Carotenoids - Food Sources, Production and Health Benefits

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NUTRITION AND DIET RESEARCH PROGRESS
CAROTENOIDS
FOOD SOURCES, PRODUCTION
AND HEALTH BENEFITS
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CAROTENOIDS
FOOD SOURCES, PRODUCTION
AND HEALTH BENEFITS
MASAYOSHI YAMAGUCHI
EDITOR
New York

Copyright © 2013 by Nova Science Publishers, Inc.
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Contents
Preface vii
Chapter I Carotenoids as Bioactive Compounds 1
Aline Inácio Alves, Afonso Mota Ramos,
Paulo César Stringheta and Ellen Silva Lago Vanzela
Chapter II Food Sources: Production and Health Benefits of Carotenoids 35
Lucia Maria Jaeger de Carvalho,
Lara de Azevedo Sarmet Moreira Smiderle,
Ediane Maria Gomes Ribeiro, Gisela Dellamora Ortiz,
Patrícia Barros Gomes and José Luiz Viana de Carvalho
Chapter III Carotenoids with κ-End Group 49
Enrique Murillo, Veronika Nagy, Attila Agócs
and József Deli
Chapter IV Development of a Method to Induce ROS Resistant High-Yielding
Mutant Lines of Sugarcane, Saccharum officinarum cv NiF8
and NiTn18 79
Kohji Hasunuma, Yusuke Yoshida, Md Emdadul Haque,
Yosuke Fukamatsu, Yoshibumi Terajima and Makoto Matsuoka
Chapter V Molecular Base for Carotenoids Antioxidant Activity in Model
and Biological Systems: The Health-Related Effects 93
Dragan Cvetkovic, Leszek Fiedor, Joanna Fiedor,
Anna Wisniewska-Becker and Dejan Markovic
Chapter VI Carotenoids in Herbal Medicines 127
Mamta Kshipra Misra, Kshipra Misra, Satinder Kaur Brar
and Mausam Verma
Chapter VII Microalgae: A valuable Source of Natural Carotenoids
with Potential Health Benefits 143
Faruq Ahmed, Kent Fanning, Holger Schuhmann,
Michael Netzel and Peer Schenk

vi Contents
Chapter VIII Sodium Taurocholate Optimum Amount of Blood Parrots

on Astaxanthin Availability 165
Xidong Mu, Huiyun Yang, Yinchang Hu
and Jianren Luo
Chapter IX Carotenoid-Based Ornaments As Signals of Health Status in Birds:
Evidences from Two Galliform Species, The Red-Legged Partridge
(Alectoris rufa) and the Red Grouse (Lagopus lagopus scoticus) 173
Lorenzo Pérez-Rodríguez, Jesús Martínez-Padilla
and François Mougeot
Chapter X Dietary Carotenoid Pigments and Human Health Benefits 199
Hasna El Gharras
Chapter XI Health Benefits of Carotenoids for Human Skin 229
Maxim E. Darvin and Juergen Lademann
Chapter XII Nanoparticles of Chitin Nanofibril-Hyaluronan Block Polymer
Entrapping Lutein as UVA Protective Compound 237
P. Morganti, H. D. Chen, X. H. Gao, P. Del Ciotto,
F. Carezzi and G. Morganti
Chapter XIII Bixin and Norbixin: Chemistry, Production and Health Benefits 261
Miguel Roehrs and Rafael Roehrs
Chapter XIV Biology of Carotenoids and their Potential Cardiovascular Health
Benefits 271
Assunta Pandolfi
Chapter XV Effects of Carotenoids on Mammalian DNA Polymerase Inhibition
and Anti-Inflammatory Activity 297
Yoshiyuki Mizushina, Isoko Kuriyama, Toshifumi Takeuchi,
Fumio Sugawara and Hiromi Yoshida
Chapter XVI Dietary β-Cryptoxanthin Stimulates Osteogenesis:
Role in Prevention and Treatment of Osteoporosis 319
Masayoshi Yamaguchi
Index 337

Preface
Carotenoids are the most widespread pigments in nature. One or more carotenoids in
combination give rise to colors ranging from yellow to red in birds, fishes, crustaceans,
microorganisms, fruits and plants, including the dark green ones. There are over 600 known
carotenoids and they are divided into two classes, xanthophylls, which contain oxygen, and
carotenes, which are purely hydrocarbons and contain no oxygen. Carotenoids have many
health benefits including antioxidant properties, free-radical scavengers, decreased risk of
cardiovascular disease, anti-inflammatory properties, osteoporosis prevention and other
diseases. This book, an entitled ―Carotenoids: Food Sources, Production and Health
Benefits‖, has been written to outline the recent topics and it is composed from Chapter 1 to
15.
Chapter 1: Carotenoids are among the most studied bioactive compounds and contribute
to the health-promoting properties of fruits and vegetables. Health benefits include
antioxidant action related to the decrease of degenerative illnesses. Therefore, this chapter
aims to describe the main carotenoid bioactive compounds in terms of their chemical
structure, natural sources, bioavailability and bioconversion and analyze their effects on
health and nutrition, with a focus on recent studies and a perspective to the future. This
chapter emphasized the importance of the carotenoids, showing their sources, chemical
structures, principal biological functions, as well as the factors that influence carotenoid
composition in foods and their stability. This knowledge is of great importance since, after
acquired, it is possible to recommend measures to increment these compounds in diets by
ingestion of food considered as source of carotenoids or their addition in the form of food
additives (natural colorants).
Chapter 2: Carotenoids can be divided in two classes: carotenes and xanthophylls.
Xanthophylls represented by zeaxanthin, lutein, cryptoxanthin, violaxanthin and astaxanthin,
and carotenes, such as licopene, β-carotene and -carotene, are easily found in food, with the
exception of zeaxanthin, and β-carotene is the most widespread of carotenoids. The highest
carotenoid contents are found in fruits and vegetables, especially those with pro-vitamin A
and antioxidant activities. High contents of  and β-carotene, as well as β-cryptoxanthin, are
found in fruits, such as mango, papaya and apricot, and vegetables of orange color or orange
pulp, such as carrot, pumpkin and yellow sweet potato. Carotenoid composition can vary
qualitative and quantitatively among vegetables. Leafy or unleafy green vegetables show a
specific qualitative profile, in which lutein, beta-carotene, violaxanthin and neoxanthin are the
main carotenoids. Carotenes are more abundant in root vegetables, such as carrot and sweet

viii Masayoshi Yamaguchi
potato, and squash, such as pumpkins, whereas oxygenated carotenoids are mostly found in
grains such as corn. Vegetables and fruits have more complex and diversified carotenoid
compositions. Biotechnological processes have been used to produce carotenoids from
microorganisms, such as the fungus B. trispora, microalgae Dunaliella to obtain β-carotene
and xantaxanthin from microalgae and yeasts [Haematococcus sp and P. rhodozyma] for
pharmaceutical purposes. Production of carotenoids is also being evaluated using yeasts such
as X. dendrorhous, R. glutinis, R. mucilaginosa, Sporobolomyces and P. rhodozyma.
Chapter 3: Carotenoids with θ-end group occur typically in certain Capsicum species.
Capsanthin is the main carotenoid of paprika but also occurs in other plants in much lower
amount. Capsorubin and cryptocapsin are minor components accompanying capsanthin.
These red pigments are biosynthetised from carotenoid-5,6-epoxides by means of specific
enzymes. A new special family of carotenoids bearing 3-dehydroxy-θ-end group were found
in tropical fruits of extremely high pigment content. Some of them contains β-end group as
well which means potential vitamin A activity. Here we describe the first isolations and
structure elucidations of carotenoids with θ-end group, their occurrence and biosynthesis, the
chemical synthesis and some transformations of θ-carotenoids, as well as their biological
activities.
Chapter 4: This chapter introduces recent research findings as to the development of a
method to induce ROS resistant high yielding mutant lines of sugar cane, Saccharum
officinarum cv NiF8 and NiTn18. Stems of Saccharum officinarum cv NiF8 and NiTn18 were
treated with liquid medium containing various concentrations of Paraquat for 1 week, and
they were transplanted to planters. The authors obtained candidates of mutant lines, R4-6-1a
and R80-13-3a, showing very good growth from NiF8 and NiTn18 original lines. The green
leaf color of these mutant lines was thicker than those of original lines. The total number and
weight of stems of mutant lines were around 2-fold compared with those of the original lines.
The SPAD values of leaf estimating chlorophyll contents were greater in the mutant lines
compared with the original lines. Punch out discs of leaf contained chlorophyll a and b,
carotenoids and anthocyanins with higher concentrations in mutant lines compared with those
from original NiF8 and NiTn18 lines. Singlet oxygen evolved will make flight out side of
emitted chloroplast, and react with unsaturated fatty acids forming malonedialdehyde. The
amounts of malonedialdehyde under strong sunlight were reduced in the mutant lines
compared with original NiF8 and NiTn18 lines. The punch out discs from leaf obtained from
NiTn18 original and mutant lines grown in Tanegashima floated on the medium containing
80 µM PQ showed resistant characteristics in the nutant. The mutant lines showed higher
tillering and were suggested to make precise planting plan to obtain larger yielding.
Chapter 5: There is a positive correlation between both, a higher dietary intake and tissue
concentration of carotenoids (Crts) and a lower risk of chronic diseases. Antioxidant activity
is one of the most important functions of Crts responsible for their beneficial effects. A
detailed mechanism of Crt antioxidant action, especially in vivo, is not fully understood. The
antioxidant activity is more effective at low pO2, while at higher pO2 (e.g. in cancerous
tissues) the generated Crt radicals have shown prooxidant activity by reacting with oxygen
and producing Crt-peroxyl radicals. It has been shown that lycopene and lutein may induce
formation of hydroperoxides in vitro, while β-carotene induces their formation and
decomposition. The alternative pro-oxidant mechanisms have been also proposed, such as
Crt-radical adducts formation, followed by decomposition into reactive alkoxyl-radicals or a
direct reaction of the formed adducts with triplet oxygen that can yield peroxyl-radicals. On

Preface ix
the other hand, the Crts prooxidant activity in vivo has been very recently attributed to the
chemical quenching of singlet oxygen by these compounds. Crts react with singlet oxygen,
giving relatively (un)stable cyclic mono- and diendoperoxides as the products. These species
can promote autooxidation of Crts and oxidation of other species initiating in vivo harmful
peroxidation of lipids. Bearing this in mind, a growing number of in vivo studies of
mechanisms of Crts anti-/pro-oxidant activity and health-related effects are to be undertaken.
A better knowledge of Crt properties and reactions in model systems appears as an useful and
necessary step for determination of conditions that predetermine the anti-/pro-oxidant activity
of these pigments in vivo.
Chapter 6: Medicinal herbs are gaining importance in modern medicines for the
prevention and cure of various chronic and pathogenic diseases. Carotenoids being oil soluble
compounds help in preventing the lipid peroxidation in the body which is associated with
atherosclerosis and cardiovascular disease and also protect the cell membrane damage.
Besides, carotenoids are also important as dietary precursors of vitamin A, which help in
maintaining the good vision. Vitamin A and β-carotene are also believed to enhance the
immune system functioning. Research also indicates that diet with low level of carotenoids
causes oxidative damage from free radicals and increases the risk of chronic diseases. Hence,
increased consumption of fruits and vegetables as well as supplementation of the carotenoids
may help in combating adverse health effects on the body. This chapter deals with the various
aspects of the carotenoids such as types, sources supplementation and bio-efficacy studies.
Chapter 7: Microalgae can produce large amounts of a wide variety of carotenoids,
including β-carotene, astaxanthin, lutein, canthaxanthin, violaxanthin and neoxanthin.
Moreover, microalgae have the added benefit that they can be grown on non-arable land,
avoiding competition with food production and precious biodiverse landscapes. Several
microalgae strains are currently being used commercially for production of astaxanthin and β-
carotene and research is ongoing to optimise production of other commercially important
carotenoids. This chapter will review the recent advances in the production of carotenoids
from microalgae, the potential health benefits discovered by the use of carotenoids from
microalgae, and the current status of biotechnological application of these compounds. The
chapter will also discuss future direction of research to enhance our understanding of the
carotenoid biosynthesis pathways in microalgae and the potential use of microalgae as
nutraceuticals and food supplements.
Chapter 8: Body color of ornamental fish is crucial for their market value. Previously, the
authors found that sodium taurocholate ranging from 1000~ 2000mg/kg can effectively
improve body color through higher utilization of astaxanthin in blood parrot (Cichlasoma
synspilum ♀× Cichlasoma citrinellum ♂), one of the most popular ornamental fish, but
accurate requirements of sodium taurocholate to aquaculture remain unclear. Furthermore, the
authors continued to study the optimum amount of sodium taurocholate supplement ranging
from 1000~1800 mg/kg, by observing body color with concentration of total carotenoid in
blood parrots. The total carotenoid content in the skin/caudal fin of 7 groups (total 462 blood
parrots) was determined at 15d, 30d, 45d, and 60d. The results showed that all fish adding
sodium taurocholate had improved body color by better pigmenting efficacy of astaxanthin
compared to fish (groups A (control groups) or B (only astaxanthin)). Significant differences
in the total carotenoid content in the skin/caudal fin between the groups with 400 mg/kg
astaxanthin plus sodium taurocholate level and groups A (control groups) or B (only
astaxanthin) were detected from day 15 to day 60. A supplement of 1600 mg/kg sodium

x Masayoshi Yamaguchi
taurocholate was the most effective. Based on the current findings, the authors suggest that
400 mg/kg astaxanthin plus 1600 mg/kg sodium taurocholate improves pigmentation quickly
over a 45 day period in the cultured blood parrot, which may be an effective, lower cost
option for a color-enhancing supplement.
Chapter 9: Carotenoids are large lipophilic compounds that can only be produced by
plants, fungi and bacteria. Although animals cannot synthesize them de novo, many taxa
accumulate them in exposed parts of the body for communication purposes. In many birds,
carotenoids are responsible for the bright yellow-red coloration of integuments or plumages,
and these have often been shown to advertise an individual‘s superior health. Some of the
hypotheses aimed at explaining the function of carotenoid-based traits rely on the assumption
that carotenoids have key physiological roles in the organism. Specifically, they are
hypothesized to be significant antioxidants and immune-stimulants, being therefore intimately
linked to the anti-parasite defenses and health. The mechanisms and functions of the
expression of carotenoid-based traits have been particularly well studied in birds. In this
chapter we first review the current hypotheses linking carotenoids, oxidative stress, immune
response and parasites, together with the current evidences in support of these key
physiological functions. Secondly, we focus on two gallinaceous bird species that have been
intensively studied in recent years, both in captivity (the red-legged partridge, Alectoris rufa)
and in the wild (the red grouse, Lagopus lagopus scoticus). In both species, ingested
carotenoids are metabolized and transformed before being used to color bright red ornaments.
The redness of the sexual ornaments relates to the concentration of carotenoids in blood, and
varies with infection levels by intestinal parasites and the ability of individuals to mount an
immune response to pathogens. Circulating levels of carotenoids through the blood stream are
also negatively affected by physiological stress (oxidative stress and stress hormones), which
is partly a consequence of parasite infections and immune responses. The evidence for
carotenoids to act as antioxidant and reduce oxidative stress is still mixed and debated, but
some studies highlighted that carotenoid-based ornaments can signal an individual´s ability to
resist oxidative stress. Finally, we identify key open questions that deserve further
investigation in this field.
Chapter 10: Carotenoid pigments are a group of bioactive compounds that are of interest
to the food scientists, nutritionists and food industries due to their positive impact on human
health and their economic benefits. Carotenoids are responsible for the attractive colour of
many plant food (mainly fruit and vegetables), which is perhaps the first attribute that
consumers assess when determining the quality and appearance of a product, and therefore
conditions its acceptability. In addition, carotenoids have diverse biological functions and
activities, such as the well known provitamin A activity, antioxidant capacity and
enhancement of the immune system. There are an extensive number of factors affecting the
efficient incorporation of these phytochemicals from the diet, although in many cases no
biological activity will be put in action within the consumer body (animal or human) without
a first visual attraction.
Chapter 11: Human skin is rich in different antioxidants. Amongst a variety of
antioxidants, fat-soluble carotenoids play an important role in the protection of human skin
against the action of most reactive free radicals, such as singlet oxygen and other reactive
oxygen species. Recent studies performed with electron paramagnetic spectroscopy show that
epidermal carotenoids could serve as marker substances for the whole antioxidant capacity of
human skin. This means that the individual concentration of carotenoids in human skin

Preface xi
reflects the current antioxidative status of human skin. Non-invasive optical methods are well
suited for in vivo investigation of kinetics of carotenoids in human skin and irreplaceable for
this task. Findings obtained on a large group of volunteers with resonance Raman
spectroscopy and reflection spectroscopy show that the concentration of cutaneous
carotenoids reflects the current lifestyle of volunteers. A high concentration of carotenoids in
human skin is always associated with a carotenoid-rich supplementation and lifestyle with
reduced stress. A low carotenoid concentration is attributed to an unhealthy lifestyle and
increased stress. Smoking, alcohol abuse, inflammation, illness, sun radiation, environmental
conditions, heavy sport loads, insomnia, fatigue, etc. are stress conditions giving rise to the
generation of free radicals in the skin and subsequent reduction of the antioxidative status.
Volunteers with a higher concentration of cutaneous lycopene were shown to have a reduced
skin roughness value, which is determined by the depth and density of furrows and wrinkles.
Thus, a high concentration of carotenoid lycopene in the skin is associated with a young-
looking visual appearance of the skin.
Chapter 12: As the exterior barrier of human body, the skin in direct contact with the
environment is exposed to oxygen and light generating reactive oxygen species (ROS) and
consequently photoaging and photocarcinogenesis. Thus, the mainstay of skin protective
strategies is UV photoprotection by inside and outside and the use of antioxidant compounds,
such as carotenoids. These natural compounds, in fact, are a class of molecules found in
plants, algae and in some fungi, used as food, that protect human body against excess light.
Besides of their specific activity, carotenoids are used as natural photo-protectant compounds,
being involved in the dissipation of excess light energy through the xanthophyl cycle,
quenching excited triplet state molecules, and singlet, oxygen. The use of these natural
molecules as antioxidant compounds may prevent, in fact, the oxidative damage of proteins,
lipids and DNA, modulating also the cell' signaling-initiating mechanism of the oxidative
stress. Essential for the effectiveness and safeness of carotenoids and chitin derivatives, is
their transport to the target site. Promising candidates as safe innovative carriers for both
endogenous and topical photoprotection are found Chitin nanofibril-hyaluronan block
polymers (CN-HA). Both Chitin and Hyaluronan are, in fact, natural ingredients
biocompatible and environment-friendly. The activity of CN-HA, as carrier, and carotenoids,
as skin photoprotectants, are reported in this chapter.
Chapter 13: Bixin is an polyunsaturated, norcarotenoid, red dye from the main fruit of
annatto, its seeds reduced to powder are widely used to color food and sunscreen. The annatto
seeds contain about 5% pigment, which consist of 70-80% of bixin. The bixin is soluble in
fats but insoluble in water. Brazil is one of the major producers of annatto and its planting
occurs primarily in the North and Northeast regions, although its cultivation in the last
decades expanded to other regions, in the Southeast, especially Sao Paulo and Rio de Janeiro.
The international market growing demand is justified by the prohibition of synthetic dyes due
to studies that have proven their toxicity. Like many carotenoids, bixin and norbixin also
present significant antioxidant properties. Both carotenoids can reduce levels of
malondialdehyde (biomarker of lipid peroxidation) when induced by cyclophosphamide
(immunosuppressant), as well as protect the DNA from oxidative damage in vitro. Bixin
inhibited in vitro generation of superoxide and the generation of reactive oxygen species such
as hydrogen peroxide and hydroxyl radicals and with protective action against mutagenicity
in human lymphocytes. Besides the antioxidant property, these carotenoids also present
important metabolic actions in lipids and sugars. Bixin acts as PPAR-γ agonist (receptor

xii Masayoshi Yamaguchi
activated by peroxisome proliferators range), a nuclear receptor that acts in the metabolism of
lipids and carbohydrates and has anti-inflammatory, immunomodulatory and anti-
atherosclerotic action. This interaction of BIX and the PPAR gamma receptor may increase
the sensitivity to insulin in adipocytes so that there is a greater glucose uptake. Furthermore,
the interaction with the receptor regulates lipid metabolism improving metabolic syndrome
present in diabetic patients.
Chapter 14: Carotenoids have been proposed to act as free-radical scavengers, even
though they can act as pro-oxidant molecules, at least at high oxygen concentration. However,
more recently, several biological beneficial activities related to their ability to regulate
different cellular functions have been proposed. Of note, a number of epidemiological studies
have shown a correlation between elevated dietary carotenoids intake and circulating levels
and decreased risk of cardiovascular disease (CVD). In fact, recently, it has been
demonstrated that circulating serum carotenoids were associated, in an apparently beneficial
direction, with markers of inflammation, oxidative stress, and endothelial dysfunction, which
are known to be associated with CVD. At present, it is widely accepted that one of the earliest
detectable pathogenic events in both human and experimental atherosclerosis is represented
by vascular inflammation associated to the activation of NF-θB pathway triggering, in turn,
the up-regulation of the expression of the vascular cell adhesion molecules (VCAM-1),
intercellular cell adhesion molecules (ICAM-1) and E-Selectin. Moreover, nitric Oxide (NO),
constitutively generated by endothelial cells, plays an important role in the maintenance of
vascular homeostasis and in the pro-inflammatory response that characterizes the early stages
of atherosclerosis. It is known that NO inhibits the vascular inflammatory response down-
regulating NF-θB-dependent expression of adhesion molecules. The maintenance of
endothelial NO bioavailability is therefore considered beneficial to endothelial functions and
more in general to vascular health. However, in the dysfunctional endothelium, NO may
rapidly react with superoxide anion (O2-) to form a stable potent oxidant peroxynitrite
(ONOO-) resulting in decreased vascular relaxation, and contributing to the up-regulation of
NF-θB dependent cellular response. Thus, the general effect of anti-oxidant molecules on the
biological function of NO is likely to be due, at least in part, to a direct removal of O2-.
Within this scenario, carotenoids may be considered potential anti-oxidant modulators of
endothelial response to pro-oxidant/inflammatory stimuli. Even though it has been recently
demonstrated in in vitro and in vivo experiments that carotenoids are able to reduce
inflammation and epidemiological studies indicate a strong correlation between dietary
carotenoid consumption and decreased risk of CVD, the mechanism underlying carotenoid‘s
cardiovascular protective activities, is still poor known.
Chapter 15: The authors screened for selective inhibitors of mammalian DNA
polymerase (pol) species from food components, focusing on carotenoids in this review. Ten
major carotenoids in food, including α-carotene (1), β-carotene (2), lycopene (3), β-
cryptoxanthin (4), zeaxanthin (5), lutein (6), canthaxanthin (7), astaxanthin (8), capsanthin
(9), and fucoxanthin (10), were investigated for their inhibitory effects on in vitro mammalian
pol activities. Compounds 5, 6, and 9 strongly inhibited the activities of mammalian pols β
and ι, which are DNA repair- and/or recombination-related X-family pols. In contrast, all
carotenoids tested had no influence on the activities of mammalian pols α, γ, and θ, which are
members of the B-, A-, and Y-families, respectively. Lutein (6) was the strongest inhibitor of
mammalian pols β and ι of the 10 carotenoids that were tested, and lutein showed
noncompetitive inhibition with both the DNA template-primer substrate and the dNTP

Preface xiii
substrate. The tendency for β and ι pol inhibition by these carotenoids showed a positive
correlation with suppression of tumor necrosis factor-α production induced by
lipopolysaccharide in cultured mouse macrophage RAW264.7 cells and 12-O-
tetradecanoylphorbol-13-acetate-induced inflammation in mouse ear in vivo. The relationship
between the structures and bioactivities of carotenoids is disccused in this chapter.
Carotenoids containing lutein may be particularly useful due to their anti-inflammatory
properties based on the ability to inhibit mammalian β and ι pol activities.
Chapter 16: Bone homeostasis is maintained through a balance between osteoblastic bone
formation and osteoclastic bone resorption. Aging induces bone loss due to decreased
osteoblastic bone formation and increased osteoclastic bone resorption. Osteoporosis with its
accompanying decrease in bone mass is widely recognized as a major public heath problem.
Among various carotenoids (carotene and xanthophylls including β-cryptoxanthin, lutein,
lycopene, β-carotene, astaxanthin, and rutin), β-cryptoxanthin, which is abundant in Satsuma
mandarin orange (Citrus unshiu MARC.), has been found to uniquely have a stimulatory
effect on osteoblastic bone formation and an inhibitory effects on osteoclastic bone resorption
in vitro, thereby increasing bone mass. β-Cryptoxanthin has an effect on the gene expression
of various proteins that are related to osteoblastic bone formation and osteoclastic bone
resororption in vitro. Intake of β-cryptoxanthin has been found to have the preventive effect
on bone loss in animal models for osteoporosis and healthy human or postmenopausal
women. β-Cryptoxanthin may be important as osteogenic factor in preventing osteoporosis in
human subjects. This chapter will discuss a role of dietaryβ-cryptoxanthin in prevention of
osteoporosis and treatment.
The editor believes that this book, which has been written to outline the recent advances
of carotenoid research, will be of interest to scientists focused in the fields of chemical and
biomedical research concerning carotenoids.

In: Carotenoids ISBN: 978-1-62808-622-5
Editor: Masayoshi Yamaguchi © 2013 Nova Science Publishers, Inc.
Chapter I
Carotenoids as Bioactive Compounds
Aline Inácio Alves1, Afonso Mota Ramos1, Paulo César Stringheta1

and Ellen Silva Lago Vanzela2
1
Universidade Federal de Viçosa – UFV, Peter Henry Rolfs, s/n,
Campus Universitário, Viçosa, Minas Gerais, Brazil
2
Universidade Estadual Paulista - UNESP, Cristovão Colombo,
Jardim Nazareth, São José do Rio Preto, São Paulo, Brazil
Abstract
The emphasis on searching for foods that contribute to adequate health has increased
worldwide. Scientific evidence has shown that foods containing physiologically active
substances are as necessary for health and disease prevention as are essential nutrients.
Carotenoids are among the most studied bioactive compounds and contribute to the
health-promoting properties of fruits and vegetables. Although hundreds of carotenoids
have been identified, the most studied are beta-carotene, lycopene, letein, zeaxanthin and
cryptoxanthin. Health benefits include antioxidant action related to the decrease of
degenerative illnesses (e.g. cancers, cardiovascular diseases, macular degeneration and
cataracts). Therefore, this chapter aims to describe the main carotenoid bioactive
compounds in terms of their chemical structure, natural sources, bioavailability and
bioconversion and analyze their effects on health and nutrition, with a focus on recent
studies and a perspective to the future.
Keywords: Bioactive compounds, pro-vitamin A, antioxidant, natural pigments
Introduction
Knowledge of food composition is fundamental for assessing nutrient availability,
nutritional adequacy of a diet and food consumption frequency. Moreover, knowledge of food
composition can contribute to research related to diet and human health as well the

2 Aline Inácio Alves, Afonso Mota Ramos, Paulo César Stringheta et al.
development of new technologies and processing techniques that preserve the maximum
nutritional value of food [1]. This is because of increased consumer demand for foods that
contribute additional health benefits and contain fewer chemical additives.
Fruits and vegetables are the main food products associated with functional claims.
Epidemiological and intervention studies have shown an inverse relationship between the
consumption of fruits and vegetables and incidence of degenerative diseases such as cancer
and cardiovascular disease. There is strong evidence of synergistic action between several
bioactive compounds present in these foods, including vitamin C, vitamin E, carotenoids and
flavonoids, with these bioactive compounds being responsible for health benefits [2]. As the
chemical structures of these bioactive compounds are very diverse, and as such they can act in
different ways regarding physiological targets and action mechanisms. Furthermore, the
possible beneficial effects of these compounds depend on the quantities consumed and their
bioavailability [3].
The bioavailability and bioconversion of carotenoids are influenced by the quantity and
structure of carotenoid intake and the food matrix in which the carotenoids are located [4,5].
Carotenoid bioavailability and bioconversion are also affected by factors intrinsic to the
individual, such as nutritional status and disease conditions that may interfere with carotenoid
absorption [6].
The functional importance of these compounds on human health has led researchers to
study bioactive compound concentrations in fruits and vegetables and search for new sources
of these compounds [7, 8]. Among bioactive compounds, carotenoids are of particular
interest. Carotenoids are present in all photosynthetic organisms. They are important natural
pigments responsible for the color, from yellow to red, of many fruits and vegetables in
addition to flowers, tubers and some animals, including birds, insects, fish and shellfish, and
microorganisms such as yeast. Animals do not synthesize carotenoids, but acquire them by
absorption through diet. This process may or may not be selective and carotenoids can
accumulate with or without minor modification [9].
Animals cannot synthesize carotenoids, but acquire them through diet (either selectively
or not) and may accumulate carotenoids with or without slight modification. This is contrast
to higher plants and some microorganisms, which are biochemically capable of carotenoid
synthesis, presenting complex composition enriched by biosynthetic precursors and derived
from the principal carotenoids [9]. α-Carotene, β-carotene, β-cryptoxanthin, lycopene, lutein
and zeaxanthin are the most studied carotenoids due to their involvement in human health and
frequently found in traditional Brazilian food, with the first three carotenoids being
considered pro-vitamin A [10].
In addition to coloring power, some biological functions are attributed to carotenoids,
with the best known being the physiological function of pro-vitamin A. Over a significant
period of time, insufficient intake of vitamin A or its precursors causes blindness and a high
(60%) rate of mortality, especially in children [11]. In developing countries, most of the
population cannot afford animal products, which are sources of preformed vitamin A. In these
areas, the vitamin A in the diet comes mainly from pro-vitaminic carotenoids such as α-
carotene, β -carotene and β-cryptoxanthin. Since the 1990s, other biological functions, such
as reduced risk of cardiovascular disease and certain cancers, a strengthened immune system,
the prevention of macular degeneration and cataract formation have been attributed to
carotenoids, with these biological functions being independent of the activity of pro-vitamin
A [11, 12, 13]. Such beneficial effects on human health have largely been attributed to the

Carotenoids as Bioactive Compounds 3
antioxidant properties of carotenoids by deactivating free radicals and sequestrating singlet

oxygen [14]. Free radicals are atoms or molecules produced continuously during metabolic
processes that act as intermediaries in the transfer of electrons in various biochemical
reactions and play an important role in human metabolism [15]. Free radicals react with
DNA, RNA, proteins and other oxidizable substances. They may contribute to aging and
development of degenerative diseases such as cancer, atherosclerosis, rheumatoid arthritis and
others [15, 16]. Antioxidants can act directly by neutralizing the action of free radicals, or
indirectly by participating in enzymatic systems with this function. Hence, antioxidants
combat free radicals and help to maintain human health [17, 18].
Carotenoids present in fresh food are naturally protected by the complex structure of
plant tissue. However, the cell structure is inevitably broken up by various processing
operations such as peeling, cutting and disintegration prior to thermal food processing. These
operations increase the surface area and therefore the oxygen exposure of the carotenoids,
putting them in contact with oxidative enzymes and light, which trigger a series of
degradation reactions (isomerization, oxidation and epoxidation). The isomerization of trans-
carotenoid compounds, the most abundant arrangement in nature, to the “cis” isomers is
caused by contact with acids, exposure to heat and light, resulting in slight losses of color and
biological activity [8]. Thus, carotenoids can undergo quantitative and qualitative changes
during the various steps that they are subjected to between harvest and consumption [19]. On
the other hand, some studies indicate that food processing can increase the bioavailability of
carotenoids by rupturing the cell wall and denaturing proteins complexed with carotenoids,
facilitating their release from the food matrix.
In this way, lycopene from tomatoes was absorbed less absorbed from fresh food and
juice (not subjected to heat treatment) than in processed tomato products [20], including juice
tomato stew with corn oil [21], tomatoes cooked with olive oil [22] and tomato paste [23].
This review emphasizes the importance of carotenoids, presenting their sources, chemical
structures, main functional property claims as well as factors that influence their composition
and stability in food products. This knowledge allows the recommendation of measures to
increase these bioactive compounds in foods, as well as establishing processing operations for
fruits and vegetables to significantly increase carotenoid retention.
Chemical Structure
Carotenoids are micronutrients present at very low levels (micrograms per gram), widely
distribution in nature and with varied chemical structures and functions [8]. Knowledge of
carotenoids is important due to the biological functions of these bioactive compounds and the
direct relation to their chemical structure.
Chemically, carotenoids are C40 tetraterpenoids formed by the tail-head union of eight C5
isoprenoid units, except in the center position where the junction occurs toward tail-tail, thus
reversing the order and resulting in a symmetric molecule. The central methyl groups are
separated by six carbon atoms, whereas other groups are separated by five carbons [8]. The
basic skeleton of this molecule family can be modified in many ways, including cyclization,
hydrogenation, dehydrogenation, introduction of oxygen-containing groups, rearrangements,

polymeric chain shortening or combinations thereof, resulting in a wide variety of structures

[24, 25]. The successive transformations are schematically illustrated in Figure 1.
Figure 1. Main carotenoid formation in plants. Reactions: 1. desaturation, 2. cyclization, 3. hydroxylation and
4. epoxidation.
Carotenoids can be classified into two large groups: carotenes, polyene hydrocarbons
with varying degrees of unsaturation, and xanthophylls, hydrocarbons with oxygenated
functional groups [26, 27]. Other modifications, such as epoxidation and cyclization of
carotenes and xanthophylls, result in a large number of epoxicarotenoides [9, 28].

During the life cycle of fruit and vegetables, the function of carotenoids is mainly to
promote tissue color. The characteristic structure of conjugated double bonds generates a
system of  electrons that moves along the polyene chain. This system constitutes the light
absorbing chromophore that gives carotenoids their attractive coloration and provides a
visible absorption spectrum in the range of 400-500 nm, which serves as the basis for their
identification and quantification. At least seven conjugated double bonds are required for a
carotenoid to be colored and as the length of the chromophore is extended, the resonance of
the molecule increases; i.e. the number of possible electronic transitions that can help to
reduce the energy of the molecular orbital of the compound is greater, resulting in
stabilization. Consequently, the wavelength at which the molecule will absorb energy will be
larger. Cyclization puts the conjugated double bonds, which are found within the rings, out of
the plane of the polyene chain, causing a hypsochromic shift (a shift of the maximum
absorption wavelength (max) to a shorter wavelength, a hypochromic effect (decrease in
absorbance) and the loss of fine structure [29].
Thus, the -carotene with seven double bonds imparts a yellow color, while lycopene,
with eleven conjugated double bonds, presents a red color. The monocyclic -carotene, as
well as the dicyclic -carotene, despite having eleven conjugated double bonds like
lycopene, present reddish-orange and orange coloration, respectively [9].
Main Sources and Applications in Food Industry

In the human diet, carotenoids are provided mainly by the intake of fruits and vegetables
as well as a small proportion of animal foods and food additives, such as natural dyes. In
photosynthetic tissues, carotenoids absorb light at wavelengths where chlorophyll absorbs
little amounts of light, allowing light collection over a more extensive range of wavelengths.
Also, carotenoids play an essential role as photo-protectants. When the energy stored in the
chlorophylls in an excited state is not rapidly extinguished by transfer of excitation, these can
react with molecular oxygen to form singlet oxygen, which is highly reactive and can damage
many cellular components, especially lipids [30, 31].
Microorganisms and higher plants are biochemically capable of synthesizing carotenoids,
since they have a complex composition enriched by biosynthetic precursors and derivatives of
the principal carotenoid. Unlike plants, animals, including humans, do not synthesize
carotenoids; therefore they must acquire these bioactive compounds through their diet [9]. Of
50 carotenoids in the human diet that can be absorbed and metabolized, only six of them (β-
carotene, α-carotene, β-cryptoxanthin, lycopene, lutein and zeaxanthin) represent more than
95 % of total carotenoids in the blood and have been studied in the context of human diet and
health [32]. However, vegetable products vary in carotenoid quality and quantity. Carotenoid
composition is also affected by factors such as plant variety, degree of ripeness, climate, soil
type, growing conditions, geographical area, unequal distribution in a particular food product
or vegetable part consumed, harvesting conditions, processing and storage [33, 34].
Green vegetables (leafy or not) have a similar qualitative profile, with lutein, β-carotene
violaxanthin and aneoxantina being the major carotenoids. The relative proportions of these
carotenoids are fairly constant, but not their absolute concentrations, which vary considerably.
Lettuce contains lactucaxantina as its major carotenoid. Green vegetables also contain minor

carotenoids, such as α-carotene, α- or β-cryptoxanthin, zeaxanthin, antheraxanthin and lutein-

5,6-epoxide [8].
Fruits and vegetables have a more complex and diverse carotenoid composition than
leafy vegetables, with considerable variation even within major carotenoids. Typically, fruits
contain few carotenoids in high concentrations, along with a number of minor components
present in small amounts or traces. The main profiles found in fruits and vegetables are as
follows: (a) negligible levels of carotenoids, as found in pears; (b) low carotenoid content,
usually chloroplast pigments, as found in grapes; (c) significant amounts of lycopene, as
found in tomatoes; (d) predominance of β-carotene, as found in acerola; (e) β-cryptoxanthin
as the main carotenoid, as found in apricots and peaches; (f) substantial amounts of epoxides,
as found in mango; and (g) a preponderance of rare or species-specific carotenoids, as found
in red pepper. Some fruits also exhibit some combination of these profiles [8]. Carotenoids
structures, characteristics and their main sources are presented in Table 1.
Fruits and vegetables are quite perishable due to their high water content and low
resistance to mechanical damage; necessitating the use of some preservation technique(s) to
ensure their stability and supply throughout the year to guarantee that the population will be
able to consume carotenoids important for health in satisfactory amounts on a regular and
continuous basis.
The same system of conjugated double bonds that is responsible for the coloring power of
the carotenoids is also responsible for their instability, since the high degree of unsaturation
subjects the molecule to diverse degradation reactions [52].
Carotenoids present in fresh food are naturally protected by the complex structure of
plant tissue. However, the breakup of the cell structure of the food, which is inevitable for
performing various food processing operations such as peeling, cutting and/or disintegration,
increases the surface area and therefore the exposure of the carotenoid to oxygen, as well as
oxidative enzymes and light, triggering a series of degradation reactions, including
isomerization, oxidation and epoxidation. Initially, the extent of these changes depends on
physical damage and possible excess temperature during handling, as well as in the time
period between harvest and processing [53].
Table 1. Structures, characteristics and main sources of carotenoids in food
Common name Characteristics Main sources

Tomato [35], Watermelon[36], Guava
Lycopene Acyclic, red color
[37]
Pumpkin [38],
-carotene Bicyclic, two -rings, orange color
Kale [39], Carrot [40]
-carotene Bicyclic,  ring and  ring, yellow color Pumpkin [38], Buriti [41], Carrot [40]
Bicyclic, two  rings and a hydroxy Papaya [42], Peach [43],
-cryptoxanthin
group, orange color Pitanga/Brazilian Cherry [44]
Bicyclic, two  rings and two hydroxy
Zeaxanthin Maize [45], Pumpkin [46], Buriti [39]
groups, yellow-orange color
Bicyclic,  ring and  ring and two Lettuce [47], watercress [47], Kale
Lutein
hydroxy groups, yellow color [48]
Bicyclic, two hydroxy groups and epoxy Mango [49], Parsley [50], Yellow
Violaxanthin
groups, yellow color Pepper [51]

The isomerization of trans-carotenoid compounds, the most abundant arrangement in

nature, to “cis” isomers is caused by contact with acids, heat and light exposure, resulting in
slight losses of color and biological activity. Oxidation, the main cause of carotenoid
degradation, occurs when they are in contact with oxygen, exposed to light or heat or in the
presence of enzymes, metals or peroxides. This reaction can be inhibited by antioxidants. It is
generally accepted that the initial stages of oxidation involving epoxidation and cleavage
result in apocarotenoids and low molecular weight compounds, similar to those produced in
the oxidation of lipids. In addition to color loss and biological activity, these compounds are
responsible for the development of off-flavor in some foods such as carrots and sweet
potatoes [54].
The scientific literature has shown that carotenoids retention can be affected in a variety
of ways, depending on time and, temperature, and used during the processing of the food, as
well as e the preliminary operations used [55]. In this way, by reducing the time between
peeling, cutting or shredding, carotenoid retention can be significantly improved [54].
In particular, when food is subjected to thermal treatment, such as bleaching,
pasteurization, baking, frying or drying, carotenoids content may decrease. In some cases,
however, the disruption of the food matrix (cell walls and membranes) has beneficial effects,
since this process enables the release and solubilization of carotenoids [55]. Moreover,
carotenoid retention is reduced by high temperatures and long processing time, independent
of the selected processing method. In this way, emerging technologies such as high pressure
processing [56, 57] and pulsed electric field [58, 59] are currently contributing to improve the
retention of carotenoids in processed foods. The use of antioxidants, sulphitation, oxygen
exclusion, light protection and storage at low temperatures can prevent significant carotenoids
degradation [8].
In the food industry, carotenoids are used primarily as dyes to restore color lost after
processing and during storage, to color pale food and to standardize the color of food
products. However, carotenoids are generally replaced by artificial colors due to the low
stability of carotenoids [27]. The increasing demand for new products that add functional
features related to health benefits, as well as consumer rejection of food products containing
artificial chemical additives, has encouraged the food industry to invest in research to replace
artificial dyes by natural pigments of food products. Food products using natural pigments as
colorants include powder preparations for refreshments, children's drinks and jellies, and
others.
Some natural extracts have been used as colorants for centuries, especially annatto
(bixin), turmeric (crocin), tomatoes (lycopene) and paprika (capsanthin). In tropical and
subtropical climates, many countries have a wide variety of carotenogenic sources including
fruits, vegetables, and oils extracted from palm fruits [34]. Overall, the colorants commonly
used in foods, drugs and cosmetics include β-carotene, canthaxanthin, lutein, astaxanthin,
zeaxanthin, capsanthin, capsorubin, and some rodoxantin and apocarotenoids. Research of
sources rich in carotenoids has increased in recent years.
In addition to pigment, carotenoids serve as a substrate for the formation of aroma in
plants and vegetables. Industrial application of flavoring after the biotransformation of
lycopene has been verified. In vitro studies have indicated that many noncyclic
noroisoprenoid compounds found in tomatoes and watermelon can be obtained by lycopene
hydrolysis [60].

