264.7 cells. Consequently, COX-2 protein expression inhibition reduces the
level of pro-inflammatory mediators alike inducible NO (iNO), PGE-2, interleukins,
and ROS [87]. Inflammation negatively affects bone health and compromises quality
of life through pannus formation and bone destruction that might consequently lead
toward disability. Long-term use of pharmacological preparations for bone rehabil-
itation alike cyclooxygenase inhibitor or nonsteroidal anti-inflammatory drugs
(NSAIDs) contributes in cardiac complications or gastrointestinal discomfort [88].
Botanical extracts like those derived from pomegranate are referred as potent carrier
of antioxidants and anti-inflammatory compounds that could further be exploited in
bone health restoration. Rheumatoid arthritis affecting about 0.5–1% world popula-
tion is a systematic autoimmune bone disorder of synovial joints. Higher infiltration
rate of inflammatory cells in synovial fluid of patients with rheumatoid arthritis
progresses toward irreversible cartilage and bone damage. Ellagic acid inhibits foot
paw edematous inflammation [89]. The study conferred ellagic acid to attenuate
etiology of adjuvant-induced arthritis (AIA)-linked pathogenesis by downregulation
of pro-inflammatory cytokines IL -1β, TNF-α, and IL 17 and upregulation serum
level of anti-inflammatory cytokines IL-10 and interferon γ (IFN-γ). Pomegranate
fruit extracts offer protection against osteoarthritis by improving cartilage stiffness
and physical fitness, decreasing level of cartilage catabolizing enzymes, and
strengthening antioxidative defense system [14]. Osteoporosis is the structural
deterioration of the microarchitecture array of the bone during remodeling phase
and progressively leads toward bone fragility and bone loss. Pomegranate peel
extract-enriched diet significantly improves bone mineralization by increasing
osteogenic transcription factors and stimulating activity of bone maker protein, i.e.,
alkaline phosphatase (ALP) [90]. Pomegranate seed oil (PSO) on account of its
punicic acid (conjugated linolenic acid)-rich profile offers osteblastogenesis devel-
opment and suppression of osteoclastogenesis in animal models fed with 5% PSO-
supplemented diet [91]. Consequent upon pomegranate antioxidants and anti-
inflammatory compounds bearing features, the fruit and its biomolecules can be
suggested as functional food to prevent bone loss and associated disorders.
3.7 Microbial Pathogenesis Inhibitory Role of Pomegranate
Multidrug-resistant bacteria, pandemic viruses, and opportunistic fungi are well-

known illnesses and life-threatening infections causing microorganism. Microbio-
logical pathogenesis and infections urge to find an alternative therapy like botanical
extracts for contending with widespread multidrug resistance among microorgan-
isms of human health concern. Phytonutrients including polyphenols, flavonoids,
tannins (condensed and hydrolysable), terpenoids, and phytosterols are extensively
explored and exploited for their potential antimicrobial activity [92, 93]. Aqueous
and methanolic extracts of pomegranate pericarp and rind holding up appreciable
concentration of anthocyanins, punicalagin, ellagic acid, gallic acid, and other poly-
phenols have been widely explored for antimicrobial efficacy and in treating a wide
range of contagions [6, 94]. Pomegranate peel methanolic extract (80%) presents
inhibitory effect against hemorrhagic Escherichia coli, Yersinia enterocolitica,
Staphylococcus aureus, and Listeria monocytogenes with minimum inhibitory con-
centration (MICs) ranging between 6 and 12 mg/ml [95, 96]. The toxicity of these
phytonutrients against bacterial cell is due to complex formation with enzymes
cofactors and sulfhydryl group of proteins that alter permeability of membrane and
disturb respiratory chain [97, 98]. In vitro and in situ application of 80% methanolic
extracts of pomegranate whole fruit, seed, and rind presented antifungal activity
against Penicillium citrinum, Aspergillus niger, Trichoderma reesei, Rhizopus
oryzae, and Mucor indicus [99]. The crude extract of pomegranate rind shows
cytotoxicity at conidial and hyphal stage against dermatophytes, e.g., Microsporum
canis and Trichophyton mentagrophytes, with MICs values of 250 μg/ml and
125 μg/ml, respectively. Polyphenols including punicalagin, catechin, and epigallo-
catechin being potential antifungal agents induce precipitation of microbial cell
membranous proteins and intercellular leakage and alter composition of cytoplasm
and outer cell membranes that inhibit fungal growth [100, 101]. Pomegranate
hydroalcoholic extracts have also been reported to offer antiviral activity against
human immunodeficiency virus type 1, herpes simplex virus 1, influenza A, adeno-
virus, and hepatitis C virus. The possible mechanism behind viral cell damage is a
complex formation between condensed tannins and viral glycoprotein that affect
viral attachment with host cell. This further suppresses viral transcription/translation
and prevents viral replication [102–104].
3.8 Anthelmintic Activities of Pomegranate
Parasites (ecto- and endoparasites) depend on host for food and shelter rendering a
compromised immune system to host, increased disease burden in human and
domestic animals, and huge economic loss. Crude pomegranate rind, bark, and
root extracts gained a lot of attention in curing parasitic infections since ages
[105–107]. Pomegranate fruit extracts have been explored as anti-cestoda,
vermifugal, and antiprotozoan mainly due to its tannins, triterpenes, sterols, glyco-
sides, and alkaloids [108, 109]. Oral administration of 40 mg/ml of pomegranate rind
crude extract to animals infested with tapeworms (Raillietina spiralis) and round-
worms (Ascaridia galli) induced parasites paralysis and reduced death time in
comparison with anthelmintics piperazine and albendazole. Ethanolic and aqueous
extracts of pomegranate exhibit ovicidal and larvicidal properties against
Gastrothylax indicus and Hymenolepis nana manifesting it a novel source of anthel-
mintic agent [7, 110, 111]. Pomegranate peel extracts show protective role against
Plasmodium-induced parasitosis (malaria). Alongside its hepatoprotective effects,
pomegranate extracts reverse signs of anemia provoked by plasmodium infestation
through the hemoglobin, and erythrocytes count back to the normal level [112, 113].
3.9 Gut Health Promotion Properties of Pomegranate
Gut microbiota represent complex and mutualistic interplay of the human’s largest
microbial biome that considerably influence homeostasis and healthy conditions of
the host. Dietary and other nutritional interventions have been shown to exert a
modulatory effect on the composition of gut microbiota. Polyphenols-rich pome-
granate juice and fruit extracts demonstrate beneficial effects on gut health and
microbiome. The gut microbes transform intact phenolic compounds, e.g.,
ellagitannins and anthocyanins, into bioactive metabolites such as ellagic acid and
urolithins. Intact ellagitannins and anthocyanins act as prebiotic and have a syner-
gistic effect in promoting probiotics properties of Lactobacilli and Bifidobacteria. It
further inhibits growth of pathogenic microbes and preserves gut microbiome
balance [114, 115]. Incorporation of pomegranate peel at 10% of the animal diet
exhibit modulatory effect on sodium oxide dismutase 1 (SOD1) and glutathione
activity and reduced level of malondialdehyde (MDA) in the small intestine of the
ruminant as compared to control diet. Pomegranate polyphenols particularly
ellagitannins, punicalagin, and ellagic acid attenuate small intestine lipid peroxida-
tion by strengthening free radicals (•O2¯ and H2O2) scavenging system and regulat-
ing enzymatic antioxidative pathways as the first-line defense system of the small
intestine [116]. Pomegranate peel extracts and seed oil prevent chronic inflammation
and necrotizing enterocolitis-mediated intestinal damage. Pomegranate polyphenols
especially ellagitannins protect from ulcerative colitis through reducing ERK1/2
activity by downregulation of mTOR downstream pathway and inhibit production
of pro-inflammatory cytokines at molecular level [117, 118]. Pomegranate seeds
have been reported to be exploited as traditional remedy for the management of
diarrhea. Antidiarrheal activity is also supported by the findings of Souli et al. [2]
suggesting methanolic extracts of pomegranate juice to significantly reduce gastro-
intestinal transit time and number of defecation against castor oil-induced diarrhea in
dose-dependent manner. Pomegranate seed oil is a rich source of minerals that might
help in regulating mineral balance in diarrheal episodes. Hydrolysable tannins offer
protein denaturation properties and form a protein-tannate complex that encourages
more resistance to intestinal mucosa, inhibits gastrointestinal transit, and reduces
excretion favoring its antidiarrheal activity [119, 120].
3.10 Role of Pomegranate in Modulating Renal Disorders
The importance of kidney function in clearing blood wastes is very evident from the
fact that they receive 20% of total blood supplies with each cardia beat to clear the
waste. Kidneys eliminate xenobiotics including endogenous metabolites, exogenous
chemicals, and drugs through urine and prevent loss of nutrients such as glucose,
inorganic ions, and oligopeptides. While in elimination and detoxification, kidneys
are utmost exposed to xenobiotics that might cause oxidative stress, nephrotoxicity,
and lipid peroxidation [121, 122]. Pomegranate aril and peel extracts show protective
effect against nitrosamine and CCl4-induced apoptosis and kidney injury via
inhibiting generation of reactive oxygen species, lowering lipid peroxidation, and
enhancing host antioxidative defense mechanisms [123]. Cisplatin – a platinum
based inorganic drug – is widely used in the treatment of carcinoma. The drug is
reported to cause renal functions impairment through ROS generation, inhibition of
protein synthesis, and mitochondrial dysfunction. Pomegranate seed oil being rich in
polyunsaturated fatty acids and antioxidants has been suggested as potent dietary
approach to avert the risks of cisplatin-induced kidneys dysfunction. Pomegranate
seed oil also reduces serum urea, renal nitric oxide, and malondialdehyde levels and
upregulate thiol contents [124]. Use of pomegranate accessions like flower extracts
(25 mg/kg b.w.) also improves gentamicin-induced weight loss and renal toxicity
[125]. Pomegranate juice consumption by the heavy smokers has also shown
remarkable ameliorative effects against nicotine-induced hepatorenal toxicity [126].
Pomegranate juice and peel extracts orally administrated to animal models at the rate
of 3 ml/kg b.w. and 200 mg/kg b.w., respectively, for a period of 8 weeks substan-
tially reduced cytokines markers and significantly improved steroid-induced kidney
inflammation [11]. The clinical trials on exploring renal protective role of pomegran-
ate demonstrate bioactive constituents of the fruit and waste fraction as precursors to
activate immune system and enhance cellular antioxidative enzymes production that
protect kidneys from inflammation-induced injury and dysfunction [4].
3.11 Pomegranate Perspective Potential in Improving Fertility
Pomegranates have potent beneficial effects in strengthening reproduction through

improving spermatogenesis and semen production, maturation of epididymal
spermatozoa, and increasing blood testosterone level [13]. Consumption of phytos-

terols, punicalagin, quercetin, ellagic acid, and vitamin A-rich natural beverages
alike pomegranate fruit juice improves sperm cell abnormalities induced by oxida-
tive stress, bisphenol A, CCl4, and lead acetate toxicity [127, 128]. Pomegranate
juice significantly improves changes in female sex hormone levels and reduces
symptoms of polycystic ovarian syndrome [129]. Oral administration of iso-
flavonoid-rich extracts of pomegranate rind to the animal models at 0.5 g/kg b.w.
increased fertility through improved sperm count, sperm motility, and sperm quality.
Cautious intake of pomegranate whole fruit extracts up to 5 mg/mL avoiding higher
doses has potential to promote fertility [130, 131].
3.12 Wound Healing Properties of Pomegranate Accessions
Wound healing is a distinct process of biochemical and cellular events that involves
immediate inflammation after injury to achieve homeostasis, tissue granulation, and
collagen formation that imparts tensile strength in the remodeling phase of skin
regeneration [132, 133]. Pomegranate pericarp and epicarp are historically used in
folk medicines for their ameliorative effects in various ailments including excised
and incised wound healing. Biochemical and histological examination revealed that
pomegranate comprises tremendous antimicrobial and antioxidant features that aid in
epithelialization and production of hydroxyproline to regenerate wounds [134, 135].
Pomegranate pericarp and epicarp extract-based ointment and gels have been exten-
sively explored in the recent past to calculate their wound healing potential. The
findings of the studies suggest that topical application of pomegranate polyphenols
and lipophilic fractions-based ointments on cutaneous wounds (sores and lesions)
result in significant wound recovery notably in diabetic patients [136, 137]. Topical
administration of 100 mg/kg pomegranate extracts for a period of 15 days to incised
wounds in experimental animals reduces 95% wound area as compared to 85% in
control petroleum jelly-based ointment [138]. Pomegranate extracts (10–20%) when
topically applied to burn wounds have also significantly reduced period of epitheli-
alization analog to standard drug [139]. Pomegranate pericarp and epicarp extracts
contain effective bioactive ingredients that aid in skin epithelialization and regener-
ation and may be recommended as a natural remedy in wound healing.
3.13 Cosmo Care Properties of Pomegranate
The skin is the most targeted and exposed site for pathogenesis from exogenous
factors especially ultraviolet radiations (UVA and UVB) and microbiological infec-
tions. Exogenous factors like UV radiations induce genotoxicity and oxidative stress
consequently creating skin disorders including immunosuppression, skin carcinoma,
sunburn, and photoaging [140]. Pomegranate a mystical fruit famed as a pharmacy
unto itself owing to its polyphenolic compounds, hydrolysable tannins, and organic
acid fractions has a tonic effect in skin care [141, 142]. Fluorescence microscopy and
anisotropic measurements present skin-protective effect of pomegranate seed oil and

peel extracts nanoemulsions entrapped with punicic acid and polyphenol-rich ethyl
acetate fraction against reactive oxygen species and UVB-mediated DNA and lipid
bilayer biomembrane damage [143, 144]. Acne – a plaguing skin disorder – is
recognized by sebaceous glands hyperactivity and inflammation leaving scars due
to loss in equilibrium of symbiotic skin microbiota. Pomegranate fruit rind and bark
rich in hydrolysable tannins (especially punicalagin) exhibit multiple anti-acne
abilities including blood purification, antibacterial, anti-inflammatory, and anti-
keratinocyte proliferation activity [145, 146]. Owing to photoprotective and eco-
friendly effects of pomegranate, seed oils and rind extracts of the fruit can be utilized
in skin beautification, combination cosmo care products, and as skin protection
factor sunscreen [20, 147].
3.14 Pomegranate Role in Maintaining Oral Hygiene
Orodental health is mostly affected by plaque-forming biofilms and cariogenic

bacteria that seek habitat in oral cavity. Several chemotherapeutic agents including
chlorhexidine, bisbiguanides, and fluorides containing mechanical floss and gels
have been formulated to ensure orodental hygiene [148]. However, long-term use of
such chemotherapeutic agents develops resistance in bacterial strains and promotes
tooth staining. Such a situation calls for the development of herbal products as an
alternative therapy to orodental chemotherapeutic agents [149]. In vivo and in vitro
studies have confirmed flavonoids, tannins, vitamins, and minerals-rich fractions of
pomegranate as anticariogenic and anti-plaque natural formulations. Pomegranate
polyphenols have strong tendency toward inhibiting growth of plaque-forming
bacteria including Streptococcus sanguis and Pseudomonas aeruginosa [150,
151]. Use of pomegranate mouthwash effectively reduced gingivitis (gum bleeding)
and periodontitis (gum infection) in comparison with 0.2% chlorhexidine standard
mouth rinse [15, 152]. Topical application of oral gel containing 10% pomegranate
extracts relieves pain and reduces time for wound healing in aphthous stomatitis
(oral ulcer) disorder [153]. Pomegranate fruit extracts efficiently reduce production
of bacterial secondary metabolites, sucrose catabolizing enzyme α-glucosidase
activity, aspartate aminotransferase activity, and upregulation of ceruloplasmin that
inhibits oral oxidative stress [93, 154]. Promising features of pomegranate extracts
against several orodental distresses encourage development of pomegranate and its
extracts-based natural oral care formulations as alternate to synthetic and chemo-
therapeutic products.
4 Conclusions
Clinical trials in human subjects and animal models have demonstrated significant
effect of pomegranate consumption in improving the body’s inherited defense
against various forms of infections, inflammatory, and non-inflammatory disorders.
In comparison with the fruits of the same and other classes, pomegranate has shown
broader applicability against serious maladies. Unlike synthetic drugs, heteroge-
neous biochemical composition of pomegranate and its extracts has ability to
manipulate multiple biochemical pathways that can further be exploited for the
treatment of various complex disorders. Controlled and clinical trials on pomegran-
ate and its hydroalcoholic extracts have been regarded as safe for human health at
orally administrated doses supplying up to 2000 mg polyphenols per day. However,
exploitation of pomegranate and derived bioactive compounds as therapeutic agent
either alone or in combination calls for careful experimentation to rule out genotoxic
response of the plant material.
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Part VII
Functional Foods
Antidiabetic Functional Foods with
Antiglycation Properties 43
Mutiu Idowu Kazeem, Habeeb Adebodun Bankole,
Azeez Ayomide Fatai, Abiola Fatimah Adenowo, and
Theophilus Clavell Davies
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285
2 Formation of Advanced Glycation End Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286
3 Antiglycation Agents from Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
3.1 Banana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
3.2 Cornelian Cherry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290
3.3 Guava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1291
3.4 Apples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1291
3.5 Bael . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292
3.6 Bitter Melon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292
3.7 Common Juniper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292
3.8 Grapevine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293
3.9 Saucer Berry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293
3.10 Lingonberry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293
3.11 Longan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294
3.12 Passion Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294
3.13 Pomegranate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294
3.14 Shaddock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1295
3.15 Rambutan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1295
3.16 Water Apple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296
3.17 Kokum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296
M. I. Kazeem (*) · H. A. Bankole · A. A. Fatai

Department of Biochemistry, Faculty of Science, Lagos State University, Ojo, Lagos, Nigeria
A. F. Adenowo
Department of Biochemistry and Microbiology, University of Zululand,
Kwadlangezwa, South Africa
T. C. Davies
Department of Geology, University of Nigeria, Nsukka, Nigeria

1284 M. I. Kazeem et al.
4 Antiglycation Agents from Beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296

4.1 Black Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296
4.2 Green Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297
4.3 Luobuma Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297
4.4 Yerba Mate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1297
4.5 Dendrobii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1298
5 Antiglycation Agents from Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1298
5.1 Mung Bean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1298
5.2 Buckwheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1299
5.3 Sickle Senna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
6 Antiglycation Agents from Vegetables and Spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
6.1 Black Nightshade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
6.2 Sangwhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
6.3 Turmeric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1301
6.4 Lotus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1302
6.5 Ginger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1302
6.6 Cumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1303
6.7 Clove . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304
7 Possible Mechanisms of Antiglycation by Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1304
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1305
Abstract
Diabetes mellitus is a disease that requires long-term management and sometimes
last throughout the lifetime of the patient. Persistent hyperglycemia experienced
in diabetes over long period results in diabetic complications, such as nephrop-
athy, neuropathy, retinopathy and cardiovascular diseases. One of the mecha-
nisms involved in the development of diabetic complications is glycation which
caused the production of advanced glycation end products (AGEs). Therefore,
any agent that prevents the formation of AGEs may be suitable for the manage-
ment of diabetes and its complications. There is a plethora of studies on the
antiglycation properties of many foods and their products, but there is no repos-
itory of their information. This is an attempt to review the available information
on the role of antiglycation agents from functional foods in the management of
diabetic complications. We hope this information will assist diabetic patients in
the choice of their diets and stimulate further research on these foods.
Keywords
Glycation · Advanced glycation end products · Functional foods · Diabetes ·
Diabetic complications
AGE Advanced glycation end products
BSA Bovine serum albumin
GOLD Glyoxal-lysine dimer
MOLD Methyglyoxal-lysine dimer
RAGE Receptor for advanced glycation endproduct
43 Antidiabetic Functional Foods with Antiglycation Properties 1285
1 Introduction
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia due to

defects in insulin secretion and/or action [1]. It causes alteration in the carbohydrate,
lipid, and protein metabolism of its sufferers leading to a variety of micro- and
macrovascular complications, which include nephropathy, neuropathy, and retinop-
athy [2]. Diabetes is a global problem affecting 382 million people in 2013, and this
number is projected to increase to 572 million in 2035 [3]. The disease caused 5.1
million deaths and more than 21 million live births were affected by it during
pregnancy, making it the third “killer” of mankind after cancer and cardiovascular
diseases [3].
Persistent hyperglycemia experienced by diabetic patients promotes an unhealthy
interaction between the excess blood glucose and some proteins like collagen,
hemoglobin, and lens crystallins [4], and this is called glycation. Glycation is a
spontaneous nonenzymatic reaction between the carbonyl group of reducing sugars
and amino group of amino acids, peptides, or proteins to form a freely reversible
Schiff base that rearranges to a more stable ketoamine called Amadori products [4].
This is then followed by complex series of reactions such as condensation and
oxidative modification to produce several heterogenous products known as
advanced glycation end products (AGEs) [5] (Fig. 1). Glycation is also referred to
as nonenzymatic glycosylation or Maillard reaction because the process was first
described by Louis Camille Maillard in 1912 [6].
Fig. 1 Chemistry of glycation reaction to form advanced glycation end products [6]
The formation of AGEs may alter the physicochemical properties of the proteins,
and in turn adversely affect their functional properties [7]. Excessive generation of
AGEs is implicated in several pathological conditions, such as cardiovascular
diseases, neurodegenerative diseases, inflammation, aging, and diabetes mellitus
[4]. Previous studies have also established the role of AGEs in the development of
diabetic complications [8–10]. Therefore the mitigation of protein glycation will be
an effective medium of preventing or ameliorating these complications. There is a
continuous demand for agents with antiglycation properties, as they may be useful in
preventing diabetic complications [11, 12]. Though some synthetic antiglycation
agents like aminoguanidine and pyridoxamine have been discovered, they have
serious toxicity issues [8]. Therefore, food-derived antiglycation agents will be
more appropriate in the management of diabetic complications due to their safety
and availability to the general populace.
This review attempts to compile a repository of plant foods, which have been
validated to display antiglycation potential (Table 1). This will serve as a guide to
future references for researchers in institutions and pharmaceutical industries, for
further studies aimed at isolation of their active components and large-scale produc-
tion. It will also assist diabetics and their families in the choice of foods that will be
consumed, in order to ameliorate the debilitating effects of diabetes and its
complications.
2 Formation of Advanced Glycation End Products
AGEs are complex, heterogeneous molecules that cross-link proteins, exhibit brow-
ning, and generate fluorescence. The generation of reactive intermediate products,
such as α-dicarbonyls, α,β-unsaturated aldehydes, dialdehydes, and keto-aldehydes,
is very important in the formation of AGEs [6]. α-Dicarbonyls include
3-deoxyglucosone, glyoxal, and methylglyoxal. 3-Deoxyglucosone is formed by
nonoxidative rearrangement as well as hydrolysis of Amadori product, and by
fructose-3-phosphate. It rapidly reacts with protein amino groups to form AGE
such as imidazolone, pyrraline, and carboxymethylysine [4, 13]. Methyglyoxal can
be produced nonenzymatically from the spontaneous decomposition of triose phos-
phates, autoxidation of carbohydrates, glucose degradation, and also by several
minor metabolic pathways, including the Maillard reaction [10]. In addition to
reaction with arginine residues to form imidazolone adducts, methyglyoxal reacts
with lysine residues in protein to form carboxyethylysine and the imidazolium
crosslink, methylglyoxal-lysine dimer (MOLD).
Due to the variety of AGEs, they are broadly classified on the bases of their
formation into three:
(i) Fluorescent cross-linking AGEs, e.g., pentosidine and crossline

(ii) Nonfluorescent cross-linking AGEs, e.g., Glyoxal-lysine dimer (GOLD) and
methyglyoxal-lysine dimer (MOLD)
(iii) Noncross-linking AGEs, e.g., carboxymethylysine and pyralline
Table 1 List of functional foods with antiglycation potential

Name Part Extract Test Method(s) Ref(s)
Bael Fruits Methanol In vitro BSA-glucose [14]
Apple Fruits Water In vitro BSA-fructose [15]
Banana Inflorescence Methanol In vitro BSA-glucose [16]
Inflorescence Ethanol In vitro BSA-fructose [17]
Flower Diet In vivo STZ-diabetic rats [18]
Black tea Leaves Water In vitro BSA-glucose [19]
Buckwheat Hull Water In vitro BSA-glucose [20]
Seed Ethanol In vitro BSA, carbonyl level [21]
Shaddock Fruit pulp Methanol In vitro BSA-fructose [22]
Cornelian cherry Fruit Water In vitro BSA-fructose [23]
Fruits Water In vivo STZ-diabetic rats [24]
Fruits Glycosides In vivo STZ-diabetic rats [25]
Fruits Morroniside In vivo STZ-diabetic rats [26]
Cumin Seeds Water In vitro/ BSA/STZ-DM rat [27]
vivo
Seeds Methanol In vitro/ BSA/STZ-DM rat [12]
vivo
Seeds Cymene In vitro/ BSA/STZ-DM rat [28]
vivo
Dendrobii Whole plant Polysaccharides In vitro BSA-glucose [29]
Black nightshade Leaf Ethanol, water In vitro BSA, carbonyl level [30]
Ginger Rhizome Ethylacetate In vitro BSA-glucose [31]
Rhizome Polyphenol In vitro BSA-glucose [32]
Rhizome Polyphenol In vivo STZ-diabetic rats [33]
Guava Leaf Water In vitro BSA, fructosamine [34]
Leaf Water In vitro Plasma glycation [35]
Leaf Methanol In vivo STZ-diabetic rats [36]
Leaf Methanol In vivo STZ-diabetic rats [37]
Yerba mate Leaf Water In vitro BSA-methglyoxal [38]
Common juniper Fruits Essential oil In vitro Insulin glycation [39, 40]
Longan Fruits Polysaccharides In vitro BSA-glucose [41]
Lotus All parts Methanol In vitro BSA glucose [42]
Saucer berry Whole plant Compounds In vitro BSA glucose [43]
Mung bean Seed Ethanol In vitro BSA glucose [44]
Passion fruit Leaf Water In vitro BSA glucose [45]
Pomegranate Fruit Polysaccharides In vitro BSA glucose [46]
Grape vine Fruit Water In vitro BSA, carbonyl level [47]
Water apple Leaf Ethanol In vitro BSA glucose [48]
Turmeric Rhizome Ethylacetate In vitro BSA glucose [49]
Lingonberry Fruit Ethanol In vitro BSA glucose [50]
Bitter melon Fruits Capsule In vivo Diabetic subjects [51]
Green tea Leaf Ethanol In vivo STZ-diabetic rats [52]
(continued)
Table 1 (continued)
Name Part Extract Test Method(s) Ref(s)
Rambutan Fruit Ethanol In vitro BSA glucose [53]
Fruit Geraniin In vitro BSA glucose [54]
Sickle senna Seeds Glucosides In vitro BSA glucose [55]
Seeds Anthraquinones In vitro BSA glucose [56]
Kokum Fruits Garcinol In vitro BSA fructose [57]
Luobuma tea Leaves Flavonoids In vitro BSA glucose [58]
Sangwhang Fruiting body Isolates In vitro HbA1c formation [59]
Clove Buds Water In vitro BSA-fructose [60]
However, AGEs can also be classified on the basis of their lifespan into long-
lived and short-lived molecules. In addition, most AGEs are formed endoge-
nously but some can also be derived from exogenous sources such as foods and
tobacco [9].
3 Antiglycation Agents from Fruits
3.1 Banana
Banana (Musa paradisiaca) is an herbaceous plant used as foods by man worldwide.

The flower and pseudostem are used for the culinary purpose in some countries [61],
while the fruit is consumed raw, cooked, or fried in all parts of the world. The leaves
are also used in the wrapping of foods which adds characteristic aroma to the food
and prevents burning. The flower and inflorescence have been reported with anti-
diabetic activity [62, 63]. Methanolic extract and different solvent fractions of
banana inflorescence were evaluated for their antiglycation activities using the
BSA-glucose antiglycation model [16]. Methanol extract as well as ethanol, water,
and butanol fractions displayed good antiglycation activities with IC50 44, 31, 55,
and 68 μg/mL respectively, and are comparable to standard antiglycation agent,
aminoguanidine (IC50: 61 μg/mL).
Also, the antiglycation effect of ethanol extract of banana flower and two
compounds isolated from it was investigated [17]. Ethanol extract of banana flower,
lupeol (Fig. 2a), and umbelliferone (Fig. 2b) inhibited the formation of early-stage
glycation product (fructosamine) by 85–90% and protein carbonyl compounds by
80–86%. However, fluorescence study on the late stage glycation moieties (AGEs)
showed that the extract and compounds inhibited it by 71–82%. The rate of
inhibition by this extract and compounds were significantly higher than the standard,
aminoguanidine.
To ascertain the antiglycation effect of banana in animal model, the effect of
banana flower and pseudostem on serum fructosamine and advanced glycation end
Fig. 2 Structures of some antiglycation compounds from fruits. (a) Lupeol; (b) Umbelliferone;
(c) Morroniside; (d) Loganin; (e) 7-O-galloyl-D-sedoheptulose; (f) Protocatechuic acid; (g)
Rosmarinic acid; (h) Quercetin-3-O-galactoside; (i) Geraniin; (j) Myricetin-3-O-rhamnoside;
(k) Phloretin; (l) Garcinol
products (AGEs) in streptozotocin-induced diabetic rats was reported [18]. Supple-

mentation of Wistar rat’s diet with banana flower and pseudostem significantly
decreased the serum fructosamine levels (2.15 and 2.37 moles/mg protein) compared
with diabetic untreated rats (3.19 moles/mg protein). The concentration of serum
AGEs was also reduced by 61.5% and 51.2% in the banana flower and pseudostem
fed rats compared with the diabetic control. This showed that banana exhibited
antiglycation property in both in vitro as well as in vivo model, and this property
may be due to the presence of umbelliferone and lupeol.
3.2 Cornelian Cherry
Cornelian cherry (Corni fructus or Cornus officinalis) is a common plant in China,

Korea, and Japan, where it found usage as a functional food and as a component of
traditional medicine [25]. Several studies have been carried out on the in vivo
antiglycation potential of Cornelian cherry in rats. The beneficial effect of Cornelian
cherry on AGE-mediated renal injury in streptozotocin-induced diabetic rats was
reported [24]. Administration of 100 and 200 mg/kg of Cornelian cherry extract to
diabetic rats for 10 days significantly reduced the renal AGE level to 5.32 and
4.92 IU compared with 6.19 IU in the diabetic control group. The renal AGE level
of the diabetic rats administered 200 mg/kg cornelian cherry was similar to the group
that received standard antiglycation agent, aminoguanidine.
Administration of 20 mg/kg iridoid glycoside and low molecular weight poly-
phenol fractions of Cornelian cherry to streptozotocin-induced diabetic rats for
10 days also showed that the iridoid glycosides fraction significantly reduced
serum glycosylated protein (19.7 nmol/mg protein) and renal AGE (3.90 IU) com-
pared with diabetic control (21.5 nmol/mg protein and 4.45 IU respectively), while
the polyphenol fractions caused downregulation of receptor for advanced glycation
end products (RAGE) to a value of 1.58 and 1.21-fold lower compared
with the normal rats [24]. The active components of the iridoid glycoside fraction
were identified to be morroniside (Fig. 2c), loganin (Fig. 2d), mevaloside,
5-hydroxymethyl-2-furfural, and loganic acid while the only component of the
polyphenol fraction was 7-O-galloyl sedoheptulose (Fig. 2e).
In order to identify the compounds responsible for the antiglycation property of
this plant, aqueous extract of Cornelian cherry and some of its active compounds
(loganin, morroniside and 7-O-galloyl-D-sedoheptulose) were tested for their
antiglycation activities using BSA-fructose model [23]. Out of this compounds,
only 7-O-galloyl-D-sedoheptulose displayed good antiglycation activity (61.90%)
at 25 μg/mL. Elsewhere, another component of the cornelian cherry fraction,
morroniside, was also evaluated for its protective effect on renal damage in
streptozotocin-induced diabetic rats [26]. Administration of 100 mg/kg of
morroniside to diabetic rats for 20 days caused a significant reduction in the
level of serum glycosylated protein (HbA1c) and thiobarbituric acid reactive sub-
stances, compared with the diabetic control. It also reduced the expression of AGE-
related proteins such N-carboxylmethylysine and RAGE of the diabetic rats. It,
therefore, implies that cornelian cherry exhibited antiglycation potential, which
may due to the presence of its phytochemicals, 7-O-galloyl-D-sedoheptulose and
morroniside.
3.3 Guava
Guava (Psidium guajava L.) is a widely cultivated plant in tropical and subtropical
countries. The fresh fruit is eaten raw while the combination of its dried leaves and
fruits are used in the preparation of drink known as guava tea [36]. Several
compounds have been isolated from different parts of this plant, which include
guavanoic acid, guavacoumaric acid, β-sitosterol-3-O-β-D-glucopyranoside, and
jacoumaric acid [64]. The antiglycation potential of ethylacetate fraction of guava
leaf extract in streptozotocin-induced diabetic rats was investigated [36]. Oral
administration of 100 mg/kg of ethylacetate fraction of guava leaf extract to diabetic
rats for 30 days caused about a two-fold reduction in the plasma glycated hemoglo-
bin (HbA1c) (3.94%) compared to the diabetic control group (7.62%). Significant
decrease was also witnessed in the serum fructosamine level of the guava-extract fed
diabetic rats (227.97 μmol/L) compared with diabetic untreated group (258.30 μmol/L).
This report is also corroborated by a similar study [37].
In order to evaluate the in vitro antiglycation effect of guava, guava leaf extracts and
some of its active compounds were tested on α-dicarbonyl compounds-induced blood
coagulation [35]. Guava leaf extract (0.001 mg/mL) significantly inhibited hyper-
coagulation induced by methylglyoxal but does not have an effect on the prothrombin
clotting time. Guava leaf extract and its active compounds (gallic acid, ferulic acid, and
quercetin) also protected antithrombin III from methylglyoxal-induced loss of activity.
Guava leaf extract at the concentration of 50 μg/mL also exhibited strong inhibition
(over 90%) of α-dicarbonyl compounds formation [34]. Catechin, gallic acid, and
quercetin (from the guava extract) also displayed over 80% inhibitory effect on α-
dicarbonyl compounds formation while ferulic acid did not. Both the extract and active
compounds of guava leaf inhibited the formation of Amadori products (fructosamine)
and AGEs from albumin in the presence of glucose. This α-dicarbonyl compounds
formation was confirmed when the ethyacetate extract of guava leaf exhibited an IC50
value of 38.95 μg/mL for the inhibition of protein glycation, which is similar to the
activity of standard antiglycation agent, aminoguanidine (IC50: 33.82 μg/mL) [36].
Therefore, guava is a potent source of antiglycation agent, which may be due to the
presence of phytochemicals such as gallic acid, quercetin, and ferulic acid.
3.4 Apples
Apples (Malus domestica) are one of the most important fruits in the world that are
rich in phytochemicals, which include catechins, epicatechin, chlorogenic acid,
p-coumaric acid, and protocatechuic acid [65]. Though apple is eaten raw as food,
it is also processed into different products such as juice, nectar, and puree due to its
health-promoting potentials. In a BSA-fructose antiglycation assay, aqueous extract
of apple and green-tea fortified apple displayed anti-AGE formation activities of 15
and 48 mg/kg dry weight, respectively [15]. This implies that aqueous extract of
apple displayed good antiglycation potential in-vitro, however fortification of apple
with green tea increased its ability to inhibit AGEs formation.
3.5 Bael
Bael (Aegle marmelos L. Corr.), also known as golden apple, bael apple, wood
apple, and stone apple, is native to India. It is a commonly used food with varieties of
biological properties [66]. Several compounds isolated from the bael fruits, include
aegelin, eugenol, marmelosin, lupeol, and fagarin [67]. It also contains tartaric acid,
flavon-3-ols, anthocyannins, and flavonoid glycosides [68]. The fresh and dried
fruits are used in the preparation of lemonade-like “soft drink” [14] while the leaves
are eaten as salads. Methanolic extract of bael apple exhibited strong antiglycation
potential with an EC50 value of 60.14 μg/mL in a BSA-glucose antiglycation
experiment [14]. This suggests that consumption of bael fruits could help in the
prevention and or amelioration debilitating effect of diabetes and its complications.
3.6 Bitter Melon
Bitter melon (Momordica charantia L.) is a plant which is consumed as food and used
as an ingredient in some Asian curries. The stem and fruits could be a source of teas
while the fruits are eaten raw mostly in Asia [69]. It is one of the popular plants used in
the management of diabetes mellitus and its complications in different parts of the
globe [70]. Several compounds have been isolated from its fruit, which includes
charantin, momordicins, cucurbitacin, gentisic acid, and diosgenin [69]. A pilot
study on the hypoglycemic and antiglycation potential of bitter melon was investigated
in type 2 diabetic patients for 16 weeks [51]. The study involved the consumption of
6.0 g dried fruit powder of bitter melon daily by the diabetic patients throughout the
duration of the experiment, which resulted in significant reduction in the levels of
blood glucose, glycated hemoglobin (HbA1c), and serum AGEs of the patients by
9.49%, 6.69%, and 6.65%, respectively. It implies that consumption of bitter melon
fruit can ameliorate diabetes mellitus and prevent the onset of its complications.
However, there is a need for in vitro evaluation of the antiglycation activity of bitter
melon and subsequent bioassay-guided fractionation of its active components.
3.7 Common Juniper
Common juniper (Juniperus communis and oblonga) is an evergreen shrub which is

endemic to Europe, Asia, and North America. Common juniper is used as spice for
food such as meat and as flavor in the preparation of alcoholic beverages [71].
Inhibition of protein glycation by essential oils of branchlets and fruits of common
juniper was investigated [39]. Essential oils from the branchlet of male tree, female
tree, and fruits inhibited the glycation of hemoglobin at all concentrations (200, 400,
and 600 μg/mL) tested. However, the rate of inhibition of glycation is inversely
proportional to the concentration of the oils which accounted for the highest inhib-
itory activities occurring at the lowest concentration (89.9%, 74.7%, and 62.8% for
male branchlet, female branchlet, and fruit oils, respectively). Essential oil from the
fruit also displayed its highest inhibitory activity on insulin glycation at 200 μg/mL
(78.4%), while that of the male (64%) and female branchlet (81%) inhibited it most
at 600 μg/mL [40]. The antiglycation activity of common juniper may be due to the
variety of components present in its essential oil. These include α-pinene, limonene,
α-thujene, terpinen-4-ol, and isoterpinolene [39].
3.8 Grapevine
Grapevine (Vitis vinifera L.) is one of the largest food crops in the world due to its
widespread consumption among people [72]. Its fruit is a berry which can be green,
red, or purple in color, and can be taken fresh, processed to make juice and wine, or
dried to produce raisins. It is a rich source of many phenolic compounds ranging
from catechins, epicatechin, epicatechin-3-O-gallate, and procyanidins [72]. The
inhibition of AGEs by red grape skin extract was investigated [47]. After 4 weeks
of incubation, the grape extract (0.031–0.5 mg/mL) inhibited the formation of AGE
by 55.23–63.52%. The extract (0.5 mg/mL) also reduced the level of fructosamine
and carbonyl content generated by 10.5% and 41.7%, respectively. Incubation with
0.5 mg/mL extract also caused significant improvement (50.4%) in the level of
protein thiol group and concomitant decrease (58.1%) in the carboxylmethylysine
content. The study suggests that red grape extract inhibited all the stages (early,
intermediate, and late stage) involved in the formation of AGEs, which makes it a
veritable source of antiglycation agents.
3.9 Saucer Berry
Grey-leaved saucer berry (Cordia sinensis Lam.) is found in Eastern African and
Middle Eastern countries of the World. The fruits and gum are consumed as food,
while the fruits are also taken in many cuisines. Several chemical compounds have
been isolated from this plant, which include transcaffeic acid, methylrosmarinate,
protocatechuic acid (Fig. 2f), kaempferide-3-O-β-D-glucopyranoside, kaempferol-
3-O-β-D-glucopyranoside, rosmarinic acid (Fig. 2g), and quercetin-3-O-β-D-
glucopyranoside [43]. The evaluation of isolated compounds from grey-leaved saucer
berry for their in vitro antiglycation potential was also reported [43]. All the compounds
tested showed good antiglycation activities by exhibiting higher percentage inhibition
of glycation which ranges between 68.0% (for protocatechuic acid) and 88.4%
(methylrosmarinate). Though these compounds displayed good antiglycation activities,
there is need for more tests to determine their IC50 values. This will enable comparison
with similar plants and with standard antiglycation agents.
3.10 Lingonberry
Lingonberry (Vaccinium vitis-idaea L.) is a low shrub bearing bright red fruits which
is most common in Russia and the Nordic countries. The fruits are eaten raw or
processed into jam, juice or syrup, and as an ingredient in dishes [73]. It is also used
by the Cree population of Canada in the treatment of diabetic symptoms like frequent
urination, abscesses, and snow blindness [74]. The inhibitory effect of lingonberry
and its active compounds on the formation of AGEs was investigated [50]. Ethanol
extract of lingonberry exhibited concentration-dependent inhibition of BSA-
glycation which culminated in IC50 of 13.50 μg/mL. Four compounds isolated
from this extract were also tested for their antiglycation activities. Quercetin-3-O-
galactoside (Fig. 2h), cyanidin-3-O-galactoside, and catechin exhibited good anti-
glycation potential with IC50 2.86, 3.10, and 8.35 μg/mL respectively, while
p-coumaric was not active. This study demonstrated lingonberry as an inhibitor of
AGEs formation, however there is a need for validation of this property in animal or
human model. The antiglycation activity may be due to the presence of phytochem-
icals like quercetin-3-O-galactoside, cyanidin-3-O-galactoside, and catechin.
3.11 Longan
Longan (Dimocarpus longan Lour.) is a tropical tree that produces exotic, attractive,
and edible fruits. The fruit is a thin and indehiscent pericarp with succulent edible
mesocarp and dark brown seed [75]. The fruits and seeds of this plant are rich in
phenolic compounds, such as ellagic acid, corilagin, kaempferol, grevifolin, and
gallic acid [76]. The fruit is also eaten by consumers globally due to its sweet juicy
mouth feel and aromatic flavor [77]. The antiglycation activity of polysaccharides of
longan fruit was investigated [41]. The study showed that polysaccharides obtained
after ultrasonic treatment under different conditions exhibited a potent antiglycation
effect in BSA-glucose glycation system. However, detailed characterization of the
possible antiglycation property of this fruits is imperative.
3.12 Passion Fruit
Passion fruit (Passiflora manicata) is a tropical plant which is characterized with

brightly colored flower and edible fruit. Its fruit is also used in the preparation of
beverages, while the leaves are used for medicinal purpose as sedative and tranquilizer.
The presence of chemical compounds such as vitexin, isovitexin (Fig. 4a), and orientin
in the leaf extract of passion fruit was reported [78]. Coincubation of aqueous extract
of passion fruit (1–100 μg/mL) with BSA-glucose medium caused significant reduc-
tion in the total glycated protein, and these results were similar to that of the standard
phenolic compound, tannic acid [45]. HPLC analysis of the extract also showed the
presence of active compounds such as isoorientin, vitexin, and isovitexin, which might
be responsible for the antiglycation property of the plant.
3.13 Pomegranate
Pomegranate (Punica granatum L.) is a shrub or small tree which grows between 5
and 8 m tall, and possesses large berry fruits. The plant originated from the Middle
East and extends throughout the Mediterranean to Southern America [79]. All parts
of the plant are useful, especially the fruit which is used in the preparation of juicy
drink while the seeds are used as spice for food. Several chemical compounds are
reportedly present in this plant, which include gallic acids, ellagic acid, caffeic
acid, luteolin, apigenin, dephinidin, and pelargonidin [79]. The antiglycation
property of polysaccharide fraction isolated from the fruit rind of pomegranate
was investigated [46]. The polysaccharide fraction (10–20 μg/mL) was able to
inhibit BSA glycation by only 28% as against 41% displayed by vitamin C. It also
reduced the formation of fructosamine by 50% after 7 day incubation, which
suggested mild antiglycation potential. The presence of polyphenols, such as gallic
acid, ellagic acid, and caffeic acid, may be responsible for the antiglycation activity
of this plant.
3.14 Shaddock
Shaddock (Citrus grandis L. Osbeck), also referred to as pomelo or pummelo, is one

of the most cultivated crops in South-East Asia due to its consumption as food and
usage in traditional medicine [80]. Previous study has showed this fruit is rich in
bioactive compounds such hesperidin, naringenin, and naringin [81]. The effect of
shaddock fruit on the concentration of AGEs, fructosamine, carbonyl carbonyl, and
carboxymethylysine was reported [22]. The shaddock extract (0.25–2.00 mg/mL)
significantly inhibited the formation of AGEs in concentration-dependent manner to
a maximum of 90.68% on the 28th day. The extract also reduced fructosamine
concentration by 3.7, 9.9, 17.5, and 30% at 0.25, 0.50, 1.00, and 2.0 mg/mL,
respectively. Concomitant reduction was also witnessed in the formation of carbonyl
and thiol compounds, following incubation with shaddock extract. The antiglycation
activities of shaddock may due to the presence of phytochemicals like neo-
hesperidin, naringin, hesperidin, and naringenin [22].
3.15 Rambutan
Rambutan (Nephelium lappaceum L.) is a tropical fruit which is native to South East
Asia. It is mostly found in Malaysia, Indonesia, Thailand as well as Philippines, and
spread to other parts of the world. Its fruit has a refreshing flavor and exotic appearance
and can be consumed fresh or in processed form [82]. Some chemical compounds
have been isolated and identified from this fruit, such as ellagic acid, corilagin, and
geraniin [83]. The potency of rambutan in the management of hyperglycemia was
investigated [53]. Ethanolic extract of rambutan inhibited the formation of AGEs by
43% in a BSA-glucose glycation model. This result was better than that of green tea
(38%), which was earlier reported as better antiglycation agent than the standard,
aminoguanidine [34]. Isolation and purification procedures on rambutan fruits yielded
a pure compound, geraniin (Fig. 2i), which exhibited a very high (98%) inhibition of
AGE formation [54]. It can therefore be concluded that rambutan displayed anti-
glycation activity, which is due to the presence of geraniin in it.
3.16 Water Apple
Water apple (Syzygium aqueum Alston) is a tropical plant which originated from
Malaysia and Indonesia. It is cultivated for its sweet tasty, bell-like fruits called apples
that are consumed fresh, and its leaves which are used as vegetables and in wrapping
other foods [84]. The inhibitory effect of ethanol leaf extract of water apple on the
formation of AGEs and the isolation of its active components was investigated [48]. The
extract displayed strong inhibitory potential against the formation of AGEs (89%),
which was higher than green tea (45%) which served as positive control. Six pure
compounds were also isolated from the extract which included 4-hydroxylbenzadehyde,
myricetin-3-O-rhamnoside (Fig. 2j), europetin-3-O-rhamnoside, phloretin (Fig. 2k),
myrigalone-G, and myrigalone-B. Therefore, water apple exhibited good antiglycation
potential, which may be due to the presence of the chemical compounds present in it.
3.17 Kokum
Kokum (Garcinia indica Choicy) is an underexploited tree distributed throughout

the tropical Asian and African countries. Its fruit is characterized by pleasant flavor
and sour taste, and is used as an acidulant in dishes as well as in the production of
beverages and butter [85]. Phytochemical study showed that the fruits contain
cyanidin-3-glucoside, cyanidin-3-sambubioside, garcinol (Fig. 2l), isogarcinol, and
hydroxycitric acid [68]. The leaves and fruits are traditionally used in the treatment
of different ailments like inflammation, diarrhea, dysentery, ulcers, and indigestion.
The antiglycation activity of garcinol isolated from the fruit rind of kokum was
investigated in BSA fructose model [57]. They showed that garcinol suppressed
fluorescence and protein cross-link formation in the reaction system, better than
aminoguanidine. Therefore, kokum leaves inhibited the formation of AGEs in vitro,
which may be attributed to its phytochemical compounds especially garcinol.
4 Antiglycation Agents from Beverages
4.1 Black Tea
Black tea (Camellia sinensis L.) is one of the most popular beverages consumed
worldwide, and is made by fermentation and drying of the leaves of the plant,
Camellia sinensis. The fermentation of this tea converts catechins to theaflavins
and thearubigins, thereby decreasing catechin content [86]. The main components of
black tea include catechins (e.g., epigallocatechin gallate), quercetin, rutin, caffeic
acid, theaflavins, and thearubigins [87]. The in vitro antiglycation and cross-link
breaking potential of black tea from Sri Lanka was reported [19]. Black tea brew
exhibited strong antiglycation (IC50: 19.04 μg/mL) and AGEs cross-link breaking
(IC50: 82.89 μg/mL) activities, and these activities were similar to that of popular
antiglycation agent, rutin.
4.2 Green Tea
The steaming of freshly harvested leaves of Camellia sinensis yields dry and stable
product referred to as green tea [86]. Though it is obtained from the same plant as
black tea, its chemical composition is quite different because it has higher quantity of
catechins (epicatechin, epicatechin-3-gallate, epigallocatechin-3-gallate, and
epigallocatechin) and do not possess theaflavins and thearubigins [86]. The reduc-
tion of protein glycation by ethanol extract of green tea in the cardiac tissue of
streptozotocin-induced diabetic rats was reported [52]. Oral administration of
300 mg/kg green tea extract to diabetic rats for 4 weeks significantly reduced the
extent of cardiac protein glycation from 235.70 mg/mg protein in diabetic control
rats to 185.30 mg/mg protein in the green tea–treated diabetic rats. The study also
revealed the extract contained 33.5%, 28.5%, 13.5%, and 3.8% of epigallocatechin,
epigallocatechin-3-gallate, epicatechin-3-gallate, and epicatechin, respectively. This
antiglycation property may be connected to the presence of catechins in the tea.
4.3 Luobuma Tea
Another tea plant with antiglycation potential is luobuma (Apocynum venetum L.).
This plant is native to China but widely distributed in the temperate zones of Eurasia
and North America. Its leaves are used in the preparation of beverage called luobuma
tea in China, Japan, and the USA. It is also used in Chinese traditional medicine in
the treatment of inflammation, hypertension, hepatitis, and diuresis [88]. Several
phytochemicals have been isolated from this plant, which includes apocyanins A, B,
C, and D, as well as apocynosides I and II [88]. Aqueous extract of Luobuma leaves
exhibited better antiglycation activity than the standard, aminoguanidine with IC50
37.2 and 59.2 μg/mL, respectively [58]. Furthermore, all the seven isolated com-
pounds (catechin (Fig. 3a), epicatechin (Fig. 3b), gallocatechin (Fig. 3c), epigallo-
catechin, epicatechin-(4β-8)-gallocatechin, epigallocatechin-(4β-8)-epicatechin and
procyanidin B-2) from this extract also displayed excellent antiglycation activities,
with IC50 values ranging between 9.1 and 19.8 μg/mL. Therefore, antiglycation
compounds from luobuma tea could be explored further as possible candidates for
the development of antiglycation drugs in the treatment of diabetic complications.
4.4 Yerba Mate
Yerba mate (Ilex paraguariensis St. Hil.) is a popular tree in the South American
countries of Paraguay, Uruguay as well as parts of Argentina and Brazil. It is used in
the preparation of tea called Mate tea by the infusion of dried leaves of the plant. It is
commercially packaged in tea bags or concentrates, for usage in the food and dietary
supplement industries [89]. Due to its nutraceutical properties, mate is used as an
ingredient in energy drinks, candy, and beer [90]. Coincubation of bovine serum
albumin (BSA) and methylglyoxal with yerba mate extract caused a dose-dependent
Fig. 3 Structures of some antiglycation compounds from beverages. (a) Catechin; (b) Epicatechin;
(c) Gallocatechin
inhibition of glycation reaching up to 40% at 20 μL/mL of the extract, which

implies good antiglycation effect [38]. This may be due to the presence of some
phytochemicals, such as methylxanthines, matesaponnins, chlorogenic acids, and
4,5-dicaffeoylquinic acid [89], in the plant.
4.5 Dendrobii
Dendrobii (Dendrobium huoshanense) is another antiglycation tea plant which is

endemic to Huoshan town, Anhui Province of China. It is used as spice in the preparation
of foods such as soups and as herbal tea or functional beverage for the protection of the
eye, liver, and stomach [91]. Phytochemical analysis revealed the presence of many
compounds like polysaccharides, 6,8-di-C-glycosyl flavonoids, shikimic acid, and
salicylic acid [92]. The antiglycation activity of polysaccharides extracted from dendrobii
has been reported [29]. Polysaccharides from dendrobii inhibited the formation of
amadori product in a dose-dependent manner to maximum of 72.54%, which was
about 1.95 fold greater than that of aminoguanidine. The formation of intermediate
dicarbonyl compound and AGEs was also decreased to a minimum of 51.9%, which
was lower than that of aminoguanidine. The inhibition of glycation by these polysac-
charides of dendrobii may be due to their antioxidant properties.
5 Antiglycation Agents from Legumes
5.1 Mung Bean
Mung bean (Phaseolus radiata L. Wilczek), also referred to as green gram or moong
bean, is a legume which is native to the Indian subcontinent but widely cultivated in the
Southeast Asian countries. The inhibitory effect of mung bean extract and some of its
active constituents on the formation of AGEs was investigated [44]. Hydroethanol extract
of mung bean displayed the highest inhibitory activity (80.4%) against AGEs formation
compared to other variety of beans such as black bean (72.1%), soybean (70.1%), and
cowpea (67.3%). Two active components isolated from mung bean, vitexin (Fig. 4a)
Fig. 4 Structures of some antiglycation compounds from legumes. (a) Vitexin; (b) Isovitexin;
(c) Cassiaside; (d) Rubrofusarin-6-O-β-D-gentiobioside; (e) Toralactone-9-O-β-gentiobioside
and isovitexin (Fig. 4b) at 100 μM, also exhibited a strong inhibitory effect on fluorescent
AGEs formation induced by methyglyoxal similar to the activity of 1 mM
aminoguanidine [44]. However, both compounds have weaker direct methylglyoxal-
trapping activities than aminoguanidine. Therefore, mung bean displayed antiglycation
effect which may be due to the presence of phytochemicals like vitexin and isovitexin.
5.2 Buckwheat
Buckwheat (Fagopyrum sp.) is a plant that is cultivated for its seeds, which is used
as food. There are two major varieties of buckwheat: common buckwheat
(Fagopyrum esculentum) which is sweet, and tartary buckwheat (Fagopyrum
tataricum) which has a bitter taste [93]. However, the chemical composition of
both is similar which include chlorogenic acid, orientin, isoorientin, vitexin,
isovitexin, and rutin [94]. Buckwheat is also a rich source of starch, protein,
minerals, vitamins, dietary fiber, and antioxidants. The in vitro antiglycation
activity of common buckwheat hull tea infusion was investigated [20]. The
ready-to-drink buckwheat hull tea displayed lower inhibition (34.90%) of AGEs
in BSA-glucose system compared to green tea.
Ethanol extract (200 μg/mL) of tartary buckwheat exhibited strong inhibition of
AGEs (83%), fructosamine (50%), and α-dicarbonyl compounds (65%) formation
[21]. It, therefore, reduced the generation of AGEs through the suppression of
fructosamine and α-dicarbonyl compounds formation. This implies that tartary
buckwheat possessed higher antiglycation activity than the common buckwheat,
and this may be due to variation in the quantities of phytochemicals present in them.
5.3 Sickle Senna
Sickle senna (Cassia tora L.) is a leguminous plant found in the South East Asia and
South West Pacific. The young leaves of this plant are cooked as vegetable while the
roasted seeds are consumed as tea. It is used in the treatment of several diseases, such
as bronchitis, leprosy, dyspepsia, hypertension, and cardiac disorders [95]. Three
naphthopyrone glucosides, namely cassiaside (Fig. 4c), rubrofusarin-6-O-β-D-
gentiobioside (Fig. 4d), and toralactone-9-O-β-D-gentiobioside (Fig. 4e), isolated
from sickle senna seeds displayed antiglycation property and showed better inhibi-
tion of AGE formation (IC50: 6.4–32.2 μg/mL) than the standard antiglycation agent,
aminoguanidine (IC50: 34.6 μg/mL) [55]. Nine anthraquinones isolated from the
seeds of sickle senna were also evaluated for their antiglycation activity, out of which
only obtusifolin (IC50: 28.9 μM) and emodin (IC50: 118.0 μM) displayed significant
inhibition of AGE formation, which were also more potent than aminoguanidine
(IC50: 961.0 μM) [56]. Therefore, the antiglycation activities of sickle senna may be
due to the presence of naphthopyrone glucosides, as well as obtusifolin and emodin.
6 Antiglycation Agents from Vegetables and Spices
6.1 Black Nightshade
Black nightshade (Solanum nigrum L.), also called hounds berry or stubble berry, is
a common food plant in Africa, Asia, and Europe. Its leaves and fruits are used in the
preparation of foods such as soup, jam, salad, and fruit juices [96]. This plant is
reported to contain steroidal glycosides (e.g., solarmargine, desgalactotigonin) and
steroidal saponins (e.g., solanigrosides as well as nigrumnins I and II) [97]. The
antiglycation property of ethanolic extract of the leaves of black nightshade and two
of its isolated compounds, solamargine (Fig. 5a) and solasonine (Fig. 5b), were
investigated [30]. The result revealed that 95% ethanolic extract of black nightshade
inhibited the generation of AGEs in a dose-dependent manner, via the lowering of
fructosamine and α-dicarbonyl compounds formation. Solasonine also exerted stron-
ger antiglycation activity for attenuating AGEs, fructosamine, and α-dicarbonyl
compounds formation than solarmargine. This implies that black nightshade leaves
possess antiglycation effects which may be due to the presence of solasonine.
6.2 Sangwhang
Sangwhang (Phellinus linteus), also called black hoof mushroom, is one of the
mushrooms that are considered functional foods in Japan, China, and South Korea,
though it is also found in tropical America and Africa. It is used in Asian herbal
medicine for the management of several diseases such as liver disorder, oral ulcer,
allergies, and diabetes mellitus [98]. Nine compounds (protocatechuic acid, pro-
tocatechualdehyde, caffeic acid, ellagic acid, hispidin, davallialactone (Fig. 5c),
Fig. 5 Structures of some antiglycation compounds from vegetables and spices. (a) Solamargine;
(b) Solasonine; (c) Davallialactone; (d) Interfungins A; (e) Inoscavin A; (f) p-Cymene
hypolomin B, interfungins A (Fig. 5d), and inoscavin A (Fig. 5e)) isolated from the
ethylacetate fraction of sangwhang were evaluated for antiglycation activities [59].
Davallialactone (50.2%), interfungins A (63.1%), and inoscavin A (45.7%) exhibited
inhibitory effect on HbA1c formation, while protocatechualdehyde (IC50: 144.28 μM),
davallialactone (IC50: 158.66 μM), and inoscavin A (IC50: 213.15 μM) prevented
methylglyoxal-mediated protein modification. However, only interfungin A (IC50:
1.15 mM) inhibited protein cross-link formation even better than aminoguanidine
(IC50: 11.93 mM). Therefore, sangwhang displayed good inhibitory potential against
AGEs formation which is due mainly to the presence of interfungin A.
6.3 Turmeric
Turmeric (Curcuma longa L.) is a rhizomatous plant which is distributed in the

Asian continent especially India. It is used in the preparation of foods as spice,
preservatives, and coloring agent [99]. It is also used in folk medicine for the
treatment of various ailments like anorexia, cough, rheumatism, hepatic disorder,

and diabetic wounds [100]. Several compounds have been isolated from this plant,
which includes curcumin, demethoxycurcumin, bisdemethoxycurcumin, and
ar-turmerone [101]. The antiglycation potential of hexane, ethylacetate, methanol,
and aqueous extracts of turmeric rhizome was reported [49]. Ethylacetate
(IC50: 0.09 μg/mL) displayed highest inhibitory effect, followed by methanol
(1.48 μg/mL), and aqueous (10.42 μg/mL) extract, while the hexane extract had no
effect. In fact, the antiglycation potential of the ethylacetate extract was about 800
times higher than that of ascorbic acid, which is the standard. Therefore, turmeric is
an excellent inhibitor of glycation and AGEs formation.
6.4 Lotus
Lotus (Nelumbo nucifera Gaertn.) is an aquatic plant cultivated mostly in China and
India but distributed throughout Asia. The young leaves, seeds, and rhizome of the plant
are used as vegetables while the matured leaves are used as functional foods [102].
The leaf is also used as ingredients in the preparation of healthy beverages and tea bags
in China [103]. It is rich in several phytochemicals, which include dauricine, lotusine,
nelumboside, pronuciferine, anonaine, and arbutin [104]. The inhibitory effect of lotus
leaves, stamens, seeds, embryos, and rhizomes on the formation of advanced glycated
end products was investigated [42]. Only the methanol extract of the leaves and stamens
demonstrated good antiglycation activities with IC50 values of 110.5 μg/mL and
125.5 μg/mL, respectively, compared to the standard, aminoguanidine. However,
only the ethylacetate fraction of the leaf effectively inhibited AGE formation
(IC50 28.18 μg/mL). Though lotus exhibited good antiglycation effects, further study
is required to isolate the bioactive compound(s) from its ethylacetate fraction.
6.5 Ginger
Ginger (Zingiber officinale Roscoe) is one of the commonly used plants as spice or
condiments in food preparation. It is a functional food which provides medical benefit to
its consumers, in addition nutritional function [105]. This is why its rhizome is widely
used in food processing and pharmaceutical industries. Major chemical constituents of
this plant include 6-gingerol, 6-shogaol, 6-paradol, α-zingiberene, and cineole [106].
The inhibition of protein glycation by ginger extract in an in vitro model was investi-
gated [31]. Ethylacetate extract of ginger at varying concentrations (100–500 μg/mL)
displayed dose-dependent inhibition of albumin glycation, which culminated in its IC50
value 290.84 μg/mL for the antiglycation activity. This is in conformity with our study
on the antiglycation potential of polyphenol extract of ginger, where the IC50 value for
the antiglycation effect was 285 μg/mL [32]. We also investigated the antiglycation
effects of polyphenols extracted from ginger in streptozotocin-induced diabetic rats [33].
Oral administration of 500 mg/kg of free and bound polyphenols of ginger to diabetic
rats for 42 days significantly reduced the glycated hemoglobin (HbA1c) level (7.10%)
compared to the diabetic control (10.33%). The formation of AGEs in the serum was
also drastically decreased from 6.80 μg/mL in the diabetic control rats to 1.98 μg/mL in
the ginger-treated diabetic animals. These reports suggest that ginger possessed anti-
glycation property both in vitro and in vivo, which may be due to the presence of
polyphenols such as gingerol and zingiberene.
6.6 Cumin
Cumin (Cuminum cyminum L.), commonly called jeera, is a widely used spice
condiment in vegetarian and nonvegetarian foods mostly in Asian countries [107].
It is also used in folklore medicine as an analgesic, antihypertensive, carminative,
purgative, and antidiabetic [108]. Its essential oil components include α- and
β-pinene, p-cymene (Fig. 2f), limonene, safranal, as well as α- and γ-terpinene.
The antiglycation potential of aqueous extract of cumin was tested on fructose-
induced glycation [27]. Coincubation of fructose with cumin extract resulted in
dose-dependent reduction of AGE fluorescence and prevented the formation of
predominant AGE, carboxymethylysine. The presence of the extract also prevented
the formation of glycation mediated protein cross-links, thereby displaying potent
antiglycation activity. Methanolic extract of cumin seed was also tested for its
inhibitory effect on glucose-induced BSA glycation [12]. Cumin extract exhibited
concentration-dependent inhibition of BSA glycation and subsequent formation of
fluorescent glycation products, culminating in low IC50 of 1.17 mg/mL.
The in vivo antiglycation activity of cumin in streptozotocin-induced diabetic rats
was reported [27]. Supplementation of diabetic rats’ diet with 0.5% cumin powder for
8 weeks significantly decreased glycated hemoglobin (HbA1c) level of the rats (5.1%)
compared to the diabetic control rats. It also caused protein carbonyl concentration to
reduce from 8.1 to 6.3 nmol/mg protein in the cumin-fed rats. Feeding diabetic rats
with cumin-supplemented diet also prevented the hyperglycemia-mediated formation
of protein cross-links and improved the total and soluble protein levels. Administration
of methanolic extract of cumin seed to diabetic rats for 28 days also caused significant
reduction in their serum glycated hemoglobin (8.39%) and renal AGE (2.25 IU/mg
protein), compared to the glycated hemoglobin (15.39%) and renal AGE (10.09 IU/mg
protein) of the diabetic control respectively [12]. Treatment with the extract also
reduced glycated and rat tail tendon collagen of the diabetic rats.
In order to identify the antiglycation agents present in cumin, one of its active
components, cymene, was investigated for its effect on glycation [28]. The
coincubation of 100 μM cymene in BSA-glucose system caused drastic reduction
in fructosamine formation and its effect was similar to that of 2 mM aminoguanidine.
This implies that cymene is 20 times more potent as an antiglycation agent than
aminoguanidine. Varying concentrations (25–100 μM) of cymene also elicited a
significant decrease in the protein-bound carbonyl compound formation. Cymene
(100 μM) also caused 56.6% and 49.16% reduction in the formation of total AGEs
and pentosidine in the BSA-glucose system. Administration of 20 mg/kg cymene to
streptozotocin-induced diabetic rats for 60 days also caused significant reduction in
their glycated hemoglobin (HbA1c) level (6.1%) compared to the diabetic control
(11.3%). The concentration of serum fructosamine was also decreased significantly
from 3.98 mM/L to 2.32 mM/L, which means 58.29% reduction was obtained. It can
be concluded that cumin possesses antiglycation property in vitro and in vivo, which
may be due to the presence of cymene.
6.7 Clove
Clove (Syzygium aromaticum (L.) Merr. & Perry) is a plant which is native to
tropical America and Australia. It is popularly used as spice in food preparation
and in the treatment of diseases, including inflammation, infections, toothache, and
respiratory and gastrointestinal disorders [109]. Chemical compounds present in this
plant include eugenol, limonene, β-pinene, ferulic acid, and kaempferol [110]. The
inhibition of AGEs formation by clove extract was investigated [60]. Clove extract
(0.25–1.00 mg/mL) inhibited the formation of AGEs to a maximum of 95.2% as
against the standard, aminoguanidine (91.5%). It also caused 72.8% reduction in the
formation of N-carboxymethylysine [60]. Coincubation of the clove extract with the
glycation reaction system also decreased the formation of protein carbonyl content
by 73.7%, as compared to aminoguanidine (60.0%). However, it significantly
elevated the level of protein thiol group better than the standard. This implies that
clove extract inhibited all the stages involved in the formation of AGEs, and
therefore can be consumed to prevent diabetic complications.
7 Possible Mechanisms of Antiglycation by Foods
The inhibition of glycation and subsequent formation of AGEs by food plants may
occur through one or more mechanisms. This includes cleavage of the AGE-
crosslink or blocking of the AGE receptor, trapping of reactive dicarbonyl species,
as well as through antioxidant activities [9, 11]. Other possible mechanisms of
antiglycation are inhibition of aldose reductase and reduction of blood glucose
level using hypoglycemic agents, such as acarbose and metformin [4, 6]. Anti-
glycation agents either function as AGE inhibitors, AGE breakers, or receptor for
AGE (RAGE) blockers. AGE inhibitors include aminoguanidine, benfotiamine, and
pyridoxamine. N-phenacyl thiazolium compound and alagebrium are AGE breakers
while cerivastatin is an example of RAGE blocker [6]. Some antioxidant compounds
have also been validated as antiglycation agents, which include vitamin C and E [8].
8 Conclusion
The incidence of diabetic complications ranging from diabetic foot, kidney dysfunc-
tion, and cardiovascular disease, which may be due to nonenzymatic glycation, is on
the increase. Synthetic drugs manufactured to ameliorate these conditions are
bedevilled with serious side effects and nonavailability, which necessitated the
search for antiglycation agents from food plants. Beverages, fruits, legumes, vege-
tables, and spices, with antiglycation potential, are presented in this review. How-
ever, further study will be required to isolate the bioactive component(s) of these
foods for possible drug development.
Acknowledgments The support of Lagos State University, Ojo, Lagos, Nigeria, is gracefully
acknowledged.
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Nutraceutical Potential of Apiaceae
44
Milica G. Aćimović
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1312
2 Bioactive Compounds of Apiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313
2.1 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313
2.2 Polyacetylenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316
2.3 Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316
3 Nutraceutical Potential of Apiaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
3.1 Anethum graveolens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
3.2 Angelica archangelica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
3.3 Apium graveolens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1320
3.4 Carum carvi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1320
3.5 Coriandrum sativum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1321
3.6 Cuminum cyminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1321
3.7 Daucus carota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1322
3.8 Foeniculum vulgare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1322
3.9 Levisticum officinale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323
3.10 Pastinaca sativa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1323
3.11 Petroselinum crispum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1324
3.12 Pimpinella anisum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1325
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1325
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326
Abstract
Apiaceae family is large, with over 3.000 species worldwide cultivated for many
purposes. Some plants in this family such as carrots, parsley, parsnip and celery are
common vegetable crops, while other members like anise, caraway, coriander,
cumin, fennel, lovage, angelica and dill are famous for their medicinal and
M. G. Aćimović (*)
Department of Alternative Crops and Organic Agriculture, Institute of Field and Vegetable Crops,
Novi Sad, Serbia

1312 M. G. Aćimović
aromatic properties. Usage of these plants is very popular in everyday diet because
of their documented health benefits. Apiaceae are a very important source of
phytochemicals – chemicals with biological activity. However, phytochemicals
are non-nutritive plant chemicals, also called nutraceuticals. They are widely used
for prevention, treatment or cure of conditions or diseases. Bioactive compounds
with nutraceutical potential are polyphenolic compounds, polyacetylenes and
terpenoids. The aim of this review is to represent selected plants of Apiaceae
family currently used as nutraceuticals and describe their nutritional benefits.
Keywords
Vegetable · Spices · Biological activity · Food · Nutrition · Phenolics ·
Polyacetylenes · Terpenoids
Abbreviations
CAE Caffeic acid equivalent
CE Catechine equivalent
DW Dry weight
FW Fresh weight
GAE Gallic acid equivalents
QE Quercetin equivalent
TFC Total flavanoids content
TPC Total phenolic contents
1 Introduction
Apiaceae (Umbelliferae) family is large, with over 3.000 species worldwide culti-
vated for food, as vegetables, herbs, spices or for medicinal purposes. Some plants in
this family such as carrots, parsley, parsnip and celery are common vegetable crops,
while other members like anise, caraway, coriander, cumin, fennel, lovage, angelica
and dill are famous for their medicinal and aromatic properties [1].
The plants from Apiaceae family are mainly temperate herbaceous annual (anise,
caraway, coriander, cumin, dill, sweet fennel), biannual (carrot, parsley, parsnip,
celery) or perennial (angelica, lovage, bitter fennel). Leaves are alternate on stem or
arranged in leaf rosette (mainly in the first year of development in biannual and
perennial members). In all the mentioned species, stem is erect and hollow, and in the
upper part is branch. Each branch finishes with an inflorescence. The small and
simple flowers are generally arranged into compound umbels. They have five petals
that are usually white or yellow, with five stamens, and an ovary with two carpels.
The fruit that develops from this ovary varies considerably between the spices.
Generally the fruit are schizocarps, which contain two seeds.
All Apiaceae plants contain a well-developed secretory system in all plant parts,
such as schizogeneus secretory cavities in the root, phloem in the stem and leaves
and clearly-delimited tissue known as vittae in the fruit. These structures are
important for depositing essential oils, which give the specific odor and flavor to
44 Nutraceutical Potential of Apiaceae 1313
each plant. Due to their flavor, a large number of plants from this family are used as
vegetables or spices [2].
Plants from the Apiaceae family are a very important source of nutraceuticals.
Their usage is very popular in everyday diet because of their documented health
benefits. The figure below shows members of Apiaceae family and their parts which
are usually used. For example, caraway, cumin, aniseed are exclusively used as seed,
while dill and coriander are used as seed and leaves, and from carrot seed and root. In
case of fennel, apart from seed and leaves, succulent leaf stalks are also used. Celery
is used in a similar way. From lovage, parsley and angelica, the root is also used
together with above-ground parts (Fig. 1).
2 Bioactive Compounds of Apiaceae
Bioactive compounds can be divided into two groups: phytochemicals which are
non-nutritive plant chemicals, also called nutraceuticals, and nutrients which include
minerals, proteins, fibers, carbohydrates, fats, etc. However, nutraceuticals possess
biological activity, while nutrients affect the growth, development and function of
the human body.
Nutraceuticals can be designated as food with medical benefits; as indicated by its
name which is derived from “nutrition” and “pharmaceutics”. They include poly-
phenolic compounds, polyacetylenes and terpenoids [3]. Nutraceuticals are widely
used for prevention, treatment or cure of conditions or diseases [4, 5, 6]. The high
antioxidant activity of nutraceuticals is the basis for many potential benefits, among
which many for degenerative and chronic diseases [7].
Phenolic compounds, as one of major bioactive nutraceutical ingredients in plants,

are responsible for the organoleptic characteristics of plant-derived foods and bev-
erages, particularly color and taste properties and they also contribute to the nutri-
tional qualities of fruits and vegetables [8]. The phenolic metabolites include:
anthocyanins, anthochlors, benzofurans, chromones, coumarins, flavonoids,
flavonones and flavonols, isoflavonoids, lignans, phenols and phenolic acids, phe-
nolic ketones, phenylpropanoids, quinonoids, stilbenoids, tannins and xanthones [9].
The antioxidant property in many plants is related to the presence of phenolic
compounds. Nutritionally, these compounds are responsible for increasing the
shelf life of foods as well as slowing the lipid, protein and enzymatic oxidation, as
well as for providing protection against development of cancers, cardiovascular and
liver diseases, diabetes, osteoporosis and neurodegenerative diseases in humans [10,
11, 12, 13, 14]. However, the content of biologically active substances, among them
polyphenols in plants, depends on various factors such as: area in which the plant is
grown (agrochemical characteristic of soil), climatic conditions in the region during
the growing season, cultivation technology but also the variety [15]. Apart from this,
Fig. 1 Plants from Apiaceae family with their parts which are usually used in nutrition
total phenolic contents depend also on postharvest processing (fresh, dry, freeze
herb), solvents used for extraction (water, ethanol, acetone, etc.) and significantly
varies due to plant material (seed, herb, root) etc. Review of total phenolic content
according to literature is shown in Table 1.
Table 1 Phenolic compound from selected plants from Apiaceae family

Plant Phenolic compounds
Anethum TPC in dill seed methanolic extract is 773.14 mg GAE 100 g 1 dw, while
graveolens TFC is 231.84 mg QE 100 g 1 dw [16]. On the other side, TPC in fresh dill
herb acetone extract is 35.23 mg GAE g 1 dw, and content of TFC is
30.39 mg CAE g 1 dw [17]. However, TPC in dry dill herb varied from
55.46 to 71.29 mg GAE g 1 dw depend on solvents [18]. Further, fresh dill
herb acetone extract contains 19.49 mg QE g 1 dw phenolic acids, and the
dominant are chlorogenic and benzoic acids [17]
Angelica TPC in angelica roots is 11.8–17.3 mg GAE g 1 extract, while content of
archangelica coumarins is 0.91 mg 100 g 1 extract [19]. The dominant are coumarin
derivatives among which isoimperatorin, oxypeucedanin, imperatorin,
ostruthol, angelicin, bergapten, scopoletin, isopimpinellin, and xanthotoxin
[20, 21]
Apium TPC in celery seed methanol extract is 825.85 mg GAE 100 g 1 dw, and
graveolens TFC is 177.57 mg CE 100 g 1 dw, while content of tanins is 243.36 mg CE
100 g 1 dw [22]. The dominant flavonoids are apigenin, luteolin, and
kaempferol, while the dominant phenolic acids are caffeic, p-coumaric and
ferulic acid [23]
Carum carvi TPC in caraway seeds is 3.99 mg GAE g 1 dw with 42 phenolic compounds
[24]. The dominant flavonoid constituent from caraway seed is quercetin
3-glucuronide, isoquercitrin, quercetin 3-O-caffeylglucoside and kaempferol
3-glucoside [25]. Phenolic acid content in caraway seed is about 65 μg g 1
dw, with dominant chlorogenic, p-coumaric and caffeic acid [24]
Coriandrum TPC in coriander leaves extracts is 1.12 mg GAE 100 mL 1 [26], and the
sativum main flavonoids are quercetin-3-O-rutinoside, kaempferol and acacetin,
while identified phenolic acids are vanilic, ferulic and p-coumaric acid [27,
28]. Coriander seed contain TPC 0.72 g GAE 100 g 1 extract [29]. Rutin,
quercetin, chlorogenic and caffeic acid were separated and identified
flavonoids in the methanolic and ethanolic extracts of coriander seed [30]
Cuminum Acetone extract of cumin seed contains TPC between 16.50 and 18.60 mg
cyminum GAE g 1 dw, TFC between 4.99 and 5.91 mg CE g 1 dw and tannin
80.23–83.23 mg CE g 1 dw [31]. The main phenolic compounds from
cumin are quercetin, p-coumaric, rosmarinic, vanillic and trans-2-
dihydrocinnamic acids, as well as resorcinol [32]
Daucus carota TPC in the carrot root varied considerably from 19.8 to 342.2 mg GAE
100 g 1 fw depend on root color [33], and from 81.25–113.69 mg GAE kg 1
fw depend on variety [15]. However, carrot contained high amounts of
phenolic acids, flavonoids, and carotenoids. Content of ß-carotenes vary
between 24.58 and 124.28 mg kg 1 fw [15] while total ascorbic acid ranged
from 41.12 to 58.36 mg 100 g 1 fw, whereas 5-caffeolquinic acid ranged
from 30.26 to 65.39 mg 100 g 1 fw [34]
Foeniculum TPC in seed methanolic extract was 1017.29 mg GAE 100 g 1 dw, while
vulgare TFC is 695.52 mg QE 100 g 1 dw [16]. Further, also TFC in seed
methanolic extract is 9.325 mg QE g 1 dw, with dominant gallic acid
(277.131 μg g 1 dw), caffeic acid (166.062 μg g 1 dw), ellagic acid
(99.476 μg g 1 dw), quercetin (781.986 μg g 1 dw) and kaempferol
(92.856 μg g 1 dw) [35]. From the other side, fennel herb contains two
phenolic compounds, 3.4-dihydroxy-phenethylalchohol-6-O-caffeoyl-β-D-
glucopyranoside and 3΄0.8΄-binaringenin [11]
(continued)
Table 1 (continued)
Plant Phenolic compounds
Levisticum TPC in lovage leaves ranged between 359.75 and 1601.87 mg GAE 100 g 1
officinale dw, while TFC varied between 551.01–3548.33 mg CE 100 g 1 dw,
depending on postharvest treatment (fresh, frozen, dry). Phenolic
compounds present in lovage leaves are: rutine, catechin, caffeic,
chlorogenic, coumaric, sinapic, and ferulic acid [36]
Pastinaca sativa The total phenolic acid in parsnip is 5.7 mg 100 g 1 fw, while the major
soluble phenolic acid is chlorogenic acid [37]. Total content of coumarins
ranged from 115.7 to 408.5 mg 100 g 1 dw. In vegetative plant parts the
dominant are isopimpinellin and psoralen, while imperatorin was dominant
in fruit [38]
Petroselinum TPC in parsley seed is 0.62 g GAE 100 g 1 extract, while in leaves it is
crispum 0.92 g GAE 100 g 1 extract [29]. However, another study shows that TPC in
parsley leaves ranged from 15.20 to 54.20 mg CE g 1 extract, while TFC is
between 4.50 and 42.1 mg QE g 1 extract [39]. Flavonoids isolated from
aqueous extract of parsley leaves: apigenin, apigenin-7-O-glucoside or
cosmosiin, apigenin-7-O-apiosyl-(1–2)-O-glucoside or apiin and the
coumarin 2,3-dihydroxyfuranocoumarin or oxypeucedanin hydrate [40]
Pimpinella TPC in anise seed is 46.17 mg GAE 100 g 1 dw, while TFC is 17.43 mg CE
anisum 100 g 1 dw [41]. Another study shows that TPC is 42.09 mg g 1 extract, and
identified mainly flavonoids (28.08 mg g 1 extract) and phenolic acids
(14.01 mg g 1 extract). Morerover, apigenin and luteolin derivatives, as well
as caffeoylquinic acid derivatives were determined [12]
2.2 Polyacetylenes
Polyacetylenes are a group of phytochemicals that have attracted significant interest

in medicine and pharmaceutical industry in recent years due to their range of
potential health-promoting bioactivities. Polyacetylenes isolated from Apiaceae
plants include antifungal and antibacterial activity [42, 43], as well as anti-
inflamatory [44] and anticancer [45, 46] properties. They also display antidiabetic
[47] effects and have potential in the treatment of endotoxemia and inflammation
accompanied by the overproduction of NO [48]. Apart from this, they possess
neurotoxic, anti-platelet-aggregatory activity and are responsible for allergic skin
reactions [49, 50, 51]. However, polyacetylenes have a major impact on the bitter
taste in roots of parsnip, celeriac, parsley and carrot, as well as fennel bulbs [52, 53,
54, 55, 56, 57, 58, 59]. The review of polyacetilenes present in plants from Apiaceae
family according to literature is shown in Table 2.
2.3 Terpenoids
Terpenoids, especially monoterpenes and sesquiterpenes are the main constituents in

essential oils which, as volatile compounds, give fragrance to many aromatic plants
[64]. Some terpenoids are very typical for plant species from the Apiaceae family as
Table 2 Polyacetylenes from selected plants from Apiaceae family

Plant Polyacetylenes
Anethum Dill roots contain polyacetylenes such as panaxynol (C17H24O) and
graveolens falcarindiol (C17H24O2) [60]
Angelica Many species from genus Angelica contain polyacetylenes. For example,
archangelica polyacetylene from A. purpuraefolia is (+)-9(Z), 17-octadecadiene-
12,14-diyne-1,11,16-triol [61], while A. furcijuga contain ( )-falcarinol
and falcarindiol [62]. A. gigas contains: octadeca-1, 9-dien-4, 6-diyn-3, 8,
18-triol (1), 18-acetoxy-octadeca-1, 9-dien-4, 6-diyn-3, 8-diol (2) and 3, 8,
18-triacetoxy-octadeca-1, 9-dien-4, 6-diyn (3) [48]
Apium graveolens Celery root contains polyacetylenes: falcarindiol, falcarinol, panaxydiol
and 8-O-methylfalcarindiol [44, 49]
Carum carvi Aliphatic C17-polyacetylenes of the caraway are falcarinol, falcarindiol,
falcarinolone and falcarindione [52]
Coriandrum In coriander, polyacetylenes are detected but not identified [52]
sativum
Cuminum cyminum No data in the available literature
Daucus carota The most abundant polyacetylenes in cultivated orange carrots is
falcarindiol (16–84 mg kg 1 fw, followed by falcarinol (8–27 mg kg 1 fw)
and falcarindiol-3-acetate (8–40 mg kg 1 fw) [59]
Foeniculum The amount of polyacetylenes in fennel bulb ranged from 0.04 to 0.24 mg
vulgare g 1 of freeze-dried plant material [49]. Polyphenoles detected in fennel
bulb are falcarindiol, falcarindiol-3-acetate, and falcarinol [57]
Levisticum Polyacetylenes from lovage roots are: 3(R)-Falcarinol (3(R)-( )-
officinale 1,9-heptadecadien-4,6-diin-3-ol] (1) and 3(R)-8(S)-falcarindiol [3(R)-8
(S)-(+)-1,9-heptadecadien-4,6-diin-3,8-diol] (2) [43]
Pastinaca sativa Polyacetylene compounds from parsnip root are falcarinol and falcarindiol
occurring in the highest concentrations (1600 and 5770 mg kg 1 freeze-
dried material, respectively), followed by falcarinone and falcarinolone.
Moreover, parsnip seeds contain polyacetylenic C18 ketoaldehyde [55]
Petroselinum Polyacetylene from parsley root are falcarindiol (up to 2320 mg kg 1
crispum freeze-dried material), falcarinol, 8-O-methylfalcarindiol (350 mg kg 1
freeze-dried material) and panaxydiol (120 mg kg 1 freeze-dried
material) [55]
Pimpinella anisum Polyacetylenes have been reported to be present in some Pimpinella
species, such as falcarinol in P. pruatjan, pentadeca-2,4,6,8-tetraene (1),
2,8-decadiene-4,6-diene-1-al (2), 2,8-tridecadiene-4,6-diene-10-ol (3)
2,8,10-tridecatriene-4,6-diene (4) in P. major, and 2-tridecaene-4,6-diene-
8-ol-10-on in P. saxifraga [63]
they are the source of the familiar taste, for example carotol in carrot, trans-anethole
in anise and fennel, carvone in caraway and dill [65, 66, 67]. Due to their aromatic
qualities, these plants are used as supplements in everyday food in order to enhance
the smell, taste and biological values. For this reason this group will be in the focus.
Essential oils have been known to possess antioxidant and antimicrobial activities,
thereby serving as natural additives in foods and food products [68, 69]. Anti-
oxidative properties of essential oils are responsible for healing or improving
degradation processes in many diseases such as cancer, rheumatoid arthritis,
Table 3 Terpenoids from selected plants from Apiaceae family

Plant
Anethum Dill seed essential oil contains carvone and limonene as the dominant
graveolens compounds [73], while the main compounds in the herb essential oil are
α-phellandrene, apiole, dill ether, limonene, geraniol and p-cymene [74]
Angelica The main components of angelica roots essential oil are α-pinene,
archangelica δ-3-carene, β-phellandrene and limonene, while in seed it is β-phellandrene,
α-phellandrene, α-pinene, myrcene and α-copaene [75, 76]
Apium graveolens Celery essential oil contains limonene and selinene. However, the
important flavor constituents of the oil responsible for the typical aroma
are phtalides (3-n-butyl-4-5-dihydrophthalide (sedanenolide), 3-n-butyl
phthalide, sedanolide, and sedanonic anhydride) [77]
Carum carvi Caraway seed essential oil is comprised from carvone and limonene,
constituting more than 90% [66, 78, 79]
Coriandrum Coriander seed essential oil contains mainly linalool [80, 81, 82], while
sativum coriander herb oil has a significantly different composition with decanal,
trans-2-decenal, 2-decen-1-ol, cyclodecane and cis-2-dodecenal as the
main compounds [83]
Cuminum cyminum The distinctive flavor and aroma are originate from essential oil, the
dominant compounds of which are γ-terpinene-7-al, cumin-aldehyde,
β-pinene and γ-terpinene [84, 85, 86, 87]
Daucus carota Carrot root essential oil contains mainly geranyl, linalool, myristicine,
pentacosane, spathulenol and trans-γ-bisabolene. The major compounds of
aerial parts’ essential oil is alismol, trans-β-caryophyllene, myrcene,
α-humulene and β-ionone [88]. The major constituents of essential oil from
carrot seeds are carotol, sabinene, α-pinene followed by aromadendrene,
β-farnesene, sesquisabinene, trans-caryophyllene and myrcene [67]
Foeniculum The dominant compound in fennel seed and herb essential oil is trans-
vulgare anethole which gives them similar scent as to aniseed [89]. However,
fennel has two varieties: sweet and bitter. Sweet fennel (var. dulce) apart of
trans-anethole (more than 80%), contains estragole (less than 10%) and
fenchone (less than 7.5%). Bitter fennel (var. vulgare) apart of trans-
anethole (55–75%) contains fenchone (12–25%) [90]
Levisticum The lovage flavor, like the celery one, originates from essential oil where
officinale the dominant compound is β–phellandrene, while phthalides are present in
small amounts and give the characteristic fragrance [91]
Pastinaca sativa Root essential oil had the two major constituents, myristicine and terpinolene
[92]. Aerial parts contain essential oil with dominant cis-β-ocimene, hexyl
butanoate, trans-β-farnesene and lavandulyl acetate [93]
Petroselinum Major compounds in parsley essential oil are apiol, myristicin and
crispum β-phellandrene [94, 95]
Pimpinella anisum Anise contains essential oil with the most abundant component, trans-
anethole, above 96% which exhibits sweet and licorice taste with a herbal
anise and fennel nuance [89, 96]
cirrhosis and arteriosclerosis as well as in degenerative processes associated with

aging [70]. Essential oils also showed antimicrobial properties, which makes them
efficient alternative antibiotics and antimycotic agents [71, 72]. Review of terpe-
noids present in Apiaceae according to literature is shown in Table 3.
3 Nutraceutical Potential of Apiaceae
The aim of this review is to represent members of Apiaceae family currently used as
nutraceuticals and describe their nutritional benefits. Investigation of nutraceuticals
from this family is very attractive because of their extensive application in everyday
diet. Review of their chemical composition and biological activity highlighted their
importance as food with health benefits.
3.1 Anethum graveolens
Dill seed and leaf are the parts which are mainly used. Dill seed is used in pickled
cucumbers, bread, processed meats, sausages, cheese and condiments. Dill leaves
are used in pickles, while fresh are used for garnish or to flavor salads, vegetable
dishes, sea food, soups, yogurt and mayonnaise [2]. Dill has been used in traditional
medicines worldwide since the ancient times. It is used to relieve colic pain in babies
and flatulence in young children, as carminative (improves appetite), mild diuretic,
galactogogue, stimulant and stomachic. It is also used for treatment of diarrhoea,
astma, neuralgia, dysuria, dysmenorrhoea, gallbladder disease and insomnia [97,
98]. However, a great number of pharmacological studies show that dill possesses
significant biological activity. Because of the high antioxidative potential [18, 99],
dill can be used to improve biochemical processes in patients who suffer from
diseases in relation to metabolic syndrome [100]. Studies show that dill can be
used for managing diabetes and cardiovascular diseases because it possesses hypo-
glycemic properties [101, 102, 103]. It is documented that dill decreased total
cholesterol without any side effects [104, 105, 106]. Apart from this, dill has
hepatoprotective properties [107, 108, 109, 110, 111, 112]. Dill exhibited great
anti-cancer activity on oral cavities and breast cancer cells lines [113]. Dill is a
good antimicrobial agent [114, 115], which makes it a very significant plant in herbal
medicine, especially as a base for the development of novel antimicrobial
phytoremedies [116].
3.2 Angelica archangelica
Angelica root is used in herbal liqueurs and bitter spirits, in flavoring meat and canned
vegetables, while the seed is used in alcoholic distillates. However, chopped angelica
green parts (leaves and herb) can be added to fruit salads, fish dishes and cottage
cheese, they are used for decorating cakes and pastry and to flavor jams and jellies,
confectionaries and liqueurs [2]. The species is well known and has been cultivated
since the ancient times for treating certain diseases, such as gastrointestinal problems,
like a carminative or in flatulent colic, as well as diaphoretic and diuretic [117].
Applied externally, angelica is good as a counter-irritant, for treatment of rheumatic
diseases [118]. New investigations of this plant show that it possesses good anti-
oxidative [119, 120, 121] and antimicrobial activity [72, 75, 122, 123]. This indicates
that angelica can be used as a botanical preservative against molds, aflatoxin con-
tamination and oxidative deterioration of walnut samples [124], as well as a control
agent for plant pathogenic fungi in natural formulations [125]. Angelica root water
extraction revealed a significant antioxidant role beside its chelation potency of lead
ions, so it can be used as a natural chelator in case of lead poisoning [126]. Clinical
investigations show that angelica expressed hepatoprotective activity [119, 127] as
well as cytotoxicity in human pancreas cancer cells and mouse breast cancer cells
[128]. Angelica also shows anxiolytic activity, so it can be used for nervous disorders
and cerebral diseases, for example for epilepsy treatment [129, 130].
3.3 Apium graveolens
Celery has different forms and uses. Turnip-rooted celery, also called celeriac, is used
mainly as grated raw salad, as well as cooked vegetable in stews and soups. Leaf
celery, called smallage, is chopped and used for garnishing and flavoring, either fresh
or dried. The succulent leafstalk, often with a part of leafblade, is used for the
preparation of sauces, vegetable juices, stews, soups, salads, etc. Celery seed is
used as condiment, in pickling vegetables, salad dressings, breads, biscuits, soups,
spice mixed with salt, as bouquet garnish [2]. In traditional medicine, celery is used to
treat many diseases. Traditionaly, celery is mainly used as a diuretic and as a treatment
for arthritis and rheumatism. Celery also has sedating effect and has been used in
herbal medicine to treat nervousness, hysteria and various other conditions [131, 132].
However, investigations show that celery possesses good antioxidant activity [133,
134, 135] as well as antimicrobial activity [136, 137]. Significant hepatoprotective
activity [138, 139, 140, 141, 142], and anti-inflammatory effect of celery are reported
[143, 144]. Hypoglycemic effect of celery is also reported, as well as that its potent
role in ameliorating stressful complications accompanied by diabetes mellitus [145,
146]. Celery acts as an intestinal smooth muscle relaxant in the digestive tract [147].
Also, it has antihypertensive properties, and can be considered as an antihypertensive
agent in chronic treatment of elevated blood pressure [148]. Celery showed a signif-
icant diuretic effect that accentuates the excretion of urinary calcium [149].
3.4 Carum carvi
Caraway seed is used in creams, cakes, baked goods, cheese, confections, fresh
cabbage, meat dishes, rye bread, salads, while essential oil obtained from seed is
used to flavor chewing gum, candy, soft drinks and alcoholic beverages [2, 150].
Caraway seed essential oil has been reported to have potential therapeutic effects,
mainly due to its high antioxidant activity [151]. Considering the radical scavenging
[152] and good antioxidant profile [153, 154], it has been recommended for its
multifaceted pharmacological properties [155]. Caraway has been used to treat
digestive disorders for a long time. It can be used successfully for establishing
normal intestinal motility, for healing chronic constipation, gastric ulcer, dysbiosis,
dyspeption and heartburn [156, 157, 158, 159, 160, 161, 162]. The application of
caraway fruits significantly inhibits the increase of total cholesterol and levels of
triglycerides [163, 164, 165]. Apart from this it can be used in the treatment of
hyperglycemia [166, 167]. Caraway fruit shows strong antibacterial and antifungal
activities [168, 169, 170]. It also has anti-inflammatory properties [157] and anti-
stress activity [171]. Caraway significantly increases urine output, and the total
volume of urine excreted [172]. In addition, the aqueous extract of caraway fruits
decreases the level of glucose in serum, urea, creatinine, total urinary protein and
microalbuminuric levels. Caraway possesses strong anti-oxidant activity which pro-
vides renoprotection against diabetes and its complications [173]. It is established
that the essential oil also protects the kidneys from damage which occur as a
consequence of diabetic nephropathy [174].
3.5 Coriandrum sativum
Coriander leaf is used to make chutneys and sauces, green salsas, dips, snacks,
soups, while the seed is used in couscous, stews and salads, as a condiment in pickle
spices, seasonings, curry powders, sausages, cakes, pastries, biscuits and buns. Seed
essential oil is used in beverages, baked goods, condiments, relishes and meat
products [2]. However, leaf and seed have different aromas because of the different
chemical composition, but both oils possess good antioxidative [175, 176], as well as
antimicrobial activity [177, 178]. Studies indicated that coriander enabled develop-
ment of a novel broad spectrum of antibacterial herbal formulations, and that it has
potential for new natural antifungal formulations [179]. However, bioactive com-
pounds present in coriander are used in traditional and modern medicine, as well as
in everyday nutrition [69, 180, 181]. In folk medicine, coriander seed is used as an
aromatic, carminative, stomachic, antispasmodic and against gastrointestinal com-
plaints such as dyspepsia, flatulance and gastralgia [179]. It is often recomanded for
insomnia and anxiety [182, 183, 184]. Its use is recommended for healing the urinary
system, ie uretritis, cystitis and urinary tract infections [185]. It has also been used in
heavy metal detoxification [186]. It is used as an analgetic and antirheumatic agent
[187]. Coriander is effective against hyperlipidemia [188, 189, 190] and hypergly-
cemia [191, 192]. It also acts as a hepatoprotectant [193, 194, 195, 196, 197, 198]
and anticancer agent [199, 200, 201]. Apart from this, coriander can also be used as
an anthelmintic [202, 203].
3.6 Cuminum cyminum
Cumin seed is used as a flavoring component in beverages, confectioneries, baked

goods, meat and meat products, condiments and relishes, gravies, snack foods,
gelatins and puddings [2]. It is generally used as a food additive, popular spice,
and flavoring agent in many cuisines [204]. Cumin seeds have also been widely used
in traditional medicine for treatment of several health disorders and diseases, such as
toothaches, dyspepsia, diarrhea, epilepsy and jaundice. Hovewer, the literature pre-
sents ample evidence for the biological and biomedical activities of cumin, among
which strong antioxidative [205, 206] and antimicrobial activity [207, 208]. Because
of this, it is an important natural food preservative. Further, there is scientific proof
that cumin shows antistress, antioxidant, and memory-enhancing activities, that its
traditional use as a culinary spice in foods is beneficial in combating stress and
related disorders [209]. Cumin possesses hypolipidemic [210, 211], as well as
hypoglycaemic potential [212, 213]. Investigations show that cumin possesses
hepatoprotective properties against drugs and chemically induced hepatotoxicities,
by increasing the level of antioxidant enzymes in the liver [214, 215, 216]. The
finding also suggests that it possesses anticancer activity against several carncer cell
lines. These studies convey the use of cumin as a helper in the therapy or the control
of colon, liver and prostrate cancer. Also, the use of cumin in diet may reduce the risk
of cancer [217, 218]. Apart from this, phytochemical constituents from cumin seed
show analgesic and anti-inflammatory activities [219].
3.7 Daucus carota
The carrot is mainly consumed as a root vegetable, primarily raw, in juices, salads, or
for pickling, while it is used cooked in soups and stews, as well as for cakes. Carrot
seed is used for essential oil distillation. Obtained oil contains carotol as the
dominant compound, and is used as a flavoring agent in food products, mainly in
beverages, baked goods, condiments, relishes and meat products [2]. Lately, a
number of studies pointed out that the aerial parts are also a source of phytochem-
icals and could be economically important, for example antioxidants and for blood
pressure lowering [220, 221, 222]. The ethnobotanical uses of this species also
included applications in the treatment of cough, diarrhea, dysentery, cancer, malaria,
tumors, as an antiseptic, abortifacient, aphrodisiac, carminative, stimulant, sto-
machic and tonic [223]. Studies show that root, as the part mainly used, act as an
antioxidant [224, 225, 226], hepatoprotectant [227, 228, 229] and gastroprotectant
[230, 231, 232, 233, 234]. Carrot is a good antiinflammatory [235, 236] and
anticancer agent [46, 237, 238, 239, 240]. Also, it possesses hypoglycaemic and
hypolipidemic properties [47, 241, 242, 243]. Carrot seeds appear to be a promising
candidate for improving memory and it would be worthwhile to explore the potential
of this plant in the management of Alzheimer patients [243]. Apart from this, carrot
seed shows antinoceptive and antiinflamatory [244], as well as hypoglycaemic and
hypolipidemic properties [245, 246, 247].
3.8 Foeniculum vulgare
Fennel seed is used in meat dishes, curries, spice blends, soups, vegetables, breads.
Fresh and chopped leaves can be used as garnish for fish dishes, sauces, salads, stews
and curries. Leaf stalk, also called pseudobulb, is used raw in salads, stuffing, soups,
sauces; it can be baked or blanched. Seed and herb essential oil is used in beverages,
condiments, relishes, baked goods, frozen dairy, gelatines, puddings, meat products
and candies [2]. Fennel has a wide range of bioactivity and has proved to be a good
source for traditional medicine. Mainly, it is used as galactagogue and emmenagogue.
Because of its diuretic activity, it is useful for kidney and bladder diseases. It is also
relieves nausea and vomiting. It is useful for chronic fever and eliminates obstruc-
tions of internal organs especially those in liver, intestine and respiratory tract [248].
It provides a noteworthy basis in pharmaceutical biology for the development and
formulation of new drugs and future clinical uses [249, 250]. However, investigations
show that fennel has efficient antimicrobial activity against bacteria [35, 251, 252],
fungi [253, 254, 255] and viruses [256]. Fennel also possesses good antioxidative
potential [257, 258], because of hat it is used as food additive to provide protection
against oxidative degradation of foods by free radicals, but also used to protect
humans from oxidative stress damage [249, 259]. Fennel also possesses anti-
inflammatory [260, 261], anticancer [262] and hepatoprotective activity [263, 264].
Further, hypolipidemic and hypoglycaemic potential are also proven [263, 265], as
well as diuretic potential and beneficial effect on renal function [266, 267].
3.9 Levisticum officinale
The lovage root is used for producing soup seasonings, finished flavorings in
liqueurs; the leaf is used for seasoning sauces, meat dishes, while the seed is used
as spice, for flavoring cakes, soups, salads, for pickled vegetables (especially
cabbage and cucumbers) [2]. Lovage has the strong, characteristic seasoning-like
principle of the herb, aromatic odor and taste [268]. Lovage is one of the herbs that
have been traditionally used for treatment of many diseases, as diaphoretic, expec-
torant, stomachic and stimulant [269]. Apart from this, lovage is used in treatment of
kidney stones and urinary tract infections [270]. These uses are approved by
pharmacological studies, i.e. lovage significantly decreased levels of urine cysteine,
creatinine and volume [271]. Hovewer, clinical experience shows that lovage in
combination with other plants can be used for treatment of urogenital and gestroin-
testinal diseases [272]. Lovage exhibited significant antimicrobial [43, 273, 274] and
antioxidative activity [36, 275]. Investigations show that lovage is an inexpensive
source of natural antibacterial substances for use in pathogenic systems to prevent
the growth of bacteria and extend the shelf life of processed foods [276]. Apart from
this, lovage possesses anticancer activity, i.e. inhibits human head and neck squa-
mous carcinoma cells growth [277] as well as human liver cancer cell and breast
cancer cell lines [278]. Lovage also shows neuroprotective activities, i.e. alcoholic
extract has both repair and restoration effects on peripheral nerves [270], as well as
antiinflamatory activity [278].
3.10 Pastinaca sativa
The parsnip is a root vegetable resembling white carrot. The root is used in soups,
stews, cakes, pies and puddings, while leaves and young leafstalks can be used
cooked with other greens as a vegetable or added to soups. The seed is used as a
condiment in making beer, wine or distilled spirits [91, 279]. Parsnip has a sweet,
distinct, aromatic flavor, similar to nutmeg and cinnamon. This plant is used in
traditional medicine worldwide, mainly as a carminative, spasmolytic and diuretic.
Also, their usage in treatment of epilepsy is mentioned. Investigations show that
parsnip shows anticonvulsant activity due to the presence of a furanocoumarin
compound, xanthotoxin [280]. Apart from this, parsnip has anticancer activity,
because it contains falcarinol which proved to be the most active compound with a
pronounced toxicity against acute lymphoblastic leukemia cell line [49]. Also,
parsnip possesses antimicrobial activity against the most common human gastroin-
testinal pathogenic microbial strains [93]. Parsnip shows significant antibacterial
activities on phytopathogenic bacteria, so it might potentially be used as a biological
pesticide [92]. The results showed that the addition of parsnip could effectively
reduce lipid oxidation, maintain or improve sensory attributes and extend the shelf-
life of beef burgers during refrigerated storage. Therefore, it is suggested that
parsnip, as a natural herb, could be used to extend the shelf-life of meat products,
providing the consumer with food containing natural additives, which might be seen
as a healthier option than those of synthetic origin [281].
3.11 Petroselinum crispum
Parsley, like celery, has different forms and uses. Roothy form is mainly used as a
vegetable, while the leafy form has two varieties, broad-leaved and curly-leaved.
The parsley root is used as a vegetable to enhance flavor in soups, stews and
condiments, while the leaf is used as a garnish (for salads, soups, boiled potatoes
and egg dishes), blended in dips and cooked sauces. Seed is usually used for
essential oil extraction. Obtained oil is used to flavor meat sauces, pickles, spice
blends, baked goods, oils and fats, processed vegetables, soups, gelatines and
puddings [2]. Parsley can be used fresh and dried. Parsley has a pungent, warm,
spicy, herbaceous-scent. However, smell and taste in dried spices are different from
the fresh ones due to changes in volatile profile during the drying process [282].
Parsley is mainly used in food industry as a vegetable and spice, while in etnophar-
macology application as an appetizer, carminative and diuretic. However, investi-
gations show that parsley shows potential to prevent oxidative stress-related
diseases and can be developed into functional food or alternative natural antioxi-
dants [283, 284]. Parsley showed a hepatoprotective effect against acute liver injury
induced by chemical agents [285, 286, 287, 288], as well as injuries due to the
complication of diabetes [289] or chronic changes induced in non-alcoholic fatty
liver disease [290]. Parsley is a good diuretic and has antihypertensive effect [291,
292]. Apart from this, parsley shows antiurolithic effects against calcium oxalate
stones [293].
3.12 Pimpinella anisum
Anise is used in beverages, baked goods, condiments, relishes, oils and fats, frozen
dairy, gravies, meat products and soft candy. Seed essential oil is used in chewing
gums, gelatines, puddings, soft and hard candies [2, 150]. Anise, like fennel, has an
aromatic and sweet taste. It is used in folk medicine in many countries for treatment
of digestive, respiratory and neurological diseases, as well as natural estrogen [294,
295]. New investigations show that anise is rich in phytochemical contents, which
possess high antioxidant [296, 297] and antimicrobial activities [71, 299]. A new
interesting approach to develop plants as natural source and preservative for the food
industry is considered. Treatment with anise is effective in reducing the level of some
of biochemical parameters and ameliorate behavior of intoxicated by lead [298].
Apart from this, anise possesses strong anticancer activity on human prostate cancer
cell line [300], as well as on gastric cancer cell line [301]. Thus, anise could be one of
the foods that attribute to cancer prevention and treatment. It could also be a natural
source of novel anticancer compounds with anti proliferative and/or apoptotic
properties. Anise also possesses hypoglycaemic and hypolipidemic properties
[302, 303]. Investigation shows hepatoprotective activity, too [304, 305]. High
range of active potentials leads to various applications of anise, such as health
supplement and pharmaceutical benefits [41]. This is conditioned by significant
content of synergistic action of the bioactive compounds present in the seed [12, 306].
4 Conclusion
Phytochemical screening of spices and vegetables which are usually used in diet,
shows that they possess bioactive constituents of pharmaceutical importance. Ther-
apeutic activity is especially important in prevention and treatment of modern
diseases which are directly related to oxidative stress, such as aging, cancer etc.
Nutritionally induced acute and chronic diseases such as diabetes, hyperlipidemia,
liver diseases and others, can be prevented or relieved by using plants. Thereby,
promoting optimal health, longevity and quality of life can all be achieved by plants,
i.e. nutraceuticals present in them.
It is known that many plants have been used as food additives and in folk
medicine for treating numerous diseases worldwide since the ancient times. How-
ever, only recently has there been a huge number of pharmacological and clinical
studies about the positive effects that plants have on the human health. These studies
have awakened people’s interest in the plant’s usage. Plants from Apiaceae family
are used in almost all national cuisines, both as a vegetable (carrot, celery, parsley,
parsnip, fennel) or as spice or condiment (dill, cumin, anise, coriander, lovage,
angelica). Due to the complex chemical composition, they show significant thera-
peutic activity, while their aromatic properties enable their wide application in
everyday nutrition. All economic studies imply that nutraceuticals will play an
important role in the future development of food with therapeutic properties.
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Functional Components and Medicinal
Properties of Food 45
Christian Izuchukwu Abuajah
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1345
2 An Overview of Functional Components of Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1346
2.1 Non-starch Carbohydrates (Examples Dietary Fibers, Fucoidans) . . . . . . . . . . . . . . . . . . 1348
2.2 Carotenoids, Phenolics, Phytosterols, Tocopherols/Trienols and Organo-Sulfur
Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1354
2.3 Probiotics and Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1359
3 Food as Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1361
4 Healing Powers of Functional Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1362
4.1 Anti-oxidation Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1362
4.2 Anti-cancer Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363
4.3 Anti-diabetic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1364
4.4 Anti-inflammatory Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1364
4.5 Cardio-vascular Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1364
4.6 Anti-hypertensive Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1365
5 Optimal Health and Well-Being . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1365
6 Mode of Action of Functional Components and How they Impact on Optimal
Health and Well-Being . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1366
7 Effect of Processing on Functional Components of Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1367
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1370
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371
Abstract
There is growing evidence that functional components (bioactives and phyto-
chemicals) of food play an integral role in the link between food and the
prevention of diseases. Although some functional components were labelled
anti-nutrients, their role as potential healthy biochemical components of diets
for the prevention of degenerative pathologies have been scientifically elucidated.
C. I. Abuajah (*)
Department of Food Science and Technology, University of Uyo, Uyo, Nigeria

1344 C. I. Abuajah
Some properties which link functional components to potential health-modulat-

ing roles and functions can be classified into anti-oxidation, anti-cancer, anti-
diabetic, anti-inflammatory, cardiovascular, anti-microbial, immunomodulatory
and anti-hypertensive. However, the mechanisms through which they impact on
human health are not completely clear. Food processing techniques exercise
effects on functional components of food. While some processing techniques
increase their concentration in food, others decrease them. Therefore, in this era
when the role of a healthy diet in preventing degenerative, non-communicable
and chronic diseases is well accepted, the borderline between food and medicine
is becoming very thin. Thus, the concept of food has obviously gone beyond
basic nutrition only. While products intended to cure diseases are classified as
medicine, a healthy diet consisting of foods with functional components can help
optimize health and promote well-being as well as reduce or prevent the risk of
developing certain diseases.
Keywords
Bioactives · Diet · Food · Functional components · Health · Medicine · Nutrition ·
Phytochemicals · Well-being
Abbreviations
AK1 Adenylate kinase1 encoding gene
CAT Catalase
CLA Conjugated linoleic acid
CR3 Complementary receptor3
DF Dietary fiber
DP Degree of polymerization
EGC3G Epigallocatechin-3-gallate
FOS Fructo-oligosaccharide
GPX Glutathione peroxidase
GR Glutathione reductase
HD High density lipoprotein
HG-2 Human hepatoma cells G2
HPP High pressure processing
HTLV-1 Human T-celllymphotroic virus type1
HWE Hot water extract
IL-1 Interleukin-1
LAB Lactic acid bacteria
LacCer Lactocylceramide (a bioactive lipid)
LDL Low density lipoprotein
MAPK Mitogen activated protein kinases
MP2 Microphage inflammatory protein2
mRNA Messenger ribonucleic acid
45 Functional Components and Medicinal Properties of Food 1345
MyD88 Myeloid differentiation primary receptor gene 88

NFAT Nuclear factor of activated T cells
NF-kB Nuclear factor kappa light-chain-enhancer of activated β cells.
NK Natural killer
Nrf2 Nuclear factor2 pathway
NTP Non-thermal processing
PEF Pulsed electric field
Pi3k Phosphatidyl inositol-4,5-biphosphate-3-kinase
PKC Protein kinase C
PUFA Poly-unsaturated fatty acid
RNS Reactive Nitrogen species
SIGNR3 Specific ICAM (intracellular adhesion molecule)-3 grabbing non-
intergrin-related antigen
syk Spleen tyrosine kinase encoding enzyme gene
TLR Toll-like receptors
TNF-α Tumor necrosis factor alpha
TRAF6 TNF receptor associated factor6
WBC Water binding capacity
1 Introduction
Food is any substance consumed to nourish and refresh the body [1]. It is the most
imaginable sets of complicated biochemical substances ingested into the body to
provide nutritional support and refreshment necessary for the health, growth, and
normal functions of every living organism. Essentially, food is a mixture of
chemicals or nutrients which can be separated into different components with one
or more different functions in the body including energy generation; growth and
repair of worn out cells and tissues; regulation of processes and protection against
diseases and invasion by pathogens. The two major components of food are nutritive
or primary metabolites (consisting of the macronutrients that are present in relatively
large amounts, such as proteins, carbohydrates, lipids and micronutrients present in
small amounts, such as vitamins, minerals, and water which are essential for the
sustenance of normal body functions, e.g., energy generation, growth, repair, regu-
lation, etc.) and non-nutritive or secondary metabolites (these are bioactives or
phytochemicals which possess properties with potentially positive effect on health
beyond basic nutrition. These non-nutritve substances are known as functional
components. A combination of the right types of food optimizes health and impacts
positively on wellness. Dietary guidelines around the world recommend increased
consumption of fruits and vegetables, as good sources of beneficial plant chemicals
and essential nutrients to improve health and reduce the risk of chronic diseases [2].
Accordingly, daily intake of at least 400 g of vegetables and fruits have been
recommended by experts [3].
1346 C. I. Abuajah
The non-nutritive components of food are variously referred to in different

contexts as functional or bioactive components (biomolecules present in food that
exhibit the capacity to modulate one or more metabolic processes, which results to
health benefits and promotion of well-being) or phytochemicals (plant-derived,
biologically active chemicals that function in the body to prevent certain disease
processes) [4, 5]. There are over a 1000 phytochemicals found in foods, and one
serving of about 120 g of a fruit or vegetable may have as many as 100 different
phytochemicals [6]. Research has established a relationship between functional
components in food, health and well-being [7]. Consequently, functional compo-
nents have health-promoting roles at various stages of disease control that are
associated with multiple progressive steps, from initiation to development. Thus,
they can be effectively applied in the treatment and prevention of diseases [8]. These
days, as a result of the acceptance of the role of a healthy nutrition based on the right
choice of diet in preventing diseases, the difference between food and medicine is no
longer obvious [9].
Previously, it was thought that functional components occur only in plant foods
including whole grains, nuts, seeds, spices, fruits, and vegetables. However, pro-
biotics, polyunsaturated fatty acids (PUFA) such as conjugated linolenic acid (CLA),
long-chain omega-3, -6 and -9 fatty acids including eicosapentaenoic acid (EPA),
docosahexaenoic acid (DHA) and bioactive peptides are equally found in animal
products such as milk, fermented milk products and cold-water fish. They also have
multiple metabolic activities allowing for beneficial effects on several diseases and
target tissues in the body [5]. Table 1 presents some functional components of food,
their common sources, and potential benefits.
This chapter therefore presents functional components of food in the light of their
medicinal properties, impact and mode of action in optimizing health and well-
being. In addition, the different types, nature, functions, source, and the effect of
processing techniques on functional components of food are discussed.
2 An Overview of Functional Components of Food
Functional components are mainly the non-nutritive secondary metabolites in food.