In addition to their coloring power, some of these compounds have the ability to convert
vitamin A, which is related to bioactivities, such as vision, bone growth and tissue
differentiation [6, 61]. Within the provitaminic A compounds are β-carotene, α-carotene and
β-cryptoxanthin. These compounds act in conjunction with lycopene to reduce the risk of
diseases such as cancer, cardiovascular diseases and immune system disorders [62, 63, 64].
Lutein and zeaxanthin may help prevent macular degeneration and cataract formation [32].
These and other allegations of functional properties are described below.
Carotenoids and Health Benefits

Carotenoids play a key role in human health. Although many hypotheses have been
confirmed, many bio-functions have not been fully elucidated in vivo.
One of the best known physiological functions is the activity of pro-vitamin A. Vitamin
A is a nutrient found only in food of animal origin in various forms including retinol, retinyl,
retinal and retinoic acid. Vegetables provide provitamin A that may be biologically
transformed into vitamin A in animals [65]. Carotenoids that can be converted to vitamin A
are those with at least one non-substituted β-ionone ring, bonded to a conjugated polyene
chain of at least 11 carbons. Thus, out of more than six hundred naturally occurring
carotenoids identified to date, only about fifty have the ability to be precursors of vitamin A
[66]. Among carotenoids, -carotene is the most active (100 % activity), while -carotene, -
carotene, -zeacarotene, -cryptoxanthin and -cryptoxanthin present 50% activity. The
central cleavage (main mechanism) divides the -carotene in the central double bond (15-15'),
and the resulting product is retinal, which can be reversibly converted to retinol (vitamin A)
and irreversibly to retinoic acid. In asymmetric cleavage -apocarotenoids are formed, which
can eventually be converted to retinal and are precursors of retinoic acid (Figure 2) [67].
Prolonged vitamin A deficiency causes a severe disease called hypovitaminosis A, which
if not treated can cause blindness and xerophthalmia. In addition to its importance in vision,
vitamin A is associated with many physiological processes, such as cell proliferation and
differentiation, spermatogenesis, fetal development, immune response, taste, hearing, appetite
and growth [68]. Vitamin A deficiency impacts public health because it affects populations
worldwide. The main strategies to combat vitamin A deficiency in developing countries
include iron supplementation, food fortification and dietary changes, such as increased
consumption of vegetables rich in carotenoids [69].
There are discrepancies in the literature regarding the efficacy and bioavailability of pro-
vitamin A carotenoids in combating vitamin A deficiency. In 2001, the U.S. Institute of
Medicine (IOM) released new conversion factors of carotenoid pro-vitaminics A, including a
new concept related to Retinol Activity Equivalents (RAE), corresponding to 1 g retinol, 12
g of -carotene or 24 g of other provitamin A carotenoid. These new factors make it
difficult to achieve the recommended quanitities of vitamin A by consuming vegetables rich
in carotenoid pro-vitaminics A. However, these factors were based on studies of healthy
populations, whereas individuals deficient in vitamin A have higher bioconversion of
carotenoids [67, 69, 70]. Furthermore, the type of carotenoid, amount ingested, molecular
union, the matrix in which they are incorporated, genetic factors, food preparation, fat and

fiber content and the varied interactions are some factors that influence the bioavailability of
carotenoids and the bioconversion to retinol [71].
Figure 2. Symmetric and asymmetric cleavage of -carotene.
Independent of their provitamin A activity, carotenoids have been associated with other
benefits to human health. The antioxidant activity of carotenoids occurs through the
sequestration of singlet oxygen, a highly energetic and reactive form, or interaction with free
radicals. These are the most suggested mechanisms to prevent or minimize problems resulting
from degenerative diseases such as various cancers [28, 72, 73, 74, 75, 76] and cardiovascular
diseases [77, 78].
Some epidemiological studies indicate that β-carotene also contributes to the prevention
and treatment of cardiovascular disease by reducing LDL-cholesterol oxidation and
atherosclerotic plaque. A Action mechanisms, such as carcinogen metabolism modulation,
cell proliferation inhibition, increased cell differentiation and the stimulation of gap junctional
communication, retinoid dependent signaling, impact on cell growth regulation and the
induction of detoxifying enzymes, have been proposed [64, 79, 80]. Although some animal
studies show that supplementation with β-carotene protects against cancer in some situations,
other population studies show negative or null effects. The population study CARET (The
Beta Carotene and Retinol Efficacy Trial) investigated 18,000 North American smokers
taking β-carotene and vitamin A supplements was expected to last five years but ended up
finishing 21 early due to a tendency of increased development of lung cancer and mortality
[81, 82].
Among carotenoids, lycopene appears to be the most potent antioxidant, being
recommended to prevent atherogenesis [78] and carcinogenesis [16]. There is scientific
evidence that lycopene has action against certain cancers (lung, esophagus and prostate)
through different action mechanisms that reduce the proliferation of epithelial cells and
carcinogenic cells, thus decreasing DNA damage and defending against oxidative stress [8].

Studies have indicated that consuming tomatoes and their byproducts, which are rich in
lycopene, is associated with reduced incidence of cancer, especially prostate [74, 76].
Recently, Tang et al. [83] conducted a study aiming to demonstrate the effect of lycopene
consumption on inhibiting the growth and progression of colon cancer in rats. They observed
chemopreventive effects of lycopene in preclinical studies, indicating its potential application
in preventing cancer. Therefore, lycopene supplementation could inhibit the growth and
progression of tumors. However, other studies have shown inconsistent results regarding the
antioxidant effect of lycopene. This may be partially explained by the variability related to the
concentration and bioavailability of lycopene in food. Furthermore, other studies suggest that
the protective effect is caused not only by the presence of lycopene, but a synergism with
other carotenoids already present in food, such as -cryptoxanthin, -carotene, -carotene,
lutein and zeaxanthin [73]. Lycopene may also play an important role in reducing the
incidence of cardiovascular disease [8].
Astaxanthin is another carotenoid with antioxidant activity. This carotenoid is found
predominantly in aquatic animals such as salmon, trout and lobster, as well as some birds
[84]. Previous studies have indicated that astaxanthin can have beneficial effects on skin,
preventing changes induced by ultraviolet rays that could cause DNA damage. However, the
effectiveness of astaxanthin depends on several factors, including concentration, the location
and type of cell on which it is acting, exposure time to ultraviolet rays and interactions with
other carotenoids such as lutein and zeaxanthin [84, 85]. In addition, other potential human
health benefits have been recently associated with these carotenoids. These benefits include
preventing cardiovascular disease, strengthening the immune system and preventing of
cataracts [86].
In particular, lutein and zeaxanthin were identified as the major components of the
macular pigment of the eyes. The biological function of these carotenoids is explained by two
mechanisms of protection: acting as antioxidants, which limits retinal oxidative stress, and
filtering blue light, which is considered the most energetic light related to the production of
optic nerve damage [79, 87]. The photochemical reactions originating in the retina and retinal
pigment epithelium make these structures highly susceptible to damage caused by oxidative
stress. The defense mechanisms of these layers of the eyeball include the presence of
antioxidants, such as carotenoids. Also, these substances may be replenished with vitamin
supplements or an appropriate food diet since they are not synthesized by humans [88]. The
main sources of lutein and zeaxanthin are egg yolk, corn, orange juice and green leafy
vegetables like spinach, kale and broccoli [89]. Additionally, zeaxanthin and lutein have
excellent antioxidant activity in liposoluble environments [90]. Studies have shown that
supplementation with foods rich in lutein and zeaxanthin can increase the concentration and
density of the macular pigment [89-98]. The levelse carotenoids are approximately 50 %
higher in subjects with healthy eyes than in individuals suffering from age-related macular
degeneration [88]. Despite the evidence, however, no direct correlation has been established
between the presence of lutein and zeaxanthin and the reduced risk of macular degeneration
and cataracts [99].
Other carotenoids, such as -cryptoxanthin, fucoxanthin, crocetin, capsanthin and
phytoene have been studied and show promising potential; hence, future studies are needed
regarding these carotenoids. This is because protective and beneficial responses are not
exclusively associated with a single factor, but with the presence of multiple factors acting in

a coordinated or synergistic manner, reinforcing the importance of variety in meal

composition and highlighting the limitations of diets with a unique and isolated component of
the nourishment [90, 100, 101].
Carotenoids appear to have an effect on immune response and intracellular
communication, presenting beneficial effects against age-related diseases. Furthermore, there
is evidence that carotenoids, in combination with other components of fruits and vegetables,
have a protective effect against some chronic diseases. In this way, the synergistic effect
between β-carotene and vitamins C and E was observed in cell protection, probably due to the
ability of β-carotene to destroy free radicals and repair the tocopherol radical produced by the
action of α-tocopherol [60, 102].
A good diet based on fruits and vegetables rich in carotenoids, along with healthy
lifestyle, which includes avoiding risk factors such as smoking and obesity [87], is more
strongly associated with reduced risk of certain diseases than a diet supplemented with
specific carotenoids [103]. A diet rich in natural antioxidants including carotenoids, vitamin
E, vitamin C and flavonoids has been associated with the prevention and treatment of
atherosclerosis, since these antioxidants can reduce the susceptibility of lipids and low density
lipoproteins (LDL) to oxidative damage, especially in the early stages of this disease [77].
In this way, Prabhu et al. [104], evaluated the chemopreventive efficacy of astaxanthin on
peroxidation, the antioxidant capacity of lipids and the proliferation of cells in 1,2-
dimethylhydrazine, colon carcinogenesis induced using mice. The results of this study
suggest that astaxanthin has beneficial effects against the progression of colon cancer induced
in rats.
Wang et al. [105], investigated the effectiveness of lycopene (pure or in tomato extract)
against hepatocarcinogenesis induced in rats. Results showed that both the lycopene and
extract can inhibit induced hepatocarcinogenesis, mainly as a result of reduced oxidative
stress.
Rock et al. [106], examined the relationship between plasma carotenoids in women over
different periods of time: less than 1 year, and after 1, 2, 3, 4 and 6 years free of breast cancer.
An average carotenoids concentration over time was calculated for each participant.
Biological exposure to carotenoids, when evaluated over the period of time of the study, was
associated with a higher probability of surviving free of breast cancer, regardless of the group
studied.
For that reason, prevention is emerging as the best and safest form of intervention. This
should be done through nutritional guidance, targeting a daily consumption of foods rich in
carotenoids [107, 108].
Absorption and Bioavailability

When ingested, carotenoids, which are soluble mainly in lipids, follow a similar path of
intestinal absorption and transportation as lipids [109]. The absorption of carotenoids by
intestinal mucosal cells is assisted by the formation of mixed micelles comprised of bile
acids, free fatty acids, monoglycerides and phospholipids. The amount of carotenoids
incorporated in the micelles depends on carotenoid polarity, as well as the composition and
saturation of micelar fatty acids [110]. Moreover, the degree of food processing is significant

for micellization efficiency. In this way, techniques such as mechanical homogenization,

thermal treatments and the addition of fat during food processing can maximize the amount of
compound incorporated and thus the efficiency of this phase [111].
Absorption occurs without cleavage in carotenoids such as β-carotene and cryptoxanthin
and the hydrolysis occurs in the interior of intestinal cells [67]. Possible factors that affect
carotenoid absorption include physical-chemical properties and source of ingested
carotenoids, the matrix in which the carotenoid is incorporated, the type and amount of lipid
ingested, the presence of factors that inhibit or facilitate ingestion, nutritional state and
individual age range, genetic factors, intestinal parasitosis and other factors related to each
individual and the interactions among these variables [5].
In this way, studies have shown that pure carotenoids dissolved in oil or aqueous
dispersion are well absorbed (> 50 %), while carotenoids such β-carotene are poorly absorbed
in cooked vegetables (e.g. carrots), as is lycopene in tomatoes juice without oil. Moreover,
other studies have shown that the type of lipid present in the diet can influence the
bioavailability of carotenoids, since meals rich in medium chain triglycerides are absorbed via
the portal vein, which diminishes the formation of chylomicrons [112]. The degree of
unsaturation in edible oils also affects carotenoid absorption. According to Clark et al. [113],
oral administration of lycopene and astaxanthin with emulsion of olive oil in mice resulted in
higher absorption compared to oral administration of these carotenoids with corn oil.
Another factor that affects carotenoid absorption is the form of their cis or trans isomers.
Most carotenoids in nature occur predominantly in the trans form [114]. In addition to the
natural occurrence of carotenoids in plants, the cis isomers are promoted mainly by contact
with acids, heat and exposure to light, resulting in slight losses of color and biological
activity. In the dehydration of mangos (Kent and Tommy Atkins varieties) [115] and pumpkin
slices [116], increases were observed in the intensity of cis-β-carotene derivative compounds.
It should be highlighted that trans-cis isomerization reduces provitamin A activity, with less
provitaminic A activity to 13-cis-β-carotene (53%) and 9-cis-β-carotene (38%) being
observed when compared to the corresponding trans-isomer [117]. This also affects the
bioavailability of carotenoids. In the case of β-carotene, trans isomers are preferentially more
absorbed than cis isomers in human beings [118].
After the absorption and during the postprandial period, carotenoids are incorporated into
chylomicrons and transported to the liver. The liver is the main storage organ of carotenoids
and controls their distribution to other tissues. Carotenoids are secreted by the liver in very
low density lipoproteins (VLDL) and are subsequently recovered in other lipoproteins, such
as low density lipoprotein (LDL) and high density lipoprotein (HDL) [119]. The
transportation of carotenoids in plasma by different types of lipoproteins is determined mainly
by carotenoid properties [67].
Some carotenoids such as β-carotene and cryptoxanthin are partially converted to retinal
during the absorption process. Subsequently, retinal is converted to retinol and transported by
the lymph vessels to the liver by the chylomicrons in the form of retinol esters [67]. The liver
contains 90 % of the vitamin A in the human body. Approximately 40 % of retinol is used,
while the rest remains stored. In the liver, retinol is released from retinyl palmitate, as a result
of the action of the enzyme retinyl ester hydrolase. Subsequently, after its release, retinol can
be linked to a plasmatic retinol binding protein (RBP) in order to move to the plasma, or it
can be captured by cytoplasmic RBP and taken to the storage sites, which are the adipocytes
and hepatocytes [67].

The storage in the form of retinol ester is done through the action of retinol
acyltransferase. After its arrival in the plasma, the complex retinol-RBP binds to prealbumin,
which protects retinol from degradation in the kidney. Retinol then enters the cellular sites
and RBP returns to the circulation to be degraded and recycled. In the cell, retinol binds to a
molecule cytoplasmic transport molecule to the RBP (specific to the organ) which carries it to
the site of action. Furthermore, retinol can be oxidized and turned into retinoic acid that binds
to specific nuclear receptors. Therefore, it has been verified that carotenoids are found in
various tissues. However, the mechanisms by which they are distributed in these tissues are
not precisely known. It is presumed that the receptors for lipoproteins and membrane proteins
are involved in this process [120].
In the absence of a functional marker of carotenoid biological activity in intact
organisms, the ―bioavailability‖ of carotenoids has been widely considered as ―assimilation
and efficiency‖ or the ability to accumulate in a defined pool of the body. These pools can be
highly dynamic, like chylomicrons in the plasma after a meal rich in carotenoids, total plasma
or lipoproteins following a subchronic or a chronic dietary supplementation, or more static
pools, such as adipose tissue or the macular pigment of the eye. Xanthophylls, lutein and
zeaxanthin comprise the macular pigment of the human retina, suggesting a macular function
and preventing macular degeneration. Thus, the density of the macular pigment can be
evaluated noninvasively by physical methods to assess its relationship with carotenoids [91].
This may be considered an indication of the bioavailability of lutein and zeaxanthin.
Thus, carotenoid bioavailability, bioconversion and bioefficacy are important terms used
in the literature and should be clearly understood. Bioconversion is the proportion of
bioavailable carotenoids converted into retinol, while bioefficacy is the efficiency with which
ingested carotenoids are absorbed and converted to retinol. Frequently, these terms are
confused, and often the term bioavailability includes both bioconversion and bioefficacy [67].
Factors that influence the bioavailability of carotenoids related to human diet have been
the most studied. The main factor is the type of food in which the carotenoids are present [5,
70]. This is because carotenoids are situated in different locations on the same plant or on
different plants. Accessing β-carotene in the plant cell can be easy or difficult and depends on
its location within the plant. In leaves, carotenoids are located at the thylakoid membranes of
chloroplasts forming a pigment-protein complex, whereas in fruits the carotenoids are located
in chromoplasts often linked to lipids or proteins.
The release of β-carotene from the cell goes through a few steps: initially the cell needs to
be broken. Then, the chrome or chloroplast must be accessed. If the pigment is linked, it must
be released. However, the bond pigment-protein is more complex and more difficult to break
than the bond pigment-lipid complex. Some authors also suggest that the β-carotene present
in fruit has higher bioavailability than in leaves [121]. Furthermore, the presence of fibers can
also affect carotenoid bioavailability [5].
Carotenoids are less bioavailable than pre-formed vitamin A since they are bonded to the
vegetable matrix. Thus, the requirements for intestinal absorption are superior to those of
provitamin A, which must be cleaved enzymatically and stored as vitamin A or carotene in
various tissues [6].
Contrary to prior belief, some forms of food processing may increase carotenoid
bioavailability [5, 122, 123]. ROCK et al. [124], found an increase in the bioavailability of β-
carotene in processed vegetables.

Food processing has also been shown to increase the bioavailability of lycopene due to its
release from the food matrix. Thus, tomato sauce and tomato puree are considered some of
the best sources of bioavailable lycopene compared to other uncooked foods, such as raw
tomato [21]. Rodriguez-Amaya [9], investigated diverse processing conditions, including:
cooking, such as scalding in water and steaming, cooking with and without a cover, boiling
and sautéing. Results indicated that carotenoids are lost in domestic preparations according to
the following cooking sequence: microwaves < steamed < boil < stew. Food processing such
as frying by immersion, prolonged boil, baked and marination cause considerable losses [8].
In this context, some studies regarding the analysis of carotenoids content in food
indicate greater ease of extraction after processing, causing an increase in the total amount of
carotenoids compared to fresh vegetables [116, 125, 126, 127]. This is probably due to a more
efficient denaturation of the protein-carotenoid complex [126]. As a result, carotenoid
retention greater than 100% has been observed in some studies, such as on the effect of
osmotic dehydration of tomatoes [128] and drying of pumpkin slices with forced air at 70 ° C
[116] on the carotenoid content. According to Rodriguez-Amaya [9], considering the fact that
carotenoids cannot be produced during processing, the possible explanation for this difference
is incomplete extraction of carotenoids from fresh samples due to greater extraction difficulty,
since they are physically protected or mixed with other food components.
Thus, the specific identification of positive and negative effects of food matrices on the
bioavailability of carotenoids should be taken into consideration, including the effects of
different food processing techniques, as well as the effects of the nutritional status on the
individual [129].
Scientific information about carotenoid bioavailability is based mainly on the
determination of serum levels, plasmatic levels or lipoprotein fractions after consumption.
Other methods for determining carotenoid bioavailability may also be used. These include the
method of balance, which consists of measuring carotenoid content by feces analysis,
measuring radioactive carotenoids in the lymph system or kinetic studies using isotope-
labeled carotenoids labeled [67].
Some methods used to evaluate the bioavailability of carotenoids in diets or dietary
supplements include: plasmatic response, human serum after carotenoid intake; assays in
fractions of triglycerides rich in lipoproteins, mixtures of chylomicrons and VLDLs; mass
balance and stable isotopes. The first is a simple procedure; however, it has some limitations:
serum response to oral doses is variable; the concentration of carotenoids in the serum
represent the balance between intestinal absorption and release of the corporal stock; the
human serum contains an endogenous concentration of carotenoids that may interfere with the
obtained values, and the pro-vitaminic A carotenoids, which are metabolized to retinyl esters
during intestinal absorption [130].
Mass balance includes the comparison of carotenoid consumption and fecal excretion to
estimate carotenoid absorption. This method has some limitations due to degradation, as well
as endogenous carotenoid secretion. The stable isotope method allows distinguishing between
carotenoids from a food source and those secreted endogenously, allowing the evaluation of
intestinal conversion of carotenoids to vitamin A, to estimate the absolute absorption, post-
absorptive metabolism and low doses, sufficient to prevent influences of endogenous pool
[130].

Carotenoids Analysis
Due to the fact that carotenoid biological functions and actions are directly related to their
structure, improving analytical methodologies for obtaining complete separation and reliably
quantifying these compounds has been intensely studied in recent decades.
This is a delicate procedure that is subject to many factors, such as: the variable nature of
the food matrix, the existence of large numbers of detected carotenoids; qualitative and
quantitative variability of food composition; wide variation in the amounts of carotenoids of
the same food; non-uniform distribution of carotenoids between samples and within the same
sample, and susceptibility to isomerization and oxidation of carotenoids during analysis or
during storage before analysis [8, 9, 65].
All the stages of carotenoid analysis, from sample preparation to quantification, must be
done appropriately. Essentially, carotenoids must be protected from light, oxygen and heat; a
vacuum or inert atmosphere must be used whenever possible and high temperatures must be
avoided. In addition, several natural factors allow the loss of carotenogenic composition in
food, evidencing the importance of sampling and sample preparation in order to obtain
reliable data [34]. Some important factors that affect the quality of the analytical data
obtained include sample selection, number of samples, handling and sample preparation
before extraction. The general stages of carotenoid analysis include sample preparation,
extraction and saponification, separation, detection and quantification [55].
Carotenoid Extraction
A good extraction procedure must release all carotenoids from the food matrix, bringing
them into the solution without any alteration. During the extraction and analysis of
carotenoids, the artifacts most commonly found are the geometric isomers formed in solution
by stereoisomerization, caused by exposure to heat, light, acids, etc. Carotenoids with
aliphatic chain are more susceptible to the stereoisomerization process. Undesirable
isomerization reactions can be prevented by conducting a careful and rapid extraction in the
absence of light and at low temperatures [131]. Since carotenoids are found in a variety of
foods, the extraction procedure must be adapted accordingly to the food analyzed [132].
When using liquid-liquid extraction, several techniques are applied, with the most
common being extraction with solvents in steps [133]. Lycopene is more soluble in
chloroform, acetone, benzene and other organic solvents. However, other solvents may be
used, including ethyl acetate, ethanol, methanol, among others, as well as their mixtures
(ethanol/hexane, acetone/ethanol, hexane/ethyl acetate, acetone/hexane, methanol
/tetrahydrofuran) [134].
In general, the solvent must be selected according to the polarity of the pigments present
in the sample. If this characteristic is unknown, a mixture of acetone/hexane (1:1, v/v) can be
used. When the carotenoids in the sample are non-polar or esters, hexane is a good selection
for extraction, whereas ethanol will extract polar carotenoids and a non-polar solvent such as
hexane promotes crystallization [135]. MERCADANTE [136], affirmed that acetone is
efficient in the extraction of carotenoids in fresh foods and ethyl acetate or diethyl ether in
lyophilized or dried foods. Rehydration is necessary for dehydrated food. It should be noted

that carotenoids may be extracted using solvents followed by an alkaline saponification [137],
which aims to hydrolyze esters of carotenoid and to stimulate the removal of lipids and
chlorophyll. This makes the posterior stages of separation, identification and quantification
easier [138]. The saponification is not necessary when the samples do not have compounds
considered interferents [55].
Xanthophylls, which are present in fruits, are normally found esterified with different
fatty acids, and the use of saponification allows easier chromatographic separation,
identification and quantification [136]. However, treatment with alkali can cause undesirable
reactions such as the formation of artifacts like cis isomers, epoxides and products from
reactions with alkali during this process. In addition, treatment with alkali can result in
degradation and loss of some carotenoids [131, 139]. Carotenoid loss can vary during
saponification according to the type of food analyzed, with higher loss of carotenoids such as
capsaxantine and β-carotene in foods that are industrialized, processed and richer in fat than
fresh food [137].
The use of conventional carotenoid extraction methods can result in the consumption of
high amounts of organic solvents, which are toxic, expensive and dangerous. Moreover,
residues of extracting solvents can contaminate the final product, making it improper for use
in the food, pharmaceutical and cosmetics industries. To avoid this inconvenience,
supercritical fluid extraction with CO2 has been used to extract carotenoids from different
types of raw material [133]. The stage of sample preparation before the supercritical fluid
extraction is the same as the one used for the extraction with organic solvents. Thus,
precautions must be taken in order to avoid chemical changes promoted by light and heat,
which favors the formation of cis isomers, modifying the biological activity of carotenoids
[140]. Although there are numerous advantages to using supercritical fluid extraction
compared to the traditional solvent extraction, Gutiérrez and Castro [140], reported that
lycopene extraction, alone or in conjunction with β-carotene, using supercritical CO2,
presents low reproducibility due to degradation and the isomerization of lycopene during
extraction.
As a result of these difficulties, some vegetables oils have been used as stabilizers. They
are also known as co-solvents. These vegetable oils, such as hazelnuts, almonds, peanuts and
sunflower, avoid or minimize the degradation of lycopene during extraction [141]. To help in
the liberation of the carotenoids in the food matrix, some enzymes may help in the digestion
of the plant material, facilitating carotenoid extraction [142]. Similarly, ultrasound can help in
the carotenoid extraction process [143]. The main effects of ultrasound in carotenoid
extraction are increased permeability of the cell wall and the production of cavitation, i.e. the
formation of bubbles in a liquid under its boiling point. These results in a strong dynamic
stress of cells (interfacial friction) that may increase pore size of the cell wall and disrupt
cellular tissue. As a consequence, the diffusion process and mass transfer are optimized,
increasing the extraction efficiency and reducing the time to extract the compounds of interest
[144]. This method has been used to extract carotenoids from different sources with good
yields, such as corn [145], tomatoes [146], palm oil [143], e.g.
Michelon et al. [147] studied carotenoid extraction from Phaffia rhodozyma using
different extraction methods, one of them being the use of ultrasound. This process, in
conjunction with the freezing of the biomass, allowed an extraction of 88.3 µg/g carotenoids.
In this context, although high levels of carotenoids may be produced from
microorganisms using biotechnology, their use is limited due to resistance in the

microorganism cell wall, which constitutes a barrier for carotenoid bioavailability. Therefore,
more studies need to be done aiming to improve the techniques of recovering carotenoids
from microorganisms for their application in the food industry, with emphases on the
enzymatic, mechanical and chemical techniques of cell disruption [147].
Separation
There is no standard method for analyzing carotenoids in foods; however, the methods
used for their separation are essentially: high performance liquid chromatography (HPLC),
open column chromatography (OCD) (used in accordance with the polarity, silica gel and
aluminum oxide and currently used for separating carotenoid patterns) and thin layer
chromatography (TLD) (used in differentiate solvents in the mobile phase, may be used to
analyze various samples simultaneously; but, due to the greater contact surface with
carotenoids, increase the losses by oxidation and isomerization) [136, 148].
The demand for rapid and reliable analytics combined with the need to create more
efficient and reliable methods of extraction and quantification led to the preference for the
HPLC technique. Among the advantages of this technique are high resolution power, high
reproducibility, the use of reusable columns, in addition to rapid separation with lower
carotenoid exposure to degradation (used under controlled conditions, without undue
exposure to air or light), allowing subsequent individual identification and quantification
[149].
HPLC analysis has presented wide application in the determination of carotenoid profiles
which may be separated by using reverse phase columns [150, 151, 152] or normal phase
[153,154]. In the work of Amorim et al. [155], with fruits commercialized in a street market,
the separations were done in an HPLC system (Shimadzu, SCL 10AT VP) equipped with a
diode array detector (DAD) (Shimadzu, SPD-M10A), chromatographic column RP-18
Lichrospher. As in the work cited, the modality of HPLC most used by researchers is the
reverse phase with monomeric column C18. This type of column presents broad diversity and
great commercial availability and lower cost, making it possible to use common solvents,
which are reproducible and may provide good separation of the principal carotenoids
naturally found in foods, with logic order of elution to facilitate the identification of the
compounds [156].
However, in processed samples, where geometric isomeres are present in more elevated
numbers and concentrations, the quantification of the carotenoids may be difficult because of
possible co-elutions of cis and trans, compounds, principally when structurally similar
carotenoids such as pairs of α- carotene and β-carotene (Figures 3 and 4, respectively) and
lutein and zeaxantina are present.
Although the efficiency of separation of these columns has been improved due to the
development of polymeric stationary phases, reducing the size of the particles and the
diameter of the columns, such advances have not overcome the elevated selectivity presented
by the column with polymeric stationary phase C30 developed by Sander et al. [157] The
potential offered by the polymeric column C30, with a stationary phase of 5 µm, was first
demonstrated in the separation of a mixture of polar carotenoid patterns (astaxanthin,
capsanthin, canthaxanthin, equinenona, lutein, zeaxanthin and β- criptoxanthin) and apolar

carotenoid patterns (trans-α-carotene, trans-β-carotene and the isomeres 15-cis-, 13-cis- and
9-cis-β-carotene, δ-carotene and lycopene) using the compound mobile phase of
methanol:ether methyl tert-butyl:water and linear gradient of 81:15:4 for 6:90:4 in 90 min,
with an output of 1 mL/min).
Figure 3. Principal geometric isomeres of α-carotene.
Figure 4. Principal geometric isomeres of β-carotene.
The high selectivity focused for polar and non-polar carotenoids, as well as their cis
geometric isomers, have been attributed in great part to the dimensions of the stationary phase
(25-30 Å) that are comparable to the dimensions of some carotenoids, e.g. the trans-β-
carotene providing a more complete interaction among the molecules [157, 158, 159]. A long
analysis time (90 min) was necessary for the chromatographic separation due to the greater
interaction of the analytes, principally of the polars, with the free silanol groups of the non-
capped stationary phase [157]. The authors emphasized, however, the chromatographic
conditions employed in this study were selected only to facilitate the comparison of the
results obtained by the C30 column with those obtained by the C18 columns and may be
modified in accordance with the compounds of interest to reduce analysis times.
Another variable that influenced the selectivity and recuperation of the carotenoids in
column with stationary phase C30 is the temperature [160, 161, 162]. When the separation of
only one class of carotenoids is required, the lowest temperatures are recommended. On the
other hand, higher temperatures must be used when a better selectivity for different
carotenoid classes with different behaviors is necessary [161].

With respect to the mobile phase, both isocratic systems [163, 164] have been employed
in elution gradients [165, 166]. In general, however, the methods of the gradient of solvents
achieve a better definition when compared with isocratic systems. The disadvantage is that
some methods can have a greater analysis time due to the necessity of rebalancing the column
after each injection [164]. Nunes and Mercadante [138], tested various conditions for
separation of isomers of lycopene using C30 and C18 columns and observed that the C30
column polymeric presented higher resolution. The best condition found by these authors was
obtained by the use of the C30 column with mobile phase methanol (0.1% triethylamine)/
ether methyl-tert-butyl alcohol of isocratic mode (50:50) and in gradient (from 60:40 to 50:50
in 20 min), both with output of 1 mL/min, column temperature of 33 ºC. However, the
gradient condition presented difficulty in the reconditioning and low repeatability of the trans-
lycopene peak, with the isocratic condition being considered better.
The choice of the combination mobile column-phase is the principal limiting factor for
the success of the chromatographic separation and recovery of the carotenoids [132]. The
most important properties considered for the choice of the mobile phase are the cost, polarity,
selectivity, viscosity, volatility and toxicity. The mobile phases normally used are comprised
of methanol or acetonitrile, with the addition of small quantities of other solvents such as
modifiers and additives.
Methanol has the advantage of being the cheapest, least toxic option and presents the
greatest carotenoid recovery. However, its viscosity is higher, which increase the pressure of
the system. Acetonitrile presents better efficiency for separating the xantophylls and is less
viscous, but presents lower recover, which may be improved by the addition of trietilamine
[167, 168].
As a modifier, the following solvents may be used: tetrahydrofuran (THF),
dicloromethane, propanol, ethyl acetate, methyl tert-butyl ether and other ethers. The
efficiency of each one depends on the carotenoids to be separated and of the stationary phase
used [156]. For the C30 column, the most used modifier is the methyl tert-butyl ether. This
modifier presents good selectivity and recovery of the carotenoids of the column, however its
high cost and currently low commercial availability limiting the use of this column modality.
Even when an adequate separation of the compounds has been obtained, it is still
necessary to identify and quantify the compounds of interest. It has been considered that this
stage of the carotenoid analysis is not easy, due to the great number of compounds detected in
nature, high qualitative and quantitative variation of these compounds in foods, the wide
range of concentrations of these pigments and their instability during analysis or food storage.
Identification and Quantification

The chromatographic behavior of carotenoids has a defined relation with its structures;
however, the identification of the carotenoids is not conclusive just considering the
chromatographic data (absorption spectrum and time of retention), especially due to the lack
of reproducibility of the retention times. The use of co-chromatography with patterns may
help in the identification, however, on certain occasions, different carotenoids may present
the same retention time in a chromatographic data system, further complicating the
confirmation of the carotenoid identities [132].

The analysis of the carotenoids via high performance liquid chromatography (HPLC)
uses, in most cases, ultraviolet-visible detectors (UV- Vis) or photodiode array [169]. The
majority of the carotenoids exhibit absorption in the visible region of the spectrum, between
400 and 500 nm. They obey the law of Beer-Lambert (e.g. absorbance is linearly proportional
to concentration) and the measurements of absorbance may be used to quantify the
concentration of a pattern of carotenoids, or to estimate the total carotenoid concentration in a
mixture of extract of carotenoids in a sample, using analytic curves, since the coefficient of
absorption is in the desirable solvent [135]. This absorption is due to conjugated double bonds
of carotenoids. The unsaturated conjugated part of the carotenoid molecule contains displaced
electrons called ―chromophore‖ and is responsible for the absorption of light in the visible
region.
The differences in the spectral characteristics of individual carotenoids are frequently
small, but are of great importance in their identification [135]. In addition to the maximum
absorption of the carotenoids, the "shape" of the spectrum provides important information for
carotenoid identification. The spectral characteristics of a pattern may be monitored with
HPLC using the detector photodiode array detector and a directory of standard spectra may be
stored, allowing the additional identification of the sample peaks. Figure 5 illustrates some
spectrums of absorption of geometric isomeres of α-carotene and β-carotene, obtained by the
detector photodiode array detector. However, some carotenoids that have the same
chromophore may consequently present the same spectrum of absorption [114]. As such,
numerous authors recommend the association of the techniques cited above with mass
spectrometry (MS) and Nuclear magnetic resonance spectroscopy (NMR Spectroscopy) to
confirm the identity of the molecule when possible. In complex matrices, when HPLC
detector photodiode array is not sufficient to acquire an unequivocal identification of the
compounds, the coupling of HPLC to a mass spectrometry (EM) usually presents optimal
results [170].
The methods of chromatography with mass spectrometry developed for carotenoid
analysis include the technique of chemical ionization at atmospheric pressure [171, 172] or
ionization by electrospray, among others [173, 174]. Some authors have used chromatography
coupled to a mass spectrometry to confirm identification and purification peaks in complex
matrixes [175, 176] while others make use of the fragmentation of the molecule to help in the
differentiation of structural isomers, such as lutein and zeaxantine [135].
Although these technologies are available, the identification and quantification of
individual carotenoids is difficult because of the great number of carotenoid structures that
exist and the difficulty in obtaining commercial patterns of the majority of the compounds
that often have a high cost. The patterns that are not commercialized are normally obtained
from natural sources rich in these patterns.
Amorim et al. [155], to help in the identification of the carotenoids, injected a mixture of
carotenoid patterns (α- and β-carotene), with the retention times and absorption spectrums of
the patterns being compared to those found in the samples. The external calibration curve was
used quantification of the carotenoid found in the samples. However, considering these
difficulties, it is common that, in various studies, the absolute quantification of each pigment
was not presented and the results were expressed as a relative percentage of area or total
concentration.

Figure 5. Absorption spectra of the geometric isomeres of α-carotene and β-carotene, obtained by the detector
photodiode array: a) trans-α-carotene, b) 9-cis-α-carotene, c) 13-cis-α- carotene, d) trans-β-carotene, e) 9-cis-
β-carotene, f) 13-cis-β- carotene.
The techniques of Raman have recently been used to analyze carotenoids in plants. The
carotenoids present two strong peaks in distinct regions of the Raman spectrum, resulting in

the stretching vibrations of the carbon-carbon bonds in the polyene chain, which may be used
to identify and quantify carotenoids in diverse plant tissues and complex food matrixes [177].
Baranska et al. [149], verified the application of Raman spectroscopy modified from Fourier
(FT-Raman), attenuated total reflection infrared spectroscopy (ATR-IR) and near infrared
spectroscopy (NIR) in rapid and direct determinations of lycopene in samples of tomato and
its by-products. The authors demonstrated that the FT-Raman spectroscopy may be
successfully applied in the quantification of lycopene in plant tissues and food products
without any previous treatment of the sample. On the other hand, the techniques of NIR and
ATR-IR presented significant water interference that did not permit the observation of the
characteristic bands of the carotenoids.
Also in this line of research, Nardo et al. [178], proposed the use of attenuated total
reflection infrared spectroscopy combined with multivariate analysis in the rapid and
simultaneous determination of lycopene and β-carotene in tomato juice. The authors
compared two distinct preparations methods of the samples analyzed (direct measurements of
the tomato paste and the method of extraction with hexane for the isolation of the
carotenoids) and used high performance liquid chromatography as a reference method to
validate the method proposed. The authors verified that the estimated content of lycopene and
β-carotene obtained by the ATR-IR technique presented good correlation with that obtained
by the reference method (HPLC). The developed methodology presented an advantage in
relation to the conventional method of UV-visible spectrophotometry regarding the
simultaneous analysis of these two carotenoids. Although this is a technique of relatively low
cost, simple and fast for lycopene analysis, the spectrophotometry in the UV-visible spectrum
does not permit the determination of β-carotene due to interference with lycopene.
It is clear that the analysis of carotenoids has been one of the goals pursued over the
years, culminating in the refinement of the analytic methods, the identification of the source
of errors and the adequate means to avoid them. These efforts reflect not only the advances in
the analytic methods and instrumentation, but also the deep knowledge of the role of these
compounds in human health [8].
Conclusion
This chapter emphasized the importance of the carotenoids, showing their sources,
chemical structures, principal biological functions, as well as the factors that influence
carotenoid composition in foods and their stability. This knowledge is of great importance
since, after acquired, it is possible to recommend measures to increment these compounds in
diets by ingestion of food considered as source of carotenoids or their addition in the form of
food additives (natural colorants).
In general, the carotenoids are compounds that present a significant importance for
diverse areas of knowledge, due to their functions and applications. Their principal sources
are fruits and vegetables, with a small proportion from foods of animal origin and food
additives, with β-carotene, α-carotene, lycopene, lutein, zeaxanthin and criptoxantine being
the most studied, aiming their use in the improvement of the quality of life of the population.
Although there is a need of new extended clinical assays, various carotenoids seem to be
promising regarding the beneficial effects related to health, such as the prevention of cancer,

cardiovascular disease, cataracts, macular degeneration and others disease, with many of
these effects being reliant on the capacity to convert these compounds in vitamin A.
Although the bioactive potential of the carotenoids has been presented, many studies
remain necessary in order to clarify the functional properties, as well as for a better use of
pigments as colorants by the food industry.
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Chapter II
Food Sources:
Production and Health Benefits
of Carotenoids
Lucia Maria Jaeger de Carvalho1,

Lara de Azevedo Sarmet Moreira Smiderle1,
Ediane Maria Gomes Ribeiro1, Gisela Dellamora Ortiz1,
Patrícia Barros Gomes1 and José Luiz Viana de Carvalho2
1
Federal University of Rio de Janeiro, Faculty of Pharmacy,
Ilha do Fundão, Rio de Janeiro, Brazil
2
Embrapa Food Technology, Rio de Janeiro, Brazil
Abstract
Carotenoids are the most widespread pigments in nature. One or more carotenoids in
combination give rise to colors ranging from yellow to red in birds, fishes, crustaceans,
microorganisms, fruits and plants, including the dark green ones. Carotenoids can be
divided in two classes: carotenes and xanthophylls, both unsaturated and unstable when
exposed to light, oxygen and heat. These compounds are synthesized by cyanobacteria,
some non-photosynthetic bacteria, fungi, algae and vegetables. Xanthophylls represented
by zeaxanthin, lutein, cryptoxanthin, violaxanthin and astaxanthin, and carotenes, such as
licopene, β-carotene and -carotene, are easily found in food, with the exception of
zeaxanthin, and β-carotene is the most widespread of carotenoids. The highest carotenoid
contents are found in fruits and vegetables, especially those with pro-vitamin A and
antioxidant activities. High contents of  and β-carotene, as well as β-cryptoxanthin, are
found in fruits, such as mango, papaya and apricot, and vegetables of orange color or
orange pulp, such as carrot, pumpkin and yellow sweet potato. Carotenoid composition
can vary qualitative and quantitatively among vegetables. Leafy or unleafy green
vegetables show a specific qualitative profile, in which lutein, beta-carotene, violaxanthin
and neoxanthin are the main carotenoids. Carotenes are more abundant in root vegetables,
such as carrot and sweet potato, and squash, such as pumpkins, whereas oxygenated

36 L. M. J. de Carvalho, L. de A. Sarmet Moreira Smiderle, E. M. Gomes Ribeiro et al.
carotenoids are mostly found in grains such as corn. Vegetables and fruits have more
complex and diversified carotenoid compositions. Biotechnological processes have been
used to produce carotenoids from microorganisms, such as the fungus B. trispora and
microalgae Dunaliella to obtain β-carotene and xantaxanthin from microalgae and yeasts
(Haematococcus sp and P. rhodozyma) for pharmaceutical purposes. Production of
carotenoids is also being evaluated using yeasts such as X. dendrorhous, R. glutinis, R.
mucilaginosa, Sporobolomyces and P. rhodozyma.
Keywords: Carotenoids, sources, production, health
Introduction
1. General
Carotenoids are pigments, with colors that range from yellow, through orange, to intense
red, due to the presence of conjugated double bonds most commonly found in the structure,
which is of the tetraterpenoid type (Fontana et al., 2000). In green plant tissues, the color of
carotenoids is masked by chlorophyll, the dominant pigment. However, during chlorophyll
degradation it is possible to observe the characteristic color of carotenoids. Carotenoids are
isoprenoids biosynthesized by tail to tail linkage of two C20 geranyl diphosphate molecules.
Thus, all carotenoids are derived from the C40 carbon skeleton consequently formed. This
structure can be modified in three ways: by changes in hydrogenation levels, by cyclization at
one end or both ends of the molecule to give different end groups and by addition of oxygen
containing functional groups (Dutta, Chaudhuri and Chakraborty, 2005). Due to their
unsaturated nature, enzymatic and non-enzymatic oxidation is the major cause of carotenoid
destruction during processing and storage of food. Factors such as temperature, light and pH
can also cause changes that may affect the color and nutritional value of foods (Meléndez-
Martínez and Heredia, 2004). The most investigated carotenoids for their involvement in
human health are β-carotene (β, β-carotene), α-carotene (β, ε-carotene), β-cryptoxanthin (β, β-
caroten-3-ol), lycopene (ψ, ψ-carotene), lutein (β, ε-carotene-3, 3'-diol) and zeaxanthin (β, β-
carotene-3, 3'-diol) (Rodriguez-Amaya, Amaya-Farfan and Kimura, 2008). In general, the
content of carotenoids in food is not changed to a great extent by common household cooking
methods such as the use of microwave oven, steam and boiling, but heating can result in
extreme oxidative destruction of these bioactive compounds (Thane and Reddy, 1997). They
are synthesized by plants and microorganisms and animals are unable to produce them. Thus,
organisms such as salmon must ingest carotenoids through food. In salmon, the most common
carotenoid is astaxanthin (Bellona, 2009). In plants, these microcomponents contribute to
photosynthesis and protect against damages caused by light. Therefore, the main sources of
carotenoids in food are fruits and vegetables (Rao and Rao, 2007).
2. Food Sources
The main carotenoids found in foods are: β-carotene, α-carotene, β-cryptoxanthin,

lycopene, lutein and violaxanthin. These carotenoids are also the most commonly found in the

Food Sources 37
human plasma, except violaxanthin. So, together with zeaxanthin, they are the most studied in
terms of health promoting effects. Brazil is one of the countries with the richest natural
sources of carotenoids (Rodriguez-Amaya et al., 2008). Nearly 90% of carotenoids in the diet
and the human body are represented by β-carotene, α-carotene, lycopene, lutein and
cryptoxanthin (Rao and Rao, 2007).
Carotenoids are present in many common foods used for human consumption and highly
pigmented fruits, juices and vegetables are the main sources. Yellow-orange fruits and
vegetables are the sources of most of the α- and β-carotene. Orange fruits, in turn, are the
main reservoirs of β-cryptoxanthin. Generally, dark green plants contain lutein, while
tomatoes and its products have high lycopene contents (Rao and Rao, 2007).
β-carotene is the most widely distributed carotenoid in foods (Rodriguez-Amaya et al.,
2008). It can be found in many dark-colored fruits and vegetables, including carrots, peas,
sweet red peppers, asparagus, tomatoes, papaya, turnip greens, kale, collard greens, spinach,
broccoli, apricots, mango, cantaloupe, pumpkin, sweet potatoes, winter squash, iceberg
lettuce, romaine, endive, peaches and watermelon (http://www.vitamina.org/
vitaDietarySources.php). Lutein and zeaxanthin are present in high concentrations in egg
yolks (Rao and Rao, 2007).
In a study covering 11 major Brazilian urban centers, carrot, tomato and lettuce were
found to be among the major foods that significantly contribute to the carotenoid supply for
the population (Niizu and Rodriguez-Amaya, 2005).
Typical dietary sources and carotenoid contents of some plant foods in the North
American diet are shown in Table 1.
Table 1. Example of the major contributors of carotenoids

in the North American diet
Carotenoid Food source Amount

β – carotene dried apricot 17600
cooked carrots 9771
cooked spinach 5300
collard greens 5400
cantaloupe 3000
beet greens 2560
cooked broccoli 1300
raw tomato 520
α – carotene cooked carrots 3723
lycopene raw tomato 3100
tomato juice 10000
tomato paste 36500
tomato ketchup 12390
tomato sauce 13060
β - criptoxanthin tangerine 1060
papaya 470
lutein cooked spinach 12475
collard greens 16300
beet greens 7700
cooked broccoli 1839
cooked green peas 1690
Source: Rao and Rao, 2007.