Although some functional components are labelled as anti-nutrients, their role as
potential healthy biochemical constituents of diets for the prevention of degenerative
pathologies have been scientifically elucidated [10]. Functional components were
mostly ignored until recently when their potential metabolic effects were first
detected. For example, flavones were found to protect against heart diseases and
soy-based estrogens against cancer [10]. The bioactivity and chemistry of other non-
nutritive compounds in foods such as red wine, coffee, nuts, seeds, whole grains,
spices, herbs, fruits and vegetables have been investigated to determine their bio-
chemical effects and potential health benefits [11]. They are essential for normal
growth, development and defense of plants [12] and seem to replicate the same
functions in humans. To date, different types of secondary metabolites have been
identified in plants [13]. Chemically, these compounds are either nitrogen-containing
alkaloids or nitrogen-deficient terpenoids and phenolics [14].
Table 1 Some functional ingredients of food, their sources, and potential benefits
Bioactive components Source Potential benefits
Carotenoids
Alpha-carotene/beta-carotene Carrots, fruits, Neutralize free radicals which may cause
vegetables damage to cells
Lutein Green vegetables Reduce the risk of muscular degeneration
Lycopene Tomato products Reduce the risk of prostate cancer
(ketchup, sauces)
Non-starchy polysaccharides
Fucoidan Mushrooms Immune modulation; apoptosis of cancer
(maitake and reshi), cells; stimulates brain development;
brown seaweeds anticlotting effect; lower blood
cholesterol levels; decrease high blood
pressure, stabilize blood sugar
Insoluble dietary fiber Wheat bran Reduces risk of breast or colon cancer
Soluble dietary fiber (β-glucans) Oats, barley, Reduces risk of cardiovascular disease;
psyllium protects against heart disease and some
cancers; lower LDL and total cholesterol
Fatty acids
Long chain omega-3 fatty Salmon and other Reduce risk of cardiovascular disease;
acids (DHA/EPA), fish oils improve mental and visual functions.
Conjugated linoleic acid Cheese, meat Improve body composition; decrease risk
(CLA) products of certain cancers
Phenolics
Anthocyanidins Fruits Neutralize free radicals; reduce risk of
cancer
Catechins Tea Neutralize free radicals; reduce risk of
cancer
Flavonones Citrus Neutralize free radicals; reduce risk of
cancer
Flavones Fruits/vegetables Neutralize free radicals; reduce risk of
cancer
Lignans Flax, rye, vegetables Prevention of cancer; renal failure.
Tannins (proanthocyanidins) Cranberries, Improve urinary tract health; reduce risk
cranberry products, of cardiovascular disease
cocoa, chocolate
Phytosterols
Stanol ester Corn, soy, wheat, Lower blood cholesterol levels by
wood oils inhibiting cholesterol absorption
Prebiotics and probiotics
Fructo-oligosaccharides (FOS); Jerusalem Improve quality of intestinal microflora;
artichokes, shallots, gastrointestinal health
onion powder,
Lactobacillus; Yogurt, other dairy Improve quality of intestinal microflora;
Biofidobacterium products gastrointestinal health
Soy phytoestrogens
Isoflavones: Daidzein Soybeans and soy- Menopause symptoms such as hot flashes;
genistein based foods protection against heart disease and some
cancers; lowering of LDL and total
cholesterol
1348 C. I. Abuajah
Groups of plant secondary metabolites are diverse and usually occur in multi-
ple and complex forms such as methylated, ethylated, glycosylated, esterified,
thiolated, or hydroxylated [15]. These include about 8000 varieties of phenolics
including flavonoids and non-flavonoids; about 25,000 terpenoids, carotenoids,
xanthophylls, and iridoids; about 12,000 alkaloids; and several sulfate-containing
compounds such as isothiocyanates [11]. To date, numerous studies of the capac-
ities of functional components as phytochemicals including glucosinolates, phy-
tosterols, tocopherols, tocotrienols, allyl sulfides, and non-starch carbohydrates
including soluble and insoluble dietary fiber and fucoidan, as antioxidation,
anticancerous, anti-inflammatory, antiviral, immune modulating, and antihyper-
tensive agents are widely reported in literature [16–18]. The list is never exhaus-
tive as new compounds beneficial to human health are constantly being
discovered [19].
Functional components exhibit the capacity to modulate one or more metabolic
processes, which results to optimal health benefits and promotion of wellness in
humans. In addition, they have been shown to exert multiple bioactive functions
including scavenging of reactive oxygen species (ROS), dynamic regulation of
metabolic functions of proteins, enzymes, transporters, receptors, and signal trans-
duction processes related to various lifestyle-related diseases. Thus, allowing for
beneficial effects on several degenerative and non-communicable and chronic dis-
eases as well as target tissues [11, 15]. Therefore, functional components have
potential positive impact on health beyond basic nutrition. An overview of the
types, functions, medicinal properties and sources of some food functional compo-
nents as reported by Abuajah et al. [20] is as follows:
2.1 Non-starch Carbohydrates (Examples Dietary Fibers,

Fucoidans)
Basically, these are structural and storage carbohydrate polymers of simple sugars
including glucose, galactose, fructose, xylose, arabinose, etc., but are not starchy
in nature (i.e. their sugar units are not linked by either α-(1 ! 4) or α-(1 ! 6)
glycosidic bonds. Thus, they are not hydrolysable by the human digestive
enzymes but undergo fermentative modification by the probiotic microbes in the
colon. There are several kinds of non-starch carbohydrates including dietary fibers
and fucoidans.
(a) Dietary fibers: Dietary fibers (DF), which could be either soluble or insoluble,
are non-starchy polysaccharides and structural materials of the cell walls of
cereals and microorganisms. They are the indigestible part of plant foods
composed of long linear and branched chains of glucose molecules held together
by bonds that cannot be hydrolyzed by human digestive enzymes. Chemically,
DFs are glucose polymers in hetero-structural configuration of β-(1 ! 3:1 ! 4)
or β-(1 ! 3:1 ! 6) bonds depending on their sources (Fig. 1). Cereal and
bacterial DFs are primarily linear with large regions of β-(1 ! 4) linkages
a CH2OH CH2OH
O O O O
β-(1.6)-D-glucose branch
O
OH OH
OH OH
CH2OH CH2OH CH2

O O O O O O O
OH OH OH
OH OH OH
β-(1.3)-D-glucose β-(1.3)-D-glucose β-(1.3)-D-glucose
CH2OH
b O
OH
CH2OH CH2OH
O O O O
OH
OH
CH2OH
O O OH
OH OH
CH2OH OH
β-(1.4)-D-glucose β-(1.3)-D-glucose
O O
OH
OH
β-(1.4)-D-glucose
O
OH
Fig. 1 Structures of β-glucans. (a) Fungi β-glucan (b) Cereal β-glucan
separating shorter stretches of β-(1 ! 3) structures. Fungi β-glucans (e.g.,

mushroom) have short β-(1 ! 6)-linked branches coming off the β-(1 ! 3)
backbone whereas those of yeast have β-(1 ! 6) branches that are further
elaborated with additional β-(1 ! 3) regions. These structural differences do
have large implications and impact on their properties, e.g., water solubility.
Also, differences in the length of the polysaccharide chain, extent of branching,

and the length of those branches result in differences in hot water extracts (HWE),
level of viscosity and differences in molecular weights [9, 21–23]. The water-soluble
fibers are mainly β-glucans, gums, pectin, mucilage and arabinoxylans while the
water-insoluble fibers are composed of lignin, cellulose and hemicellulose [24–26].
Specifically, the mixed-linkage structure of β-glucans and length of the polysaccha-
ride chain or degree of polymerization (DP) are responsible for their physical
properties and characteristics including viscosity, solubility, molecular weight,
1350 C. I. Abuajah
water binding capacity (WBC), and foamability (foam capacity and stability). These
linkages prevent compact folding of β-glucan chains, making them soluble in water
[27, 28]. To maintain their functional attributes, it is important that the processing of
β-glucans does not destroy their structure because these large macromolecules are
mechanically sensitive and can be broken at high shear rates [28].
(b) Fucoidans: Fucoidans are non-starchy but sulfated polysaccharides which occur
in various species of brown algae and brown seaweed such as mozuku, kombu and
bladderwrack. Different forms of fucoidan have also been discovered in animal
species, including sea cucumber [29]. Fucoidans have a complex structure which
varies according to its source. It is primarily a polymer of α-(1 ! 3) linked fucose
pyranose sugar units with sulfate groups substituted at C-2 and C-4 positions on
some fucose residues. In addition, fucoidan backbone could be of α-(1 ! 3)-
linked α-l-fucopyranose sugar or of alternating (1 ! 3)- and (1 ! 4)-linked α-l-
fucopyranose sugar residues, which may also include sulfated galactofucans with
backbones built of β-(1 ! 6)-d-galacto- and/or β-(1 ! 2)-d-mannopyranose sugar
units with fucosepyranose or fuco-oligosaccharide units forming branched points
(one for every 2–3 fucosepyranose residues within the chain) with glucuronic acid,
xylose or glucose substitutions [29, 30]. Two structural features which distinguish
fucosepyranose from other six-carbon pyranose sugars present in mammals are the
lack of a hydroxyl group on the carbon at the 6-position (C-6) and its l-configu-
ration [31]. Basically, fucosepyranose is equivalent to 6-deoxy-l-galactose. A
simple structure of fucoidan polymer and a non-substituted fucosepyranose
sugar unit are shown in Fig. 2.
O O O
a CH3 CH3
CH3 OH•H
HO3SO HO3SO
O O
HO3SO
HO3SO
HO3SO O
HO3SO
O O O
CH3 CH3
HO3SO HO3SO CH3
O O
b
HO3SO
O
OH
HO3SO HO3SO O HO OH
O HO
HO OH
CH3
HO
OSO3H
Fig. 2 Structures of (a) Fucoidan (b) Non-substituted fucose pyranose unit

2.1.1 Functions
The long fibrous structures of dietary fiber allow them to entrap harmful toxins and
carcinogens in the digestive tract. Cereal β-glucan (a soluble dietary fiber) has
gained special attention for their many health benefits including its viscous nature
that slows down the passage of food through the gut thereby reducing the rate of
glucose absorption in the intestine, thus lowering the glycemic index (blood
glucose level) and serum cholesterol. In addition it possesses the characteristics
of good water retention capacity, gelling ability and hydro-colloidality and these
properties have influenced their use as substitutes for fat [32]. β-Glucans act as
anticytotic, antimutagenic, antitumoregenic and immunomodulatory agents in the
human body. Fungi β-glucans, a family of diversified structures found in the cell
wall of yeast and molds, enhance leucocytes activity that is responsible for
enhancing body defense mechanism. They have been reported to act as immune
system activators and cell response modifiers. Innulin has successfully replaced fat
in dairy products [6, 22, 23, 28, 33]. Soluble dietary fiber can dissolve in or absorb
water and is effective in binding toxins and cholesterol in the intestinal tract. Based
on data from in vitro and in vivo animal studies, β-glucans enter the proximal small
intestine rapidly and are captured by the macrophages after oral administration.
The β-glucans are then internalized and fragmented into smaller sizes and are
carried to the marrow and endothelial reticular system. The small β-glucans
fragments are then released by the macrophages and taken up by the circulating
granulocytes, monocytes and dendritic cells (Fig. 3). This turns on the immune
response system [22, 34].
β-Glucans are captured by macrophages via the Dectin-1 receptor with or without
toll-like receptors 2 and 6 (TLR-2 and 6). The large β-glucan molecules are then
internalized and fragmented into smaller sizes within the macrophages. They are
carried to the marrow and reticulo-endothelial system and subsequently released.
These small β-glucan fragments are eventually taken up by the circulating
β-Glucans
Monoclonal
Dectin-1 Tumor
TLR-2/6 Antibody
Specific
with iC3b
Ag
Endosome with
β-Glucan β-Glucans
fragment bind
CR3
Tumor Cells
Granulocytes
Macrophages β-Glucan Fragments
Fig. 3 β-Glucans and resistance to infections

1352 C. I. Abuajah
granulocytes, monocytes or other macrophages via the complementary receptor 3

(CR-3). This turns on the immune response; one of its actions being the phagocytosis
of the monoclonal antibody tagged tumor cells.
Similarly, β-glucans can bind and act on a diversity of immune specific and
related receptors including Dectin-1, complement receptor cells (CR3), cytokines,
chemokines, transcriptional factors and growth factors to trigger a wide spectrum of
immune responses. The targeted immune cells of β-glucans include macrophages,
neutrophils, monocytes, natural killer (NK) cells and dendritic cells. Thus, a cascade
of information transduction system is initiated that stimulates the entire immune
system against unwanted cell growth. The immunomodulatory functions induced by
β-glucans involve both innate and adaptive immune responses [21, 22, 35, 36].
Simplified illustrations of these mechanisms are shown in Fig. 4. Insoluble dietary
fiber, on the other hand, cannot dissolve in water and is effective in adding to fecal
bulk and increasing the rate of passage of food through the intestinal tract. Insoluble
dietary fiber also dilutes out potential carcinogens and decreases contact of toxins
and carcinogens with the intestinal tract and speeds up their passage out of the body
[23, 27, 28].
β-Glucans act on a variety of membrane receptors found on immune cells. By
their actions various signaling pathways are activated and their respective simpli-
fied downstream signaling molecules are shown. The reactor cells include mono-
cytes, macrophages, dendritic cells, natural killer cells and neutrophils. Their
corresponding surface receptors are listed. β-Glucans also trigger a cascade of
cytokines release, such as tumor necrosis factor alpha (TNF)-α and various types of
interleukins (ILs). The immunomodulatory functions induced by β-glucans
involve both innate and adaptive immune responses.
Fucoidans offer various potential bioactive and functional benefits. It is used as an
ingredient in some dietary supplement products. The bioactive properties may vary
depending on the source of seaweed, the compositional and structural traits, the
content (charge density), distribution, and bonding of the sulfate substitutions, and
the purity of the fucoidan product. Fucoidan inhibits the spread of cancerous cells by
preventing the adhesion of tumor cells to the extracellular matrix as well as induce
apoptosis, or programmed self-destruction, in human T-cell leukemia virus type I
(HTLV-1) which is responsible for adult T-cell leukemia. The polysaccharide paves
way for apoptosis by inactivating NF-kB, a naturally occurring substance that
regulates anti-apoptotic proteins [31].
Fucoidan have also been shown to stimulate the phagocytic action of macro-
phages and synthesis of several immune cell types, which increase protection
against infection [21, 22, 37]. The nutritional makeup of fucoidan could be likened
to that of breast milk which is the most perfect immune-supporting food known.
The polysaccharide gives the immune system a big boost by enhancing phagocy-
tosis, the process through which white blood cells attack and destroy pathogens.
Fucoidan also increases the number of mature white blood cells that are circulating
in the body, activity against hepatopathy, uropathy and renalpathy, protective
effects on gastric organ and gastric mucosa as well as therapeutic potential in
surgery [29], bolstering the first line of defense against infections and diseases
β-glucan
Dectin-1
nd CR3
+ ? Liga SIGNR1 Scavenger
LacCer
6
TLR2 /
4
TLR
syk iC3 MIP2 PI3K

MyD88
PKC Akt
TRAF-6
card9 MAPK
NFAT
NF-κB
Dectin-1 Dectin-1 Dectin-1

TLR TLR LacCer
SIGNR1 CR3
Scavenger
SIGNR1
Dectin-1
TLR
Neutrophil
Monocyte
Macrophage
Cytokine Dendritic cell

(TNFα, IL-2,
IL-10, IL-12)
CR3 T cell
Natural killer B cell

cell
Targeted cells Humoral immunity Cell-mediated Phagocytosis

lysis immunity of
targeted cells
Adaptive immunity Innate immunity
Fig. 4 Immune activation induced by β-glucans
[37]. In addition, several other bioactivities associated with fucoidan have been
reported in literature including anti-coagulant and anti-thrombotic activity of blood
plasma, antivirus, anti-inflammatory, reduction of blood lipids, anti-oxidation, and
anti-complementary properties.
1354 C. I. Abuajah
2.1.2 Sources
Foods rich in soluble dietary fiber (DF) include apples, cranberries, mango, oranges,
asparagus, broccoli, carrots, peanuts, walnuts, most legumes, oats, and psyllium
while those rich in insoluble dietary fiber are apples, bananas, berries, broccoli, green
peppers, spinach, almonds, sesame seeds, most legumes, brown rice, whole-wheat
breads and cereals. In addition, cereals (e.g., oats and barley) and bacteria are rich
sources of β-(1 ! 3:1 ! 4)-glucan (a DF with strong colloidal properties which is
considered as good functional ingredient in foods for its cholesterol-lowering and
low-glycemic index functions). β-(1 ! 3:1 ! 4)-Glucan is present in cereal bran
(e.g., 2.2–7.8% in oat and 2.5–11.3% in barley). Brown seaweeds and some medic-
inal mushrooms are high in fucoidan [38]. Polysaccharides of mushrooms and
yeasts such as β-(1 ! 3:1 ! 6)-glucan have been in focus for their anti-tumor
activity and the chemical diversity of these glycans ranges from homopolymers to
highly complex heteropolymers. Varieties of sugars such as glucose, galactose,
mannose, xylose, arabinose, sucrose ribose, glucouronic acid etc. are involved in
the formation of such polysaccharides. Some of the glycans which form conjugates
with proteins and peptides show higher potent anti-tumor activity [21, 22, 39].
2.2 Carotenoids, Phenolics, Phytosterols, Tocopherols/Trienols

and Organo-Sulfur Compounds
Carotenoids, phenolics, phytosterols, tocopherols/trienols and organo-sulfur com-

pounds are classes of phytochemicals commonly referred to as antioxidants. Antiox-
idants are bioactive compounds which neutralize free radicals, reactive oxygen
species (ROS), and reactive nitrogen species (RNS) in the cell. A free radical is an
atom that has an unpaired electron and is highly charged and unstable and causes
damage to the cell. Free radicals can form in lipids, proteins, and carbohydrates.
Examples of antioxidants are as follows:
(a) Carotenoids (examples Lycopene, Lutein): The carotenoids are lipid-soluble

plant pigments that are either oxygenated or non-oxygenated hydrocarbons
containing at least forty carbons and an extensive conjugated double bond
system. Alpha-carotene, beta-carotene, and lycopene are the predominant non-
polar functional carotenoids and lutein is the primary polar functional caroten-
oid. Carotenoids can be found esterified to fatty acids or un-esterified in plant
tissues. Lycopene are the most active oxygen neutralizer with potential chemo-
preventive activities (Fig. 5). The total carotenoid content of fruits and vegeta-
bles varies with age and storage [40].
(b) Phenolics (examples Apigenin, Rutin): Phenolic compounds, commonly known
as polyphenols, are the most numerous and widely distributed group of functional
molecules. They are diverse groups of plant substances that contain one or more
(benzene) rings and varying number of hydroxyl (OH), carbonyl (CO) and car-
boxylic acid (COOH) groups. They commonly exist in conjugated forms with one
or more attached sugar residues. They are classified as flavonoids which comprise
a H3C
H3C CH3 CH3 CH3
H3C CH3
CH3 CH3
CH3
b OH
H3C
H3C CH3 CH3 CH3
CH3 CH3 H3C CH3

HO CH3
Fig. 5 Structures of carotenoids (a) Lycopene (b) Lutein
Fig. 6 Structures of polyphenols (a) Flavonoids (b) Nonflavonoids
of flavonols (kaempferol, quercetin), flavones (apigenin, luteolin), isoflavones

(daiazein, genistein), flavanones (naringenin, hespertin), anthocyanidins (malvidin,
cyaniding) and flavan-3-ols (catechin, epicatechin), and non-flavonoids including
chlorogenic acids such as quinic acid and tartaric acid [40, 41] (Fig. 6). Phenolics
content can vary tremendously between food sources and within foods of the same
type. The following ranges were reported for total polyphenol content in some food
materials and fruits: barley and millet (590–1500 mg 100 g1 dry matter); oats and
corns (8.7–30.9 mg 100 g1 dry matter); fresh onions and leeks (20–20.25 mg
100 g1 dry matter), fresh brussel sprouts (6–15 mg 100 g1 dry matter), blue-
berries, strawberries, cranberries, and raspberries the total polyphenol content is
about 37–429 mg 100 g1 dry matter [42].
(c) Phytosterols (example Stigmasterol): Phytosterols are the plant equivalent of
cholesterol in animals. Their structures are similar. However, the side-chain in
plant sterols contains additional double bonds and methyl and/or ethyl groups.
1356 C. I. Abuajah
a b
H
H
H H
H H H H
HO HO
Fig. 7 Structures of phytosterols (a) campesterol (b) Beta-sitosterol
a
R1
HO
CH3
R2 O H H
CH3 CH3 CH3 CH3
R3
b
R1
HO
CH3
R2 O
CH3 CH3 CH3 CH3
R3
Fig. 8 Structures of (a) Tocopherol (b) Tocotrienol
The most common bioactive phytosterols are beta-sitosterol, kampesterol and

stigmasterol (Fig. 7). A daily non-vegetarian diet contains approximately
250 mg of unsaturated phytosterols while a vegetarian diet contains over
500 mg. The saturated derivatives of plant sterols are plant stanols such as
sitostanol [5].
(d) Tocopherols/Tocotrienols (example Vitamin E): The tocopherols and
tocotrienols are lipid-soluble functional components which contain a phenolic-
chromanol ring linked to an isoprenoid side chain that is either saturated
(tocopherols) or unsaturated (tocotrienols) (Fig. 8). There are also four primary
forms of tocopherols and tocotrienols: alpha, beta, gamma, and delta that differ
in the number and position of methyl groups on the phenolic-chromanol ring. In
addition, tocopherols have three asymmetrical carbons at positions 2, 4, and 8 of
the isoprenoid side chain. Consequently, there are eight isomeric forms of
tocopherols, of which RRR-a-tocopherol has the greatest bioactivity and is
also the most abundant in human blood and tissues [6, 40].
Fig. 9 Structures of organo- a b

sulfur compounds (a) Benzyl S
isothiocyanate (b) Diallyl C H2C S
N S CH2
disulfide
(e) Organo-sulfur compounds (example Diallyl sulphide): The organo-sulfur

compounds are commonly found in cruciferous vegetables such as broccoli,
cauliflower, and brussel sprouts or allium vegetables (vegetables in the same
family or class with cabbage, onions and garlic) such as leeks. Organo-sulfur
compounds contain sulfur atoms that are bound to a cyanate group or a carbon
atom in a cyclic or non-cyclic configuration. The functional ingredients of foods
containing organo-sulfur compounds are obtained only after cutting, chewing or
crushing has disrupted the cells to expose them. In cruciferous vegetables,
various isothiocyanates such as sulforaphane, phenyl isothiocyanate and benzyl
isothiocyanate (Fig. 9a) are formed from glucosinolyates by the action of
myrosinase. In alliums, allicin is formed from allin and then rapidly converted
to diallyl sulfide, diallyl disulfide (Fig. 9b) or diallyl trisulfide by the action of
allinase. In both cruciferous and allium vegetables, these hydrolytic breakdown
products are the health-promoting functional components [5].
2.2.1 Functions
The primary functions of antioxidants include the regulation of the redox potential
within a cell and the reduction of potential initiators of cell death and carcinogenesis.
Hence antioxidants are anti-carcinogenic agents. The redox potential refers to the
balance of the reducing and oxidizing reactions that occur within the cell. Redox
changes within a cell are able to trigger various molecular responses such as
induction of cell death and activation of signal transduction (the transfer of messages
between cells and within a cell). Therefore, redox regulation of physiological and
pathological processes is important in optimizing health and disease prevention [40,
43]. Other functional antioxidant compounds are able to bind to toxins or carcino-
gens in the intestinal tract, such as the binding of N-nitroso compounds by poly-
phenols in tea, thereby preventing their transformation or even absorption [40].
The lipid-lowering mechanism of phytosterol and stanols occurs by sequestering
cholesterol in the intestinal tract and reducing its absorption. Epidemiological and
experimental studies suggest that dietary phytosterols may offer protection from
most of the common cancers in western societies such as colon, breast and prostate
cancers [44]. The possible mechanism by which phytosterols offer this protection
include its effects on membrane structure, tumor and host cell tissues, signal
transduction pathways that regulate tumor growth and apoptosis, immune functions
of the host and cholesterol metabolism by the host [21, 22]. Phytosterols are pre-
cursors for lactogogues. Lactogogues are generally prescribed to lactating mothers to
augment milk production and also acts as a precursor for hormones required for
1358 C. I. Abuajah
reproductive growth. Other phytosterols including stigmasterol, sitosterol and

kampesterol are also precursors for hormones. These compounds increase the
estrogen production, which in turn stimulates the proliferation of the mammary
gland ducts to produce milk [45].
The structural similarity between several isoflavone metabolites and those of
estrogens and estradiols suggests the possibility of estrogen-like biological activities
in isoflavones. Isoflavones or phytoestrogens, however, exhibit antagonist estrogen
activity resulting in lower overall exposure to estrogen in premenopausal women and
reducing breast cancer risk [46–48]. In postmenopausal women, phytoestrogen-rich
diets reduce hormone-sensitive plasma cholesterol levels and bone loss [49, 50].
Similarly, the induction of enzyme systems that detoxify toxic chemicals includ-
ing the phase I (example, cytochrome P450 group of oxidases) and phase II (e.g.,
N-acetyl transferase, glutathione S-transferase, UDP-glucoronyl transferase, etc.)
detoxifying enzymes is thought to reduce one’s susceptibility to mutagenic effects.
Functional food components with antioxidant functions are able to activate phase II
detoxifying enzymes via the antioxidant responsive element pathway such as the
nuclear factor [erythroid-derived 2]-like 2 also known as Nfe2l2 or Nrf2 signaling
pathway [40, 42, 51]. Organo-sulfur compounds such as isothiocyanates, in partic-
ular sulforaphane, are potent mono-inducers of phase II detoxifying enzymes [52]
while diallyl sulfides from garlic preparations are inducers of both phases I and II
detoxifying enzymes [53].
A primary mechanism for immune-modulation is the multiple antioxidant capability
of polyphenols, tocopherols, carotenoids, isothiocyanates, and allyl sulfides, lycopene
being the most active oxygen neutralizer with potential chemo-preventive activities.
Together, these compounds are able to reduce the deleterious effects of reactive oxygen
species (ROS) and free radicals, which cause premature death of immune cells [54].
Garlic is found to be a superior phytochemical in the reduction of total cholesterol levels
[55] and is also reported to control arterial stiffness by increasing the good cholesterol:
high density lipoprotein (HDL) and decreasing the bad cholesterol: low density lipo-
protein (LDL). It also inhibits inducible nitric oxide synthase by reducing the protein and
mRNA and thus promotes vasodilatation of blood vessels. Garlic has strong
immunopotential capacity and enhances the natural killer (NK) activity and proliferation
of T-lymphocytes by delaying the hypersensitivity reaction [40].
Aged garlic extract is found to be a promising immune modifier with internal
body regulatory functions, particularly in the control of sarcoma-180 and lung
carcinoma as well as inhibition of platelet aggregation. Both the oil- and water-
soluble components of garlic extract have shown health benefits. Specifically, its oil
extract reduces serious mental disorder and prevents blood coagulation even in
diabetics while its water extract is effective in cell cycle and viability of human
hepatoma cells (HG2) [6]. Since garlic extract reverses oxidant responses it seems
likely that it protects tissues from oxidative damages [56].
2.2.2 Sources
Carrots, squash, sweet potato, and spinach contain abundant beta-and alpha-carotene
and the dark green leafy vegetables such as kale, spinach, mustard greens, and green
beans are good sources of lutein. Lycopene is found predominately in tomatoes.

Other bioactive components in tomato are kaempferol or chlorogenic acid, which
have anti-mutagenic activities. This suggests that tomato suspension have a protec-
tive effect on colon cancer which is mediated by the modulation of different
biological pathways during carcinogenesis [57]. Typical dietary sources which are
rich in tocopherol and tocotrienols include vegetable oils, nuts and the germ portion
of grains [58].
Among the foods that have been shown to have beneficial immunomodulatory
effects are broccoli, garlic, onions, vegetable oils, almonds, and walnuts [5, 6].
Similarly, garlic, soy bean, cabbage, ginger, licorice root extract (extract of the
root of Glycytthiza glabra) and umbelliferous vegetables (vegetables that grow or
produce its plant beneath the ground, e.g., carrots) have been identified as foods and
herbs with the highest anticancer activity. Citrus in addition to providing an ample
supply of vitamin C, folic acid, potassium and soluble fiber contains a host of active
phytochemicals [58].
Green tea enhances humoral and cell-mediated immunity while decreasing the
risk of certain cancers and cardio-vascular disease. On the other hand, ginseng
enhances the production of macrophages and T-cells, natural killer cells and colony
forming activity of bone marrow [59]. Soy bean, garlic, ginger and green tea which
have been suggested, in epidemiological studies, to reduce the incidence of cancer
may do so by inducing programmed apoptosis (cell death). Soybean extract has been
shown to prevent the development of polycystic kidneys [60]. Turmeric is most
potent against skin tumors [61].
Moringa oleifera is a rich source of a variety of functional components (phyto-
chemicals) in its leaves, pods and seeds. Moringa is reported to contain 7 times more
vitamin C than oranges, 10 times more vitamin A than carrots, 17 times more
calcium than milk, 9 times more protein than yoghurt, 15 times more potassium
than bananas and 25 times more iron than spinach [62]. In addition, it contains other
phytochemicals such as tannins, phenolics such as flavonoids, sterols, terpenoids,
saponins, anthraquinones, alkaloids and reducing sugar as well as other anti-cancer-
ous agents like glucosinolates, isothiocyanates, glycoside compounds, glycerol-1-9-
octadecanoate and about 76% poly-unsaturated fatty acids such as linoleic acid,
linolenic acid and oleic acid [45].
2.3 Probiotics and Prebiotics
(a) Probiotics are the beneficial live microorganisms, which, when administered in
adequate amounts, confer health benefits on the host [63], examples Lactic acid
bacteria (LAB), Biofidobacterium.
(b) Prebiotics are the non-digestible food ingredients that stimulate the growth and
activity of probiotics in the digestive system in ways claimed to be beneficial to
health. Thus, prebiotics are healthy non-digestible food ingredients that make
their way through our digestive system and help the beneficial or good bacteria
grow and flourish [63, 64].
1360 C. I. Abuajah
2.3.1 Functions
Probiotics are believed to protect humans in two major ways. The first is the role that
they play in human digestive tract. Human digestive tracts need a healthy balance
between the good and bad microorganisms. But our lifestyles such as poor food
choices, emotional stress, and lack of sleep, antibiotic over-use, other drugs, and
environmental influences can shift the balance in favor of the bad microorganisms
[65]. When the digestive tract is healthy, it filters out and eliminates things that can
damage it, such as harmful microbes, toxins, chemicals, and other waste products.
On the flip side, it takes in the things that the human body needs (nutrients from food
and water) and helps deliver them to the cells where they are needed. The idea is not
to kill off all of the microbes. Human bodies do have a need for the bad ones and the
good ones. The problem is when the balance is shifted to have more bad than good.
An imbalance has been associated with diarrhea, urinary tract infections, muscle
pain, and fatigue [65].
The second major benefit of probiotics is the impact they have on the immune
system. The immune system protects against pathogens. When it doesn’t function
properly, one can suffer from allergic reactions, autoimmune disorders (for example,
ulcerative colitis, Crohn’s disease, and rheumatoid arthritis), and infections (for
example, infectious diarrhea, Helicobacter pylori, skin infections, and vaginal
infections). By maintaining the correct balance from birth, these ailments are
prevented [63, 66]. During delivery through the birth canal, a newborn picks up
the beneficial bacteria from his/her mother. These good bacteria are not transmitted
when a Caesarean section is performed and have been shown to be the reason why
some infants born by Caesarean section have allergies, sub-optimal immune sys-
tems, and lower levels of gut micro-flora [65].
Some of the specific mechanisms by which probiotics exclude undesirable
microorganisms include the production of inhibitory substances, blocking of adhe-
sion sites, competition for nutrients, degradation of toxin receptors, and stimulation
of immunity [66].
Prebiotics stimulate the growth of beneficial and healthy microorganisms (pro-
biotics) in the gut with a resultant increased resistance to invading pathogens. This
positive impact of prebiotics, in an unaltered form, in the human intestine is known
as the prebiotic effect. However, such prebiotic effect is manifested when there is
increase in the number and activity of probiotics. This effect is induced by consum-
ing functional foods that contain prebiotics. The prebiotic definition does not
emphasize a specific microbial group.
2.3.2 Sources
Dietary supplements and fermented food products have been advertised as
containing beneficial cultures. These cultures are what would now be considered
probiotics [67]. Other foods currently claimed to provide probiotics are cereal juice,
frozen yogurt, granola, candy bars, and cookies. While they may contain probiotics,
there is no guarantee that they have them in optimal levels. Only the manufacturer of
the product can confirm if there are any studies to support his specific claims [65].
The most common types of prebiotics are non-starchy carbohydrates such as

soluble dietary fiber (e.g., β-glucans, inulin, etc.) and other oligosaccharides such as
fructo-oligosaccharide (fructans), galacto-oligosaccharide, etc. Many of the plants
frequently eaten as vegetables – asparagus, garlic, leek, onion, artichoke – are
excellent sources of inulin. β-Glucans and inulin are common in many plants
containing dietary fiber and fructans. Traditional dietary sources of prebiotics also
include soybeans, raw oats, unrefined wheat and unrefined barley. Some of the
oligosaccharides that naturally occur in breast milk are believed to play an important
role in the development of a healthy immune system in infants through the prebiotic-
probiotic relationship.
3 Food as Medicine
Since ancient times, plants have always been the common sources of food and
medicines, either in the form of traditional herbal preparations or as pure active
principles [68]. Many herbal extracts have been tested in numerous systems to assess
their chemopreventive and chemotherapeutic efficacy [69]. Similarly, there is grow-
ing evidence that functional components (phytochemicals) play an integral role in
the link between food and health thereby providing disease-fighting foods for the
prevention of chronic degenerative pathologies or in support of traditional remedies
[18]. Most of the observed therapeutic effects of plants have been linked to their
potent phytochemical content especially anti-oxidants. Healing of diseases or main-
tenance of a healthy lifestyle based on antioxidant activity could be the scientific
basis of traditional herbal medicines [70].
In this regard, the eastern civilizations of Asia and the oriental cultures
including the Chinese, Japanese, Indians and Egyptians were the few civilizations
that explored these healing powers of food phytochemicals for thousands of years
and provided evidences which supported claims that functional phytochemicals in
foods can be effectively used to prevent the risk of developing diseases and
promote well-being [71, 72]. However, a diet can only be adjudged healthy if
its combination of individual food types is appropriate and their nutritive and non-
nutritive metabolites are biologically available for cell absorption and utilization.
Table 2 gives the effect of some physiological factors on the bioavailability of
functional components. Limiting certain food types or components such as sugars,
salts and saturated or trans-fatty acids alone or simply delivering an intake of pure
nutrients alone may not be regarded entirely as a healthy diet. Therefore, while it
is easy to recommend a healthy diet, it is much harder to define it for a particular
need [9].
Hence, in this era when the role of a healthy diet in preventing non-communicable
diseases are well accepted, the borderline between food and medicine is becoming
very thin [9]. Thus, the concept of food has obviously gone beyond basic nutrition
only. While products intended to cure diseases are classified as medicine, a healthy
diet consisting of foods with functional components can help promote well-being
and even reduce the risk of developing certain diseases [70].
1362 C. I. Abuajah
Table 2 Effect of some physiological factors on the bioavailability of functional componentsa

S/N Factors Effect
1. Food matrix Functional components are
compartmentalized, bound in cell walls and
are unavailable
2. Food preparation Cutting, mashing, grinding, peeling,
trimming activate enzymes, e.g., polyphenol
oxidase, allinase. Improves availability of
antioxidants
3. Food processing: Thermal/non-thermal Mild heat treatment (steaming, blanching)
processing, (NTP example high pressure over short period improves antioxidant
processing (HPP), pulsed electric field content but this decreases with severe
(PEF)) treatment (boiling, cooking). NTP has
positive effect on polyphenols and negative
effect on carotenoids
4. Food mixtures Enhances availability of polyphenols with
sugars, ascorbic acid, fat and stabilizing
effects of polyphenols on other
phytochemicals
5. Gastric digestion De-polymerization of large polyphenols,
hydrogen bonding of polyphenols to
proteins
6. Small intestine Micelle formation, increased role of uptake
and efflux transporters (cell to gut lumen to
basolateral to cell), transition from matrix to
oil phase
7. Colon Phase I/II interactions, influence of micro-
flora on phytochemicals, colonic absorption,
increased formation of metabolites and
phase I/II products
8. Tissues Biotransformation, phase I and II
metabolism, interaction with transporters in
certain tissues (blood-brain barriers,
placenta, testis) or excretory organs (liver,
kidney)
a
Bohn et al. [15]
4 Healing Powers of Functional Components
Some properties which link functional components to potential health-modulating

roles and functions can be classified into antioxidation, anti-cancerous, anti-diabetic,
anti-inflammatory, prevention of cardio-vascular diseases, anti-microbial, immune-
modulatory and anti-hypertensive [20, 73, 74].
4.1 Anti-oxidation Properties
Free radicals in the form of reactive oxygen species (ROS) or reactive nitrogen
species (RNS) are the usual by-products of body metabolic activities which involve
oxidation and reduction reactions in cells. It is expected that at low concentrations,

these reactions have beneficial effects on cellular responses and immune functions
[75]. However, at high concentrations they cause the condition known as oxidative
stress which is harmful to cell structures. In simple terms, oxidative stress is defined
as the condition whereby the balance between formation and removal of free radicals
is shifted towards formation of more free radicals rather than their removal [76]. It is
generally believed that oxidative stress plays a major role in the development of
chronic and degenerative diseases such as cancer, diabetes and cardio-vascular
diseases [75, 77]. The human body has its own endogenous anti-oxidant system
designed to combat oxidative stress which is supported and strengthened by exog-
enously supplied anti-oxidants in diets [75]. In this regard, phytochemicals such as
phenolic compounds are believed to play key role in protecting the body against
oxidative stress and its effects due to their well-known anti-oxidant properties. The
antioxidant capacity or activity of phytochemicals, in vitro, is well reported in
literature and serves as an indicator of the potential ability of dietary antioxidants
to combat oxidative stress [78, 79]. This has been determined mostly through free-
radical scavenging ability although other assays have been used such as inhibition of
lipid peroxidation and metal ion chelating capacity. Similarly, the potential of anti-
oxidants to combat oxidative stress have been demonstrated in vivo, mostly in rats.
This involved the monitoring of activities of anti-oxidant enzymes or anti-oxidant
molecules within the experimental animal on feeding it with the phytochemical of
choice [79–81]. Overall, anti-oxidant supplementation reduced lipid peroxidation
and enhanced anti-oxidant capacity of blood (plasma), heart, kidney, testes, lung and
pancreas. It is therefore acceptable that oxidative stress is a precursor for the
development of various chronic and degenerative diseases. It has been suggested
that free radicals are involved in the pathology of more than 50 human diseases,
including aging [82]. However, it is also important to note that free radicals are not
harmful at all times, rather, their toxicity depends on several factors including the
type of ROS/RNS, their concentration and localization, and the kinetics of their
production and elimination [83].
4.2 Anti-cancer Properties
Various reports provide some evidence of potential anticancer properties of func-

tional components of food. These properties have been demonstrated in vitro
through assays including their effects on oxidative stress (free-radical scavenging
activity), inhibition of DNA damage, anti-proliferative effects on cancer cell lines
and phase II enzyme induction [84]. Methoxylated 3-deoxyanthocyanins was found
to be the most potent inducer of quinine oxidoreductase, a phase II detoxifying
enzyme [85]. The effects of functional components on oxidative DNA damage have
been reported [86]. Higher total phenolic contents including flavonoids and non-
flavonoids such as rutin and quercetin increased the inhibition of oxidative DNA
damage. Similarly, the inhibition of hydroxyl radical-induced DNA damage by an
anthocyanin has also been reported [87].
1364 C. I. Abuajah
4.3 Anti-diabetic Properties
Diabetes is associated with various conditions including oxidative stress, impaired

insulin secretion and insulin resistance due to malfunctioning of β-cells of the pancre-
atic islets of langerhan, leading to defective utilization of carbohydrates as energy
source [88]. Due to their anti-oxidant properties, phenolic compounds may reduce
oxidative stress conditions and also protect pancreatic β-cells. Another widely used
way of demonstrating anti-diabetic effects of functional components is by determining
their inhibitory effects against starch-degrading enzymes such as α-amylase and
α-glucosidase. Phytochemicals have been shown to possess inhibitory effects
against α-amylase and α-glucosidase [89, 90]. Some in vivo studies on the anti-
diabetic effects of phytochemicals have also been reported. Diets high in phytochem-
icals could protect against hyperglycemia and alloxan-induced oxidative stress
through the restoration of levels of endogenous enzymatic superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase
(GR) and non-enzymatic (vitamins E and C) anti-oxidants in diabetic rats [91].
4.4 Anti-inflammatory Properties
Inflammation may be described as an immune response to cellular or tissue injury or

infection by pathogens [92]. The condition of inflammation itself is not considered a
disease. However, if left unchecked to become chronic, there can be exacerbated
tissue damage and modulation of various cell-signaling pathways [93]. Chronic
inflammation causes life style diseases, such as obesity, metabolic syndrome, arterial
sclerosis, and cancer [94–98]. The process of inflammation consists of a wide and
complex range of cellular and molecular pathways and reactions involving a host of
enzymes [92]. These enzymes include cyclooxygenase, lipoxygenase, phospholi-
pase A2 and nitric oxide synthase. Cytokines such as IL-1, IL-2 and TNF-α and
nitric oxide are important pro-inflammatory products of cellular reactions [16, 73].
Phytochemicals can modulate inflammatory processes by inhibiting pro-inflamma-
tory enzymes [92] which influence the production of the cytokines and nitric oxide.
The anti-inflammatory properties of dietary phytochemicals may be determined by
their inhibitory activities against the pro-inflammatory enzymes and by monitoring
the production of pro-inflammatory cytokines. Simple nitric oxide radical scaveng-
ing capacity could also be used as an indicator of anti-inflammatory properties. In
vitro and in vivo anti-inflammatory activities have been widely reported [80, 81, 99].
4.5 Cardio-vascular Properties
The oxidation of LDL seem to trigger-off the onset of coronary heart disease [100].
The oxidized LDL is taken up by macrophages (foam cells) and smooth muscle cells
leading to the formation of fatty streaks and eventual development of arteriosclerosis
[101]. The prevention of the oxidation of LDL may be a potential prevention of
cardiovascular disease. The ability of functional components including phenolics to

prevent LDL oxidation by exerting anti-oxidant effects such as cholesterol lowering
effects through the suppression of LDL-cholesterol and the elevation of HDL
cholesterol that is important for the prevention of cardiovascular disease has been
reported in literature [102]. The effect of supplementation of phytochemicals from a
pseudocereal (quinoa) in a fructose containing diet on some cardiovascular disease
parameters in male Wistar rats was studied by Pasko et al. [103]. Rats fed with
quinoa had reduced serum total cholesterol, LDL, triacylglycerol, glucose and
plasma total protein. Those fed fructose had decreased HDL but this decrease was
reversed on feeding with quinoa. The reduction in LDL and prevention in decrease in
HDL on feeding with quinoa suggests potential ability of the phytochemicals in
quinoa to prevent cardio-vascular disease [73]. The observed hypo-cholesterolemic
effects may be due to synergistic effects of high crude fiber and high anti-oxidant
concentrations such as quercetin. Thiol-containing compounds and phenolics were
shown to inhibit rat microsomal lipid peroxidation which may be significant for the
prevention of LDL oxidation and hence prevention of arteriosclerosis and cardio-
vascular disease [104]. Oxidative stress-mediated conditions such as cardiovascular
disease are also accompanied by loss of endogenous antioxidant compounds such as
glutathione-glutathione disulfide ratio [77].
4.6 Anti-hypertensive Properties
Functional components possess therapeutic capacity to decrease blood pressure

[105]. These non-nutritive food factors such as flavonoid polyphenols (flavonols
example quercetin; flavones example lutelin; and isoflavones example coumesterol)
and carotenoids are scavengers of free radicals like superoxide anions, lipid proxy
radicals, and lipid oxidation of LDL cholesterol, hence are anti-atherogenic (decreas-
ing the formation of atherosclerotic plaques and arterial stiffness) thereby making
arteries more responsive to vasodilation thereby lowering blood pressure [106]. Low
risk of stroke is associated with high intake of plant food materials including fruits
and vegetables and thus prevents hypertension [105]. In addition, several plant
sources in the forms of herbs and spices including Allium sativum (Garlic), Camellia
sinensis (Tea), Hibiscus sabdariffa (Roselle), Nigella sativum (Black cumin), Panax
(Ginseng), Cymbopogon citratus (Lemon grass), Zingiber officinale (Ginger) con-
tain non-nutritive secondary metabolites which have been demonstrated to possess
vaso-relaxant, anti-oxidant, anti-proliferative, anti-inflammatory, and diuretic func-
tions which play anti-hypertensive roles [107].
5 Optimal Health and Well-Being
Health and well-being or wellness are terms which are often used interchangeably,
but their meanings are no doubt different. According to the World Health Organi-
zation, health is a state of “complete” physical, mental and social well-being and not
1366 C. I. Abuajah
merely the absence of disease or infirmity. Health is therefore a dynamic condition

resulting from a body’s constant adjustment and adaptation in response to stress and
changes in the environment for the maintenance of a homeostatic inner equilibrium
[108]. Accordingly, the primary determinants of health include social, economic, and
physical environments, as well as the individual’s personal characteristics and
behaviors. This definition has attracted serious criticisms for being overtly inclusive
and practically unattainable, especially as it relates to the word “complete.” How-
ever, “complete” medically expands the definition of health beyond the mere absence
of diseases [109]. The maintenance and improvement of health, accordingly, depends
not only on external or environmental factors (including the health care systems) but
also on the efforts and lifestyle choices of the person involved [110]. On the other
hand, optimal health is a level of health where the body’s natural defense mechanisms
are activated to function maximally. Optimal health turns off all chronic infections and
oxidative stress. It prepares the body to respond quickly and effectively to any invasion
by pathogens, toxins and other infections thereby preventing the onset of degenerative
diseases including cancer, hypertension, stroke, diabetes, etc.
Although variously defined, depending on the context of usage, well-being
generally depicts a healthy balance of the mind, body and spirit (state of
comfortability or happiness) which results in an overall feeling of wholesomeness.
However, according to the National Wellness Institute, wellness is considered as an
active process through which people become aware of, and make choices towards, a
more successful existence based on a conscious, positive and affirming, self-directed
and evolving process of achieving full potential; a multidimensional and holistic
lifestyle, encompassing mental and spiritual well-being, and the environment. There-
fore, in understanding the difference between these two terms, health could be
regarded as a state of being – both physical, mental, and social – whereas wellness
aims to enhance this state of being by living a healthy lifestyle [110].
6 Mode of Action of Functional Components and How they

Impact on Optimal Health and Well-Being
The mechanisms through which functional components impact on human health are
not completely clear and seem to be multiple [18, 111]. Some identified multiple
molecular targets of functional components include pro- and anti-apoptotic proteins,
cell cycle proteins, cell adhesion molecules, protein kinases, transcription factors,
metastasis and cell growth pathways [112–114]. Phytochemicals such as epigallo-
catechin-3-gallate (EGC-3-G) from green tea, curcumin from turmeric, and resver-
atrol from red wine tend to aim at a multitude of molecular targets. These multiple
characteristics may have contributed to the unavailability of definitive mechanisms
of action despite decades of research [115]. Specifically, the main mechanisms
suggested for phenolics include an improvement in the lipid profile of plasma, an
increase in its antioxidant activity as well as an enhancement of the endothelial
function by exerting anti-inflammatory effects, reducing LDL oxidation, inhibiting
endothelial NADPH oxidase, modulating nitric oxide synthase activity/expression

and increasing nitric oxide status [116, 117].
Data from in vitro studies suggest a protective interaction of flavonoids with
lipid bilayers against oxidative damage, as a result of their localization in
lipoprotein domains and cell membranes thus explaining the possible in vivo
role phenolics in protecting LDL from oxidation. Moreover, it has been hypoth-
esized that bioactive compounds, once absorbed and metabolized, may accumu-
late inside the cell membrane modifying the membrane composition, fluidity and
functionality [83, 118]. In vivo, plant antioxidants are generally assessed for their
effects on the activity of endogenous antioxidant enzymes or oxidative damage
biomarkers before and after induction of oxidative stress in experimental ani-
mals. Some of these commonly used methods directly evaluate the enzymatic
activity of endogenous anti-oxidants such as SOD, CAT, GPx and GR, while
other methods involve quantification of oxidative damage biomarkers. The for-
mation of specific end products resulting from interaction of ROS with biolog-
ically important macromolecules such as DNA, protein and lipids is measured by
quantifying oxidative damage biomarker methods. DNA damage is determined
by measuring the 8-hydroxydeoxyguanosine content. Carbonyl and aldehyde
such as malondialdehyde contents are measured as biomarkers of protein and
lipid oxidation, respectively [119]. Potential health benefits of functional com-
ponents of food are summarized in Fig. 10.
Several claims have been made, evaluated and approved by various regulatory
agencies in different parts of the world in respect of the health benefits and positive
effects of functional components of food such as soluble DF including β-glucans on
health and well-being [120]. Such approvals include those by the US Food and Drug
Administration (FDA) in 1997 [121]; the Panel on Diabetic Products and Nutrition and
Allergies of the European Commission in 2009 and 2011, respectively [122, 123]; the
Joint Health Claims Initiative (JHCI) expert committee and council of the UK [124] and
in Sweden [28]. These approvals were based on recommended dose of 3.0 g day1 of
β-glucans to attain health benefits including maintenance of gut health and normal
blood cholesterol level, reduction of energy and post-prandial glycemic responses.
Processed β-glucans are effective food supplement which has no side effects
irrespective of level of consumption and therefore generally recognized as safe
(GRAS) [125].
7 Effect of Processing on Functional Components of Food
Processing exercises some effects which could be positive or negative on phyto-

chemicals and other functional components of food. While some food processing
techniques increase the concentration of these functional components in food,
others decrease them (Table 2). It has been shown that the length of post-harvest
storage, steam blanching, and thermal processing all influence the retention of
functional compounds in allium vegetables [126]. However, losses of about
30-80% of bioactive isothiocyanates through heat processing have been reported
1368
Vascular system
Anti-hypertension, anti-
diabetic, anti-oxidant
activities
Immune system/Cancer Increased vasodialation
and blood flow
Activation of Beneficial lipid profile
immune response Endothileal homeostasis
system
Anti-coagulant and anti-
Enhance leucocyte thrombotic activity
activity Lower serum cholesterol
Anti-tumor properties
Detoxification of Inflammation
cancer precursors
Apoptosis of Inhibition of neuro-inflammation
cancerous cells Inhibition of pro-inflammatory
Inhibition of DNA cytokines and chemokines production
oxidation Reduction in adhesion molecule
expression
Fig. 10 Potential health benefits of functional components

C. I. Abuajah
[120]. In addition, high temperatures (100 C and above) inactivates key enzymes
such as myrosinase in cruciferous vegetables and allinase in allium vegetables,
thereby reducing the amount of functional components in these food materials.
However, temperatures associated with normal cooking have shown little evi-
dence of substantial loss of isothiocyanates. Leaching of glucosinolates and their
hydrolysis products also results in a reduction in total phytochemical content
following cooking. Research has shown that heating garlic in a microwave oven
for 30-60 s or heating it to a temperature of 60-100 C results in significant losses
of its anti-inflammatory, anti-cancerous, anti-microbial and anti-oxidant activi-
ties [127].
The bioavailability of carotenoids and other lipid-soluble functional food com-
ponents have been shown to improve with processing techniques that increase
surface area, such as size-reduction, example, cutting and chopping, as well as
heat treatment that breaks down protein and carbohydrate matrix [128, 129]. Also,
the brewing of tea leaves, whether black or green, releases 69-85% of their bioactive
flavonoids within 3-5 min in hot water [129].
In contrast, drying pre-treatment before boiling was found to reduce cooking time
thereby leading to less leaching of anti-oxidants in edible Irish seaweed
Homanthalia elongata. In terms of extract, drying followed by boiling of
Homanthalia elongata had the most significant effect on phytochemicals as its
total phenolic content increased by 174%. However, this processing treatment
reduced its anti-microbial activity compared to extracts from fresh samples [130].
Table 3 below gives a summary of the effect of some processing techniques on the
anti-oxidant content of some foods.
Table 3 Effect of processing on antioxidant content of some foods compared to nonprocessed

foodsb
S/N Food Type of processing Effect (%)
1. Apple Peeling () 33–66%
2. Carrots Steaming (+) 291%
3. Carrots Boiling (+) 121–159%
4. Cucumbers Peeling () 50%
5. Asparagus Steaming (+) 205%
6. Broccoli Steaming (+) 122–654%
7. Green cabbage Steaming (+) 448%
8. Red cabbage Steaming (+) 270%
9. Green pepper Steaming (+) 467%
10. Red pepper Steaming (+) 180%
11. Potatoes Steaming (+) 105–242%
12. Tomatoes Steaming (+) 112–164%
13. Spinach Boiling (+) 114–184%
14. Sweet potatoes Steaming (+) 413
b
Hadvorsen et al. [131]
1370 C. I. Abuajah
8 Conclusion
It is a widely held view that the consumption of a healthy diet can help prevent non-
communicable degenerative diseases. This is as a result of the presence of non-
nutritive or secondary metabolites known as functional components especially in
plant based foods. Similarly, there is also growing evidence that functional compo-
nents including phytochemicals and bioactives play an integral role in the link
between food, optimal health and well-being. Epidemiological data support this
preventive and protective effects of food against chronic degenerative pathologies
such as cancer, diabetes, and cardiovascular diseases as a result of their functional
components. Such functional components include dietary fibers, fucoidans, caroten-
oids, phenolics, phytosterols, tocopherols/trienols, organo-sulfur compounds, pre-
biotics and probiotics.
Increasing scientific evidence indicate that insufficient functional components
in diets through the low consumption of foods such as fruits, vegetables, nuts,
whole grains, spices is among the risk factors which contribute to increased
incidence of noncommunicable “silent killer” diseases in the world today.
Some properties which link functional components to potential health benefits
can be classified into antioxidation, anti-cancerous, anti-diabetic, anti-inflamma-
tory, cardiovascular, anti-microbial, immunomodulatory and anti-hypertensive,
anti-coagulant and anti-thrombotic activity of blood plasma, anti-virus, reduction
of blood lipids including cholesterol, anti-complementary properties, activity
against hepatopathy, uropathy and renalpathy, protective effects on gastric
organ and gastric mucosa.
Thus, the idea of food these days has obviously gone beyond mere provision of
biochemical building blocks necessary to refresh and nourish life in the form of
primary nutrients to cells of an organism. While products intended to cure diseases
could be classified as medicine, a healthy diet consisting of appropriate food types
with functional components can serve as a scientific alternative to reduce the risk of
developing certain diseases. Obviously, functional components of food will play an
important role in health maintenance in the future as a result of their medicinal
properties. However, their bioavailability and levels required in humans are critical
factors necessary to optimize their potential benefits. Hence, in this era when the role
of a healthy diet in preventing non-communicable diseases is well accepted, the
borderline between food and medicine is becoming very thin.
Acknowledgements The author expresses grateful acknowledgement to Dr. Elena Barascu of

Department of Food Engineering, Valahia University, Targoviste, Dambovita, Romania and
Prof. Lavinia Buruleanu, the Editor-in-Chief, Annals Food Science and Technology for granting
kind permission to reuse the original article, Current developments on β-glucans as functional
components of food: a review published in volume 14, issue 2, and pages 217–229 of its 2013
edition in this work. It is further acknowledged that the journal article, Functional components and
medicinal properties of food: a review which was first published in the Journal of Food Science and
Technology, volume 52, issue 5, pages 2522–2529 in 2015 also contributed to this book chapter
project.
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Development of Functional Dairy Foods
46
Natália Martins, Maria Beatriz P. P. Oliveira, and
Isabel C. F. R. Ferreira
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1378
2 Dairy Food Products: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1379
3 Functional Foods: Innovative Trends on Dairy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1381
4 Functional Dairy Foods: Agro-Industrial and Biotechnological Prospects . . . . . . . . . . . . . . . 1384
5 Mushrooms and Plant-Food Bioactives: Key Factors on Functional Dairy Products
Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1387
6 Bioactive Effects of Functional Dairy Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390
7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1392
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393
Abstract
There has been a growing interest on functional foods, markedly recognized as
being able to provide additional benefits on health promotion, wellbeing mainte-
nance, and disease prevention. Based on this scenario, food industries have been
increasingly focused in developing added-value foodstuffs, being dairy foods one
of the most currently used food products for functional purposes. Different
N. Martins
Mountain Research Centre (CIMO), Polytechnique Institute of Bragança, Bragança, Portugal
M. B. P. P. Oliveira
I. C. F. R. Ferreira (*)
Mountain Research Centre (CIMO), Polytechnique Institute of Bragança, Bragança, Portugal
Instituto Politécnico de Bragança, Bragança, Portugal

1378 N. Martins et al.
extraction and encapsulation technologies have been used to obtain target food
bioactive ingredients and to ensure an effective functionalization of dairy prod-
ucts, respectively. Probiotics, prebiotics, mushrooms, and plant food bioactive
extracts comprise the most commonly used food ingredients to produce func-
tional dairy foods, mostly fermented milk, yogurt, and cheese. In fact, dynamic
and promissory biological effects have been documented for these functional
dairy foods, among them antioxidant, cardioprotective, antihypertensive, immu-
nomodulatory, antimicrobial, antidiabetic, anti-inflammatory, neuromodulatory,
and even bone protection. However, besides the impact of health benefits on
consumers’ acceptance and subsequent consumption of functional dairy foods,
other factors, such as consumers’ familiarity with new products and functional
ingredients used on their formulation, consumers’ knowledge and awareness
about the credibility of shared health effects, and finally the organoleptic and
sensory evaluation of the developed functional dairy foods, have also a determi-
nant role. Anyway, consumers are considered self-contributors for this promising
food innovation. Thus, the concept of functional dairy foods may represent an
upcoming multiniche market and sustainable trend to be exploited.
Keywords
Functional foods · Dairy products functionalization · Food ingredients ·
Extraction/encapsulation technologies · Consumers’ acceptance
1 Introduction
Consumers’ perception about the real importance of a balanced diet and healthy
lifestyle on disease prevention, health maintenance, and longevity promotion has
driven the development of multiple and increasingly deepen studies [1, 2]. Foods,
food ingredients, and even bioactive molecules with health-promoting abilities
present a large demand by worldwide consumers [3]. In fact, biotechnological and
agro-food industries have been increasingly focused on the development of highly
specific methodological procedures to incorporate certain bioactive molecules on
daily products, making them more safe, effective, bioavailable, and even able to
confer certain additional biological attributes [4, 5]. Despite the inexistence of a
general consensus, these food products are commonly referred as functional foods,
i.e., “foods that when included in the normal diet display one or more target
functions in the human body, being able to improve the health status and/or to
reduce the likelihood of some disorders occurrence” [6]. Thus, functional foods
make part of daily diet, both as natural, whole, or unmodified foods; however, they
may also be used and/or incorporated as food constituents, added and/or removed by
technological or biotechnological procedures that modulate their bioavailability,
focusing on the improvement of their biological effects [6, 7]. Several food groups,
such as meat and fish products, ice cream, cheese, yogurt, and other dairy products
present very interesting chemical characteristics and nutritional composition that
46 Development of Functional Dairy Foods 1379
triggers the focus of researchers to the incorporation and application of bioactive

molecules in those food matrices [3].
Dairy products are amongst the most commonly tested and selected food groups
toward the incorporation of food ingredients for functional purposes. Milk, cream,
yogurt, kefir, powdered milk, condensed milk, ricotta, butter, casein, fermented dairy
drinks, infant milk formula, colostrum, cheese, and ice creams are the most common
traditional dairy products considered as functional foods [8, 9]. All these products
have on their chemical composition recognized ingredients that provide, besides the
nutritional benefits, a considerable improvement on human health and well-being
[8]. Numerous nutrients are present on dairy food products, being vitamins, min-
erals, and proteins the most abundant ones. However, some preparations present
active ingredients with low availability and/or nutritional deficits of these constitu-
ents, being milk fortified with calcium and vitamin-D (fortified product) one of the
most representative examples of this situation [10, 11]. Moreover, the incorporation
of milk supplements and milk-based food products, such as probiotic bacteria and
prebiotic polysaccharides, omega-3 fatty acids, and other compounds, is becoming
increasingly common (enriched product) [10, 11]. Moreover, there are some situa-
tions in which some deleterious components need to be removed, reduced, or
replaced by another substance with beneficial effects, for example, fibers as fat
releasers in ice cream products (altered products) [10, 11]. Curiously, functional
dairy foods are virtually found in all these types of functional food categories,
despite they are not homogeneously scattered over all segments of the growing
market.
In this sense, the present chapter aims to provide an overview on functional dairy
foods, emphasizing the role of biotechnological and agro-industrial prospects in their
development, describing the most commonly used plant food bioactives on func-
tional dairy foods formulation, and lastly their bioactive and health-promoting
abilities.
2 Dairy Food Products: An Overview
Innovations in the agro-food sector are mostly focused on the improvement of the
currently available food products, always taking into account some specific criteria,
such as the quality, safety, and even distinct characteristics related with bioactive and
health promoter effects conferred by these matrices [12–15]. Besides, the informa-
tion about nutrients, ingredients, and food additives should also be prioritized
[12, 14]. Specifically, the food industry has been the target of intensive and impres-
sive innovative processes along with different parts of the food chain, being the most
evident ones classified in five distinct sectors: (1) new food ingredients and mate-
rials; (2) innovation in fresh foods; (3) innovation in food quality; (4) new food
process techniques; and (5) new packaging methods [12]. All these innovative
aspects result from consumers’ demand that trigger the search and development of
highly effective products, techniques, and services by food science and technology
researchers in articulation with the food industry (Fig. 1). Within the scope of food
Consumers
Researchers
Agro-
industries
New food ingredients and materials

Innovation in fresh foods Food quality formulation
Innovation in food quality Nutritional directions
New food process techniques Additive regulations
New packaging methods
Functional Dairy Food
Yogurt Cheese Butter Ricotta
Milk Ice-cream Kefir
Fig. 1 Main actors and factors involved in the development of functional dairy foods
science and technology, the market of dairy products has gained a renowned niche of
demand and recognition by consumers, besides of their increasing research interest
and innovation procedures.
Milk is one of the most widely consumed dairy product, being used over centuries
as part of daily diet [15, 16]. Despite a growing evidence showing that their
unlimited ingestion is associated with several disorders, including some forms of
cancer, asthma, diabetes, obesity, cardiovascular diseases, and even osteoporosis,
more recent clinical data have demonstrated that calcium supplementation helps to
promote bone health besides other benefits [9, 15, 17]. Nevertheless, there are some
data that still remains inconclusive concerning to the linkage between high dietary
calcium intake from milk and prevention of osteoporosis and bone loss [16]. On the
other hand, and considering that many people are lactose-intolerant or even allergic
to milk, the interest was progressively driven to milk fermented products, such as
yogurt and cheese [16, 18, 19]. Fermented dairy products contain live beneficial
microorganisms that can digest lactose and other nontolerable milk constituents,
exert cholesterol-lowering effects, and also contribute to cancer risk reduction
[15]. These promising modulatory effects are mainly reached through action of
those microorganisms, namely probiotic bacteria, that significantly enhances the
health-promoting abilities of dairy products, contributing for a longer and healthier
lifetime [9, 16]. Not least important to highlight is that the benefits of probiotic
bacteria may be achieved both directly and/or indirectly, being all these aspects
carefully described in a specific section of this chapter.
Considering the recent findings on this field, milk and other dairy products, and
more specifically fermented dairy products, may have a crucial and determinant
functional role in contributing to health and well-being of worldwide population.
This aspect has driven an increasing interest of researches in developing functional

foods based on milk and other dairy products, which are known by humanity since
medieval times due to their health benefits. More recently, the biologically active
constituents have been increasingly studied and their benefits reported, remaining,
however, the interest to deepen knowledge on this field, in order to promote their
strengthening and valorization.
3 Functional Foods: Innovative Trends on Dairy Products
Functional foods have been the target of an intense investigation by researchers,

highly demanded by consumers as also with increasingly strict regulatory processes
applied to agro-industries. Despite dairy products present a broad history of use, in
the last years a high demand by consumers for enhanced-dairy foods with additional
health-promoting abilities has been observed [20, 21]. The large evidence that foods
in general, and specifically dairy foods with balanced-enriched nutritional composi-
tion, may confer additional health benefits has stimulated the development of novel
formulations of dairy products containing active ingredients [9, 18, 22]. Until now,
prebiotics, probiotic bacteria, vitamins and minerals, flavoring agents, plants, fruits,
and even its derived extracts are amongst the most frequently used food ingredients
in the design of functional dairy food formulations (Fig. 2). In fact, the development
and formulation of functional dairy foods are on the top of the current studies, being
yogurt, followed by cheese, butter, milk through different preparations (powdered,
Fig. 2 Most commonly used

ingredients in the formulation
of functional dairy foods
Plants
Fruits
Extracts Minerals
Flavoring agents Vitamins
Probiotic
Prebiotics bacteria
processed, condensed, and even milk-based foods), ice cream, kefir, and ricotta the
most widely investigated toward the incorporation of the previous mentioned ingre-
dients for different functional purposes (Fig. 1). This information is briefly summa-
rized in Table 1 and described in detail in the following paragraphs.
Studies with probiotics are on the top of dairy products’ investigations, including
the use of microorganisms, such as Bifidobacterium, Lactobacillus, Streptococcus,
and Saccharomyces, individually or in cocultures [4, 14, 18]. Very interesting results
have been reported related with food quality characteristics, effectiveness, and
acceptance by consumers [4, 18]. There is an increasing evidence related with the
effective biological potential and health benefits of probiotic bacteria, namely on the
prevention of gastrointestinal disorders, microbiota modulation, selective inhibition
of opportunistic microorganisms, on the metabolism of toxic substances, as anti-
carcinogenic/antimutagenic agents, and even on the reduction of cholesterol levels
and of lactose intolerance [3, 4, 7]. This last one has received a pivotal attention by
food industries, due to the increasing rates of lactose intolerance, which directly
interfere with food selection by worldwide consumers [4]. Different levels of lactose
intolerance require specific dietary recommendations, including to avoid dairy
products and derivatives [16]. Therefore, the incorporation of probiotic bacteria in
various matrices of dairy products to reduce lactose intolerance is very important.
Yogurts and cheeses are amongst the most investigated dairy products toward the
incorporation of probiotic bacteria for functional purposes [4].
The incorporation of vitamins and minerals on dairy foods is not less common,
both in bioavailable, stable, and soluble forms to enrich the nutritional value as also
to extend the shelf life of dairy products [17]. Calcium, iron, and vitamins C, D, and
Table 1 Types of food ingredients commonly incorporated in the formulation of functional dairy
foods
Types of food Dairy products
ingredients Detailed food ingredients incorporation
Prebiotics Milk proteins, lactose, saccharose, lactulose, fructo-, Cheese, yogurt
glyco- and galactooligosaccharides, arabinose,
galactose, inulin, raffinose, mannose, lactulose,
staquiose, xylooligosaccharides, isomaltulose
(palatinose), isomaltooligosaccharides, and soy
oligosaccharides
Probiotics Species: Bifidobacterium, Lactobacillus, Cheese, yogurt
Streptococcus and Saccharomyces
Vitamins and Calcium, iron, vitamins C, D, B2, B9, and B12 Milk, cheese,
minerals yogurt
Flavoring agents Polyphenols, flavonoids, carotenoids Milk, cheese,
yogurt
Plants, fruits, and its Chamomile, fennel, honey, mushrooms, chestnut, Cheese, yogurt
derived extracts lemon balm, rosemary, basil
Others Omega-3 fatty acids are linoleic acid (LA), Milk, yogurt
eicosapentaenoic acid (EPA), and docosahexaenoic
acid (DHA); β-carotene
B12 are among the most frequent ones [9]. Other B vitamins, such as folate and
riboflavin, have been also used; some of them are even synthesized from various
nonvitamin precursors through action of certain bacteria in plant and dairy foods
[14]. Although several years ago synthetic formulas were the most common, pres-
ently the incorporation of natural ingredients rich on these nutrients are on the top of
agro-industrial selection and consumers’ preference [3]. A similar scenario is
observed for the addition of flavors and aromas, in which essences, plant fruits
and/or plant fruit extracts, and even honey have been increasingly used, while
artificial flavoring agents become secondary in the formulation of dairy products
[2, 23, 24]. The addition of plants and fruits and/or its derived extracts seems to
present modulatory effects on gut microbiota, i.e., favors the growth of beneficial
bacteria and limits the growth of opportunistic microorganisms [14]. Still, these
active ingredients are also able to contribute for the improvement of the dairy
products shelf life at large extent, being therefore currently used for different
purposes [25].
Plants and fruits are per definition, composed by a rich and complex pool of
chemical molecules, most of them already characterized and properly identified as
having important bioactive effects at different levels [26]. Within the different
classes of bioactive molecules, phenolic compounds have received a special atten-
tion, due to their well-known antioxidant properties [26]. Nevertheless, most of these
bioactive molecules present inactive forms, being transformed into active forms by
metabolic processes; in this field lactic acid bacteria (Lactobacillus and Streptococ-
cus) display a very important role, favoring the conversion of inactive phenolic
compounds into their biologically active forms, via expression of glycosyl hydro-
lase, esterase, decarboxylase, and phenolic acid reductase enzymes [14]. These
biochemical reactions lead to the formation of several biologically active molecules,
namely pyranoanthocyanidins and 3-desoxypyranoanthocyanidins that display
determinant and regulator effects as health promoters and disease preventers
[14]. Once again, probiotic bacteria seem to exert multiple contributing effects
when combined with other food ingredients in dairy functional products’ formula-
tion. However, besides plant extracts, several other ingredients, such as milk pro-
teins, inulin, oligosaccharides, and lactulose, have been incorporated for prebiotic
purposes, i.e., to promote the growth of Bifidobacterium and other probiotic bacteria,
to ensure gut health, to keep harmful bacteria under control, and to enhance immune
system [9, 27]. Besides prebiotic effects, these active ingredients also show other
important functions, such as improvement of minerals and vitamins absorption,
decrease of triglycerides and cholesterol levels, and due to their high contents in
dietary fiber, improvement of fecal transit time and stool weight, crucial to prevent
constipation [9, 22]. Not least important to point out is that the above referenced
health conditions are amongst the most common disorders observed in modern
society.
The latest studies performed on this area have investigated different parameters
related with the general quality and acceptability of the incorporation of honey to
produce functional yogurts [2], mushrooms on functional cheese formulations [23],
and even phenolic extracts derived from different plant parts on dairy functional
yogurts and cheese formulation [25, 28–36]. Nevertheless, further studies are nec-
essary to evaluate the feasibility, efficacy, effectiveness, and bioavailability of
bioactive ingredients used to functionalize dairy products, and not least important
to evaluate the consumers’ acceptance.
4 Functional Dairy Foods: Agro-Industrial

and Biotechnological Prospects
A balanced and healthy diet is a key determining factor for overall quality of life,
which has been increasingly evident for worldwide consumers [6]. In fact, the
perception about the real importance of some specific foods and even food ingredi-
ents in the daily diet is increasingly common, up to a point that the consumers’
demand of healthy and functional foods reached exponential levels [13, 14]. Being
consumers directly involved on food selection, development, and evaluation of its
overall quality and acceptability, the food industry is increasingly focused on the
formulation of foods with abilities to improve the health and well-being of con-
sumers, at the same time that favor digestive system (which is considered a key
factor for overall quality of life), besides other physiological and metabolic effects
[9, 11]. Therefore, the development of functional dairy foods provides a great
opportunity to contribute to the improvement of food quality and consumers’ health
and well-being.
Among the wide variety of food ingredients, there are three main groups that have
become increasingly common in functional foods formulation, such as: probiotics
(live bacteria), prebiotics (compounds as fibers), and antioxidants [27, 37]. The latest
group of antioxidants mainly includes vitamins, minerals, plants and fruits, and even
its derivatives (Table 1). Most of the food ingredients currently incorporated in
functional dairy foods formulation naturally occurs on these dairy matrices, such
as bioactive peptides, probiotic bacteria, antioxidants, vitamins, specific proteins,
oligosaccharides, organic acids, highly absorbable calcium, conjugated linoleic acid,
and other biologically active components [9, 37]. Notwithstanding, and despite their
natural occurrence, a limited content of bioactive compounds is present, which can
compromise the effective value of these products [31]. Therefore, the incorporation
of these active ingredients in the functionalization of milk and dairy products is of
the utmost importance. Using these food ingredients, new functional dairy products
may be formulated, through modification of the traditional formulas, adding, elim-
inating, or even substituting some constituents, and even though the addition of
some wholesome compounds.
For example, Caleja et al. [31] observed that the incorporation of antioxidants
from natural origin (from chamomile and fennel extracts) in yogurts not only
improved their biological activity but also satisfied consumer demands, in compar-
ison with the synthetic additive, potassium sorbate [31]. Also important to highlight
is that the yogurt functionalization did not alter significantly its nutritional profile,
external appearance, pH, and even fatty acids content, being therefore suitable for
upcoming use both in food industry and in the dairy sector, where synthetic additives
are commonly used [31]. On the other hand, Heleno et al. [36] performed an
experiment using Agaricus bisporus extracts obtained by ultrasound-assisted extrac-
tion, and ergosterol, to incorporate in dairy beverages at concentrations mimicking
those that commercially occur in phytosterol-added yogurts. The authors observed
that samples incorporated with the extract and with ergosterol showed a higher
antioxidant capacity at same time that protected yogurt from oxidation, improving
therefore their shelf life, without modifying the nutritional value in comparison with
the original product [36]. Similar findings were reported by Carocho et al. [34] using
chestnut flowers, lemon balm, and its respective aqueous extracts (decoction) to
incorporate into “Serra da Estrela” cheese, aiming not only to assess their ability to
maintain its nutritional value but mainly to promote additional characteristics toward
new dairy foodstuffs formulation [34]. Caleja et al. [25] and Carocho et al. [34]
incorporated, respectively, fennel in cottage cheese [38] and basil in “Serra da
Estrela” cheese [35], aiming to assess their functional and preserving abilities. The
authors found that both samples did not alter significantly the nutritional value of
cheeses, at same time that improved their antioxidant properties, preserved the
contents in unsaturated fatty acids and proteins, as also exerted a prominent
functionalizing and preservative effect.
Concerning the incorporation of prebiotics and probiotics in functional dairy
products formulation, multiple findings have reported that, by one hand the incor-
poration of prebiotics not only conferred protection but also improved the viability
of probiotic bacteria, presupposing therefore there action as symbiotic ingredients
[39]. On the other hand, it was also shown that the incorporation of stingless bee
honey in goat yogurt containing Lactobacillus acidophilus positively affected sev-
eral characteristics, among them the color, syneresis, viscosity, sensory acceptance,
and even purchase intention [2]. But besides to these aspects, probiotic bacteria also
exert other highly valuable biological effects, such as being responsible for
reinforcing natural intestinal flora, decreasing serum triglycerides, cholesterol, trans-
aminase, and total bilirubin levels, exerting immunomodulatory effects, protecting
against gastrointestinal pathogens, contributing for lactose metabolism, infantile
diarrhea, besides reducing the incidence of urogenital and respiratory diseases and
preventing the occurrence of some cancers [3, 40].
It is clearly evident that these food ingredients may provide considerable nutri-
tional and sensory attributes of quality and acceptability, despite having a great
market potential to be exploited considering the functional properties. In this sense,
and considering that the top research areas in the Food Science and Technology
comprises the extraction and characterization of new natural ingredients with bio-
logical effects for further incorporation into functional foods formulation, it is very
important to use proper extraction methodologies and even effective technologies in
functional foods preparation. Therefore, there is not only interest in ensuring a
proper extraction of the bioactive constituents, but also the use of effective encap-
sulation methodologies toward the preservation of all the characteristics presents in
the developed functional food until the physiological site of action is reached.
Considering the existence of a wide variety of bioactive molecules with suitable
characteristics for upcoming use as food ingredients on functional foods
formulation, it is mandatory to select the most appropriate extraction methodology

aiming to achieve the highest extraction yield, without affecting both the chemical
composition and even the final bioavailability and subsequent biological activity.
The quality of the bioactive compounds is closely dependent on the suitability of the
extraction process and the proper selection of separation steps, which will also affect
the subsequent identification and characterization of those biomolecules [3, 4]. In
this sense, the selected extraction technique will also need to convert bioactive
compounds into more suitable forms both for detection and subsequent characteri-
zation, but also to provide a strong and reproducible method to be used in other
molecules belonging to the same group [3, 4]. There are a wide variety of extraction
techniques, but the most commonly used are briefly described in Table 2, namely
Soxhlet extraction (SoE), Ultrasound-assisted extraction (UAE), Supercritical-fluid
extraction (SFE), Accelerated solvent extraction (ASE), and finally Shake extraction
(ShE).
Table 2 Most commonly used extraction technologies in functional foods formulation

Extraction techniques
Soxhlet extraction (SoE) Commonly used to extract lipophilic compounds (hexane or
petroleum ether as solvents), but can be also used to extract polar
compounds, such as some specific phenolic compounds that are
effectively extracted using methanol. More recently has been also
applied to extract volatile compounds. Bioactive molecules:
carotenoids, phenolic compounds, sterols, and aromatic
compounds.
Ultrasound-assisted Based on ultrasonic vibrations, being a fast and simple method,
extraction (UAE) allowing the extraction of multiple samples at same time. The
solvent extraction used (mainly methanol, acetone, water, and ethyl
acetate) displays a crucial role, but also sample size, pH,
temperature, and pressure determines extraction quality and yield.
Bioactive molecules: carotenoids, polysaccharides, proteins,
phenolic compounds, aromatic compounds, and sterols.
Supercritical fluid Currently considered a “green” methodology, this technique is used
extraction (SFE) on the extraction of multiple nutraceutical ingredients, usually
performed using carbon dioxide under high pressure. This technique
allows the use of low temperatures, presents a high selectivity, needs
low solvent volume, small samples, short extraction times, and great
automation procedures. Bioactive molecules: carotenoids, sterols,
tocopherols, and phenolic compounds.
Accelerated solvent This technique is carried out under high pressure and temperatures,
extraction (ASE) presenting, however, some advantages in relation to the previous
ones, such as enable the extraction of fresh samples, short extraction
times, higher automation procedures, and good extraction rates.
Bioactive molecules: vitamins, phenolic compounds, and
carotenoids.
Shake extraction (ShE) This technique needs the use of a shaking device, allowing more
effective extractions at short times. The main advantage is to
increase the surface where solvent extraction interacts with the
sample material. Bioactive molecules: phenolic compounds.
Encapsulation technique is a complex process that includes the creation of a

barrier, more or less complex, that acts covering bioactive components inhibiting the
occurrence of chemical interactions, protecting against environmental factors (i.e.,
temperature, pH, enzymes, and oxygen), and even allowing the progressive release
of the active components under certain conditions [3, 4].
There are three main groups of encapsulation techniques, i.e., microencapsula-
tion, nanoencapsulation, and even emulsions, as briefly described in Table 3. The
selection of a specific encapsulation procedure mainly depends on two distinct
characteristics: the type of core material and characteristics of the final product
where the technique will be applied. Moreover, the material of capsule wall, size,
shape, and structure of capsule particles also determines the stability of the bioactive
molecules during production, storage, and even releasing during consumption
[3, 4]. However, the encapsulation technologies mainly aspire in ensuring a proper
stabilization of the bioactive compounds, warranting that they reach consumers in
appropriate levels, maintaining their bioavailability, avoiding the formation of harm-
ful and unpleasant compounds and even to mask some undesirable sensorial attri-
butes characteristic from some bioactive substances [3, 4].
5 Mushrooms and Plant-Food Bioactives: Key Factors

on Functional Dairy Products Formulation
The world of plant-food bioactives has received a pivotal attention in the last years,
being the assessment of their pharmacological effects and related biotechnological
applications an exponential niche of market to be exploited. Regarding particularly
their biotechnological applications, the sector of food industry and specifically the
subsector of development of new food ingredients and materials are under dynamic
innovation and overall exploitation. Inside this subsector, the area of functional
foods’ formulation is of largest demand not only by consumers but also of the
utmost interest of researchers and industries (Fig. 1). Moreover, being the group of
dairy products one of the most widely recognized food categories for functional
purposes, considering the presence of promissory molecules with multiple benefits, a
pivotal attention and intensive research work has been carried out on this field. On
the other hand, and despite probiotics and prebiotics have been on the top of the
latest studies on this area, a great attention has been given to plants, fruits, and its
derived extracts on fortification of dairy food products and subsequent functiona-
lization. As briefly described in Table 1, different plant, fruits, derived extracts, and
even other food constituents have been used to functionalize dairy products. Yogurt
and cheese are the most commonly selected to be investigated. Also interesting to
point out is that these food ingredients are not only used to improve the nutritional
value, health-promoting effects, and to confer additional benefits to dairy food
products but also to improve their shelf life and even to remove some unpleasant
and even nonbeneficial, nontolerated, and unaccepted characteristics by consumers.
Concerning the use of plant food bioactives in the development of functional
dairy foods, chamomile and fennel have been incorporated both in yogurt [31] and
Table 3 Most commonly used encapsulation technologies in functional foods formulation

Encapsulation technologies
Microencapsulation
Spray drying This is the most commonly used preparation, destined to prepare dry and
stable food additives and flavors. It is based on the injection of a liquid
suspension of the bioactive compound at the top of a vessel, drying the
droplet into a fine powder particle concomitantly with hot air. Then, liquid
droplet solidified and entraps the bioactive molecule. The technique is
economic, flexible, and suitable to be used in a continuous operation, besides
to provide a high protection to short exposure periods to acids, humidity, and
oxygen.
Freeze drying Commonly known as lyophilization or cryodesiccation. This technique is
great to be used in a wide variety of heat-sensitive ingredients. It consists in
homogenize core materials from the initial solution and then originates
colyophilizes, i.e., irregular particles. This technique is suitable to
encapsulate water-soluble essences, natural aromas, and even drugs.
Coacervation This technique is based on the phase separation of hydrocolloids present in
an initial solution and further deposition of the newly formed coacervates
around the suspended or emulsified bioactive molecule. It is considered an
expensive method, being particularly suitable to be used in high-value and
sensitive functional ingredients (i.e., polyphenols).
Liposome This technique consists in the formation of a lipid membranous system that
entrapment encapsulates a hydrophilic space, leading to the formation of a suitable place
to encapsulate water-soluble, lipid-soluble, and amphiphilic functional
ingredients. With this technique, it is possible to control the release rate and
exact point of incorporated materials delivery. Encapsulated materials
through this technique are protected from stomach digestion, so ensuring
their subsequent bioavailability and bioactivity in the next section of the
gastrointestinal tract.
Cocrystallization This technique consists in the modification of the sucrose structure, leading
to the formation of an irregular agglomerated crystal. Bioactive ingredient is
therefore incorporated into the porous matrix of the newly formed microsized
crystals. This technique improves the solubility, homogeneity, dispersibility,
hydration, anticaking, stability, and flowability of the encapsulated functional
ingredients. It also allows the conversion of liquid materials into powders,
which facilitates their subsequent application in pharmaceutical industry.
Yeast- This technique is based on the use of yeast cells (Saccharomyces cerevisiae)
encapsulation as the encapsulating material, being also usually applied to essential oils,
flavors, and polyphenols. The target ingredients can pass across cell wall and
remains inside the cells, being also ensured the control of the diffusion of the
incorporated functional ingredients. Finally, this technique is also embroiled
with green chemistry principles, once no additives besides water, yeast, and
target functional ingredients are used. It is also a low-cost technique that
allows the processing of high volumes of bioactive ingredients.
Cold gelation This is a recent method, consisting from an alternative way to develop protein
microparticles in food industry. No organic solvents are necessary, being
encapsulation achieved under mild conditions, minimizing the destruction of
sensitive nutraceutical compounds. Moreover, it is also possible to denature,
dissociate, and even aggregate under different conditions of pH, ionic
strength, and temperature.
(continued)
Table 3 (continued)
Encapsulation technologies
Nanoencapsulation
This technique is more complex that microencapsulation, mainly because of the intricate
morphology of the capsule and core material, and the demand in controlling the release rate of the
nanoencapsulates. It involves the incorporation, absorption, or dispersion of the bioactive
compounds and subsequent formation of the functional ingredients encapsulated in small vesicles
(<100 nm). Nanoparticles ensure a greater surface area, increase the solubility and enhance the
bioavailability of the target ingredients, due to their subcellular size and improved controlled
release. They can also easily penetrate tissues through fine capillaries, crossing epithelial lining
fenestration, being therefore efficiently taken up by cells and allowing and efficient delivery of the
active compounds in the target sites of body.
Emulsions
In a broad sense, an emulsion is a mixture of at least two immiscible liquids, usually oil and water,
where one of the liquids is dispersed as small spherical droplets in the other. Therefore, emulsions
are turbid, with small droplets sizes ranging from 0.2 mm to 10 mm, may remaining stable for a
considerable time-period, despite being classified as thermodynamically unstable systems.
Emulsions may be also classified into two distinct groups: microemulsions and nanoemulsions.
Microemulsions are systems consisting from a mixture of water, hydrocarbons and amphiphilic
compounds, forming kinetically and thermodynamically stable compounds, transparent,
homogeneous, and isotropic solutions, ranging particle sizes from 5 nm to 100 nm.
Nanoemulsions consists from fine dispersed emulsions and submicron emulsions, that in contrary
of microemulsions, are not thermodynamically stable. They are produced by microfluidization or
micelle formation techniques, exhibiting poor solubility and low bioavailability.
cheese [25, 30, 38] samples for subsequent analytical and methodological studies.
Lemon balm [33, 34], rosemary [32], chestnut [33, 34], and basil [35] were incor-
porated in cheese samples, while honey [2], wild blackberry [41], and seaweed
extracts [42] were used to functionalize yogurt samples. More recently, dairy
beverages were tentatively functionalized with pure ergosterol and mycosterols
from Agaricus bisporus extracts [36], as an alternative to phytosterol-based bever-
ages; and also incorporated antioxidant extracts from two mushrooms species,
namely Suillus luteus (L.: Fries) and Coprinopsis atramentaria (Bull.), acting as
food ingredients in the functionalization of cottage cheese [23]. Interestingly, in all
the above described experiments, prominent results were achieved both in terms of
protection against oxidation, improving consequently the shelf life of the
functionalized dairy products, as also in terms of nutritional value, stronger antiox-
idant activity, moisture and sensory attributes, such as external color, syneresis, and
viscosity [31, 32, 34]. Moreover, organoleptic characteristics also display a crucial
role in overall sensory acceptance and purchase intention, which was achieved
though the functionalization of these dairy products that provide beneficial charac-
teristics both for consumers (contributing for healthier lifestyles) and producer
industries (commercialization of added-value products) [2, 33]. From the point of
view of consumers, two additional aspects should be also considered. The first one is
related with the healthier substitution of artificial preservatives from natural ones,
improving consequently the acceptability, quality, and security of the final product
[11]. In fact, modern consumers are increasingly focused on the consumption of

naturally-derived and safer products and formulated with natural food ingredients.
The second one is related with the health-promoting abilities attributed to the
consumption of these functional dairy foods; it has been increasingly documented
and widely recognized that daily diet exerts a determinant role in health maintenance
and disease prevention [8, 22].
The last aspect that is not least important is related with the high interest by food
industry in always searching for new products, being this goal markedly driven by
the production of cheaper food products, but healthier for consumers also looking for
longer storage periods [7, 14, 33]. Therefore, consumers’ acceptance and subsequent
selection of a specific functional dairy product is markedly determined by a host of
factors. The first one is related with the primary health concerns, as already
highlighted, the consumers’ familiarity with the new functional dairy food and
with the functional food ingredients used on their formulation (i.e., nature of the
carrier product, delivery of health effects, etc.) [11, 43]. On the other hand, con-
sumers are awareness about the health effects of multiple substances and food
ingredients, being thus essential to provide specific and credible information about
the latest documented studies on the area. Finally but also determinant is the overall
organoleptic and sensory evaluation carried out by consumers about the new prod-
uct. In fact, different surveys have been applied to consumers, being clearly evident
the key conditions for acceptance, such as taste, product quality, price, convenience,
and even the reliability of health claims [11, 43, 44]. Therefore, during the devel-
opment of a functional dairy food, all these aspects should be taken into consider-
ation, besides to ensure their added value in terms of biological activity.
6 Bioactive Effects of Functional Dairy Foods
There is a widespread knowledge about the real benefits of functional dairy foods at
the level of health promotion, well-being maintenance, and longevity [27, 44]. By
themselves dairy products can confer several biological effects due to the naturally
occurrence of biologically active constituents on its chemical composition, such as
bioactive proteins, lipids, oligosaccharides, immunoglobulins, enzymes, hormones,
antimicrobial peptides, cytokines, growth factors, among others [37]. Nevertheless,
these constituents exist in limiting quantities, being therefore extremely usefulness
their incorporation in dairy products functionalization [31]. Furthermore, it has been
increasingly clear that some fermented dairy foods promote human health not so
much directly attributable to the starting materials, but instead from the outcomes of
fermentation, thus conferring additional properties beyond basic nutrition [14].
In fact, the formulation of dairy food products able to promote health and well-being
is one of the main key research priorities by food industries, that has mostly favored the
selection and subsequent consumption of foods enriched with physiologically active
components, such as probiotics, prebiotics, and more recently plant food bioactives [3,
14, 37]. One of the main and most widely recognized biological effects of functional
dairy products are their recognized modulatory activities, through the action of probiotic
Table 4 Biological activity of functional dairy foods

Functional dairy
Bioactive effect Detailed activity food
Cardiovascular Improvement of total cholesterol, non-HDL-C, and Fermented milk
disease LDL concentrations
Hypertension Antihypertensive peptides acting as angiotensin- Fermented milk,
converting-enzyme (ACE) inhibitors Cheese, Yogurt
Immunomodulatory Stimulation of the natural immunity, modulation of Yogurt, Cheese,
the production of cytokines, inhibition of Kefir
carcinogenic effects, gut fermentation, metabolites
Infection control Peptides exerting antimicrobial effects, reduction of Fermented milk
the incidence of fever and constipation
Inflammatory Reduction of symptoms of flatulence, abdominal Fermented milk,
bowel syndrome pain, cramps, and stomach rumbling yogurt
Mood and brain Modulation of the activity of brain regions Fermented milk
activity responsible for controlling central processing of
emotion and sensation
Muscle soreness Suppression of muscle soreness Fermented milk
Obesity Improvement of body fat percentage, waist Yogurt,
circumference and waist-to-hip ratio, lean body mass Fermented milk
Osteoporosis Increase of bone mineral density and short-term Kefir
changes in turnover
Type-2 diabetes Decrease of insulin resistance and increase of insulin Yogurt,
sensitivity, improving glucose tolerance, and Fermented milk
metabolism
bacteria, also existent in fermented milk products [14, 37]. Besides other aspects, they
exert an important role in gut fermentation metabolites, and therefore contributes to local
and systemic beneficial effects in humans, not only at a level of digestion, absorption,
and metabolism but also elimination, once favor a healthy bowel movement [14,
15]. More interestingly is that the functional effect of dairy products may be achieved
directly through the action of probiotic bacteria or even indirectly because of their
metabolic activity, i.e., microbial metabolites, such as vitamins, proteins, peptides,
oligosaccharides, organic acids, and bacteriocins [14, 37]. In fact, the modulatory effects
conferred by probiotic bacteria are both related with the stimulation of the natural
immunity as also modulation of the production of cytokines, antimicrobial peptides,
and inhibition of the carcinogenicity of multiple substances [45]. Another pronounced
bioactive effect of functional dairy products is their ability to minimize the incidence of
hypertension [14, 46]. In fact, dairy products, such as milk and cheese, contain
significant amounts of antihypertensive peptides, including lactopeptides isoleucine-
proline-proline and valine-proline-proline, that mainly acts as antihypertensive
angiotensin-converting-enzyme (ACE) inhibitors [14, 46, 47]. On the other hand,
whey and casein fractions of yoghurt have also shown strong antihypertensive effects
being documented by several studies their role in curtail several syndromes among
which in controlling blood pressure [46]. Other studies have even revealed that other
peptides from dairy fermented products also exert antithrombotic, satiety, opioid,
immunomodulatory, antimutagenic, osteogenic, and antioxidative effects [14, 48]. Addi-

tionally, interesting reports have revealed a positive association between fermented dairy
foods’ consumption and weight maintenance and body fat reduction, besides their
ability to reduce the risk of type 2 diabetes and other metabolic disorders, and overall
morbidity and mortality, mainly from yogurt consumption. The effects on glucose
metabolism seems to be mainly related with the improvement of glucose metabolism
and insulin sensitivity, and reduction of muscle soreness [14, 46]. The positive effects on
inflammatory bowel diseases and other immune-related pathologies, including arthritis,
sclerosis, fibromyalgia, migraine, and depression have been also increasingly evident, as
also their modulatory effects at a level of brain and mood [14, 49], still, however, being
essential to deepen knowledge on this field. All the above described biological effects
are briefly summarized in the Table 4.
7 Concluding Remarks
The world of functional foods development has led to one of the most promising and
dynamic development on a specific segment of food industry. The increasing consumer
awareness and demands for healthy products, in association with the latest advances on
the area of food science and technology are the most important factors supporting the
new revolution on food science and innovation. Nevertheless, consumers’ acceptance
and preferences are not homogenous, being observed a large distribution and differ-
ences on functional foods acceptance. Specifically, the development and commercial-
ization of functional dairy products is not easy, it involves complex, expensive, and
specific requirements. Besides the consumers’ demand, during the development of a
dairy functional product, the technical conditions as also legislation conditions should
also be considered. Special requirements stablished to apply during the development
and marketing of functional dairy foods should be met, as also their economic
potential; health impact, acceptance, and overall interest should not be forgotten.
Particularly, consumers’ acceptance has been considered a key success factor on
market orientation, brand new product introduction, and consumer-led product devel-
opment, besides to be successful in negotiating market opportunities. Therefore, by
purchasing functional dairy foods, consumers may have the overall health benefits of
functional foods, besides to achieve a positive and valuable impression of themselves.
In fact, they are self-contributors for this achievement, once they interfere both on
selection, formulation, evaluation, and overall qualification of these products. Besides
to the highlighted aspects, consumers may follow a balanced diet and acquire a healthy
lifestyle, which differ from the conventionally stablished healthy diet. Finally and not
least important is that the concept of functional dairy foods may represent a sustainable
trend in a multiniche market to be exploited.
Acknowledgments The authors are grateful to Foundation for Science and Technology (FCT,
Portugal) for financial support to CIMO (UID/AGR/00690/2013) and REQUIMTE (UID/QUI/
50006/2013 - POCI/01/0145/ FEDER/007265) research centres.
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Nutrients and Nutraceuticals from Seafood
47
V. Venugopal
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1399
2 Nutraceuticals and Functional Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1399
3 Nutrients from Seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1400
3.1 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1400
3.2 Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1403
3.3 Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1404
3.4 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1406
3.5 Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1407
3.6 Minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1409
3.7 Evaluation of the Nutritional Value of Fishery Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1409
4 Nutraceuticals and Bioactive Compounds from Seafood Discard . . . . . . . . . . . . . . . . . . . . . . . . 1411
4.1 Nutraceuticals of Nitrogenous Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412
4.2 Lipid-Based Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1417
4.3 Polysaccharide-Based Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1421
4.4 Mineral-Based Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1429
4.5 Marine Biotechnology for the Isolation of Nutraceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . 1429
4.6 Commercial Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1430
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432
V. Venugopal (*)
Former Scientific Officer Food Technology Division, Bhabha Atomic Research Center,
Mumbai, India
Visiting faculty, Department of Food Science and Technology, Kerala University of Fisheries and
Ocean Sciences (KUFOS), Kochi, Kerala, India

1398 V. Venugopal
Abstract
Seafood comprising of finfish and shellfish significantly contribute to world food
security. Seafood species are nutritious since they are rich in proteins and other
nutrients including peptides, essential amino acids, long-chain omega-3 polyunsat-
urated fatty acids, carotenoids, vitamins including vitamin B12, and minerals such
as calcium, copper, zinc, sodium, potassium, selenium, iodine, and others. Com-
mercial fish processing generates about 30 million metric tons of discards
consisting of shell, head, bones, intestines, fin, skin, etc. These discards are rich
in several nutraceuticals and biologically active compounds, which include oils
containing omega-3 PUFA; carotenoids such as astaxanthin and β-carotene; pro-
teins including myosin, collagen, and gelatin; enzymes; essential amino acids and
peptides; polysaccharides and their derivatives including chitin, chitosan, glucos-
amine, and glycosaminoglycans; and mineral-based compounds. These com-
pounds, depending on their nature, can have varying physiological functions
including antioxidant, anti-inflammatory, anti-allergic, antitumor, anti-obesity, anti-
coagulant, antimicrobial, immunomodulatory, and other activities, which are valu-
able in healthcare. Marine biotechnology offers several techniques to isolate these
nutraceuticals from seafood and their processing discards. This article briefly
surveys nutrients and nutraceutical contents of seafood and their potential benefits
in human nutrition and healthcare and current commercial status.
Keywords
Seafood · Processing discards · Nutrients · Nutraceuticals · Health benefits ·
Marine biotechnology
Abbreviations
AAS Amino acid score
CHD Coronary heart disease
CS Chondroitin sulfate
EAA Essential amino acids
FAO Food and Agriculture Organization of the United Nations
FDA Food and Drug Administration, USA
FSA Food Standard Agency, UK
MUFA Monounsaturated fatty acid
NMFS National Marine Fisheries Service, USA
PER Protein efficiency ratio
RDA Recommended Dietary Allowance
SFA Saturated fatty acid
USDA United States Department of Agriculture
WHO World Health Organization
47 Nutrients and Nutraceuticals from Seafood 1399
1 Introduction
Seafood, in a broader perspective, comprises of both finfish and shellfish items from
marine, estuarine, brackish, and freshwater habitats and forms a sizeable component of
the total world food production. According to the State of World Fisheries and Aqua-
culture, published by the FAO, in the year 2014, an amount of 167.2 million metric tons
(MMT) of seafood was globally available, supplying about 20 kg of fish per capita [1].
The major finfish species include anchovies, mackerel, tuna, herring, cod, whiting, and
others, while shellfish include two major groups, crustaceans and mollusks, based on
their structural features. Shrimp, lobster, crayfish, crab, and krill belong to crustaceans,
while mollusks are composed of bivalves (consisting of mussels, oysters, clams, and
scallops), cephalopods (squid, cuttlefish, and octopus), and gastropods (abalone, sea
snail, cockle, and whelks). Increasing consumer demand has resulted in aquaculture of
73.8 MMT of preferred seafood items in 2014, consisting of 49.8 MMT of finfish, 16.1
MMT of mollusks, and 6.9 MMT of crustaceans [1]. Commercial fishing operations
often result in large volumes of bycatch having poor consumer appeal, which is mostly
used as landfill, fertilizer, or animal feed. Commercial seafood processing leads to huge
volume of discards weighing as high as 50% of total landings, consisting of shell, head,
bones, intestines, fin, skin, and others [2, 3]. High-value seafood, bycatch, and pro-
cessing discards are all rich in many nutrients including peptides; essential amino acids;
long-chain omega-3 polyunsaturated fatty acids (PUFAs); carotenoids; vitamins A, B3,
B6, B12, and D; and various minerals such as calcium, copper, zinc, sodium, potassium,
selenium, iodine, and others [4]. Besides, they contain many nutraceuticals, which may
be nitrogen, lipid, polysaccharide, or mineral based. These compounds may be proteins
including collagen and gelatin; protein hydrolyzates; peptides having interesting bio-
activities; lipids rich in long-chain PUFA; hydrocarbons such as squalene; carotenoids;
polysaccharides such as chitin, chitosan, glycosaminoglycans, and their derivatives; and
mineral-based products such as bone powders. These, depending upon their nature, have
interesting bioactivities offering potentials for development of functional foods and uses
as natural food additives, medicinal drugs, and encapsulation as well as carrier materials
for nutraceuticals [4–7]. With changing consumer interests toward natural bioactive
compounds for healthcare and rapid developments in biotechnology, there is a vast
scope for secondary processing of seafood and their processing discards for isolation of
nutraceuticals. Such an attempt also helps total utilization of seafood for human
consumption favoring minimizing seafood-associated environmental problems. This
article discusses various nutrients, nutraceuticals, and other bioactive compounds pre-
sent in both edible portions and processing discards of seafood products. At the onset, a
brief discussion on nutraceuticals and functional foods is provided.
2 Nutraceuticals and Functional Foods
The term nutraceutical is a contraction form that links both the words nutrition and
pharmaceutical, coined in 1989 by Dr. Stephen L. DeFelice, founder and chairman of
the Foundation for Innovation in Medicine (FIM), Crawford, New Jersey. It is
1400 V. Venugopal
defined as any substance that may be considered a food or part of a food which
provides medical or health benefits including the prevention and treatment of disease
and includes isolated nutrients and compounds that are used as dietary supplements
[8–10]. The term functional food was coined in Japan in the mid-1980s and is
defined as “foods for specified health uses.” These foods contain ingredients that
can address diseases such as hypertension, allergy, etc. [11]. A food can be regarded
as functional if it is satisfactorily demonstrated to benefit one or more target
functions in the body, beyond adequate nutritional effects, in a way that is relevant
to either improved health or well-being or to a reduction in the risk of disease [12].
Plaza et al. [13] emphasized three important characteristics of a functional food,
namely: (i) the functional effect is different from that of normal nutrition, (ii) the
functional effect will be demonstrated satisfactorily, and (iii) the benefit can result in
an improvement of physiological function or in a reduction of risk of developing a
pathological process. It is now well recognized that dietary habit is one of the most
important determinants of chronic diseases. This has led to an increased interest of
the consumers toward natural bioactive compounds as nutraceuticals. Fish and
shellfish are well recognized to possess appreciable nutraceutical potential to protect
human health. This aspect will be broadly covered in this chapter. At the beginning,
nutrient contents and nutritional value of seafood are considered.
3 Nutrients from Seafood
The percentage of edible lean tissue in aquatic species is appreciably greater than that
in beef, pork, or poultry. About 50–60% of the weight of fish is constituted by its
muscle. The proximate composition of fish muscle gives a general idea on its
nutrient contents. The proximate composition of fish muscle varies greatly among
species, their age, sex, habitats, and season. Information on proximate composition
of fish and shellfish are provided by various databases. These include the global
database of FAO/INFOODS [14], the USDA [15], the US National Marine Fisheries
Service [16], the Department of Health UK [17], and FSANZ [18]. Besides, several
authors have also discussed proximate compositions and nutrients in various fish and
shellfish [19–24]. Information is also available on the website, SELFNutritionData
[25]. The muscle contains water (52–82%), proteins (16–21%), lipids (0.5–2.3%),
ash (1.2–1.5%), and a small proportion (about 0.5%) of carbohydrates. The various
nutrients present in seafood are briefly discussed below.
3.1 Proteins
Seafood items are rich in Proteins. More than 50% of the world’s seafood catch come
from developing countries. Therefore, people from these countries derive much of
their protein requirements from fishery products [1]. In the year 2014 an amount of
146.3 MMT of seafood was used as human food, giving a per capita seafood
consumption of 20.1 kg, which contributed to about 20% of total average per capita
intake of animal protein [1]. Proteins in finfish are distributed in both light and dark
muscle. Demersal fish such as cod and haddock have low percentage of dark muscle,
which is present under the skin on both sides of the body. On the other hand, in
pelagic fish, such as herring and mackerel, the proportions of dark muscle are high,
with more vitamins and fats, providing energy for their rapid movement. Light
muscle is more abundant in demersal fish. There are three main fractions of muscle
proteins in fish and shellfish, namely, myofibrillar (structural proteins) consisting of
myosin, actin, and others; sarcoplasmic (soluble proteins) consisting of myoglobin,
hemoglobin, globulins, albumins, and various enzymes; and stroma (connective
tissue) proteins consisting mainly of collagen, elastin, and gelatin. These fractions
constitute 70–80%, 20–30%, and 3% of total muscle proteins, respectively. Muscle
proteins are conveniently characterized by their solubility properties. Sarcoplasmic
proteins are defined as those soluble when the muscle is extracted with water or
solutions of low ionic strength, physiological or less. Myofibrillar proteins are
soluble at elevated ionic strengths (> 0.3). Myofibrillar proteins correspond to
65–75% (w/w) of the total protein in fish and shellfish muscle [4, 22]. These proteins
consist mainly of myosin (which account for 65–78%) and also actin (F and G
types), tropomyosin, M protein, α-actinin, β-actinin, C protein, as well as troponins I
and C, which are involved in muscle contraction. The myosin molecules resemble a
thread (2 nm x 160 nm) with two globular heads (19 mm long) attached at one end to
a tail portion. Fish myosins, compared to myosins from terrestrial animals, are more
unstable. A novel protein, paramyosin (molecular weight 200 to 250 kDa), is also
found at varying levels in invertebrate myofibrils, but not in vertebrate myofibrils.
The protein is involved in catch contraction of bivalves [22]. Fish meat, unlike
bovine meat, contains much less stroma proteins, which are the least soluble. When
heated, fish muscle connective tissue proteins dissolve more readily and soften,
contributing to softness of the cooked meat, in comparison with the tough connective
tissue of land animals [24].
The contents of proteins vary from 18% to 23% depending on the seafood items,
on the habitats (marine water, freshwater, or brackish water), and on the depth, i.e.,
pelagic (fast-moving species on surface waters such as tuna, herring, mackerel, sprat,
anchovy, and sardine) or demersal (slow-moving species in deep waters such as cod)
[21, 24, 26]. Shellfish, in general, contain slightly more proteins than finfish. The
reported average protein contents (g%, raw meat) of various shellfish are shrimp,
17.0–22.1; scallop, 14.8–17.7; squid, 13.2–19.6; crab, 15.0–18.4; lobster,
18.2–19.2; krill, 12.0–13.0; clam, 9.0–13.0; mussel, 12.6–13.0; cuttlefish,
16.6–17.3; and oyster, 8.9–14.3 [16–22]. Myofibrillar proteins are the major frac-
tions of the foot and mantle of the clam, constituting 31 to 40% [26]. The claw meat
and hepatopancreas of the green crab (C. mediterraneus) had protein levels of
17.8–18.2% and 13–14%, respectively [27]. The raw meat of brown shrimp
(Crangon crangon) from the Black Sea has a protein content of about 18.5% [28].
The white shrimp has higher protein contents than black tiger shrimp, the former
having higher amounts of stromal proteins with greater pepsin-soluble collagen [29].
Marine snail meat and hepatopancreas are important sources of protein [30]. The
edible portion of common octopus (O. vulgaris) has appreciable level of proteins
1402 V. Venugopal
[31]. Protein contents of some raw shellfish species from Indian waters vary from
14.0 to 21.6% [32].
An adequate diet must contain an appropriate level of nutritive protein to support
long-term health. Fish muscle proteins are considered nutritionally equivalent or
slightly superior to meat proteins due to their lower collagen contents and higher
sensitivity to proteolytic digestion [33–35]. The nutritive value of a protein is
determined by its primary structure, amino acid composition, contents of essential
amino acids, susceptibility to enzymatic digestion, and chemical changes during
processing such as thermal treatment. Most seafood proteins show a digestibility
above 90% associated with release of copious proportions of essential amino acids
(EAAs) (lysine, methionine, threonine, tryptophan, isoleucine, leucine, phenylala-
nine, and valine), suggesting high nutritive value of the proteins [35, 36]. Enzymatic
digestion of proteins in the human digestive system gives rise to many peptides and
amino acids, which can be absorbed in the intestine in the form of single amino acids
and oligopeptides [25, 33, 34]. Animal feeding experiments are used to determine
nutritive value of proteins. These include the nitrogen balance method based on
protein digestibility, determination of protein efficiency ratio (PER, weight gained
per g of protein consumed), net protein utilization (NPU, ratio of amino acid
converted to protein to the ratio of amino acids intake), and biological value
(a measure of absorption and utilization of protein by the living organism). These
studies have suggested the high nutritive value of fish proteins [37]. It is generally
accepted that the relative content of EAAs is the major factor determining the
nutritional value of a food protein. Most animal proteins have a satisfactory essential
amino acid (EAA) pattern required to satisfy amino acid requirements. The amino
acid score (AAS) of a protein is indicative of its nutritional quality; an AAS score of
100 means high protein quality. Proteins of seafood including shellfish, cooked
under moist heat, have AAS scores as high as 100 indicating their high nutritional
quality [25, 38]. The protein digestibility-corrected amino acid score (PDCAAS) is
based on the amino acid content of food protein, its digestibility, and its ability to
supply EAAs according to requirement [19, 25]. The PDCAAS of shrimp is 1,
indicating its good protein quality [38]. Shellfish proteins have PER values that are
slightly above that of casein, the major milk protein [22, 24].
Dietary fish proteins, rich in arginine, glycine, and taurine, may have ability for
improved resolution of inflammation in the muscle, attributed to their ability to
decrease the production of major pro-inflammatory cytokines (TNF-α, IL-6) and to
limit the accumulation of pro-inflammatory macrophages at the site of injury. Cod
protein promotes growth and regeneration of the skeletal muscle after trauma, partly
because of the improved resolution of inflammation [39]. Krill meat concentrate has
78% protein and 8% fat on dry weight basis. The protein has PDCAAS and PER
equal to casein and hence is a promising protein source for human consumption [40].
Spray-dried protein powder from threadfin bream (Nemipterus japonicus) has a PER
value of 3.52 0.27 and trypsin and pepsin digestibility up to 80% [41]. In view of
their nutritional properties, seafood proteins are able to enhance the nutritive value of
proteins from plants, legumes, grains, nuts, seeds, and vegetables, which may be
deficient in one or more of the essential amino acids [42, 43]. Fish meat and also
other commonly consumed protein foods are also significant sources of nutrients
such as calcium, vitamin D, potassium, iron, and folate [35, 44].
3.2 Amino Acids
Seafood items are important sources of both EAAs and nonessential amino acids.
The predominant EAAs in seafood are lysine and leucine, and within the nonessen-
tial, aspartic and glutamic acids are more abundant. The nonprotein nitrogen fraction
of fish and shellfish muscle, which is usually higher than that of terrestrial animals,
contains amino acids in addition to small peptides, creatin, creatinine, tri-
methylamine oxide, and nucleotides. Glycine, taurine, alanine, and lysine are gen-
erally present in relatively higher amounts. Certain nonessential amino acids, such as
aspartic acid, glycine, and glutamic acid, necessary for growth and maintenance in
humans, are present in seafood, as revealed by the amino acid compositions of 60
commercially important fish and shellfish [45].The whole body tissues of Atlantic
halibut, yellowtail flounder, and Japanese flounder contain all the EAAs [46]. The
freshwater fish, carp, has high content of free amino acids, irrespective of the area of
breeding, the dominant amino acids being histidine, methionine, cysteine, phenyl-
alanine, tyrosine, lysine, and threonine [24].
Crustaceans have more amino acid contents than finfish. The amino acids alanine,
glutamic acid, and glycine are mostly responsible for the flavor of cooked shellfish.
Alanine and glycine contribute to sweet tastes and glutamic acid to the “umami”
taste of crustaceans [47]. Shrimp has good contents of glycine (up to 1% of the fresh
muscle), alanine, and proline [16]. The major EAAs of shrimp are arginine, lysine,
leucine, and methionine, while the nonessential amino acids are glutamic acid,
aspartic acid, proline, and glycine [48]. Glutamic acid and glycine contents were
greater in black tiger shrimp meat, while white shrimp had good levels of hydroxy-
proline. Arginine, leucine, isoleucine, and proline were predominant in both shrimps
[29]. The Norway lobster has threonine, leucine, valine, lysine, and arginine, each at
levels of about 40 mg% of raw meat. It has also glycine, alanine, and glutamic acid at
58, 57, and 31 mg%, respectively. Besides, the lobster also contained the dipeptide
anserine at 53 mg% of raw meat [49]. Edible tissues of the marine snail are good
sources of EAAs, particularly aspartic acid [30]. Green crab meat is balanced in
EAA contents, except for tryptophan [50]. Fish sauces are products that contain
significant amounts of peptides and amino acids [52].
Cooking, in general, enhances the digestibility of fish proteins. Mild cooking
causes little loss of protein with only a slight loss in available lysine, whereas drastic
heating can significantly reduce the protein quality. Boiling has little effect in the
composition of shellfish. Moisture, protein, fat, ash, and carbohydrate contents of the
cooked horse mackerel ranged from 56 to 61%, 21 to 24%, 13 to 20%, 1.7 to 2.5%,
and 1 to 4%, respectively. The changes in amino acids in the fish were significant
after frying, grilling, and steaming. Cooking increased the contents of essential and
nonessential amino acids compared to raw fish. Amino acid contents of grilled horse
mackerel were higher than those found in fried and steamed fish [52]. Literature on
1404 V. Venugopal
the influence of processing on nutritional value of seafood has been reviewed by