3. Carotenoids Production
The natural production of carotenoids is estimated at 100 million tons per year the highest
of which corresponds to fucoxanthin obtained from brown photosynthetic algae. β-carotene
can be obtained from genetically modified strains of the filamentous fungi Phycomyces
blaskeleeanus and Blakeslea trispora as an alternative to chemical synthesis (Fontana et al.,
2000).
The biotechnological production of astaxanthin from Mucor circinelloides was evaluated
by Anjos et al. (2010) as an alternative to the chemical route, using alternative substrates such
as molasses and obtaining 7.4 g.g-1 of astaxanthin.
Gondin Neto et al. (2009) evaluated β-carotene production by Rhodotorula glutinis using
cashew juice as substrate. They obtained 5.0 to 120 µg of β-carotene /g of dry cell.
Microbiological production of astaxanthin, β-carotene and lycopene was carried out by
Andrade (2003) using three strains of the Mucor genus (M. circinelloides, M. javanicus and
M. racemosus) and two strains of Cunninghamella (C. bertholletiae and C. elegans).
Astaxanthin (19.8 κg.g-1) was observed for the first time in a Mucor species and β-carotene
(13.5 κg.g-1) content was higher in the M. javanicus mycelium. Optimization of the process
promoted an increase in yield from 145.9 to 1,297.0 κg.L-1 and confirmed, thereby, the
potential of M. javanicus as a source of astaxanthin.
De Lourdes et al. (2012) described the production of phytoene, β-carotene, lycopene,
derivatives of bacterioruberin, and salinixanthin by the halophilic microoganism Halomonas
elongate, obtaining a strain able to produce practically pure β-carotene.
On the other hand, Yimyoo et al. (2011) optimized the conditions for carotenoid
production using Rhodosporidium paludigenum.Carotenoid concentration reached 3.42 mg.L-
1
with a dry cell mass of 7.59 g.L-1 at 132 h.
Silva et al. (2011) used pure glycerol and raw glycerol as additional carbon sources for
the production of carotenoid from Phaffia rhodozyma NRRL Y-17268, concluding that both
carbon sources could be used for carotenoid production.
The use of agricultural wastes (molasses) as carbon source to produce carotenoids from
Rhodotorula mucilaginosa was studied by Aksu and Eren (2005). The highest carotenoid
concentration (89.0 mg total carotenoids per liter of fermentation broth) was obtained when
20 g.L−1 sugar cane molasses was used as carbon source; while the highest product yield
(35.0 mg total carotenoids per gram of dry cells) was achieved when 13.2 g.L−1 whey lactose
was the carbon source in the broth.
Kim et al. (2007) produced astaxanthin using the red yeast Xanthophyllomyces
dendrorhousto. The addition of Perilla frutescens (final concentration, 5%) or Allium
fistulosum extracts (final concentration, 1%) enhanced the pigment production to 32 mg.L-1.
The results suggest that plant extracts can effectively enhance astaxanthin production.
Guedes et al. (2011) reported the production of lutein, astaxanthin and β-carotene from
microalgae such as Dunaliella salina (astaxanthin, cantaxanthin, lutein), Chlorella vulgaris
(cantaxanthin, astaxanthin), Coelastrella striolata var. multistriata (canthaxanthin,
astaxanthin, β-carotene) and Scenedesmus almeriensis (lutein, β-carotene), among others.
In Table 2 can be observed the biological functions and carotenoids in animals.

Food Sources 39
Table 2. Biological functions and carotenoids in animals
Nervous system Immune system/ Digestive system Endocrine and Transport functions
tissue repair / and nutrition reproductive and biochemical
maintenance systems interactions
Chemoreception Cancer prevention Liver function Protection of Oxygen supply
reproductive within cells
tissues Transfer of
pigments
Perception of General immunity Protection of

light digestive
enzymes
Electron Wound healing Support of

acceptor steroid synthesis
Calcium Antioxidant Vitamin A Water balance

transport precursor
Cataract Cardiovascular Sexual Reduction in blood

prevention disease prevention maturation cholesterol titre
Protection from high Endocrine Gap junction
temperatures support communication
Protection from UV Growth and cell
radiation differentiation
Stabilization of
proteins and
membranes
Stabilization of chitin
Source: Olson and Owens, 1998.
4. Health Benefits
The major health benefits attributed to carotenoids are mainly due to pro-vitamin A and
antioxidant activities presented by these bioactive compounds. Although the antioxidant
properties of carotenoids have been suggested as the main mechanism by which beneficial
effects are provided, there are also others. Several in vitro and in vivo experiments, in animals
and humans, have demonstrated the antioxidant properties of carotenoids such as β-carotene
and lycopene (Stahl and Sies, 2003).
Astaxanthin, for example, has a 250 times greater capacity than α-tocopherol in
combating free radicals (active oxygen species). Photoemission measurements indicate that
the ability of singlet oxygen sequestration by carotenes and xanthophylls is maximal for
lycopene, high for astaxanthin, intermediate for β-carotene or lutein and lower for bixin and
crocin (Fontana, et al., 2000).
Astaxanthin is the major carotenoid of the xanthophyll group with various biological
functions, including protection against the effects of UV light and increased immune
response. A large variety of microorganisms is capable of synthesizing astaxanthin, including
fungi such as Mucor circinelloides (Anjos et al., 2010).

More than 600 carotenoids have been identified in nature. However, about only 40 are
present in typical foods. Of these 40, only 20 carotenoids have been identified in human
blood and tissues (Rao and Rao, 2007).
According to epidemiological studies, there is a positive relationship between high
intakes of carotenoids and concentrations in tissues and low risk of chronic diseases (Elliott,
2005).
In more recent years, other health promoting effects have been attributed to carotenoids:
immunomodulation and reduced risk of chronic degenerative diseases such as cancer,
cardiovascular diseases, and age related muscular degeneration as well as cataract formation.
Carotenoids have also been identified as potential inhibitors of Alzheimer‘s disease. Such
biological activities bear no relationship to vitamin A activity, but have been attributed to
their antioxidant properties, specifically the ability to sequester singlet oxygen and interact
with free radicals (Palozza and Krinsky, 1992).
Therefore, other mechanisms of action of carotenoids against chronic diseases have been
reported, such as modulation of the metabolism of carcinogens, inhibition of cell
proliferation, enhancement of cell differentiation, stimulation of gap junction communication,
blue light-filtering (Astorg, 1997; Olson, 1999), regulation of cell growth, modulation of gene
expression (carotenoids are also present intracellularly), enhancement of the immune system,
modulation of Phase I and II drug metabolizing enzymes and effects on cell functions like
inhibition of monocyte adhesion and platelet activation.
Furthermore, carotenoids such as α-and β-carotene and β-cryptoxanthin have the
additional advantage of being able to convert vitamin A and thus prevent diseases.
The intake of β-carotene and lycopene has been shown to be inversely related to the risk
of cardiovascular diseases and certain cancers, whereas the intake of lutein and zeaxanthin
has a correlation with prevention of diseases related to the eye.
The minimum requirement for a carotenoid to possess pro-vitamin A activity is to have
an unsubstituted β-ring with a polyene chain of 11 carbons. Thus, α-carotene and β-
cryptoxanthin present about 50% of the activity of β-carotene, while lutein, zeaxanthin and
lycopene have no activity.
In animals, the concentration of carotenoids in certain tissues (hair, feathers, shell), plus
the antioxidant protection, integrates the complex network of communication signals involved
in attraction, camouflage and warning. Vitamin A or retinol (direct derivative of β-carotene)
is involved in vision, reproduction and normal development of the skin.
β-Carotene
β-carotene is the foremost precursor of vitamin A. Basically, the structure of vitamin A
(retinol) is half of the β-carotene molecule with a molecule of water added at the end of the
polyene chain. Consequently, β-carotene is the carotenoid vitamin A higher power, to which
100% vitamin A activity is assigned.
So, it has an important role in processes involved in vision, cell differentiation,
glycoprotein synthesis, mucus secretion from epithelial tissues, reproduction, overall growth
and bone development. There are also claims that β-carotene would act as a suppressor of the

Food Sources 41
human immunodeficiency virus and help in protective immunity against infectious diseases
such as measles.
However, in the late 80's and early 90's, numerous retrospective studies (case-control)
and prospective (cohort) conducted in several countries have consistently shown a strong
inverse association between dietary intake of β-carotene, or their plasma concentration, and
the incidence of cancer, particularly lung cancer (Ziegler, 1991; Block et al. 1992; van Poppel
and Goldbohm, 1995).
The pro-oxidant effect of β-carotene was also demonstrated in mice, which showed
increased activity of phase I enzymes in the liver, kidney and intestine, as well as increased
oxidative stress (Paoline et al., 2001). Recently, studies in humans also demonstrated pro-
oxidant properties of β-carotene. Supplementation of β-carotene in pharmacological levels
increased the incidence of lung cancer in smokers in the essay "Alpha-Tocopherol Beta-
Carotene" (ATBC) and increased cardiovascular mortality in a group of smokers, ex-smokers
and individuals exposed to asbestos in test efficiency of β-carotene and retinol ("β-carotene
and retinol efficiency industry") (Omenn et al., 1996).
This inverse relationship was also observed with the incidence of cardiovascular diseases
(Gaziano and Hennekens, 1993; Manson et al., 1993; Kohlmeier and Hasting, 1995).
Therefore, β-carotene fell into disrepute when intervention studies began to show that, instead
of diminishing, it increased the incidence of lung cancer in smokers and asbestos workers
(ATBC Study Group, 1994; Omenn et al., 1996).
Given these results, Mayne (1996) concluded that while the recommendations to increase
consumption of carotenoid rich fruits and vegetables remain valid, the use of β-carotene
supplements in order to prevent cardiovascular disease and lung cancer, particularly for
smokers was no longer recommended. It was later recognized that in the intervention studies,
β-carotene was administered at much higher doses (20-30 mg) than the optimum daily intake
levels established in epidemiological studies (approximately 4 mg), in addition to diets
containing other carotenoids and other food constituents, which may indicate the existence of
synergism with β-carotene (CARIG, 1996).
Furthermore, participants of the intervention studies, in which increases in the incidence
of cancer were registered, were smokers or persons exposed to asbestos (a well known
carcinogen) for excessive lengths of time, and the process of oxidative stress and/or cancer
could have reached a stage where the carotenoid would be more effective. Thus, carotenoids
regained their importance in health, but the emphasis shifted towards other carotenoids than
β-carotene, whereas recognition that the protective effect of foods is not only due to a class of
compounds, but rather results from the action of a number of bioactive compounds in each
food, increased. However, even in the absence of synergism, and considering that the health-
promoting substances present in food have different mechanisms of action, one can count at
least with complementary or additive effects of several compounds acting at different stages
of disease development.
The increased incidence of lung cancer in smokers and asbestos workers, mediated by the
consumption of high doses of supplemental β-carotene (ATBC Study Group, 1994; Omenn et
al., 1996), led many authors to consider this carotenoid to express a pro-oxidant effect.
According to Palozza (1998), there was already evidence of pro-oxidant activity of β-carotene
and other carotenoids, both in vitro and in vivo, with the conversion of antioxidant to pro-
oxidant depending on the redox potential of the molecule and its biological environment.

However, it was noted that oxygen pressure and concentration of carotenoids in in vitro
experiments were much higher than those encountered in physiological conditions (Krinsky,
2001). Young and Lowe (2001) also found no direct evidence to prove the hypothesis that
carotenoids could behave as a pro-oxidant in a biological system. The latter authors found
that more likely, considering the behavior of carotenoids in vitro, a number of factors will
reduce their effectiveness as an antioxidant in vivo, making them ineffective against certain
reactive oxygen species.
These observations suggest a possible biphasic response of β-carotene that promotes
health benefits when ingested in food levels, but may have adverse effects when ingested in
larger quantities. Over a long period of time, β-carotene was used as "gold standard" to study
the relationship between oxidative stress and chronic diseases. The research focus has now
changed to another carotenoid with antioxidant activity: lycopene.
However, due to some inconsistencies in the research data and the apparent differences in
effectiveness of different carotenoids, besides the possible synergistic or, at least, additive
effects, the current recommendation of increased consumption of a variety of carotenoid rich
fruits and vegetables remains.
Licopene
Little is known about the in vivo metabolism of lycopene. The action of lycopene in
human health has received great prominence in recent years (Stahl and Sies, 1996; Gerster,
1997; Clinton, 1998; Sies and Stahl, 1998; Giovannucci, 1999; Rao and Agarwal, 1999;
Khachik et al., 2002), with the strongest scientific evidence being in relation to lung,
esophagus and prostate cancer. Highest emphasis has been given to its action against prostate
cancer (Hadley et al., 2002; Giovannuci et al., 2002; Wertz et al., 2004; Stacewicz-
Sapuntzakis and Bowen, 2005), via different mechanisms that lead to reduction of the
proliferation of normal epithelial and prostate cancer cells, decreased DNA damage and
improved defense against oxidative stress.
In 1999, studies showed that the intake of lycopene and lycopene blood levels were
inversely related to many cancers including prostate, breast, cervical, ovarian, liver and other
organs (Giovannucci, 1999). Many other studies since then have shown that the increased
intake of lycopene and serum levels of lycopene was significantly reduced in affecting the
risk of cancer (Giovannucci et al., 2002; Kucuk et al., 2001; Rao, Ray and Rao, 2006).
The antioxidant properties of lycopene have been the main focus of research to study its
biological role. However, it has been shown to exert its effects through other mechanisms,
which include regulation of gene function, intercellular communication (gap junctions),
hormone and immune modulation, carcinogen metabolism and metabolic pathways involving
drug metabolizing enzymes (phase II) (Agarwal and Rao, 2000; Wertz, Siler and Goralczyk,
2004).
Furthermore, baseline levels of serum lycopene have been associated with increased risk
of mortality from coronary diseases. The findings that lycopene has a stimulatory effect on
cell proliferation and alkaline phosphatase, a marker of osteoblast differentiation and its
inhibitory effects on osteoclast formation and resorption are evidences of the involvement of
lycopene on bone health and need more research trials or investigation (Rao and Rao, 2007).

Food Sources 43
Lycopene can also play a role in preventing cardiovascular diseases (Arab and Steck,
2000; Rissanen et al., 2002). A multicenter study involving 10 European countries found that
lycopene, or some related substance, could contribute to the protective effect of vegetables
against the risk of myocardial infarction, this association was not observed with α-and β-
carotene (Kolhmeier et al., 1997). A prospective study of women, which lasted 7.2 years,
indicated that dietary lycopene and other phytochemicals in tomato products confer
cardiovascular benefits (Sesso et al., 2003).
Another prospective study conducted for 12 years in the U.S., however, showed reduced
risk of coronary artery disease in women, associated with high consumption of α- or β-
carotene rich foods, but not with the consumption of lycopene, β-cryptoxanthin or lutein /
zeaxanthin rich foods (Osganian et al., 2003).
Lycopene is a carotenoid that contributes to the red color of food. It is a phytonutrient
with antioxidant role in the organism, preventing the formation of free radicals or inhibiting
the damage caused by them. Science has proven that lycopene can help prevent the onset of
cardiovascular disease, as well as prostate, pancreatic, stomach and colon cancer.
Tomato (Lycopersicon esculentum Mill.) and its products are the richest sources of
lycopene from foods consumed by the population in general. This fruit of the Solanaceae
family has high levels of this component, which apparently has its availability increased with
processing and heating tomato. Besides the nutritional factor, the concentration of lycopene in
tomatoes is associated with improved visual perception of the products, thus there is a strong
demand to increase the levels of this pigment in fruit cultivars for both fresh consumption and
industrial processing. The cultivars with highest levels of nutritional factors, including
lycopene, depend on the availability of methods for simple yet accurate. Raw tomato has on
average 30 mg lycopene/Kg of fruit, tomato juice, about 150 mg lycopene/liter; ketchup
contains on average 100 mg.Kg-1 product. Tomato production in Brazil is approximately 3
million tons/year, being 65% in natura and 35% for industrial processing. This is equivalent
to say that about 1,500 tons of lycopene molecule are produced annually in Brazilian crops
(assuming average levels of lycopene of 50 ug.g-1).
Lutein and Zeaxanthin

Lutein and zeaxanthin are the yellow pigments of the macula of the human retina (Bone
et al., 1988; Handelman et al., 1988; Landrum and Bone, 2001) and are considered
responsible for the protective effect of carotenoids to the eye, acting both as antioxidants and
as high energy blue light-filters (Krinsky et al., 2003). Although not all studies show such
relationship, the consumption of these carotenoids by eating foods such as spinach,
watercress, corn and egg, or their serum, exhibited an inverse correlation with the risk of
macular degeneration (EDCC 1993; Seddon et al., 1994; Snodderly, 1995; Moeller et al.,
2000), the leading cause of vision loss in the elderly. It has been shown that high
concentrations of lutein and zeaxanthin, measured in the central region of the retina, made its
holders 82% less likely to develop macular degeneration (Bone et al., 2001). According to the
reviews by Moeller et al. (2000) and Alves-Rodriguez and Shao (2004), the lack of lutein has
also been consistently associated with increased risk of cataracts. The extraction of cataracts
is one of the most frequently performed surgeries in the elderly.

Mandelli (2012) identified the carotenoids from Thermus filiformis strain ATCC 43280
through different growth conditions and verified the antioxidant activity of the carotenoid rich
extract. Zeaxanthin (14.4%) was the most abundant carotenoid, followed by zeaxantin
monoglycoside (4.7%) and thermozeaxanthins with branched fatty acids with 11 (2.3%), 12
(5.2%), 13 (25.4%) and 14 (9.0%) carbon chains, as well as derivatives of thermozeaxanthin-
13 (0.8%) and thermozeaxanthin-15 (2.8%). β-caroten-3-ol represented 35.3% of the total
content of carotenoids (1,517 mg.g-1).
Mandonade (2003) also identified the yeasts Rhodotorula mucilaginosa and L125 as
Rhodotorula graminis isolated from tomato sauce and leaves of sugar cane. The yeast
Rhodotorula glutinis had the highest total carotenoid concentration (881 mg.L-1), followed by
Rhodotorula graminis (594 mg.L-1), Rhodotorula mucilaginosa-137 (590 mg.L-1) and
Rhodotorula mucilaginosa-135 (545 mg.L-1). The main pigments found in these yeasts were
torulene and β-carotene. β-carotene predominated in Rhodotorula graminis, Rhodotorula
glutinis and Sporobolomyces.
Conclusion
Carotenoids are biosynthesized by plants, algae, bacteria, and fungi, but not by animals,
which must obtain them from food. These compounds are divided into two major classes,
based on their structural elements: carotenes (constituted by carbon and hydrogen) and
xanthophylls (constituted by carbon, hydrogen, and additionally oxygen). Carotenoids have
positive effects on human health, such as pro-vitamin A, antioxidant and anticancer activities,
anti obesity effects, and anabolic effects on bone components. Despite the possible existence
of synergism of β-carotene with other carotenoids or food constituents and the effects it can
cause in smokers, the current recommendation to increase the consumption of varied
carotenoid rich fruits and vegetables remains.
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prevention. Amer J. Clin Nutri. 62(6), 1393S-1402S.
Wertz, K.; Siler, U.; Goralczyk, R. 2004. Lycopene: modes of action to promote prostate
health. Arch. Biochem. and Biophys. 430, 127-134.

Yimyoo, T., Yongmanitchai, W. and Limtong, S. 2011. Carotenoid production by

Rhodosporidium paludigenum DMKU3-LPK4 using glycerol as the carbon source.
Kasetsart J. 45, 90 – 100.
Ziegler, R.G. 1991. Vegetables, fruits, and carotenoids and the risk of cancer. Amer J. Clin.
Nutri.53, Supplement 1, 251S-259S, http://www.vitamina.org/vitaDietarySources.php.
Acessed in february, 15, 2013.

Chapter III
Carotenoids with κ-End Group
Enrique Murillo1, Veronika Nagy2, Attila Agócs2

and József Deli2
1
University of Panamá, Biochemistry and Nutrition Laboratory
2
University of Pécs, Medical School,
Department of Biochemistry and Medical Chemistry
Abstract
Carotenoids with θ-end group occur typically in certain Capsicum species.
Capsanthin is the main carotenoid of paprika but also occurs in other plants in much
lower amounts. Capsorubin and cryptocapsin are minor components accompanying
capsanthin. These red pigments are biosynthetised from carotenoid-5,6-epoxides by
means of specific enzymes. A new special family of carotenoids bearing 3-dehydroxy-θ-
end group were found in tropical fruits of extremely high pigment content. Some of them
contain β-end group as well which means potential vitamin A activity.
Here we describe the first isolations and structure elucidations of carotenoids with θ-
end group, their occurrence and biosynthesis, the chemical synthesis and some
transformations of θ-carotenoids, as well as their biological activities.
Introduction - Historical Background
The first carotenoids containing -end group were isolated from red paprika (Capsicum
annuum). Red paprika pigments were studied as early as the nineteenth century, but the
crystallization of pigments was not successful, because paprika extracts always contain fatty
acids, oils and other lipids. [1] Zechmeister and Cholnoky succeeded in separating the
pigments from these other compounds by saponification in the early 1930s. They were able to
crystallize a red pigment, which they called capsanthin, [2] however, it consisted of several
components. Later Zechmeister and Cholnoky separated some other pigments from red
paprika extract by using column chromatography, namely -carotene, cryptoxanthin and

50 Enrique Murillo, Veronika Nagy, Attila Agócs et al.
zeaxanthin. They also found another red pigment, which was called capsorubin. [3] They tried
to establish the structure of capsanthin and capsorubin, and formulated their structures as 1
and 2 [3d].
At the beginning of the 1950s Cholnoky and coworkers began to investigate the fresh
paprika fruits. First, red tomato-shaped paprika (Capsicum annuum var. lycopersiciforme
rubrum) was studied. [4] This kind of paprika is green when unripe, and becomes red during
ripening. Both stems and fruits of unripe and ripe paprika were investigated. In unripe paprika
-carotene, -carotene monoepoxide, zeaxanthin, lutein, violaxanthin and, in traces,
antheraxanthin were found. The ripe fruits, however, contained -carotene, cryptoxanthin,
zeaxanthin, antheraxanthin, violaxanthin, capsanthin, capsorubin as main pigments, and some
of their cis-isomers. A new red pigment called cryptocapsin was also found in ripe paprika.
[5] Cholnoky et al. also made attempts to elucidate the carotenoid composition of yellow
tomato-shaped paprika (Capsicum annuum var. lycopersiciforme flavum). [6] This subtype is
green in its unripe state, and the ripe fruit is yellow or orange, but its colour never turns red. It
was found that the unripe paprika contained -carotene, -cryptoxanthin, lutein, violaxanthin,
and foliaxanthin (neoxanthin). The ripe fruits contained - and -carotene, - and -
cryptoxanthin, lutein, violaxanthin, foliaxanthin, and their cis-isomers. Red pigments
capsanthin and capsorubin, however, could not be detected. -Cryptoxanthin was discovered,
crystallized and characterized first from this source. [7] Based on the analytical data,
Cholnoky proposed the scheme for the biosynthesis of paprika carotenoids. [8] It was
supposed that zeaxanthin was converted into antheraxanthin and violaxanthin by epoxidation,
then capsanthin and capsorubin were formed from antheraxanthin and violaxanthin,
respectively, by ring opening and rearrangement (Figure 1).
Figure 1.

Carotenoids with θ-End Group 51
Based on these findings Cholnoky suggested that the structure of capsanthin and
capsorubin should be corrected. Subsequently, the structure of these red pigments was
investigated for several years by english (Weedon), swiss (Karrer) and hungarian research
groups. [9] Eventually the group of Karrer clarified the structure of capsanthin using infrared
spectroscopy. [10] Chemical evidence obtained by oxidative degradation was given by
Cholnoky and Szabolcs [11] for the structure of capsanthin and capsorubin. Capsanthin and
capsorubin were shown to contain one and two cyclopentane rings, respectively, adjacent to
the keto groups which are parts of the conjugated double bond system. Cyclopentane ring had
not been known previously to occur in carotenoids. The full evidence for this structure was
obtained by X-ray study of capsanthin-bis(p-bromo-benzoate). [12] The total synthesis of
capsanthin (3), capsorubin (4) and cryptocapsin (5) was achieved in 1983 by Rüttimann et al.
with the cooperation of Weedon et al. [13].
Occurrence of Carotenoids with -End Group in

Higher Plants
Carotenoids with 3-Hydroxy--End Group
Capsanthin and capsorubin are the most thoroughly studied and most widespread
carotenoids with -end-group. Capsanthin, isolated first from the red fruit of Capsicum
annuum [2], has also been found in the pollen anthers of Lilium tigrinum [14] and Aesculus
hippocastanum [15], in the fruit of Berberis spp. [16] and Asparagus officinalis [17], and in

Lilium amabile petals. [18] Capsorubin has been isolated from the petals of Cajophora
lateritia [18a], the pollen anthers of Aesculus hippocastanum [15], and the fruits of
Asparagus officinalis [17]. In the 1980s and 1990s the carotenoid composition of some of the
above mentioned plants was reinvestigated, paying special attention to the minor carotenoids
with interesting end groups. New pigments containing -end group were isolated from red
paprika: capsanthin 5,6-epoxide (6), its furanoid capsochrome (7), capsanthin 3,6-epoxide (8)
contained a 3,6-epoxy--end-group, [19] and 5,6-diepicapsokarpoxanthin (9), which
contained a 3,5,6-trihydroxy--end group.

The carotenoid composition of ripe and unripe fruits of Asparagus officinalis were
studied by HPLC, [20] where the major θ-carotenoids were capsanthin (3), capsorubin (4),
and capsanthin 5,6-epoxide (6).
Reinvestigation of the petals of Lilium tigrinum by HPLC and the isolation of some
minor compounds were reported: capsanthin 5,6-epoxide (6) and 5,6-diepicapsokarpoxanthin
(9) were isolated by HPLC-controlled preparative column chromatography [21].
From the ripe fruits of Asparagus falcatus capsanthin (3), capsorubin (4), capsanthin 5,6-
epoxide (6), and capsochrome (7) were isolated by preparative column chromatography and
characterized by spectroscopic methods. [22] Two minor carotenoids were also isolated and,
based on their spectral data, identified as (all-E,3S,5R,6R,3‘S,5‘R)-6,7-didehydro-5,6-
dihydro-3,5,3‘-trihydroxy-,-caroten-6‘-one (capsoneoxanthin, 10) [23] and (9Z)-capsanthin
5,6-epoxide ((9Z)-6) [24].
Carotenoids with -end group have recently been found in relatively large amounts in
many tropical plants. Our preliminary investigations showed that capsorubin (4) was the main
carotenoid in the brown leaves and seeds of zamia (Zamia dressleri), as well as in the fruits of
jipi-japa (Carludovica palmata), and papua fruit (Pandanus conoideus). Cryptocapsin (5) was
found to be the main pigment component in the fruits of red mamey (Pouteria sapota), and
large amount of it can also be found in the fruits of maracuja chino (Cionosicyos
macrunthus). All mentioned tropical fruits also contain more or less of capsanthin (3). From
red mamey, a new epoxy-carotenoid, cryptocapsin 5,6-epoxide (11) was isolated and
characterized [25].
Carotenoids with 3-Oxo--End Group
Based on UV-VIS spectra, chemical and chromatographic properties, the occurrence of

various carotenoids with the -end group, especially capsanthone (12) was postulated, in the

anthers of flowers of different species of Aesculus (horse chestnut) a long time ago. [15, 26]
As main pigments, capsanthin, capsanthone, capsorubin, zeaxanthin, and -carotene were
found, accompanied by small amounts of cryptoxanthin, lutein, cryptocapsin (5),
cryptocapsone (14), and capsanthin monoepoxide (6). [15].
Further studies were carried out on the carotenoid composition of leaves in various
phases of growth, floral buds in various stages of development, and flowers of Aesculus
parviflora and A. Rubicund [27].

In reinvestigation of buds, pollen and petals of two species of Aesculus (hippocastanum

and pavia), [27] no carotenoids with the θ-end group were isolated, which was in contrast to
earlier reports. [15,28] From the pollens of Aesculus hippocastanum, a new carotenoid was
isolated as a main carotenoid and, based on the spectroscopic data, identified as (all-E,3R)-3-
hydroxy-6'-apo--caroten-6'-al (aesculaxanthin, 15) [28].
During the isolation of minor components from red paprika, HPLC investigation showed
that one of the fractions always contained a red carotenoid in significant amounts. This
compound was isolated and characterized as capsanthone (12) [29].
The natural occurrence of capsanthone (12) in different species of red paprika was also
proved. Semisynthetic capsanthone (12), capsorubone (13) and cryptocapsone (14) were
prepared by Rüttimann et al., [13b] but the natural compounds 13, 14 have not been isolated
and characterized before.
The next report about the isolation from natural sources and characterization of
carotenoids containing 3-oxo--end group was published in 2001, when Maoka et al. reported
the isolation and characterization of capsanthone 3,6-epoxide (16) from red paprika
(Capsicum anuum L.) [30].
Carotenoids Containing -End Group without Hydroxy Group
The above mentioned carotenoids (3-10) contain one, and in the case of capsorubin (4)
two 3-hydroxy-6-oxo--end groups. Two carotenoids with unique structures, 3‘-deoxy-
capsanthin (17) and 3,4-dehydroxy-3‘-deoxycapsanthin (18) have been reported as very
minor components in paprika by Maoka et al. [31] These carotenoids have a 6-oxo--end
group without hydroxy group.

In 2011, the isolation and structure elucidation of a new carotenoid, sapotexanthin (19),
was reported from red mamey (Pouteria sapota). [32] 3‘-Deoxycapsanthin (17) and
3‘-deoxycapsanthin 5,6-epoxide [25] (20) were also isolated and characterized from this
source. Furthermore, 3‘- and 3-deoxy-derivatives of capsorubin, namely 3‘-deoxycapsorubin
(21) and 3,3‘-dideoxycapsorubin (22) could be isolated in small amounts [33].

Apocarotenoids
Eleven apocarotenoids including two compounds with θ-end group (23 and 24) were
isolated from the fruits of the red paprika Capsicum annuum L. [34] Structures of the new
apocarotenoids were determined to be apo-12‘-capsorubinal (23), and apo-8‘-capsorubinal
(24) by spectroscopic analysis. These carotenoids were assumed to be the products of
oxidative cleavage of C40 carotenoids such as capsanthin in paprika.
Biosynthesis of -Carotenoids in Higher Plants
The biosynthesis of carotenoids [35] is a branch of the well-known isoprenoid pathway,

that begins with the conversion of mevalonic acid into the five-carbon compound isopentenyl
diphosphate. In a series of prenyltransferase reactions, the 40-carbon carotenoid phytoene is
assembled from eight molecules of isopentenyl diphosphate. Phytoene subsequently
undergoes a series of four desaturation or dehydrogenation reactions that results first in the
formation of phytofluene and then, in turn, -carotene, neurosporene and lycopene [36].
Xanthophylls are formed enzymatically as oxidation products of α- and β-carotene. The
most common oxygen containing groups, originate from molecular oxygen, found in plant
xanthophylls are hydroxy at C(3), epoxy at the 5,6 position of the ring, and keto-group.
Zeaxanthin and lutein are formed by the hydroxylation of the 3 and 3' carbon atoms of β,β-
carotene or β,ε-carotene, respectively. Since the absolute configuration of carbon atoms
bearing the hydroxyl group is opposite on ε and β rings of lutein, different enzymes are
thought to catalyse their formation. Antheraxanthin with one epoxy group and the diepoxide
violaxanthin are formed starting from zeaxanthin, by introduction of the epoxy groups into
5,6 and 5',6' positions. Epoxidation of zeaxanthin, which requires NADPH and O2, [37] and
cyclic deepoxidation of violaxanthin represent key mechanisms in the adapting plants and
green alga. Enzymes involved in these reactions are assumed to be mixed function

oxygenases. Xanthophyll epoxides serve as precursors of the plant hormone abscisic acid
(Figure 2).
lycopene
lycopene lycopene
-cyclase -cyclase
(CrtL-e) (CrtL-b)
-carotene -carotene
lycopene
lycopene
-cyclase
-cyclase
(CrtL-b)
(CrtL-b)
-carotene
C-4-oxygenase
-carotene -carotene (CrtO, bkt)
-carotene
-carotene -karotin
hydroxylase
hydroxylase
(CrtZ, CrtR)
(?)
hydroxylase echinenone
(CrtZ, CrtR)
-carotene
C-4-oxygenase
-cryptoxanthin zeinoxanthin -cryptoxanthin (CrtO, bkt)
-carotene -carotene
-carotene
hydroxylase
canthaxanthin
hydroxylase hydroxylase (CrtZ, CrtR)
(?) (CrtZ, CrtR) canthaxanthin
hydroxylase
lutein zeaxanthin (?)
zeaxanthin violaxanthin zeaxanthin violaxanthin astaxanthin

de-epoxidase epoxidase de-epoxidase
epoxidase
(?) (aba2) (vde)
(?)
lutein 5,6-epoxide anteraxanthin capsanthin

capsorubin
synthase (CCS)
zeaxantin violaxantin
epoxidase de-epoxidase
(aba2) (vde)
capsanthin
capsanthin
violaxanthin capsorubin
synthase (CCS)
neoxanthin
synthase
(NES)
capsorubin
ABA neoxanthin
Figure 2.
In earlier works, Deli et al. suggested a plausible pathway of biosynthetic transformations

of 5,6-epoxy end group in paprika (Figure 3). [38] According to this, six different
biosynthetic routes exist. Out of these, pinacol rearrangement leads to θ-end group of
carotenoids.
In addition, the 5,6-epoxy end group may undergo allenic rearrangement, epoxide ring
opening, endo epoxide rearrangement and furanoid-oxide rearrangement resulting in 3,5-
dihydroxy-allenic, 3,5,6-trihydroxy-, 3,6-epoxy- and 5,8-epoxy end groups, respectively.

C H
O H
HO HO O HO
OH
O
HO
?
- H2O - H2O
O OH OH
OH ? HO ?
OH
Figure 3.
Capsanthin-capsorubin synthase (CCS), which catalyses the conversion of 5,6-epoxy end

groups into -end groups, was isolated and characterized by Bouvier et al. [39] The fact that
CCS also exhibits lycopene cyclase activity is likely to be related to the chemical mechanisms
leading to the formation of β-rings in β-carotene and θ-rings in capsanthin and capsorubin. In
both mechanisms a carbenium ion at C(5) as intermediate is formed. [40] In addition, both
reactions are likely to be initiated by a protonic attack on either a double bond or an epoxy
group (Figure 4).
Figure 4.

Figure 5.
Figure 6.
On the basis of the reaction mechanism for CCS described above, we suggested a
mechanism for the formation of 3,5,6-trihydroxy-carotenoids isolated from red paprika
(Figure 5). [41] Either the (3S,5S,6S)- or the (3S,5R,6S)-end group may be formed via the
carbenium ion at C5. Therefore, during the enzyme-catalyzed hydrolysis of carotenoid-5,6-
epoxides, the configuration at C5 may change, and the configuration at C6 remains
unchanged.
For a long time it had not been clarified whether the 3,5,6-trihydroxycarotenoids are
intermediates or byproducts of pinacol rearrangement. The clearing up of this question
demanded biochemical investigations. Based on enzymatic investigations, it is assumed that
3,5,6-trihydroxy-carotenoids are by-products of pinacol rearrangement [42].

It seems likely that θ-carotenoids without hydroxy-group are biosynthesized from β-

carotene via β-carotene 5,6-epoxide in an analogous way to the formation of capsanthin from
zeaxanthin via antheraxanthin (Figure 6).
The presence of θ-carotenoids without hydroxylated rings in the flesh of red mamey is
due to the coincidence of two rare metabolic events: 1) high activity of enzymes that catalyze
the epoxidation of non-hydroxylated -ring, and 2) the high activity of enzymes that catalyze
the pinacol rearrangement of epoxides.
Occurrence of κ-Carotenoids in Other Sources
Mytiloxanthin (25) has a unique cyclopentyl enolic -diketone group conjugated to a

polyene chain in its structure. It was isolated as a major carotenoid from the edible mussels
Mytilus edulis [43] and coriescus, [44] and as a minor carotenoid from other marine
invertebrate animals including Halocynthia roretzi, [45] Amaricum pliciferum [46] and seven
species of tunicates [46].

The 4-hydroxy derivative of mytiloxanthin (26) was isolated as a minor carotenoid from
starfish Asterina pectinifera and A. amurensis. [47] 19-Hydroxymytiloxanthin (27) was
isolated as 19 butanoate and 19-hexanoate from the sponge Phakellia stelliderma. [48] The
19-hexanoate has also been isolated from the mussel Mytilus edulis [43b].
Agelaxanthin (28) is the methoxy derivative of 19-hydroxymytiloxanthin, and was found
in the marine sponge Agelas schmidtii [49].
Mytiloxanthinone (29) containing cyclopentanone ring, was isolated from tissues of the
sea squirt Halocynthia roretzi [47, 50] and from Styela clava, S. plicata and Botryllus
schlosseri [47].
4-Oxomytiloxanthin (30) was isolated as a minor carotenoid from samples of
carotenoprotein astearubin obtained from the starfish Asterisa rubens. It was liberated from
the carotenoprotein with acetone [51].


Bastaxanthin D (31) and its 3‘-oxo derivative, bastaxanthin C (32) contain sulphate ester.
Both carotenoids were isolated from the marine sponge Lanthella basta. [52] The non
esterified derivative of 32 is bastaxanthol C (33), which was also isolated as minor carotenoid
from Lanthella basta. [53] An other interesting minor carotenoid, bastaxanthol B (34),
containing aldehyde group at 17‘ position, was isolated Lanthella basta too [52].
Two θ-carotenoids with aromatic ring were found in marine sponges. Trikentriorhodin
(35) was isolated as a major carotenoid from Trikentrion helium, [54] Ageles schmidtil, [49]
Microciona prolifera, [55] Clathria frondifera and Tedania digitata [56].
Synthesis of Carotenoids with -End Group

The first total synthesis of capsanthin (3), capsorubin (4), and cryptocapsin (5) was
achieved in 1983 by Rüttimann et al. with the cooperation of Weedon‘s group [13].
Capsanthin (3) and capsorubin (4) were also synthesized via the C15-cyclopentyl ketone
prepared by Lewis acid-promoted regio and stereoselective rearrangement of a 5,6-epoxy
dienal (Figure 7) [57].
Figure 7.
Figure 8.

One year later, the synthesis of 3,6-epoxy carotenoids cucurbitaxanthin A,

cycloviolaxanthin and capsanthin 3,6-epoxide 3 was accomplished via C15-3,6-epoxides
prepared by the regioselective ring opening of 3-hydroxy-5,6-epoxides [58].
Reactions of Lewis acids with epoxides bearing various olefinic groups at C-6 showed
that the way of oxirane ring cleavage depended upon both the length of the conjugated double
bond system and the electron-withdrawing ability of the substituents adjacent to the double
bond. As shown in Figure 8, in the case of electron-withdrawing diene-ester or diene-nitrile
having a hydroxyl group at C-3 position, this reaction resulted in the predominant formation
of 3,6-epoxides (route b).
Treatment of epoxides possessing electron-withdrawing groups (diene-ester or diene-
nitrile) with SnCl4 or aminium salts resulted in the production of 3,6-epoxides, which were
transformed into cucurbitaxanthin A, cycloviolaxanthin, and capsanthin 3,6-epoxide (8),
respectively. [59] Regio and stereoselective rearrangement of the optically active C15-epoxide
possessing a conjugated dienal group at C-6 resulted in the formation of θ-end group in good
yields, which was transformed to capsanthin (3) and capsorubin (4), respectively.
The biomimetic synthesis of mytiloxanthin was published by Ito‘s group [60].
Chemical Transformations of -Carotenoids

Capsanthin and capsorubin contain hydroxy and keto-group(s) as functional groups. For
the reduction of the carbonyl function of carotenoids metal hydrides are generally used, either
LiAlH4 in Et2O or in tetrahydrofuran, or NaBH4 in EtOH or in EtOH/benzene. Upon reduction
of the carbonyl groups, a hypsochromic shift (to shorter wavelengths) and increased spectral
fine structure can be seen in the UV/VIS spectrum. Reduction products are usually readily
separated from the starting compound because the carotenol product is more polar than the
initial ketone. However, the reduction of keto-groups of carotenoids produces two
stereoisomers, which may be very difficult to separate.
Oppenauer oxidation is a gentle method for selective oxidization of secondary alcohols to
ketones. In this reaction the hydroxy group on cyclopentane ring is oxidized and the other one
on the cyclohexane ring remains unchanged. Capsanthone (12) was prepared from capsanthin
(3) by this method [61]. In the sixties and seventies the reduction of capsanthin (3) was
studied by our research group. [61-62] The reaction of 3 with NaBH4 gave the two
steroisomeric capsanthols 36 and 37 ((3R,3‘S,5‘R,6‘S)- and (3R,3‘S,5‘R,6‘R)-,-carotene-
3,3‘,6‘-triol, resp.). The Oppenauer oxidation of these capsanthols yielded the corresponding
capsanthol-3‘-one epimers (3,6'-dihydroxy-,-caroten-3'-ones, 38, 39).

Later the (3R,5‘R,6‘S)- and (3R,5‘R,6‘R)-capsanthol-3‘-ones (38, 39) were reduced by

different complex metal hydrides to give triols 40 and 41. The diastereoisomeric (3‘S)- (36,
37) and (3‘R)- (40, 41) capsanthols were fully characterized [63].

With a similar method, (6‘S)- and (6‘R)-capsorubol-6-one (42, and 43 resp.) (6S,6‘S)-,
(6S,6‘R)- and (6R,6‘R)-capsorubol (44, 45, and 46, resp.) and (6‘S)- and (6‘R)-cryptocapsol
(47 and 48, resp.) were prepared in crystalline from by the reduction of capsorubin and
cryptocapsin [64].

In a study on the reduction of oxo-carotenoids by NaBH4, the transformation of

carotenoid esters to free hydroxy-carotenoids was observed. Thus, the reduction of capsanthin
3,3‘-diacetate resulted in the corresponding capsanthol 3,3‘-diacetates, as well as free
capsanthol epimers (36, 37). This observation led us to develop a method for the preparation
of partially acetylated carotenoids. The 3,6‘-diacetate (49), 3‘,6‘-diacetate (50), and 6‘-acetate
(51) of (6‘R)-capsanthol (37) were obtained from (6‘R)-capsanthol-3,3‘,6‘-triacetate (52), and
the 3- and 3‘-acetates 53 and 54 of (6‘R)-capsanthol (37) were synthesized from (6‘R)-
capsanthol 3,3‘-diacetate (55) [65].