Venugopal [22].
Taurine (2-aminoethanesulfonic acid) contents were relatively high in flatfish and
ray and low in silver pomfret, yellow croaker, and baby croaker. Although humans
synthesize taurine from methionine and cysteine, aging lowers production of taurine
in the body, necessitating supplementation of the amino acid. Shellfish and other
seafood are good sources of taurine; its contents in the muscle of mussel, oyster,
cuttlefish, and squid vary between 7.5 and 12.0% of total amino acids [32]. Taurine
acts beneficially on glucose metabolism and also lowers blood pressure by reducing
cholesterol absorption [43, 53, 54]. Processed fishery products may be supplemented
with taurine, the added taurine retaining in the product without adversely affecting
the sensory acceptability [55].
3.3 Lipids
The contents of lipids in fishery products vary depending on the species, season,
geographic location, age, gender, and diet. Feed has a strong influence on the lipid
contents of fishery products including cultured items [20]. The lipid content can vary
from 4% to more than 30% in Atlantic mackerel and from 2 to 25% in Atlantic
herring, depending on the season. Species living in temperate waters have more lipid
contents in their flesh than leaner tropical fish [24]. Contrary to terrestrial animals,
which deposit their lipids in the adipose tissue, fish have lipids in the liver, muscle,
and perivisceral and subcutaneous tissues. Lipid content in dark muscle is much
higher than in white muscles. Pelagic fish, which contain more dark muscle, have
more fat than the demersal species such as cod, which have higher proportions of
white meat. Depending on the fat content, fish are classified as lean (less than 2%
fat), low-fat (2–4% fat, such as cod and hake), medium-fat (4–8% fat), and fatty fish
(more than 8% fat, such as mackerel and herring).
The fatty acid composition of seafood is different from red meat, vegetable, and
dairy products and also showing marked variability within and between species. The
fatty acids are present as triglycerides, which are prone to hydrolysis by lipases. The
phospholipids of tropical fish are more saturated than those from cold water. The
major phospholipids include phosphatidylcholine, phosphatidylethanolamine, and
sphingomyelin. In contrast to red meat, marine fish lipids have appreciable pro-
portions of n-3 (omega-3) long-chain PUFA, particularly eicosapentaenoic acid
(EPA) (C20:5w3 cis-5,8,11,14,17) and docosahexaenoic acid (DHA) (C22:6w3, cis-
4,7,10,13,16,19). Fish obtain these from PUFA-rich phytoplankton used as feed by
the animal. Fatty fish such as herring and mackerel contain more omega-3 PUFAs
(particularly EPA and DHA) than leaner species. Saturated fatty acid (SFA) content
is comparatively less. Freshwater fish generally contain lower proportion of n-3
PUFA than marine fish [47]. Marine steroids are composed of cholesterol, which is
present at 20 to 50 mg per 100 g raw meat of finfish and 250 to 650 mg per 100 g in
roe [21].
Shellfish items have low crude lipid contents, generally up to 2% (w/w) of their
raw muscle. The contents of EPA and DHA in shellfish usually range between 300
and 500 mg% of raw muscle; their contents are generally lower than those of oily
finfish such as Atlantic mackerel and sardine [19, 21]. Shellfish lipids, like those of
finfish, are rich in PUFA generally exhibiting a ratio of n-3 PUFA to n-6 PUFA above
1.0 [22, 56]. The Mediterranean giant red shrimp (Aristaeomorpha foliacea) has
good levels of n-3 PUFA, particularly EPA and DHA [57]. The lipids of the shrimp
spp. (P. longirostris and A. antennatus) and Norway lobster (N. norvegicus) contain
42–48% PUFA, 26–35% MUFA, and 23–27% SFA [48]. The crude fat (1%, w/w) of
brown shrimp (C. crangon L) meat consisted of 33% SFA, 22% MUFA, and 29%
n-3 PUFA. The SFA and MUFA fractions have 21% palmitic acid and 14% oleic
acid, respectively. The PUFA consisted of EPA and DHA at 41% and 32%, respec-
tively [28]. The n-3 PUFAs of white shrimp (P. vannamei) and black tiger shrimp
(P.monodon) are 42 to 44% of crude lipids. PUFAs of the white and black shrimps
have DHA-to-EPA ratios of 1.05 and 2.15, respectively [29]. Ozogul et al. [58]
reported that the common cuttlefish (S. officinalis), European squid (L. vulgaris),
common octopus (O. vulgaris), and musky octopus (E. moschata) had PUFA, SFA,
and MUFA contents of 43.6–56.5%, 28.2–35.3%, and 4.4–9.5%, respectively. The
fatty acids of the common octopus (O. vulgaris) consisted of 58.6% of n-3 PUFA
(composed of 20.1% EPA and 26.3% DHA), 25.9% SFA, and 15.4% MUFA [31].
The mussel (M. coruscus) has higher contents of PUFA than SFA and MUFA, with
DHA and EPA comprised of 12–18% and 10.8–14.6% of total fatty acids, respec-
tively [59]. Crustaceans, bivalves, and cephalopods may contain total sterols at
150–250 mg%, w. wt. [16, 19, 28].
The changes that take place in fats during heat processing greatly depend on the
fatty acid composition. Unsaturated lipids are very susceptible to oxidation.
Enzymes such as lipoxygenase, peroxidase, and microsomal enzymes can poten-
tially initiate lipid peroxidation producing hydroperoxides, which decompose to a
wide range of compounds. These compounds, which include aldehydes, ketones,
alcohols, small carboxylic acids, and alkanes, give rise to very broad odor spectrum
and also yellowish discoloration to the product. These substances are formed slowly
at normal frying temperatures in pure fats, but their formation is catalyzed by traces
of metals such as iron and copper present in the fish. The common dietary antiox-
idants vitamin E (tocopherol), vitamin C, polyphenols including flavonoids, and
carotenoids are able to control lipid oxidation (see also Sect. 3.4).
The human diet contains long-chain PUFA belonging to the n-6 and n-3 families.
Major dietary n-6 PUFAs include linoleic, C18:2w6 (18:2); γ-linolenic, C18:3w6 (18:3);
and arachidonic, C20:4w6 (20:4) acids, whereas major dietary omega-3 PUFAs
include α-linolenic, C18:3w3 (18:3), EPA, and DHA. Linoleic, α-linolenic, and
γ-linolenic acids are present in large quantities in foods of plant origin. Arachidonic
acid originates from muscle and organ meats or may be synthesized within the body
from linoleic acid. In comparison with n-6 PUFA, n-3 PUFA can have significant
health benefits. The cardioprotective role of n-3 PUFA has been determined by
several studies [60, 61]. Other benefits include their antidepressive, antiaging, and
antiarthritic effects, among others [61] (see also discussion on Sect. 4.2.1). The mode
1406 V. Venugopal
of action of EPA and DHA is attributed to their ability to give rise to a class of
pharmacologically important groups of compounds, such as prostaglandins, prosta-
cyclins, thromboxanes, and leukotrienes (collectively called eicosanoids). Both n-3
and n-6 PUFAs are precursors of eicosanoids. Whereas eicosanoids derived from n-6
PUFA, such as arachidonic acid, have pro-inflammatory functions, eicosanoids
derived from n-3 PUFA have anti-inflammatory properties. The n-3 PUFA-derived
eicosanoids antagonize the formation of inflammatory eicosanoids such as prosta-
glandin E2, derived from arachidonic acid and other n-6 PUFAs. The n-3 PUFAs
impart their anti-inflammatory effects via reduction of nuclear factor-κB activation.
This transcription factor is a potent inducer of pro-inflammatory cytokine produc-
tion. Through these actions, the n-3 PUFAs alter cell and tissue functions that favor
disease prevention and maintenance of health [61, 62].
3.4 Carotenoids
Carotenoids are a group of pigments that contribute to the yellow, orange, and red
color of aquatic organisms. Carotenoids exist in mollusks (oyster and scallop),
crustaceans (shrimp, lobster, crayfish, and crab), and finfish (salmon, trout, sea
bream, and tuna). They are also present in their processed products influencing
consumer acceptability. Animals, including humans, do not synthesize carotenoids
de novo and rely upon diet as the source of these compounds. Fish and shellfish
accumulate carotenoids in their body tissues from carotenoid-rich plants, which are
used as their feeds. The carotenoids may be either hydrocarbons or xanthophylls (the
oxygenated derivatives) and include astacene, astaxanthin, canthaxanthin,
cryptoxanthin, fucoxanthin, lutein, neoxanthin, violaxanthin, zeaxanthin, allo-
xanthin, and β-carotene [4, 63, 64]. Astaxanthin, perhaps, is the important pigment
present in fishery products. The red-orange color of salmonid fish is due to the
presence of astaxanthin. The carotenoids β-carotene and β-cryptoxanthin ingested by
the animals may be converted to different compounds, including astaxanthin and
vitamin A. In shellfish carotenoid contents vary depending on body parts; they are
high in their carapace, followed by the head, while their meat has minimum contents.
For example, the total carotenoid contents (mg g1) of raw meat, head, and carapace
of shallow water shrimp (P. monodon) are 17.4, 58.4, and 86.6, respectively. Similar
pattern is observed in carotenoid contents in the body parts of shallow water shrimps
(M. dobsoni, P. stylifera, and P. indicus), deep-sea shrimps, S. indica, and A. alcocki
[64]. Carotenoid content in raw body tissues of scallop ranged from 7 to 60 μg g1.
The pigment contents varied in this order: gonad > mantle > adductor > gill [65].
Antioxidants, defined as substances capable of delaying, retarding, or preventing
the development of rancidity or other flavor deteriorations, can inhibit or retard
oxidation either by scavenging the free radicals that initiate oxidation or by breaking
the oxidative chain reactions. The mechanisms also involve binding of metal ions,
scavenging of oxygen, converting hydroperoxides to non-radical species,
deactivating singlet oxygen, and thereby suppressing the generation of free radicals
and reducing the rate of oxidation. Autooxidation of unsaturated lipids leads to
formation of reactive oxygen species (ROS), which include peroxy radicals (ROO.),
superoxide anion (O2), hydroxyl radical (HO), and alkoxy radical (RO), among
others. Non-radical derivatives are hydrogen peroxide (H2O2), ozone (O3), and
singlet oxygen (1O2). The hydroxyl radical is the most reactive ROS, followed by
singlet oxygen. Reactions of ROS with food components destroy nutrients; change
the functionalities of proteins, lipids, and carbohydrates; and lead to the formation of
undesirable volatile compounds and carcinogens. Carotenoids are natural antioxi-
dants, which are able to quench singlet oxygen and act as in vivo scavengers of ROS.
The antioxidant activities of carotenoids have been attributed to the presence of
conjugated double bonds in their structures [66, 67]. The singlet oxygen scavenging
ability is the highest for β-carotene, followed by tocopherol, riboflavin, vitamin D,
and ascorbic acid [66]. Free radical stress can cause tissue damage and result in an
inflammatory response. Adequate intakes of carotenoids and also other antioxidants
such as vitamin A, vitamin C, and vitamin E may help prevent free radical-induced
adverse oxidative processes in living systems. Carotenoids, in general, also possess
anti-inflammatory properties, presumably due to their effects on intracellular signal-
ing cascades, thereby inhibiting production of inflammatory cytokines [67, 68].
Astaxanthin, the major shellfish carotenoid, has the ability to protect body tissues
from oxidative damage by UV light. The antioxidant activity of astaxanthin is higher
than that of β-carotene, lutein, lycopene, ɑ-tocopherol, and canthaxanthin. It also has
anti-inflammatory and cardioprotective properties [69]. The potential health benefits
of astaxanthin also include its anticancer, antiaging, and immunostimulating activ-
ities. The carotenoid also possesses antidiabetic properties, controls cataracts, and
inactivates gram-negative bacteria such as Helicobacter pylori, responsible for
chronic gastritis [63, 68, 69]. Canthaxanthin and β-carotene protect macrophage
receptors from ROS, while β-carotene protects neutrophils, a major class of white
blood cells, which use ROS to kill phagocytized bacteria [70].
3.5 Vitamins
Vitamins and minerals are essential for normal physiological functions. Their con-
tents in fishery products vary with the species, age, season, sexual maturation, and
geographic area. Fatty fish are excellent source of fat-soluble vitamins A, D, and E,
which are more concentrated in their liver, viscera, and eyes but in less amounts in
their flesh [21]. The flesh of lean fish contains about 25 to 50 IU of vitamin A, while
in fatty fish such as mackerel and herring, the content ranges between 100 and
1500 IU. Vitamins A and D are also present in copious levels in the liver of lean
species like cod. In freshwater fish, vitamin A exists mainly in the form of
3,4-dehydroretinol. Considerable amount of vitamin A, as retinol, is found in
shark and oysters, as well as in oily fish such as small sardines, herring, and horse
mackerel. Fish liver oil is an excellent source of vitamin A. Shrimp, blue mussel,
oyster, and scallop are good sources of vitamin A [15–17, 21, 23].
Vitamin D mainly occurs in nature as ergocalciferol (vitamin D2) or cholecalcif-
erol (vitamin D3). Fish obtain their requirements of vitamin D3 through phyto- and
1408 V. Venugopal
zooplanktons used as food. Fatty fish like horse mackerel or herring have a higher
content of vitamin D than lean species like flounder or marine trout. Contrary to the
general belief, there was no significant correlation between contents of fat and
vitamin D. Pelagic fish with high fat content provides excellent dietetic sources of
vitamin D3. Shrimp recorded about 0.06 μg vitamin D3 per 100 g [71]. Fish liver and
oils are also rich sources of vitamin D3 containing up to 300 μg of the vitamin per kg
oil [24]. The anticancer properties of vitamin D have been pointed out recently [72].
Vitamin E comprises of four tocopherols, α-, β-, γ-, and δ-tocopherol, all of them are
present in seafood and aquacultured products, particularly fatty fish items [73].
Through its potent antioxidant activity, the vitamin quenches free radicals formed
by oxidative processes, thereby controlling inflammatory processes; heating pro-
cesses, boiling, grilling, and frying, caused a decrease in tocopherol contents in
fishery products [74]. Fish flesh seems to be considerably rich in the antihemorrhagic
vitamin K [24].
Water-soluble vitamins are distributed along the whole fish, with niacin, thiamine,
riboflavin, pyridoxine, and B12 being the most representative in aquatic organisms
[4, 24]. The average content of thiamin varies from 4 to 400 μg per 100 g meat.
However, much of thiamin is destroyed by heat and oxygen or is lost in cooking
water or when exposed to ionizing radiation. Modest amounts of riboflavin are
present particularly in the dark flesh of some species like canned herring, mackerel,
and pilchard. Ideal sources of pyridoxine (vitamin B6) are salmon and tuna and, to
some extent, shellfish. Niacin, like thiamine, forms part of a coenzyme essential in
the production of energy. The vitamin ranges from 0.9 mg to 3.1 mg per 100 g raw
fish flesh. Vitamin B12 (cobalamin) is found in significant quantities in fish, partic-
ularly fatty fish. In general, seafood items contain 0.89 to 42 μg of vitamin B12 per
serving (3 oz). Anchovies, clams, herring, oysters, pilchard, and sardines have
vitamin B12 ranging from 25 to 40 μg per 100 g raw meat. Shellfish species contain
all the vitamins, particularly vitamin B12. Oyster, mussels, and short-necked clam
have a content of 46.3, 15.71, and 87.0 μg per 100 g, respectively. Contents of
vitamin B12 are generally higher in muscle tissues of crab and lobster [14]. Certain
fish like salmon, sardine, trout, and tuna have a B12 content varying from 3.8 to
8.9 μg 100 g1; however, mollusks and clams have an average of 98 μg 100 g1 of
B12 [75]. The flesh of Atlantic salmon, European hake, and sardine has folic acid
concentrations of 10, 27, and 24 μg 100 g1, respectively [76].
Vitamins are considered the most susceptible to loss during heat treatment; the
magnitude of loss depends on the specific vitamins and the conditions employed.
Water-soluble vitamins may be lost due to leaching. A combination of oxygen, light,
and heat causes a greater loss of vitamins than any one of these factors individually.
Folate and vitamin B6 are susceptible to destruction due to oxidation. Riboflavin is
reasonably stable during cooking but is sensitive to light as it decomposes on
exposure to ultraviolet rays. The fat-stable vitamin A and also carotenes are rela-
tively stable at normal cooking temperatures, but the high temperatures used in
frying can produce oxidative losses and isomerization of the carotenes, with signif-
icant losses of biological activity. Vitamin E is slowly destroyed during frying and is
decomposed by light.
3.6 Minerals
Several minerals are essential for life. The process of bone formation, for example,
requires adequate and constant supply of nutrients such as calcium, magnesium,
vitamin D, vitamin K, and fluoride, besides proteins. The ash contents up to 2.0%
(w/w) of seafood carry macroelements such as calcium (Ca), magnesium (Mg),
sodium (Na), potassium (K), iron (Fe), and phosphate (Pi) and microelements such as
zinc (Zn), copper (Cu), chromium (Cr), manganese (Mn), selenium (Se), fluorine (F),
and iodine (I), which are important for human nutrition [15, 16, 19, 35]. Fe, Zn, Cu, and
Mn concentrations are generally higher in the liver than in muscle tissues, either being
wild or farmed fish. The minimum contents (μg g1 edible fish flesh) of Na, K, Mn, Ca,
Cu, Pi, and I are 250, 250, 100,100, 1.1, 250, and 0.1, respectively [21]. Fish is a good
source of Zn and Cu that ranged from 7 to 34 μg g1 in wild sea bass and from 3.7 to
76.2 μg g1 in the farmed ones [21]. An average serving of fish can grossly satisfy the
total human requirements for essential microelements [21, 24].
Most shellfish are good sources of Na, K, Pi, Fe, Zn, Se, and Cu. Most fresh
marine fish may be considered moderately low-sodium foods delivering approxi-
mately 140 mg sodium per serving. However, the sodium content of most processed
fish and seafood products (frozen, canned, smoked, cured) can be substantially high,
ranging from 3 to 9 mg g1. Seafood is a source of calcium, its contents varying from
60 to 1200 μ per g1 depending upon the species. The iron content of edible tissue
may vary as 90 μg g1 in cod, flounder, and pollock and 90–200 μg g1 in carp,
catfish, salmon, and trout. Copper, which is necessary for normal blood formation
and maintenance of blood vessels, tendons, and bones, is sufficiently available from
shellfish. Mollusks and crustaceans contain appreciable levels of Cu and Zn [19].
Oysters are rich in zinc, iron, and copper [21]. Mg, Ca, and Fe are in appreciable
levels in white and black shrimps [29]. The contents of Ca, Mg, Pi, and Na in blue
crab vary in claw, breast meat, and hepatopancreas [77]. The edible portions of clam
are rich in Na, K, Ca, Mg, Fe, Zn, and Cu [26]. The contents (mg 100 g1) of iron,
zinc, and calcium in 55 samples of fresh fishery products from Bangladesh ranged
from 0.34 to 19, 0.6 to 4.7, and 8.6 to 1000, respectively. Shrimp had maximum Cu
and I contents of 12 and 1.2 μg g1, respectively [71]. Fish and shellfish are rich
sources of iodine, being highest in oysters, followed by clams, lobster, shrimp, and
crab followed by ocean fish. Oceanic fish has 0.3 to 3.0 μg g-1iodine and freshwater
fish, 0.02 μg to 0.04 μg g1 [20].
3.7 Evaluation of the Nutritional Value of Fishery Products
It is interesting to evaluate the nutritive value of fishery products against the US

2015–2020 Dietary Guidelines, which provides consumers of different age and sex
guidance to choose a healthy eating pattern to prevent diet-related chronic diseases.
The Guidelines are designed to meet the Recommended Dietary Allowances (RDA)
and the Adequate Intakes for Essential Nutrients set by the Institute of Medicine
1410 V. Venugopal
(IOM), US National Academies [78]. The Guidelines recommend daily intake of the
following amounts of nutrients by males aged 19–30, namely:
Protein: 56 g
Total fat: 20–35 g (saturated fat, <10% of total fat)
Macro-minerals (in mg): Na, 2300; Ca, 1000; K, 4700; Pi, 700; and Mg, 400
Micro-minerals (in mg except vitamin D and vitamin B12): Fe, 18; Zn, 11; Mn, 2.3;
Cu, 0.9; and Se, 0.055
Vitamins (in mg except vitamin B12 which is in μg and vitamin D, which is in IU): A,
900; B1, 1.2; B2, 1.3; B6, 1.3; B12, 2.4 μg; niacin, 16; D, 600 IU; E, 15; K, 0.1; and
folate 400
Calories: 2400 to 3000
The daily nutritional goals for other age and sex groups are also given in the
Guidelines [78]. Seafood including shellfish can significantly satisfy requirements of
almost all of the nutrients, mentioned above. Dayal et al. [38] calculated percent
direct value (%DV) (representing ability to satisfy dietary requirement of each
nutrient as per recommended values) for various nutrients present in seafood. They
reported %DV values of 75 for total EPA and DHA contents and a value of 70 for the
essential amino acids methionine, tryptophan, and lysine present in tiger shrimp
(P. monodon) [38]. Consumption of 100 g of most shellfish can meet at least 50%
requirement for proteins by the adult [23]. Compared with suggested amino acid
requirements by the FAO/WHO, the hydrolyzates of the little Loligo squid
(U. chinensis) have high nutritional value and are a potential nutritious supplement
used in various food products [79]. Selenium has a %DV as high as 110, suggesting
that the shrimp fully satisfied the dietary requirement for this mineral [38]. Shellfish
and other seafood provide good measures of vitamin B12 and vitamin D. A 100 g
serving of shellfish, except squid, scallop, and crayfish, can provide appreciable
amounts of the vitamin B12 necessary to satisfy its dietary requirement. The giant red
shrimp, as well as Norway lobster, is a valuable source of nutrients, including
proteins and antioxidants, among others, for the human diet [48]. The muscle and
gonads of female crab (C. pagurus) have favorable n-3 to n-6 PUFA ratios and a
well-balanced essential amino acid composition [80, 81]. American and European
lobsters have nutritive values compatible with nutritional foods [82]. The mussel
P. viridis has balanced ratios of essential to nonessential amino acids and also of n-3
to n-6 PUFA contents [83]. Furthermore, the consumption of mollusks can make an
important contribution to the daily dietary intake requirement of Se, Cu, and Zn [84].
The presence of appreciable levels of PUFA (including EPA and DHA), vitamins,
minerals, and amino acids qualifies the oyster (C. madrasensis) as a potential
“health” food. The shellfish has also atherogenic and thrombogenicity indices
together with a good hypocholesterolemic to hypercholesterolemic ratio, pointing
out its health benefits [85]. The blue crab could be used as a dietary supplement to
balance human nutrition [77]. Nutritional claims have also been made with respect to
common octopus [31] and Asian hard clam [26]. The nutritional value of seafood has
also been pointed out by many studies [19, 23, 35]. The Second International
Conference on Nutrition (ICN2), held in Rome in November 2014, confirmed the

importance of seafood including shellfish as a source of nutrition and health [1].
4 Nutraceuticals and Bioactive Compounds from Seafood

Discard
Industrial processing of seafood generates voluminous discards such as scales,

shells, frames, backbones, viscera, head, liver, skin, belly flaps, dark muscle, roe,
and others. Processing discards of crustaceans are composed of the cephalothorax,
carapace, and tail; shell discards of shrimp, krill, and crab constitute as high as 50%
of the shellfish. Increased aquaculture production of popular shrimp and other
shellfish items has led to a significant amount of discards. Depending upon finfish,
discards may range from 25 to 50% of the raw material. The backbone accounts for
about 15% of the wet weight of fish such as Atlantic cod. In addition, approximately
50,000 tons of scale is produced from the current global fish processing operations
[3]. Currently most of these discards are used as landfill or converted into fertilizer or
used for animal feed and ensilage. For example, the Norwegian fisheries annually
produce more than 615,000 tons of discards; most of it is converted into fish silage
and fish meal as animal feed [52]. Furthermore, commercial fishing operations
results in a large proportion of bycatch, which has poor consumer value due to
inherent problems related to unattractive color, flavor, texture, small size, and high
fat content. Most of these underutilized fish belong to the abundantly available
pelagic species and are considered as bycatch [86]. Secondary processing of seafood
discards can significantly reduce the environmental hazards associated with the
industry, besides yielding valuable ingredients for diverse applications.
The important ingredients that can be recovered from seafood discards include
proteins including collagen and gelatin and their hydrolyzates, peptides, enzymes,
oil, carotenoids, bone calcium, chitin, chitosan, and other compounds, many of
which have nutraceutical potentials [2, 36, 87–89]. During the recent years, there
is increased consumer interest toward nutraceuticals and natural bioactive com-
pounds as functional ingredients to prevent, alleviate, or treat diverse diseases
[88]. An example may be cited. The processing of the Black Sea anchovy gives
rise to approximately 32% (w/w) discards consisting of the head, frame, and viscera.
These have protein and fat contents ranging from 12 to 17% and 10 to 23.9%,
respectively. The discards are also rich sources of lysine and leucine, constituting
6–7% and 5–6% of total amino acids, respectively, PUFA (about 32–40% of total
fatty acids, with n-3 fatty acids comprising about 27–34% of total fatty acids), and
also various minerals. The anchovy discards are raw material for protein powder,
protein hydrolyzates, fish oils, and mineral supplements [90].
Interests in seafood- and other marine-derived compounds stem from the fact that
most aquatic organisms have inbuilt mechanisms to survive hostile oceanic envi-
ronments such as varying degrees of salinity, pressure, temperature, and illumina-
tions. Most marine organisms produce several secondary metabolites, which
although are not directly involved in central physiological functions, yet contribute
1412 V. Venugopal
to their survival. They synthesize novel compounds with interesting bioactivities,

which facilitate them to adapt to these conditions. Seafood species, for example, in
the extremely low temperatures of the polar region possess enzymes having novel
features relating to isozyme distribution, substrate binding, amino acid sequence,
low activation energies, high-specific activities, and thermal sensitivities [91, 92].
Besides, most marine organisms also produce several secondary metabolites such as
sulfated polysaccharides, peptides, alkaloids, terpenoids, and others, which although
are not directly involved in central physiological functions, yet contribute to their
survival [4]. These novel compounds may possess a wide spectrum of bioactivities
encompassing anticancer, anti-inflammatory, antiviral, antimicrobial, antioxidative,
antihypertensive, anti-atherosclerotic and anticoagulant, immunomodulatory, anal-
gesic, antidiabetic, appetite-suppressing, neuroprotective, and other interesting func-
tions. Therefore, during the recent years, the search for nutraceuticals and other
medicinal compounds from seafood and other marine organisms such as sponges,
tunicates, bryozoans, mollusks, and sea slugs and other organisms has intensified for
the potential control of various diseases such as cardiovascular disease (CVD),
diabetes, osteoporosis, arthritis, Alzheimer’s disease, and others [5, 88, 91]. Recent
studies have indicated that specific compounds from fish such as cod, anchovy, eel,
and shellfish like mollusks (mussel, oyster, clams, and abalone) as well as sea
cucumbers may possess in vivo/in vitro anticancer/antitumor activities, supporting
health benefits of these organisms. Some of the compounds can also function as
hypotensive agents, cardioactive substances, muscle relaxants, antibiotics, and anti-
viral agents [4, 92]. Nguyen [93] observed that about 50,000 tons of lobster
processing discard by-products can be valuable sources of bioactive compounds
having potential applications in nutraceutical, pharmaceutical, and medicine, besides
water treatment, agriculture, and other industries. Table 1 shows major nutraceuticals
and bioactive components available from seafood.
The remaining part of this chapter is intended to briefly discuss various
nutraceuticals and other bioactive compounds from seafood processing discards
and underutilized fish. For the convenience of discussion, these compounds are
grouped as those of nitrogenous origin, lipid-based compounds, polysaccharides
and their derivatives, and mineral-based compounds [6].
4.1 Nutraceuticals of Nitrogenous Origin
4.1.1 Proteins
Seafood proteins, besides notable nutritive properties, as discussed above, also
have excellent functional properties, which include solubility, viscosity, water-
holding capacity, gelation, adhesion, elasticity, foaming, and oil emulsification
capacities, which make them interesting food ingredients [4]. These properties
emanate from their compositional and structural features, which include hydro-
philic character, hydrodynamic size and shape, and ability for hydrogen bonding
and ionic hydration, among others [4]. Seafood processing discards are therefore
valuable sources of functionally active proteins. Techniques for isolation of these
Table 1 Major nutraceuticals and bioactive components from seafood

Finfish
Bioactive peptides
Biological calcium
Carotenoids
Enzymes including cold-adapted enzymes
Glycosaminoglycans including chondroitin sulfate, dermatan sulfate, and hyaluronic acid
Long-chain omega-3 polyunsaturated fatty acids (PUFAs)
Phosphopeptide from fish bone
Protein hormones such as calcitonin
Protein isolates including collagen and gelatin
Squalene and squalamine
Shellfish (crustaceans and mollusks)
Bioactive peptides
Carotenoids
Chitin, chitosan, and chitosan derivatives
Enzymes
Glucosamine
Long-chain omega-3 polyunsaturated fatty acids (PUFAs)
Mussel polysaccharides, lipids, and other products
Protein isolates including collagen and gelatin
proteins involve extraction by dilute acids, bases, or isoelectric solubilization/

precipitation [94, 95]. Filleting operations of large fish such as hake, seer, and
others give rise to frames that carry large amounts of meat portions. Mechanical
deboning helps to recover meat from the frames. Thorough washing of the recov-
ered meat gives surimi, which because of its excellent gel-forming properties, is an
ideal raw material for the development of restructured imitation seafood [94].
Generally fish protein isolates maintain their properties for about 6 months when
stored at 5 C but loses them rapidly at 30 C. Deterioration during storage is
prevented by lowering the moisture content of the product and by eliminating
oxygen from the package [96]. The protein isolate can be used as an ingredient to
enhance protein contents of food products and also as a nutraceutical. Fortification
of extruded corn snacks with up to 18% fish protein powder and 17% omega-3 fish
oil did not influence the sensory attributes of the products when served within
12 weeks of production [96]. Soluble powders recovered from processing discards
of Alaska pollock had protein contents ranging from 65% to 79% and nitrogen
solubility value as high as 86%. Emulsion capacity (mL of oil emulsified per mg1
protein) and emulsion stability of the pollock protein powders ranged from 29% to
35% and 65% to 78%, respectively, with a maximum fat-adsorption values of
10.6 mL of oil g1 protein. The protein powder was also a good source of K, P, Mg,
and also amino acids. Emulsions made with soluble protein powders from pollock
exhibited viscoelastic characteristics, suggesting its use as a food fortificant and
functional additive [97]. The nutraceutical potential of protein isolates has also
1414 V. Venugopal
been indicated. For instance, the protein isolate from striped bass (Morone
saxatilis) has a positive effect in control of CVD [95].
Preparation of fish protein hydrolyzates (FPHs) is a plausible method for utiliza-
tion of proteins from seafood discards. Currently hydrolyzed fish proteins are used as
condiments and sauces in the East Asian countries. FPHs have potential as a
functional ingredient and protein supplement for developing formulated ready-to-
eat products, which can help increase in protein consumption by the general public
for health benefits [98]. In the year 2014, a landing of 7.7 MMT of tuna was recorded
[1]. A sizeable portion of tuna is used for canning, which generates as much as 70%
solid discards from the fish, consisting of muscle (after loins are taken), viscera, gills,
dark flesh, head, bone, and skin. Proteins from these discards can be recovered as
FPH for use as food supplement to enhance functional effects such as whipping,
gelling, and texturing properties [99]. FPH has also been recognized as potential
nutraceutical essentially due to the activity of antioxidant peptides present in the
product [99] (see Sect. 4.1.2.). Both in vitro and in vivo studies have shown that
certain peptide fractions in FPHs may stimulate the non-specific immune defense
system [51]. Preliminary studies have indicated that expression levels of tumor
necrosis factor (TNF-α) had a tendency to be lower after the addition of FPH.
Furthermore, the combination of FPH and omega-3 fatty acids synergistically
decreased expression levels of TNF-α, compared to individual effects of omega-3
fatty acids or FPH [100]. Animal feeding study conducted at Norway suggested the
effect of fish protein hydrolyzate as a cardioprotective nutrient. The fish protein
treatment reduced plasma cholesterol level and altered the fatty acid composition in
liver, plasma, and triglycerol-rich lipoproteins in obese Zucker rats. The ratio of
HDL cholesterol to total cholesterol was greater in the animals, compared with
casein-fed animals [101]. Although not a nutraceutical, mussels possess specialized
proteinous glue, collectively referred to as mussel adhesive proteins (MAPs), which
adhere well to surfaces despite the presence of water. The protein is rich in the amino
acid, L-3,4-dihydroxyphenylalanine (DOPA). MAPs may be used for surgical tissue
adhesion or as components of muco-adhesive drug delivery systems. Another
protein, green fluorescent protein (GFP), isolated from the jellyfish in 1962 has
interesting applications in biotechnology and cell biology [4].
Frames discarded from fish filleting operations, fins, scales, air bladder, and fish
heads are also good sources of collagen. Collagen and its partially hydrolyzed form,
gelatin, contain more than 80% nonpolar amino acids such as glycine, alanine,
valine, and proline and hence are different in their amino acid contents, in compar-
ison with fish muscle proteins. Collagen-based biomaterials are extensively used as
food additives and in ophthalmology, dermatology, drug delivery, cosmetics, and
other industries [102, 103]. Fish collagen has lower denaturation temperatures than
porcine collagens. Therefore, collagen from fish offal can be a better alternative to
mammalian collagen for use in foods, cosmetics, and medicine. Collagen hydroly-
zates are beneficial for the treatment of osteoarthritis and other joint disorders. Orally
administered collagen hydrolyzate is absorbed intestinally and is accumulated in the
cartilage. Ingestion of collagen hydrolyzate stimulates synthesis of macromolecules
by chondrocytes in extracellular matrix [104]. Collagens can be easily converted into
gelatin by hot water treatment. Gelatin has found extensive application in the
pharmaceutical industry. It is also being used as a matrix molecule for nanoparti-
cle-based drug delivery applications. Most of the current production of gelatin is
from porcine and bovine hides, bones, and hooves. The incidents of mad cow disease
have made adverse impact on the use of mammalian gelatins. Fish gelatins could be
plausible substitutes to mammalian gelatins, since they have properties such as
bloom strength (the gel strength equivalent referred to in the industry), viscosity,
and solubility comparable with those of mammalian gelatins [102, 103].
Fishery discards such as viscera, liver, and head are sources of proteases, lipases,
transglutaminases, amidases, lipases, phospholipase, chitinases, alginate lyases,
β-1,3-glucanase and carrageenases, and other enzymes. These can have potential
applications in diverse fields such as food processing, pharmaceuticals, textiles, and
protein engineering. Enzymes from fish and shellfish from cold habitats are partic-
ularly useful since they can function effectively at lower temperatures, thereby
saving energy and protecting the food products [105, 106]. For example, pepsin
from the gastric mucosa of polar cod has high specific activity at low temperature,
and a fish trypsin exhibits high salt stability [107]. Cold-active proteases are useful
for controlled protein digestions and extraction of carotenoproteins, which are
known to be thermolabile. Transglutaminase is an enzyme that catalyzes cross-
linking of glutamine and lysine in proteins through acyl-transfer reaction involving
the γ-carboxamide group of peptide-bound glutamine residues and the ε-amino
group of lysine residues. The enzyme from cold-adapted organisms can modify
texture of protein foods at low temperatures [107, 108]. A number of seafood
enzymes can replace conventional seafood processing operations such as isolation
and modification of fish proteins and marine oils, production of bioactive peptides,
acceleration of traditional fermentation, peeling and deveining of shellfish, scaling of
finfish, removal of membranes from fish roe, extraction of flavors, shelf life exten-
sion, texture modification, removal of off-odors, and quality control either directly or
as components of biosensors; non-fishery applications include use of trypsin from
the stomach of tuna for milk clotting, cheese ripening, meat tenderization, and other
processes [109]. Table 2 indicates various enzymes from fishery sources and their
potential applications.
4.1.2 Bioactive Peptides

Bioactive peptides are protein fragments that have various physiological function-
alities in the human body following consumption. These peptides are derived
through enzymatic hydrolysis of proteins including food proteins including seafood
muscle and the stromal proteins, collagen and gelatin [97, 110]. Enzymatic hydro-
lysis of fish processing discards under controlled conditions gives rise to peptides,
which are separated by ultrafiltration or other suitable techniques [98]. Peptides from
marine by-products have high nutraceutical potentials to address important public
health issues like obesity, stress, hypertension, and others. These arise from their
interesting bioactivities, which are dependent on their amino acid composition
and sequences, and include their antimicrobial, antiviral, antitumor, antioxidative,
antihypertensive, cardioprotective, anti-amnesiac, immunomodulatory, analgesic,
1416 V. Venugopal
Table 2 Enzymes from fishery sources and their potential applications

Enzymes Fishery sources Characteristics and potential applications
Proteases including Carp, capelin, Milk clotting activity at alkaline pH. Degrade
chymotrypsin and carp, proteins including
Cathepsin D Herring, Atlantic Myofibrillar proteins. Preparation of FPH
Cod, rainbow
trout,
Scallop, dogfish,
squid, prawn,
sardine
Collagenases Carp, catfish, cod, Comparable to mammalian
Crab species, metalloproteinases
pacific
Rock fish,
bivalves
Chymosin (rennin) Carp, seal Optimal pH 2.0–3.5, possesses high milk
clotting activity
Lipases Atlantic cod, seal, Production of omega-3-enriched triglycerides,
Salmon, sardine, flavor enhancement
Indian mackerel,
Red sea bream
Transglutaminases Various fishery Comparable to mammalian
products metalloproteinases
Chitinases Shellfish, squid Optimal pH 2.0–3.5, possesses high milk
Liver, octopus clotting activity
saliva
β-1,3-glucanase, Abalone, scallop, Production of omega-3-enriched triglycerides,
β-galactosidase Tilapia, sea flavor enhancement
cucumber
Lysozyme Arctic scallop Can produce low molecular weight
shell, crab shell antibacterial chitosans in coordination with
cellulase and chitinase
Catalase, Marine mussel and Antioxidants
Glutathione other organisms
Peroxidase
Carbohydrases such as Finfish such as Deacetylation of chitin
β-galactosidase tilapia
Adapted from Ref. [109]
antidiabetic, antiaging, appetite-suppressing, and neuroprotective activities [7, 70,

98, 110–113]. A major bio-function of many peptides is in the control of CVD. High
blood pressure is one of the relevant independent risk factors for CVD. Angiotensin
I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure
and is a multifunctional enzyme, which promotes conversion of angiotensin I to the
potent vasoconstrictor angiotensin II. Inhibition of ACE is considered to be a useful
therapeutic approach in the treatment of hypertension. A number of seafood-derived
peptides can inhibit angiotensin I-converting enzyme (ACE) (EC3.4.15.1). ACE-
inhibitory peptides have been produced from salmon, oyster, squid, sea urchin,
shrimp, snow crab, seahorse, jelly fish, Alaska pollock, bigeye tuna muscle, sea
cucumber, sea bream, yellowfin sole, and others [112, 113]. A number of peptides
from fishery sources are capable of scavenging free radicals and reactive oxygen
species, thereby preventing oxidative damage [110, 112]. Fish and shellfish are also
good sources of antimicrobial peptides, which are described in the hemolymph of
spider crab, oyster, American lobster, shrimp, and green sea urchin [110]. A novel
anticancer peptide from the shellfish C. gigas exhibited cytotoxic activity, inducing
death of prostate, breast, and lung cancer cells [114]. Table 3 gives peptides from
various fishery sources and their bioactivities.
4.2 Lipid-Based Compounds
4.2.1 Fish Oils

Processing discards, particularly the liver of fish such as albacore, cod, salmon,
shark, haddock, and tuna, are good sources of oil and rich in omega-3 PUFA and
vitamins A and D. The oil of fish species, such as Atlantic mackerel, shark,
anchovies, menhaden, and Atlantic sardine, can have up to 35% omega-3 fatty
acids, with EPA and DHA at around 10% of the oil. The European fish species
such as capelin, herring, sand eel, and sprat are intermediate in oil contents, ranging
between 18% and 25%. The demersal fish such as cod and halibut have a lower oil
content of 15% to 20% [115–117]. Oil from whole fish or their discards are
traditionally recovered by the wet reduction process. It involves steaming of the
whole fish or processing discards and pressing of the cooked material to separate the
solid portion as press cake for use as fish meal. The oil and water released during the
pressing stage are pumped through screens and decanters to remove suspended
solids and then centrifuged to separate the oil, which is then filtered and stabilized
with antioxidants for storage [118]. Extraction of oil from various fish such as cod,
herring, salmon, and other fish has been discussed [4]. The traditional cooking
process for oil extraction has been replaced by isoelectric precipitation of oil-
bound proteins [119], enzymatic treatment [120], or supercritical extraction using
CO2 [121]. Principal fatty acids in some fish oils are shown in Table 4.
The liver of shark is 22 to 30% of body weight, and the oil content in the liver may
be as high as 90% of its weight. Recovery of oil from shark consists of natural
decomposition of the liver, acid ensilage in the presence of formic acid, alkali
digestion, and steam rendering (90 C for 30 min) [4]. Shark oils could be attractive
sources of n-3 fatty acids, specifically DHA, with content between 13% and 18%
[116]. The oil of Echinorhinus brucus (bramble shark) has DHA (18%), EPA (16%),
palmitic acid (15%), oleic acid (12%), stearic acid (8%), and squalene (38.5%),
besides fat-soluble vitamins such as vitamin A, 17 mg%; vitamin D, 15 mg%; and
vitamin K, 11.5 mg%. The oil showed high in vitro cytotoxic effect against the
human neuroblastoma cell line (SHSY-5Y) [123]. Oil from the liver of black shark
(Labeo chrysophekadion), Mako shark (Isurus oxyrinchus), and hammerhead shark
(Sphyrna spp.) is also rich in vitamins A and D [4]. Salmon oil is an excellent source
of EPA (18%) and DHA (12%) and is commercially available [7]. Oil of Antarctic
1418 V. Venugopal
Table 3 Bioactive peptides from some fishery sources and their hydrolyzates (FPHs)
Fish/Shellfish Sequence and other characteristics Functions/activities
Alaska pollock Gly-Pro-Leu ACE inhibitory
Wakame Tyr-Asn-Lys-Leu ACE inhibitory
Bigeye tuna Gly-Asp-Leu-Gly-Lys-Thr-Thr-Thr-Val-Ser- ACE inhibitory
frame Asn-Trp-Ser-
Pro-Pro-Lys-Try-Lys-Asp-Thr-Pro
Shrimp Le-Phe-Val-Pro-Ala-Phe ACE inhibitory
(protease)
Shrimp Peptidea (molecular size, Anticancer
<10, 10 to 30, >30 kDa)
Wakame Tyr-Asn-Lys-Leu –
(papain)
Oyster Cys, Leu, Glu, Asp, Phe, Tyr, Ile, Gly Antimicrobial
American Gln-Tyr-Gly-Asn-Leu-Leu-Ser-Leu-Leu-Asn- Antimicrobial
lobster Gly-Tyr-Arg
Jumbo squid Gelatin peptidea Antioxidant
Blue mussel Glu-Ala-Asp-Ile-Asp-Gly-Asp-Gly-Gln-Val- ACE inhibitory,
(fermentation) Asn-Tyr-Glu-Glu-Phe-Val- anticoagulant, antioxidant
Ala-Met-Met-Thr-Ser-Lys
Scallop γ-glutamyl-valyl-glycine Flavoring agent
Spider crab Proline-arginine-rich peptidea Antimicrobial
Snow crab Cationic peptidea Anticancer
(protamex)
Oyster Peptidea Anticancer, immune-
stimulant
Clam Peptidea ACE inhibitory
(thermolysin)
Clam Various peptidesa Hypocholesterolemic
(protamex) effect
Oyster Peptidea Antioxidant
(thermolysin)
Oyster Pro–Val-Met-Gly-Asp and Glu-His-Gly-Val Antioxidant
(subtilisin) peptides
Squid skin Peptidea ACE inhibitory
collagen
Krill Various peptidesa ACE inhibitory
Naturally present peptides in raw muscle
Crab, shrimp Crustin or crustin-like, callinectin, tachyplesin Antimicrobial
Crayfish Astacidina Antimicrobial
Lobster Crustin or crustin-likea Antimicrobial
Mussel Tyr-Pro-Pro-Ala-Lys Antioxidant
Mussel Mytilin, mytimycin, myticin, pernina Antimicrobial
Shrimp Penaeidin, crustin-like peptide Antimicrobial,
antioxidant
Scallop γ-glutamyl-valyl-glycine Flavoring agent
Summarized from various sources
a
Sequence not reported
Enzymes used for FPH given in parentheses
Table 4 Principal fatty acids in some fish oils

Fatty acid Capelin Atlantic mackerel Atlantic sardine Anchovies
Myristic (14.0) 7 8 8 9
Palmitic (16:0) 10 14 18 19
Palmitoleic (16:1) 10 7 10 9
Oleic (18:1) 14 13 13 13
Eicosanoid (20:1) 17 12 4 5
Cetoleic 14 15 3 2
EPA (20:5) 8 7 18 17
DHA (22:6) 6 8 9 9
Values are % of total lipids
Source: Adapted from Ref. [122]
krill (E. suberba) contains omega-3 fatty acids, phospholipids and also natural
pigments, and vitamins. However, since the oil content is only about 3% of the
body weight, a large amount of krill needs to be processed for oil recovery [124].
The hydrolyzate of squid processing by-products had phospholipids fraction (45.6%
of oil) which contained EPA and DHA at 16.9% and 29.2%, respectively. Peptides
isolated from the soluble fraction exhibited angiotensin I-converting enzyme (ACE)
inhibitory activity. The presence of both omega-3 fatty acids and ACE inhibitory
peptides suggested the nutraceutical potential of the squid hydrolyzate [125].
Appreciable amounts of PUFA particularly EPA and DHA in fish oils denote
significant therapeutic value of the oil. Moderate consumption of fish oil has shown
to decrease the risk of major cardiovascular events including coronary heart disease
(CHD) and atrial fibrillation [60, 126–128]. Shark oil has been an age-old remedy for
various diseases as well as a source of strength and virility [116]. The favorable
nutritional composition of bramble shark oil makes it a nutritional supplement [123].
While fish is the preferred source of omega-3 PUFAs, for those who do not consume
fish, omega-3 PUFA supplementation is a feasible alternative [126]. This also holds
good for consumers of Western-type diet, which is generally deficient in n-3 fatty
acids [12]. It has been suggested that the daily intake of combined EPA and DHA
should be at least 500 mg for individuals without underlying overt cardiovascular
disease and at least 800 to 1000 mg for individuals with known coronary heart
disease [128]. DHA is crucial for development of the brain and the central nervous
system in infants and also to suppress neuro-inflammation and oxidative stress [70].
Studies indicate that long-chain n-3 PUFA can also have anticancer, anti-depressive,
antiaging, and antiarthritic effects as well as neurodevelopment in children and can
address a number of chronic diseases, including a spectrum of liver fat-related
conditions [61, 62, 129–133]. Regular consumption of whole milk fortified with
omega-3 along with oleic acid, minerals, and vitamins has been reported to reduce
cell adhesion besides having anti-inflammatory effects in healthy children [131]. The
mode of action of the PUFAs, particularly EPA and DHA, is attributed to their ability
to give rise to a class of pharmacologically important groups of compounds,
collectively called eicosanoids, as discussed in Sect. 3.3. In view of their therapeutic
1420 V. Venugopal
benefits, many international and national health organizations recommend daily

intakes of EPA and DHA in the range of 250 to 500 mg for general health and
higher amounts for people diagnosed with CVD [126, 128]. Professional bodies
such as the American Heart Association, Department of Health UK, and Food Safety
Authority (FSA), among others, suggest daily total intake of EPA and DHA varying
from 250 to 1000 mg/day [4, 35]. Microencapsulation technologies (spray-drying,
spray-chilling, extrusion, co-crystallization, freeze-drying, and others) provide supe-
rior process to make odorless capsules containing the PUFA in gelatin or other
capsules [4]. In view of the sensitivity of PUFA to oxidation, it is advisable that those
who consume omega-3 may take adequate amounts of antioxidant nutrients, espe-
cially vitamin E, vitamin C, and selenium.
4.2.2 Squalene, Squalane, and Related Compounds

Squalene is a hydrocarbon (C30H50), having structures 2,6,10,15,19,23-Hexa-
methyltetracosa-2,6,10,14,18,22-hexaene. Livers of deep-sea shark species contain
high contents of squalene and also other hydrocarbons like pristine, presumably for
buoyancy, since they lack swim bladders. Liver oils of deep-sea sharks, mainly
Centrophorus spp., found under depths in the Pacific, North Atlantic, and Indian
Oceans contain about 85–90% unsaponifiable matter, which is essentially squalene
[117]. The liver oil of shovelnose dogfish contains 60% hydrocarbons [4]. The liver
oil of Echinorhinus brucus (bramble shark) has 39% squalene [123]. Squalene is a
skin rejuvenating agent and possesses antilipidemic and membrane-stabilizing prop-
erties. Both squalene and its hydrogenated product, squalane, are considered to
possess huge potential in nutraceutical, pharmaceutical, and cosmeceutical indus-
tries [134]. Pristane (C19 H40) (2,6,10,14-tetramethylpentadecane), a natural satu-
rated terpenoid alkane, and squalamine, an amino sterol antibiotic, are also found in
shark liver oil. Squalamine has remarkable anti-angiogenic, antitubercular, and
antiviral properties [4].
Other lipid-based bioactive compounds have been identified, particularly from
mussels. A lipid extract of hard-shelled mussel (Mytilus coruscus) possesses strong
anti-inflammatory activity and has the potential to treat rheumatoid arthritis [135].
The immune-strengthening properties of lipid extracts of the New Zealand green-
lipped mussel (Perna canaliculus) have made the shellfish an alternative to conven-
tional drugs in the treatment of rheumatic diseases like osteoarthritis, joint pain, and
also atopic asthma [136, 137] .
4.2.3 Carotenoids
Shells of shrimp, prawn, krill, crab, and lobster contain astaxanthin ranging from 40
to 200 mg kg1 dry weight, which is present as free or bound to proteins and/or
chitin [63, 64]. Other shellfish carotenoids include canthaxanthin present in crayfish,
mytiloxanthin in mussel, and mactraxanthin and fucoxanthin present in clams [2, 63,
64, 138]. The main pigment in the muscle tissues of the Yesso scallop, one of the
important farmed scallops in China, was identified as pectenolone (3,30 -dihydroxy-β,
β-caroten-4-one) [139]. The process of extraction of carotenoids from seafood
discards involves initial digestion with proteolytic enzymes such as pepsin, trypsin,
or alcalase, followed by drying of the shell discard and recovery of the carotene
pigments by organic solvents such as acetone, hexane, or isopropyl alcohol. If
vegetable oil such as soybean oil is used for extraction, the isolate is pigmented
oil. In recent years supercritical CO2 has been successfully used for extraction of the
pigments [64]. While the extracted carotenoids are used in aquaculture to impart
color to the farmed fish and shellfish, in recent years there has been awareness on
their bio-functions in human health. These include their antioxidant activities, their
role as provitamins, and their anticancer activities, among others [63, 64, 66]. There
is an immense scope to make use of these pigments for prevention of various
diseases related to oxidative stress and resulting tissue damage, as discussed earlier
(see Sect. 3.4).
4.3 Polysaccharide-Based Compounds
Polysaccharides are emerging ingredients against many chronic diseases. These

macromolecules are biodegradable and characterized by water- and fat-binding
capacities, gelation, viscosity, and other functional properties, which offer them
various biomedical and food applications. Functions of polysaccharides in food
and medicine encompass adhesive action, coating, emulsification, encapsulation,
film formation, foam stabilization, and as swelling and thickening agents. Poly-
saccharides are amenable to chemical modifications, providing derivatives having
enhanced and specific functions. While agricultural products are the traditional
sources, marine polysaccharides are gaining importance because of recognition of
their interesting therapeutic and other functions. These facilitate medicinal appli-
cations of marine polysaccharides and their derivatives in drug delivery, tissue
regeneration, wound healing, dental implants, blood plasma expanders, vaccines,
non-viral gene delivery, and many others [140, 141]. The major polysaccharide-
based compounds from seafood and their various applications are briefly discussed
below:
4.3.1 Chitin, Chitosan, and their Derivatives

Chitin is the second most abundant natural biopolymer derived from exoskeletons of
crustaceans and also from cell walls of fungi and insects [142]. In lobster, crab, krill,
shrimp, and prawn, chitin forms the outer protection coatings of the animal through a
covalently bound network with proteins. The dry shell discards of crab, shrimp, and
lobster may contain chitin up to 70% on dry weight basis, while its contents in dry
squid skeleton pen and deproteinized shell of krill may be at a lower value of about
40%. Although not a nutraceutical, chitin offers various applications essentially
through its deacetylated form, chitosan, as well as its numerous derivatives in
medicine, food, and many other areas. The literature is voluminous on the topic
and only a very brief discussion will be attempted here.
The process of isolation of chitin from crustacean discards consists essentially of
three steps: demineralization of dried and pulverized shell discards usually by dilute
hydrochloric acid, deproteinization by dilute alkali, and finally decoloration by sun
1422 V. Venugopal
a CH3
H H H H H C OH
CH2OH
O CH2OH O
HO NH2 HO NH
O O O
O O
HO HO O
HN
CH2OH O CH2OH
HN
H H H H H H
C O C O
CH3 CH3
b
H H H H H
CH2OH O CH2OH O HO
HO NH2 NH2
O O
O HO O
HO CH2OH O
NH2 CH2OH O HN
H H H H H H
C O
CH3
Fig. 1 Structure of chitin (a) and chitosan (partially deacetylated) (b)
drying, followed by washing and drying. Demineralization needs to be done under

controlled conditions to prevent deacetylation of chitin. Proteins which exist as
caroteno-protein complexes may be separated by trypsin treatment in the presence
of ethylenediaminetetraacetic acid (EDTA). Decolorization can be performed by
solvent extraction employing acetone or ethanol [142, 143]. An integrated process
for isolating bioactive molecules including protein, chitin, carotenoids, and glycos-
aminoglycans from Pacific white shrimp (Litopenaeus vannamei) processing waste
has been reported. Autolysis of 1 kg of the shrimp head followed by lyophilization of
the autolyzate gave about 120 g of protein hydrolyzate. The recoveries of chitin and
carotenoids were 25 2 mg g1 and 195 g1 wet processing waste [144].
Chitin is a cationic polymer formed by units of N-acetyl-D-glucosamine, joined
by ß-linkages. The structure of chitin is ß-(1–4)-N-acetyl-D-glucosamine, which is
ß-(1–4)-N-acetyl-2-amino-2deoxy-D-glucose (Fig. 1a). It may be also regarded as
a derivative of cellulose, in which, the C-2 hydroxyl group is substituted by an
acetyl amino group. Chitin occurs in three polymorphic forms, the most common
α-chitin and also β- and γ-chitins, which differ in their arrangement of the molec-
ular chains. Chitin structure has an extensive intermolecular hydrogen bonding
associated with exclusion of water, leading to its stability. Chitin is a very light,
white or yellowish, powdery/flaky product. It is insoluble in water as well as
almost all common organic solvents and acidic and basic aqueous solutions. Chitin
swells in cold alkali when some deacetylation takes place. Chain lengths of chitin
and degree of acetylation differ according to sources and recovery conditions.
Chemical derivatives of chitin include carboxymethyl chitin, hydroxyethyl chitin,
ethyl chitin, chitin sulfate, glycol chitin, and glucosylated chitin, which have
varied chemical and functional properties [142].
Chitin upon deacetylation yields chitosan, which is a collective name

representing chitin deacetylated to various degrees. Generally chitosan is produced
by deacetylation of chitin using 30–60% (w/v) sodium or potassium hydroxide at
80–140 C followed by drying to get chitosan flakes. Chitosan with a variable degree
of deacetylation ranging from 60 to 80% was recovered at 17 4 mg g1 head
discard (wet weight) of white shrimp (L. vannamei) [144]. Chitosan has also been
extracted by various enzymes such as lysozyme, neutral protease, and chitin
deacetylase. The average molecular weight of chitosan obtained using the enzymatic
method is 268 kDa [145]. Higher extraction temperatures enhance deacetylation and
also result in fragmentation of the chitosan. Chitosan polymers are thought to contain
more than 5000 repeating acetylglucosamine and glucosamine units. The molecular
weights of chitosan range from 300 to over 1000 kDa and its degree of deacetylation
from 30% to 95%. Both molecular weight and degree of deacetylation have influ-
ence on the functionality of chitosan. A minimum deacetylation of 70% is required
for chitosan to be acceptable for various purposes.
The structure of chitosan is β-(1–4)-linked-D-glucosamine, i.e., poly [β-(1–4)-
linked-2-amino-2-deoxy-D-glucose] (Fig. 1b). Chitosan is a cationic polyelectrolyte
white solid, insoluble in pure water, but unlike chitin, it is soluble in weakly acidic
aqueous media. Chitosan derivatives in the form of acetate, ascorbate, lactate, and
malate are water-soluble. Chitosan is soluble in aqueous acid and crystallizable from
aqueous alkaline solution. The value of pKa for the positively charged ammonium
group of chitosan is about 6.2. When the pH is raised to about 6.5, chitosan
precipitates in a gel form. The net cationic charge as well as presence of multiple
reactive functional groups in the molecule makes chitosan a valuable compound for
practical uses.
Chitosan can be easily converted to various derivatives for diverse applica-
tions, as shown in Fig. 2. These derivatives, depending on their chemical nature,
have interesting characteristics such as biocompatibility, biodegradability to
harmless products, nontoxicity, physiological inertness, and antimicrobial and
gel-forming properties, among others. Chitosan derivatives, depending on their
chemical nature, can have bioactivities such as antioxidant, anti-inflammatory,
anti-allergic, antitumor, anti-obesity, antidiabetic, anticoagulant, antiviral, immu-
nomodulatory, cardioprotective, and others. These properties make chitosan and
its derivatives valuable compounds in several fields including food, nutrition,
medicine, agriculture, biotechnology, and material science [146–150]. Due to its
cationic nature, chitosan binds dietary fat in the stomach, leading to decreased
intestinal absorption of fat and cholesterol, thereby reducing low-density lipo-
proteins in the blood and liver. For dietary purpose, chitosan needs to be intro-
duced through food and should be soluble at acidic pH. Chitosan can be a food
preservative due to its antimicrobial activity against a wide variety of pathogenic
and spoilage-causing microorganisms, including fungi and gram-positive and
gram-negative bacteria. Chitosan films can be used as a packaging material for
the preservation of foods [145]. Potential medicinal applications of chitosan and
its derivatives include drug delivery, hemodialysis membranes, artificial skin,
hemostatic agents, hemoperfusion columns, wound healing and blood
1424 V. Venugopal
OH
O I
OH
O O OH O O
O OH O O OH O
NH
Organic
OH H N H C O Synthetic NH
O HO Cu OH R Intermediate C O
O OH O OH CH3 OCH2.COOH
Removal of O
N-Acetylation
Toxic metals O
O OH
Emulsifer NH-R
Deoxyhalogenation
Cosmetics R=CH2 Motal chelation
C CH3 Moisture retainer
NH
(Skin care
OH Hydroxypropylation products) C O
OH OH O–/N–Carboxy
O alkylation CH2COOH
O
O
O OH Photoactivation O
N3 O OH OH
Deamination O
CI
Protein NH F
NH2 O OH
immobilizer N Chitosan CHO
Phosphorylation or
Nitration O-Acylation Depolymerization (low
OH MW chitosan)
Cyanoethylation O
O
Sulfation OC R
O OH O Schiff’s base O
O OH O
O
OH NH
OCH2.CH2CN O S OH
R P OH R O
OO NH2 or NH
O OH O Emulsifier C O
Adsorbant of uraniumO OH O OH O
from sea water O R
or O
NH2 O OH
R=NO2 Micro- NH2
Explosive comp. filtration Anticoagulant
membrane Immobilization NH
systems
CH
R
Fig. 2 Various chemical derivatives of chitosan and their uses (Source: Ref. 142 with permission
from Taylor & Francis)
coagulation, and also gene therapy. The property of chitosan to form gel at
slightly acid pH provides antacid and anti-ulcer activities [4, 140].
Chitosan and also other polysaccharides such as hyaluronic acid among others
have received attention in the fields of regenerative medicine and tissue engineering,
as ideal materials for artificial extracellular matrices [151]. Various physical forms of
chitosan include nanoparticles, nanofibrils, microspheres, composite gels, fibers,
films, and bandages. Chitosan-carrageenan composite nanoparticles have shown
promise as carriers of therapeutic macromolecules. Nanocomposite films of chitosan
and other marine hydrocolloids such as agar, alginate, and carrageenan can be a good
carrier for nutraceuticals including omega-3 PUFA in the future [4]. Chitosan-based
hydrogels and wound healing bandages have found commercial acceptance.
Chitosan-based bone graft substitutes are biocompatible and biodegradable, with
structural similarity to the bone, having excellent mechanical strength. Derivatives

of chitin and chitosan have applications as cosmetic ingredients. These include
O-carboxymethyl chitin and O-hydroxypropyl chitin, carboxymethyl chitosan,
chitosan acetate, adipate, glycolate, hydrolyzed chitosan, pyrrolidone carboxylic
acid (PCA), hydroxyl methyl, hydroxyl propyl, formate, ascorbate, and salicylate
of chitosan, among others [4, 103, 152]. Methods of manufacture of chitosan
derivatives and biomedical applications have been recently discussed [153]. A
number of these products are presently commercially available [91, 151,
153–155]. Salient biomedical applications of chitosan are pointed out in Table 5.
The biodegradation of chitosan by chitinases leads to formation of nontoxic
oligosaccharides of variable length [149, 156]. While these oligosaccharides are
not digested by gastrointestinal enzymes, they modify the viscosity and freezing
point of foods, affect emulsification and gel formation, possess bacteriostatic prop-
erties, and act as humectants. Chitosan oligosaccharides are used as antioxidant,
antitumor agent, and antimicrobial agents. They stimulate growth of lactobacilli
and other probiotic organisms. Low molecular weight chitosans (25–50 kDa)
suppress H. pylori and also have been recognized to protect normal cells from
apoptosis [156].
4.3.2 Glucosamine
Glucosamine is the end product of hydrolysis of chitosan. It is an amino sugar that is
naturally produced in humans and a key building block in the synthesis of glycos-
aminoglycans. Glucosamine is a recognized nutraceutical for joint pain relief. It
promotes synthesis of cartilage proteoglycans and synovial production of hyaluronic
Table 5 Salient applications of chitosan in biomedical applications

Applications Salient property
Wound healing, burn therapy Forms tough, water-absorbent, biocompatible films that
promote tissue growth
Hemodialysis membranes Chitosan-cellulose blended membranes have improved
dialysis properties in artificial kidney due to improved
permeability
Drug delivery Inexpensive carrier encapsulating nutraceuticals and
drugs. It also helps transdermal delivery of drugs
Removal of toxins Chitosan-encapsulated activated charcoal has potential to
remove toxins
Hemoperfusion Chitosan and its oligomers can satisfy the requirements of
specificity and blood compatibility
Artificial scaffolds Chitosan along with chondroitin sulfate supports
proteoglycan production in cartilages
Anticholesterol drug Reduces lipid absorption and hence reduces cholesterol
Dental bioadhesive biodegradable Collagen-chitosan membrane has potential for treatment
sutures of periodontal defects in dentistry
Composite films and nanoparticles Various processes in tissue engineering for drug delivery,
with other polysaccharides skin recovery, scaffolds, and others
Adapted from various references
1426 V. Venugopal
acid (HA), which has anti-inflammatory and analgesic properties. Glucosamine

along with chondroitin sulfate (see Sect. 4.3.3) is a highly effective treatment for
arthritis and osteoporosis [148, 149].
4.3.3 Glycosaminoglycans
Glycosaminoglycans (GAGs) are heteropolysaccharides defined by a repeating
disaccharide unit without branched chains, in which one of the two monosaccharides
is an amino sugar (N-acetylgalactosamine or N-acetylglucosamine) and the other
one is a uronic acid. GAGs are present on all animal cell surfaces and in the
extracellular matrix where they exist as proteoglycans by covalently linking to
proteins (such as growth factors, enzymes, and cytokines). Based on the disaccharide
composition, linkage type, and presence of sulfate groups, GAGs may be chondroi-
tin sulfate (CS), hyaluronic acid (HA), dermatan sulfate (DS), heparin, or keratan
sulfate (KS). GAGs are significantly present in the cartilage, which serves as a
cushion between bones and joints. The cushion structures are formed by a matrix
of collagen and elastin associated with proteoglycans consisting of CS, KS, DS, and
heparan sulfate. Hyaluronic acid (HA) is the only non-sulfated GAGs, which is not
covalently bound to the protein in any tissue, although specific HA-protein interac-
tion is shown. CS and HA are the most commercially valued GAGs because of their
physiological functions and high activity. CS plays an important role in the elasticity
and function of the articular cartilage and is mainly attached covalently to core
proteins in the form of proteoglycans. CS is composed of an alternating sequence of
sulfated and/or unsulfated D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine
(GalNAc) residues linked through alternating β (1–3) and β (1–4) glycosidic bonds
and sulfated in different carbon positions, as shown in Fig. 3. Some GlcA residues
are epimerized into iduronic acid, the resulting disaccharide being referred as
dermatan sulfate. The classification and type of CS are dependent on sulfate group
placing, namely, sulfate at carbon 4 (CS-A), at carbon 6 (CS-C, more common), both
at carbon 4 and 6 (CS-E), at carbon positions 6 of GalNAc and 2 of GlcA (CS-D),
and at carbon 4 of GalNAc/carbon 2 of GlcA (CS-B). Sulfation in different positions
confers specific biological activities to chondroitin [157, 158].
CS from terrestrial and marine sources contains diverse chain lengths and
sulfation. Examples are shark, CS-D; dogfish, CS-A and CS-D; squid and salmon,
CS-E and CS-E; and ray, CS-A and CS-C [157]. The endoskeletons of shark are
composed of cartilage, which contains up to 29% CS. Processes for isolation of CS
from shark cartilage and also from the cartilages of ray, swordfish, dogfish, fins of
shark and skate, and salmon nasal cartilage have been reported [158–160]. CS was
extracted from fish head cartilage employing hydrolysis by alcalase (55.7 C,
pH 8.2) and chemical treatment of the hydrolyzates by alkaline-alcoholic saline
solutions (0. 54 M NaOH, 1.17 V EtOH, and 2.5% NaCl) for selective dissolution
followed by ultrafiltration [158, 159]. Optimization of skate (Raja avirostris) carti-
lage hydrolysis for the preparation of chondroitin sulfate has been reported [159].
A low-cost two-step process recovery of CS in non-denaturing conditions was
reported. It consists of an enzymatic extraction followed by tangential membrane
filtration [160]. Figure 4 provides an overview of chondroitin sulfate recovery and
Fig. 3 Chondroitin sulfate

derivatives
purification processes from marine cartilage by-products. CS is quite stable to

heating at temperatures as high as 121 C for several min. Shark cartilage powder
is traditionally used for wound healing and as anti-angiogenic, antiarthritic, and
antitumor agent. CS is considered a nutritional supplement that provides support for
strong, healthy cartilage and joints [161].
Hyaluronic acid (HA) is a linear, high molecular weight unbranched and non-
sulfated GAG made by alternating disaccharide units of N-acetyl-D-glucosamine
and D-glucuronic linked by β-(1 ! 3) and β-(1 ! 4) glycosidic bonds. It is used in
ophthalmological surgeries and cosmetic regeneration of soft tissues. Marine dis-
cards have been also explored for HA [158].
The presence of heparin has been reported from different shrimp species. Sulfated
glycosaminoglycans that exhibited electrophoretic migration pattern similar to mam-
malian heparin were recovered (79 2 μg g1 wet processing waste) from Pacific
white shrimp (L. vannamei) shell discards. Their degradation products suggested the
presence of C6-sulfated heparin sulfate [144].
Sea cucumbers are found in shallow water areas of the sea to deep ocean floors,
where they live on decaying organic matter. The body wall of the sea cucumber
contains high amounts of sulfated glycans, which differ in structure from glycos-
aminoglycans of animal tissues. The cucumber cell wall polysaccharide was com-
parable in backbone structure with the mammalian chondroitin sulfate, but some of
the glucuronic acid residues displayed sulfated fucose branches. These sulfated
glycans exhibit a wide range of biological activities, which include anticoagulant
activity, venous antithrombic activity, and recombinant HIV reverse transcriptase
activity [4].
1428 V. Venugopal
CHONDRICTHYES SKELETONS
Warming
Mechanical cleaning
Milling
Solid wastes (flesh rests) CARTILAGE
Enzyme proteolysis (alcalase, papain...)
Centrifugation
Fish meal SED SUP

Alkaline-EtOH proteolysis
SED SUP EtOH-Rectification
Redissolution
Neutralization
CS solution
Permeation, diafiltration, concentration UF/DF (10–30 kDa)
PER RET
Drying
Milling
CS powder (99.5% purity)
Fig. 4 Overview of chondroitin sulfate recovery and purification processes from marine cartilage
by-products. SED, Sediment; SUP, supernatant; PER, permeate; and RET, retentate (Courtesy:
Vázquez JA, Ref. 158)
4.3.4 Other Seafood Polysaccharides

Bioactivities and nutraceutical potentials of other polysaccharides from seafood
have also been reported in recent times. Mollusks have a wide range of uses in
pharmacology; several compounds from mollusks having pharmacological value
have been described in the literature [4]. Mytilan, a mussel polysaccharide, pos-
sesses antibacterial, antioxidant, and immune-modulating activities. Another mus-
sel polysaccharide, a (1–4)-D-glucan, is known to exhibit antioxidant activity and
a protective effect on acute liver injury in mice [137]. The high radical scavenging
capacity and total phenolics suggest the nutraceutical potential of the mussel
P. viridis [83]. Abalone is a source of bioactive compounds having antithrombotic,
anticoagulant, anti-inflammatory, antioxidant, and anticancer activities. Polysac-
charides and also proteins and fatty acids of abalone provide health benefits
beyond basic nutrition [162]. The glucan extract from China white jade snail
(Achatina fulica) has significant antioxidant activities, suggesting its potentials as

dietary antioxidant [163].
4.4 Mineral-Based Compounds
4.4.1 Bone Powders

Fish bone is a potential source of calcium for use as a fortificant of the mineral. To
prepare the product, fish is treated with hot water followed by hot aqueous acetic
acid, which softens the bone and converts into an edible form. Superheated steam is
preferred to reduce the loss of soluble components from fish tissue, which enables
better recovery of the bone within a shorter period. The treated bones are subjected to
saponification, degreasing, and degumming. The bone preparation is a good source
of bioavailable calcium [164]. Tuna powder is a value-added product that contains a
proper balance of calcium and phosphorus; it can be used as a food supplement and
for treating osteoporosis [165]. The powder can also combat calcium deficiency in
children [166]. The bone preparations of channel catfish have antibacterial activities,
with pepsin hydrolyzate of the bone showing the greatest antibacterial activity [167].
A fish bone phosphopeptide (FBP) containing up to 24% of phosphorus has a
molecular weight of 3.5 kDa and has high calcium-binding activity, which could
be utilized as a nutraceutical [168].
4.5 Marine Biotechnology for the Isolation of Nutraceuticals
Marine biotechnology explores aquatic organisms for the recovery of nutraceuticals,

pharmaceuticals, cosmeceuticals, and other products through a multitude of pro-
cesses. Whereas the conventional processes include liquid–liquid or solid–liquid
extraction and enzyme-assisted extraction, advanced biotechnological methods are
pressurized liquid extraction, subcritical and supercritical extractions, and micro-
wave- and ultrasound-assisted extractions, among others [93, 169–174]. Membrane
separation is beneficial to extract, concentrate, separate, or fractionate bioactive
compounds. Membrane bioreactors integrate reaction vessels with membrane sepa-
ration units for producing materials such as peptides, chito-oligosaccharides, and
PUFA from seafood discards [175]. Supercritical carbon dioxide (SC-CO2) extrac-
tion at optimal conditions of 40 MPa and 65 C was employed to concentrate PUFA
from fish oil [176]. Vázquez et al. [158] suggested environment-friendly and sus-
tainable processes combining various microbial, chemical, enzymatic, and also
membrane technologies for the recovery of chitin/chitosan, chondroitin sulfate,
and hyaluronic acid from marine discards. In this, the conventional process for
chitin/chitosan was supplemented with protease treatment and/or lactic acid fermen-
tation for protein removal and also use of fungal deacetylase for conversion of chitin
to chitosan. The process appears to have scope for large-scale processing of seafood
discards in eco-friendly conditions. The process is depicted in Fig. 5. The authors
also reported a pilot plant scale processing of Penaeus vannamei by-products using a
1430 V. Venugopal
Fig. 5 Eco-friendly process for the preparations of chitin and chitosan from crustacean shell
discard (Courtesy: Vázquez JA, Ref. 177)
combination of enzymatic and chemical technologies, which gave chitin with 30%
yield. Deacetylation under optimal conditions yielded chitosan having a molecular
weight of 82 kDa and 92% deacetylation [177].
4.6 Commercial Status
Interests in marine bioactive products are shown by the fact that in the past 8 years,
annually, more than 1000 new compounds from marine organisms have been
described [178]. A number of by-products from seafood have already entered
commercial markets. Fish trypsin, chymotrypsin, and cold-active chlamysin (lyso-
zyme) are available commercially. Some of the other commercial products include
Neptune Aquatein, krill extract (http://ingredientsnetwork.com/neptune-technolo
gies-bioressources-comp249137.html), heat-labile shrimp alkaline phosphatase, ura-
cil-DNA glycosylase from Atlantic cod (http://arcticzymes.com), seafood protein
extracts (http://novozymes.com), and protein blends from CIFT, Kochi, India (www.
cift.res.in). Animal proteins can be used as a source of bioactive hydrolyzates and
peptides with potential for use as functional food ingredients industry [179]. Fish
gelatin is available commercially. Collagen or gelatin peptides with a molecular
weight around 1–5 kDa containing tripeptides, dipeptides, and also free amino acids
are marketed as nutraceuticals for the maintenance of normal bone and tendon
integrity, improving joint health. Calcitonin is a hormone (32-amino acid peptide
containing a single disulfide bond), known to participate in calcium and phosphorus

metabolism in mammals. The major source of calcitonin is from the parafollicular or
C cells in the thyroid gland. Calcitonin produced from salmon is currently commer-
cially available. Ziconotide is a 25-amino acid peptide derived from the ω-conotoxin
of cone snail (Conus magus) found in tropical waters. Ziconotide has been approved
by the US FDA for analgesic use. Other products from fish which are under clinical
trials include fish gelatin and Gabolysat and Seacure ® (a hydrolyzate of fish proteins)
[103, 114].
Annually around 1 million tons of fish oil is produced, with a current global
market valued at US$3.9 billion [180]. At present the main use of fish oil is for
aquaculture. Current consumption level of fish oil for human nutrition is not
adequate [181]. In order to increase the consumption, foods fortified with omega-3
are made available in the markets all over the world. Developments in fortification
technology have encouraged more than 200 omega-3 PUFA-fortified foods, such as
bread, dairy products, eggs, pasta, biscuits, margarines, and other spreads available
commercially in Japan and other countries [182, 183]. Capsules such as Lovaza®
which contains ethyl esters of EPA and DHA are available commercially (https://
www.drugs.com/pro/lovaza.html). There is, however, concern that because of sea-
food production reaching a plateau in recent years, the commodity may not be able to
fully satisfy future needs. Therefore, other sources such as algae may be required to
produce EPA and DHA to meet the increasing demand [181].
Chitosan and its derivatives are finding several end users which, apart from
healthcare, medical, food, and beverages, include water treatment, agriculture,
biotechnology, and others, encompassing about 30 companies (www.strategyr.
com/Chitin_and_Chitosan_Market_Report.asp) with a projection of a market
worth of US $4.2 billion by 2020. Anti-inflammatory and antiarthritic dietary
supplements such as “Seatone” and “Lyprinol” from mussel are commercially
available [137]. Tetrodotoxin (TTX) and saxitoxin are neurotoxins found in many
marine organisms including mussels and fish. TTX is currently under clinical trials
due to its specific and reversible activity on voltage-dependent sodium channels
[181]. CS, hyaluronic acid, and also chitosan have attracted increasing commercial
attention in the formulation of cosmeceuticals, nutraceuticals, and food ingredients
[103, 158]. Some fish-based cosmeceutical products include Penzim, a natural skin
care product from Iceland, derived from natural marine enzymes from the Arctic
Ocean from North Atlantic cod (http://www.andra.is/), extracts from male sturgeon
gonads, zonase enzyme and gelatin from fish eggs for treatment of psoriasis and
eczema, cod sperm as water binder in body lotions, shark cartilage containing
chondroitin sulfate used in various creams, squalene from shark liver oil as mois-
turizer in body lotions, and guanine from fish such as herring and ribbon fish, used in
nail polish [103]. A product depicted as “skin food from the sea” contains blend of
marine-derived nutrients and is suggested for antiaging (www.nutreeplus.com/anti
aging.html). A number of companies like Aquapreneur (www.aquapreneur.com),
Sederma (http://www.sederma.fr), NutraIngredients (https://www.nutraingredients.
com), SpecialChem (http://www.specialchem4cosmetics.com), Fortitech (fortitech-
premixes.com), Copalis (http://www.copalis.fr/), and others have been active in
1432 V. Venugopal
developing marine ingredients such as collagen, elastin, various peptides, GAGs,

alkyl glycerols, fish oil, hydrolyzed proteins, calcium supplements, glucosamine,
hyaluronic acid, and chondroitin sulfate for food, pharmaceutical, and other
purposes.
5 Conclusions
Seafood items are rich in nutrients that can have positive influence on human health.
Nutrient contents depend on the species, and thus consumption of individual species
significantly determines the nutritional benefits derived by individual consumers.
The health benefits derived from seafood items also depend on the frequencies of
their consumption, as well as quantities consumed. Seafood items and also their
processing discards are good sources of many nutraceuticals. In recent times, marine
biotechnology has made rapid advancements, which could be used to recover
nutraceuticals and other bioactive compounds from seafood discards. There is robust
evidence that fish oil and its component fatty acids, especially EPA plus DHA, are
beneficial in maintaining cardiovascular health in normal adults. Fish protein hydro-
lyzates contain peptides with significant antihypertensive and other bioactivities.
Fish cartilage products such as shark cartilage and chondroitin sulfate, glucosamine,
and other glycosaminoglycans are able to alleviate rheumatoid arthritis. Fish skin
collagen and gelatin have a potential to replace bovine collagen and gelatin in food,
pharmaceuticals, and cosmetics. Chitosan and its derivative offer immense applica-
tions in human health and cosmeceutical fields. There is large potential for devel-
opment of functional foods fortified with nutraceuticals such as fish oils, bioactive
peptides, and others. Gelatin, chitosan, and its derivatives can be carriers of
nutraceuticals and drugs and food packaging materials. Uses of marine
nutraceuticals are increasing in countries such as Japan and others. As Borresen
[184] pointed out, there is a need for nutritionists and other scientists to evaluate the
nutritional value of fishery items that can lead to total utilization of the commodity
for human welfare.
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Roots and Tubers as Functional Foods
48
Anoma Chandrasekara
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443
2 Starchy Roots and Tuber Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.1 Potato (Solanum tuberosum) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.2 Sweet Potato (Ipomea batatas L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446
2.3 Cassava (Manihot esculenta) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1446
2.4 Yams (Dioscorea sp.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1447
2.5 Aroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
3 Nutrients in Roots and Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
3.1 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
3.2 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1451
3.3 Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4 Nonnutrient Compounds in Roots and Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.1 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.2 Saponins and Sapogenins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1453
4.3 Steroidal Glycoalkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1453
5 Bioactivities of Phytochemicals in Roots and Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1454
5.1 Hormonal Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1454
5.2 Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1454
5.3 Antiulcerative Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1457
5.4 Anticancer Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1457
5.5 Hypoglycemic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1458
5.6 Hypocholesterolemic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1459
5.7 Antiobesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
5.8 Immunomodulatory Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1461
5.9 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463
6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1464
A. Chandrasekara (*)
Department of Applied Nutrition, Faculty of Livestock Fisheries and Nutrition, Wayamba
University of Sri Lanka, Gonawila, Sri Lanka

1442 A. Chandrasekara
Abstract
Starchy roots and tuber crops are important components in the human diet.
There are number of roots and tubers belonging to several species and make
an extensive biodiversity even within the same geographical location. From
the ancient time of human evolution starchy roots and tubers have been a
part of food choices and add variety to the modern diet in addition to
offering numerous desirable nutritional and health benefits such as anti-
obesity, antioxidative, hypoglycemic, hypocholesterolemic, antimicrobial,
and immunomodulatory activities, among others. There are a number of
bioactive constituents, namely, phenolic compounds, saponins, bioactive
proteins, glycoalkaloids, phytic acids, and hydroxycoumarins, reported in
tuber crops. Except the common potatoes, sweet potatoes, and cassava, other
starchy tuber crops are yet to be explored for their nutritional and health
benefits to use as functional foods. Some edible tubers are served for
traditional and alternative medicinal sources. Tubers and roots are potential
functional foods and nutraceutical ingredients to manage a number of ail-
ments and to ensure general wellness.
Keywords
Antioxidative · Hypoglycemic · Hypocholesterolemic · Phenolic compounds ·
Saponins
AMPK Adenosine monophosphate-activated protein kinase
ACC Acetyl coenzyme A carboxylase
DPPH 2,2, diphenyl-1-picrylhydrazyl
ERK Extracellular signal-regulated protein kinase
FAO Food and Agriculture Organization
GAE Gallic acid equivalents
GGT Glutamyltransferase
LPS Lipopolysaccharide
MTT Microculture tetrazolium treatment assay
NCDs Noncommunicable diseases
NASA National Aeronautics and Space Administration
NO Nitric oxide
IFN-γ Interferon- γ
ORAC Oxygen radical absorbance capacity
OGTT Oral glucose tolerance test
SHBG Sex hormone binding globulin
TPC Total phenolic content
t-BHP Tert-butylhydroperoxide
WSSP White skinned sweet potatoes
48 Roots and Tubers as Functional Foods 1443
1 Introduction
Roots and tuber crops are important cultivated staple carbohydrate sources,
second to cereals, generally in tropical regions in the world. During fast few
decades the roles of roots and tubers in the economy and food systems of
developing countries have been changed. They are subsistence crops of pivotal
importance in consumers and producers in low-income countries. In addition,
they provide a substantial input for animal feeds and industrial products such as
distilled spirits, starches, fermented foods, and other minor products. Economi-
cally important roots and tubers include potatoes, cassava, sweet potatoes, yams,
and aroids.
An important agronomic advantage of root and tuber crops as staple foods is
their favorable adaptation to diverse soil and environmental conditions and a
variety of farming systems with minimum agricultural inputs. Further, the
variation in the growth pattern and production requirements of root and tubers
make their way into distinct production systems and varied consumption uses.
However, roots and tuber crops are bulky in nature with high moisture content
of 60–90% leading them to be associated with high transportation cost, short
shelf life, and limited market margin in developing countries where they are
produced.
The contribution of starchy roots and tubers to the energy supply in different
populations varies with the region and the country. Their annual global production
is approximately 845 million tons [1]. Asia is the main producer of starchy roots
and tubers followed by Africa, Europe, and America. Asian and African regions
produced 43% and 33%, respectively, in the global production of roots and tubers
[1]. Cassava, potatoes, and sweet potatoes consist of 90% global production of root
and tuber crops though a number of species and varieties are consumed [1]. Roots
and tubers provide dietary energy in the form of carbohydrates, dietary fiber,
vitamins, and minerals (Table 1; [2]). It is interesting to note that high yields of
roots and tubers give more energy per land unit per day than those of cereal grains
[3]. In general, roots and tubers provide low protein contents ranging from 1% to
2% on a dry weight basis [3]. However, potatoes and yams contain high amounts of
proteins among other tubers. Sulfur-containing amino acids, namely, methionine
and cystine, are the limiting ones in root crop proteins. Cassava, sweet potatoes,
potatoes, and yam contain some vitamin C, and yellow varieties of sweet potatoes,
yam, and cassava contain β-carotene. Roots and tubers are deficient in most other
vitamins and minerals but contain significant amounts of dietary fiber [3]. Nutri-
tional value of roots and tubers vary with variety, location, soil type, and agricul-
tural practices, among others.
Noncommunicable diseases (NCDs) are in rise in developed as well as devel-
oping countries on concurrence with increasing aging population worldwide.
Dietary and lifestyle factors play a prominent role in oxidative stress which
contributes immensely to the etiology of NCDs as well as the aging process.
Several epidemiological studies showed the association between plant food
intake and reduced NCDs [4–7]. Furthermore, identification of specific plant
Table 1 Nutrient composition of major starchy roots

Cassava Potato Sweet potato Yams
Crude analysis
Dry matter (DM) (g/100 g fresh weight (FW) 31.3 22.2 30.8 31.1
Protein (g/100 of DM) 2.7 9.2 5.3 6.4
Protein (g/100 of FW) 0.8 2.0 1.6 2.0
Fat (g/100 of DM) 0.62 0.5 1.95 0.42
Available carbohydrates (g/100 of DM) 86.9 66.7 78.2 72.8
Fiber (g/100 of DM) 7.9 9.3 10.2 17.9
Energy (kcal/100 g of DM) 364 316 351 318
*Amino acid composition (mg/g crude protein)
Ile 28 39 37 37
Leucine 40 59 54 65
Lysine 41 60 34 41
Methionine þ Cystine 27 30 28 28
Phenyl alanine þ tyrosine 41 78 62 80
Threonine 26 39 38 36
Tryptophan 12 14 14 13
Valine 33 45 45 47
Minerals (mg/100 kcal)
Calcium 23.8 8.8 32.3 25.3
Phosphorus 28 71 42 44
Magnesium 48 28 23
Iron 0.88 0.57 0.78 0.91
Zinc 0.41 0.49 0.83 0.11
Vitamins
Vitamin A (μg eq ret./100 kcal) 3.7 1.2 1321.1 1.7
Vitamin B1(μg /100 kcal) 45 157 59 91
Vitamin B2 (μg /100 kcal) 22 67 46 30
Nicotinamide (μg /100 kcal) 446 1737 554 607
Vitamin C (μg /100 kcal) 22.3 24.2 27.7 10.1
Sources: Souci et al. [118]; * FAO [119]
constituents which convey health benefits is of much interest. Foods of plant

origin consist of a wide range of nonnutrient phytochemicals which are synthe-
sized as secondary metabolites and serve a wide range of ecological roles in
plants [8].
Tubers and root crops contain several bioactive compounds such as saponins,
phenolic compounds, glycoalkaloids, phytic acids, carotenoids, and ascorbic acid
among others. A number of bioactivities, namely antioxidant, immunomodulatory,
antimicrobial, antidiabetic, antiobesity, and hypocholesterolemic activities are
reported for tubers and root crops. This chapter discusses the distribution and
bioactivities of several selected nutrient and nonnutrient phytochemicals in starchy
roots and tuber crops.
Table 2 Common tuber crops worldwide

Botanical name Family Common name
Potatoes Solanum tuberosum Solanaceae
Country potato Solenostemon Lamiaceae (mint Innala, ratala (Sri Lanka)
Hausa potato rotundifolius family)
Cannas Canna edulis Cannaceae Buthsarana (Sri Lanka)
Maranta arundinacea L Marantaceae Arrow root
Hulankeeriya (Sri Lanka)
Aru aru,Arawak (India)
Taro Xanthosoma Araceae Kiriala (Sri Lanka)
sagittifolium Keladi (Malasia)
Phueak (Thailand)
Khoai mon (Vietnam)
Sato-imo (Japan)
Yam Dioscorea alata Dioscoreaceae Purple yam; Greater yam
Guyana;Water yam
Winged yam;
Raja ala (Sri Lanka);
Ube (Philippines)
Sweet potatoes Ipomoea batatas Convolvulaceae Camote; Batata
Shakarkand
Cassava Manihot esculenta Euphorbiaceae Yuxco; Mogo; Manioc
Mandioca; Kamoteng
kahoy
Elephant foot Amorphophallus Araceae White pot giant arum;
yam paeoniifolius Stink lily
2 Starchy Roots and Tuber Crops
Roots and tubers are belonging to different botanical families but are grouped
together as all types produce and store edible food in underground parts. Starchy
roots and tubers store starch in subterranean stems, roots, rhizomes, corms, and
tubers. Potatoes and yams are tubers whereas taro and cocoyams are derived from
corms, underground stems, and swollen hypocotyls. Cassava and sweet potatoes are
storage roots whereas canna and arrowroots are edible rhizomes. Vegetative propa-
gation is a common characteristic of roots and tubers. These plant parts include
tubers (potatoes and yams), stem cuttings (cassava), vine cuttings (sweet potatoes),
and side shoots, stolons, or corm heads (taro and cocoyam). Table 2 presents
commonly consumed starchy tuber and root crops worldwide.
2.1 Potato (Solanum tuberosum)
Potato is ranked currently at the fourth place of the highly consumed crops through the
world followed by maize, wheat, and rice. The average annual potato production is about
381 million tones [1]. Potato has a highland origin and has been domesticated in the high
Andes of South America. It is a popular food crop in the cool highland areas of South
America, Asia, and Central and Eastern Africa [9]. China is the biggest potato producer
followed by Russia and India. The average energy contribution from potato in the world
ranged from 41 to 130 Kcal per day for developing and developed countries, respectively
[10]. Potato is a high yielding cash crop with a short cropping cycle of 3–4 months.
Further, potato is a significant source of carbohydrates and they can be processed into a
variety of foods such as mashed potato, chips and fries, deep frozen and dehydrated
products. Starch derived from potato has shown a great economic importance. The
productivity of potato in terms of nutrients is higher compared to maize, wheat, and rice.
Potato produces high amount of calories, protein, and calcium from the same land
than those of other major crops leading its significance as a food security crop.
Potatoes have several secondary metabolites which demonstrated antioxidant as well
as other bioactivities [11].
2.2 Sweet Potato (Ipomea batatas L.)
Sweet potato originated from the Central America and the cultivation has been spread
through the tropical, subtropical, and warm temperate countries. In Asia it is predominate
in lowland conditions. Sweet potato is known as the seventh largest food crop with an
annual production of 106 million tones [1]. They are known as an insurance crop as the
cultivation can be extended throughout the year under suitable climatic conditions and its
greater capacity to withstand the adverse climatic changes. In addition, the crop can be
harvested for an extended period of time giving advantages in the poor communities.
Further, the short growing cycle of sweet potato is an advantage for the flexible planting
and harvesting conditions in spite of the climatic changes. Rice and sweet potato can be
grown with crop rotation. China is the largest producer of sweet potato [12]. National
Aeronautics and Space Administration (NASA) has selected sweet potatoes as a potential
crop to be grown and incorporated into the menus for astronauts on space missions due to
its unique features and nutritional value [13]. It can be consumed in many ways such as
boiled, baked, fried, boiled canned or frozen [14]. The biofortified sweet potatoes provide
considerable amounts of beta-carotene, and it has been proved as a cost-effective strategy
to provide vitamin A for the vulnerable communities including young children, pregnant
and lactating mothers. Sweet potato is an important source of many vitamins, minerals,
and dietary fiber. Further it is a good source of nonnutritive bioactive compounds such as
phenolic acids and anthocyanins [15–19]. The different flesh colors such as cream, deep
yellow, orange, and purple are attributed to the degree of anthocyanin and carotenoids
content of sweet potatoes [15–18, 20].
2.3 Cassava (Manihot esculenta)
Cassava is a perennial shrub belonging to the family of Euphorbiaceae. Nigeria,

Thailand, Indonesia, and Brazil are the leading cassava producers in the world. The
Manihot genus comprises of 98 species and the most prominent species is M.

esculenta [21]. The crop is originated in South America and has speared through
tropical and subtropical regions of Africa and Asia [22]. Cassava is the sixth most
important food crop in the world with about 268 million tones annual production [1].
It is the most widely cultivated root crop and contains nearly the maximum theoret-
ical concentration of starch on a dry weight basis among food crops. The edible roots
can store the foods underground for up to 3 years contributing for the food security
consistently.
Different range of food products are derived from cassava such as traditional and
novel food products, fermented products, ethanol, and livestock feeds. The starch
extracted from cassava is used as the raw material for the production of bread,
crackers, pasta, and tapioca. Cassava leaves are also a nutritious food addition.
Through the west, steamed and boiled cassava roots are pounded in to sticky
dough and consumed as fufu. The Philippines consume cassava pulp made into
pellet as landing or cassava rice [23]. Cassava starches are popular in the industry
due to its high availability (75–80% of the dry matter content of roots) and the easily
extractable nature due to the lower content of protein and fat. Further, cassava is used
for the manufacturing of paper, textiles, adhesives, and high fructose corn syrup [14].
Cassava is well known for the production of hydrogen cyanide (HCN) in toxic
quantities. The presence of two types of cyanogenic glycosides such as linamarin
(93% of the cyanide content) and lotaustralin has been reported [24]. High doses of
HCN about 0.5–3.5 mg/Kg of body weight can exert lethal effects. Further, non-
cyanogenic glycosides, hydroxycoumarins such as scopoletin, terpenoids, and fla-
vonoids, have been investigated in cassava roots. Both roots and leaves contain the
cyanogenic glycosides and the content is not significantly different from the roots.
Despite that cassava leaves are a good source of calcium and protein [23].
2.4 Yams (Dioscorea sp.)
Yams are a group of many species of plants bearing rhizomes or carbohydrate rich
underground tubers belonging to the genus Dioscorea. They are mainly distributed
through the tropics and only a few numbers can be found in temperate regions of the
world [25]. Yams are staple foods in West Africa, Southeast Asia, and the Caribbean
regions in the world and help to ensure the food security and livelihood of at least 60
million people in West Africa [26]. They rank as the third important source of calorie
and protein contributor for the livelihood of Benin and Ghana populations. The
contribution for the protein intake is even higher than the widely grown and
available cassava. They are classified into the genus Dioscorea of the family of
Dioscoreaceae, which is one of the oldest members of the monocot group [27, 28].
Even though there are many varieties present, only few numbers of species are
popular throughout regions in the world. About 603 species have been identified and
from them 50 species are edible. Dioscorea alata, Dioscorea esculenta Dioscorea
rotundata, Dioscorea hispida, Dioscorea bulbifera, Dioscorea trifida, Dioscorea
nummularia, and Dioscorea pentaphylla are prominent yam species, among others.
A wide variation in morphological characteristics presents within the same species of

yams and these include size of tuber, color of flesh, presence of roots, and thickness
of peel (Fig. 1).
The yams are usually grown in intercropping systems with coconut palms.
Further, yams can be easily grown due to their specific ability to thrive under low
agricultural inputs, having tolerance to stress environmental conditions, ability to
resist pest attacks, and high adaptability to mixed cropping systems. They are
considered as seasonal crops which are generally planted in the end of March to
April [29]. The harvesting season begins usually during the period of December to
February. After harvesting, these yams would be on a dormancy period until the
favorable conditions are met for the growth of new plant.
Dioscorea alata is called as winged yam, greater yam, or water yam and includes
a wide array of tubers referred in several local names in different countries. It has
been reported that D. alata are originated in Asia [30]. They are widely cultivated in
India, Sri Lanka, South East Asia, and Pacific Islands and they prefer the low lands
up to 800 m. The tubers are famous for the sweetness and the delicacy of the tubers.
Dioscorea alata contains only a single large tuber per vine [30]. The shapes of the
tubers have significant differences. They may be globular, long, and flattened or
palmate. Sometimes they may be branched or lobed but usually large in size.
Yam tubers have various bioactive components, namely, mucin, dioscin,
dioscorin, allantoin, choline, polyphenols, diosgenin, and vitamins such as caroten-
oids and tocopherols [31, 32]. Mucilage of yam tuber contains soluble glycoprotein
and dietary fiber. There are evidences to show hypoglycemic, antimicrobial, and
antioxidant activities of yam extracts [33, 34]. Yams may stimulate the proliferation
of gastric epithelial cells and enhance digestive enzyme activities in the small
intestine [35]. Dioscorea esculenta (Chinese yam, lesser yam, sweet yam) is one
of the main yam species found in Sri Lanka [29]. They are native to Indo-China and
at present they have widely been distributed through Madagascar and New Guinea.
Usually the yams are cultivated in the lowland up to 700 m. Their stems contain a
large number of tubers and they are generally ovoid and cylindrical in shape [29]. In
addition, the tubers may be intermingled and covered with numerous spiny roots.
The color of the tuber flesh varies from white to yellowish white. Leaves are broadly
ovate with a chordate base. The raw tubers are known for a number of medicinal
properties [30].
Dioscorea pentaphylla is known as five-fingered yam and buck yam. These tubers
are variable in shape such as finger shaped, cylindrical, or shapeless. The skin of the
tuber is brown in color and the flesh ranges from white to yellow. Sometimes there
may be violet spots in the flesh. The species is native to South East Asia and is grown
in teak forests or forest borders with altitudes of 500–1000 m. Tubers are consumed
as staple food especially as an alternative to maize in the dry areas. It is used as a raw
material for the production of starch and alcohol. The origin of Dioscorea bulbifera
L. is evidenced from Asia, Africa, and Oceania and are known as air potato, aerial
yam, or potato yam. The characteristics of the vine are documented as stem round,
twining to the left, without spines subterranean tuber small or absent, and spongy.
Dam ala Ini ala
Kombu ala Kahata ala
Kirikondol Kidala
Fig. 1 Different accessions of Dioscorea alata

Leaves are alternate, broad, round, heart-shaped, and leaf blade embossed with well-
marked veins. In addition, less known Dioscorea species have been reported and
they include Dioscorea rotundata Poir.; D. cayenensis (African yam, white yam);
Dioscorea trifida L. (Cushcush, Indian yam); and Dioscorea nummularia Lam.
(Pacific yam, hard yam).
2.5 Aroids
Aroids are tuber or underground stem-bearing plants belonging to the family