The above mentioned semisynthetic capsanthol epimers and different capsanthol acetates
were used for the study of the chirality of supramolecular carotenoid self-assemblies by CD
spectroscopy [66].
Biological Effects of Carotenoids with -End Group

Mainly capsanthin and capsorubin, being the most abundant θ-carotenoids, have been
studied from biological aspects.
The antioxidant activities of capsanthin and its natural fatty acid esters were compared to
that of other abundant carotenoids, as β-carotene, lutein, and zeaxanthin. Matsufuji et al.
measured the formation of methyl linoleate hydroperoxides during free radical oxidation of
methyl linoleate. Capsanthin suppressed hydroperoxide formation as well as the other
carotenoids, however, capsanthin decomposed more slowly than the other carotenoids, and its
radical scavenging effect was found to last longer. Capsanthin mono- and/or diesters showed
similar behavior, esterification did not affect the radical scavenging ability of capsanthin.
Capsanthin and its fatty acid esters were proved to be more effective radical scavenger than β-
carotene [67].
Capsorubin, possessing two carbonyl groups at 6- and 6‘ positions, showed more potent
antioxidant activity than capsanthin [68].
These findings suggests that the antioxidant ability of capsanthin would not be influenced
by hydroxyl groups at the 3- and 3‘ positions in the β-ionone and cyclopentane rings, the
effect is due to the polyene chain, and especially enhanced by the conjugated keto group [67].
Capsanthin and capsorubin have also been reported to quench singlet oxygen effectively.
[69] Murakami et al. screened several natural carotenoids (among them capsanthin,
capsorubin, capsanthin 3,6-epoxide) for their modifying effects on superoxide (O2-) and nitric
oxide (NO) generation from activated human and mouse leukocytes, respectively [70].
Excessive and prolonged generation of superoxide and nitric oxide from inflammatory
leukocytes means oxidative stress, which is closely associated with carcinogenic processes,
and with several lifestyle-related diseases. Cytotoxicity was not observed in any carotenoids,
however, the inhibitory activities of capsanthin and capsanthin 3,6-epoxide in O2- generation

were among the best ones, and were significantly higher than those of β-carotene and a green
tea polyphenol, epigallocatechin gallate. Capsorubin was found to be less active [70].
With respect to the structure-activity relationship, the presence of a single 3-hydroxy-θ-
end group could be important for the activity. This is supported by the fact that an exchange
of the 3-hydroxy-β-end group present in cucurbitaxanthin A by a 3-hydroxy-θ-end group in
capsanthin 3,6-epoxide resulted in a signifcant increase of inhibitory activity in O2-
generation.
Figure 8.
The inhibitory activity of capsanthin on NO generation was comparable to that of β-

carotene, however, capsorubin and capsanthin 3,6-epoxide showed remarkably higher
inhibition. The 3-hydroxy-θ-end group may also be important for NO generation suppression,
when comparing the activity of capsanthin 3,6-epoxide to that of cucurbitaxanthin A. [70]
Kim et al. showed that capsanthin significantly preserved the activity of superoxide dismutase
in carbontetrachloride-treated rats, and also inhibited lipid peroxidation [71].
Anti-tumor-promoting activity of capsanthin and related carotenoids were examined in
vitro on the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by a
tumor promoter. [72] Fatty acid diester of capsanthin and capsorubin diester showed the
strongest inhibitory effects, although capsorubin and capsanthin 3,6-epoxide were also more
active than β-carotene. The esterification of the hydroxyl groups with fatty acids enhanced the
inhibitory activity toward EBV-EA activation. Capsanthin, capsanthin 3‘-ester and capsanthin
3,3‘-diester, major carotenoids in paprika, also exhibited potent anti-tumor activity in an in
vivo mouse skin two-stage carcinogenesis assay. As esterification enhances the liposolubility
and stability of carotenoids, it may increase the absorption of carotenoids into skin tissue [72].
Tsushima et al. reported the inhibitory activity of other θ-carotenoids toward EBV-EA
activation: mytiloxanthinone and mytiloxanthin exhibited moderate activity [73].
The multidrug resistance (MDR) proteins are present in a majority of human tumors and
are an important final cause of therapeutic failure. Compounds which inhibit the function of
the multidrug resistance-efflux proteins may improve the cytotoxic action of anticancer

chemotherapy. Molnár and coworkers have examined the multidrug resistance (MDR)
modulating activity of several carotenoids in tumor cells, and shown that capsanthin and
capsorubin enhanced the drug accumulation most potently. α- and β-carotenes, however, were
proved to be inefficient. [74] Paprika extracts of high capsanthin content showed similar
favourable effect [75].
Ascorbic acid was shown to influence the inhibitory effect of certain carotenoids on the
multidrug resistance of cancer cells. Ascorbic acid improved the effect of capsorubin in a
dose-dependent manner, but the effect of capsanthin was not modified. A special complex
formation between membrane-bound capsorubin and ascorbic acid was suggested, which can
be exploited in experimental chemotherapy [76].
Capsorubin was found to accumulate selectively in the nuclei of cancer cells, suggesting
that the membrane affinity of capsorubin is stronger than the other carotenoids. Furthermore,
capsorubin had a remarkable antiproliferative effect on lung cancer cells, and reduced
immediate-early tumor antigen expression. Capsanthin, however, proved to be not effective
under similar conditions [76].
Conclusion
Studies on carotenoids with -end group have a long history. They are carotenoids that
with some exceptions can be found in little amounts among other pigments. As this review
shows, their chemistry was quite thoroughly investigated over the years, nowadays the focus
is - as with other carotenoids - laid on biochemical investigations or on antioxidant activity of
these pigments. Although until recently the discovery of new -carotenoids was not to be
expected, the determination of the carotenoid profile of some tropical fruits gave some
interesting results including new -carotenoids that are present in considerable amounts. This
discovery can open a new area in the research of -carotenoids and also can deliver new
information about the biosynthesis of -carotenoids in general.
Acknowledgments
This study was supported by the grant OTKA K 83898 (Hungarian Scientific Research
Foundation).
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[75] Molnár, P.; Kawase, M.; Satoh, K.; Sohara, Y.; Tanaka, T.; Tani, S.; Sakagami, H.;
Nakashima, H.; Motohashi, N.; Gyémánt, N.; Molnár, J., Biological activity of
carotenoids in red paprika (PM1), valencia orange and golden delicious apple.
Phytother. Res. 2005, 19, 700-707.
[76] Molnár, J.; Serly, J.; Pusztai, R.; Vincze, I.; Molnár, P.; Horváth, G.; Deli, J.; Maoka,
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by carotenoids and xanthophylls with ascorbic acid to reverse multidrug resistance in
cancer cells. Anticancer Research 2012, 32, 507-518.

Chapter IV
Development of a Method to Induce

ROS Resistant High-Yielding Mutant
Lines of Sugarcane, Saccharum
officinarum cv NiF8 and NiTn18
Kohji Hasunuma1, Yusuke Yoshida1, Md Emdadul Haque1,

Yosuke Fukamatsu1, Yoshibumi Terajima2 and Makoto Matsuoka2
1
Kihara Institute for Biological Research, Yokohama City University
641-12 Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, Japan
2
Sugarcane Experimental Station in Tanegashima, KONARC, Japan.
NARO Kyushu Okinawa Agricultural Research Center
Nishino-omote-shi, Kagoshima Japan
Abstract
Stems of Saccharum officinarum cv NiF8 and NiTn18 were treated with a liquid
medium containing various concentrations of Paraquat (PQ) for 1 week, and they were
transplanted to planters in Dec, 2006, and placed in a green house at 23C. From April
2007 the seedlings with good growth were transplanted to larger planters. We obtained
candidates of mutant lines, R4-6-1a and R80-13-3a, showing very good growth
characteristics from the original lines of NiF8 and NiTn18, respectively. The green
mutant leaves were deeper in color than those of the original lines. Total stem numbers
and stem weights of mutant lines in 2008 and 2009 were around 2-fold compared with
those of the original lines. The values of SPAD photometer of leaves estimating
chlorophyll contents were greater in mutant lines compared with those of the original
lines. Punch out discs of leaves contained chlorophyll a and b, carotenoids and
anthocyanins with higher concentrations in mutant lines compared with those from the
original NiF8 and NiTn18 lines. Singlet oxygen evolved will make flight outside of
emitted chloroplast, and react with unsaturated fatty acids in the membrane systems
forming malondialdehyde. The amounts of malondialdehyde in leaves under strong

80 Kohji Hasunuma, Yusuke Yoshida, Md Emdadul Haque et al.
sunlight were reduced in mutant lines compared with those of the original NiF8 and
NiTn18 lines. The punch out discs from leaves obtained from NiTn18 original and the
mutant line, R80-13-3a grown in Tanegashima were floated on a medium containing 80
µM PQ, which showed resistant characteristics in mutant leaf discs compared with those
of the original line. The mutant lines showed higher tillering characteristics and were
suggested to make precise planting plan to field to obtain larger yielding.
Keywords: Anthocianin, Carotenoids, Chlorophyll a and b, High yielding mutants,

Malondialdehyde, Paraquat treatment, Reactive oxygen species (ROS) resistant,
Saccharum officinarum, Tillering
Abbreviations
CAT Catalase
DMSO Dimethyl sulfoxide
H 2O 2 Hydrogen peroxide
MDA Malondialdehyde
NADH Reduced form of nicotinamide adenine dinucleotide
NDPK Nucleoside-diphosphate kinase
1
O2 Singlet oxygen, exited state
3
O2 Triplet oxygen, basal state
O2.- Superoxide (Superoxide anion radical)
ppm Part per million
PQ Paraquat (Mehtyl viologen)
R4-6-1a A mutant line obtained from the original line, NiF8
R80-13-3a A mutant line obtained from the original line, NiTn18
ROS Reactive oxygen species
SOD Superoxide dismutase
Introduction
In the history of the earth during the last 650,000 years, human beings showed
evolutionary processes from Homo erectus to Homosapiens sapiens [1]. During this period,
ancestors of human beings received 6 glacial periods and 6 warm periods (interglacial
periods) with period lengths of around 100,000 years. In the warm periods the concentration
of carbon dioxide in the air on the earth became around 290 ppm and in the glacial periods the
carbon dioxide concentration was around 180 ppm [2]. In the warm periods with high
concentrations of carbon dioxide plants were growing thic on the earth reducing the
concentration of carbon dioxide, which led to the lowering of carbon dioxide concentrations
on the earth. The results of lowering the carbon dioxide concentration had led to a decrease in
surface temperature on the earth, which led to the freezing circumstances of glacial periods.
These results imply the very important roles of plants to reduce the concentrations of carbon
dioxide on the earth [3].

Development of a Method to Induce ROS Resistant High-Yielding Mutant Lines … 81
Maintaining the circumstances on earth is very important in order to keep crop fields in
good condition. Depending on the change of ambient circumstances showing increases in the
temperature on the earth, global warming will result in the formation of dried areas and a vast
area of the earth will be desert, which cannot be used for a field of crop plants [4].
Further it is reported that the global warming on the earth increased the amount of rain
fall on the Indian continent, which had proportional relationship with the increase in the rate
of drift of the continent to Eurasian continent [5, 6]. The global warming will result in an
increase in the movement of plates on the surface of the earth, which will lead earth quake in
2004 caused in the southern part of Sumatra, and also in Japan Northeast part of Pacific
Ocean caused large earthquake in March 11, 2011. We are facing a very critical period in the
history of the earth. Such a result can cause unexpectedly large earthquakes followed by
unexpectedly large Tsunamis from the unlimited usage of fossil fuels, such as petroleum,
natural gases and coals. To reduce the unrestricted usage of fossil fuels, providing
unexpectedly high yielding crop plants are necessary to incorporate carbon dioxide on the
earth by increasing the rate of photosynthesis by crop plants under strong sunlight. Providing
large amounts of carbohydrates by creating mutant crop plants with high yields will supply
huge amounts of carbohydrate sources for biofuels such as bio-ethanol, bio-gases and bio-oils
in place of fossil fuels. Therefore, a method to create mutant crop plants with unexpectedly
high yielding will give an essential method to reduce the carbon dioxide on the earth [7].
In the previous report using Pisum sativum cv Alaska we reported a high yielding mutant
R3-1, which showed resistant phonotype to Paraquat (PQ) [8]. Strong sunlight will cause a
triplet state of chlorophyll in chloroplasts and transfer the energy to ambient triplet oxygen
(3O2), and the resulting singlet oxygen (1O2) will make flight with a path length of 63~500 µm
going out of chloroplast [9, 10]. Singlet oxygen captured by catalases (CATs) in cytosol.
CATs binding singlet oxygen will make protein-protein interaction with precursor of NDPK-
2, which has capacity to bind NADH [8, 11]. Electrons from bound NADH on NDPK-2 will
reduce the bound singlet oxygen on CATs releasing super oxide (O2.-) in the cytosol.
Superoxide will be reduced by Mn SOD (superoxide dismutase), which showed increase in
the enzymatic activity [8]. The resulting hydrogen peroxide (H2O2) will be detoxified by
enhanced enzymatic activity of CATs by getting electron from NADH bound to precursor of
NDPK-2 attaching on the surface of chloroplast [8] releasing H2O, triplet oxygen (3O2) and
thermal emission. In the present report we have established a method to induce mutations in
the Succharum officinarum with characteristics of resistant phenotype to PQ, with high
yielding. The characteristics from several point of view of biochemistry were checked on the
chlorophyll contents, photo-pigment contents such as carotenoids and anthocyanin and
oxidation decomposition of unsaturated fatty acids in plasma membrane.
Materials and Methods
Strains Used in this Experiment
Succharum officinarum cv NiF8 and NiTn18 were used in Okinawa prefecture and the
southern districts of Kyushu in Japan, respectively. These lines were provided from the
NARO Tanegashima Experimental Station in Nov. 2006.

The treatment of stem sections with a medium containing Paraquat (PQ: Methyl
viologen, Sigma USA); fifteen stems with 6 septa with good budding of NiF8 and NiTn18
were cut into 3 parts with 2 septa. Total of 45 stem parts with two septa were washed by tap
water, and then treated with 1 litter of MS medium containing 0, 4, 8, 40 and 80 µM of PQ,
and each treatment by PQ included 9 stems with 18 buddings, respectively. Since the stems
include sugar the medium was fermented by fungus. Every 2 days, the medium was replaced
with fresh medium containing the same concentrations of PQ. After 7 days of treatment the
stem section was planted in a large planter with two stems of total of 4 buddings. The upper
one was designated as a, and the second was as b. Nine stems were included in a bowl
containing 1 litter of MS medium with various concentrations of PQ. Two bowls were used
for one concentration of PQ for two original lines.
Estimation of Chlorophyll Contents by SPAD Photometer
SPAD values were determined by checking 10 positions of each leaf blade of the first, the
second and the third opening leaves. Three stems were checked by the same methods. SPAD
photometer (502-Plus) was manufactured by Konica-Minolta. The average of the results of
three stems were presented in Fig.3.
Extraction of Pigments by DMSO and OD Determination of their Amounts
Ten punch out discs were obtained from one leaf blade to plastic Petri dish and the fresh
weight in g of the discs were measured. Then the leaf discs were placed in a flat bottom tube
containing 7 ml of preheated DMSO with cap, which was heated at 60 C for 30 min. The OD
was read by a Beckman DU530 spectrophotometer and the amounts of chlorophyll a and b,
carotenoids and anthocyanins were determined, principally according to the method described
[12].
Determination of PQ resistant characteristics
The stems of the original lines and mutant lines were sent to Tanegashima Research
Station at the end of the first cultivating year in January 2008. The mutant lines of R80-4 and
R80-5 grown in Tanegashima Research Station indicated NiTn18 R80-13-3a mutant line with
the stem No of 4 and 5. Ten Petri dishes containing 10 punch out discs were prepared. One of
them contained 25 ml of MS medium containing 0 µM of PQ and used for the control
experiment. The remaining 9 dishes contained 25 ml of MS medium containing 80 µM of PQ.
The Petri dishes containing 10 discs with MS medium with or without PQ were incubated at
25 ℃ under 250 µmol/m2/sec of continuous fluorescent light for 24 h.

Determination of membrane lipid oxidation
The determination of oxidation of a membrane lipid prepared from membrane lipids was
described [8, 13] by use of membrane lipid from No 5 opening leaves under strong sunlight.
The method was applied to leaf blades of original lines and the mutant lines grown in
Tanegashima Research Station.
Results
Figure 1. Growth patterns of original lines and the mutant lines in 2007 in Yokohama. Growth of NiF8
original line and the high yielding mutatnt line, R4-6-3a. Author standing is 163 cm in height. Growth of
NiTn18 original line (left side) and the high yielding mutant line, R80-13-3a (right side).

Figure 2. Growth patters of original lines and the mutant lines in 2008 in Yokohama. Growth of second year
NiF8 original line and the high yielding mutant line, R4-6-3a. Growth of NiTn18 original line and the high
yielding mutant line, R80-13-3a.

Table 1. Induction of mutations to high yielding by various concentrations of PQ by use

of stems from NiF8 and NiTn18
A Growth of primary plants treated by PQ by use of stems of NiF8.
B Growth of primary plants treated by PQ by use of stems of NiTn18
The two treated stem sections containing 2 buddings were planted in a large planter and
each seedling showing good growth was transferred to a round large planter. From the 18
buddings treated by 5 different concentrations of PQ we obtained a candidate of mutant from
NiF8 and also from NiTn18 as the original lines, respectively. From NiF8 we obtained a
candidate of mutant R4-6-3a at the concentration of 4 µM PQ presented in Fig. 1a, which
showed very good growth compared with that of the original WT line as shown in Table 1a.
By use of NiTn18 we obtained a candidate of mutant line R80-13-3a with very good growth
and tillering by a PQ concentration of 80 µM, which showed extraordinary good growth and

tillering as shown in Fig.1b, and in Table 1b. We focused to these lines with high yielding.
The plants became wilting when the sunlight was strong with no rain, thus we made a
frequent irrigation system with water spray, and a 200 g/planter of compound organic
fertilizer (Wako) was also used every year. In January the stems were cut and the weight (g)
and height (cm) were measured. In the winter period the planters were placed in a green
house, because winter period was so cold showing low temperatures as low as –2 C.
Figure 3. SPAD values of leaf blades of original lines and the mutant lines in 2008 in Yokohama. SPAD
values of leaf blades of the first, the second and the third open leaves of original NiF8 line and the high
yielding mutant line, R4-6-3a. SPAD values of leaf blades of the first, the second and the third open leaves of
original NiTn18 line and the high yielding mutant line, R80-13-3a.
The growth patterns of NiF8 original line and the R4-6-3a mutant line in 2008 were
presented in Fig. 2a and those of NiTn18 original line and the R80-13-3a mutant line were as
shown in Fig. 2b. In both mutant lines we could detect the increase in the green color of leaf
blades in the mutant lines with increasing tillering. We estimated the differences of the
concentrations of chlorophyll by SPAD values. The measurement of the leaves of three stems
from the first opening leaf, the second leaf and the third leaf were made with a SPAD
photometer. As presented in Fig. 3a and in Fig. 3b, both of the mutant lines showed higher
values of SPAD measurement, indicating that under strong sunlight the concentrations of

chlorophyll in these mutants showed higher values compared with those of original lines.
From the changes of SPAD values form the first, the second and the third leaves in the
mutants showed increase in the concentrations of chlorophyll. However, under strong sunlight
in the case of original wild type line the concentrations of chlorophyll in these leaves did not
show an increase, but rather showed reduced concentrations.
Figure 4. Extraction of chlorophyll a and b, carotenoids and anthocyanins from leaf discs of original lines and
the mutant lines in 2008 by use of DMSO. Extraction of chlorophyll a and b, carotenoids and anthocyanins
from original line of NiF8 and the high yielding mutants R4-6-3a. Extraction of chlorophyll a and b,
carotenoids and anthocyanins from original line of NiTn18 and the mutant R80-13-3a.
We measured the concentrations of chlorophyll a and b, carotenoids and anthocyanins by

extracting these pigments from 10 punch out of leaf blades by preheated DMSO. As shown in
Fig. 4a and Fig. 4b both mutant lines contained higher concentrations of these pigments.
However, the estimation of the concentrations of chlorophyll a and b from the extraction
using DMSO seemed to be unstable, and therefore further measurement of the concentrations
of chlorophyll a and b by a different method is required.

Figure 5. Resistant phenotypes of NiTn18 R80-4 and R80-5 compared with leaf discs from the original
NiTn18 line.
On these mutant lines we investigated whether the 90 pieces of punch out discs of leaf
blade of these mutant lines were resistant to PQ used for the induction of mutation. In the case
of NiF8 R4-6-3a the PQ concentration caused the high yielding mutation was 4 µM, and not
so high and therefore the 4µM of PQ was not so effective to induce the difference between
original line and the mutant line. We could not detect the difference in these testing.
However, in the case of NiTn18 R80-13-3a, the concentration of PQ was high enough to
cause damage to punch out discs of the original line. As expected we could detect the
differences between leaf discs of original line and those of mutant lines. R80-4 and R80-5
indicate the original stems came from No 4 and No 5 stems of former mutant lines, indicating
the mutant lines of these stems include much of mutant cells showing highly resistant to 80
µM of PQ. The results were presented in Fig. 5, supporting the prediction above described.
Figure 6. Amounts of malondialdehyde in the membrane preparations of NiF8 original line and the mutant,
R4-1, and in NiTn18 original line and the mutant lines, R80-4 and R80-5.
The high resistant characteristics of the leaf discs of NiTn18 R80-13-3a compared with
those of the original line indicate that whole body of the sugarcane was constituted by the
cells resistant to 80 µM PQ, indicating highly resistant phenotype of mutant line.
The leaves of NiF8 original line and those of the mutant line, and also the leaves of
NiTn18 original line and the NiTn18 mutant R80-4 and R80-5 lines were supplied to

investigate the concentrations of malondialdehyde formed by the function of singlet oxygen

to break double bonds of unsaturated fatty acids in the plasma mambrane. The formation of
malondialdehyde (MDF) was presented in Fig. 6. In NiF8 original line the concentration of
MDF was high compared with those of the mutant line. However, the difference
corresponded to a slight decrease. In case of NiTn18 original line the leaf membrane
preparation contained roughly same concentrations of MDF as that in the NiF8 orginal line.
However, in the R80-4 and R80-5 leaves the concentrations of MDF were so reduced
corresponding to 80% of that in the original line. These results indicate that PQ resistant
characteristics of R80-4 and R80-5 lines will be the result of high activity to detoxify the
singlet oxygen, and possibly also high activity to detoxify superoxide and hydrogen peroxide
reducing toxic activity of ROS.
Discussion
The fundamental information obtained by this work is that the photo-inhibition caused by
strong sunlight resulting in the emission of singlet oxygen is enough to reduce the rate of
photosynthesis. By the gain of function mutation causing increase in the detoxification
process of ROS including singlet oxygen the rate of photosynthesis will be raised up,
although the actual demonstration of the process is not yet performed.
By the acceptance of strong sunlight chlorophyll will be excited to triplet state and
transfer the energy to triplet oxygen emitting singlet oxygen. The emitted singlet oxygen will
show the lifetime of 0.5 to 4 µs with average of 2 µs [10]. From the estimate of photodynamic
therapy (PDT), the flight path length of singlet oxygen caused by the two laser beams exiting
a modified form of porfirin will emit singlet oxygen with the flight path length of 500 µm
[11]. We could calculate that by the 0.5 to 4 µs of lifetime singlet oxygen make the flight path
length of 500 µm, and therefore the flight path length should be 63 to 500 µm. Because the
sizes of chloroplasts are 3 to 8 µm, we could consider that the singlet oxygen emitted from a
chloroplast will go outside of the chloroplast. There is no need of acceptor for singlet oxygen
such as CAT in the inside of the chloroplast [8, 14].
Rather singlet oxygen will be accepted by the unsaturated fatty acids in the membrane
forming MDF. The principal existence of unsaturated fatty acids in the membrane of plants
will have the protecting function from singlet oxygen inevitably evolved by strong sunlight.
The severe reduction of the amounts of MDF in the mutant line NiTn18 R80-13-3a leaf
membrane will be the result of the reduction of the amounts of singlet oxygen by efficient
detoxification via the capture of it by CATs in the cytosol [8].
Membrane destruction by singlet oxygen will lead to a profound reduction in the rate of
photosynthesis, and a reduction in the amounts of MDA will indicate the increase in the rate
of photosynthesis. However, there is no direct evidence of the increase in the rate of
photosynthesis. The concentration of chlorophyll a and b should also be rechecked by the
different extraction method, because the extraction method by use of DMSO needs heating to
65 ℃ for 30 min, which will associate to some extent the decomposition of chlorophyll a and
b.
These mutants of R4-6-3a from NiF8 and R80-13-3a from NiTn18, respectively, will be
obtained as the results of efficient mutation induction by this method. The stem budding will

contain 1million cells, and if one of the cells induce mutation to be resistant to ROS, the
resistant cell has a capacity to develop to a plant. Among 18 million cells in 18 buddings one
of the cell will cause very good mutations leading to point mutations to a resistant phenotype
to ROS. The mutations will contribute to the removal of toxic characteristics of singlet
oxygen as shown in Pisum sativum cv Alaska by inducing mutations in both leader, I12L, and
C-terminal, E205K, sequences of NDPK-2 [7, 8]. We estimate that such very efficient
characteristics of mutation inductions will be made systematically, forming some of the
mechanisms to return to the ancient forms of genes, which are required in the early periods in
the history of the earth. In the ancient period of the earth the sunlight was too strong and
severe without the filter function of oxygen to cut ultraviolet light on the earth. Even under
such conditions, plants had come up to lands enduring to the irradiation by ultraviolet light
from strong sunlight. Therefore, plants may have fundamental capacity to be alive under very
severe conditions of strong sunlight, and such functions may be furnished by changing the
activity of some gene products such as NDPK. In the case of the Alaska pea, we could detect
a ROS resistant mutant, R3-1 with two-fold increase in NDPK-2 and autophosphorylation
activities, and such mutants showed around a 2-fold increase in the yielding of pods [8].
These mutants of NiF8 and NiTn18, R4-6-1a and R80-13-3a have large characteristics of
increase in their tillering numbers. Under such characteristics, the yield test of these mutant
lines should be done by changing the distance of planting stems, such as using zigzag
planting to obtain maximum yield in a field.
Conclusion
1. We obtained R4-6-3a and R80-13-3a mutant lines from Saccharun officinarum cv NiF8
and NiTn18 original lines, which showed high yielding, and high tillering capacities.
2. These mutants showed deeper green leaves, which contained larger amounts of
chlorophyll a and b, carotenoids, and anthocyanins than those in the original lines.
3. These mutants included lower concentrations of oxidized unsaturated fatty acids, MDA,
in the leaves under strong sunlight.
4. NiTn18 R80-13-3a mutant line, R80-4 and R80-5 showed higher capacity to show
resistant characteristics to 80 µM PQ, indicating that the whole plant cells include PQ
resistant characteristics.
5. To obtain high yielding crops by using these mutant lines and the planting plan, such as
the distance of the shoots, should be made precise.
Acknowledgments
We are grateful to Nissan Co., Ltd. for financial support and their co-work. We are also
grateful to Yamada Science Foundation and Mishima Kaiun Foundation for financial
supports. We are also grateful to A. Komamine, Y. Ogihara and stuffs of Kihara Institute for
Biological Research for valuable criticisms. We are indebted to K. Sonoike of Waseda
University for valuable discussion on the photoinhibition under strong sunlight. We are
indebted to H. Terauchi in Tanegashima Station for his continuous support for this work. This

work was supported by Grant in Aid from Ministry of Agriculture, Fishery and Forestry
(1421-1) and by the fund from YCU.
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Miller, H. L. (2007) Climate Change 2007; The physical Science Basis, Contribution of
working group I to the Forth Assessment Report of the Intergovermental Panel on
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[5] Ottto-Bliesner, B. L. (1995) Continental drift, runoff and weathering feedbacks:
implication from climate model experiments. J. Geophys. Res. 100: 11,537-11,548.
[6] HP: Climate change affects movement of continents.
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Lee, B. (2011) Global warming, plant paraquat resistance, and light signal transduction
through nucleoside diphosphate kinase as a paradigm for increasing food supply.
Naunyn-Schmidberg‘s Arch Pharmacol. 384 (4-5) : 391-395.
[8] Haque, Md. E., Yoshida, Y. and Hasunuma, K. (2010) ROS play an important role in
the plant growth and production in ROS-resistant Pisum sativum cv. Alaska. Planta,
232: 367-382.
[9] Wohre, D., Wendt, A., Weitemeyer, A., Stark, J., Spiller, W., Schneider, G., Muller, S.,
Michelsen, U., Kliesh, H., Heuermann, et al. (2010) Reviews: metal chelates of porfirin
derivatives as sensitizers in photooxidation process of sulfur compounds and in
photodynamic therapy of cancer. Chemistry and Materials Science, Russian Chemical
Bulletin. 43: 1953-1964.
[10] Poliacki, M., Javonovic, M., Lugonja, B., Gajic, B., Ros, T. (2006) Review article:
Topical photodynamic therapy. Arch. Oncol. 14: 39-44.
[11] Wang, N., Yoshida, Y. and Hasunuma, K. (2007) Catalase-1 (CAT-1) and nucleoside
diphosphate kinase-1 (NDK-1) play an important role in protecting conidial viability
under light stress in Neurospora crassa. Mol. Genet. Genomics, 278: 235-242.
[12] Richardson, A. D., Duigan, S. P. and Berlyn, G. P. (2002) An evaluation of noninvasive
method to estimate foliar content. New Phytol. 153: 185-195.
[13] Buege, J. A. and Aust, S. D. (1972) Microsomal lipid peroxidation. Methods Enzymol.
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Chapter V
Molecular Base for Carotenoids

Antioxidant Activity in Model
and Biological Systems: The Health-
Related Effects
Dragan Cvetkovic1, Leszek Fiedor2, Joanna Fiedor3,

Anna Wisniewska-Becker2 and Dejan Markovic1
1
Faculty of Technology, University of Nish, Leskovac, Serbia
2
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Cracow, Poland
3
AGH-University of Science and Technology,
Faculty of Physics and Applied Informatics, Cracow, Poland
Abstract
There is a positive correlation between both, a higher dietary intake and tissue
concentration of carotenoids (Crts) and a lower risk of chronic diseases.
Antioxidant activity is one of the most important functions of Crts responsible for
their beneficial effects. Their long system of conjugated C=C bonds, in which the π-
electrons are highly delocalized, is for this antioxidant action a crucial structural feature.
Crts are therefore among the most effective scavengers of free radicals and quenchers of
singlet oxygen, which makes them efficient chain-breaking agents, or preventive
antioxidants, respectively. In the latter case, they suppress destruction of vital cellular
components such as lipids, proteins and DNA. These antioxidant actions of Crts have
been primarily investigated in homogenous solutions to avoid the problems with
solubility and aggregation, but also in models of biomembranes, such as micelles and
artificial lipid membranes.
The oxidative stress is considered as the most common cause of lipid peroxidation in
cells leading to numerous chronic diseases in humans. The so called reactive oxygen
species can lead to a series of various diseases, such as heart diseases, osteoporosis and

94 Dragan Cvetkovic, Leszek Fiedor, Joanna Fiedor et al.
different types of cancers. Crts in human plasma, particularly β-carotene and lycopene
constitute the first line of protection of plasma lipoproteins against oxidation. There is
also an evidence that Crts prevent the initiation and development of various kinds of
cancers, in particular of skin, prostate, bladder, and the digestive tract. Also
photooxidative processes induced by sunlight, especially by its UV part, take a big part in
patho-biochemical changes in the skin and eyes. The damage to the retina, in particular to
the macula lutea, done by light and oxygen, is a major cause of age related macular
degeneration (AMD), a widely spread vision malfunction in developed countries. Crts,
mainly lutein and zeaxanthin, act as photo-protectors of the retina by their antioxidant
activity and at the same time as photo-filters (by absorbing high-energy blue light). Also,
there is an evidence from the in vitro and in vivo studies that β-carotene, located in the
skin epidermis, acts preventively against photooxidative damage induced by UV-
radiation.
Yet, a detailed mechanism of Crt antioxidant action, especially in vivo, is not fully
understood. The antioxidant activity is more effective at low partial oxygen pressure
(pO2), while at higher pO2 (e.g. in cancerous tissues) the generated Crt radicals have
shown prooxidant activity by reacting with oxygen and producing Crt-peroxyl radicals. It
has been shown that lycopene and lutein may induce formation of hydroperoxides in
vitro, while β-carotene induces their formation and decomposition. The alternative pro-
oxidant mechanisms have been also proposed, such as Crt-radical adducts formation,
followed by decomposition into reactive alkoxyl-radicals or a direct reaction of the
formed adducts with triplet oxygen that can yield peroxyl-radicals. On the other hand, the
Crts prooxidant activity in vivo has been very recently attributed to the chemical
quenching of singlet oxygen by these compounds. Crts react with singlet oxygen, giving
relatively (un)stable cyclic mono- and diendoperoxides as the products. These species can
promote autooxidation of Crts and oxidation of other species initiating in vivo harmful
peroxidation of lipids. Bearing this in mind, a growing number of in vivo studies of
mechanisms of Crts anti-/pro-oxidant activity and health-related effects are to be
undertaken. A better knowledge of Crt properties and reactions in model systems appears
as an useful and necessary step for determination of conditions that predetermine the anti-
/pro-oxidant activity of these pigments in vivo.
1. Introduction
Carotenoids (Crts) are C40 tetraterpenoids, often symmetrical, made up of eight
isoprenoid (C5) units joined head-to-tail, except at the molecule center where a tail-to-tail
linkage occurs. The ―backbone‖ chain may have cyclic structures at both ends, which can be
substituted with oxygen-containing functional groups. There are about 700 known Crt
structures, including bacterial Crts (Britton et al. 1995, Britton et al. 2008). Most Crts found
in higher plants are plain hydrocarbons, termed as carotenes (Cars), such as β-carotene (β-
Car) and lycopene (Lyc, Figure 1), or oxygenated Car derivatives, e.g. xanthophylls, such as
hydroxyl-substituted lutein, or epoxy-substituted violaxanthin. The unique physicochemical
properties of Crts are related to the conjugated structure of the polyene chain, and can explain
the presence of these molecules in most biochemical systems, stretching from photosynthetic
apparatus to human tissues and organs, and biosensors and biomimetic devices as well (Stahl
& Sies 2003). The intensive attractive coloration of Crts is due to an extended -electron
delocalization in the conjugated polyene chain, giving rise to strong light absorption in the
visible part of the spectrum.

Molecular Base for Carotenoids Antioxidant Activity in Model … 95
The same conjugated system is a crucial feature of Crts, responsible for their antioxidant
activity, regardless of the particular reactive oxygen species (ROS) involved. The latter one
may involve very reactive, electron-unpaired free radicals (like superoxide anion-radical, ·O2-
, or hydroxyl radical, ·OH), or toxic singlet oxygen, 1O2, which reacts with Crts (and other
substrates, like lipids) by a non-radical mechanism. Crts are one of the most effective
quenchers of singlet oxygen and/or excited triplet states of photoactive molecules.
Multiplicity of potential reactive sites with 1O2 and free radicals in the Crt structures is one of
the most important factors that crucially determines their antioxidant capacity (Haila 1999).
They can act as (free radical) chain-breaking agents (Edge & Truscott 2000; Krinsky & Yeum
2003), but also in prevention by quenching 1O2 (Tatsuzawa et al. 2000; Montenegro et al.
2002), suppressing possible destruction of vital biomolecules such as lipids, proteins and
DNA.
However, Crts do not necessarily act as antioxidants under all circumstances,
independent of many involving factors, which (besides its own chemical structures) may
include reactivity of ROS involved, or mobility of the interactive species (Crts-ROS) in the
system, dominantly determined by molecular organization of surrounding environment (Haila
1999; Tsuchihashi et al. 1995). Moreover, a few previous (Fiedor et al. 2001; Fiedor et al.
2005) and some recent studies (Ramel et al. 2012a) revealed that chemical quenching of 1O2
may result in formation of very reactive endoperoxides of Crts which actually can promote
further oxidation reactions. Moreover, the in vivo studies have shown that partial oxygen
pressure (pO2) in blood may be a factor governing the anti/pro-oxidant behavior of Crts (the
latter one occurring at higher pO2 values - Burton & Ingold 1984; El-Agamey et al. 2004). In
addition, there are many controversial results concerning anti- vs. pro-oxidant Crts behavior
since they have been obtained using different experimental approaches (in vitro, ex vivo or in
vivo studies), and different model systems.
1.1. Antioxidants
All organisms at the cellular level are susceptible to ROS, such as ·O2-, ·OH and H2O2,
formed during the metabolism of oxygen, and also 1O2. Free radicals are constantly produced
in living organisms and they can attack surrounding oxidizable substrates (lipids, proteins,
carbohydrates and DNA) and thus initiate harmful chain-reactions such as lipid peroxidation.
Free radical chain reactions lead to malfunctions of the cell biomembranes, which may
become fatal in the absence of preventing or defense mechanisms. That is why antioxidants
play an important role in natural defense mechanisms in living organisms. As long as there is
a balance between free radical level and the prevention and/or their inhibition, there are no
harmful effects on the cell membranes, cells, tissues and to the whole organism. The free
radical quenchers neutralize free radicals, by pairing with the unpaired electrons through
direct interaction, or by suppressing reactions and mechanisms that lead to free radicals
generation (preventive action). Britton defined an effective free radical quencher as a
molecule which removes radicals from the system either through a direct reaction to yield
harmless products, or by disrupting free radical chain reactions (Britton 1995). The
antioxidant capacity is determined by intrinsic chemical reactivity of the antioxidant toward
the radicals, site of generation and reactivity of the radicals, concentration and mobility of the

antioxidant in the system, stability of antioxidant-derived radicals, and interaction with other
antioxidants (Haila 1999; Tsuchihashi et al. 1995).
Preventive antioxidants suppress oxygen radicals formation and represent first line of
defense against free-radicals. This group includes antioxidative enzymes (superoxide
dismutase, catalase, glutathione peroxidase, reduced glutathione and peroxidase) and
glutathione reductase, glucose-6-phosphate dehydrogenase. It also includes other proteins,
e.g. transpherine, ceruloplasmin and albumin, and small molecules such as uric acid and
bilirubin (Vega & Pizarro 2000) – and it serves to protect proteins, but lipids and DNA as
well (Montenegro et al. 2002; Palozza & Krinsky 1992; Miki 1991; Tatsuzawa et al. 2000;
Stahl & Sies 2003).
Chain-breaking antioxidants are usually a broad class of compounds that scavenge free
radicals, but also act as electron donors, decompose lipid peroxides (formed in the
termination phase). They quench singlet oxygen, inhibit the action of some enzymes, and act
synergistically with other antioxidants. There is a large number of natural antioxidants
belonging to this group. Some of them are vitamins (α-tocopherol - vitamin E, ascorbic acid -
vitamin C, retinol - vitamin A, riboflavin and thiamine - vitamin B1 and B2, etc.), flavonoids,
phenolic compounds, nitrogen compounds (alkaloids, amines and amino acids), and even Crts
and Chls (Upston et al. 1999; Niki et al. 2005; Fukumoto & Mazza 2000; Wall 2000; Kitts
1997; Beuttner 1993; Deng et al. 1997).
1.1.1. Mechanisms of Carotenoids Antioxidant Activity

This action of Crts could be related to: (i) the multiplicity of closely spaced energy levels
between excited and ground states of the Crt molecule wherefore they can dissipate energy
via small collisions with the system components; (ii) the minimal reactivity of excited-state of
Crts to sensitize other molecules; (iii) the fact that resonance states in the excited Crt
molecule allow delocalization and its stabilization; and (iv) the multiplicity of potential
1
reactive sites for O2 in the Crt structure (Haila 1999).
1.1.2. Singlet Oxygen Quenching by Carotenoids

1
Crts are able to quench O2 physically and chemically. If physical quenching occurs,
there are no changes in the chemical structure of Crt because of the excess energy dissipation
in the form of heat to the surrounding medium (Edge et al. 1997; Krinsky & Deneke 1982;
Haila 1999).
a) Physical quenching of singlet oxygen by carotenoids
Crts are able to protect systems from harmful singlet oxygen, by an energy transfer
1
process, i.e. by quenching either a triplet sensitizer or O2:
3
S  1Crt  S  3Crt  (1)
O2 1Crt 3O2  3Crt 

1
(2)
3
Crt  1Crt  heat (3)

Crt triplet formed according to the equations (1) and (2) - 3Crt* - can easily lose its
energy to the environment and return to its ground state - 1Crt (eq.3) (Hailla 1999; Frank &
Cogdell 1996; Palozza & Krinsky 1992). The number of conjugated double bonds in a Crt
molecule is important for their ability to quench 1O2. The maximal protection has been shown
by Crts having 9 or more conjugated C=C bonds, and the end-groups (Edge et al. 1997).
b) Chemical quenching of singlet oxygen by carotenoids
More and more evidence is being obtained that Crts also act as chemical quenchers of
ROS (Foote & Denny 1968; Burton & Ingold 1984; Edge & Truscott 1999). Recently, it was
shown that in plant leaves Crts are also able to quench 1O2 by a chemical mechanism leading
to their oxidation. In low-light-grown A. thaliana, the 1O2-specific endoperoxides (Figure 1)
were found and their levels rapidly elevated during high-light stress, indicating chronic
oxidation of Crts by 1O2. The major product, β-Car endoperoxide, undergoes a fast turn-over
in leaves, having a half-life time of 6 h. Its accumulation can be regarded as a marker of 1O2
production in leaves, precedes the accumulation of fatty acid oxidation products (Ramel et al.
2012a; Ramel et al. 2012b).
The importance of reactive endoperoxides of Crts, the products of their chemical
reactions with 1O2 is currently hotly debated also in the context of pro- and antioxidant
functioning of these pigments as diet components. Our model studies showed that the
mechanism of the Crt photoprotective action is strongly medium-dependent (Fiedor et al.
2001; Fiedor et al. 2005). For instance, β-Car in methanol has no protective effect towards
self-sensitized photodegradation of Chla whereas in acetone it prevents degradation of Chla,
at the cost of rapid photodegradation of the Crt.
lycopene
all-trans-carotene
OH
lutein
HO OH
zeaxanthin
HO
5,8-endoperoxide
O
O
O
O
5,8,5',8'-diendoperoxide
O
O
Figure 1. Structural formulae of lycopene, all trans -carotene, xanthophylls (lutein and zeaxanthin) and
endoperoxides of -carotene, possibly involved in the prooxidant activity of carotenoids.