Araceae. Aroids can be grown in both rain-fed and irrigated climates. Taro is used
as the generic name for the four related species belonging to Araceae including
edible tubers such as taro (Colocasia), giant taro (Alocasia), tannia or yautia
(Xanthosoma), elephant foot yam (Amorphophallus), and swamp taro
(Cyrtosperma) [36]. They serve as a staple food for many islands of the South
Pacific, such as Tonga and Western Samoa, and in Papua New Guinea. The cultiva-
tion has been extended to Asia, Africa, Pacific and Caribbean islands. Aroids
originated from the Indo–Malayan region between Myanmar and Bangladesh.
They are known to have thousands of different cultivars and most of them prefer
the humid environments. The young leaves are eaten as vegetables. They are well
cultivated in the low lands and highlands up to 2.7 m above the sea level. Aroids are
considered as the staple in Irian Jaya, the Moluccas, and Mentawai Islands in West
Sumatra. They can be consumed as chips as well as steamed forms among others.
3 Nutrients in Roots and Tubers
Starchy roots are major contributors to the supply of carbohydrates in the diet. The
protein content of the tubers varies from 1% to 2% on fresh weight basis. The
limiting amino acids of the tubers are identified as sulfur-containing amino acids,
namely, methionine and cystine, whereas sweet potato contains lysine [3]. Vitamin C
are available in cassava, sweet potato, and potato. Further, potato is a dietary source
of micronutrients such as zinc and iron. Sweet potato cultivars are known for the
dietary fiber, mineral, and vitamins including β-carotene and tocopherols. Especially
the yellow-fleshed sweet potato varieties are known for their high β-carotene con-
tent. Taro and potato have appreciable level of potassium. All most all the tuber
species are rich with dietary fiber (Table 2).
3.1 Proteins
Roots and tubers are not considered as appraisable sources of proteins and the
contents are variable. In populations worldwide, global contribution of proteins
from roots and tubers in the diet is less than 3%. However, this contribution
varies from 5 to 15% depending on the quantity consumed as a staple in African
countries [9]. Cassava contains only 1–2% of protein on dry weight basis with low
content of sulfur-containing amino acids [37].
Dioscorin is the main storage protein known to present in Dioscorea yams and is
about 90% of water extractable soluble proteins in a majority of Dioscorea species.
Among many activities reported dioscorin has shown carbonic anhydrase and
trypsin inhibitor activities [38]. Further, dehydroascorbate reductase (DHA) and
monodehydroascorbate reductase (MDA) and immunomodulatory activities of
dioscorin in the presence of glutathione have been reported [39]. Dioscorin from
fresh yam (Dioscorea batatas) showed 2,2, diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging activity [40]. Further, dioscorin demonstrated angiotensin
converting enzyme (ACE) inhibitory and antihypertensive activities on spontane-
ously hypertensive rats [31, 41, 42].
Sporamin is a soluble protein and serves as the main storage protein of sweet
potato tubers. It accounts for 60–80% of tubers total proteins [43]. The sporamin of
sweet potatoes was initially known as ipomoein and is a nonglycoprotein without
glycan. The sporamin is generally stored in vacuoles in the monomeric form. This
protein is initially produced as preprosporamin, which is synthesized by the mem-
brane-bound polysomes in the endoplasmic reticulum (ER) [44]. Sporamin is a
trypsin inhibitor with a kunitz-type trypsin inhibitory activity which has potential
application in the transgenic insect-resistant plants [45]. Furthermore, sporamin
showed various antioxidant activities related to stress tolerance, such as DHA and
MDA reductase activities [46].
Patatin is a storage protein in potato tubers and contributes to about 40% of
soluble proteins [47]. It is a glycoprotein in the storage of parenchyma cells and
exhibited a number of bioactivities [48].
3.2 Carotenoids
Carotenoids serve as the most extensively available natural pigments with yellow,
orange, and red colors in plants. Carotenoids, either isolated from natural sources or
chemically synthesized, have been widely utilized as natural nontoxic colorants in
manufactured foods, drinks, and cosmetics due to the distinctive coloring properties.
The majority of carotenoids are unsaturated tetraterpenes with the same basic C 40
isoprenoid skeleton resulting from the joining of eight isoprene units in a head-to-tail
manner with the exception of the tail-to-tail connection at the center. They are
hydrocarbons and are soluble in nonpolar solvents such as hexane and petroleum
ether. However, the oxygenated derivatives of carotenes, such as xanthophyll,
dissolve better in polar solvents such as alcohols. Carotenoids are important mole-
cules in living organisms. They participate in a variety of photochemical reactions in
photosynthetic systems of higher plants, algae, and phototrophic bacteria [49].
Carotenoids possess numerous bioactivities and are well known for provitamin A
activity. In addition, they play important roles in human health and nutrition, namely
antioxidant activity, regulation of gene expression, and induction of cell-to-cell
communication [50]. It has been demonstrated that zeaxanthin and lutein are stable
throughout artificial digestion, whereas β-carotene and all-trans lycopene are

degraded in the jejunal and ileal compartments. Among the isomers, the stability
of 5-cis lycopene is superior to that of all-trans, and 9-cis lycopene [51]. Yellow to
orange varieties of sweet potatoes and yams are good sources of carotenoids
and lutein, zeaxanthin, violaxanthin and neoxanthin are major carotenoids in pota-
toes [52]. Further, digestive stability of lutein and zeaxanthin of yellow-fleshed
potatoes were reported to be high ranging from 70% to 95% [53].
3.3 Ascorbic Acid
Ascorbic acid, also known as vitamin C, is a water-soluble vitamin. Ascorbic acid

naturally occurs in plant tissues, commonly in fruits and vegetables but considerable
quantities also present in several root crops. However, the level could be reduced
during cooking of roots unless skins and cooking water are utilized [54]. Root crops,
if carefully prepared, can make a significant contribution to the vitamin C content of
the diet. Potatoes serve as a principal source of vitamin C in British diets, providing
19.4% of the total requirement [3]. In general, yams contain 6–10 mg of vitamin
C/100 g and may vary up to 21 mg/100 g. Further, the vitamin C content of potatoes
is very similar to those of sweet potatoes, and cassava. The concentration of ascorbic
acid varies with the species, location, crop year, maturity at harvest, soil, nitrogen
and phosphate fertilizers [3].
4 Nonnutrient Compounds in Roots and Tubers
Bioactive compounds in plants are secondary metabolites having pharmacological or

toxicological effects in humans and animals. Secondary metabolites are produced
within the plants besides the primary biosynthesis associated with growth and
development. These compounds perform several essential functions in plants,
including protection from undesirable effects, attraction of pollinators, or signaling
of essential functions, among others. Roots and tubers are rich with nonnutritive
phytochemicals. Yam tubers contain mucin, dioscin, dioscorin, allantoin, choline,
polyphenols, diosgenin, and saponins [32]. Potatoes are rich with phenolic acids
including chlorogenic acids and flavonoids and some varieties are rich sources of
anthocyanins [55]. Cultivation location, climatic factors, soil type, agricultural
practices, and environmental stress are the key important determinants of the
nutritional and phytochemical profile of tubers.
Phenolic compounds are important molecules with an aromatic ring attached with
one or more hydroxyl groups. They are derived from biosynthetic precursors such as
pyruvate and acetate, amino acids such as phenyl alanine and tyrosine, and acetyl
CoA and malonyl CoA by following the pentose phosphate, shikimate, and
phenylpropanoid metabolism pathways [8, 56]. Among groups of phenolic com-
pounds abundantly found in plants simple phenolics, phenolic acids, flavonoids,
coumarins, stilbenes, tannins, lignans, and lignins are reported to possess myriad of
health benefits [8]. Cultivar, environmental conditions, cultural practices, post-
harvest practices, processing and storage conditions affect the quantity of phenolic
compounds present in a given species of plant material, among others [57]. Phenolic
acids found in plants are categorized as hydroxybenzoic and hydroxycinnamic acids.
Hydroxybenzoic acids include gallic, p-hydroxybenzoic, vanillic, syringic, and
protocatechuic acids, among others. The hydroxycinnamic acids include p-
coumaric, caffeic, ferulic, and sinapic acids. These latter compounds with a phenyl
ring (C6) and a C3 side chain are known as phenylpropanoids and serve as precursors
for the synthesis of other phenolic compounds. Flavonoids are common and known
constituents synthesized by condensation of a phenylpropanoid compound with
three molecules of malonyl coenzyme A. The subclasses such as flavones, flavonols,
flavonones, flavononols, isoflavones, flavans (catechins and anthocyanidins), and
flavanols are common molecules of flavonoids, among others. Flavones and flavo-
nols are present as aglycones in foods [57]. The phenolics present in tubers render
several health benefits, namely, antibacterial, anti-inflammatory, and antimutagenic
activities.
4.2 Saponins and Sapogenins
Saponins are high molecular weight secondary metabolites and are glycosides
consisting of a sugar moiety linked to a triterpene or steroid aglycone. The common
biological feature of saponins include ability to lyse erythrocytes and foam [58]. The
aglycone portion of the saponin molecule is called sapogenin which is hydrophobic.
The hydrophilic sugar moiety present in saponins include one to three straight or
branched sugar chains and they are composed of D-glucose, L-rhamnose, D-galactose,
D-glucuronic acid, L-arabinose, D-xylose, and D-fucose [59]. Depending on the type
of genin present, saponins are divided into three groups, namely, triterpenoid
saponins, steroidal saponins, and steroidal alkaloid saponins. Steroidal saponins
are precursors for the chemical synthesis of birth control pills (with progesterone
and estrogen), similar hormones, and corticosteroids [60]. According to recent
findings steroidal saponins could be a novel class of prebiotics to lactic acid bacteria
and are effective candidates for treating fungal and yeast infections in humans and
animals [61].
4.3 Steroidal Glycoalkaloids
Glycoalkaloids are belonging to phytochemicals found in species of the genus

Solanum and Veratrum [62, 63]. Alkalioids are nitrogen-containing secondary
metabolites and are found in several higher plants as well as microorganisms and
animals [64]. The skeleton of alkaloids is derived from amino acids and moieties
from other pathways, such as those originating from terpenoids. The alkaloids in
plants act as phytotoxins, antibactricides, insecticides, fungicides, and as feeding
deterrents to insects, herbivorous mammals, and mollusks [65]. The two main
glycoalkaloids present in commercially cultivated potatoes include α-chaconine
and α-solanine which are glycosylated derivatives of the aglycone solonidine.
Wild potatoes (Solanum chacoense) contain leptinine 1, leptinine 11, leptine 1,
leptine 11, and dehydrocommersonine [66]. The major glycoalkaloid reported in
tomatoes is α-tomatine which is a glycosylated derivative of aglycone tomatidine.
Steroidal alkaloids and their glycosides present in several species of Solanum are
known to possess a variety of biological activities such as antitumor, antifungal,
teratogenic, antiviral, and antiestrogenic activities. Certain glycoalkaloids are used
as anticancer agents [67, 68]. The steroidal alkaloid glycosides showed cytotoxic
activity against various tumor cell lines [69].
5 Bioactivities of Phytochemicals in Roots and Tubers
5.1 Hormonal Activities
Some roots and tubers especially yams belonging to Dioscorea have demonstrated
health effects due to the bioactivities affected on hormones. According to Wu et al.
[70] yam diet has ability to reduce the risk of cancer and cardiovascular diseases in
postmenopausal women. They showed that the levels of serum estrogen and sex
hormone binding globulin (SHBG) increased significantly after subjects had been on
a yam diet for 30 days. Further, serum hormone parameters such as estrogen,
estradiol, and SHBG did not change in controls who had sweet potatoes compared
to those who had yam diet. The risk of breast cancer increased by estrogens might be
balanced by the elevated SHB and the ratio of estrogen plus estradiol to SHBG. The
study further showed that increased SHBG levels had a protective effect against the
occurrence of type 2 diabetes mellitus and coronary heart diseases in postmeno-
pausal women [70].
Chen et al. [71] showed that chronic administration of Dioscorea may enhance
the bone strength. Further it shed light on the role of Dioscorea in bone remodeling
and osteoporosis during the menopause [71]. Administration of Dioscorea to ovari-
ectomized rats decreased the porosity effect on bones and increased the ultimate
force of bones. The changes in biochemical and physiological functions seen in these
animals were similar to those in menopausal women [71].
5.2 Antioxidant Activity
Antioxidants play a pivotal role in attenuating several chronic diseases such as

different types of cancer, cardiovascular diseases, arthritis, diabetes, autoimmune
and neurodegenerative disorders, and aging in which oxidative stress plays a major
role in the development of the diseases. Several studies provide evidences for the
antioxidant activities of roots and tuber crops.
Potato is a rich source of a number of phytochemicals and exhibit antioxidant
activities to varying degrees. Methanolic extract of potatoes demonstrated high
antioxidant activities as determined by DPPH radical scavenging activity [72].
Further, the total phenolic content (TPC) ranged from 16.6 to 32 mg gallic acid
equivalents (GAE)/100 g dry matter, and EC50 of DPPH radical scavenging activity
of potatoes extract was 94 mg/mL [72]. The major phenolic compound reported in
potato was chlorogenic acid which constituted more than 90% of its phenolics [73].
Extracts of flavonoids and flavones of potatoes showed high scavenging activities of
reactive oxygen species. Potatoes scavenged 94% of hydroxyl radicals [74]. In
addition, a range of hydrophilic oxygen radical absorbance capacity (ORAC) of
200–1400 μmol trolox equivalents/100 g fresh weight (FW) and the lipophilic
ORAC range of 5–30 nmol alpha-tocopherol equivalents/100 g FW were reported
for potatoes [75]. Further the TPC of water soluble and insoluble extracts of 15
Andean potato cultivars ranged from 2.4 to 28 μmol GAE/g (dm) and TFC varied
between 0.9 and 4.8 μmol CE/g (dm) [76]. Antioxidant activities varied between
cultivars. In addition, five phenolic compounds, namely, gallic acid, protocatechuic
acid, cholorogenic acid, syringaldehyde, and epicatechin, were identified as major
compounds.
Han et al. [77] showed that ethanolic extracts of purple-fleshed potato flakes had
effective free radical scavenging activity and inhibition of linoleic acid oxidation
in a rat model. Further, potato extracts enhanced hepatic manganese superoxide
dismutase (SOD), Cu/Zn-SOD, and glutathione peroxidase (GSH-Px) activities as
well as mRNA expression, suggesting a reduced hepatic lipid peroxidation and an
improved antioxidant potential [77]. Potatoes contain water-soluble antioxidant
compounds such as glutathione and ascorbic acids [52].
Ji et al. [78] reported the contents of phenolic compounds and glycoalkaloids of
20 potato clones and their antioxidant, cholesterol uptake, and neuroprotective
activities in vitro. Peels of purple and red pigmented potato clones showed higher
phenolic content than those of yellow and un-pigmented clones [78]. Chlorogenic
acid (50–70%) and anthocyanins, namely, pelargonidin and petunidin, were identi-
fied as major phenolic compounds present in potatoes. Major glycoalkaloids
reported to present in potatoes included α-chacoine and α-solanin and their contents
were reduced by granulation process. Peels of potato clones showed the highest
DPPH radical scavenging activity followed by flesh and granules [78]. Thus, potato
peels can be utilized as a potential source of nutraceuticals. In addition, carotenoids
such as lutein, zeaxanthine, and violoxanthine present in potatoes contribute to the
antioxidant activity. The contents of carotenoids range from 50 to 100 μg in white-
fleshed varieties to 2000 μg per 100 g FW in orange-fleshed cultivars [79].
The peels of sweet potato possessed a potent wound-healing effect which appears
to be related to the free radical scavenging activity of the bioactive constituents and
their ability in lipid oxidation inhibition [80–82]. According to Suzuki et al. [80]
sweet potato fiber was effective in healing of burns or decubital wounds in a rat
model. They further observed the reduction of wound area and changes of the quality
of the wounds after treating with sweet potato fiber compared to those of the control.
Later, Chimkode et al. [81] also showed that petroleum ether extract of sweet potato
had a significant effect on the closure of scar area for the complete epithelialization
compared to the control.
The methanolic extracts of the peels and peel bandage of sweet potato tubers were
screened for wound-healing effect by excision and incision wound models on Wistar
rats [82]. It was found that hydroxyproline content was significantly increased in the
test group than that of wounded control. This may improve collagen synthesis which
affects the increased wound healing. Further, the content of malondialdehyde
decreased in the test groups compared to that of wounded control, indicating lipid
oxidation inhibitory effect of sweet potato peels [82].
The flavonoid contents of different selected tuber crops such as sweet potato,
potato, coco yam, dasheen (Colocasia esculenta), St Vincent yam, yellow yam
(Dioscorea cayenensis), and water yam (Dioscorea alata) varied from 71.5 to
390.7 CE/100 mg. Water yam (Dioscorea alata) reported the highest DPPH radical
scavenging activity of 96% among others [83].
Phenolic content and antioxidant activities of yam vary with the cultivar. Cornago
et al. [84] showed that purple yam (Dioscorea alata) and lesser yam (Dioscorea
esculenta) had a TPC ranging from 69.9 to 421.8 mg GAE/100 g dry weight. The
purple yam variety Daking showed the highest TPC and antioxidant activities as
measured by DPPH radical scavenging activity, reducing power and ferrous ion
chelating capacity whereas varieties Sampero and Kimabajo showed the least.
Water and ethanolic extracts of yam peel showed the antioxidant activity on tert-
butylhydroperoxide (t-BHP)-induced oxidative stress in mouse liver cells, namely
Hepa 1–6 and FL83B [85]. Ethanolic extracts (EE) of yam peel exhibited a better
protective effect on t-BHP-treated cells than that of water extracts (WE). Further, EE
had increased catalase activity whereas WE decreased it.
Chen and Lin [86] showed that heating affected the TPC, antioxidant capacity,
and the stability of dioscorin of various yam tubers. Cooked yams showed lower
TPC than their raw counterparts. Further, the DPPH radical scavenging activities
declined with increasing temperature. TPC and dioscorin content of yam cultivars
(Dioscorea alata L. var. Tainung No. 2) and Keelung yam (D. japonica Thunb. var.
pseudojaponica (Hay.) Yamam) correlated with DPPH radical scavenging activity
and ferrous ion chelating activity [86]. Phytochemicals of yams seem to enhance
the activities of endogenous antioxidant enzymes. The administration of yams
decreased the levels of γ-glutamino transpeptidase (GGT), low-density lipopro-
tein, and triacylglycerol in serum of rats in which hepatic fibrosis was induced by
carbon tetrachloride [87]. Treatment of rats with yams increased the antioxidant
activities of hepatic enzymes, namely, glutathione peroxidase and superoxide
dismutase [87].
Limited studies have reported the antioxidant activities of cassava roots. The
antioxidant activities of organically grown cassava tubers were higher than those of
mineral-base fertilized roots. They further found that TPC and flavonoid content
(FC) were significantly higher for organic cassava compared to that of cassava
grown with inorganic fertilizers [88].
5.3 Antiulcerative Activities
The antiulcerative activity of sweet potato tubers was studied in a rat model [89]. The
extract of sweet potatoes did not show any toxic or deleterious effects by oral route
up to 2000 mg/kg. Further, superoxide dismutase (SOD), catalase (CAT), glutathi-
one peroxidase (GPx), and glutathione reductase (GR) activities were significantly
elevated by administration of tuber extracts in treated rats, indicating the ability of
restoring enzyme activities compared to the control. Kim et al. [16] showed that
butanol fraction of sweet potato could be a better source for treating gastric ulcers
induced by excessive alcohol intake.
5.4 Anticancer Activities
In the contemporary society cancer has been a leading cause of death mainly due to
unhealthy food habits and lifestyle. Cancer is a multistage disease and tapping at any
initial juncture could help to attenuate the disease condition. There are evidences to
show that dietary components, specifically present in plant foods, are important to
reduce and prevent the risk of cancers. A number of phytochemicals found in root
and tubers have demonstrated anticancer effects in several types of carcinoma cell
lines and animal models.
Extracts of sweet potatoes demonstrated antiproliferative activity in human
lymphoma NB4 cells [90]. Further it has been shown that aqueous extract of sweet
potatoes had higher antiproliferative activity than that of ethanol extracts. Cell
proliferation was analyzed at 48 h after in human lymphoma NB4 cells which had
been cultured with several concentrations of extracts 0, 25, 50, 100, 200, 400, 800, or
1000 μg dm/mL in the media using the microculture tetrazolium treatment assay
(MTT).
Further it has been shown that the phytochemicals present in sweet potato roots
may exert a significant effect on antioxidant as well as anticancer activities [90].
Antioxidant activity is directly related to the phenolics and flavonoid contents of the
sweet potato extracts. There was an additive role of phytochemicals contributing
significantly to the potent antioxidant activity and antiproliferative activity in vitro
[90]. Two anthocyanin pigments, namely, 3-(6,60 -caffeylferulylsophoroside)-5-glu-
coside of cyanidin (YGM-3) and peonidin (YGM-6), purified from purple sweet
potato inhibited the reverse mutation induced by mutagenic pyrolysates of trypto-
phan (Trp-P-1, Trp-P-2) and imidazoquinoline (IQ) in the presence of rat liver
microsomal activation systems [91]. Red colored potatoes demonstrated higher
inhibition of carcinogenesis compared to counterparts of white in breast cancer-
induced rats [92]. In addition, the red cultivar of potatoes reported high levels of
anthocyanins and chlorogenic acid derivatives which could be contributors for
observed effects.
Further Madiwale et al. [93] demonstrated that purple-fleshed potato had higher
potential in suppressing proliferation and elevated apoptosis of HT-29 human colon
cancer cell lines compared to those of white-fleshed potato.
Storage of potatoes affected their antioxidant and anticancer activities and TPC.
The extracts of fresh and stored potatoes inhibited cancer cell proliferation and
elevated apoptosis. However, anticancer effects were higher in fresh potatoes than
those of stored tubers. The investigators further demonstrated that storage duration
of potatoes had a strong positive correlation with antioxidant activity and percentage
of viable cancer cells but a negative correlation with apoptosis induction. These
findings further elaborated that antioxidant activity and phenolic content of potatoes
increased with storage, but antiproliferative and proapoptotic activities were
decreased [93].
Apart from phenolics in roots and tubers, saponins play a pivotal role as antican-
cer/antitumor agents. A number of groups of saponins, namely, cycloartanes,
ammaranes, oleananes, lupanes, and steroids, demonstrated antitumor effects on
different types of cancers. Auyeung et al. [94] showed that cycloartanes possess
antitumor properties in human colon cancer cells and tumor xenografts. They
downregulated expression of the hepatocellular carcinoma (HCC) tumor marker
α-fetoprotein and suppressed HepG2 cell growth by inducing apoptosis and modu-
lating an extracellular signal-regulated protein kinase (ERK)-independent NF-κB
signaling pathway [94]. Furthermore, oleananes exerted their antitumor effect
through various pathways, such as anticancer, anti-metastasis, immunostimulation,
and chemoprevention [94].
A number of studies reported that glycoalkaloids, namely, α-chaconine and α-
solanine, present in some tubers are potential anticarcinogenic agents [95].
Glycoalkaloids showed antiproliferative activities against human colon (HT29)
and liver (HepG2) cancer cells as assessed by the MTT assay [95].
The aqueous extract of Dioscorea alata inhibited the H2O2-CuSO4-induced
damage of calf thymus DNA, and protected human lymphoblastoid cells from
CuSO4-induced DNA damage [96]. Phenolic compounds saponins and polysaccha-
ride mucilage present in yams are responsible for the observed bioactivities of yams
extract. Water-soluble mucilage polysaccharides are the most important copper
chelators in the extract of water yam. Thus Dioscorea alata aqueous extracts
could serve as potential agents in the management of copper-mediated oxidative
disorders [96].
5.5 Hypoglycemic Activities
Diabetes mellitus is among the NCDs marked by elevated levels of glucose in the
blood leading to complications that can untimely cause death. Evidences are emerg-
ing that a number of phytochemicals in foods are effective agents in the prevention
as well as management of type 2 diabetes. The extract of white-skinned sweet
potatoes (WSSP) reduced hyperinsulinemia in Zucker fatty rats by 23, 26, 60, and
50%, after 3, 4, 6, and 8 weeks of oral administration, respectively, of WSSP similar
to troglitazone (insulin sensitizer) [97]. Further lipid parameters such as blood
triacylglycerol (TG) and free fatty acid (FFA) lactate levels were also lowered by
the oral administration of WSSP. Further histological examinations of the pancreas
of Zucker fatty rats showed a remarkable regranulation of pancreatic islet of B-cells

in the WSSP and troglitazone treated groups after 8 weeks. These evidences further
reinforced that WSSP was likely to improve the abnormal glucose and lipid metab-
olism in insulin-resistant diabetes mellitus. In addition, supporting these observa-
tions extracts of sweet potato peels have shown reduced plasma glucose levels of
diabetic patients [98]. Ingestion of 4 g of Caiapo, the extract of WSSP, per day for
6 weeks reduced fasting blood glucose and total as well as low density lipoprotein
(LDL) cholesterol in male Caucasian type 2 diabetic patients who were previously
treated by dietary management alone [98]. The improvement of insulin sensitivity, as
determined by the frequently sampled intravenous glucose tolerance test (FSIGT),
indicated that Caiapo extracts showed beneficial effects via reducing insulin resis-
tance. Further Ludvik et al. [98] confirmed the beneficial effects of Caiapo on
glucose and serum cholesterol levels in type 2 diabetic patients treated by diet
alone for 3 months after recruitment for the study. Improved fasting blood glucose
levels and glucose levels during an OGTT and in the postprandial state as well as
improvement in long-term glucose control was also observed as expressed by the
significant decrease in HbA1c [98].
The ethanolic extract of Dioscorea alata showed antidiabetic effects on alloxan-
induced diabetic rats [99]. It was observed that diabetic rats who administered yam
extract exhibited significantly lower creatinine levels which could be a result of
improved renal function by reduced plasma glucose level and subsequent glycosyl-
ation of renal basement membranes. A number of bioactives, including phenolics,
was identified in the ethanolic extract of D. alata. These include hydro-Q9
chromene, γ-tocopherol, α-tocopherol, -feruloyl glycerol, dioscorin, cyanidin-3-
glucoside, catechin, procyanidin, cyanidin, peonidin 3-gentiobioside, and alatanins
A, B, and C [99].
5.6 Hypocholesterolemic Activity
Diet plays an important role in the regulation of cholesterol homeostasis and CVDs
are among the leading causes of death worldwide. External agents possessing anti-
cholesteromic activities continuously show beneficial effects on risk reduction and
management of CVDs.
Diosgenin, a steroidal saponin of yam (Dioscorea), demonstrated antioxidative
and hypolipidemic effects in a rat model [100]. Rats fed with a high-cholesterol diet
were supplemented with either 0.1 or 0.5% diosgenin for 6 weeks. The lipid profile
of the plasma and liver, lipid peroxidation and antioxidant enzyme activities in the
plasma, erythrocyte, and gene expression of antioxidative enzymes in the liver and
the oxidative DNA damage in lymphocytes were measured. Diosgenin showed
pancreatic lipase inhibitory activity, protective effect of liver under high cholesterol
diet, reduced total cholesterol level, and protection against the oxidative damaging
effects of polyunsaturated fatty acids [100].
Steroidal saponins of yams are used for industrial drug processing. Saponins,
such as dioscin and gracilin, and prosapogenins of dioscin have long been identified
from yam. The content of the dioscin was about 2.7% (w/w). Diosgenin content was
about 0.004 and 0.12–0.48% in cultivated yams and wild yams, respectively. The
anti-hypercholesterolemic effect of yam saponin is related to its inhibitory activity
against cholesterol absorption [101].
The hypocholesterolemic effect of yam diosgenin had been reported by an in vivo
study. Rats fed with yam (Dioscorea) showed a decreased cholesterol absorption,
increased hepatic cholesterol synthesis, and increased bilary cholesterol secretion
without affecting serum cholesterol level [102]. In agreement with this finding,
several studies showed that diosgenin, present in some Dioscorea, could enhance
fecal bile acid secretion and decrease intestinal cholesterol absorption [103, 104].
The relative contribution of bilary secretion and intestinal absorption of cholesterol
in diosgenin-stimulated fecal cholesterol excretion were studied in wild-type (WT)
and Niemann-Pick C1 Like 1 (NPC1L1) knockout (LIKO) mice. NPCiL1 was
recently identified as an essential protein for intestinal cholesterol absorption
[105]. Diosgenin significantly increased bilary cholesterol and hepatic expression
of cholesterol synthetic genes in both WT and LIKO mice. In addition, diosgenin
stimulation of fecal cholesterol excretion was primarily attributable to its impact on
hepatic cholesterol metabolism rather than NPC1L1-dependent intestinal cholesterol
absorption [105].
Native protein of dioscorin purified from D. alata (cv. Tainung No. 1) (TN1-
dioscorin) and its peptic hydrolysates showed ACE inhibitory activities in a dose-
dependent manner [41]. The kinetic analysis of dioscorin showed a mixed
noncompetitive inhibition against ACE. Their results suggested that dioscorin iso-
lated from Dioscorea might be beneficial in controlling high blood pressure
[105, 106].
Chen et al. [35] reported the effects of Taiwanese yam (Dioscorea alata Cv
Tainung No.2) on mucosal hydrolase activities and lipid metabolism in male Balb/
c mice. High level of Tainung No.2 yam in the diet (50% w/w) reduced plasma and
hepatic cholesterol levels and increased fecal steroid excretions in the mice model.
This could be due to the loss of bile acid in the enterohepatic cycle to fecal excretion
[35]. They further suggested that the increased viscosity of the digest and the
thickness of the unstirred layer in the small intestine caused by Tainung No.2 yam
fiber (and/or mucilage) decreased the absorption of fat, cholesterol, and bile acid.
Short-term (3 week) consumption of 25% Tainung No.2 yam in the diet could reduce
the anthrogenic index, but not total cholesterol level in nonhypercholesteromic mice.
However, additional dietary yam (50% yam diet) could consistently exert hypo-
cholesterolemic effects in these mice [35]. However, diosgenin was not elucidated in
Tainung No.2 used in this study [35]. Thus, authors suggested that diosgenin might
not be involved in the cholesterol lowering effect of Tainung No.2 yam. Dietary fiber
and viscous mucilage could be active components for the beneficial cholesterol
lowering effects of yam. Further, short-term consumption (3 week) of 25%
uncooked keelung yam effectively reduced total blood cholesterol levels and the
atherogenic index in mice. Authors indicated that the active components for the lipid
lowering effects may be attributed to dietary fiber, mucilage, plant sterols, or
synergism of these active components [35].
5.7 Antiobesity
Obesity resulting from the chronic disruption of energy balance in the body has
become an epidemic globally. Hypertrophy and hyperplasia of fat cells is clearly
explained for their unique contribution in pathology of obesity. There are a number
of behavioral, metabolic, and biological influences leading to obesity [107]. In
addressing the management of obesity phytochemicals may be potential agents
targeting increasing energy expenditure, fat oxidation or inhibiting adipose tissue
proliferation.
Differentiation and proliferation inhibitory activities of sporamin of sweet
potato tubers were reported in 3 T3-L1 preadipocytes [41]. It should be noteworthy
that sporamin did not exhibit any cytotoxic activity toward the model cell line
3 T3-L1 preadipocytes which have frequently been used to study differentiation of
adipocytes in vitro. Concentration range of 0.025–1 mg/ml sporamin showed
antidifferentiation and antiproliferation effects on 3 T3-L1 cells similarly to
0.02 mg/ml berberine. Berberine is a traditional Chinese medicine used as an
antimicrobial and antitumor agent [41]. Hwang et al. [108] demonstrated that
purple sweet potato is a potential food, which can be used for the prevention of
obesity. Anthocyanin fractions of purple sweet potato inhibited hepatic lipid
accumulation through the induction of adenosine monophosphate activated protein
kinase (AMPK) signaling pathways. AMPK plays an important role in the regu-
lation of lipid synthesis in metabolic tissues [108]. Anthocyanin dose of 200 mg/kg
of body weight per day reduced weight gain, hepatic triacylglycerol accumulation
and improved serum lipid parameters in mice fed for 4 weeks with purple sweet
potatoes. Furthermore, anthocyanin administration increased the phosphorylation
of AMPK and acetyl coenzyme A carboxylase (ACC) in the liver and HepG2
hepatocytes. These authors further suggested that anthocyanin found in purple
sweet potatoes may improve high fat diet induced fatty liver disease and regulate
hepatic lipid metabolism [108].
5.8 Immunomodulatory Activities
Phytochemicals are reported to possess a number of bioactivities in management and

risk reduction of NCDs and immunomodulatory and anti-inflammatory properties
are reported among others. The mechanisms of action include effects on enzyme
function involved in the generation of inflammatory mediators such as nitric
oxide (NO), prostanoids and leukotrienes, regulation of gene, and protein expression
[109].
The effects of purified dioscorin from yam tubers on native BALB/c mice spleen
cell proliferation were assayed by MTT assay [26]. Dioscorin stimulated mouse
macrophage-like cells RAW264.7 to produce nitric oxide (NO), in the absence of
lipopolysaccharide (LPS) contaminations. Yam dioscorin exhibited immunomodu-
latory activities by the innate immunity which is a nonspecific immune system which
comprises the cells and mechanisms that defend the host from infection by other
organisms in a nonspecific manner. Dioscorin was reported to stimulate cytokine

production and to enhance phagocytosis. Furthermore, the released cytokines may
act synergistically with phytohemagglutinin (PHA) which is a lectin found in plants
that stimulate the proliferation of splenocytes [26].
Yam mucopolysaccharides (YMP) demonstrated the immune activity in sev-
eral cell lines. In vitro cytotoxic activity of murine splenocyte against leukemia
cells was increased in the presence of YMP of Dioscorea batatas at 10 μg/ml
[110]. Furthermore, the production of interferon-γ (IFN-γ) was significantly
increased in the YMP-treated splenocytes, suggesting their capability of inducing
cell-mediated immune responses. In addition, YMP at a concentration of 50 μg/ml
increased uptake capacity and lysosomal phosphatase activity of peritoneal macro-
phages [110].
Bioactive compounds from Dioscorea enhanced murine splenocyte proliferation
ex vivo and improved regeneration of bone marrow cells in vivo [111]. Mice which
were fed with a Dioscorea extract recovered damaged bone marrow progenitor cells
that had been depleted by large doses of 5-flourouracil (5-FU). In addition, they
found that the compound(s) responsible for these bioactivities had a high molecular
weight (100 kDa) and were most likely polysaccharides. The high molecular
weight polysaccharides in Dioscorea crude extract–II (DsCE-II) may act on specific
target cell types such as dendritic cells, intestinal epithelial cells, and T-cells in the GI
tract to mediate a cascade of immune regulatory activities. This may lead to the
recovery of damaged cell populations exposed to 5-FU or other chemical insults in
the bone marrow, spleen, or other immune cell systems [111].
Oral administration of Dioscorea tuber mucilage from Taiwanese yams
(Dioscorea Japonica Thunb var) showed significant effects on the innate immunity
and adaptive immunity on BALB/c mice [112]. It was interesting to note that the
specific antibodies rapidly responded against foreign proteins (or antigens) in the
presence of yam mucilage. Mucilage from these yam varieties exhibited a stimula-
tory effect on phagocytic activity by granulocyte and monocyte (ex vivo), on
peritoneal macrophages, and on the RAW 264.7 cells (in vivo) of mice [112].
Yams (Dioscorea esculenta) demonstrated anti-inflammatory activities on carra-
geenan-induced edema in the right hind paw of Wistar rats [113]. However, it was
noted that the activity was short lived as it was quickly removed from the system
after reaching the peak within 2 hours. Phytochemical screening of D. esculenta
confirmed the presence of saponins, β-sitosterol, stigmasterol, cardiac glycosides,
fats, starch, and diosgenin, which could be responsible for the observed activity
[113]. Diosgenin contained in Chinese yam was an immunoactive steroidal saponin
which also showed prebiotic effects. Diosgenin had also beneficial effects on the
growth of enteric lactic acid bacteria [61].
In a human trial the impact of the consumption of pigmented potatoes on
oxidative stress and inflammatory damages has been demonstrated [114]. In this
study participants were administrated white-, yellow- (high concentrations in phe-
nolic acids and carotenoids), or purple-fleshed (high content of anthocyanin and
phenolic acids) potato once per day in a randomized 6-week trial which reported
good compliance. The results showed that the consumption of pigmented potatoes
was responsible for elevated antioxidant status and reduced inflammation and DNA
damage, which was observed through the reduction of inflammatory cytokines and
C-reactive protein concentrations [114].
Methanolic extracts of Amorphophallus campanulatus (Suran) tuber also showed
immunomodulatory activities in Wistar albino mouse model [115]. The mice were
immunized with sheep red blood cells to assess delayed type hypersensitivity and
charcoal clearance test. Active phagocytosis is the major defense mechanism against
infection. They indicated that the presence of steroids and flavonoids in
Amorphophallus campanulatus (Suran) tuber may be responsible for the observed
immunomodulatory activity [115].
5.9 Antimicrobial Activity
Yam varieties with their phenolic compounds are potential agents with antimi-
crobial efficacy. Sonibare and Abegunde [116] reported that the methanolic
extracts of Dioscorea yams (Dioscorea dumetorum and Dioscorea hirtiflora)
showed antioxidant and antimicrobial activities. Antimicrobial activity was deter-
mined by the agar diffusion method (for bacteria) and pour plate method (for
fungi). Nonedible D. dumetorum showed the highest in vitro antibacterial activity
against Proteus mirabilis. The methanolic extracts from D. hirtiflora demon-
strated antimicrobial activity against all tested organisms, namely Staphylococcus
aureus, E-Coli, Bacillus subtilis, Proteus mirabilis, Salmonella typhi, Candida
albicans, Aspergillus niger, and Penicillium chrysogenum. Saponin present in
yams (Dioscorea) also has tendency to ward off microbes [117]. These com-
pounds serve as natural antibiotics, helping the body to fight against infections
and microbial invasion [117].
6 Conclusions
Foregoing evidences support the fact that roots and tubers are important components
in the everyday diet for humans adding variety in the plate and a number of health
implications to attenuate NCDs. They perform a pivotal role as an energy contributor
while providing many desirable nutritional and health benefits, namely, anti-
oxidative, hypoglycemic, hypocholesterolemic, antimicrobial, and immunomodula-
tory activities among others. There are a wide array of roots and tubers which can be
utilized in preparation of numerous commercial foods though usage may vary with
the country and region. Thus, tubers may serve as functional foods and nutraceutical
ingredients to attenuate NCDs and to maintain wellness.
Acknowledgment This research was supported by the Research Grant Scheme of Wayamba
University of Sri Lanka through a grant (SRHDC/RP/04/13-09) to AC. The author wishes to
thank members of the research team Apeksha Herath, Jayani Wijerathne, Upuli Dahanayake,
Thamilini Joshepkumar, and Saman Ranasinghe at Wayamba University of Sri Lanka.
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Betalains: Application in Functional Foods
49
Wee Sim Choo
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472
2 Chemical Properties of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1474
3 Biological and Pharmacological Properties of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1478
3.2 Anticancer Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1481
3.3 Anti-Lipidemic and Lipid Oxidation Inhibition Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483
4 Application of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1486
4.1 Natural Colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1486
4.2 Other Applications of Betalains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1488
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1488
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489
Abstract
Betalains are plant derived natural pigments that are presently gaining popularity
for use as natural colorants in the food industry. Although betalains from red beet
are one of the most widely used food colorant, betalains are not as well studied as
compared to other natural pigments such as anthocyanins, carotenoids, and
chlorophylls. This chapter describes the chemical properties of betalains includ-
ing their biosynthesis. Two main classes of betalains, betacyanins and
betaxanthins, are described. Current reports on the biological and pharmacolog-
ical properties of betalains from four main sources such as red beet, amaranth,
prickly pear, and red pitahaya are also described. The biological and pharmaco-
logical properties of betalains that are covered in this chapter include antioxidant,
W. S. Choo (*)
School of Science, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway,
Selangor, Malaysia

1472 W. S. Choo
anticancer, anti-lipidemic, and antimicrobial activities. Lastly, this chapter

describes the application of betalains as natural colorants and other potential
application in functional foods.
Keywords
Amaranth · Beetroot · Betacyanin · Betaxanthin · Cactus pear · Colorant · Prickly
pear · Red dragon fruit · Red pitahaya
1 Introduction
Betalains are one of the most common plant pigments found in nature besides
carotenoids, chlorophylls, and anthocyanins. Unlike anthocyanins or chlorophylls
which are ubiquitous in the plant kingdom, betalains are found in a much smaller
group of plants. It has been established that in the plant kingdom there is mutual
exclusivity with respect to betalains and anthocyanins whereby both pigments have
never been reported in the same plant [1]. The occurrence of betalains are restricted
to the order Caryophyllales and some higher fungi such as Amanita muscaria
(fly agaric) [2]. Betalains can be found in the families of the Caryophyllales
(Achatocarpaceae, Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Chenopodiaceae,
Didiereaceae, Halophytaceae, Hectorellaceae, Nyctaginaceae, Phytolaccaceae,
Portulacaceae, and Stegnospermataceae). Only two families of the Caryophyllales,
the Caryophyllaceae and Molluginaceae produce anthocyanins [3].
The most common and widely known source of betalains are those belonging to
the Amaranthaceae (namely, Beta vulgaris L. and Amaranthus sp.) and Cactaceae
families (namely, Opuntia sp. and Hylocereus sp.) [4, 5]. Beta vulgaris subsp.
vulgaris or commonly known as red beet or beet root (Fig. 1a) is mostly cultivated
in North America, Central America, and Europe. Red beet is the major commercially
exploited betalain crop and a vegetable characteristic of the Eastern and Central
European diet. Red beets are not only consumed as fresh vegetables but are also
processed to obtain desiccated or frozen products, juices, and their concentrates as
well as natural pigments used as food additives [6]. Amaranthus or collectively
known as amaranth (Fig. 1b) is a cosmopolitan genus of annual or short-lived
perennial plants made up of about 60 species. Some amaranthus are cultivated as
leafy vegetables, grains, or ornamental plants, while others are weeds. Amaranthus
are cultivated and consumed as a leafy vegetable in many parts of the world. Certain
Amaranthus species are consumed as cooked or fresh [7, 8]. Several Amaranth
species are raised as grains in Asia and America. Opuntia sp. fruits or commonly
known as prickly pears or cactus pears (Fig. 1c) are widely distributed in Mexico,
Central and Southern America, Australia, South Africa, and the Mediterranean.
Prickly pears have edible cladodes, but its sweet and colored fruits are the most
desirable and commercially valuable parts of this plant and present economic
relevance throughout arid and semiarid areas of the world [9, 10]. Hylocereus
polyrhizus or commonly known as red pitahaya or red dragon fruit (Fig. 1d) is
well known for its aesthetically pleasing deep purple color pulp with numerous small
49 Betalains: Application in Functional Foods 1473
Fig. 1 Common sources of betalains (a) Red beet, (b) Amaranth (Source: iStock photos),
(c) Prickly pear (Source: Peterson [11]), (d) Red pitahaya (Source: iStock photos)
soft seeds. This fruit has high appeal in the European and United States market and is
cultivated in Asia and Australia.
Stenocereus sp. is an emerging common source of betalains belonging to the
Cactaceae family. S. pruinosus, S. stellatus, and S. queretaroensis are endemic of
Mexico. These species produce fruits named pitayas [12]. Fruits are spherical or
ellipsoid, spiny, green or red peeled berries [13] with juicy flesh of different colors
such as white, yellow, pink, orange, red, or purple [12]. In several references
[14–17], the name pitaya has also been given to fruit of the species Hylocereus
sp., but there are notable differences between the fruits from these two genera. The
fruits of Hylocereus sp., named pitahayas, do not have spines, possess certain type of
bracts, and their size is higher than that of the fruits of Stenocereus sp. [12].
1474 W. S. Choo
Betalains are present in other plant families in interesting amount, such

as Baselaceae, Asparagaceae, Nictaginaceae, Pandanaceae, Phytolaccaceae, and
Portulacaceae. Basella rubra L. and Ullucus tuberosus are the most studied sources
of betalains belonging to the from the Basellaceae family; Cordyline fruticosa
belonging to the Asparagaceae family, Mirabilis jalapa L. from the Nyctaginaceae
family, Pandannus amaryllifolius from the Pandanaceae family, Rivina humilis L.
from the Phytolaccaceae family, and Talinum triangulare from the Portulacaceae
family. In a plant, betalains can be present in seeds, leaves, flowers, sprouts, aerial
parts, roots, and even in fruits [18]. The occurrence of betalains in different parts of
plants has been discussed comprehensively in a recent review by Martin et al. [18].
Betalains play important roles in plant physiology and visual attraction for pollina-
tors and seed dispersers [19]. In some plants they also have specific functions.
Betalains were discussed to provide protection from the harmful effects of ultraviolet
radiation in the ice plant (Mesembryanthemum crystallinum L.) [20]. In cactus thorns
betalains provide insect-repelling signals whereas for underground plant like red
beet, they can improve the resistance to soil pathogens [19]. In red beet, betalains act
as reactive oxygen species scavengers, limiting damage caused by wounding and
pathogen infiltration in the plant tissues [21]. In food, betalains serve two functions.
Betalains improve the aesthetic value of foods and contribute to consumers’ health
and well-being [19].
In this chapter, the chemical properties of betalains including their biosynthesis
are described. The biological and pharmacological properties of betalains from the
four main common sources, namely, red beet, amaranthus, prickly pear, and red
pitahaya and their application in functional foods or foods that potentially offer
health benefits beyond basic nutrition are also covered in this chapter.
2 Chemical Properties of Betalains
Betalains are water-soluble nitrogen-containing pigments which can exist as the red-
violet betacyanins or yellow betaxanthins. These two main classes of betalains can
occur in the same plant part, despite the difference in coloration. However,
betacyanins occur with greater frequency than betaxanthins. In early days,
betacyanins were addressed erroneously as nitrogenous anthocyanins whereas
betaxanthins were called flavonoids [22]. The term “betalains” was first introduced
by Mabry and Dreiding in 1968 [23]. Betalains are immoniun derivatives of
betalamic acid (Fig. 2) that is based on the protonated 1,2,4,7,7-pentasubstituted
1,7-diazaheptamethin system with a betalain color being attributable to the resonat-
ing double bonds (Fig. 2) [24].
When the basic structure is substituted with an aromatic nucleus, a change in the
absorption maximum from 540 nm (red-violet betacyanins; Table 1) to 480 nm
(yellow betaxanthins; Table 2) is observed [24]. To date, there are 51 and 23 different
betacyanin and betaxanthin structures identified, respectively [18]. Common names
of betacyanins or betaxanthins are assigned in relation to the plant from where they
were first isolated. Examples are given in Tables 1 and 2 [22, 24, 25]. Betacyanin
Fig. 2 Resonance structure of betalain
Table 1 Examples of betacyanins

R1 O
H
+ COOH
N
R2O
HOOC N COOH
H
Substituent group
Name R1 R2 Plant source
Betanin β-glucose H Beta vulgaris
Amaranthin 20 -O-(β-glucuronic acid)-β-glucose H Amaranthus
tricolor
Hylocerenin 3-methyl-3-hydroxy methyl glutaryl H Hylocereus
polyrhizus
Phyllocactin 60 -O-(malonyl)-β-glucose H Phyllocactus
Hybridus
Celosianin-I 20 -O-[O-(p-coumaroyl)-β-glucuronic acid]-β- H Celosia
glucose cristata
Gomphrenin-I H β- Gomphrena
glucose globosa
Iresinin-I 20 -O-(β-glucuronic acid)-60 - O –(3-hydroxy-3- H Iresine herbstii
methylglutaryl)-β-glucose
Rivinianin 30 -O-(SO3H)-β-glucose H Rivinia humilis
1476 W. S. Choo
Table 2 Examples of betaxanthins

R1 R2
+
N
HOOC N COOH
H
Substituent group
Name R1 R2 Plant source
Indicaxanthin Proline Proline Opuntia ficus-indica
Portulacaxanthin-I Hydroxyproline Hydroxyproline Portulaca grandiflora
Vulgaxanthin-I Glutamine H Beta vulgaris
Vulgaxanthin-II Glutamic acid H Beta vulgaris
Miraxanthin-II Aspartic acid H Mirabilis jalapa
Humilixanthin Hydroxynorvaline H Rivinia humilis
a b
Fig. 3 (a) Betanidin (b) cylco-DOPA
structures show variations in their sugar or acyl groups whereas betaxanthins show
conjugation with a wide range of amines or amino acids in their structures [26].
The aglycon betanidin (Fig. 3a) is the backbone of all betacyanins. The occur-
rence of different betacyanin structures is due to the glycosylation and acylation
of the resulting 5-O- or 6-O-glucosides. The most common glycosyl moiety is
glucose, although sophorose and rhamnose could also occur, but less frequently.
The most common acyl groups are sulfuric, malonic, 3-hydroxy-3-methylglutaric,
citric, p-coumaric, ferulic, caffeic, and sinapic acids [27]. Betanin (betanidin 5-O-ß-
glucoside) from red beet was the most studied betacyanin pigment [22]. Betacyanins
can be further classified into four groups: betanin, gomphrenin, amaranthin, and
Table 3 General Betacyanin Betaxanthin

classification of betalains
Betanin-type group Amino-acid-derived conjugate
Betanin group
Prebetanin Portulacaxanthin II
Neobetanin Portulacaxanthin III
Phyllocactin Tryptophan-betaxanthin
20 -apiosyl-phyllocactin Tyrosine-betaxanthin
Hylocerenin
Lampranthin I, II
Rivinianin
Amaranthin-type group Amine-derived conjugate
Amaranthin group
Iresinin Vulgaxanthin-I
Celosianin 3-Methoxytyroamine-
betaxanthin
Gomphrenin-type group
Gomphrenin class I, II, III,IV
Betaninidin 6-O-sophorosides
derives
Bougainvillein-type group
Bougainvillein r-I, r-III
Bougainvillein-v
Mammillarinin
Adapted from Slimen et al. [22] and Pavokovic and Krsnik-Rasol [28]
bougainvillein (Table 3). These groups differ by the attachment of glucosyl groups to
the oxygen atoms in the ortho position on the cyclo-DOPA (Fig. 3b) moiety. The
betanin-type group has a hydroxyl group linked to the C6 carbon and a glucosyl
group on the C5 one. Gomphrenin- type group is known as structural isomers of
betanins with a hydroxyl group attached to the C5 carbon and a glucosyl linked to
the C6 carbon [2]. Amaranthin-type group differs from the betanin-type group by
having two contiguous glucosyl groups attached to the C5 carbon [28].
Bougainvillein-type group is known as carboxylated or decarboxylated betanins
[22]. Betacyanins are known to display two absorption maxima, one in the UV-
range between 270 and 280 nm due to the cyclo-DOPA structure and a second one in
the visible range at about 540 nm [19].
In betaxanthins, the cyclo-DOPA unit of betacyanins is replaced by an amino acid
or amine. Hence, betaxanthins can be classified into two groups: amino-acid-derived
conjugate group and amine-derived conjugate group (Table 3). Indicaxanthin
from cactus pear was the first yellow pigment structurally characterized, having
proline as substituent. It is also the most commonly studied betaxanthins besides
vulgaxanthin I with glutamine as substituent. Vulgaxanthin I is the most abundant
betaxantin found in Beta vulgaris [22]. Condensation of betalamic acid with various
amino acids or amines in betaxanthins produce hypso- and bathochromic shifts [19].
The maximum absorption of betaxanthins varies from 460 to 480 nm and is
associated with the structure of the amino or amine conjugates. Amine conjugates
display a lower absorption maximum than their respective amino acids [29]. The
1478 W. S. Choo
highest absorption maximum was displayed by indicaxanthin and portulacaxanthin I

with proline and hydroxyproline as a substituent, respectively [29, 30].
The biosynthesis of betalains was studied since the 1950s and their biosynthesis is
summarized in a number of reviews [2, 31–36]. Betalains arise from arogenate of the
shikimate pathway whereby arogenate is converted to L-tyrosine, an amino acid, via
arogenate dehydrogenase [37, 38]. Betalains are therefore secondary metabolites
derived from the amino acid L-tyrosine. The pathway involved in the biosynthesis of
betalains begins with the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine
or better known as L-DOPA [39] through the monophenolase activity of the enzyme
tyrosinase (or polyphenoloxidase) [31, 40]. Betalamic acid is derived from L-DOPA
by spontaneous intramolecular condensation through the activity of the enzyme 4,5-
DOPA-extradiol-dioxygenase [31]. Condensation of betalamic acid with another
DOPA derivative such as cyclo-DOPA produce red colored betacyanins whereas
condensation of betalamic acid with an amino acid or amine produce yellow
betaxanthins [1]. Figure 4 shows the biosynthetic pathway of betanidin, a betacyanin
and indicaxanthin, a betaxanthin as examples of betalains [1].
3 Biological and Pharmacological Properties of Betalains
Betalains play an important role in human health because of their biological and
pharmacological properties/activities such as antioxidant, anticancer, anti-lipidemic,
antimicrobial, etc. [5]. Although various pharmacological activities of betalains have
been observed in vivo, the use of extracts limits the conclusions drawn, the hypoth-
esis on the mechanisms involved, and the therapeutic potential of the assays. To date,
studies with purified pigments are scarce, but they provide exciting conclusions [41].
Also, the likely optimum concentration for daily intake may only be decided after
systematically addressing stability and bioavailability issues of betalains [42]. There
are lack of stability and bioavailability studies on different groups of betalains.
Current knowledge on the stability or bioavailability of betalains has been discussed
in a number of reviews [4, 18, 42–46]. Current reports on the antioxidant, anti-
cancer, anti-lipidemic, and antimicrobial activities of betalains from red beet, prickly
pear, amaranth, and red pitahaya are covered in this section.
3.1 Antioxidant Activity
A broad definition of antioxidant is “any substrate that, when present at low

concentrations compared to those of an oxidizable substrate, significantly delays
or prevents oxidation of the substrate” [47]. The term “oxidizable substrate” includes
almost everything found in foods and in living tissues including proteins, lipids,
carbohydrates, and DNA. The target chosen and the source of oxidative damage
therefore play paramount importance in characterizing an antioxidant [47]. The
bioavailability of betalains is at least as high as flavonoids, which are well-accepted
natural antioxidants. Betalains, as natural antioxidants, may provide protection
Fig. 4 Biosynthetic pathway of betanidin and indicaxanthin
against oxidative stress-related disorders [6, 48]. The antioxidant activity of

betalains has been studied extensively and shown in several chemical and biological
models [6, 22, 49–54] including the ex vivo oxidation of human low-density
lipoproteins (LDL). However, ex vivo oxidation studies of human LDL or lipid
inhibition studies are covered in a separate following section. The antioxidant
activity of betalains in vitro was investigated using the DPPH (1,1-diphenyl-2-
picrylhydrazyl) free radical scavenging assay, ABTS (2,20 -azinobis(3-ethylben-
zothiozoline-6- sulfonic acid) diammonium salt) radical scavenging assay, electron
spin resonance spectroscopy, etc. Some studies measured the TEAC (trolox equiv-
alent antioxidant capacity) assay that is based on the ability of an antioxidant to
1480 W. S. Choo
scavenge ABTS radical cation relative to that of the water-soluble vitamin E

analogue, Trolox [55].
In vitro studies of betalains from red beets have demonstrated that they possess
high antiradical and antioxidant activity. The free radical-scavenging activity of
betanin from red beet measured in a TEAC assay at pH 7.4 and in a DPPH assay
was about 7.5- and 3.0-fold higher, respectively, than that of vitamin C, which is
commonly accepted as an effective natural water soluble antioxidant [50, 56].
Betalains from red beet in a TEAC assay were also found to have 1.5–2.0-fold
greater free radical scavenging activity than some anthocyanins [56] such as
cyanidin-3-O-glucoside and cyanidin, above pH 4 [57]. Purified betanin and
indicaxanthin from prickly pear were more effective than Trolox at scavenging
ABTS radical [49]. Similarly, EC50 values determined using the DPPH radical-
scavenging activity method for pulp and peel extracts of red pitahaya containing
betalains were 22.4 0.29 and 118 4.12 mol vitamin C equivalents/g, respec-
tively [58]. However, betalain extracts of red pitahaya also have high content of
ascorbic acid which acts as potential antioxidant. Isolated betacyanins such as
betanin, phyllocactin, hylocerenin, and their isomers are therefore required to assess
their antioxidant capacity separately [59]. Betalains from Amaranthaceae deter-
mined using DPPH method were found to have EC50 values ranging from 3.4 to
8.4 μM. Gomphrenin type betacyanins and betaxanthins assessed using the DPPH
method demonstrated the strongest antioxidant activity which was three to four times
stronger than ascorbic acid and also stronger than other potent antioxidants such as
rutin and catechin [50]. In addition, betalain pigments from various plants of the
family Amaranthaceae exhibited strong antioxidant activity presented as mean EC50
value (μM): amaranthine or isoamaranthine (8.37 or 8.35), iresinins (8.08),
celosianins (7.13), betanin or isobetanin (4.88 or 4.89), 3-methoxy-tyramine-
betaxanthin (4.21), (S)-tryptophan-betaxanthin, dopamine-betaxanthin (4.08), acyl-
ated gomphrenins (4.11), and simple gomphrenins (3.35) [50]. Although free radical
scavenging activity investigated using the DPPH- or TEAC-assay can provide
valuable insights on the mechanisms of action, it is important to note that in vitro
chemical assays bear no similarity to biological systems including the absorption of
antioxidant compounds by the human body [60]. The measurements of the in vitro
antioxidant activity of betalains therefore needs to be carefully interpreted [5].
Betanin from red beet was reported to dose-dependently scavenge DPPH-,
galvinoxyl-, superoxide-, and hydroxyl-radicals in electron spin resonance spectros-
copy and spin trapping studies and prevented hydrogen peroxide induced cellular DNA
damage in cultured HT-29 cells. Furthermore, betanin treatment induced the transcrip-
tion factor Nrf2 and resulted in an increase of heme oxygenase 1 (HO-1) protein levels,
PON1-transactivation and cellular glutathione (GSH). Nrf2 is a transcription factor
regulating the expression of genes encoding proteins important in antioxidant defense.
The antioxidant enzyme HO-1 is a Nrf2 target gene. The hepatic enzyme PON1 also
exhibits antioxidant and antiatherogenic activity. GSH is an important cytosolic anti-
oxidant centrally involved in redox signaling and stress response. These studies
suggested that betanin is both a free radical scavenger and an inducer of antioxidant
defense mechanism in cultured cells [61]. Red beet pomace containing betalains was
evaluated on three different free radical species: DPPH, OH and O2 using ESR
spectroscopy and was found to show better scavenging activity (EC50

OH = 0.0655 mg/mL, EC50DPPH = 0.0797 mg/mL, and EC50O2 = 1.0625 mg/
mL) than those of butylated hydroxyanisole (BHA) (EC50DPPH = 0.180 mg/mL,
EC50OH = 1.505 mg/mL, and EC50O2 = 2.680 mg/mL) [62].
The antioxidant capacity of betacyanins from red pitahaya was evaluated using
the peroxyl radical generating system in the presence of AAPH and the peroxyl
radical scavenging capacity was dose-dependent in the low concentration range
(25–100 nM). The mol-Trolox equivalent activity/mol compound (mol-TEA/mol-
compound) as an index of the antioxidant capacity indicated 3.31 and 2.83 mol-
TEA/mol-compound for betanin and phyllocactin, respectively [54]. Betalains from
red beets inhibited peroxynitrate-mediated tyrosin nitration and DNA strand cleav-
age [63]. It was also found that pre-treatment of HT-29 cells with betalains signif-
icantly reduced hydrogen peroxide induced DNA damage [61]. The nitrogen
scavenging activity of betacyanins was examined using NOR3 as an NO donor,
and it was found that the IC50 values of nitrogen radical scavenging activity were
24.48, 17.51, 6.81 for betanin, phyllocactin, and betanidin, respectively [54]. The
reduction of the nitrite level when treated with betacyanins was high in comparison
to flavonoids such as aromadendrin, quercitrin, and loliolide [64]. These studies
demonstrated the potential DNA-protective effect of betalains against reactive oxy-
gen and nitrogen radicals.
Sources of betalains such as cacti fruits and red beet exhibited strong antioxidant
activities in biological environment [4] by inhibiting lipid peroxidation and heme
decomposition at very low betalain concentrations. Betalains have been shown to
protect vascular endothelium cells, which are direct targets of oxidative stress in
inflammation from cytokine-induced redox state alteration [65]. Betalains also
counteract lipoperoxidases which may damage gastrointestinal cells during food
digestion [6, 50]. Human ingestion of a single dose of red beet juice resulted in an
increase of antioxidants in the system with only 0.28% of betalains excreted in urine
within the 24 h after consumption [66]. The oral administration of betalains from red
beets (5, 20 or 80 mg/kg body weight) in mice exposed to gamma irradiation
significantly enhanced the activity of antioxidant enzymes such as superoxide
dismutase (SOD) and gluthathione peroxidase (GPx) in the liver, spleen, and kidney,
in a dose-dependent manner [67]. Opuntia ficus-indica extracts containing betalains
were also found to counteract the release of acetaldehyde in blood due to chronic
alcohol intake by restoring the malondialdehyde (MDA) and GSH level in the
erythrocytes [68]. The use of in vivo assays using mice models and human trials
permit the detection of gross effects resulting from multiple mechanisms but they
reveal little about the individual mechanisms of activity [69]. A combination of in
vitro and in vivo studies may be able to provide an efficient and effective approach to
screen the potential antioxidant activity of betalains [5].
3.2 Anticancer Properties
Cancer is a growing problem worldwide that had surpassed common infectious

diseases as the second leading cause of death globally [70]. Different approaches
1482 W. S. Choo
are being explored for the prevention and treatment of cancer [71]. Exploring natural
compounds for their anticarcinogenic properties is one of the major approaches to
prevent or treat cancer. In this context, the establishment of health benefits of edible
fruits through in vitro anticancer assays would substantiate their potential as func-
tional foods [72].
Betanin from red beet showed excellent growth inhibition of MCF-7 (breast),
HCT-116 (colon), AGS (stomach), SF-268 (CNS), and NCI-H460 (lung) cancer cell
lines with IC50 values of 162, 142, 158, 164, and 147 μg/mL, respectively [73].
Betanin/isobetanin concentrate from red beet was reported to significantly decrease
cancer cell proliferation and viability of MCF-7-treated cells. The expressions of
apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and
the mitochondrial membrane potential was altered, demonstrating the involvement
of both intrinsic and extrinsic apoptotic pathways [74]. Betanin from red beet
resulted in a 49% inhibition of HepG2 cell proliferation at 200 μg/mL, and betaine
yielded a 25% inhibition at 800 μg/mL [75]. The in vitro inhibitory effect of red beet
extract (betalains) on Epstein-Barr virus early antigen (EBV-EA) induced by the
tumor promoter 12-O-tetradecanoylphorbol-I3-acetate revealed a high order of
activity compared to paprika (capsanthin), cranberry (anthocyanins), red onion
skin (anthocyanins), and short and long red bell peppers (carotenoids). An in vivo
anti-tumor promoting activity evaluation against the mice skin and lung bioassays
also revealed a significant tumor inhibitory effect [76]. Red beet extract containing
betalains exhibited a lower cytotoxic profile compared to doxorubicin (a red colored
anticancer antibiotic drug) in in vitro studies utilizing two cancer cell lines of human
origin (prostate PC-3 and breast MCF-7). However, it had minimal effect on the
growth of normal human skin (FC) and liver (HC) cells in contrast to doxorubicin. It
was suggested that as a dietary supplement, the red beet extract may have the
potential to lower the serious adverse effects associated with doxorubicin
(adriamycin) therapy [77]. Red beet extracts containing betalains were also found
to counteract the lethal effects of the chemical carcinogen 7,12-dimethylbenz[a]
anthracene (DMBA) in the livers of Sprague- Dawley rats. This was achieved by a
reduction in activity of EROD (CYP1A1 marker) and MROD (marker of CYP1A2)
and an increase in the activities of hepatic phase II enzymes, gluthathione
S- transferase (GST) and NAD(P)H:quinone oxidoreductase-1 (NQO1) [78]. Simi-
larly, the effect of red beet extracts containing betalains on MCF-7 and MRC-5 (fetal
lungs) cell lines determined by using the sulforhodamine B (SRB) assay resulted in
inhibiting concentration (IC50) values in the range of 362–504 and 383–588 μg/mL
for MRC-5 and MCF-7, respectively [79]. Red beet pomace extract containing
betalains exhibited cytotoxic properties against Ehrlich carcinoma (EAC) cells in
vivo. The EAC cell numbers were decreased in all extract-treated groups compared
with the untreated EAC control group. The largest decreases in EAC cell numbers
were observed in the pretreated male (approximately 53%) and female (approxi-
mately 47%) mice, and also the EAC cell viability was decreased after administra-
tion of red beet pomace extract containing betalains [62]. In three different
experimental tumor models in mice, a very low dose of betanin (0.0025%) from
red beet acted as a potent chemopreventive agent [80]. Red beet extract containing
betalains consistently reduced multiorgan tumor formations in various animal

models when administered in drinking water (25–78 μg/mL, equivalent to a daily
intake of about 6–20 μg of beetroot betacyanins/kg body weight) [76, 81]. A long-
term daily exposure to low doses of red beet extract through diet was postulated to be
safe and sufficient to produce cancer chemopreventive effect in humans [82].
Immortalized ovarian and cervical epithelial cells as well as ovarian, cervical, and
bladder cancer cells that were exposed to prickly pear (Opuntia ficus-indica) extracts
containing betalains displayed a significant increase in apoptosis and growth inhi-
bition in a dose- and time-dependent manner. The extracts containing betalains
affected cell cycle of cancer cells by increasing G1 and decreasing G2 and S phases,
significantly suppressed tumor growth in nude mice, increased annexin IV expres-
sion, and decreased VEGF by modulating the expression of tumor-related genes
[83]. Betanin from Opuntia ficus-indica fruits were found to possess an anti-
proliferative activity in K562 cells with an IC50 of 40 μM. Betanin induced apoptosis
in K562 cells through the intrinsic pathway and is mediated by the release of
cytochrome c from mitochondria into the cytosol and poly (ADP) ribose polymerase
(PARP) cleavage [84]. Another study showed that prickly pear extracts containing
betalains exhibited a significant growth inhibitory effect on human ovarian cancer
cells similar to a synthetic chemopreventive agent, 4- HPR, in nude mice [83].
Extracts containing betalains from prickly pears was found to be less effective in
inhibiting colon cancer cell (HT29) growth compared to doxorubicin, an anticancer
drug. Doxorubicin induced HT29 cycle arrest in G2/M phase [85] and thus com-
bining the drug treatment with extracts from prickly pears that arrested cells on
different cell cycle checkpoints (G1 and/or S phases) could be a promising strategy
to improve the anticancer effect and diminished chemoresistance as an adjuvant of
chemotherapy [86]. Screening of antiproliferative activity of extract containing
betalains from the fruit of red pitahaya on AGS (a human gastric adenocarcinoma
cell line), HeLa (a human cervical adenocarcinoma cell line), MCF-7 and B16F10
melanoma cell [87] was carried out but the results were not significant. To date, there
are no reported studies on the anticancer or antiproliferative activity of extract
containing betalains from Amaranthus. The use of cancer cell lines allowed the
investigation of potential anticancer compounds in a simplified, controlled, and
reproducible environment [88, 89]. However, the inability of cell cultures to behave
like tumors and their interactions with the host [89] means that the efficacy of
betalains as an anticancer agent in human clinical trials may not be the same. Further
in vivo studies in a model that closely approximates the targeted human cancer are
therefore needed [5].
3.3 Anti-Lipidemic and Lipid Oxidation Inhibition Effect
Betalains exhibited antioxidant effect to inhibit lipid oxidation. In a linoleate perox-

idation assay by cytochrome c, linoleate peroxidation was inhibited by betanin,
betanidin, catechin, and α-tocopherol with IC50 values of 0.4, 0.8, 1.2, and
5.0 mM, respectively. Betacyanins from red beet were therefore more potent
1484 W. S. Choo
antioxidants than catechin or tocopherol in inhibiting linoleate peroxidation [6].

Betaxanthins from red beet were found to inhibit lipid peroxidation by cytochrome
c at an IC50 of ~1.0 μM. In addition, betanin was almost twice as effective as catechin
in preventing LDL oxidation by H2O2-activated metmyoglobin [6]. Betanin from red
beet was found to inhibit lipid peroxidation in a model liposome oxidation assay
using fluorescence spectroscopy [73]. Betalains from prickly pears inhibited the
myeloperoxidase/nitrate-induced oxidation of human LDL by scavenging the initi-
ator radical nitrogen dioxide and lipoperoxyl radical [90]. A short-term supplemen-
tation study on healthy volunteers with prickly pear fruit showed a significant
decrease in 8-epiprostaglandin F2α (8-epi-PGF2α) and malondialdehyde in plasma,
the ratio of reduced to oxidized glutathione (GSH:GSSG) in erythrocytes, and lipid
hydroperoxides in LDL (these are biomarkers of oxidative stress) whereas supple-
mentation with 75 mg vitamin C did not significantly affect any marker of oxidative
stress [91]. Betalains in prickly pear were suggested to be responsible in the
protection of oxidative damage to lipid [6]. The lipid peroxidation inhibitory effects
of prickly pear fruit extracts containing betalains on fish oil, fish oil-in-water
emulsion, and linoleic acid were investigated using conjugated diene hydroperox-
ides, weight gaining, peroxide value, and thiobabituric acid reactive substances
assays. Prickly pear extracts containing betalains were able to show multiple lipid
peroxidation inhibitory effect on different stages of lipid oxidation products gener-
ation in oils and emulsion model systems [92]. In an ex vivo study in which pooled
human plasma from 10 healthy volunteers was incubated with either 25–100 μM
betanin or indicaxanthin isolated from prickly pear, incorporation of both com-
pounds in LDL was observed, with a maximum binding of 0:52 and 0:51 nmoles
of indicaxanthin and betanin, respectively, per mg LDL protein. Indicaxanthin-
enriched and betanin-enriched LDL were also more resistant than homologous
native LDL to copper-induced oxidation [93].
Lipid-lowering drugs are used for primary and secondary prevention of cardio-
vascular diseases. The use of plant extracts to treat or regulate the conditions of
lipidemia are gaining interest as they are known to have no side effects or toxicity
compared to common synthetic lipid lowering drugs [5]. Administration of an
aqueous extract of Amaranthus tricolor containing betalains at 200 mg/kg and
400 mg/kg body weight to diabetic rats significantly reduced blood cholesterol,
triglyceride, and LDL levels, and increased HDL level [94].
A study on hypercholesterolemic rats found that force feeding of 300 mg/kg
(body weight) of betalain containing extracts from pulps of Hylocereus polyrhizus
reduced their total cholesterol level by 43.5% [95], probably by increasing excretion
of bile acids. Feeding of betalains enriched beet crisps in mice resulted in a
significant decrease in serum glucose level, atherogenic index, isovaleric acid
concentration in the caecal, and body weight in rats. Furthermore, oral administra-
tion of betalains suppressed production of short-chain fatty acids, prevented the rise
in serum total cholesterol and triacylglycerol levels in dyslipidemic rat models [96].
Rats fed with a control diet containing mixture of powders containing red beet as a
source of betalains showed a significant reduction in their plasma total lipids, total
cholesterol, LDL, triglycerides, and the ratio of total cholesterol/HDL in different
degrees, while HDL increased significantly [97]. Betalains may therefore have poten-
tial application in the management of hyperglycemia and associated lipidemia [5].
3.4 Antimicrobial Activity
Betalains have been reported to exhibit antimalarial and antimicrobial effects, but
their effects are dependent on the dose and type of betalains present. Betalain rich
Amaranthus spinosus showed significant antimalarial activity in mice due to high
levels of betanin and amaranthin. These compounds were able to chelate the
indispensable inner cations (Ca2+, Fe 2+ and Mg2+) in the parasite and block the
parasites choline intracellular transport which is crucial for malarial parasite growth
[98]. Beetroot pomace containing betalains induced zones of reduced growth in
Salmonella Typhymurium, Staphylococcus aureus, and Bacillus cereus [99]. Beet
root pomace containing betalains was also found to inhibit Gram-negative bacteria
(Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, Citrobacter
youngae, Enterobacter cloacae, S. Typhimurium) with S. Typhimurium and C.
freundii being most susceptible [100]. Red beet pomace extract containing betalains
showed inhibitory activity against Gram-negative bacteria in which the most sus-
ceptible strains are S. Typhimurium and C. freundii. Among the Gram-positive
bacteria, S. aureus, Staphylococcus sciuri, and B. cereus showed high susceptibility.
However, the extract did not exert any activities towards wild strains Bacillus sp.,
Enterococcus faecalis and Listeria monocytogenes and did not inhibit the growth of
microorganisms with eukaryotic type of cells such as Saccharomyces cerevisiae,
Candida albicans, Aspergillus niger, and Penicillium aurantiogriseum [62]. Slight
antibacterial activity of methanolic extracts of red beet was also obtained against
E. coli and S. aureus and absence of antifungal activity against A. niger and
C. albicans were also reported by Rauha et al. [101].
Extracts of xoconostle pear (Opuntia matudae) containing betalains had a signif-
icant inhibitory effect on the growth of E. coli O157:H7 with an increase in
xoconostle pear extract concentration associated with a slower growth rate and
reduction in bacterial populations [102]. Betalains-rich fraction extracted from the
pulp of red pitahaya exerted a broad antimicrobial spectrum by inhibiting Gram-
positive bacteria (B. cereus, S. aureus, Escherichia faecalis, L. monocytogenes) at
7.8 μg/mL, Gram-negative bacteria (E. coli, Proteus mirabilis, Proteus vulgaris,
P. aeruginosa, Salmonella typhi Ty2, Yersinia enterocolitica, Klebsiella pneumonia,
Enterobacter cloacae, Enterobacter aerogenes) at 15.6–62.5 μg/mL, yeasts
(C. albicans, Rhizoctonia solani) at 125–250 μg/mL and molds (Fusarium
oxysporum, Cladosporium herbarum, Botrytis cinerea, Aspergillus flavus) at
500 μg/mL [103]. Betacyanin rich extract from red spinach (Amaranthus dubius)
(minimum inhibitory concentration [MIC] values: 0.78–3.13 mg/mL) demonstrated
a better antimicrobial activity profile than that of red pitahaya (MIC values:
3.13–6.25 mg/mL) against nine Gram-positive bacterial strains (S. aureus, Entero-
coccus sp., Bacillus sp.). Similarly, betacyanin rich extract from red spinach
(MIC values: 1.56–3.13 mg/mL) was more active than that of red pitahaya (MIC
1486 W. S. Choo
values: 3.13–6.25 mg/mL) against five Gram-negative bacterial strains (Shigella

flexneri, S. Typhimurium, K. pneumoniae, E. coli, P. aeruginosa) [104]. The antimi-
crobial activity of betalains may be due to their adverse effects on the structure,
function, and permeability of the cellular membranes of the microbes which eventually
leads to cell death [100]. Although betalains were found to inhibit a wide antimicrobial
spectrum, very little is reported in the literature on the mechanism of microbial
inhibition by betalains. The specific underlying cellular and molecular mechanism of
antimicrobial activity of betalains should be investigated in future studies [5].
4 Application of Betalains
The main application of betalains to date is as natural colorants. Other potential

application of betalains in functional foods is also covered in this section.
4.1 Natural Colorants
Colorants or color additives can be used to serve several purposes in food pro-
cessing, including standardizing raw ingredient colors, providing color identities to
otherwise colorless foods, and accounting for loss during processing or storage
[105]. There is growing interest in the use of natural pigments for food coloring
which are considered to be harmless or even healthy. Synthetic colorants are
becoming more and more critically assessed by the consumer. There were numerous
regulation changes worldwide in terms of permitted colorings throughout the years.
Many advanced countries have their own regulations and a list of approved food
colors that can be used, including maximum daily intake. For instance, the USA
permitted list of synthetic colorants was reduced from 700 to only seven until the
beginning of the twenty-first century [106]. The last synthetic food colorant to be
approved by the Food and Drug Administration (FDA) was FD&C Red No. 40
(Allura Red) in 1971 and there are not likely to be any more in the near future [107].
Colored natural extracts are preferred over purified colors because declaration of the
former allows clean labeling [108]. Betalains are less commonly used than antho-
cyanins in food processing despite being more pH stable than the anthocyanins [39].
Betalains are water-soluble pigments that are stable between pH 3 and 7 and well
suited for coloring from sour (low acid) to neutral foods [4, 19] whereas the use of
anthocyanins is not possible due to the instability at pH values over 3 [28]. Betalains
can be effectively stabilized by ascorbic acid, which on the other hand is known to
facilitate anthocyanin degradation [109]. Application of betalains instead of antho-
cyanins for coloring food with high vitamin C content or vitamin C-supplemented
products may be of particular interest [44].
Betalains have been used as food colorants at least since the turn of the twentieth
century [24]. The early application involved the use of pokeberry (Phytolacca
Americana) juice to improve the color of red wine [24, 110]. The addition of
pokeberry juice to wine was forbidden in France in 1892 because the juice also
contains a purgatory and emetic saponin called phytolaccatoxin [110, 111]. The red
pigment from pokeberry was called phytolaccanin until research established that it
was identical to betanin in red beets [111, 112]. Today, the food colorant known as
“beetroot red,” extracted from red beet is commercialized in European Union and
USA as food colorant. This food colorant is also called betanin or known as E-162 in
the European Union and as 73.40 in the 21 CFR section of the FDA in the USA
[113]. Betanin from red beet is one of the successful natural colorants used in the
food industry [114] and approved for use as colorants in dairy products, sauces,
soups, cosmetics, and pharmaceuticals by the US FDA and European Union [115].
Red beet colorants can be effectively used to color dairy products such as yoghurt
and ice cream, salad dressings, hard candies and fruit chews, frostings, cake mixes,
gelatin desserts, starch-based puddings, meat substitutes, poultry meat sausages,
gravy mixes, soft drink and powdered drink mixes [116, 117]. Commercial red
beet colorants are available as either juice concentrates (produced by vacuum-
concentration of red beet juice to 60–65% total solids) or powders (produced by
freeze or spray drying), containing from 0.3% to 1% of pigment [24, 117, 118]. The
concentration of pure pigment required to obtain the desired hue is relatively small,
rarely exceeding 50 mg/kg, calculated as betanin [26] as betalains have good
tinctorial strength [108]. Betanin from red beets are, however, afflicted with a narrow
color spectrum [108]. The typical earthy flavor caused by geosmin [119] may also
deter the commercial use of E-162 in food application. Furthermore, the risk of
carry-over earth bound microorganism from the raw material red beetroot is an
important consideration [120]. Red beets are notorious accumulators of nitrates
and nitrites during growing, and there is a necessity to reduce these levels in red
beets [110].
The use of Opuntia sp. as colorants has several advantages such as neutral smell
and/or taste, low nitrate content [115, 121], a broad color range [19, 121], and
represents a lower risk for microbiological carry-over [9]. In particular, the yellow
orange prickly pear fruits are of promising potential because of the scarceness of
yellow water-soluble pigments [121]. Moreover, the low levels of colorless phenolic
compounds in prickly pear fruits make them very promising since potential interac-
tions of betalains with these phenolics are avoided [9]. However, prickly pears
contain high amounts of reducing sugars and amino acids and thereby may enhance
Maillard product formation [9]. Opuntia ficus-indica is the most extensively studied
Opuntia species. Recent research carried out with various Opuntia species has
revealed Opuntia stricta to be a promising source of betacyanin pigments with
high betanin levels (800 mg/kg) which are five times higher than those found in
red fruits of O. ficus-indica and even higher than those shown by commercial red
beets used for their purple color [115]. Opuntia stricta extracts lack the yellow
betaxanthins, and betanin and isobetanin are the betacyanins present [122]. Concen-
trated betacyanin extracts from the fruits of O. stricta with its pleasant taste and
flavor showed high potential to be used as a natural colorant [113, 122]. Red- purple
powder from O. stricta fruit juice was successfully applied as a colorant in two food
model systems: a yogurt and a soft-drink [123]. On the other hand, Amaranthus
plants, which can grow in a wider range of environments are a potential source of
1488 W. S. Choo
red-violet betacyanin pigments and an alternative to the use of red beet [124]. Some
Amaranthus genotypes produce particularly high biomass and contain high levels of
pigment [125]. Betacyanins from Amaranthus showed high potential to be used as
natural colorants in jelly, ice cream, and beverage [126, 127]. Red pitahaya is high in
betacyanins and devoid of betaxanthins (which are present in E-162) providing
colorants with a better tinctorial strength. Therefore, the red pitahaya can be used
as an alternative source of betacyanins in regions where it is grown [128].
Betacyanin from red pitahaya showed promising application as a natural colorant
in dairy products such as milk [128] and yogurt [129] whereby sensory acceptability
of panelists of using colorant preparations from red pitahaya were similar or higher
than that of E-162 [128, 129].
4.2 Other Applications of Betalains
Commercially, fruits and cladodes of Opuntia species are made into products such as
juices, jams, and dehydrated fruits [130]. Many research have been carried out to
expand the use of betalains, particularly betalains from the four main sources,
namely, red beet, prickly pear, amaranthus, and red pitahaya. Effort has been made
to produce various types of red beet juice [131–138], prickly pear beverages or juices
[121, 139–144], and red pitahaya juices [16, 145, 146]. In addition, spray drying or
other drying and encapsulation techniques have been investigated to produce dried
form (powder) of betalains or to further stabilize betalains [126, 138, 147–156].
Powdered or dried red beet containing betalains has been incorporated into edible or
intelligent films [157, 158], emulsified pork sausage [159], and cheese [160]. Red
beet juice containing betalains has been investigated to produce nutritious pasta
[161] and added as part of a curing system to produce chicken frankfurters [162].
Red beet chips as healthy fried snack foods were also investigated [163]. Prickly
pear containing betalains has been investigated to produce enriched cereal-based
extrudates [164] whereas Opuntia ficus-indica cladodes as a functional ingredient
was incorporated into bread [165]. The effect of betacyanin pigments from
Amaranthus tricolor and Amaranthus cruentus on commercial wheat flours was
investigated as a basis to expand the applications of betalains as natural quality
enhancers of wheat-based foods [166].
5 Conclusions
The biological and pharmacological properties of betalains-rich foods such as red

beetroot, amaranth, prickly pear, and red pitahaya show their great potential as
functional foods. Betalains should be used regularly in the diet. Consumers are
most likely to benefit from the regular consumption of products rich in betalains
such as juice and other products made of foods colored with betalains. The lack of
commercially available standards has limited betalain analysis. There is a need for
more highly purified betalains and increasing of studies using purified compound
instead of betalain-rich extracts would help to establish the actual role played by
betalains alone or in cooperation with other compounds. The underlying cellular and
molecular mechanisms of betalains need to be addressed in more detail in future
studies. Further research is also needed to determine the concentration, extractability,
and stability of other source of betalains in order to expand the use of betalains. Plant
sources of betalains other than red beet should be grown in sufficient quantities to
ensure that betalains could be produced at a large scale for food industry use.
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Nutraceutical Potential of Guava
50
Moni Gupta, Anshu Wali, Anjali, Sachin Gupta, and
Sudheer K. Annepu
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500
2 Ecological Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501
3 Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502
3.1 Cultivars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1502
3.2 Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1504
3.3 Layout and Planting Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
3.4 Crop Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
3.5 Crop Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505
3.6 Harvesting and Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1506
4 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1506
5 Nutritional Attributes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1507
5.1 Phytochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1507
5.2 Chemical Composition of Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1510
5.3 Chemical Composition of Guava Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512
5.4 Chemical Composition of Guava Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513
6 Bioactive Potential of Guava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513
6.2 Anti-Inflammatory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515
6.3 Antidiabetic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515
M. Gupta (*) · A. Wali · Anjali

Division of Biochemistry, Faculty of Basic Sciences, Sher-e-Kashmir University of Agricultural
Sciences and Technology, Chatha, Jammu, Jammu and Kashmir, India
S. Gupta (*)
Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural
Sciences and Technology of Jammu, Jammu, India
S. K. Annepu
Divison of Crop Improvement, ICAR-Directorate of Mushroom Research, Solan, Himachal
Pradesh, India

1500 M. Gupta et al.
6.4 Antidiarrheal Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1516

6.6 Anticancer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1518
7 Value-Added Products and Nutraceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1519
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1520
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1520
Abstract
Psidium guajava commonly known as guava is one of the economical fruit crops
belonging to the Myrtaceae family and grows in tropical and subtropical region.
Largely grown as wild crop or selection variants, disease-free quality planting
materials for establishment of guava orchard is necessary. The fruit is also labeled
as super-fruit, and because of its unique flavor, taste, and health-promoting
qualities, it is regarded as functional food or potent nutraceutical. It is rich in
antioxidant compounds and contains a high level of ascorbic acid content,
carotenoids, and phenolic compounds. It is acclaimed as the “poor man’s apple
of the tropic.” Traditionally guava leaves and fruits are used in folk medicine for
the treatment of various ailments like diarrhea, flatulence, gastric pain, wounds,
rheumatism, ulcers, etc. Guavas possess antioxidant, antimicrobial, anticancer,
antidiabetic, and anti-inflammatory activities, supporting a great therapeutic
potential and a wide range of clinical applications. The important active bio-
chemical compounds in guava are essential oils, phenolics, flavonoids, caroten-
oids, triterpenoids, esters, aldehydes, etc. Guava fruit is highly perishable, so to
increase its shelf life, it may be processed into various value-added products like
guava juices, squash, nectar, leather, jam, jellies, powder, etc. This chapter
describes in detail about the guava plant, ecological requirements for its growth,
various methods of its propagations, current knowledge about its nutraceutical
properties and its application in preparing guava-based value-added products.
Keywords
Psidium guajava · Guava · Cultivation · Phytochemicals · Bioactive potential ·
Nutraceutical · Value-added products
1 Introduction
Guava (Psidium guajava Linn.) belonging to the family Myrtaceae, with about
133 genera and more than 3,800 species, is native to Mexico, Central America, the
Caribbean, and the Northern part of South America [1]. It is also grown in all the
tropical and subtropical areas of the world including India and adapts to different
climatic conditions but prefers dry climates. It is widely grown in tropics with its
fruit widely known for its exotic flavor and potent aroma. Guava is a traditionally
used plant because of its immense food and nutrition value. World production of
guava is estimated to be about 1.2 million tons; however India and Pakistan
contributes to about 50 percent of the world production [2]. Guava is also known
as poor man’s fruit or apple of tropics. The common varieties of guava are the red
50 Nutraceutical Potential of Guava 1501
(P. guajava var. pomifera) and the white (P. guajava var. pyrifera) [3, 4]. Various
parts of the guava plant, viz., root, bark, leaves, and fruits, are found to possess
many pharmacological properties as it is used in the treatment of various disorders
[5]. Guava is rich in minerals and functional components such as vitamins and
phenolic compounds which makes beneficial contribution to the human diet and is
well accepted by the consumers [6]. All parts of this tree, including fruits, leaves,
seeds, bark, and roots, have been utilized traditionally in many countries for
treatment of looseness of the bowels, menstrual disarranges, vertigo, anorexia,
stomach-related issues, gastric deficiency, aroused mucous layer, laryngitis, skin
issues, ulcers, vaginal discharge, cold, cough, cerebral ailments, nephritis, jaun-
dice, diabetes, malaria, and rheumatism [7–9]. It additionally has anticancer
properties. The leaves of the guava tree are full of antioxidants, anti-inflammatory
agents, antibacterial, and even tannins that can have significant health benefits,
from treating stomach troubles to chronic diseases like cancer [10]. The fruit is
labeled as super-fruit, and because of its unique flavor, taste, and health-promoting
qualities, it is regarded as functional food or potent nutraceutical [11]. The term
nutraceutical was coined by Stephen DeFelice in 1989 from nutrition and
pharmaceuticals. It is defined as a food that provides health benefits, including
prevention and cure of a disease [12]. The term nutraceuticals as commonly used
in marketing has no regulatory definition [13]. The concept of nutraceuticals,
functional food, designer food, phytochemicals, bioactive compounds, etc. is
misnomers and quite confusing and are often used interchangeably. The supple-
mentary diets or dietary supplements however differ from nutraceutical in many
ways. Nutraceuticals are not only supplementary food included in diets but also
conventional food which is part of the daily diet having health benefitting poten-
tials. Nutraceuticals are also referred to as natural functional food or bioactive
phytochemicals that have health-promoting, disease-/disorder-preventing, or
medicinal properties.
These nutraceutical contains the macro- and micronutrients like carbohydrates,
proteins, fats, lipids, vitamins, minerals, antioxidants, etc.; however, bioactive com-
pounds are minor components of food and are defined as having the following
characteristics: they are present in low concentrations; they are not considered to
be nutrients; and they have a proven health effect [14]. Interaction between func-
tional food components such as prebiotics, probiotics, phytochemicals, and intestinal
microflora has consequences on human health [2].
2 Ecological Requirements
Guava can be grown under a wide range of climatic conditions. Compared to other
fruit crops, guava is highly resistant to drought. As compared to the tropical areas,
those with distinct winter promote production of abundant crop with better quality.
In the tropics and subtropics, guava is found from sea level to an altitude of 1500 m
asl. Guava trees growing at lower elevation are generally vigorous with heavy fruit
set. Sandy loamy soils with the pH range of 5.5–7.5 are highly suitable for growing
guava. However, it can be grown with minimum care in marginal and soils affected
with salinity. A temperature range of 23–28 C during flowering to fruiting is found

to be optimum. Temperatures lower than 7–8 C cease the plant growth, and the
leaves turn to purple. Natural defoliation occurs during the low winter temperatures
and the flowering induced with rise in temperature and increased soil moisture. A
rainfall pattern with alternating dry and wet conditions is highly suitable for maxi-
mum fruit set and high yields.
3 Cultivation
3.1 Cultivars
Majority of the cultivars of guava have been evolved after selection from seedling
variants. Several promising genotypes from other countries have also been intro-
duced into India by researchers. Even though many varieties are available in India,
Allahabad Safeda and Sardar (L-49) occupied maximum share owing to its yield and
market acceptability. Recently ICAR-IIHR, Bengaluru, released a pink-fleshed
variety named as Lalit that has attained a wide popularity across the guava-growing
regions of the country. Apart from these prominent cultivars, some of the cultivars
grown in India and their important varietal characters are described in below table.
Developed/
S. no Cultivar released by Characters
1 Allahabad CISH, Lucknow The fruits are relatively soft and contain less
Safeda number of seeds. Suitable for both table and
processing purposes
2 Sardar CISH, Lucknow Fruits with white flesh and more number seeds.
Skin is harder than Allahabad Safeda
3 Lalit CISH, Lucknow Suitable for high-density planting. Epicarp is in
saffron yellow color, and the flesh is in red blush
color. High yielder compared to the existing
commercial varieties in India
4 Shweta CISH, Lucknow Fruits are very attractive with white creamy epicarp
and red spots of blush; flesh is snow white in color
5 Allahabad CISH, Lucknow Large and uniform pink color fruits with deep pink
Surkha color flesh. The fruit contains strong flavor with
less number of seeds
6 Pant Prabhat GBPUA&T, Fruits with smooth peel and light yellow in color.
Pant Nagar White color pulp with small seeds and high flavor
7 Arka ICAR-IIHR, Pulp is white in color with sweet taste. Keeping
Mridula Bengaluru quality is good
8 Arka ICAR-IIHR, Medium-sized fruits with white and sweet pulp.
Amulya Bengaluru Pulp contains round-shaped small number of seeds.
Keeping quality is good
9 Arka Kiran ICAR-IIHR, Fruits with firm pulp and pink in color
Bengaluru
(continued)
Developed/
S. no Cultivar released by Characters
10 Arka ICAR-IIHR, Medium-sized fruits with deep pink color pulp and
Rashmi Bengaluru soft seeds
11 Hisar CCSHAU, Hisar Medium-sized round-shaped fruits with smooth
Safeda and yellowish green surface. Pulp is creamy white
in color with few number of seeds
12 Hisar CCSHAU, Hisar Medium-sized round-shaped fruits with smooth
Surkha and yellowish green surface. Pulp is pink in color
with harder seeds
Allahabad Safeda Sardar
Arka Amulya
Lalit
Arka Kiran
Arka Rashmi
Photos courtesy: ICAR-Indian Institute of Horticultural Research, Bengaluru
Apple color, Anakapalli, Banarasi Surkha, Chittidar, Dholka, Dharwar, Habsi,

Karela, Punjab smooth, Sangam, Mizapuri seedling, Nasik smooth white, Thailand
guava, Philippine guava, Florida seedling, etc. are some of the locally grown
cultivars available in India.
3.2 Propagation
In spite of large number of private and public sector nurseries, still there is a shortage
of disease-free quality planting materials for establishment of guava orchard. Mass
multiplication through vegetative propagation results in uniform crop with relatively
short pre-bearing period in guava compared to the seed propagation. Out of the
several methods of vegetative propagation, air layering, wedge grafting, budding,
and stooling are found better for raising the productive seedlings in guava [15].
Healthy, disease-free, and vigorous mother trees should be selected for raising the
nursery stock.
3.2.1 Air Layering

In this method, the healthy branches of 1.2 cm or more diameter are selected and then
girdled by removing a strip of bark with the width of about 2 cm. The girdled area is
then covered with the sphagnum and wrapped with the polythene film. Within
3–4 weeks, the roots start developing from the layered portion. The rooted layers
are detached from the mother plant and can be used for planting. Rainy season is the
best season for air layering.
3.2.2 Wedge Grafting

The rootstock selected for grafting is split to about 4–4.5 cm with a knife, and
wedge-shaped cut slanting from both the sides is made on the lower side of the scion
material. The scion material is inserted in to the rootstock split and pressed properly
to bring both the materials into contact with each other. The union is then tied with
the polythene strip. The scion starts sprouting after 9–12 days and should be
transferred for hardening after 1 month.
3.2.3 Budding
Patch budding is considered to be the efficient method of propagation in guava with
high success rate. In this method, healthy seedlings of 1 year old are selected, and
1–1.5 cm long patch is removed from the rootstock. Unsprouted, dormant buds
selected from the leaf axils of scion variety is fitted into the rootstock patch and tied
with the polythene strip. After 2–3 weeks, the strips should be removed to examine
the success of the budding.
3.2.4 Stooling
In this method, self-rooted plants are planted 0.5 m apart in the stooling bed and then
allowed to grow for about 3 years. Then these are cut down to the ground level, and
new shoots emerges on the stumps. All these shoots are mounted with the soil to a
height of 30 cm. On the onset of monsoon, the shoots are detached from the mother
plant and can be used for direct planting.
3.3 Layout and Planting Systems
This refers to the planting of trees in an orderly manner to ensure the maximum
number of trees per unit area and to facilitate the smooth operation of intercultural
activities. Planting systems such as square, rectangular, triangular, hexagonal, and
quincuncial systems can be adopted based on the availability of land. After comple-
tion of layout, the pits of 75 cm 75 cm 75 cm are dug and left open for 15 days.
The grafts are to be placed in the center of the pits and pressed tightly all around. The
graft/bud union should remain well above the soil surface. A planting distance of
5 m 5 m is recommended for healthy and vigorous growth of the plants. With this
spacing 400 trees can be planted in one ha of land. High-density planting with the
spacing of 3.0 m 3.0 m and 1.5 m 3.0 m is also recommended for specific
varieties. Rows should be planted in North-South direction to facilitate maximum
sunlight exposure. Pits should be watered copiously immediately after planting.
Mulching can be done at the basins to conserve the moisture during the initial stages
of establishment.
3.4 Crop Regulation
Flowering and fruiting in guava follow specific pattern and occur in two major
seasons, once during March to May and the later in July–August [16]. The fruits
from March to May flowering are harvested in rainy season, and the fruits from
second flowering are harvested during winter, i.e., late October to mid-February. The
July flowering gives more number of flowers and good quality of the fruits compared
to the summer flowering. Orchard losses can be avoided by adopting effective crop
regulation practices to manage the flowering in guava and for harvesting the good
quality fruits. Spraying urea at 10–15% twice during bloom (April–May) eliminates
the rainy season fruiting [17].
3.5 Crop Management
3.5.1 Orchard Management

Guava, being a perennial crop, needs utmost care while selecting the varieties and
the quality planting material. Any mistakes committed in the early stages of the
orchard management cannot be rectified at a later stage.
3.5.2 Nutrient and Water Management

Amount of manures and fertilizers to be applied to guava tress depends upon the age
of tree and the soil characteristics. At the time of planting, each pit should be filled
with well rotten farm yard manure at 15–20 kg and 1.5 kg of single superphosphate.
A recommended dose of 260 g urea + 375 g SSP + 100 g of MOP per plant should be
supplied in the first year. The dose increases with each passing year.
The fertilizers are applied in two split doses preferably in June and September.
Guava is highly prone to micronutrient deficiency and remedial measures should be
taken immediately with foliar sprays of micronutrients. Furrow and basin method of
irrigation are most popular in guava orchards [18]. In the initial stages of plant
growth, it is imperative to maintain the optimum soil moisture by watering every
alternate day. After 2 years of age, watering may be done once in every 7 to 10 days
interval during the summers, and irrigation should be avoided during the rainy
season. During the dry season, the flowering and fruiting will be greatly influenced
by the water availability [19]. Sprinkler and drip irrigations can be adopted for
increasing the productivity and quality of fruits [20].
3.6 Harvesting and Handling
The quality of guava fruit depends on the stage of maturity. The fruits are usually
ready to harvest 4–5 months after the flowering. Guava fruits are generally
handpicked. Color change, specific gravity, total soluble solids, acidity, etc. are the
useful criteria to judge the maturity levels in guava [21]. At the time of maturity, the
specific gravity becomes <1.0, and it starts floating on the water surface. The fruits
with the specific gravity of 1.00–1.02 have better shelf life and suitable for long-
distance transport. It is desirable to pick the fruits along with 5–7 cm stalk and two to
three leaves. Proper grading and sorting increase the postharvest shelf and fetch high
prices to the growers. At room temperature, the shelf life is for a few days only. The
fruits picked during the winter season can be stored for 7–8 days at ambient
temperatures whereas rainy season picked fruits can be stored only for 2–3 days,
depending on the variety. At low temperatures of 10–15 C, the shelf life can be
extended up to 3–4 weeks [22]. For low temperature storage, fruits must be picked at
hard, green, and immature stage without color break.
4 Morphology
Guava trees are little shrubby evergreen trees with a considerable measure of wide-
spreading branches and square fleece twigs. The branches are screwy bringing
inverse clear out. The blooms are white borne independently, or in little gatherings
in axils of leaves of late development, petals are incurved; they are fragrant with four
to six petals. Each blossom bears various white needlelike stamens which oblige
smooth anthers. Self-fertilization is conceivable, yet cross-pollination by creepy
crawlies brings about higher yields [23]. The organic product is little, 3 to 6 cm
long in different shapes going from ovoid, round, to pear-formed contingent on
species. The external skin might be unpleasant having a severe taste or delicate and
sweet.
The skin shading is typically green before development however winds up

noticeably yellow, maroon, or green when ready with tissue running from white,
yellow, and pink to red differing with species. Natural product might be thin-shelled
with many seeds installed in a firm mash or might be thick-shelled with fewer seeds.
Kind of natural product shifts from sweet to profoundly corrosive. The distinctive
fragrance ranges from solid and penetrating to mild and pleasant [24].
5 Nutritional Attributes
Guava is reported to possess rich nutritional attributes ranging from carbohydrates,

minerals, vitamins, to antioxidants. The nutritional composition of guava has been
reported to vary with cultivars, season, and environment. The main carbohydrate in
the form of simple sugars has been identified as glucose and xylose [25]. Every
100 gram of fresh guava fruit servings in general contributes to 75–85% of moisture,
3–7% of total dietary fiber, 1–6% of protein, and 0.7 to 0.11% of lipids.
Among mineral contents, potassium content (352.7 mg/100gft) is reported to be
highest in fresh guava, followed by phosphorus (17.8–30 mg/100gft), calcium
(9.1–17 mg/100gft), and iron (0.4–0.7 mg/100gft) [2, 26, 27]. It is also reported to
contain many of the vitamins required by human body of which the vitamin C
content (50–300 mg/100gft) is reported to be highest in guava [28].
In a study [29], it was observed that with the advancement of fruit maturity at
different stages, the total soluble solid (TSS), sugar (total, reducing, and
non-reducing), and ascorbic acid contents increased significantly, while during
fruit ripening stage both acidity and pectin decreased. Of all the guava cultivars
studied, L-49 showed highest TSS (12.250B), total sugar (8.50%), and ascorbic acid
(265.09%) followed by Allahabad Safeda, while L-49 showed minimum acidity
(0.26%) and maximum pectin content (0.77%). Pectin methyl esterase (PME)
activity increased progressively in all the cultivars up to half ripe stage (HRS) and
subsequently decreased at full ripe stage (FRS). Maximum PME activity was found
in L-49 (56.25 units/g.f.wt) at HRS, whereas it showed a decrease at FRS
(52.25 units/g fresh weight (FW) followed by Allahabad Safeda and Lalit. Thus, it
was concluded that L-49 was superior among all the commercial cultivars of guava
grown under subtropical condition followed by Allahabad Safeda and Lalit.
5.1 Phytochemicals
Phytochemicals (phyto, plant) are biologically active chemical compounds found in

plants offering health advantages for humans in addition to those attributed to
macronutrients and micronutrients [30]. Phenolic compounds are secondary metab-
olites which are produced in the shikimic acid of plants and pentose phosphate
through phenylpropanoid pathway [31]. They contain benzene rings with one or
more hydroxyl substituent and range from simple phenolic molecules to highly
polymerized compounds [32]. The phytochemical analysis of guava leaves revealed
the presence of flavonoids, glycosides, alkaloids, steroids, and many other metabo-
lites and absence of tannins and saponins [33].
Guava is one of the promising fruits rich in lectins, saponins, tannins, phenols,
triterpenes, and flavonoids. High levels of vitamins, dietary fiber, and carotenoids,
altogether make guava therapeutically an important fruit [34]. The fruit pulp is rich
in ascorbic acid and carotenoids (lycopene, β-carotene, and β-cryptoxanthin). The
seeds, skin, and bark have glycosides, carotenoids, and phenolic compounds.
5.1.1 Polyphenols
Polyphenols are synthesized by numerous plants as secondary metabolites. Poly-
phenolic compounds serve as both functional components to the plant and the
consumer. For instance, some polyphenols serve as pigment (anthocyanins) com-
pounds to ward off insects and other herbivores such as astringent, tannins, and UV
light protectants such as carotenoids [35].These compounds are also important in
foods for their sensory attributes such as color, astringency, and bitterness, as well as
their possible nutritional properties. Polyphenols are products of three major plant
metabolic pathways. Phenolic compounds consist of a phenol and an aromatic ring
with at least one hydroxyl group attached. Phenolic compounds can be classified into
several categories based on their structures. Phenolic acids are secondary metabolites
of the shikimate pathway; the phenylpropenoid pathway produces the cinnamic acid
derivatives which are precursors of flavonoids and ligans, and the “flavonoid route”
produces the numerous and diverse flavonoid compounds [36]. Polyphenols, which
include flavonoids, have at least two phenol groups. The largest polyphenols are the
tannins which can be classified into two subgroups, the hydrolyzable and the
condensed tannins. Hydrolyzable tannins are those which are readily hydrolyzed
by acids or enzymes into gallic or ellagic acid [37]. Hydrolyzable tannins are
commonly found in foods such as guava, grapes, and wine. Both condensed and
hydrolyzable tannins have been shown to have antioxidant, enzyme-inhibiting, and
antimicrobial properties [36]. Condensed tannins, also called “vegetable tannins” or
proanthocyanidins, are flavonoid polymers which can be degraded in the presence of
acid and heat to form either cyanidin or delphinidin and are relatively stable as
compared to the hydrolyzable tannins [37]. Common condensed tannins include
polymers of catechin and epicatechin which can be found in guava, teas, and
numerous fruits and vegetables. The third phenolic group is the phenolic acids that
consist of a benzene ring and at least one carboxylic acid group. Examples of
phenolic acids are caffeic, chlorogenic, p-coumaric, gallic, and ellagic acids
(Table 1). Many phenolic acids are linked through ester, ether, and acetal bonds to
either structural components of the plant such as cellulose, proteins, or lignin; to
larger polyphenols (tannins); or to smaller organic molecules such as glucose
[38]. This leads to considerable diversity among the various classes of polyphenolics
and therefore inherent difficulty in analysis and identification. Limited information
exists on the guava phenolics; however, previous studies have shown guava to
contain a variety of polyphenolics including flavonols, phenolic acids, flavan-3-
ols, and condensed tannins [39, 40].
Table 1 Phenolics in guava

S. no Chemical name Structurea Reference
1 Gallic acid [41]
2 Catechin [41]
3 Epicatechin [39]
4 Myricetin [39]
5 Apigenin [39]
6 Kaempferol [41]
7 Ellagic acid [42]
8 Naringenin [43]
9 Quercetin [42]
a
Structure taken from ChemSpider structure draw online software
5.1.2 Ascorbic Acid

Fruits are the major source of ascorbic acid, a nutrient required for humans and one
of the most abundant antioxidants consumed. L-ascorbic acid is an excellent reduc-
ing agent, and large quantities may help stabilize phenolics and other antioxidants
during processing by the donation of hydrogen atoms. This reducing ability is due to
its 2,3-enediol moiety. Ascorbic acid is often considered as an index of nutrient
quality during processing and storage of foods because of its stabilizing nature [44,
45]. Guava contains approximately 230 mg of total ascorbic acid/100 g of edible
portion of fruit, five times more than a serving of orange. Among all fruits, it is
second to acerola cherry in vitamin C content [46].
5.1.3 Carotenoids
Carotenoids are abundant in red-, yellow-, orange-, and green-colored vegetables and
fruits. After chlorophyll, they are the second most widely occurring plant pigment
found in nature. Carotenoids are tetraterpenes that can be classified into two major
groups including carotenes (hydrocarbons) and xanthophylls (oxygenated hydrocar-
bons). Carotenoids may be straight chained, such as lycopene, or contain a five or six
carbon ring on one or both ends, such as β-carotene [47]. The high degree of hydration
and long carbon chain length of these molecules make them hydrophobic and there-
fore fat-soluble molecules. The major purpose of carotenoids in the human diet is to
serve as precursors to provitamin A. In order to serve this purpose, the carotenoid must
contain a β-ionone ring [45]. Carotenoids containing this structure include β- and
α-carotene and ß-cryptoxanthin. Carotenoids without this structure, such as lycopene,
do not possess provitamin A activity yet serve as dietary antioxidants. As an antiox-
idant, carotenoids are known to quench singlet oxygen and protect against cellular
oxidative damage [48]. A wide variety of carotenoids have been identified in guava
including phytofluene, β-carotene, lycopene, cryptoflavin, cryptoxanthin, and lutein
(Table 2) [28]. Lycopene is a fat-soluble carotenoid responsible for the red or pink
pigment in several fruits and vegetables such as tomatoes, watermelon, pink grape-
fruit, and guava. Structurally, lycopene is a linear, 40 carbon hydrocarbon containing
11 conjugated and 2 non-conjugated double bonds [49]. Of all the dietary carotenoids,
lycopene has the highest singlet oxygen quenching ability in the body. However, since
lycopene lacks a β-ionone ring, it does not provide vitamin A activity.
5.2 Chemical Composition of Leaves
5.2.1 Leaf Phenolics

All parts of guava have been used for various purposes such as hepatoprotection,
antioxidant, anti-inflammatory, antispasmodic, anticancer, antimicrobial, anti-
hyperglycemic, analgesic, endothelial progenitor cells, anti-stomachache, and anti-
diarrhea. The primary constituents of guava leaves are phenolic, isoflavonoids,
gallic acid, catechin, epicathechin, rutin, naringenin, and kaempferol having
hepatoprotective, antioxidant, anti-inflammatory, antispasmodic, anticancer, antimi-
crobial, antihyperglycemic, and analgesic actions [51].
Table 2 Structure of major carotenoids reported in guava

S. no Name Structure References
1 Beta- [28, 50]
carotene
2 Lycopene CH3 CH3 CH3 CH3 [28, 50]

CH3
H3C
CH3 CH3 CH3 CH3
3 Lutein H3C CH3 CH3 CH3

H3 C OH [28]
CH3 CH3 H3C CH3

HO CH3
4 Rubixanthin H3C CH3 CH3 CH3 [28]

CH3
CH3 CH3 CH3 CH3

HO CH3
5 Cryptoflavin CH3
CH3
[28]
O
CH3

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Reference Series in Phytochemistry

More information about this series at http://www.springer.com/series/13872

With 238 Figures and 206 Tables

ISSN 2511-834X ISSN 2511-8358 (electronic)

© Springer Nature Switzerland AG 2019

We are pleased to present a three-volume treatise on Bioactive Molecules in Food.

timely compilation of information about bioactive molecules present in our

Prof. Jean-Michel Mérillon

Part I Favorable Regimen for Good Health and

1 Health Beneﬁts of Dietary Phenolic Compounds and

2 Intake of Mediterranean Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3 Flavonoids – Food Sources, Health Beneﬁts, and Mechanisms

4 Bioactive Molecules, Nutraceuticals, and Functional Foods in

5 Challenges in Optimal Utilization of Bioactive

6 Bioactive Food Components in the Prevention of Cardiovascular

7 Capsicum annuum Bioactive Compounds: Health Promotion

8 Sesame: Bioactive Compounds and Health Beneﬁts . . . . . . . . . . . . 181

Part II Proteins and Their Biological Activity . . . . . . . . . . . . . . . . . 221

10 Plant Proteins from Legumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Part III Lipids and Their Biological Activity . . . . . . . . . . . . . . . . . . . 465

17 Bioactive Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

21 Protective Effect of Omega 3 Fatty Acids EPA and DHA in

Part IV Food Carbohydrates and Derived Compounds . . . . . . . . . 699

24 Total Dietary Fiber Intake, Whole Grain Consumption, and

Part V Food Carotenoids ................................. 865

30 Natural Food Pigments and Colorants . . . . . . . . . . . . . . . . . . . . . . 867

32 Sweet Potato: Bioactive Compounds and Health Beneﬁts . . . . . . . 919

Part VI Food Polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971

34 Bound Phenolics in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973

Part VII Functional Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281

43 Antidiabetic Functional Foods with Antiglycation Properties . . . . 1283

45 Functional Components and Medicinal Properties of Food . . . . . . 1343

46 Development of Functional Dairy Foods . . . . . . . . . . . . . . . . . . . . 1377

47 Nutrients and Nutraceuticals from Seafood . . . . . . . . . . . . . . . . . . 1397

48 Roots and Tubers as Functional Foods . . . . . . . . . . . . . . . . . . . . . . 1441

49 Betalains: Application in Functional Foods . . . . . . . . . . . . . . . . . . 1471

50 Nutraceutical Potential of Guava . . . . . . . . . . . . . . . . . . . . . . . . . . 1499

51 Cereal-Based Fermented Foods of Africa as Functional Foods . . . 1527

52 A Novel Delivering Agent for Bioactive Compounds:

53 Bioactive Molecules in Edible and Medicinal Mushrooms for

54 Bioactive Molecules of Spirulina: A Food Supplement . . . . . . . . . . 1621

55 Bioactive Compounds from Garcinia Fruits of High Economic

Part VIII Agricultural Practices and Food Quality . . . . . . . . . . . . . . 1671

56 Factors Inﬂuencing Sweet Taste in Apple . . . . . . . . . . . . . . . . . . . . 1673

57 Advances in Pseudocereals: Crop Cultivation, Food

58 Pesticide Residues in Fruits and Vegetables . . . . . . . . . . . . . . . . . . 1715

59 Banana and Plantains: Improvement, Nutrition, and Health . . . . 1755

60 Veterinary Antibiotics in Animal Diet: Effects on

61 Insecticide Residues in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1793

62 Edible Mushrooms: Cultivation, Bioactive Molecules,

63 Plants Probiotics as a Tool to Produce Highly Functional Fruits . . . 1849

64 Bioactive Compounds of the Wonder Medicinal

65 Minas Frescal Cheese as a Probiotic Carrier . . . . . . . . . . . . . . . . . 1895

Part IX Methods of Food Analysis and Quality Control . . . . . . . . . 1927

66 Dietary Phenolic Compounds in Biological Samples:

67 Rheology of Food Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

68 GLC/HPLC Methods for Saffron (Crocus sativus L.) . . . . . . . . . . . 1987

69 Use of Nanotechnology for Immobilization and

Part X Miscellaneous Case Studies ......................... 2183

74 Are Myristica fragrans L. (Myristicaceae) and Its