In this photosensitized reaction in acetone a cascade of several oxidation products of β-

Car has been observed. The identification of these products by mass spectrometry showed
that β-Car sequentially accumulates up to six oxygen atoms while its C40-skeleton remains
intact (Fiedor et al. 2001; Fiedor et al. 2005). It was shown that the photosensitized
degradation of β-Car is strongly inhibited by the 1O2-quencher, DABCO, while the radical
quencher, BHT, showed only little effect, which confirmed the crucial role of 1O2 as the
reactive intermediate. The main conclusion from these studies was that β-carotene molecules,
while retaining the main C40 skeleton, are able to chemically bind up to 6 oxygen atoms, and
that the products of these reactions have capacity of oxidizing other molecules. From the
chemical point of view, the Crt endoperoxides seem to be the best candidates as the entities
promoting oxidations of other organic substances. Such pro-oxidant activity of Crts was also
reported in lipid peroxidation studies (Burton & Ingold 1984). However, neither the
mechanisms and the products of these reactions of Crt are well established, nor the
contribution of these pro-oxidants to light-induced cellular damage and oxidative stress in
living tissue (Young & Lowe 2001). Another study showed that the cleavage products of β-
Car can be the cause of oxidative stress in mitochondria by inhibiting the respiration (Siems
et al. 2002).
In the earlier studies (Foote & Denny 1968; Burton & Ingold 1984), to account for the
formation of endoperoxides of -Car, a primary attack of peroxyl radicals to the C-7 position
had been suggested, which would be favored by the relatively high electrophilicity of this site.
The resulting radical of -Car was proposed to react with molecular (triplet) oxygen to
produce the cyclic endoperoxide (Figure 1). Based on the structural assignment of the
products by NMR as cyclic endoperoxides of β-Car (Figure 1), we have proposed a 2:4
cycloaddition as the oxygenation mechanism of β-Car. The concerted mechanism requires the
presence of an s-cis-diene conformation of the Crt educt. The formation of 5,8-endoperoxides
can proceed without prior conformational change of all-trans-β-Car because of the sterically
favored 6-s-cis conformation, while the formation of 7,10-endoperoxides requires an s-trans
to s-cis conversion at the C-8/C-9 single bond.
Because Crts and their endoperoxides are involved in the pro/anti-oxidant processes
taking place in lipid membranes, the site of their origin, the Crts endoperoxides may affect
biological systems in several ways, by modification (physically and/or chemically) of
membrane properties, and by a direct chemical damage to other vital components of the cells.
The effects on the membrane will result, like in case of unmodified Crts, from their presence
as rigid polar molecules spanning the bilayer. The effect of Crt endoperoxides, due to their
polarity, should be stronger than that of nonpolar Crts. On the other hand, considering their
reactivity, they may affect membrane properties also chemically – causing lipid peroxidation
and membrane damage. In animals, the highest concentration of Crts is found in the eye retina
of primates (Landrum & Bone 2001). High-energy, short-wave visible light promotes the
formation of ROS that can initiate lipid peroxidation (see below) in the retinal membranes, an
abundantly illuminated tissue with large respiratory demands for oxygen (elaborated in more
details in 1.4.1., ―Age-related macular degeneration‖).
As discussed above, the photosensitized oxidation of -Car leads to the formation of
colored products that could be accumulated and purified on a larger scale, which enables us to
perform a full structural analysis of these pigments by mass and NMR techniques. These facts

speak not only for the high reactivity of these compounds but also for their relatively high
stability in solution.
Figure 2. Normalized amplitude of the central peak of the EPR spectra of 16-SASL plotted as a function of
temperature (cooling experiments) in DMPC bilayer. Experiment performed for the pure DMPC (●), in the
presence of 25 mol% of cholesterol (○) and in the presence of 25 mol% of cholesterol peroxide (▼).
Their stability in lipid membranes is to be confirmed, however, our preliminary data

obtained with a chemically closely related species (cholesterol peroxide) show that such
compounds can be inserted and monitored in the model lipid membranes. Figure 2 shows the
effect of cholesterol peroxide, obtained by the photosensitized reaction, on DMPC
(dimyristoylphosphatidylcholine) phase transition, measured by an EPR spin labeling
technique, by using n-doxylstearic acid spin label (16-SASL). As it can be seen, the effect of
cholesterol peroxide is comparable to that of cholesterol, a well-known membrane modifier.
Certainly, no phase transition is observed in the presence of 25 mol% of cholesterol, or its
peroxide. This proves a good incorporation and stability of this peroxide in the membrane.
Moreover, an HPLC-EC(Hg) detection performed on the investigated membranes after EPR
measurements revealed the presence of unmodified cholesterol peroxide in the original
amount.
1.1.3. Antioxidant Activity of Carotenoids Towards Free Radicals

Crts lose their color while interacting with free radicals. This bleaching is due to the
shortening of the conjugated system of double bonds, either by their breaking or addition on
some double bond. There are three main mechanisms by which Crts can react with free
radicals and thus terminate free radical chain reactions, such as lipid peroxidation (LP).
Which of these mechanisms will be functional mainly depends on the structure of the radicals
and the Crt (Burton & Ingold 1984; Lim et al. 1992; Edge & Truscott 2000; Krinsky & Yeum
2003; Woodal et al. 1997b):

(1) Electron transfer. In this reaction, an electron exchange between Crts and free radical
(ROO·) takes place leading to free radical deactivation and Crt cation radical formation
(Crt·+):
 
ROO  Crt  ROO   Crt (4)
Crts are scavengers of propagating radicals, which is extremely important when the lipids
(and lipidic compounds) are the targets: the "neutralization" of lipid radicals prevents the
formation of lipid peroxides as a terminal stage of chain mechanism. This is important
because of instability of lipid peroxides (LOOH), which may lead to the formation of new
radicals through their decomposition, and thus switch the chain reactions on. The unpaired
electron in a Crt radical is strongly delocalized over the polyene system. This stabilizes Crt
radicals and enables their participation in further reactions (e.g. addition), permitting its
spectrophotometric monitoring as well (strong absorption in near infrared region) (Simic
1992; Polyakov et al. 2001a; Polyakov et al. 2001b; Jeevarajan et al. 1996; Krawczyk 1998;
Burke et al. 2001). Crts react with a large number of free radicals via electron transfer
mechanism: trichlormethylperoxyl (CCl3OO·), sulfonyl (RSO2·), nitrogen dioxide (NO2·) and
tocopheroxyl cation (TO· +) radicals (Mortensen et al. 2001).
(2) Addition reaction. Burton and Ingold (1984) have first proposed addition reactions of
Crt with free radicals, suggesting that a peroxyl radical can be attached anywhere on polyene
chain of Crt molecule forming a carbon-centered radical [ROO-Crt]·:
ROO   Crt  ROO  Crt 


(5)
The carbon-centered radical is stabilized by resonance so it terminates a chain reaction of

peroxidation and acts as an antioxidant in solution (Krinsky & Yeum 2003). The formed
adducts absorb light in the same region as the parent Crt, while not absorbing light above 600
nm (Frank et al. 1999; Edge & Truscott 2000).
(3) Hydrogen abstraction. Woodall and associates (1997b) have first proposed a
mechanism of hydrogen atom transfer leading to the formation of neutral Crt radical, Crt·:
ROO   Crt  ROOH  Crt   H  (6)
The neutral Crt radical is stabilized by resonance and has no strong absorption in the near
infrared region like Crt cation and Crt anion radicals (Edge & Truscott 2000; Krinsky &
Yeum 2003; Frank et al. 1999).
Among various Crt ‗reactive species‖, Crt cation radicals (Crt·+) are the most studied.
They are of special interest because of their distinctive property of being fairly strong
oxidizing agents (Böhm et al. 2012). The reduction potentials and relatively long lifetimes of
Crt cation radicals are thought to be sufficient to damage key biological compounds such as
amino acids and, in this way also proteins (Edge et al. 1998; Burke et al. 2001). These
physical properties of Crt cation radicals might also account for the unexpected change from
their antioxidant to prooxidant behavior. It is postulated that such redox-controlled reactions
may be responsible for both harmful as well as beneficial health effects. One of such
examples is the formation of the cation radicals of Crts present in macula (lutein, zeaxanthin,

meso-zeaxanthin – see more about in 1.4.1.), which is known to be almost exclusively

repaired by highly non-polar Crt, lycopene. It is suggested that their reversible oxidation to
the respective cation radicals, followed by regeneration with lycopene, is crucial for their
proper transportation and accumulation in the eye (Mares-Pearlman et al. 1995; Gouranton et
al. 2008). Lycopene is recognized as one of the most effective 1O2 quenchers among dietary
Crts (Di Mascio et al. 1989). The high antioxidant capacity of lycopene, a plain highly
unsaturated hydrocarbon (11 conjugated and 2 unconjugated double bonds), is related to the
large number of conjugated double bonds present in this molecule (Conn et al. 1991; Schmidt
2004). However, as the authors emphasize, mixtures of Crts are more effective than the single
compounds, what is especially evident in solutions containing additional lycopene or lutein.
In in vitro systems lycopene was found to be strong free radical scavenger including nitrogen
dioxide (NO2·), thiyl (RS·) and sulphonyl (RSO2·) radicals (Mortensen et al. 1997). Also its
ability to inactivate hydrogen peroxide was demonstrated (Lu et al. 1995).
1.2. Pro-Oxidant Activity of Carotenoids
The term pro-oxidant is often used in different contexts in the literature. Pro-oxidant can
be a metal ion able to reduce an activation energy of the lipid peroxidation – if the lipids
represent the oxidation target. A pro-oxidant can also be an initiator added to the reaction
mixture to start LP chain reaction, and leading to the formation and decomposition of lipid
hydroperoxides. A compound can exhibit the anti- or pro-oxidant activity depending on its
concentration, the experimental conditions, the stages of oxidation and types of other
participants in the reaction (Burton & Ingold 1984; Young & Lowe 2001).
The antioxidant activity of Crts depends on oxygen partial pressure (pO2) and Crts can
act as antioxidants suppressing LP chain reaction reacting with peroxyl radicals (Burton &
Ingold 1984). However, at increased pO2 values (non-physiological, 760 mmHg, 100% O2),
Crts demonstrate pro-oxidant activity through interaction of a Crt radical (Crt ·) with oxygen
and formation of a Crt-peroxyl radical (Burton & Ingold 1984; Young & Lowe 2001; Krinsky
2001):
 
Crt  3O2  Crt  OO (7)
This is an autoxidation process in which the created Crt-peroxyl radical may play a role
of a pro-oxidant by promoting oxidation of unsaturated lipid (RH):
 
Crt  OO  RH  Crt  OOH  R (8)
 
R 3O2  R  OO (9)
The Crt-radical adduct [ROO-Crt]· may be decomposed into a Crt-epoxide and alkoxyl-
radical which can promote LP chain reaction (Liebler & McClure 1996):

ROO  Crt  
 Crt  epoxide  RO  (10)
Alternatively, the Crt radical-adducts can react with ROO· giving a non radical product
[ROO-Crt-OOR] in the first step:
ROO  Crt  ROO  

 ROO  Crt  OOR (11)
which can then be decomposed into carbonyl compounds and two alkoxyl radicals (RO·):
ROO  Crt  OOR decomposition products  2RO 

(12)
and, finally, the adduct can react directly with oxygen producing peroxyl radicals:
ROO  Crt  3O2  ROO  Crt  OO  (13)
Burton and Ingold have concluded that β-Car act as a pro-oxidant at high pO2 (760
mmHg) and a relatively high concentration (> 0.5 mM) (Burton & Ingold 1984; Burton
1989). There are additional supporting reports referring the other Crts (Palozza 1998). There
have also been some evidence for β-Car pro-oxidant activity at physiological oxygen pressure
(150 mmHg), but at higher concentration of β-Car, when peroxidation was initiated by a
thermal initiator AAPH in rat liver microsomes (Zhang & Omaye 2001). Lowe and associates
(1999) have investigated concentration-dependent anti-/pro-oxidant activity of β-Car and
lycopene in HT29 colon carcinoma cells, with xanthine/xanthine oxidase playing a role of an
oxidation initiator. They have concluded that at physiological levels both β-Car and lycopene
prevents cellular damage, but at higher concentrations (4-10µM), the ability to protect these
cells is lost (Lowe et al. 1999; Krinsky 2001). So, it was concluded that an increased
consumption of β-carotene leads to enhanced lipid peroxidation in liver, kidney and brain of
mice exposed to methyl mercury chloride (CH3HgCl); the explanation was that the higher
concentration of β-carotene leads to the higher concentrations of β-carotenyl-radicals (eqs. 5
and 6) in the system and/or faster autoxidation of β-carotene at high pO2 (eq. 7) (Palozza &
Krinsky 1992; Kennedy & Liebler 1992; Edge et al. 1997; Krinsky & Deneke 1982).
1.3. Antioxidant/Pro-Oxidant Activity of Carotenoids in Different Molecular

Organizations and Environments
The autoxidation reaction (eq. 7) is the most pronounced at Crts high concentrations
(Young & Lowe 2001; Burton & Ingold 1984; Burton 1989; Palozza 1998; Palozza &
Krinsky 1992; Kennedy & Liebler 1992; Edge et al. 1997; Takahashi et al. 1999; El-Agamey
et al. 2004; Tsuchihashi et al. 1995; Mortensen et al. 2001; Mortensen & Skibsted 1998). The
reactivity of Crts with oxidizing and epoxidazing entities depends on the electron density
along the polyene chain; the epoxidazing effect is favored on the terminal double bonds,
while the oxidizing chain splitting is favored at C-7, 8 position of Crts (Britton 1995). Such
conclusions were drawn mainly based on in vitro studies where Crts act as antioxidants under

ambient conditions (pO2 ~ 150 mmHg, i.e. 20% O2), while the pro-oxidant function has been
observed at much higher oxygen pressures (20 - 100% O2, i.e. 150-760 mmHg) (Burton &
Ingold 1984; Haila 1999; Palozza & Krinsky 1992; El-Agamey et al. 2004; Young & Lowe
2001; Palozza et al. 1997). These and other results show a dependence of Crts anti-/pro-
oxidant behavior on the complexity of the studied systems such as solution or organized
biomembranes models (Haila et al. 1997; Heinonen et al. 1997; Palozza 1998; El-Agamey et
al. 2004). The results depend for example on differences in lipid composition of prepared
liposomes (Sarbolouki et al. 2005), the presence of other antioxidants, Crts concentration, pO2
level in the system, selected method for LP monitoring, and the method with which lipid
peroxidation was initiated (free-radicals, autoxidation, photosensitation, UV-irradiation etc.)
(Haila 1999; Krinsky & Deneke 1982; Young & Lowe 2001; Palozza 1998; Subagio &
Morita 2001; Subagio & Morita 2003).
Palozza (1998, 2005) has concluded that the antioxidant activity of Crts can be
"reversed", converted into pro-oxidant activity depending on Crts redox potential. The
reaction of Crts with peroxyl-radicals leads to its rapid bleaching of the pigment due to the
degradation and formation of oxy-products. Bleaching is a result of Crt oxidation, i.e.
breaking (shortening) of Crt conjugated systems (Britton 1995; Tsuchihashi et al. 1995;
Kennedy & Liebler 1991; Kennedy & Liebler 1992; Martin et al. 1999; Takahashi et al. 1999;
Bonnie & Choo 1999; Carail & Caris-Veyrat 2006). The shortening most commonly produces
apocarotenals and apocarotenones, but Crt-epoxides are very common, too. These compounds
with pro-oxidant activity are also formed in the reaction of Crts with singlet oxygen (Britton
1995; Fiedor et al. 2005; Fiedor et al. 2001; El-Agamey et al. 2004). Woodall and coworkers
(1997a; 1997b) have concluded that the oxidation of Crts initiated by Fenton reaction and
azo-initiators in solution leads to a wide variety of the oxidation products, suggesting that the
reaction can take place with any part of the Crt molecule (with polyene chain as well as with
endocyclic double bond). Some researchers have investigated the pro-oxidant activity of Crts
as a consequence of their instability to light and high temperatures, as well as a function of
Crts concentration and type of protective lipoidal targets (Subagio & Morita 2001; Subagio &
Morita 2003). Based on their research, the Crts decomposition products are responsible for
the initiation of LP. The products of LP (in the dark, at 40°C) are measured by conjugated
dienes test (increasing absorbance at 234 nm) - tracing an increase of lipid hydroperoxide
(LOOH) concentration – with synchronous monitoring of Crt bleaching. In their experiments
(Subagio & Morita 2001; Subagio & Morita 2003) β-carotene in concentration of 5 µg/g has
shown antioxidant activity, the concentration of 10 µg/g has shown no effect to the
peroxidation kinetics, while the higher concentrations (30 µg/g) have shown pro-oxidant
activity. Lutein has shown a proportional pro-oxidant effect for all above mentioned
concentrations.
El-Agamey and associates (2004) have shown that Crt antioxidant activity decreases due
to aggregation, with synchronous Crt autoxidation in such systems. They have noted that the
polarity and structure of generated radicals in the model systems is of great importance. The
more polar lipid-peroxyl-radicals accumulate in the lipid-water boundary phase, while Crts
(especially carotenes) remain within the lipid bilayer. Matsushita and Lim with their
coworkers (Matsushita et al. 2000; Lim et al. 1992) have reported on PC liposome
peroxidation, where LP has been initiated using azo-initiators in dark, in the Crt presence.
These results have been interpreted not only in the light of Crts scavenging capacity, but in
the light of the location and orientation of carotenoids inside PC liposomes, and their

incorporation degree within liposomes. Their interpretation is that polar Crts are as "rivets"
through the membrane, nailing down harder lipid bilayers – thus preventing the diffusion and
free movement of radical species (and so propagation step of LP). The closer inter-and
intramolecular packing of fatty acids chains in the lipid bilayer – making a "cage" that
prevents free radicals mobility – is an important factor for the resistance of liposomes to
oxidative stress (Markovic et al. 1990; Markovic & Patterson 1993; Markovic 2001; 2006).
1.3.1. Antioxidant/Pro-Oxidant Activity of Carotenoids Towards Lipids in

Solution
There are different views about mechanisms of Crts antioxidant activity in lipid
peroxidation process, concerning lipid peroxyl radicals (LOO·) capture, either trough electron
transfer (eq. 4), adduct formation (eq. 5) and/or transfer of a hydrogen atom (eq. 6). In the
cases where LP was initiated by free radical azo-generators (AMVN, AAPH, AIBN) the
adduct formation has been cited as the most probable (Burton & Ingold 1984; Palozza &
Krinsky 1992). However, some studies done by mass spectroscopy suggest that Crts can react
with L·, LO· and LOO· through hydrogen atom transfer, generating a neutral Crt radical (Crt
·
), which in combination with another radical can give a substitution product (Haila 1999).
The mechanism of Crt antioxidant activity in the LP process depends on the solvent
polarity, Crt structure and concentration as well as their tendency to build aggregates, mutual
reactivity of the created radicals, rate and mechanism of the Crts-radicals interaction (El-
Agamey et al. 2004). Crt reactions with lipid radicals are monitored by real-time techniques
tracing short-lived radical species, like laser flash photolysis (LFP) and pulse radiolysis. The
studies of Crt antioxidant activity in homogeneous solutions avoid problems with Crts
solubility and aggregation that predominantly occurs in heterogeneous systems. Lipid
peroxidation was initiated mainly by a radical generator (AIBN and AMVN) in chloroform,
benzene and hexane (Farombi & Britton 1999; Haila 1999). The stability of four selected Crts
(β-carotene, lycopene, lutein and neoxanthin) toward UV-irradiation in all three UV-ranges
(UV-A, UV-B, UV-C), as well as its antioxidant action on UV-induced lipid peroxidation of
lecithin in aqueous solution has been studied by thiobarbituric acid - malondialdehyde (TBA-
MDA) test1 (Cvetkovic & Markovic 2008a; 2008b) and by spectrophotometric conjugated
dienes test (Cvetkovic & Markovic 2011). It has been found that Crts undergo bleaching via
probable free radical mediated mechanism following first-order kinetics (Cvetkovic &
Markovic 2008a) in hexane solution, while the Crts antioxidant capacities appeared to be
strongly affected by UV-action. High energy input of the involved UV-photons plays major
governing role, though a certain impact of the Crt structures cannot be neglected. The results
suggest a minor contribution of selected Crts to prevention of lecithin peroxidation in the
studied system as a result of UV-irradiation (Cvetkovic & Markovic 2008b).
The ability of Crts to quench singlet oxygen (eqs. 2 and 3) in the presence of lipids has
been also extensively studied. There are reports showing Crt antioxidant activity in foods in
the presence of natural photosensitizers, like chlorophylls (Montenegro et al. 2002; Palozza &
Krinsky 1992; Miki 1991; Stahl & Sies 2003). The use of conventional methods (monitoring
of peroxide number, conjugated dienes and oxygen concentration) did not provide evidence
1
Thiobarbituric acid reactive substances (TBARS) are formed as a byproduct of lipid peroxidation.
Malondialdehyde is one of them, and it can be detected by the TBA-MDA test using thiobarbituric acid as a
reagent, measuring absorbance increasing at 532 nm which is maximum of TBA-MDA complex absorption.

whether Crts act as singlet oxygen quenchers or as radical scavengers (Haila 1999). As noted
above, by changing the pO2 in vitro it is possible to make a distinction between Crt
antioxidant/pro-oxidant activity. It was clearly shown that Crt antioxidant function increases
with the decrease of pO2 in the reaction system (Burton & Ingold 1984; Haila 1999; Palozza
& Krinsky 1992; El-Agamey et al. 2004). Lycopene and lutein promote formation, while β-
carotene promotes formation and decomposition of hydroperoxides in vitro (Haila 1999). Crts
have therefore shown very contradictory behavior in these systems. Initiation of lipid
peroxidation has been done by different ways (autoxidation, azo-initiators, photosensitization,
―triggering‖ mostly free-radical reactions). The most commonly used test for degree of lipid
peroxidation monitoring is TBA-MDA test (Liebler et al. 1997; Sujak et al. 1999; Tian Li et
al. 2000; Rengel et al. 2000; Goto et al. 2001; Barros et al. 2001; Stahl et al. 1988).
1.3.2. Antioxidant/Pro-Oxidant Activity of Carotenoids in Models of

Biomembranes
Location of Polar and Non-Polar Crts in the Membrane and Their Effect
on Membrane Properties
Crts are also known as modifiers of biological membranes (Gruszecki & Strzałka 2005;
Cvetkovic et al. 2013). Crts, being insoluble in water, in biological systems are either
incorporated into the lipid part of the membrane, or remain protein-bound. The shifts in their
absorption spectra observed after the incorporation into the lipid membrane, suggests that
their polyene chains are located in the environment with dielectric properties corresponding to
those of lipid hydrocarbon chains (Frank et al. 1999). Depending on the structure, Crt
molecules adopt various orientations within the membrane and dipolar terminally
dihydroxylated Crts, such as xanthophylls lutein and zeaxanthin, are oriented perpendicularly
to the membrane surface and parallel to lipid alkyl chains, spanning this way the lipid bilayer
like a molecular rivet (Gabrielska & Gruszecki 1996; Gruszecki & Strzalka 2005; Gruszecki,
2009) (Figure 3). When the thickness of the bilayer is less than the length of xanthophyll
molecule they are inclined with respect to the normal to the bilayer surface (Gruszecki &
Sielewiesiuk 1990; Wisniewska & Subczynski 1998). Xanthophylls may also modify the
thickness of the lipid bilayer. The thickness of the hydrophobic core of the membrane
increases when it is smaller than the distance between the opposite polar groups in the
xanthophyll molecule, and decreases in the reverse case (Sujak et al. 2002; Frank et al. 1999).
Non-polar Crts, such as β-Car, are oriented in the lipid bilayer rather randomly (van de Ven et
al. 1984) (Figure 3). By using resonance Raman and linear dichroism spectroscopies it was
shown that β-Car and lycopene may orient parallel to the membrane surface, and be deeply
immersed in the hydrophobic core of the lipid bilayer (Young & Lowe 2001; Gruszecki &
Strzalka 2005).
Obviously, the presence of polar hydroxyl groups ensures the perpendicular orientation of
xanthophyll molecules in the bilayer whereas the rigid system of conjugated C=C bonds is
exposed to van der Waals interactions with hydrophobic alkyl chains of lipids. The strong
interactions between polar Crts and lipids are responsible for the effects on membrane
properties. Indeed, xanthophylls strongly affect membrane properties while the effects of β-
Car on membranes are much less pronounced (Gabrielska & Gruszecki 1996; Kostecka-
Gugala et al. 2003; Wisniewska et al. 2006). Xanthophylls increase the order and decrease the

alkyl chain motional freedom in fluid-phase membranes (Subczynski et al. 1993). The
ordering effect of Crt cis-isomers on membranes has been investigated and in case of
zeaxanthin it has been shown to increase in the direction: all-trans<9-cis≤13-cis (Widomska
& Subczynski 2008). Also, they broaden the lipid phase transition and shift its temperature
down (Kostecka-Gugala et al. 2003; Wisniewska et al. 2006). They also reduce the oxygen
transport at all locations in the membrane (Subczynski et al. 1991) and significantly increase
the hydrophobicity of the membrane interior (Wisniewska & Subczynski 1998).
Figure 3. The schematic presentation of orientation of different carotenoids in the hydrophobic core of the
lipid membranes (Gruszecki & Strzalka 2005).
Relation between Crts Location in the Membrane and Their Antioxidant

Properties
The effectiveness of Crts as membrane antioxidants has often been analyzed in the
context of their structure and position in membranes. Woodall et al., (1997a) suggested that
the presence of terminal polar hydroxyl groups in Crt molecules affects their antioxidant
properties in membrane. Although dipolar zeaxanthin and nonpolar -carotene show similar
antioxidant properties in organic solutions, they differ when incorporated into membranes.
Zeaxanthin was shown to react with free radicals much more effectively than -carotene,
especially when lipid peroxidation was initiated in the aqueous phase (Woodall et al., 1997a).
It was concluded that the location of β-Car within the hydrophobic core of the membrane
prevents its reaction with free radicals at the membrane-water interface. Moreover, in case of
nonpolar lycopene, the authors observed its prooxidant activity. However, other researchers
suggested that different location of both polar and non-polar Crts in the membrane may lead
to their more potent antioxidant action: xanthophylls scavenge free radicals by their polar
groups at the membrane-water interface, while non-polar carotenes are more efficient towards
free radicals generated within the hydrophobic core of membranes (Matsushita et al. 2000;
Lim et al. 1992). This may explain a synergistic effect observed in a joint antioxidant action
of different Crts (El-Agamey et al. 2004; Stahl et al. 1998).
Relation between Crts Effects on Membranes and Their Antioxidant Activity

It has been hypothesized that the transmembrane orientation of polar Crts may enhance
their antioxidant properties also indirectly, by modifying the structure and dynamics of the
membrane so that it becomes less prone to the oxidative damage (Subczynski et al. 2010). As
discussed above, xanthophylls rigidify and stabilize the lipid membrane, thus slowing down

the diffusion. They raise the membrane hydrophobic barrier for polar molecules, free radicals
and ions which could otherwise initiate the lipid peroxidation. Finally, one of the most
important consequences of Crts incorporation into the membrane is a decrease in the amount
of oxygen penetrating the lipid bilayer, as demonstrated by monitoring the diffusion of
oxidized products (Subczynski et al., 1991; Frank et al. 1999). McNulty et al. (2007) have
shown that the ordering effect of Crts on the lipid membrane is accompanied by a strong
antioxidant action, as seen for instance, with the dipolar xanthophyll astaxanthin. On the
contrary, nonpolar Crts like -Car or lycopene disorder the membranes and act as
prooxidants. Crt cyclic endoperoxides, which are the products of chemical quenching of 1O2,
are more polar than their parent molecules (Fiedor et al. 2005). It has been shown for example
that -Car endoperoxide is more sensitive to oxidation in liposomes than -Car itself (Stratton
& Liebler 1997). However, an indirect antioxidant action of Crts via their alteration of
membrane properties requires a high concentration of Crts in the membrane (McNulty et al.
2007).
Therefore, the conclusion can be drawn that the structure of a Crt molecule is crucial for
the interaction of these pigments with lipid membranes. This interaction, in turn, may affect
Crt antioxidant activity (Subczynski et al. 2010). On the other hand, membrane itself – its
composition, structure and lateral organization – affects the organization of Crt in the lipid
bilayer, including orientation (trans-membrane vs. parallel) and localization (distribution
between membrane domains). It was recently shown that xanthophylls are selectively
accumulated in membrane domains that contain unsaturated phospholipids, and thus are
located in the most vulnerable regions of the membrane (Subczynski et al. 2010; Wisniewska
& Subczynski 2006a; Wisniewska & Subczynski, 2006b). Non-polar carotenoids are oriented
in the lipid bilayer rather randomly, and are distributed between membrane domains more
evenly (Subczynski et al. 2010).
1.3.3. Antioxidant/Pro-Oxidant Activity of Carotenoids Ex Vivo

Antioxidant activity of Crts has been intensively investigated ex vivo, too. The majority
of studies have used either low-density lipoproteins (LDL) or microsomal fractions from
various tissues. Crts are added directly to LDL or introduced into LDL by oral ingestion of
supplements from fruits and vegetables (Krinsky & Johnson 2005). Isolated lymphocytes or
neutrophils have also been investigated with the same goal. β-Car has shown anti- as well as
pro-oxidant activity in such ex vivo systems (Carpenter et al. 1997; Romanchik et al. 1997;
Dugas et al. 1998; Panasenko et al. 2000; Bowen and Omaye 1998; Krinsky & Johnson 2005;
Krinsky 2001). It emerges from these studies that Crts express antioxidant activity in isolated
LDL only in the presence of other antioxidants (vitamin E, flavonoids, polyphenols, phenolic
acids, etc.). The results obtained by feeding volunteers with carotenoids, fruits and vegetables
are also contradictory regarding isolated LDL oxidation (Lin et al. 1998; Agarwal & Rao
1998; Krinsky 2001).
Rat liver microsomes are another very common used ex vivo model, though the exact
location of Crt molecules in the microsomes fraction may be an issue (Palozza & Krinsky
1992). The results are very similar regarding the effect of oxygen partial pressure. β-Car is an
effective antioxidant at 15 mm Hg, but a pro-oxidant at 150 mm Hg, when LP was initiated
by lipid soluble azo-initiator and the TBA-MDA has been used (Zhang & Omaye 2001).
Immunological cells, human neutrophils, mouse macrophage RAW 264.7, human

Promyelocytic HL-60 and human lymphoid cells have been also used as models to study Crts
antioxidant activity toward LP. Inhibition of DNA oxidation by carotenoids is also common
model used for this propose. Addition of phorbol 12-myristate 13-acetate to monocytes
results in the release of ROS, and when these activated monocytes are incubated with
lymphocytes, DNA can be damaged. The used Crts (β-Car, lycopene and lutein) have shown
inconsistent results (Azqueta & Collins 2012; Krinsky & Johnson 2005).
1.4. In Vivo Antioxidant Studies and the Related Health Effects
Since the concentration of oxygen in human body varies from organ to organ, more and
more investigations deal with in vivo studies of potential Crt pro-oxidative effects in living
tissues. Pressure of oxygen in the alveoli of the lungs is 100 mm Hg, 40 mm Hg in the vein,
5-15 mm Hg in tissue. It has been proved that cancerous tissues have a higher pO2 than
normal tissues. Different concentration of oxygen in various organs leads to variations in Crt
antioxidant activity in certain parts of the body. However, it must be noted, that the
concentration of oxygen in the body is significantly lower than in the investigated artificial
systems where the pro-oxidant functions of Crts was observed (150 - 760 mm Hg) (Young &
Lowe 2001; Burton & Ingold 1984).
High Crts concentration leads to a large concentration of their oxidation products in the
system (Young & Lowe 2001). For example, the lungs of smokers who are continuously
exposed to oxidative stress have a high concentration of ROS which in combination with a
higher concentration of Crts leads to the formation of Crt oxidation products, potential pro-
oxidants (eqs. 8-13). Furthermore, there is an evidence that protective effect of β-Car and
lycopene towards lipid peroxidation and DNA damage in adenocarcinoma cells decreases
with increasing concentration of carotenes (Siems et al. 2005; Palozza 2005). The latter one
leads to increased permeability of cell membranes, permitting an easier penetration of ROS in
the cell aqueous phase and the consequent cell destruction. At higher concentrations Crts
undergo aggregation and the aggregates increase the membranes fluidity and permeability
leading to facilitation of Crts pro-oxidant action.
In humans and animals, Crts (mainly β-Car and lycopene) besides direct action also act
indirectly, by synergistically acting with other antioxidants in the body. For example,
vitamins from group A are produced by oxidative metabolism of β-Car (in most cases), which
is another form of Crts protection in vivo (Tapiero et al. 2004).
Direct antioxidant activity. It is considered that oxidative stress is the most common
cause of lipid peroxidation in vivo which leads to a number of chronic diseases. ROS are
formed through a series of metabolic processes and/or various external stresses which can
lead to heart disease, osteoporosis, cancer, etc. Total antioxidant capacity of plasma depends
on relative concentrations of its antioxidant components and their synergism. The presence of
over twenty Crts in human plasma has been confirmed, with lycopene in the highest
concentration (30% of the total Crts content). Beside lycopene, β-Car, zeaxanthin,
cryptoxanthin and α-carotene are also present and all together belong to a group of highly
efficient singlet oxygen deactivators (Tapiero et al. 2004; Palozza & Krinsky 1992). It has
also been shown that Crts act as antioxidants in the liver scavenging ROS, which have been
produced by misuse of alcohol. The most common targets are four double bonds of
arachidonic acid, an important constituent of phospholipids in liver cell membranes.

Lycopene has shown the highest activity in these processes (Tapiero et al. 2004). Heart
disease is the most common cause of death in the modern world. There is an agreement that
the oxidation-modified lipoproteins are the main causes of atherosclerosis. Lipoproteins are
mainly damaged by free radical action, and one of the most common promoters of free
radicals is a cigarette smoke. It has been shown that smokers have significantly lower
concentrations of β-Car in the blood. Large number of studies have confirmed lycopene and
β-Car as the first line of protection against oxidation of lipoproteins, and therefore against
atherosclerosis and myocardial infarction, as well (Tapiero et al. 2004; Palozza & Krinsky
1992). The in vivo studies have shown that Crts prevent the formation and development of
skin, prostate, bladder, digestive and other kinds of cancers; lycopene shows the highest
activity again, especially when it comes to bladder cancer (Matos et al. 2000; Tapiero et al.
2004). In addition, Crts prevent the loss of immunity in people who are infected with HIV.
The Crts protective functions increase in the presence of vitamin E, this effect is particularly
expressed for lycopene in the case of prostate cancer (Tapiero et al. 2004).
Indirect antioxidant activity. As noted above, the interaction of carotenoids with free
radicals may lead to the generation of potentially dangerous Crt-radicals in vivo (they can
oxidase tyrosine and cysteine). These reactions, for example, can modify the proteins and
affect its function. However, Crt radicals are neutralized by action of vitamins (C and E),
which actually represents the mechanism of synergistic action of Crts and vitamins: the
produced ascorbic acid- and tocopherol-radicals are then regenerated by enzymes and
vitamins, erasing a potential risk for the organism (Stahl & Sies 2003; Palozza & Krinsky
1992; Wrona 2003; Montenegro et al. 2002; Burke et al. 2001; Liebler et al. 1997;
Handelman 2001). On the other side, tocopheroxyl radicals, formed by reaction of vitamin E
with other radicals, can be regenerated by Crts (Krinsky & Yeum 2003; Haila 1999). The
reaction of Crt-radicals with vitamin C is specific because vitamin C is hydrophilic, and
located in the aqueous phase surrounding the lipoprotein biomembranes. The Crts orientation
inside the membrane has a major impact on its antioxidant efficiency (Young & Lowe 2001;
Frank et al. 1999). Some authors consider the presence of tocopherols as a crucial factor for
photoprotective effect of Crts in skin and eyes (El-Agamey et al. 2004).
Food rich in Crts has most often been used to elevate Crt levels in organism. Collins has
demonstrated that oxidative damage to lymphocytes correlates inversely with plasma Crt
concentrations, and the extent of DNA damage is susceptible to reduction by Crt-containing
foods (Collins 2001). The author concluded that Crts generally act as antioxidants in vivo, but
since food contains many other additional compounds the relation between Crts intake and
prevention of DNA damage must be considered as associative.
Various animals have been used in order to evaluate Crt antioxidant effects in vivo
despite the problem that most of experimental animals are very poor Crts absorbers (Krinsky
& Johnson 2005). Feeding hamsters and rats with lycopene leads to prevention from a
carcinogen-induced oxidative damage (Bhuvaneswari et al. 2001a; Bhuvaneswari et al.
2001b). Humans have an almost unlimited capacity to absorb dietary Crts, still, a caution with
in vivo results interpretation has been suggested (Rice-Evans et al. 1997). The use of
appropriate biomarkers may be a key issue to determine whether dietary Crts affect the
oxidative stress status in humans. Thiobarbituric acid-reactive substances (TBARS), such as
MDA, were considered for a long time as a valid measure of lipid peroxidation, but it is now
accepted that TBA is a somewhat non-specific biomarker. However, numerous investigators
have demonstrated that there is a positive correlation between Crts-sufficient diets and

decrease of MDA levels in plasma (Dixon et al. 1994; Dixon et al. 1998; Meraji et al. 1997).
Damage to DNA has also been used as a biomarker of oxidative damage. The most common
measured product to evaluate DNA damage has been 8-hydroxy-2‘-deoxyguanosine
(8OHdG) (Halliwell 2000). The number of strand breaks is another marker of DNA damage,
observed in lymphocytic DNA, however, an increased fruit and vegetable intake can decrease
that biomarker (Krinsky & Johnson 2005).
1.4.1. Specific Health Effects of Carotenoids
Skin Effects
The appearance of erythema, i.e. sunburn (erythema solare) is the first effect after the
exposure of skin to ultraviolet radiation. Besides burns, ultraviolet radiation induces oxidative
processes that lead to skin damage and premature skin aging. Photoinduced ROS damage
DNA and enzymes in the skin and destroy collagen which leads to the appearance of deep
grooves and bumps on the skin (Wlaschek et al. 2001; Biesalski et al. 2003). A prolonged
exposure to sunlight causes sunburn, pigmentation, hyperplasia, immunosuppression, DNA
damage (De Gruijl et al. 2001; Pfeifer et al. 2005), melanoma and non-melanoma skin cancer
(Young 1996; Ouhtit & Ananthaswamy 2001). Solar radiation, particularly its UV part, has
effects on cytochrome P450, glutathione, superoxide dismutase, glutathione peroxidase,
catalase, glutathione-S-transferase and ceruloplasmin (Afaq & Mukhtar 2001). There is an
evidence that each of the three main types of skin cancer, i.e. basal cell carcinoma (BCC),
squamous cell carcinoma (SCC) and melanoma is caused by excessive to a natural sunlight
exposure (Armstrong & Kricker 2001). UV-B radiation (280-320 nm) exerts highly
mutagenic and carcinogenic effects in animal experiments compared to UV-A (320-400 nm).
DNA nucleotide bases directly absorb UV-B photons, and although DNA is week UV-A
absorber, could be damaged by photosensitization reaction initiated through absorption of
UV-A by an unidentified chromophore (Ichihashi et al. 2003).
The in vitro and in vivo studies showed that β-Car acts preventively against photo-
oxidative damage and skin burns. β-Car reduces lipid peroxidation, and quench singlet
oxygen-mediated photochemical reactions in rodents skin. In cultured skin cells it decreases
photoinactivation of catalase and superoxide dismutase, as well as protein cross linking. In rat
kidney fibroblasts it diminished UVA-induced catalase deactivation and lipid peroxidation
(Biesalski & Obermueller-Jevic 2001). In the skin, β-Car is mainly located in the epidermis,
where the largest amount of UV-radiation is being absorbed; skin exposure to the sunlight
reduces its concentration. When the skin is exposed to UV radiation only, the concentration of
β-Car remains mostly unchanged, while the concentration of lycopene significantly decreases.
A higher Crts concentration in the skin means less risk of skin burns. However, a ―safe range‖
of intracellular β-Car levels has not been determined yet, and it is not known whether β-Car
accumulation in the skin may have harmful side effects (Biesalski & Obermueller-Jevic
2001).
Clinical data available on β-Car effects on skin cancer show certain contradictions. Some
studies and analysis showed that β-Car supplementation, 50 mg/on alternate days, during 12
years, has no effect on development of a nonmelanoma skin cancer and other malignant
neoplasms. It is difficult to determine any concrete relationship between plasma β-Car levels
and risk of nonmelanoma skin cancer (Frieling et al. 2000). On the other hand, the dietary β-

Car accumulated in mouse skin acts as a protective agent against UV-A induced oxidative
damage (Bando et al. 2004). An LC/MS analysis of mouse skin lipids, confirmed the
formation of β-Car 5,8-endoperoxide in vitro, a specific marker for the singlet oxygen
chemical reaction, suggesting that β-Car potentially acts as a singlet oxygen quencher in the
skin (Bando et al. 2004).
Considering the effects of increased UV fraction of solar radiation on Earth, it is clear
that intensive Crts evaluation as possible photo-protectors is certainly justified. Most
commonly, β-Car (Biesalski & Obermueller-Jevic 2001; Bando et al. 2004) and lycopene
(Stahl & Sies 2003; Tapiero et al. 2004) have been investigated in this context but these
studies left many opened questions, a wide field of future research on the role of skin Crts.
The oral supplementation of Crts may lead to an imbalance in the cutaneous antioxidant
network, and thus exert possibly harmful effects on skin exposed to sunlight.
Age-Related Macular Degeneration

Carotenoids protect different tissues against oxidative stress. The tissue particularly
susceptible to oxidative stress and LP is the eye retina, because it is abundantly illuminated,
has high respiratory demands for oxygen and a large content of polyunsaturated lipids,
including docosahexaenoyl fatty acid (DHA) with six double bonds (Beatty et al. 2000). The
damage done to the retina by oxygen and light may result in the onset of age-related macular
degeneration (AMD), the leading cause of irreversible blindness in humans in developed
countries (Hollyfield et al. 2008).
A growing number of studies indicate that two dipolar, terminally dihydroxylated Crts –
xanthophylls lutein and zeaxanhin, which selectively accumulate at extremely high
concentrations in the central part of the eye retina, macula lutea, of primates (Bone et al.
1985; Bone et al. 1988; Landrum et al. 1999), can impede the onset of AMD (Gale et al.
2003; Mares-Perlman et al. 2002; Mares 2004; Cho et al. 2004; Mares-Perlman et al. 2001;
Robman et al. 2007; Obana et al. 2008). Lutein and zeaxanthin are not uniformly distributed
within the macula. Zeaxanthin prevails in the fovea, the most central part of the macula where
only cones are located, whereas lutein dominates in the parafoveal region (Bone et al. 1988;
Landrum et al. 1999). Interestingly, other dietary Crts, such as nonpolar lycopene or β-Car are
not found in the retina. Different mechanisms are considered to explain a putative protective
role of xanthophylls in the eye retina, among them are the modulation of oxidative stress and
redox balance, the interactions with key molecules in signal transduction cascades, the
influence on gene expression (e.g. connexin or inflammation-related genes) or interactions
with membrane-bound proteins and lipids (San Giovanni & Neuringer 2012). Most studies
though focus on their antioxidant function. Macular xanthophylls are thought to combat light-
induced damage mediated by ROS indirectly, by absorbing the most damaging high energy
photons prior to the formation of ROS (Kirschfeld 1982; Wooten & Hammond 2002), and
directly, by chemical and physical quenching of ROS once they are formed (Krinsky 1989;
Edge et al. 1997). Also, xanthophylls efficiently quench excited triplet states of
photosensitizers. Lutein and zeaxanthin (and a zeaxanthin isomer – meso-zeaxanthin, a
metabolite of lutein) are accumulated in nerve fibers (so called Henle‘s fiber layer of
photoreceptor axons) and membranes of photoreceptor outer segments (POS). It is not clear
though whether xanthophylls are freely embedded in the lipid part of membranes, or bound
by special proteins. The published reports suggest that lutein and zeaxanthin are directly

incorporated in the lipid part of retinal cell membranes (Bone & Landrum 1984; Bone et al.
1992). According to the other hypothesis, macular xanthophylls are bound by membrane-
associated xanthophyll-binding proteins (Bhosale et al. 2004; Bhosale & Bernstein 2007;
Loane et al. 2008; Li et al. 2011; Vachali et al. 2012).
In both cases macular xanthophylls can show their antioxidant properties. If located in the
lipid part of membranes, they can directly protect unsaturated lipids against peroxidation in
situ. It has been shown that lutein and zeaxanthin have strong effects on various properties of
membranes, whereas nonpolar β-Car has practically no effect (Wisniewska & Subczynski
1998; Wisniewska et al. 2006; see also 1.3.2). As mentioned previously (1.3.2), the effect on
membranes can be directly and indirectly related to the enhanced antioxidant properties of
these two xanthophylls compared to carotenes. It has also been hypothesized that due to their
transmembrane location in the lipid bilayer (see Figure 3), macular xanthophylls are better
soluble in membranes compared to other dietary Crts (Subczynski et al. 2010). Their
solubility thresholds in fluid-phase model membranes, i.e. Crts concentration up to which
they exist mainly as monomers, are in the range of 2 to10 mol% (Lazrak et al. 1987;
Gabrielska & Gruszecki, 1996; Sujak et al. 2000, 2002). The nonpolar β-Car already
aggregates at concentrations as low as 0.5 to 1 mol% (Kennedy & Liebler 1992; Woodall et
al. 1995). Hence, it has been suggested that good membrane solubility of lutein and
zeaxanthin is a feature that distinguishes these two xanthophylls from other Crts found in the
blood plasma, and accounts for their selective accumulation in the membranes of the retina
(Subczynski et al. 2010). Moreover, using models of POS membranes, which are known to
have a domain structure (Seno et al. 2001; Boesze-Battaglia et al. 2002; Nair et al. 2002;
Martin et al. 2005), it has been found out that lutein and zeaxanthin are selectively
accumulated in the domains enriched in unsaturated phospholipids (Wisniewska &
Subczynski 2006a; 2006b). The location of xanthophylls in the membrane and additionally in
its most vulnerable regions (prone to LP) seems to be advantageous for their antioxidant
action. Indeed, in the experiments on LP involving ―homogeneous‖ unsaturated membranes
and domain-structured ones it has been shown that the selective accumulation of macular
xanthophylls in domains rich in unsaturated lipids significantly enhances their effectiveness
as antioxidants (Wisniewska-Becker et al. 2012). Therefore, it was suggested that the
selective accumulation of macular xanthophylls in the most vulnerable regions of
photoreceptor membranes may enhance their ability to protect these cells against oxidative
damage and prevent the development of age-related macular diseases, including AMD.
Although the results of experiments on model membranes confirm the location of
macular xanthophylls within the lipid part of the membrane, it cannot be excluded that
protein-Crt interactions are comparably significant in the selective accumulation of lutein and
zeaxanthin in the protein part of retinal cells. The Bernstein‘s group has for years been
searching for specific xanthophyll binding proteins, which could be involved in the uptake,
stabilization, and also antioxidant functions of lutein and zeaxanthin. First, Pi isoform of
glutathione S-transferase (GSTP1) has been identified as a zeaxanthin-binding protein in
human macula, with a high affinity for both dietary zeaxanthin and non-dietary meso-
zeaxanthin (Bhosale et al. 2004). Additionally, it has been shown that GSTP1 acts
synergistically to zeaxanthin in protecting model membranes against oxidative damage
(Bhosale & Bernstein 2005). Few years later the same group identified one of the
steroidogenic acute regulatory domain proteins (StARD3) as a lutein-binding protein (Li et al.
2011). Interactions of macular xanthophylls and their binding proteins have been investigated

by surface plasmon resonance (SPR) and the pigment binding constants have been determined
(Vachali et al. 2012). Based on high affinity of GSTP1 to zeaxanthin and StARD3 to lutein, it
was hypothesized that xanthophyll binding proteins can be responsible for the capture of
these xanthophylls by the retina and their selective accumulation in this tissue (Loane et al.
2008; Vachali et al. 2012).
Cancer Diseases
Carotenoids have to be dissolved in organic solvent, or in different ―carriers‖ like
lipoproteins, micelles, cyclodextrin-formulations, liposomes and water dispersible
formulations before its application to cellular systems providing penetration into it. Crts are
able to alter growth patterns and inhibit growth in tumor cell lines, but these investigations
have been done in vitro, with possible implication in vivo. β-Car, crocetin, lycopene,
canthaxanthin fucoxanthin, α-carotene, neoxanthin, phytofluene, δ-carotene, phytoene, β-
cryptoxanthin, zeaxanthin, lutein and some others Crts have been investigated in different
laboratories on different types of cancer-cells (Krinsky & Johnson, 2005; Astrog, 1997;
Naves & Moreno 1998; Elliott 2005; Tapiero et al. 2004; Hughes 2001). It was found that α-
carotene is the most active component of palm oil (Krinsky & Johnson, 2005). Lycopene has
expressed a strong inhibitory action against endometrial, mammary and lung human cancer
cells in concentration of 1-2 µM (Levy et al. 1995). β-Car, neoxanthin, fucoxanthin and
lycopene have also proved to be very effective inhibitors of prostate cancer cells growth
(Kotake-Nara et al. 2001). Crts act as modulators of apoptotic signaling in cancer cells acting
as potent antiblastic agents (Palozza et al. 2004). It should be noted that many results dealing
with the Crts pro-apoptotic effects have been demonstrated only in vitro. Furthermore, it is
not clear whether these molecules modulate the apoptotic signaling as the intact molecule or
through the products of their oxidation (i.e. retinoids) (Palozza et al. 2004; Palozza et al.
2001a; Palozza et al. 2001b).
There are several mechanisms by which Crts can function in cancer prevention: in form
of provitamin A by effecting cellular differentiation and proliferation; through antioxidant
function, by scavenging free-radicals and preventing damage of cellular DNA and other
molecules; by immunomodulatory effects and cell-cell communication which restrict clonal
expansion of initiated cells (Serpeloni et al. 2012; Rao & Rao, 2007; Matos et al. 2000;
Krinsky & Johnson 2005). Azqueta and Collins (2012) have found a clear distinction between
effects of vitamin A and provitamin A Crts (β-Car, α-carotene and β-cryptoxanthin), and
effects of non-vitamin A Crts (lycopene, lutein, astaxanthin and zeaxanthin), in cultured cells,
in experimental animals, and in humans based on reports from the last five years. The group
of non-vitamin A Crts is almost invariably assigned protection against DNA damage induced
by endogenous or exogenous agents. The provitamin A Crts show a more diversified
spectrum of effects, sometimes protecting and sometimes enhancing DNA damage. The
tendency to make an extended damage is seen mainly at high concentrations of Crts, which
may express pro-oxidant actions.
In a recently published review article Tang (2012) called chemopreventive effects of Crts
―the silver bullet for cancer prevention‖. Crts express chemopreventive capability by
interrupting several stages of cancer formation including initiation, promotion, progression
and metastasis, and proposed molecular mechanism of action through the modulation of cell-
signaling pathways and gene expression. The effects of β-Car, lycopene, fucoxanthin, β-

cryptoxanthin, astaxanthin, lutein and zeaxanthin have been studied in this review, and many
evidences suggest that Crts play important roles in the prevention of tumor growth.
Coronary Diseases
Antioxidative role of Crts is probably the main reason of their protective effect against
coronary vascular disease. Oxidation of LDL is one of the main reasons of coronary vascular
disease development via atherosclerotic lesion. The fact that LDL is major transporter of β-
Car and lycopene in the circulation, and having in mind that these Crts can scavenge peroxyl
radicals and quench singlet oxygen favors this hypothesis. There are also observational
epidemiological studies which confirm that foods reach in Crts and vitamins are associated
with reduced risk of cardiovascular disease (Mayne, 1996). However, Crts effects on
coronary artery disease are a subject of disagreement. In some case-control studies a clear
relationship between serum Crts content and risk of myocardial infarction was not found. On
the other case, an inverse association between serum β-Car content and the risk of cardio-
vascular diseases was noticed (Street et al. 1994; van de Vijver et al. 1997). Dietary lycopene
was not strongly associated with risk of cardiovascular disease in women, but a possible
inverse association was noted for higher levels of tomato-based products intake with
cardiovascular disease. A higher plasma lycopene concentration is associated with a lower
risk of cardiovascular disease in women (Krinsky & Johnson 2005). Plasma levels of lutein,
lycopene, α-carotene, β-Car were significantly lower in patients with an acute ischemic
stroke, probably as a result of increased oxidative stress (Krinsky & Johnson 2005; Lidebjer
et al. 2007).
Conclusion
It is clear that carotenoids due to their antioxidant activity are able to inhibit oxidation of
vital biological molecules such as lipids, LDL or even DNA. Due to the fact that oxidative
damage caused by ROS is involved in the pathogenesis of various human chronic diseases
Crts seem to have beneficial effects on human health. Their positive effects are various; from
the physical and chemical mechanisms involved in quenching of ROS to light absorption and
modifications of membrane properties. However, depending on the particular circumstances,
e.g. in the oxygen-rich molecular environment, from being a ―part of the solution‖ (anti-
oxidant activity), Crts may turn to be a ―part of the problem‖ (pro-oxidant activity). Certainly,
the world of Crts is fascinating and their understanding requires further investigations.
Acknowledgments
This research is a part of the project TR 34012 and the project OI 172044 financially
supported by the Ministry of Education and Science of the Republic of Serbia. D.C. is a
recipient of the Queen Jadwiga Fellowship from the Jagiellonian University (2010-2011).
Also the financial support from the Foundation for Polish Science is acknowledged (grant
TEAM/2010-5/3 to LF).

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Chapter VI
Carotenoids in Herbal Medicines
Mamta Kshipra Misra1, Kshipra Misra1, Satinder Kaur Brar2

and Mausam Verma3
1
Defence Institute of Physiology and Allied Sciences,
Lucknow Road, Timarpur, Delhi, India
2
Institut National de la Recherche Scientifique, Centre - Eau Terre Environnement,
490, rue de la Couronne, Québec, Québec, Canada
3
CO2 Solutions Inc., rue Jean-Perrin, Québec, Québec, Canada
Abstract
Medicinal herbs are gaining importance in modern medicines for the prevention and
cure of various chronic and pathogenic diseases. Almost 70% of the currently practiced
medicines are derived from the natural sources. Different parts of the plants are known to
be rich source of various phytochemicals responsible for mitigation of the physiological
damage caused to different organs of the body during exposure to different types of
stresses.
Carotenoids are one such class of bioactive compounds found in most of the fruits
and vegetables and are colorful fat soluble pigments. They mainly consist of carotenes (α,
β, δ and γ carotene) lycopene and xanthophylls. Lutein, astaxanthin, violaxanthin,
zeaxanthin and capsanthin are examples of xanthophylls which is a class of oxygenated
carotenoids derived from the natural sources. Reddish orange color foods, such as carrots,
apricots, mangoes, squash, papaya and sweet potatoes are known to possess significant
amount of β-carotene, α-carotene, and β-cryptoxanthin. Green vegetables, such as
spinach, kale and collard green contain good amount of β-carotene while lycopene is
found in tomatoes, guava and pink grapefruit. Other sources of carotenoids include
salmon, shellfish, milk and egg yolk etc.
Carotenoids being oil soluble compounds help in preventing the lipid peroxidation in
the body which is associated with atherosclerosis and cardiovascular disease and also
protect the cell membrane damage. Besides, carotenoids are also important as dietary
precursors of vitamin A, which help in maintaining the good vision. Vitamin A and β-

Corresponding author: Email: [email protected].

128 Mamta Kshipra Misra, Kshipra Misra, Satinder Kaur Brar et al.
carotene are also believed to enhance the immune system functioning. Research also
indicates that diet with low level of carotenoids causes oxidative damage from free
radicals and increases the risk of chronic diseases. Hence, increased consumption of fruits
and vegetables as well as supplementation of the carotenoids may help in combating
adverse health effects on the body. This chapter deals with the various aspects of the
carotenoids such as types, sources supplementation and bio-efficacy studies.
Keywords: Medicinal herbs, carotenoids, dietary supplements, health effects
1. Introduction
Carotenoids is a class of phytochemicals with good antioxidant properties and are found
in plants, most of the fruits and vegetables etc. and are responsible for giving colors.
Carotenoids, the oil soluble pigments, occur naturally in plants, in various microorganisms
but cannot be synthesized in animals. Hence, human can fulfill their requirement only by
supplementation. There are over 600 carotenoids found in nature but about 40 of them
constitute human diet and 20 out of these 40 have been recognized in human blood and
tissues [1]. All the carotenoids are tetraterpenoids, derived from 8 isoprene molecules having
a chain of 40 carbon atoms. These are broadly categorized in two different groups i.e.
carotenes and xanthophylls.
Carotene is the class of carotenoids which does not have any oxygen molecule and β-
carotene and lycopene are the two major carotenes belong to this group of carotenoids
because of their biological activities. While, zeaxanthin, lutein, astaxanthin etc. are the
oxygenated carotenoids and hence categorized as xanthophylls. These are mainly found in the
colored fruits, flowers, vegetables and also in various marine organisms.
Many epidemiological studies in literature have clearly shown the regular consumption of
carotenoids in the form of herbal medicines/supplements help in reducing the impact of
various chronic diseases on human health. One of the most important biological activities
known for carotenoids is their ability to convert some of the carotenoids into retinol or
vitamin A. Carotenoids also act as antioxidant by protecting the cellular component of the
body against oxidative damage. They also act as good immunomodulator and are helpful in
cell communication essential for normal growth of the cells [2-3]. A low dietary intake of
carotenoids can cause various chronic disorders such as cardiovascular disease and different
types of cancer etc. Since humans and animals are mostly incapable of naturally synthesizing
carotenoids, it must be obtained either through the diet or by supplementation in the form of
herbal formulation for the development of strong immune system in order to protect from
various chronic and degenerative diseases.
This chapter will mainly focus on the classification of carotenoids, their natural sources,
characterization, bioefficacy and their role as herbal medicine.
2. Classification and Sources of Carotenoids

Carotenoids are colored pigments found in plants, fruits, vegetables and are also
synthesized by certain microorganisms. Mainly, they are categorized into two types, carotene

Carotenoids in Herbal Medicines 129
and xanthophylls based on the presence or absence of oxygen molecule in their chemical
structure. Carotenoids are isoprenoid compounds biosynthesized by tail-to-tail linkage of two
C20 geranylgeranyl diphiosphate molecules that results in parent C40 carbon skeleton system.
This specific skeleton of C40 is the basis of all individual variations [4]. The most remarkable
and distinct feature of carotenoid structure is the long chain of alternate single and double
bonds that forms the central part of the molecule. It constitutes a conjugated system in which
the п-electrons are effectively delocalized over the entire length of the polyene chain. This
criterion is responsible for the distinct molecular shape, chemical activity and light absorbing
properties [5]. Moreover, the activity of carotenoids depends upon the size of ring substituent.
The basic chemical structure and schematic representation of the classification of carotenoids
are shown in Figure 1 and 2 respectively.
Figure 1. Basic chemical structure of carotenoid reproduced form M. Agarwal, et al.
Figure 2. Schematic representation of classification of carotenoids.
2.1. Carotenes
Carotenes are a class of carotenoids which is devoid of oxygen. Typically they are pure
hydrocarbons containing carbon and hydrogen. α, β, γ, δ-carotene and lycopene etc are the
major carotenoids covered under this group.

Apricots, cantaloupe, carrots, pumpkins, sweet potato, green beans, broccoli, brussel
sprouts, cabbage, kale, kiwi, lettuce, peas and spinach are the rich source of α and β-carotenes
[6]. In microorganism, Dunaliella Salina (microalgae) is also known as a major source of β-
carotene [7] along with some secondary carotenoids such as astaxanthin and zeaxanthin [8].
Lycopene with highest antioxidant capacity amongst the carotenoids is responsible for its
characteristic bright red color found predominantly in tomatoes, watermelons, pink grape
fruits, papaya, etc. [9] and in human the highest lycopene level is found deposited in various
glands and organs such as prostate and adrenal glands, testes, breast, liver, lungs and also in
blood and skin of the human body [1]. Shellfish, milk and egg yolks also provide good source
of carotenoids.
2.2. Xanthophylls
Xanthophylls such as Lutein, zeaxanthin, astaxanthin, violaxanthin, capsanthin,

cryptoxanthin etc. are the oxygenated carotenoids, responsible for the yellow color of leaves
and other related parts of the plants. The detail description of each one is given below.
Lutein and zeaxanthin are naturally occurring antioxidants found in various yellowish
orange colored fruits and flowers. Marigold is one of the major sources of lutein and
zeaxanthin in nature [10]. They are also present in high concentration in egg yolks [11].
Green vegetables, especially spinach, kale, and collard greens are the best sources of lutein
along with β-carotene.
Astaxanthin is quite common in nature and is found especially in marine environment of
various crustaceans and most of the fish and algae. Pink colored flesh of the salmon fish is a
prominent example of astaxanthin. Marine animals obtain astaxanthin in their diet from
zooplankton. Most concentrated well established natural source of astaxanthin is green algae
Haematococcus pluvialis (H. pluvialis) that provides approximate 10,000-40,000 ppm (parts
per million) (mg/kg) of astaxanthin and is also produced commercially from this microalgae
or crustaceans shells. Apart from astaxanthin, H. pluvialis is also a good source of β-carotene,
lutein and canthazanthin [12] and is marketed as a powerful antioxidant currently.
The orange-colored fruits and vegetables including carrots, apricots, mangoes, papaya
and sweet potatoes contain significant amounts of β-cryptoxanthin along with α and β-
carotenes.
Violaxanthin is a natural xanthophyll pigment found in a variety of plants with orange
color pigment. Nannochloropsis (Eustigmatophyceae) is a source of commercially available
pigment including violaxanthin [13]. It is biosynthesized from zeaxanthin by epoxidation
(epoxide is a cyclic ether). As a food additive it is used as a food coloring agent. Flowers and
leaves of Tropaeolum majus L. is also rich sources of violaxanthin [14].
Canthaxanthin is widely distributed in nature. It was first isolated in edible mushrooms. It
has also been found in many green algae, crustaceans and fish such as carp, seabream and
trush wrasse. In the egg, canthaxanthin is transferred from yolk to the developing embryo
where it might help to protect the developing chick against oxidative damage, particularly
during the sensitive periods of hatching and early post hatch life.
Capsanthin is the main carotenoid found in paprika (Capsicum annuum). The oil soluble
extract of Capsicum Annum Linn is also known as paprika oleoresin and as paprika extract
which is primarily used as a colouring and flavouring agent in various food products. It is

composed of capsaicin, the main flavouring compound giving pungency in higher

concentrations, and capsanthin is the main coloring compounds among other carotenoids [15].
The sources of various carotenoids to be supplemented in different forms (medicines/extracts)
for health benefits are summarized in Table 1.
Table 1. Dietary Sources for carotenoid supplementation
Carotenoids Name source References

α-Carotene Carrot, peppers,buriti, red palm oil [16-17]
Carotenes β-Carotene Apricots, Broccoli, Carrots, mango, [16-17]
leaves, red pepper, oregano, spinach
Dunaliella Salina (algae) Haematococcus [6]
pluvialis (algae) etc. [12]
Lycopene Tomatoes, pink guavas, watermelons and [1, 9, 17]
pink grape fruits, papaya, apricot.
Lutein Mango, papaya, peaches, prunes, oranges, [7, 17]
Xanthophylls green beans, broccoli, brussel sprouts,
corn, [12]
Haematococcus pluvialis algae
Zeaxanthin Buriti, corn, marigold, [10, 17]
Dunaliella Salina (microalgae) [7]
Egg product etc [18]
β- Caja, pitanga, red pepper etc. [17]
Cryptoxanthin
Capsanthin Red peppers [15]
Canthaxanthin Mushrooms [15]
Haematococcus pluvialis algae [12]
Astaxanthin Marine sources such as algae, salmon and [15]
crustaceans. Lobster, Haematococcus
pluvialis, [12]
Dunaliella Salina [7]
3. Bio-Absorption Mechanism of Carotenoids

The absorption of carotenoids in the biological system is affected by various factors. For
example food processing and cooking of the food products easily breakdown the tissues and
release the carotenoids for their better absorption in the body [19-20]. Though the presence of
dietary fat and gastric pH is also found to affect the carotenoid absorption, not much literature
is available on the absorption of carotenoids other than β-carotene and lycopene. Studies
indicate that the carotenoids are first absorbed into the gastrointestinal (GIT) mucosal cells
and remain as such there as well as in the tissues till the availability of fat in the system [21].
In intestine, carotenoids absorption takes place by passive diffusion through micelles
formation resulted from dietary fat and bile acids. Subsequently these integrated micelles are
mixed with the chylomicrons and released into the lymphatic system. Later, they are
incorporated into the lipoprotein at the site of the liver and released into the blood stream. The

schematic representation of general mechanism of bio-absorption of carotenoid is given in

Figure 3.
Figure 3. Schematic representation of bio-absorption of carotenoids.
4. Estimation of Carotenoids
There are various methods available for the qualitative and quantitative analysis of
carotenoids. The analysis depends upon the type of the carotenoid and the nature of herbal
supplement (crud/extract/preparation) from which it needs to be extracted before analysis.
Various spectroscopic (UV-VIS/ FT-IR/NMR/MS) as well as chromatographic techniques
(GC/HPTLC/HPLC) are used for the determination of carotenoids in herbal products. Some
of the standard techniques used are described in the subsequent paragraphs.
4.1. Total Carotenoid Content
A total carotenoid content is determined using colorimetric technique [66]. The

lyophilized or dried carotenoid extract is dissolved in the solvent which has been used for its
extraction and the absorbance is measured at 450 nm in the UV-VIS-spectrophotometer. The
total carotenoid content is calculated according to the formula as given below [22].

Total carotenoids content (κg/g) = A × V (ml) × 10 4

A1% 1cm × sample weight (g)
where, A= absorbance, V= total volume of the extract being analyzed, W = weight of the
sample and A1% 1cm = 2592 (β-carotene extinction coefficient in solvent).
4.2. Instrumental Techniques for Identification and Quantification

of Carotenoids
Chromatographic techniques such as High Performance Thin Layer Chromatography

(HPTLC) and High Pressure Liquid Chromatography (HPLC) alone as well as in combination
with spectroscopic techniques like Mass Spectrophotometer (MS) and Nuclear Magnetic
Resonance (NMR) are being widely used for the separation and identification of different
carotenoids in herbal medicines.
S. H. Razavi could isolate and quantify some of the difficult carotenoids such as
astaxanthin, canthaxanthin, apocarotenoic ester, torularhodin and β-carotene from a
microorganism strain of Sporobolomyces ruberrimus H110 by an on-line UV-HPLC/APCI-
MS (atmospheric pressure chemical ionization) system. They developed the method to
distinguish torularhodin and canthaxanthin which have the same molar mass but a different
chemical structure [23].
High throughput Ultra Performance Convergence Chromatography (UPC2Water
cooperation, USA, has very recently (2013) developed an improved and highly sensitive
technique, high throughput Ultra Performance Convergence Chromatography (UPC2) for the
determination of carotenoids in dietary supplemented herbal capsules. Three most important
carotenoids i.e. β-carotene, lycopene and lutein were separated within two minutes. It is about
four times faster than any traditional method of separation and hence reduces the
consumption of solvent by 85% [24].
5. Role of Carotenoids as Herbal Medicines

Herbal medicines rich in carotenoids are known to be used for improving the health in
several ways. Due to their good antioxidant property, they enhance the immunity of the
system. Some of the herbal formulations of carotenoids are known for their provatamin A
activity and improving the eye vision by protecting the macula of eye against photo oxidative
damage and macular degeneration along with several other eye related disorder [25-26].
Many epidemiological studies reveal that the most significant role performed by
carotenoids is in the process of cell communication which is an important factor to regulate
essential biological functions that helps in prevention of various chronic and degenerative
diseases such as inhibition of proliferation of uncontrolled cell growth in case of tumors and
cancers [27]. They are also found to reduce the risk of cardiovascular and other related
diseases through the inhibition of oxidation of low density lipoprotein (LDL) [28]. Further,
studies have also confirmed the role of carotenoids in other important biological activities
such as modulation of gene expression along with the modulation of drug metabolizing

enzymes and defence against the carcinogens [29]. Various biological functions of
carotenoids rich herbal products are summarized in Figure 4. Some of the case studies in
subsequent paragraphs will clearly define the important role performed by carotenoid rich
herbal medicines.
Antioxidant
Activity
UV light
protection
Other
Provitamin A
physiological
activity
Gastric role
infection Carotenoids
rich herbal
medicines
Cell signaling Photo-oxidative

damage
Anti-cancer
Prevention of Macular
Immuno- chronic and degeneration
modulation Eye care
degenerative
diseases Cataract
Cardiovascular
disorder
Retinitis
Modulation of pigmentosa
xenobiotic metabolizing
enzymes
Figure 4. Protective bio-efficacy of carotenoids rich herbal formulations
6. Case Studies
6.1. Antioxidant Activity
Following examples will give a close look at the antioxidant activity behavior of
caratnoids.
In one of the studies, a comparative antioxidant capacity of various carotenoids has been
carried out and lycopene is found to exhibit maximum singlet oxygen quenching capacity
[30].
Further, a comparative study of astaxanthin with the other carotenoids confirmed that
astaxanthin was more potential towards the protection of membrane containing phospholipids
from peroxidation compared to β-carotene, zeaxanthin and canthaxanthin [31].
Another study reveals that the level of enzymatic antioxidant i.e. catalase (CAT),
superoxide dismutase (SOD) and peroxidase in mice plasma and liver increased significantly
when supplemented with microalgae biomasses (Haematococcus pluvialis, Scenedesmus
platensis or Botryococcus) rich in carotenoids such as β-carotene, lutein, canthaxanthin,

astaxanthin indicating that these carotenoids decreased the free radical mediated oxidative
stress [32].
Some of the studies have also indicated the benefits of tomato consumption as an
antioxidant in humans. A study has clearly shown that the consumption of tomato based
formulation or product containing 15 mg of lycopene daily in the presence of other
phytonutrient of tomato enhanced the lipoprotein protection from oxidative stress [33].
6.2. Provitamin A Activity
Ability to convert carotenoids into vitamin A is one of the most important functions of
carotenoids takes place in human body. This conversion of carotenoid into retinol or vitamin
A is known as provitamin A activity. Though β-Carotene is the major provitamin A
carotenoid in human however α-carotene and β-cryptoxanthin are also metabolized to vitamin
A in body. One molecule of carotene is converted into two molecules of vitamin A in the
body [34] and vitamin A is known for the sharp and good vision. Therefore, herbal
supplements rich in carotenoid especially β-carotene must be taken in diet which has 100%
provitamin A activity [35].
6.3. Eye Care
Studies reveal the protective role of lutein and zeaxanthin against photooxidative damage
as they directly absorb the blue light and near-ultraviolet light in the retina and protect the
macula which in turn prevents the degeneration of eye vision in human [25, 36-37]
S. E. Hankinson et al. conducted a comparative study between spinach (as it contains
high lutein) and carrots consumption (high in β-carotene content). Results revealed that diet
containing spinach was more effective in comparison to carrots which further suggested that
lutein is more effective and responsible for the inverse relation to cataract extraction [38].
Retinitis pigmentosa (RP) is also an eye degenerative disease which causes damage to
photorecptor and may result in blindness. A pilot study was conducted to investigate the
effect of lutein supplementation (40 mg lutein/day for 9 weeks) on RP patients. Patients were
found to improve significantly visual acuity when self tested using computer simulation [26].
These results suggest that lutein may have a protective effect against RP and participate in
defensive function for eye health.
6.4. Prevention of Chronic and Degenerative Diseases
A few examples as given below suggest that people consuming high carotenoids in their
diet have the lower risk of chronic and degenerative diseases.
6.4.1. Anti-Cancer Activities

Based on in-vivo and in-vitro experiments, carotenoids are also found to be potent
inhibitor of proliferation of various human cancers via process of proper cell communication

[39]. Lycopene has been found to possess wide spectrum of anticancer activities amongst the
other carotenoids [40-41]. In one of the studies, inhibitory effect of lycopene on mammary
and prostate cancer cell growth was observed by inhibition of cell cycle progression from
G0/G1 to the S phase, linked with decrease in cyclin D1 protein levels which is a key
regulator of cell proliferation [27].
Many other studies have also reported that lycopene decreases prostate specific antigen
(PSA) level as well as the growth of prostate cancer in lately diagnosed prostate cancer
patients receiving 15 mg of lycopene daily for 3 weeks prior to radical prostatectomy [42-43].
Cell culture studies using human cancer cell lines have also shown to inhibit the growth of
tumors cells significantly in lycopene treated growing media [44].
In another study, astaxanthin has been found to possess antitumor activity when
examined on mice model fed with astaxanthin. Consequently, a significant reduction in the
size and weight of Meth-A tumor cells was observed in the mice fed with astaxanthin [45].
Many other carotenoids including β-carotene, astaxanthin, canthaxanthin and lycopene have
also shown reduction in size and number of liver neoplasias in-vivo model indicating anti-
tumor activity of carotenoids [46].
6.4.2. Immunomudulating Activities

Carotenoids have demonstrated significant stimulatory effect on immune system. In one
of the studies of human blood cells d astaxanthin enhanced the immunoglobulin production in
response to T-dependent stimuli [47].
According to one of the other studies, a ratio of CD4 to CD8 lymphocyte is found to
increase with the increase in doses of β-carotene supplementation. Since this ratio is very low
in patients suffering from HIV disease, it is indicative of protective efficacy of β-carotene
[48].
6.4.3. Cardiovascular Activities

According to the literature, various in-vivo, in-vitro and human clinical studies have been
conducted on effect of carotenoids on cardiovascular diseases and the results indicate that
carotenoid rich diet could minimizes LDL oxidation and lipid peroxidation hence protect
themselves from various cardiovascular disorders such as atherosclerosis, heart attack etc.
[28, 49-50].
A comparative study was undertaken between the population of Lithuania and Sweden in
which low level of lycopene was found to be linked with increased risk of coronary heart
disease (CHD) [51].
There are several other studies in support of protective effect of supplementation of
oxygenated carotenoids such as lutein, zeaxanthin, lycopene, β-cryptoxanthin, β-carotene in
the patient suffering with acute and chronic coronary syndromes [52-53]. Astaxanthin
recommended as dietary supplement to protect against atherosclerotic cardiovascular disease
[54]. Paprika is the main source of capsanthin and it has also been found to alter plasma high
density lipoprotein cholesterol levels and hepatic gene expression in rats [55].
6.4.4. Modulation of Xenobiotic Metabolizing Enzymes

Carotenoids are also known for the modulation of xenobiotic metabolizing enzymes i.e.
enzymes of phase I and phase II reactions [56-57].

Lycopene induces phase II enzyme which helps in the elimination of toxins and
carcinogens from the body by conjugating with reactive electrophiles and becoming more
preventive against a variety of carcinogens in living system.
Chemopreventive nature of lycopene has been examined in which DMBA (9,10-
dimethylbenz-a-anthracene) is used to induce hamster buccal pouch tumors. Results of this
study suggest that the lycopene increased the level of GSH and the phase II enzyme GST
which inactivates the carcinogens [29].
Effects of other carotenoids such as β-carotene, β-apo-8'-carotenal, canthaxanthin and
astaxanthin were investigated on liver xenobiotic metabolizing enzymes in mice by P Astorg
et al. [58]. Only canthaxanthin showed the increased activity of CYP 1 A (Phase I enzyme) in
comparison to remaining carotenoids.
6.5. Protection from Ultra Violet (UV) Rays
Some of the studies have demonstrated the effect of dietary intake of carotenoids in
protection against UV light exposure such as erythema, sunburn, photo induced oxidation,
inflammation, immunosuppression, aging and even the skin cancer [59-60].
One animal study showed astaxanthin supplementation protects the retinal photoreceptors
in the eyes of rats exposed to acute UV light injury [61] and one in-vitro study also showed
the protective effect of astaxanthin against UV-induced photooxidation and was found to be
more effective than β-carotene and lutein [62]. β-Carotene has been shown to produce some
degree of protection against sunburn but should be taken prior to exposure and canthaxanthin
is also used to reduce photosensitivity i.e. erythropoietic protoporphyria (EPP) [63].
6.6. Protection against Gastric Infection
Carotenoids are also known to provide protection against gastric disorders specially
caused by Helicobacter pylori (H. pylori) which can result into duodenal and peptic ulcer
along with gastric and stomach cancer [64-66].
In one of the animal based studies, astaxanthin rich Haematococcus algae meal
demonstrated that it reduces Helicobacter pylori bacterial infection resulting in significant
decrease in gastric inflammation. Moreover, other cell culture studies are also in favor of
direct antibacterial effects of Haematococcus algae meal enriched with astaxanthin against
Helicobacter pylori infection [67].
7. Toxicity of Carotenoids
Carotenoids are generally non toxic however, there are a few experimental evidences
showing prooxidant activity of β-carotene in different models under certain conditions.
An interesting study on French women revealed that, the effect of β-carotene
supplementation is associated with a higher risk of tobacco related cancers in smokers, while
the risk of these cancers decreased with increasing β-carotene intake in non smokers [68].

Likewise, in another study it was found that supplementation of β-carotene enhanced the
risk of lung cancer and mortality in chronic smokers. In this study interactions between β-
carotene and cigarette smoke condensate (tar) was analyzed which resulted in the formation
of lipid peroxidation products [69].
Conclusion
Carotenoids have been proved to be potent phtytochemical pigments found in nature as
they posses wide spectrum of bioactivities when used as herbal medicines. On the basis of
results of the studies reviewed in this chapter, carotenoids may be defined as herbal
supplements rich in nutrients which not only reduce the oxidative stress but also help in the
protection against various chronic diseases like cancer etc. Therefore, they are recommended
to be used as dietary supplement or as herbal medicines to provide better immunity and
proper maintenance of physiological functions of the body. However, more research needs to
be carried out on carotenoids in order to understand the exact mechanism of action behind
their protective efficacy.
Acknowledgments
The authors are thankful to Director, DIPAS, Delhi, for her constant support and
encouragement. One of the authors, Ms. Mamta is thankful to University Grants Commission,
Delhi, India for getting the senior research fellowship.
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Chapter VII
Microalgae: A valuable Source

of Natural Carotenoids with Potential
Health Benefits
Faruq Ahmed1, Kent Fanning2, Holger Schuhmann1,

Michael Netzel2,3,4 and Peer Schenk1,
1
School of Agriculture and Food Science, The University of Queensland, St Lucia,
QLD, Australia
2
Department of Agriculture, Fisheries and Forestry (DAFF), Coopers Plains,
QLD, Australia
3
Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food
Innovation (QAAFI), The University of Queensland, St Lucia, QLD, Australia
4
CSIRO Animal, Food and Health Sciences, Coopers Plains, QLD, Australia
Abstract
Carotenoids are receiving increased attention in recent years due to their ability to act
as antioxidants and their potential role in preventing the development of different
degenerative diseases and health conditions in humans, including age-related macular
degeneration, cataract, certain cancers, rheumatoid arthritis, muscular dystrophy and
cardiovascular problems. Microalgae can produce large amounts of a wide variety of
carotenoids, including β-carotene, astaxanthin, lutein, canthaxanthin, violaxanthin and
neoxanthin. Moreover, microalgae have the added benefit that they can be grown on non-
arable land, avoiding competition with food production and precious biodiverse
landscapes. Several microalgae strains are currently being used commercially for
production of astaxanthin and β-carotene and research is ongoing to optimise production
of other commercially important carotenoids. This book chapter will review the recent
advances in the production of carotenoids from microalgae, the potential health benefits
discovered by the use of carotenoids from microalgae, and the current status of

Corresponding author email: [email protected].

144 Faruq Ahmed, Kent Fanning, Holger Schuhmann et al.
biotechnological application of these compounds. The chapter will also discuss future
direction of research to enhance our understanding of the carotenoid biosynthesis
pathways in microalgae and the potential use of microalgae as nutraceuticals and food
supplements.
Keywords: Antioxidants; carotenoids; health benefits; microalgae
Introduction
Carotenoids are found mostly in green leafy and yellow-coloured vegetables and orange
coloured fruits. Carotenoids are lipophilic compounds containing 40 carbon chains and have
been divided into carotenes and xanthophylls based on their chemical structure. The carotenes
are hydrocarbons whereas the xanthophylls have oxygenated functional groups making them
more polar than the carotenes [1]. All xanthophylls are produced by higher plants and
microalgae, including violaxanthin, antheraxanthin, zeaxanthin, neoxanthin and lutein. Some
other xanthophylls are synthesised exclusively by green microalgae, diatoms or brown algae
e.g. loroxanthin, astaxanthin, canthaxanthin, diatoxanthin, diadinoxanthin and fucoxanthin
(Figure 1) [2]. Carotenoids are also categorised as primary and secondary due to the
distinction that the primary carotenoids are structural and functional components of the
cellular photosynthetic apparatus, making them integral for the survival of the cells, whereas
secondary carotenoids are produced only after exposure to specific environmental stimuli
through a process known as carotenogenesis [2]. Carotenoids have also been divided into
provitamin A (β-carotene, α-carotene, and β-cryptoxanthin) and nonprovitamin A (lutein,
lycopene and zeaxanthin), based on the possession of potential vitamin A activity by being a
source of retinol when dietary preformed vitamin A fails to meet the body‘s needs [3].
Carotenoid pigments provide protection to the photosynthetic apparatus in plants by
dissipating excess energy. They also have a major role in photosynthesis by harvesting light
and by stabilising protein folding in the photosynthetic apparatus [3]. More than 600 different
carotenoids are known to be present in nature and their distribution, molecular structure or the
presence of specific biosynthesis pathways have been suggested as useful tools for algae
classification. Carotenes and xanthophylls are predominantly synthesised and accumulated
within the plastids of green microalgae, however, secondary xanthophylls may accumulate in
the cytoplasm (e.g. astaxanthin in Haematococcus sp.) leading to the speculation that
xanthophylls synthesised in the chloroplast may be exported, and consequently accumulate in
the cytoplasm [2].
Carotenoids have been used as food colorants for many decades and are increasingly
becoming popular as dietary supplements [4]. Carotenoids are known as important
antioxidants and there is evidence that they are involved in other biological functions, e.g.
regulatory effects on intra- and intercellular signalling and gene expression [5]. Carotenoids
quench singlet oxygen which mainly arises from sunlight absorption by chromophores and
thus protect the chlorophylls, lipids, proteins and DNA from the singlet oxygen damage. The
mechanism of singlet oxygen quenching involves the conversion of the excess energy of the
singlet oxygen into heat via the formation of the lowest excited triplet state of the carotenoids
(3CAR)[4]. However, each carotenoid has different antioxidant properties determined by its
polarisation, affecting its orientation in the cell membrane. For example, β-carotene is

Microalgae 145
accumulated in the inner portion of the cell membrane and contributes to membrane stability
by reacting with oxygen radicals whereas lutein is localised across the membrane and
provides protection against by-products from peroxyl radicals [6]. Because of the fat-soluble
nature, the uptake pathway of carotenoids from diets is the same as other lipophilic nutrients
and consumption of dietary fat together with carotenoids improves their absorption. After
ingestion, the carotenoids initially appear in the chylomicron and VLDL fraction of the blood.
The LDL particles in the blood are the major carrier of hydrocarbon carotenoids whereas both
LDL and HDL are equal distributors of xanthophylls. Circulating carotenoids in the
bloodstream are thus transported to all tissues including the skin [7]. There are reports that
carotenoids can protect human skin against UV-induced damage. Therefore, it has been
suggested that increasing the carotenoid content in plants can lead to the improvement of the
nutritional quality of the diet as high conservation of fundamental cellular signalling
processes and protective mechanisms are observed in nature [7].
Among the various sources of carotenoids, microalgae have created a wide interest due to
the advantages that they are relatively easy to cultivate, not being in competition with food
production, can adapt to environmentally changing conditions by producing a great variety of
secondary metabolites, and the production of these compounds can be triggered by either
controlling the cultivation conditions or by using genetic engineering approaches. For
decades, microalgae have been used as food for aquaculture but they can also be a good
source of pharmaceutical products and can also be used to purify wastewater [8]. Two
microalgae strains, Dunaliella salina and Haematococcus pluvialis are well known to
accumulate β-carotene (up to 14% dry weight) and astaxanthin (2–3% dry weight)
respectively, under stress conditions [8]. Recently, there has been interest in fucoxanthin, a
carotenoid available in brown algae, due to claims that it can inhibit cell growth and induce
apoptosis in human cancer cells and that it possesses anti-inflammatory, anti-oxidant, anti-
diabetic and anti-obesity properties [9].
This chapter will review the recent advances in the production of carotenoids from
microalgae and the potential health benefits discovered by the use of carotenoids from
microalgae. Moreover, we will discuss future directions of research to enhance our
understanding of the carotenoid biosynthesis pathways in microalgae and the potential use of
microalgae as nutraceuticals and food supplements.
Induction of Carotenoid Biosynthesis

in Microalgae and Advances in Large-scale
Production
The carotenoid compound from microalgae that was first commercially exploited was β-
carotene in the 1980‘s [10]. Currently companies in Australia, Israel, India and China are
producing β-carotene from D. salina (Table 1). The different conditions known to induce β-
carotene accumulation are high photon flux densities in the wavelength range of 400–700 nm
(known as photosynthetically active radiation, PAR) which has been reported to cause an
increase in carotenoid level in the range of 1-10% dry weight [11]. High salinity (near
saturation level) is known to cause significant accumulation of β-carotene in D. salina which

has been commercially exploited by several companies (Table 1) [12]. Nitrogen deprivation
has been reported as another tool for inducing β-carotene accumulation in several species of
Dunaliella [13]. The presence of Fe2+ as well as a carbon source is also able to induce
synthesis of β-carotene in D. salina [2]. In some other trials, UV-A radiation (320–400 nm)
along with PAR resulted in increased β-carotene accumulation. Combined stress through
nitrogen starvation and UV-A radiation along with PAR caused a 2.5 fold increase in D.
bardawil [13].
Figure 1. Chemical structures of major carotenoids from microalgae.

Microalgae 147
The commercial production plants in Australia use open pond technology in large areas
(250 ha) with minimal operational control, water mixing through wind and convection, and
no CO2 addition [14] (Figure 2). The other producers use raceways where water mixing is
achieved by paddle wheels and CO2 is supplied to the raceways, achieving a productivity of
200 mg β-carotene m-2 day-1. The different large-scale production systems, e.g. semi-
continuous cultivation or the use of biphasic photobioreactors can give different results in
terms of productivity [2]. A two stage intensive cultivation technology was proposed where at
stage one algae growth is optimised for biomass production followed by transfer of the
culture to one third dilution and enhancing carotenogenesis through nitrogen deprivation [14].
However, a semi-continuous intensive cultivation approach was reported that involved 3-4
days dilution cycles under optimised conditions and keeping nitrate levels between 0.5 to 3
mM and this achieved a much higher productivity compared to the simple two-stage approach
without dilution [2]. Moreover, heterotrophic culture of D. salina enriched with 67.5 mM
acetate and 450 κM FeSO4 caused significant increase in cellular β-carotene content [15]. As
light contributes greatly to β-carotene accumulation, the effect of red and blue light on the
carotenoid biosynthesis process was studied recently and the results suggested that the
combination of red and blue light along with long-term iterative stress led to higher β-
carotene accumulation in D. salina [16].
Several studies aimed to find a substitute of D. salina as a producer of β-carotene and
several microalgae species have been reported as promising candidates. Six eustigmatophytes
(Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica,
Vischeria punctata and Vischeria stellate) were grown in bubble column photobioreactors
and were found to accumulate β-carotene at 1.5 – 5.9% of its dry weight [17]. Production was
also attempted for Eustigmatos polyphem using flat panel as well as bubble column
photobioreactors and β-carotene accumulation up to 60 mg/g intracellular biomass was
reported [18].
The second carotenoid from microalgae that has been commercially exploited is
astaxanthin. The best natural source of astaxanthin so far has been the freshwater microalga
Haematococcus pluvialis which is normally found in ephemeral pools of freshwater with
cooler temperature. It has been well known that light stress and nitrogen deprivation causes
enhancement of production of H. pluvialis aplanospores. Massive amounts of astaxanthin are
produced when the cells undergo a dormant stage under this nutrient stress [10]. Moreover,
flashing light caused a four-fold increase in astaxanthin accumulation compared to conditions
under continuous light, suggesting that light quality is more important for this purpose [19].
Besides nitrogen deprivation, addition of CO2 and iron caused two- to seven-fold higher
production of astaxanthin [20]. Moreover, high phosphorus and low nitrogen (low N/P ratio)
in the medium and low-cost hydroponic fertilisers can be useful for achieving high cell
density in the vegetative stage of H. pluvialis growth [21]. Development of a highly
photosensitive mutant strain through ethyl methansulfonate (EMS) treatment and random
selection was reported that could accumulate 1.5-fold higher astaxanthin than the wild-type
strain [22].
Some companies follow a two stage production system which involves growing algal
biomass under optimal growth conditions (known as green stage) followed by exposure of the
microalgae to adverse environmental conditions that cause the accumulation of astaxanthin
[23]. Recent studies suggested a slight modification of the two-stage production system,
involving addition of sodium acetate at the green stage at stationary (senescent) phase, can

significantly increase astaxanthin accumulation [24]. A closed culture technology called

PhytoMax PCS has been developed that automatically regulates cell culture conditions before
they are transferred to open ponds for the final stage of astaxanthin production. A separate
approach was proposed by Micro Gaia involving a single-step, continuous manufacture
process using moderate nitrogen limitation which was later tested in a model called
Aquasearch Growth Modules using enclosed, computerised photobioreactors for rapid growth
of H. pluvialis followed by daily transfer of the microalgae to a pond culture system to induce
astaxanthin accumulation [25].
Figure 2. Hutt Lagoon in Western Australia: the largest commercial β-carotene production facility (photo:
courtesy of Sabine Bauer; www.bauer-seyr.at).
Table 1. Examples of carotenoids from microalgae currently being exploited

commercially
Carotenoid Microalgae Company Name Website

Astaxanthin Haematococcus Cyanotech, www.cyanotech.com/;
pluvialis Algatechnologies, Mera www.algatech.com/;
Pharmaceuticals, www.merapharma.com/;
AstaReal AB, http://www.bioreal.se/;
Jingzhou Natural http://www.asta.cn/
Astaxanthin Inc
β-carotene Dunaliella BASF, www.basf.com/;
salina Nikken Shohonsha Co. http://www.nikken-miho.com/
Due to disadvantages of H. pluvialis, such as slow growth, low cell yield, high
contamination risk, susceptibility to adverse weather conditions, requirement of economically
unreasonable levels of irradiance and inability to grow in dark heterotrophic conditions,

Microalgae 149
another green microalga, Chlorella zofingiensis, has been proposed as an alternative. These
algae are easier to grow than H. pluvialis (three times faster growth rate than H. pluvialis),
have higher tolerance to environmental variations, and can grow under dark heterotrophic
conditions e.g. in the presence of pyruvate, citrate and malate [26]. Similar to H. pluvialis,
this strain is also reported to accumulate higher astaxanthin content when grown in the
presence of Fe+2 [27]. A mutant of Chlorella zofingiensis was developed through chemical
treatment and was reported to accumulate higher amounts of astaxanthin when grown in cane
molasses [28].
The main factors responsible for affecting lutein accumulation are temperature,
irradiance, pH, nitrogen availability, salinity for marine species, and specific growth rate [2].
Presence of oxidising substances (e.g. H2O2, NaOCl in the presence of Fe2+) has also been
reported to affect lutein accumulation [29]. Unlike the conditioning protocols for
accumulation of astaxanthin and β-carotene, lutein requires microalgae at their optimum
growing condition, so that the factors mentioned above do not affect biomass productivity as
that would reduce lutein production. To date, no microalgae have been commercially
exploited for lutein, however, Muriellopsis sp. has become a promising candidate strain and
attempts to produce it in large-scale cultivation systems are underway [29].
Table 2. Potential sources of other carotenoids of interest from microalgae not currently
being commercially exploited
Carotenoid Source References

Lutein Muriellopsis sp., Scenedesmus almeriensis, [30-35]
Auxenochlorella (Chlorella) protothecoides, Chlorella
zofigiensis
Cantaxanthin Chlorella zofigiensis, Scenedesmus komareckii, Dunaliella [36-38]
salina aplanospores
Zeaxanthin D. salina mutants [39]
Echinenone Botryococcus braunii [40]
Fucoxanthin Phaeodactyllum tricornutum [41]
Phytoene and Specific metabolic inhibitor treated D. salina [42]
phytofluene
It was also reported that the extremophile microalga Chlamydomonas acidophila

produced high concentrations of lutein (10 g/kg biomass) under mixotrophic growth
conditions with urea. A high amount of zeaxanthin was also produced in this strain when
grown on glycine and the combination of glycine and glycerol enhanced β-carotene
accumulation.
High PAR and ultraviolet radiation (UVR) are known to trigger de-epoxidation of the
xanthophyll cycle in microalgae leading to the higher production of violaxanthin and
zeaxanthin, respectively, in Nannochloropsis gaditana [43]. Similarly, more conversion to
diatoxanthin from diadinoxanthin due to exposure of PAR and UVR was reported in
Phaeodactylum tricornutum [44, 45]. Increase in total carotenoid content was also reported in
UV-A- and PAR-treated Nitzschia closterium and Isochrysis zhangjiangensis [46], however,
the study did not report which particular carotenoid was significantly increased due to the
stress.

Although many microalgae strains have been discovered to be producing different

carotenoids of commercial interest (Table 2), the dominant carotenoid typically constitutes
only 70% of the total carotenoid content, unlike β-carotene and astaxanthin which constitute
90% of the carotenoids in D. salina and H. pluvialis, respectively [10]. The presence of other
carotenoids in the extracts creates problems in the commercial exploitation of these strains for
the desired carotenoid.
One key aspect of large scale carotenoid production from microalgae is the design and
construction of photobioreactors catered to the optimum culture conditions required for the
strain based on its unique physiological and growth characteristics. The various proposed
designs were reviewed briefly by Guedes et al. [2] who concluded that flat panel photo-
bioreactors could be the standard for industrial production. Several proposed improvements to
flat panel photobioreactors were flat panels with inner walls arranged to promote an ordered
horizontal culture flow driven by a mechanical pump, glass sheets glued together with silicon
rubber, plastic bag located between two iron frames [2]. Assuring proper mixing of biomass
in the large-scale culture is important for optimum growth of microalgae. The parameters
affecting mixing in a bioreactor are the volumetric flow rate of air, height/diameter (H/D)
ratio, and diameter of an air sparger [47]. A v-shaped bottom design bioreactor was tested and
optimised for astaxanthin production and was suggested suitable for mass cultivation [47].
Recently, the same researchers introduced a novel thin film photobioreactor made of cast
polypropylene film and tested flat, horizontal and vertical tubular shapes of the bioreactors.
They concluded that a vertical tubular bioreactor with H/D ratio 6:1 and cylindrical stainless
steel spargers was the best design, achieving astaxanthin productivity as high as 218.6 mg/L
[48].
Harvesting and extraction are two critical areas of carotenoid production from microalgae
that require severe improvement in order to make it more cost-effective and an attractive
business venture. The key steps in this process are biomass drying and disruption followed by
extraction and purification. Several recent studies suggested substantial improvements in this
area and more research is underway. Among the various methods available for disrupting
microalgae cells are e.g. milling, ultrasound, microwave, freezing, thawing or chemical
extraction. Two protocols involving sonication and ball milling in the presence of alumina
and 4% KOH gave promising results [29, 33] and have the potential to become useful in the
large-scale production phases. Supercritical fluid extraction (SFE) using modified CO2 has
become a promising alternative to the classical solvent extraction process due to the fact that
the former can be less time consuming and more efficient and also because of the non-
flammable, non-toxic, and relatively inert nature of supercritical CO2 [49]. However, the
requirement of expensive equipment to produce supercritical CO2 and the tendency to recover
chlorophylls more efficiently than carotenoids, leading to the production of extracts with
relatively poor specifications, make SFE less competitive than solvent extraction [50]. A
novel method was developed recently using green Monoraphidium sp. where the chlorophyll
content was removed by the addition of acid (e.g. H2SO4 or HCl) followed by alkali treatment
to neutralise the acid [51]. An accelerated solvent extraction methodology has been proposed
that uses a special type of contactor to circulate solvent at high pressure and temperature
through a tightly packed biomass bed followed by extraction with hexane or ethanol [33].
This process yielded high amounts of lutein but the high temperature caused the formation of
a toxic compound pheophorbide raising concern for the process [33]. An environmentally-
friendly method was proposed by Kang et al. [52] for direct extraction of astaxanthin from

Microalgae 151
Haematococcus sp. using vegetable oils (e.g. olive oil). The method disrupted the cells by
vigourous stirring and then separated the oily extracts from culture medium containing cell
debris by gravity settling giving a recovery of up to 93.9% [52].
A different extraction involving wet biomass was recently reported by Kleinegris et al.
[53] who used a flat-panel photobioreactor as a turbidostat keeping the number of stressed D.
salina cells a constant through a continuous, predefined level of irradiation. The system
consisted of an organic phase containing dodecane that formed an emulsion allowing
continuous extraction of β-carotene from the aqueous phase. However, low yield of β-
carotene compared to the conventional extraction processes made it unsuitable for
commercial operations [2]. In a recent study, column chromatography coupled with modified
supercritical fluids extraction was successfully attempted in Nannochloropsis oculata
resulting in higher recovery of six carotenoids from soxhlet and supercritical fluids extraction
[54]. The recently patented ―milking‖ of microalgae technology for biodiesel production
involves stimulation of cells through electrical modulation causing increase in membrane and
cell wall permeability and consequently allowing oil droplets to be excreted while
maintaining cell viability [8] and can also be a useful tool for extracting secondary
metabolites including carotenoids.
Health Benefits of Carotenoids from Microalgae

Damage of the DNA is generally associated with a high risk of developing diseases.
DNA damage can be caused by various reactive molecules, including free radicals and singlet
oxygen which are produced in the body by various external environmental factors [4].
Carotenoids are known to provide protection against DNA damage although there is lack of
agreements between studies due to the findings that carotenoids may act as anti or pro-oxidant
depending on the carotenoid concentration and the oxygen concentration in the cellular
environment [55]. Carotenoids that occur in microalgae, but especially lycopene from
tomatoes, were found to protect the intestine against radiation-induced damage [12, 56].
Human trials revealed that β-carotenes may act as a protective nutrient against lung cancer
[57, 58]. However, this effect was not observed in a small group of male heavy smokers
which was attributed to the absence of vitamin C in them leading to the establishment of a
relationship between the presence of vitamin C and the behaviour of β-carotenes [59]. Studies
on the combined effects of ascorbic acid, α-tocopherol and carotenoids (β-carotene) showed
synergistic protective effects for lymphocyte cells [60], reconstituted human serum [61], and
uroporphyrin-mediated damage [62]. There is experimental evidence of the prevention of the
different phases of the atherosclerotic process by lycopene (prevention of endothelial injury,
modulation of lipid and cholesterol metabolism, inhibition of low-density lipoprotein
oxidation, prevention of oxysterol-induced cell damage, inhibition of foam cell formation,
inhibition of smooth muscle proliferation); however, the mechanism of such prevention is not
fully understood [63]. Several studies suggested a lower risk of amyotrophic lateral sclerosis
(ALS) in individuals with the intake of β-carotene, lutein and lycopene [64, 65]. Fitzerald et
al. (2013) attributed the associations between carotenoids and lower risk of ALS to the
antioxidant activity of the carotenoids that can help prevent mitochondrial distress,

interruptions of glial glutamate transport, protein aggregation or inflammation, thus prevent-

ing motor neuron death [66, 67].
It has been reported that excess exposure to UVA and UVB radiation from sunlight can
cause breakage of DNA strands, aberrations of the chromosomes and tumorigenic transforma-
tion in skin keratinocytes [68]. This can lead to erythema and a high risk of skin cancer in the
later part of life. Daily exposure to normal sunlight can cause loss of collagen fibres and skin
elasticity leading to skin ageing [4]. β-carotene is already well known to ameliorate
erythropoietic protoporphyria [69] and it has been reported that the combination of lipid and
water-soluble compounds, e.g. β-carotene, lycopene, vitamin E and C, selenium and
proanthocyanidins can reduce erythema from UV-B exposure [70]. In a separate study,
lycopene from tomato paste was found to provide protection against acute and potentially
long-term photo-damage by reducing reactive oxygen species (ROS) leading to less erythema
and less singlet oxygen, leading to reduction in matrix metalloproteinase (MMP)-1 [71].
These experimental results suggest that carotenoids can reduce short and long term sunlight-
induced skin damage and improve skin health by reducing skin roughness [72, 73]. Photo-
protective effects of β-carotene, lycopene, canthaxanthin, lutein, and natural/dietary sources
of those carotenoids including tomato products, carrot juice, algal or petal extracts were
investigated in human intervention studies and the results were extensively reviewed [74-76].
Studies on the effects of β-carotene on sunburn prevention showed positive responses through
the ingestion of synthetic β-carotene, extracts of natural sources high in β-carotene or
antioxidant mixtures with β-carotene as a major constituent. These studies suggested a dosage
of 10 mg/day β-carotene to obtain photo-protection [7]. In a separate study, astaxanthin
exhibited a pronounced photo-protective effect and counteracted UV-A-induced alterations,
e.g. apoptosis, increased levels of ROS, decreased antioxidant enzyme activities, membrane
perturbation, and elevated expression of heme oxygenase-1 to a significant extent [77]. Also,
fucoxanthin exhibited anti-pigmentation activity in mice by inhibiting tyrosinase activity,
melanogenesis in melanoma, and UV-B-induced skin pigmentation [78].
Lutein, zeaxanthin and meso-zeaxanthin, an isomer of lutein, are collectively known as
macular pigments because of their presence in the macular region and these pigments are
believed to play a major role in protecting the retina by quenching ROS [4]. Diets rich in
lutein and zeaxanthin are well known to reduce the prevalence of age-related macular
degeneration (AMD), the leading cause of blindness among elderly people and women, who
are especially at greater risk than men due to reduced macular pigment [79, 80]. Even in
healthy populations dietary lutein intervention has been reported as important means of
maintaining the health of the macula [81]. Daily supplementation dosages ranging from 6 mg
to 30 mg showed improved visual functions such as contrast and retinal sensitivities, and a
reduction in age-related cataracts [82-85].
More recent studies suggest that lutein supplementation can benefit driving at night and
other spatial discrimination tasks carried out under low illumination, besides increasing
macular pigments density levels and contrast and glare sensitivity [86]. It has also been
reported that the diets supplemented by lycopene and β-carotene along with lutein and
zeaxanthin can help the fight against AMD, suggesting that lycopene, although not directly
involved in preventing AMD, can protect the other carotenoids from oxidation and help
increase their bioavailability [87, 88]. Clinical trials also suggested that tomato-rich diets
containing lycopene can lower the incidence of prostate cancer [89-91], although
contradictory results were reported as well [92, 93].

Microalgae 153
Although inverse relationship between several degenerative diseases and dietary intake of
carotenoids or their blood or tissue concentrations were reported in many clinical studies, a
high inter-individual variability was observed in absorption, and the responses of blood and
tissue of dietary carotenoids [3]. Such variation was attributed to the genetic variants in genes
encoding for the proteins involved in carotenoid metabolism. It was reported that different
alleles in single nucleotide polymorphisms located in or near several of the genes encoding
these proteins caused the varied blood and tissue concentrations of carotenoids [3]. Different
mechanisms were described regarding the absorption and assimilation of carotenoids in
humans. Carotenoids may be cleaved into retinoids and apo-carotenals after their absorption
from the gut. Alternatively, the carotenoids can be assimilated into lipoproteins and later
secreted in the blood stream [94]. Two enzymes (BCMO1 and BCDO2) have been identified
that are responsible for the bioconversion of β-carotenes; the reaction mechanisms and
substrate specificity have been reviewed by [1] Lietz et al. (2012).
The series of conjugated double bonds in carotenoids make them susceptible to oxidative
cleavage. Cleavage of these double bonds causes the formation of a large number of
carbonyl-containing oxidation products called apocarotenoids [95]. Studies have suggested
that such apocarotenoids are biologically active as anticancer agents, e.g. by growth inhibition
of leukemia cells [96], estrogen-dependent breast and androgen-dependent prostate cancer
cells [95].
Additionally, carotenoid oxidation products have been reported to modulate the activity
of various transcription systems including ligand activated nuclear receptors e.g. the retinoic
acid receptor, retinoid X receptor, peroxisome proliferator-activated receptor, estrogen
receptor, and the electrophile/antioxidant response element pathway and nuclear factor-kB,
the transcription systems that have an important role in cancer [95]. Thus, it has been
concluded that apocarotenoids are natural compounds with multifunctional activity and can
be useful in the prevention of cancer and other degenerative diseases. The formation of new
blood vessels determines the growth of tumours and the development of metastases. It has
been reported that β-carotene treatment causes significant reduction in the number of tumour-
directed capillaries as well as the reduction of serum vascular endothelial growth factor the
proinflammatory cytokines [97]. The treatment of melanoma cells with β-carotene in vitro
also showed similar reduction of these cytokines [97].
Provitamin A carotenoids (β-carotenes, α-carotenes and β-cryptoxanthin) from plant
sources are additional major dietary components for vitamin A for most parts of the world,
including developed countries, where highly restrictive diets using a strong dietary regime
eliminating certain categories of food might create risk of vitamin A insufficiency [98].
Vitamin A plays a significant role in cell differentiation, embryonic and vision development,
growth hormone production, glycoprotein synthesis, and carcinogenesis [99, 100]. Besides
proven evidence of provitamin A activity, β-carotene has been hypothesised to play an
important role in body fat reserve reduction/control [101, 102]. Weber and Grune (2012)
reviewed the β-carotene intake in industrialised countries from available literature and
concluded that 35% of current vitamin A intake was coming from β-carotenes. They
recommended a combination of both components (vitamin A and β-carotenes) in the diets to
reach the recommended daily dietary intake of vitamin A.

Current Biotechnological Application

of Carotenoids
Carotenoids have long been used as food colourants and as dietary supplements in fish
and shellfish hatcheries. However, the recent discoveries of their health benefits have
extended their application to other sectors. As mentioned previously, astaxanthin and β-
carotene from microalgae have been marketed by several companies in different parts of the
world. For example, BioAstin® is being marketed by Cyanotech and sold as a dietary
supplement with approval from the U.S. Food and Drug Administration (FDA). The company
claims that BioAstin® is sold to address various health markets e.g. joint health, general
health, sports nutrition and suncare products. Algatechnologies is marketing the product
AstaPure™ which is derived from H. pluvialis. The product is available in several forms, e.g.
powder, emulsion, softgel, beadlets. On the other hand, the astaxanthin from Mera
Pharmaceuticals is called AstaFactor® and it is sold in rejuvenating and sports formula, and
also as a combination of salmon oil, fish and astaxanthin called ―salmon essentials‖. The
highest variation of astaxanthin products is marketed by AstaReal AB, a Swedish biotech
company with the product name AstaREAL® and the retail products Astaxin® for human
dietary supplementation and NOVASTA®/Astaequus® for animals.
Nikken Shohonsha markets their β-carotene products as ―Dunaliella‖ and ―Dunalliela
Hard Capsules‖ and they are sold as human dietary supplements. The other β-carotene
producer BASF has the product name Lucarotin® and mainly sells them to the aquaculture
and poultry industries. However, they are listed as a human dietary supplement as well.
Future Direction of Research

One key area for research is bioprospecting involving identification, isolation and growth
optimisation of new and locally available microalgae strains producing high amounts of
carotenoids. Besides production of high amounts of carotenoids, the strains should also be
screened for species dominance, harvesting ease, and tolerance to variable environmental
conditions. Moreover, successful scaling up of the desired strains for large-scale commercial
production for carotenoids and other secondary metabolites requires major improvements in
the design of bioreactors using inexpensive and environmentally-friendly materials providing
high productivity and accurate control of key parameters for high yield of biomass. Such
improvements should look into areas such as assuring proper mixing of culture, rate of gas
exchange, light penetration capacity and pH control.
As pointed out earlier, harvesting and extraction are two major areas of research in order
to make carotenoid production more economically sustainable and ensure competitive
advantage over other sources. Although many different approaches for harvesting have been
proposed, such as sedimentation, membrane filtration, flocculation, concentration methods
e.g. drum, spray, fluidised bed or freeze drying but no universal method is yet to be
developed. Due to the intracellular nature of the metabolites, proper cell disruption methods
also need to be developed. The proposed methods are autoclaving, bead-beating, microwave,
ultrasonication, or exposure to high concentrations of salts, acids, alkalis, or enzymes.

Microalgae 155
Another area of research focuses on the genetic modification of microalgae to obtain high
carotenoid producing transgenic strains or mutants. Among the commercially important
species, genetic modification of Haematococcus has been attempted leading to the
development of a strain with a modified version of its own phytoene desaturase gene.
Moreover, gene expression profiles associated with astaxanthin-accumulating Haematococcus
following stress have been reported. Several recently published papers characterised the
different genes involved in carotenogenesis of astaxanthin under stressful conditions. The key
genes studied so far in the carotenoid biosynthesis pathway are BKT1, PDS, CRTR-B, LYC,
IPI-1, IPI-2, PSY, BKT2, CRTO, and CZLCY [103-105]. Moreover, higher accumulation of
violaxanthin and lutein through overexpression of the phytoene synthase-encoding gene from
Chlorella zofingiensis in Chlamydomonas reinhardtii was reported [106]. The homologous
gene from D. salina and BKT1 from H. pluvialis were successfully transferred to C.
reinhardtii to help understand the mechanisms of actions of these genes in the carotenoid
biosynthesis pathway [103, 107]. Recently, cDNAs from Adonis aestivalis were identified
that could enable the production of astaxanthin and other carotenoids when operably linked to
promoters appropriate to the transgenic hosts [108]. Such research outcomes will enhance the
attempts to produce transgenic high carotenoid producing microalgae.
Although the commercially explored microalgae strains for carotenoids are autotrophs,
mixotrophic cultivation of several strains resulting in significant carotenoid accumulation
have been reported opening up avenues of more research possibilities in this area. Growth of
potential strains in the dark may solve many of the bioreactor designing constraints faced by
the industry and therefore should be explored further.
The beneficial roles of carotenoids described so far involved mostly whole or processed
food leading to the notion that such beneficial effects could be due to the synergistic effects of
the different nutritional components present in the food. Therefore, human trials are required
to corroborate the existing evidence.
Studies suggested that carotenoid oxidation products inhibit cancer cells growth at a
much higher level than intact carotenoids. Further research is required to understand the
physiological significance of these findings and the mechanism of action of these carotenoid
derivatives.
Due to high variation in absorption and blood and tissue responses of dietary carotenoids
caused by the genetic variations in the proteins involved in carotenoid metabolism, further
research is needed to identify all proteins involved the metabolism and assess the effect of
different genetic variation, e.g. copy number variants and epigenetic modifications can
modulate carotenoid status. The findings of such research can lead to the development of
personalised dietary guidelines for carotenoids based on individual genetic characteristics.
Conclusion
Microalgae are already a significant contributor in the continuously growing carotenoids
market. With the large interest in various commercial sectors, microalgae can become a major
source for carotenoids in the future, as microalgal biomass production is set to increase due to
their potential other uses as a source of biofuels and high-protein biomass in future algal
biorefineries. However, in order to increase production of microalgal carotenoids, major

advancements in such areas as low cost harvesting and extraction, genetic manipulation and
production cost reduction would be required.
Acknowledgments
We wish to thank the Australian Research Council for financial support and Sabine Bauer
for kind provision of photos from Hutt Lagoon.
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2013.

Chapter VIII
Sodium Taurocholate Optimum Amount

of Blood Parrots on Astaxanthin
Availability
Xidong Mu, Huiyun Yang, Yinchang Hu and Jianren Luo*

Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key
Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation,
Ministry of Agriculture, Guangzhou, China
Abstract
The body color of ornamental fish is crucial for their market value. Previously, we
found that sodium taurocholate ranging from 1000~ 2000mg/kg can effectively improve
body color through higher utilization of astaxanthin in blood parrot (Cichlasoma
synspilum ♀× Cichlasoma citrinellum ♂), one of the most popular ornamental fish, but
accurate requirements of sodium taurocholate to aquaculture remain unclear. In the
present study, we continued to study the optimum amount of sodium taurocholate
supplement ranging from 1000~1800 mg/kg, by observing body color with concentration
of total carotenoid in blood parrots. The total carotenoid content in the skin/caudal fin of
7 groups (total 462 blood parrots) was determined at 15d, 30d, 45d, and 60d. The results
showed that all fish adding sodium taurocholate had improved body color by better
pigmenting efficacy of astaxanthin compared to fish (groups A (control groups) or B
(only astaxanthin)). Significant differences (P< 0.05) in the total carotenoid content in the
skin/caudal fin between the groups with 400 mg/kg astaxanthin plus sodium taurocholate
level and groups A (control groups) or B (only astaxanthin) were detected from day 15 to
day 60. A supplement of 1600 mg/kg sodium taurocholate was the most effective. In
conclusion, based on the current results, we suggest that 400 mg/kg astaxanthin plus 1600
mg/kg sodium taurocholate improves pigmentation quickly over a 45 day period in the
cultured blood parrot, which may be an effective, lower cost option for a color-enhancing
supplement.
* Corresponding Author address. Email: [email protected].

166 Xidong Mu, Huiyun Yang, Yinchang Hu et al.
Keywords: Blood parrot, Sodium taurocholate, Optimum amount, Carotenoid
Introduction
The body color of ornamental fish plays an important role in determining their market
value [1-2]. Many factors affect the body color of fish, such as genes [3], hormone
concentrations [4], age, gender [5], physiological factors, external environment [6], and diet
[7]. Of these, color-enhancing additives to the diet offer a quick and successful method for
improving body color. Previous studies have demonstrated the effects of different additives
on pigmentation in many aquatic fish, including Salmo salar [8], Penaeus japonicus [9],
Oncorhynchus mykiss [10-11], Carassius auratus [12-13], Sparus aurata [14], and Clarias
fuscus [15]. Previous research has also underscored the importance of carotenoids as the
primary pigments in fish [11, 16], although aquatic animals are unable to synthesize
carotenoids. Instead, they obtain carotenoids through their diet via additives such
as astaxanthin, which is a fairly effective synthetic compound for improving fish color [17].
Skrede et al. [18] found that astaxanthin stimulated fish to produce more intense red than
canthaxanthin. Therefore, astaxanthinas are a popular diet supplement to produce a bright red
color. Due to the high price and low absorption efficiency of astaxanthin in fish, it is
necessary to better develop additives that may promote absorption and increase the
pigmentation obtained with astaxanthin at a lower cost.
The blood parrot (Cichlasoma synspilum ♀× Cichlasoma citrinellum ♂) is one of the
most popular ornamental fish. They are more valuable if they have strong pigmentation from
a diet rich in carotenoids. Li et al. [2] reported that adding 300-500 mg/kg astaxanthin was the
optimum level for improving the body coloring of the blood parrot. Previously, we examined
the effect of sodium taurocholate on the pigmentation efficacy of astaxanthin in the blood
parrot and found that adding 1000-2000 mg/kg sodium taurocholate improves the absorption
and pigmentation of astaxanthin in the blood parrot [19], but accurate requirements of sodium
taurocholate to aquaculture remain unclear. In the present study, we determined the optimum
amount of sodium taurocholate for use as an additive to improve the utilization of astaxanthin
in blood parrots by observing body color with concentration of total carotenoid in blood
parrots.
Methods
Participants
The experiment was performed at the Pearl River Fisheries Research Institute, Chinese
Academy of Fishery Science (Guangzhou, China). A total of 462 live blood parrots (initial
weight 26.0± 0.46 g) with a faded surface area of more than 95% were obtained from
Guangzhou Huadiwan ornamental fish market (Liwan District, Guangzhou). Barley-based
diets were purchased from Guangzhou Tai Feng Co., Ltd, Guangzhou, China. The astaxanthin
used in the study was 10% pure and was obtained from BASF, Germany. The sodium
taurocholate used in the experiments was produced by Andy Bio, Beijing, China.
The fish were divided into seven experimental groups, and each group was divided into
three replicates. The feeding experiments for each group were performed at the same time

Sodium Taurocholate Optimum Amount of Blood Parrots ... 167
under the same water environment conditions. The experimental groups are listed in Table 1.
The fish were cultivated in an aquarium measuring 120 cm × 50 cm × 48 cm with a sustained
supply of oxygen (above 6 mg/L). The water temperature was 25-28°C, and the water was
changed three times per week, with one-third of the water changed each time. All fish were
fed twice per day at 9:00 and 16:00 h. The duration of each feeding session was 5-10 min.
Carotenoids obtained from four points on the skin and caudal fin of blood parrots were
measured at 15 d, 30 d, 45 d, and 60 d, according to the methods developed by
Boonyaratpalin et al. [20] and Yang et al. [19].
Statistical analyses of data were performed using the Microsoft Excel 2010 and SPSS
Statistics 17.0 with the general linear model (GLM) procedure. The results are presented as
the mean (X) ± standard deviation (S.D.). The effect of treatment was determined using a one-
way ANOVA followed by Tukey test of multiple comparisons at the 95% significance level.
For each of the seven experimental groups, the total carotenoid content of the skin/caudal
fin at 15 d, 30 d, 45 d, and 60 d is shown in Table 1. The difference in the total carotenoid
content of fish fed with various sodium taurocholate concentrations proved to be significant
(P<0.05). As expected, the total carotenoid content of skin increased with time within each
group receiving the same sodium taurocholate concentration. Although all experimental
groups with added sodium taurocholate (Groups C/D/E/F/G) were explicitly pigmented and
showed a slight change in body color after 6 weeks, there was great change in the total
carotenoid content (X = 114.14~144.20 mg/kg) of the skin with increasing sodium
taurocholate concentration (Table 1, Figure 1 (a)). During the feeding period, the highest total
carotenoid content (X = 83.95 mg/kg at 30d; X = 114.90 mg/kg at 45d; X = 144.20 mg/kg at
60d) was found in Group F (1600 mg/kg) (P<0.05) after 30 days of the experimental diet.
After 15 days, the fish that received sodium taurocholate (Groups C/D/E/F/G) had
significantly higher (P<0.05) carotenoid content compared to those that did not receive
sodium taurocholate (Group B). This difference was significant at 30 d, 45 d, and 60 d (Table
1). In each period, we found that the total carotenoid content of skin was approximately 30
mg/kg higher than that of Group B. The experimental data show that sodium taurocholate not
only improved the utilization of astaxanthin but also enhanced the color of blood parrots and
that the optimum additive amount of sodium taurocholate was 1600 mg/kg (Group F).
The total carotenoid content of the caudal fin of all groups was positively correlated with
sodium taurocholate level except for the group G (1800 mg/kg), which had values slightly
lower than expected (Table 1 and Figure 1(b)). The carotenoid content was significantly
different (P＜0.05) between the groups (C/D/E/F/G) and groups A or B from 15 d to 60 d. At
day 15, Group G had the highest (P<0.05) total carotenoid content, as it did at 30 d and 45 d.
The rate of increase of the total carotenoid content of caudal fin doubled between days 15 and
30 and again between days 30 and 45, but it barely increased between days 45 and 60. At the
end of the experiment, group F showed the highest total carotenoid content (X = 392.48
mg/kg; P<0.05). As expected, the total carotenoid content of the caudal fin was much higher
than that of skin for all groups. Our results suggest that the caudal fin of blood parrots is
sensitive to astaxanthin. These preliminary analyses suggest that the recommended value for a
sodium taurocholate additive is 1600 mg/kg.

Table 1. Carotenoids content in the skin and caudal fin of blood parrots fed different experimental diets for 15, 30, 45, and 60 days
Group Astaxanthin Sodium Total carotenoids content of skin (mg/kg) Total carotenoids content of caudal fin (mg/kg)
(mg/kg) taurocholate 15d 30d 45d 60d 15d 30d 45d 60d
(mg/kg)
A 0 0 14.89±1.54a 19.84±0.94a 26.10±2.47a 37.06±3.09a 20.58±1.08a 34.77±2.66a 42.58±2.24a 56.09±0.52a
B 400 0 28.09±1.86b 43.23±2.23b 73.59±4.27b 95.78±3.63b 44.64±1.70b 79.90±1.77b 230.46±6.30b 267.19±4.28b
C 400 1000 34.11±3.01bc 60.61±1.66c 85.90±3.09c 114.14±2.56c 58.80±3.27c 124.71±2.62c 322.75±3.68c 366.65±3.98c
D 400 1200 38.41±2.71c 68.67±2.38d 96.77±3.94d 122.55±1.64c 66.10±0.79d 137.72±2.47d 344.35±3.97d 373.23±2.39c
E 400 1400 41.18±1.33cd 75.86±1.63e 107.80±2.23e 137.21±4.40d 73.91±2.64e 162.06±2.51e 371.67±3.86e 385.50±2.14d
F 400 1600 43.55±2.01cd 83.95±1.86f 114.90±1.74e 144.20±2.93d 80.08±1.30e 177.70±1.41f 385.17±3.10f 392.48±1.46d
G 400 1800 45.01±0.99d 79.28±1.90ef 103.78±3.84de 122.16±4.45c 85.16±2.04f 183.02±1.15f 366.93±4.42e 373.51±1.63c
Note: The means with different letters within the same column are significantly different at the 0.05 probability level, and the means with the same letters are
not significant differences. A: basal diet without astaxanthin. B: basal diet with 4 ‰ of astaxanthin; C: basal diet with sodium taurocholate (1000mg/kg). D:
basal diet with 4 ‰ astaxanthin plus 1200mg/kg sodium taurocholate. E: basal diet with 4 ‰ astaxanthin plus 1400mg/kg sodium taurocholate. F: basal
diet with 4 ‰ astaxanthin plus1600mg/kg sodium taurocholate. G: basal diet with 4 ‰ astaxanthin plus1800mg/kg sodium taurocholate.

160 15d 30d 45d 60d
Carotenoi ds content£ ®mg/kg £©

140
120
100
80
60
40
20
0
A B C D E F G
Gr oups
(a)
450 15d 30d 45d 60d

Carotenoids content £®mg/kg £©
400
350
300
250
200
150
100
50
0
A B C D E F G
Gr oups
(b)
Figure 1. Total carotenoid content in skin (a) and caudal fin (b) of the blood parrot for 15, 30, 45, and 60 days
in different experimental diets. A: basal diet without astaxanthin. B: basal diet with 4 ‰ of astaxanthin; C:
basal diet with 4 ‰ astaxanthin plus 1000 mg/kg sodium taurocholate. D: basal diet with 4 ‰ astaxanthin
plus 1200 mg/kg sodium taurocholate. E: basal diet with 4 ‰ astaxanthin plus 1400 mg/kg sodium
taurocholate. F: basal diet with 4 ‰ astaxanthin plus 1600 mg/kg sodium taurocholate. G: basal diet with 4
‰ astaxanthin plus 1800 mg/kg sodium taurocholate.
At the end of the experiment (6 weeks), there was no significant differences, as detected
by the naked eye, in the body color of all fish that were fed the different fixed dietary sodium
taurocholate concentrations (1000-1800 mg/kg), although this additive effectively influenced
body color.
In conclusion, sodium taurocholate (1000~1800 mg/kg) is able to efficiently improve
pigmentation and the absorption of astaxanthin in blood parrots. In particular, 1600 mg/kg
sodium taurocholate for 45 days produced rapid pigmentation and an excellent effect. Based
on the current results, we suggest that 400 mg/kg astaxanthin plus 1600 mg/kg sodium
taurocholate improves pigmentation quickly over a 45 day period in the cultured blood parrot,
which may be an effective, lower cost option for a color-enhancing supplement.
This study was supported by the program of National Science Infrastructure Platform of
China and Science and Technology Developing Fisheries Program of Guangdong Province
(2011B060400023).

170 Xidong Mu, Huiyun Yang, Yinchang Hu et al.
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Chapter IX
Carotenoid-Based Ornaments As
Signals of Health Status in Birds:
Evidences from Two Galliform Species,
The Red-Legged Partridge (Alectoris
rufa) and the Red Grouse
(Lagopus lagopus scoticus)
Lorenzo Pérez-Rodríguez1,2, Jesús Martínez-Padilla2

and François Mougeot3,4,*
1
CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos,
Campus Agrário de Vairão, Vairão, Portugal
2
Department of Evolutionary Ecology,
Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid, Spain
3
Instituto de Investigación en Recursos Cinegéticos - IREC (CSIC-UCLM-JCCM),
Ciudad Real, Spain
4
Estación Experimental de Zonas Áridas (CSIC), Ctra. de Sacramento s/n.,
La Cañada de San Urbano, Almería, Spain
Abstract
Carotenoids are large lipophilic compounds that can only be produced by plants,
fungi and bacteria. Although animals cannot synthesize them de novo, many taxa
accumulate them in exposed parts of the body for communication purposes. In many
birds, carotenoids are responsible for the bright yellow-red coloration of integuments or
plumages, and these have often been shown to advertise an individual‘s superior health.
*
Corresponding Author address: Email: [email protected]; [email protected].

174 Lorenzo Pérez-Rodríguez, Jesús Martínez-Padilla and François Mougeot
Some of the hypotheses aimed at explaining the function of carotenoid-based traits rely
on the assumption that carotenoids have key physiological roles in the organism.
Specifically, they are hypothesized to be significant antioxidants and immunostimulants,
being therefore intimately linked to the anti-parasite defenses and health. The
mechanisms and functions of the expression of carotenoid-based traits have been
particularly well studied in birds. In this chapter we first review the current hypotheses
linking carotenoids, oxidative stress, immune response and parasites, together with the
current evidences in support of these key physiological functions. Secondly, we focus on
two gallinaceous bird species that have been intensively studied in recent years, either in
captivity (the red-legged partridge, Alectoris rufa) or in the wild (the red grouse, Lagopus
lagopus scoticus). In both species, ingested carotenoids are metabolized and transformed
before being used to color bright red ornaments. The redness of the sexual ornaments
relates to the concentration of carotenoids in blood, and varies with infection levels by
intestinal parasites and with the ability of individuals to mount an immune response to
pathogens. Circulating levels of carotenoids through the blood stream are also negatively
affected by physiological stress (oxidative stress and stress hormones), which is partly a
consequence of parasite infections and immune responses. The evidence for carotenoids
to act as antioxidants and to reduce oxidative stress is still mixed and debated, but some
studies highlighted that carotenoid-based ornaments can signal an individual´s ability to
resist oxidative stress. Finally, we identify key open questions that deserve further
investigation in this field.
Keywords: Carotenoid, red grouse Lagopus lagopus scoticus, red-legged partridge Alectoris
rufa, parasite, immunity, oxidative stress
Introduction
Carotenoids are a large family of lipophilic compounds produced by photosynthetic
organisms and certain bacteria and fungi (Goodwin 1984). Animals are not able to synthesize
carotenoids de novo, but often deposite these pigments in skin, bills, eyes, scales and feathers
(Fox 1976). This results in some of the most striking visual displays (bright yellow-red
ornaments) of the animal kingdom. These carotenoid-based colorations are becoming an
increasingly popular study subject among evolutionary and behavioral ecologists, as these
traits often play key roles in communication (i.e. they are used as signals of bearer quality in
social and mate choice contexts). This has been intensively investigated in birds in the recent
years, a taxonomical group where carotenoid-based colorations are particularly prevalent and,
perhaps, the most common taxon of study for behavioral ecologists. In fact, some avian
carotenoid-based traits, such as the red plumage of the house finch (Carpodacus mexicanus)
or the red beak of the zebra finch (Taenyopigia guttata), are amongst the most deeply studied
animal sexual signals.
The evolution of carotenoid-based traits into signals of quality requires the existence of a
differential expression of such coloration (i.e. size of the color patch or color intensity) for
high- and low-quality individuals. Thus, as for any signaling trait, the expression of
carotenoid-based colorations must be linked in some way to individual quality in order to
evolve as reliable signals of quality. This drives us to the concepts of ―honesty‖ and ―costs‖.
The production or maintenance of honest (i.e. reliable) signals must entail significant costs of
different nature (i.e. behavioural, physiological, reproductive, etc.) for the bearer in order to

Carotenoid-based Ornaments as Signals of Health Status in Birds 175
prevent the evolution of individuals producing signals that do not correspond to their actual
quality, i.e. cheaters (Zahavi 1975). Thus, honest signals should work as handicaps for the
organism, so that only high quality individuals are able face the costs asociated with maximal
signal expression. Apart from these revealing handicaps, honest signaling may also result
from the expression of condition/dependent handicaps (Collins 1993; Hill 2011), which are
traits that do not impose a special cost for the bearer, but whose production is linked to
individual condition (health, parasite load, energy reserves) in such a way that only healthier
individuals are able to maximize signal expression. As we will see below, most hypotheses
addressing the function and evolution of carotenoid-based traits as honest signals of quality
refer to condition-dependent handicap mechanisms.
Carotenoid Metabolism in Birds:

From Food to Visible Integuments
The above mentioned versions of the handicap principle applied to carotenoid-based
traits require a deep knowledge of the physiological pathways followed by carotenoids. It
implies the study of the different stages involved from their acquisition via food sources to
their deposition in integuments or plumages creating brightly colored signals. Birds must first
obtain carotenoids from their diet. However, birds and other animals are able to perform some
metabolic transformations, so that the carotenoid composition of visible integuments and
internal tissues may differ substantially from that of food sources. Although more than 700
different carotenoids have been described, only a very small subset of them can be found in
the organism of birds (McGraw 2006). This is because only some specific carotenoids are
present in their diets, but also because absortion efficiency greatly differ depending on the
carotenoid type, ranging from 1 to 99% (Parker et al. 1999), as well as the bird species. In
general, birds tend to preferentially absorb and accumulate xanthophylls (carotenoids that
contain oxygen in their functional groups) over carotenes (which contain only hydrogen).
Less than 10 different carotenoids have been described in avian diets (McGraw 2006), but
their relative amounts radically differ: about 60-90% of ingested and circulated carotenoids in
birds are lutein and zeaxanthin, whereas only small amounts of β-criptoxanthin, β-carotene
and others carotenoids are found. Diet composition strongly determines the relative
proportion of circulated carotenoids. Aquatic species, for instance, circulate significantly
higher amounts of β-carotene, canthaxanthin or astaxanthin than species feeding on seeds or
plants (McGraw 2006). As expected, diet explains a significant proportion of the variability in
blood carotenoids between species (Tella et al. 2004). Species feeding on plants show higher
carotenoid-levels, whereas those feeding on vertevrate preys show lower levels of blood
carotenoids. However, beyond such effect of diet, variability in circulating carotenoids in
birds (ca. 65%) is mostly explained by phylogenetic relationships: species sharing common
ancestors tend to show similar levels of circulating carotenoids (Tella et al. 2004). Although
this phylogenetic effect could be due partly to its covariation with diet, it does stress the
importance of physiological processes, like absortion or transport efficiency, that are likely
conserved and more similar among closely related species.
In vertebrates, ingested carotenoids are extracted from food along with other lipid-soluble
molecules by passive diffusion trough the intestine (Parker 1996). In birds, however, there

seems to be some lipid-independence in the absorption of carotenoids, and evidence from

chickens indicate that carotenes and xanthophylls are absorbed at different sections of the
intestine (Tyczkowski and Hamilton 1986). Once they are retrieved from food and fractioned
with lipids in the intestinal mucosa, carotenoids are packaged into chylomicrons, enter the
lymphatic system and are circulated through the bloodstream. Lipoproteins are the exclusive
transporters of these pigments in the body, and experimental evidence in the zebra finch have
shown that increases in lipoprotein levels enhance circulating carotenoids, ultimately
increasing carotenoid-based beak coloration (McGraw and Parker 2006). Also, it has been
proposed that lipoprotein levels can be upregulated by androgens, setting a basis for the
endocrine control of carotenoid-based signals (McGraw et al. 2006). Although the link
between circulating carotenoids and androgens has been supported by several different
experimental studies in birds (Blas et al. 2006; Alonso-Álvarez et al. 2009; Martínez-Padilla
et al. 2010; Peters et al. 2012), whether such mediating effect of lipoproteins is a general rule
still awaits further studies.
Bounded to lipoproteins, carotenoids are delivered by blood stream to internal tissues, as
well as to colored external integuments in species with carotenoid-based ornamentation.
Carotenoid concentration among tissues of a given individual are overall correlated, although
some tissues and organs (i.e. fat, liver, ovaries in females during reproduction) are more likely
to accumulate carotenoids than others (Surai 2002; McGraw and Toomey 2010). The specific
carotenoids deposited in body tissues generally match those found in the blood, although
some subtle variations between organs have been described, which could reflect some
physiological discrimination or tissue specific demands (McGraw 2006).
Carotenoids present in external integuments of birds (skin, beaks, feathers) may come
directly from diet, or may have been metabolically transformed prior to deposition.
Carotenoid-based traits typically show yellow, orange or red colors depending on the specific
types of carotenoids deposited, as well as their relative and absolute concentrations. Yellow
colorations result from the deposition hydroxycarotenoids (e.g. lutein, zeaxanthin, canary
xanthophylls), which are the carotenoids most commonly acquired through diet.
Ketocarotenoids (e.g. astaxanthin, canthaxanthin, adonirubin) are red carotenoids responsible
for reddish colorations of plumages and bare parts. These are metabolized from yellow
hydroxycarotenoids or from β-carotene. In most cases, a mixture of different carotenoids,
with different origins, are found in avian integuments. Also, carotenoids may interact with
other pigments (eumelanin, pheomelanin) or with the microstructure of feathers and tissues to
produce a large variety of colors, like green or blue (e.g. Lucas and Stettenheim 1972; Prum
and Torres 2003). Interestingly, carotenoids deposited into feathers are typically in their free
forms, whereas those deposited into living tissues like skin, beaks and combs, are often
esterified (Czeczuga 1979; Casagrande et al. 2011; García de-Blas et al. 2011). Carotenoid
esterification in these tissues could be related to the dynamic nature of the color of these traits
(Pérez-Rodríguez 2008) or could play a role in stabilizing pigment properties. Unfortunately,
experimental studies on this topic are still pending.
The specific site where dietary carotenoids are metabolized into colorful integumentary
carotenoids have been the subject of debate for some time. The liver was initially proposed as
a potential site for carotenoid conversion, given its richness in carotenoids and enzimes
(Brush 1990). However, later studies failed to find in blood or liver those metabolically
derived carotenoids present in feathers or beaks of songbirds, suggesting that these metabolic
transformation might be made directly within the integument or within the follicle of

developing feathers (McGraw 2004). Nevertheless, some yellow metabolized carotenoids –

e.g. anhydrolutein and dehydrolutein– have been found in the blood of at least one bird
species before depositing them into feathers (McGraw and Schuetz 2004). Also, recent
studies have found exceptions among red metabolically-derived carotenoids: the red
carotenoid 3-hydroxy-echinenone, present in the feathers of common crossbills (Loxia
curvirostra), was found in the liver and plasma of the species, but not in its diet, suggesting
that the liver was the site of conversion (del Val et al. 2009). Clearly, more studies, covering a
larger set of species, are required to elucidate where carotenoids are transformed. Although
the role of the liver on integumentary carotenoid metabolim is debated, its role in pro-vitamin
A metabolism of ingested carotenoids is well supported, which has been recently vindicated
to propose a novel pathway for the honesty of carotenoid-based signals of health (Hill and
Johnson 2012). Given the limited knowledge about the site(s) of metabolic conversion, it is
not surprising that the identity of the enzyme(s) responsible for these transformations are still
unkown to date. The enzymatic control of these conversions remains speculative (Stradi et al.
1996, McGraw et al. 2003, McGraw 2006) and the same applies to our understanding of the
underlaying genetic mechanisms (Walsh et al. 2012).
Carotenoid-based Ornaments As Honest Signals

of Quality
Carotenoids are the second most prevalent pigment in the avian integument, melanins
being the first. Unlike these, that may lead either to mimetic or conspicuous patterns,
carotenoids mostly lead to striking yellow, orange and red traits. Thus, carotenoid-pigmented
plumages, beaks, wattles, combs and other skin patches, which evolved to be seen and play
crucial roles in bird communication. Carotenoid-based traits often function as communication
signals, but what do they signal? Handicap theory predicts that the information conveyed by a
given signal must be linked to the cost or the limitation associated to its production. Evidence
from several species support the signaling role of carotenoid-based signals, particularly in
sexual selection scenarios (reviewed by Hill 2006). Initial studies proposed that carotenoid-
based traits evolved as signals of foraging efficiency and body condition (Slagsvold and
Lifjeld 1985; Hill 1990), whose honesty was achieved by a condition-dependent handicap
mechanism. This hypothesis relies on the fact that carotenoids must be acquired through diet
and the assumption that carotenoids are a limited and scarce resource for most species. Good
foragers or dominant individuals are expected to have a better access to food sources, also
obtaining higher amounts of carotenoid to maximize signal expression. This implies that
carotenoid-based signal expression will be honestly associated with nutritional status, with
energy and carotenoid intake being positively associated (although this may not always be the
case, e.g. Sternalski et al. 2010). Recent evidence in birds indicate that individuals with
brighter carotenoid-based ornaments also show a greater capacity to solve foraging problems
(Mateos-González et al. 2011), providing behavioral support for this hypothesis.
This ―foraging ability‖ hypothesis remained for years as the main plausible mechanism to
explain the evolution and maintenance of carotenoid-based traits as signals of quality.
However, the publication of the seminal paper of Lozano (1994) opened a new window for
the study of these traits, highlighting the importance of carotenoids as mediators of several

key physiological processes, notably immune responsiveness and parasite resistance. Later,
von Schantz et al. (1999) incorporated these ideas within a broader context, highlighting the
connections between oxidative stress and immunity and paying attention to the antioxidant
properties of carotenoids. This resulted in an alternative hypothesis to explain the evolution
and maintenance of carotenoid-based signals, which relies on the assumption that carotenoid
pigments have crucial physiological functions for self-maintenance. If so, individuals should
potentially face a trade-off between allocating available carotenoids for these self-
maintenance functions or for ornamental coloration. In further detail, healthier individuals
would require lower amounts of carotenoids for health-related functions, allowing them to
allocate more carotenoid pigments to ornamental coloration, to display brighter or more
intensly colored signals, and advertise their superior health to conspecifics (Figure 1).
These carotenoid-related physiological trade-offs are usually pooled under the same
mechanism to explain honest signalling. This is understandable given that parasites, immune
function and oxidative stress are closely interconnected, and that the antioxidant and
immunostimulant roles of carotenoids are often difficult to tease appart. However, the
implications of these functions differ, and they should be considered separately in order to
fully understand the information content of carotenoid-based signals.
Carotenoid-based Traits, Immunocompetence

and Parasites in Birds
Parasites are a major force in evolutionary biology as they detract energy from their
hosts, damaging their health and ultimately their fitness (Møller et al. 1990, 1999).
Consequently, hosts should prevent pathogen infection, by behavioral or physiological means
(Møller et al. 1999). From a physiological point of view, perhaps the most efficient way of
fighting parasites off is the immune system, designed to protect the host against a variety of
pathogens, from virus to parasites. Most of the literature has been focused on the evolutionary
meaning of the host capacity to develop an effective immune system to fight against
pathogens. However, not all individuals have the same immune capacity to react to the same
pathogen. Hamilton and Zuk (1982) linked the individual capacity of the hosts to fight against
parasites within a sexual selection context. They suggested that sexual selection should favor
individuals with genetic disease resistance by assessing the full expression of those secondary
sexual traits that are dependent of individual‘s health. It implies that individuals with
enhanced genetic capacity to fight against parasites are able to display their sexual traits at
their best.
The expression of sexual characters is costly to produce and, according to Hamilton and
Zuk (1982), such a cost can be caused by pathogen infection. In relation to carotenoid
pigmented traits, parasite infection reduces the expression of these traits (Brawner et al. 2000;
Martinez-Padilla et al. 2007), and the expression of carotenoid-pigmented traits may inform
about males‘ ability to resist parasite infection (Lindstrom and Lundstrom 2000). From a
physiological perspective, immune defense is the mechanism that allows hosts to fight against
pathogen infection. A seminal paper by Blount et al. (2003) suggested that dietary carotenoid
supply produced a parallel increase in carotenoid-dependent pigmentation and immune
defense in zebra finches. Later on, it was proved that mounting an immune response is

physiologically costly to produce in birds (Horak et al. 2000; Ots et al. 2001; but see Owen-
Ashley et al.2004) resulting in negative fitness consequences (Marzal et al. 2007). Immune
capacity is intimately linked to genetic quality (Hamilton and Zuk 1982), but interestingly
carotenoids can boost immune defense (Møller et al. 2000; McGraw and Ardia 2003, 2007),
resistance to parasite infection (Baeta et al. 2008) and mounting an immune response reduces
the availability of carotenoids (McGraw and Ardia 2003; Alonso-Alvarez et al. 2004; Peters
et al. 2004; Aguilera and Amat 2007; Pérez-Rodríguez et al. 2008). However, the inter-
relations between immune capacity and carotenoid availability is more complex than initially
though (Biard et al. 2009), deserving further research. Such cost on immune response,
however, may constrain the expression of carotenoid-pigmented sexual signals. Thus, a
physiological trade-off emerges because individuals should allocate their carotenoids to
physiological functions (i.e. health or self maintenance) or to ornament pigmentation (Lozano
1994).
Figure 1. Summary of the pathways followed by carotenoid pigments from their aquisition through diet to
their deposition in ornaments. Arrows indicate the inter-relations between four main components of an
individual´s heatlh status (parasites, immune responses, oxidative stress and physiological stress), that can
reduce the amount of available carotenoid pigments that are ultimately deposited in integuments and used for
ornamental displays (combs for red grouse, below left, and red bill and eye rings for red-legged partridge,
below right). These effects or allocation priorities are also modulated by sexual hormones (in particular
testosterone) and by the environmental context (see text for further details).
Most sexual characters in males are modulated by hormones, and specifically by

testosterone (Adkins-Regan 2005). This hormone plays a crucial role in sexual selection as it
enhances the expression of secondary sexual traits, but also has immunosuppressive effects
(Folstad and Karter 1992). Folstad and Karter (1992) suggested that testosterone may mediate

the honesty of the sexual signaling based on an elegant refinement of the handicap principle
(Zahavi 1975). They suggested that only high-quality individuals are able to afford the
immunosuppressive effects of testosterone while showing the most elaborate sexual displays
(Folstad and Karter 1992), what is known as the Immunocompetence Handicap Hypothesis
(ICHH). This hypothesis predicts that testosterone and parasites act in opposite directions,
such that testosterone increases the susceptibility to parasitism, and parasites constrain
testosterone-dependent behavior or ornamentation in this case. Interestingly, testosterone may
up-regulate the bioavailability of circulating carotenoids, that can be used for ornament
pigmentation by one side or to counteract the immunosuppressive effects of testosterone on
the other (Blas et al. 2006; Martinez-Padilla et al. 2010). This trade-off has been studied in
red-legged partridges and red grouse (see below), but also in other species. Studies looking at
allocation priorities of carotenoids are however still controversial and deserve further
investigation. Peters et al. (2012) used a two-way experiment to simultaneously manipulate,
testosterone and carotenoids, and found that testosterone increased circulating levels of
carotenoids only in carotenoid-supplemented birds and that there was no effect of testosterone
on plumage coloration. While several studies have explored the effects of immunity on
carotenoid levels or carotenoid-pigmented traits, further experimental studies manipulating
testosterone and pathogen infection (or immune capacity) are needed for a deeper
comprehension of the complex interactions involved (Figure 1).
These physiological trade-offs have been deeply studied in birds mainly in captive
conditions and less intensively in free-living individuals. However, in natural conditions,
individuals are under different environmental stressors that can influence how they resolve
physiological trade-offs. This may explain some of the inconsistencies found between species
(Owen-Ashley et al. 2004). In natural populations where environmental conditions are good,
individuals may have a greater flexibility for allocating their carotenoids between
ornamentation and immune defense. Conversely, in populations where environmental
conditions are stressful (i.e. high parasite prevalence, low food abundance at population level)
individuals can be more constrained in their allocation of carotenoids to ornamentation and
immune defense, prioritizing immune defense instead of ornamental pigmentation. Examples
of this idea are scarce in birds, but there is some support of this environmental-dependent
resolution of carotenoid allocation trade-offs. In red grouse, individuals treated with
testosterone increased their carotenoid levels only in populations where population density
was low, and increases of testosterone were only positively associated with ornamental
coloration in low-density population (Martinez-Padilla et al. 2010). The authors hypothesized
that individuals living in low-density populations can store their carotenoids and use them
when needed. In captive male zebra finches, the presence of females can affect the expression
of carotenoid-based sexual traits under carotenoid-demanding conditions and lack of
pathogen infection (Gautier et al. 2008). This suggests that the expression of the signal can be
adaptive because it is modulated depending on the expected benefits in relation to the social
environment individuals live in (Gautier et al. 2008).
The response of individuals to stressful situations is modulated by stress hormones
(corticosterone in birds), which can act as mediators of sexual signal expression (Husak and
Moore 2008). Free-living animals experience many stressors (e.g. weather, predators,
parasites, social conflicts) that challenge their homeostasis (Romero 2004). A major
adaptation is the hypothalamic–pituitary–adrenal axis, which releases glucocorticoids in
response to stressors, allowing individuals to recover homeostasis in the best condition.

However, chronic or inappropriate stress responses can exert deleterious effects, such as
immunosuppression (Romero 2004). How an individual responds to or copes with stressors is
likely a major determinant of its fitness, and ornaments could reveal how individuals cope
with stressors. There is some evidence that the expression of carotenoid-based traits is
associated with stress hormone levels, although the sign of the association may vary (e.g.
Cote et al. 2010; Costantini et al. 2008; Loisseau et al. 2008; Bortolotti et al. 2009). The link
between stress hormones and carotenoid-based traits may be mediated by their effects on
immunity or oxidative stress (Romero 2004; Costantini et al. 2008).
Recently, it has been shown that choosing a parasite-free mate may reduce parasitism risk
of the choosy partner (Martinez-Padilla et al. 2012). In fact, there in an asymmetry in such
infection risk such as males have a higher increase of being parasitized than females. This
may have implications for the ICHH (Folstad & Karter 1992), which was initially developed
for males. If males mate with highly parasitized females, this extra cost may alter the
resolution of the trade-off between investing in testosterone-dependent behavior and defense
against parasites, and perhaps may also alter how carotenoids are allocated to carotenoid-
pigmented sexual traits by males. Thus, parasite levels of female mates should be considered
for a better comprehension of the effects of the ICHH.
Carotenoid-based Traits
and Oxidative Stress in Birds
Oxidative stress is the result of the imbalance between production of reactive oxigen
species (ROS) and the antioxidant defences of the organism in favour of the former (Halliwell
and Gutteridge 2007). ROS are highly reactive molecules that may damage (oxidize)
important biomolecules such as DNA, lipids and proteins. Most endogenously produced ROS
are normal by-products of aerobic metabolism. Apart from cell metabolism, another
important source of ROS is the immune response. During an infection, the immune system
cells generate ROS as a way to combat invading pathogens. Although ROS are produced as
part of the killing mechanism, this may cause oxidative damage to the host organism,
increasing the cost of immune response (Halliwell and Gutteridge 2007). During the last
decades, studies from the medical and nutrition literature summarized and highlighted the
antioxidant properties of carotenoids and the beneficial effects of carotenoid-rich diets on
several diseases attributed to oxidative stress (Krinsky 1989; Rao and Agarwal 1999; Rao and
Rao 2007; Halliwell and Gutteridge 2007).
The physiological trade-offs proposed by Lozano (1994) and von Schantz et al. (1999) to
explain the honesty of carotenoid-based signals nourished from these studies highlighting the
healthy properties of carotenoids in humans, resulting in an enthusiastic assumption of the
―antioxidant role‖ hypothesis (Møller et al. 2000) (Figure 1). However, years later several
studies questioned the key assumption of this ‗‗antioxidant role‘‘ hypothesis, claiming that
carotenoids are not as powerful antioxidants as initially proposed (Hartley and Kennedy 2004;
Isaksson and Anderson 2007; Costantini and Møller 2008; Isaksson et al. 2008). As an
alternative way to connect oxidative stress and carotenoid-based ornamentation, despite a
possible poor antioxidant capacity of these pigments, it has been suggested that carotenoids
are particularly sensitive to be altered by ROS. This may result in a spurious relationship

between carotenoid pigmentation and oxidative stress that is mediated by the quality of the
antioxidant system (Hartley and Kennedy 2004).
The antioxidant properties of carotenoids have been a subject of controversy among
ecophysiologist and behavioral ecologists during the last decade, and have been addressed by
recent reviews (Costantini and Møller 2008; Catoni et al. 2008; Monaghan et al. 2009; Pérez-
Rodríguez 2009; Svensson and Wong 2011). Although some criticisms regarding the
antioxidant properties of carotenoids already exist in the medical literature (Rice-Evans et al.
1997;Young and Lowe 2001; Krinsky 2001), most of the controversy arises from the intrinsic
differences between humans and birds, that impose restrictions to the extrapolation of the
roles of carotenoids between them (Pérez-Rodríguez 2009). For instance, birds circulate
different amounts and, most importantly, different types of carotenoids (birds circulate
xantophylls whereas humans circulate mostly carotenes). This is crucial because, at least in
vitro, carotenes show a higher ROS scavenging capacity than xanthophylls (Miller et al.
1996; Stahl et al. 1998). Also, there are important physiological differences between birds and
humans that may affect the relative importance of carotenoids as antioxidants in both groups.
For instance, birds, in contrast to mammals, are uricotelic and therefore maintain blood levels
of uric acid three times higher than humans. Also, unlike humans, most bird species are able
to synthesize vitamin C, a powerful antioxidant that interacts with carotenoids during redox
reactions (Surai 2002). Both uric acid and vitamin C may deeply affect the ‗‗antioxidant
environment‘‘ of an organism, which may in turn affect the action of carotenoids (Stahl and
Sies 2003). In addition, susceptibility to oxidative stress may differ between taxa due to
differences in metabolic rates or life histories, or due to structural factors, such as the degree
of unsaturation of lipids in cell membranes (Stahl and Sies 2003).
For the reasons mentioned above, extrapolating the antioxidant role of carotenoids from
humans or mammal models to birds seems questionable. Although some general patterns may
be conserved, elucidating the role of carotenoids can be validated using avian models. To
date, experimental and correlational studies in birds have led to inconsistent results. This may
be due, in many cases, to a simplistic conceptualization of the antioxidant system and redox
reactions, to an incomplete asessment of the antioxidant defences and oxidative damage, or to
experimental designs that do not allow to tease apart the antioxidant and immunostimulant
roles of carotenoids (Pérez-Rodríguez 2009). However, a recent phylogenetically controlled
meta-analysis performed on 88 different species of birds found that carotenoids are
significantly positively related to plasma antioxidant capacity, but not significantly related to
oxidative damage (Simons et al. 2012). The relationship between oxidative status and
carotenoid-based ornamentation was positive, though not significant.
Further research is still required to clarify the importance of carotenoids as signals of
oxidative stress in birds. Experimental designs addressing this question should take into
account several important features of carotenoids and the antioxidant network, avoiding too
simplistic interpretations of antioxidant responses. For instance, experimental designs must
consider measures of antioxidant capacity and oxidative damage simultaneously, taking a
dynamic approach and covering different tissues, in addition to blood parameters (Pérez-
Rodríguez 2009, Simons et al. 2012). Finally, alternative pathways linking carotenoids to
vital biochemical processes must be explored. In this sense, Hill and Johnson (2012) recently
proposed a biochemical model for the regulation of ornamental coloration based on
interdependencies of carotenoid and retinoid biochemistry. This model proposes that vitamin
A regulatory mechanisms, redox systems, and carotenoid pigmentation pathways link

carotenoid coloration to oxidative status and other aspects of host performance, such as
immunocompetence. Based on complex enzymatic cycles and pathways, this model offers a
novel window to study the physiological basis of the honesty of carotenoid-based signals, in
birds as well as in other taxa.
Carotenoid-based Ornaments As Signals

of Quality in the Red-legged Partridge
(Alectoris rufa)
The red-legged partridge (Alectoris rufa) is a medium sized game bird belonging to the
family Phasianidae, endemic to the western Mediterranean where its natural distribution
encompasses France, NW Italy and the Iberian Peninsula including the Mediterranean islands
of Corsica, Elba and the Balearics. It inhabits areas of a wide variety of land uses, although it
shows the highest densities in the centre-south of Spain, and is least abundant along the
Mediterranean coast (Blanco-Aguiar et al. 2003).
Both sexes spend the winter in sedentary flocks. However, from January-February, males
exhibit territorial behavior and perform visual and vocal displays to attract females. The
species is socially monogamous. Mating takes place between February and April, and egg
laying between April and early June (Cramp and Simmons 1980). Females typically lay and
incubate one clutch of 6-15 eggs, but in some cases (up to 50% of pairs in good years) they
lay a second clutch that is entirely incubated by the male (Green 1984; Casas et al. 2009).
During the first weeks after hatching, partridge chicks feed on insects, but they soon move to
a diet based on seeds and green plants that they will maintain throughout their lives.
Figure 2. Two images of a male red-legged partridge showing its melanin based plumage pattern and its
carotenoid-pigmented beak, legs and eye rings. Photo credits: Rafael Palomo.

lutein
zeaxanthin
a) plasma, liver
and fat
absorbance units
carotenoid
diesthers
astaxanthin carotenoid
monoesthers
unknown
red carotenoid
b) beak, eye ring
canthaxanthin and legs
lutein
retention time (min)

Figure 3. Illustrative High Performance Liquid Cromatograms showing the carotenoid composition of a)
plasma, liver and fat and b) any of the red traits (beak, legs and eye rings) of the red-legged partridge.
Courtesy of Esther García de Blas.
The red-legged partridge shows a slight sexual size dimorphism (males weigh ca. 480
grams, whereas females are 70 grams lighter on average; overall body size show marked
variations across the area of distribution). However, males and females show the same
plumage pattern (Figure 2), characterized by a black spotted bib and a barred flank feather
tract. The back is olive-green, whereas the chest and the belly are grey and orange,
respectively. Carotenoids are absent from this plumage pattern, which is pigmented by
pheomelanin and eumelanin instead. However, both sexes show conspicuous and carotenoid-
based red legs, beaks and eye rings that significantly differ in color intensity between sexes
(Villafuerte and Negro 1998; Pérez-Rodríguez 2008).
The red coloration of the beak, legs and eye rings of the red-legged partridge results from
the accumulation of red carotenoids in the ramphoteca (beak), podotheca (leg) and epidermis
(eye ring) of the bird. High Performance Liquid Chromatography analyses have revealed that
the main carotenoids in all three integuments are astaxanthin and a still unknown carotenoid

whose structure appears similar to that of papilioerythrinone (García-de Blas et al. 2011;
García-de Blas et al. in press - Figure 3). Also, small amounts of lutein and canthaxanthin are
found in all three ornaments. Interestingly, both astaxanthin and the unknown red carotenoid
are mostly (>80%) in esterified forms (mono- and di-esters of carotenoid). Lutein and
cantaxanthin, however, did not produce esterified compounds. Importantly, there are only two
carotenoids in the plasma and internal tissues (liver, fat) of the red-legged partridge: lutein
and zeaxanthin (Figure 3) that are acquired through diet. This implies that the red carotenoids
found in ornamental traits (and their esters) are synthesized within integuments from these
two dietary carotenoids.
Carotenoid esters have never been found in feathers of any species (McGraw 2006).
However, it may be the rule in living tissues of birds (Czeczuga 1979; Casagrande et al. 2011)
and other taxa, like fishes or crustaceans (e.g. Wade et al. 2008; Pike et al. 2011). The role of
carotenoid esterification in these tissues is still unknown. In the red legged partridge,
carotenoid esters where better predictors of integument redness than total carotenoids in the
tissue (although the latter still correlates well with overall ornament coloration). It is therefore
likely that pigment esterification could play a role in carotenoid stabilization or mobilization
to/from living tissues for signaling purposes. This hypothesis, however, requires further
experimental support.
There is a slight but significant sexual dimorphism in carotenoid-based coloration in the
red-legged partridge, males showing redder eye rings and beaks than females (Pérez-
Rodríguez and Viñuela 2008; Pérez-Rodríguez 2008). Also, the proportion of the eye ring
skin pigmented by carotenoids (eye ring pigmentation, hereafter) is greater in males than in
females (Pérez-Rodríguez and Viñuela 2008; Pérez-Rodríguez 2008). As many other
carotenoid-based ornaments, the legs, eye rings and beak of the red-legged partridge show a
certain degree of reflectance in the ultraviolet range of the visual spectrum. As found in the
fleshy ornaments of grouse (Mougeot et al. 2007a), ultraviolet reflectance of partridge
ornaments seem to increase when carotenoids deposited in the ornament are scarce and the
underlying dermis layers (eye ring) or the outermost keratinized parts of the integument
contribute relatively more to ornament reflectance (Pérez-Rodríguez 2008). One direct
consequence of this is that ornament redness and relative UV reflectance are negatively
related, and the higher the carotenoid concentration in the integument, the higher its relative
red saturation and hue, and the lower its ultraviolet reflectance is (Pérez-Rodríguez 2008).
One important feature of carotenoid-pigmented living tissues is that their color can be
dynamically adjusted according to the condition or status of the individual. In the red-legged
partridge, eye ring and beak redness mirrored seasonal variations in circulating carotenoids.
This results in higher expression of carotenoid-based ornamentation coinciding with the end
of the mating season and the start of laying, which is consistent with a signaling role of these
traits in sexual selection scenarios. These changes could be partly attributed to the action of
androgens, which increase carotenoid absorption and regulate their mobilization to
ornamental traits in this species (Blas et al. 2006; Alonso-Álvarez et al. 2008, 2009).
However, besides this dynamism in coloration, red color expression relative to others in the
study population was consistent across the breeding season and between consecutive years,
thus indicating that carotenoid-based traits in the red-legged partridge accurately reflect
consistent differences in individual quality (Pérez-Rodríguez 2008).
Further evidences are also consistent with a signaling role of carotenoid-based traits in
the red-legged partridge. For instance, when redness of the beaks and eye rings of males is

artificially enhanced by means of paint, their females lay earlier and produce more eggs than
those mated with control (un-manipulated) males (Alonso-Alvarez et al. 2012). Therefore,
females respond to male coloration and adjust their allocation to current reproduction
accordingly (Andersson 1994). Field data also support a signaling role of carotenoid-based
coloration among partridges, as males and females mate assortatively according to beak
coloration in the wild (Mougeot et al., unpublished data), which is may be considered as
evidence of mutual mate choice.
Carotenoid-based ornamentation in the red-legged partridge is related to several different
indicators of health status. Eye ring and beak redness are related to individual condition and
to the degree of physiological stress, estimated using the relative abundance of heterophils in
the blood as a proxy (Pérez-Rodríguez and Viñuela 2008; Mougeot et al. 2009a).
Experimental manipulations of body condition resulted in rapid reductions of eye ring
coloration, but were not mirrored by short-term changes in beak redness. This suggests that
different ornaments reflect changes in body condition but at different speeds or intensities:
eye ring, a fleshy ornament, appears to respond more rapidly to changes in the nutritional
status than the bill, which is a keratinized structure (Pérez-Rodríguez and Viñuela 2008).
Also, individuals with higher abundance of coccidia (an intestinal parasite) showed lower
levels of circulating carotenoids, which resulted in lower eye ring redness (Mougeot et al.
2009a). Coccidia are known to affect carotenoid absorption, mainly because of the damage
caused to the intestinal mucosa (Allen 1987). In addition, the immune response against
parasite infection, which is often associated to an increase of ROS production (Halliwell and
Gutteridge 2007), may also contribute to the negative effect of coccidia on circulating
carotenoid levels and carotenoid-based ornamentation.
Circulating carotenoids and carotenoid-based coloration of eye rings and beak are also
positively related to the cell-mediated immune responsiveness, measured as the skin swelling
response to the intra-dermical injection of a plant lectin, phytohaemagluttinin (Pérez-
Rodríguez et al. 2008; Mougeot et al. 2009a). Such an immune challenge also induced a 13%
decrease in circulating carotenoids within 24 hours although that decrease did not affect
carotenoid-based coloration, probably because of the short term effect of the T-cell-mediated
immune response. However, these results highlight a link between immunity and carotenoids,
which are consumed during immune responses.
Besides the links among carotenoids, immune response and oxidative stress, the above
mentioned decrease in circulating carotenoids in partridges exposed to an immune challenge
did not appear to be strongly associated with oxidative stress (Pérez-Rodríguez et al. 2008). In
fact, T-cell-mediated immune challenge did not affect oxidative stress markers (lipid
peroxidation, antioxidant capacity). Also, neither circulating carotenoids nor carotenoid-based
coloration were related to oxidative stress. However, individual changes in oxidative damage
during immune response were predicted by individual coloration, suggesting that carotenoid-
based traits in this species may foresee an individual´s ability to resist oxidative stress (Pérez-
Rodríguez et al. 2010). Finally, oxidative stress affects carotenoid circulation and deposition
into red ornaments: when exposed to low doses of a source of ROS (diquat) during four
months, red legged partridges showed lower levels of circulating carotenoids and paler
carotenoid-based ornaments (Alonso-Alvarez and Galván 2011). This supports the hypothesis
that carotenoids are sensitive to oxidative stress, which is reflected by the ornaments based on
these pigments, which can be considered as an adequate proxy of oxidative status in this
species.

Figure 4. Two images of a male red grouse displaying its supraorbital red com. Photo credits: Gary R.
Bortolotti and Jesús Martínez Padilla.
Carotenoid-based Ornaments As Signals

of Quality in the Red Grouse (Lagopus lagopus
scoticus)
Grouse (Tetraonidae family) are medium sized game birds of the northern hemisphere,
living in arctic, boreal and temperate region (Watson and Moss 2008). Grouse display bright
yellow red supra-orbital combs, a main sexual ornament that is pigmented by carotenoid

pigments (Egeland et al. 1993; Mougeot et al. 2007a; Siitari et al. 2007) (Figure 4). Grouse
combs reflect in the ultraviolet (UV), which is not visible to humans but is likely to be visible
to grouse (Mougeot et al. 2005). Comb UV reflectance decreases with increasing comb size
and redness, and is mainly a property of the un-pigmented skin, with the carotenoid pigments
deposited in the epidermis to increase the orange-red coloration acting as a mask that reduces
skin UV reflectance (Mougeot et al. 2007a). Combs are displayed by both sexes, although
they are typically larger and brighter in males (Mougeot and Redpath 2004; Martinez-Padilla
et al. 2011) (Figure 4). Both the size and carotenoid-coloration of combs are testosterone-
dependent; in red grouse, for example, males circulating more testosterone display larger and
redder (Mougeot et al. 2005; Martinez-Padilla et al. 2010). Moreover, males with higher
testosterone levels are typically dominant and more aggressive, and have higher mating
success, suggesting that comb size and coloration functions in intra- and inter-sexual selection
processes (Redpath et al. 2006b).
A main parasite of red grouse is the nematode Trichostrongylus tenuis, which as well
known negative effects on condition, reproduction and survival (Hudson 1986; Hudson et al.
1992; Redpath et al. 2006a). T. tenuis parasite abundance can be easily measured (Seivwright
et al. 2004) and manipulated in free-living red-grouse, by means of parasite purging (dosing
with anthelminthic to reduce parasite abundance) or by means of challenges with parasite
infective larvae, in order to increase parasite abundance (e.g. (Mougeot et al. 2005;
Seivwright et al. 2005; Martinez-Padilla et al. 2007; Mougeot et al. 2007b; Mougeot et al.
2009; Mougeot et al. 2010a; Mougeot et al. 2010b). This has allowed to test the effects of T.
tenuis parasites on carotenoid-based ornamentation (comb redness), and to demonstrate that
males with more parasites, which are typically in poorer condition, circulate fewer
carotenoids, and have less colored combs (Martinez-Padilla et al. 2007; Mougeot et al. 2007b;
Martinez-Padilla et al. 2010; Mougeot et al. 2010a). There are several, non-exclusive,
mechanisms that may explain this effect of parasites on carotenoid-based ornamentation.
First, intestinal parasites may compete with the host for the absorption of carotenoids
acquired through diet, and birds with abundant parasites may have a reduced absorption, and
fewer carotenoid pigments available overall. Second, hosts have to mount an immune
response to the parasite infection, and given that carotenoids have immune-stimulant
properties, those red grouse with more parasites would have fewer carotenoids available for
boosting their immune response, and might be forced to use available pigments for this to the
detriment of using them for displaying redder combs (Mougeot et al. 2007b). Male red grouse
with redder combs have a greater ability to mount a T-cell-mediated immune response than
males with duller combs (Mougeot 2008), and therefore might advertise that they are better
able to respond to a new immunological challenge. This might be because they are overall
healthier (i.e. have fewer parasites) and therefore more able to use available carotenoid to
respond to immune challenges. Third, parasites have been shown to cause oxidative stress, i.e.
to increase levels of oxidative damage and reduce the availability of circulating antioxidant
defenses; (Mougeot et al. 2009b; Mougeot et al. 2010a). This would imply that red grouse
with more parasites might need to use more of their available carotenoid pigments to reduce
oxidative stress and, as a result, would have fewer carotenoids available for increasing
ornamental coloration. In that respect, experiments have shown that reductions in T. tenuis
parasite abundance increase carotenoid-based ornamentation, proportionally to the change in
oxidative balance. Specifically, following parasite purging, those males that increased their
comb redness the most, were those that increased antioxidant defenses and reduced oxidative

damage most, (Mougeot et al. 2010a). This suggests that the effects that parasites have on
carotenoid-based ornaments might be mediated by oxidative stress. Finally, there is evidence
for stress hormones, specifically corticosterone, to also modulate the effects of T. tenuis
parasites on comb redness. After parasite purging, those males that experienced less
physiological stress overall (measured as the overall amount of corticosterone deposited in
feathers grown during experiment), were able to increase their comb redness the most
(Mougeot et al. 2010b).
Conclusion
Carotenoid-based ornaments are a paradigm among sexual signals in animals, especially
in birds. Honest signalling theory predicts that the production and maintenance of reliable
signals must entail costs (or limitations) for the bearer. Carotenoids seem to be a limited
resource for most species, which implies that the expression of carotenoid-based colorations
will be higher in those individuals with better foraging capacity. This implies that carotenoid-
based ornaments may serve as signals of body condition and foraging ability. In addition,
potential links between carotenoids and key physiological processes, like immune responses
and oxidative stress, may result in new pathways for the honesty of these traits.
In the red-legged partridge, the red coloration of the beak, eye rings and legs is based on
carotenoid pigments (mostly astaxanthin plus another red carotenoid still unidentified) that
are metabilized by the bird from dietary carotenoids (lutein and zeaxanthin). These changes
take place at the integuments, and >80% of deposited carotenoids are esterified. Despite the
fact that mate choice experiments have not been performed yet, there is evidence supporting a
signaling role of these traits in this species: individuals mate assortatively according to beak
redness in the wild, and beak and eye ring coloration are maximized during the mating
season. Furthermore, experimental enhancement of male redness resulted in higher breeding
effort by females, who seem to perceive trait expression as a signal of mate quality. In fact,
beak and eye ring redness are dynamic traits related to individual nutritional and health status.
The redness of these ornaments is related to circulating carotenoids, and both are positively
related to T-cell-mediated immunocompetence and negatively to intestinal parasite (i.e.
coccidia) infection levels. Although there are no sound evidences that carotenoids may exert a
powerful antioxidant action in this species yet, they are sensitive to increased oxidative stress,
which reduces their circulating levels and reduces ornament redness. Taken altogether, these
results support the hypothesis that carotenoid-based ornaments function as honest signals of
health (condition, immunocompetence, parasite infection and oxidative status) in the red-
legged partridge.
As in may other tetraonid species, the red grouse exhibit bright red supraorbital combs
pigmented by carotenoids. These ornaments are present in both sexes, but are more developed
in males than in females, and their size and color are maximized during the mating season,
and these ornemants likely play a role both in inter- and intra-sexual selection processes.
Comb redness is possitively associated with body condition and T-cell-mediated immunity,
and negatively affected by coccidia and T. tenuis infection, a main intestinal nematode
parasite. In fact, the negative impact of T. tenuis on red grouse combs has been repeatedly
demonstrated in several field experiments. This negative effect of T. tenuis on comb redness

appears to be mediated, at least in part, by parasite effects on oxidative balance, that seems to
reduce the amount of bioavailable carotenoids for physiological functions. Carotenoid-
allocation to ornaments is modulated by testosterone levels, though this effect depends on the
social or population context. Therefore, as in the red-legged partridge, these results also
support the hypothesis that carotenoid-based ornamentation advertises an individual´s overall
health status (nutritional status, parasite levels, immunocompetence) and that their expression
could be mediated by the environmental context individuals live in..
There are still many relevant questions that deserve further research. In the specific case
of the red-legged partridge, field experiments that simultaneously manipulate hormone levels
and oxidative stress would be necessary to validate the above mentioned results obtained
under captive conditions. This is relevant because wild partridges circulate greater amounts of
carotenoids than captive ones, and also display redder ornaments (Garcia-de Blas et al. in
press). Also, experiments combining carotenoid supplementation and oxidative challenges are
required to fully assess the antioxidant function of carotenoids. The link between carotenoids
and humoral immune responses, and between carotenoids and parasites other than coccidia,
are necessary to obtain a more complete view of the conections between these pigments and
the immune system. In the red grouse, experimental designs directly manipulating oxidative
stress independently of parasite and testosterone levels are also required to fully assess the
importance of this factor as a regulator of carotenoid-based ornamentation.
Beyond these two model species, our understanding of the function of carotenoid-based
signals would benefit from biochemical studies on the transformation of ingested carotenoids
(at the enzymatic and genetic level) and on the pathways involved in all steps of carotenoid
use and metabolism. Studies using radio-labeled carotenoids would be very helpful in that
respect. Furthermore, studies performed to date have paid a disproportionate attention to
plasma carotenoids, whereas other carotenoids in tissues have been neglected. A complete
understanding of the roles of these molecules in the organism, particularly regarding their
controversial antioxidant roles, would require an exploration of their effects in other tissues,
and in phases of the living cycle other than adulthood. Also, opening the mind to new
possible and testable pathways linking carotenoid-based coloration to key physiological
proceses (such as vitamin A metabolim, for instance; Hill and Johnstone 2012) will result in a
better comprehension of the evolution of these traits as indicators of health. Finally, a still
pending issue is to address the connection between ecological and physiological factors
affecting the allocation of carotenoids (among other resources) to ornament expression.
Environmental variables like age- and sex-ratios, population density, climatic conditions,
resource availability, etc. are expected to strongly influence individual trade-offs.
Establishing the connections between environmental factors and physiological trade-offs
remains a major challenge for evolutionary ecologists.
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Chapter X
Dietary Carotenoid Pigments

and Human Health Benefits
Hasna El Gharras
Laboratoire de Chimie Organique et Analytique. Unité de Chimie Agroalimentaire.
Université Sultan Moulay Slimane. Faculté des Sciences et Techniques. Béni-Mellal.
Morocco
Dedicated to the memory of my dear father
Abstract
Nutrition plays an important role in the treatment of many diseases, and the right
choice of nutrients can help to prevent disorders and improve the quality of life.
Carotenoid pigments are a group of bioactive compounds that are of interest to the food
scientists, nutritionists and food industries due to their positive impact on human health
and their economic benefits. Carotenoids are responsible for the attractive colour of many
plant food (mainly fruit and vegetables), which is perhaps the first attribute that
consumers assess when determining the quality and appearance of a product, and
therefore conditions its acceptability.
In addition, carotenoids have diverse biological functions and activities, such as the
well-known provitamin A activity, antioxidant capacity and enhancement of the immune
system. There are an extensive number of factors affecting the efficient incorporation of
these phytochemicals from the diet, although in many cases no biological activity will be
put in action within the consumer body (animal or human) without a first visual
attraction.
Keywords: Carotenoids, sources, human health benefits, cancer, cardiovascular disease

Email: [email protected].

200 Hasna El Gharras
Introduction
Carotenoids are a group of natural pigments responsible for the yellow, orange or red
colour of many foods. Besides the well-known provitamin A activity of some of these
compounds, they have also been associated with lowered risk of developing degenerative
diseases such as cancer, cardiovascular diseases, cataract and macular degeneration. Some
fruits and vegetables are rich sources of carotenoids, as well as other bioactive
phytochemicals, and many vegetables are widely available year-round.
Reliable data on carotenoids provide information to consumers and public health workers
on food sources of these compounds, and are needed to assess dietary carotenoid intake and to
serve as basis for studies on the physiological actions of carotenoids and their relationship to
health and diseases. Although more than 700 carotenoids have been described in Nature, not
all natural sources of them are present in our normal diet. It is estimated that we only have
access to about 40 carotenoids that can be absorbed, metabolised, and/or used in our bodies.
That number is reduced to 6 if we consider the carotenoid profile that is usually detected in
human blood plasma. This group includes α- and β-carotene, lycopene, β-cryptoxanthin,
zeaxanthin and lutein, which are regularly present in the foods [1].
Carotenoids, which belong to the chemical group known as isoprenoid polyenes, are
lipid-soluble, yellow-orange-red pigments found in all higher plants and some animals. Due
to the numerous conjugated double bonds and cyclic end groups, carotenoids present a variety
of stereoisomers with different chemical and physical properties. The most important forms
commonly found among carotenoids are geometric (E-/Z-). A double bond links the two
residual parts of the molecule either in an E-configuration with both parts on opposite sites of
the plane, or a Z-configuration with both parts on the same side of the plane. Geometrical
isomers of this type are interconvertible in solution. This stereoisomerism exerts a marked
influence on the physical properties.
Isomers differ not only in their melting points, solubility and stability, but also in respect
to absorption affinity, colour and colour intensity. Animals cannot synthesize carotenoids, so
their presence in the body is due to dietary intake of foods such as pink salmon flesh. The
plumage of many birds owes its colour to carotenoids. Plant, algae, fungal and synthetic
(nature-identical) carotenoids are permitted as colorants in food products, but not animal
carotenoids. Carotenoids owe their name to carrots (Daucus carota), and xanthophylls
(originally phylloxanthins) are derived from the Greek words for yellow (xanthos) and leaf
(phyllon). Together with anthocyanins, carotenoids are the most complex class of natural food
colorants with over 750 different structures identified.
This review will focus on the sources, chemistry, absorption, transport and the role of
carotenoids in human health.
Dietary Sources, Chemistry, Absorption

and Transport of Carotenoids
Carotenoids are a family of pigmented compounds that are synthesized by plants and
microorganisms but not animals.

Dietary Carotenoids Pigments … 201
In plants, they contribute to the photosynthetic machinery and protect them against
photo-damage. Fruits and vegetables constitute the major sources of carotenoid in human diet
[2–4]. They are present as micro-components in fruits and vegetables and are responsible for
their yellow, orange and red colours. Carotenoids are thought to be responsible for the
beneficial properties of fruits and vegetables in preventing human diseases including
cardiovascular diseases, cancer and other chronic diseases [5, 6]. They are important dietary
sources of vitamin A [5]. In recent years the antioxidant properties of carotenoids has been
the major focus of research [5]. More than 600 carotenoids have so far been identified in
nature.
However, only about 40 are present in a typical human diet. Of these 40, about 20
carotenoids have been identified in human blood and tissues.
Major dietary carotenoids include the hydrocarbons, ß-carotene, α-carotene and lycopene
and the xanthophylls, or oxygen-containing carotenoids, ß-cryptoxanthin, lutein and
zeaxanthin (Figure. 1). The estimation of carotenoid intakes has been made possible through
the publications of the qualitative and quantitative carotenoid content of commonly consumed
foods [1, 7, 8].
Figure 1. The structures of the predominant carotenoids found in human plasma.
All carotenoids possess a polyisoprenoid structure, a long conjugated chain of double

bond and a near bilateral symmetry around the central double bond, as common chemical
features [9]. Different carotenoids are derived essentially by modifications in the base
structure by cyclization of the end groups and by introduction of oxygen functions giving
them their characteristic colours and antioxidant properties.
Due to the presence of the conjugated double bonds, carotenoids can undergo
isomerization to cis-trans isomers. Although the trans isomers are more common in foods and
are more stable very little is known about the biological significance of carotenoid
isomerization in human health.
Although carotenoids are present in many common human foods, deeply pigmented
fruits, juices and vegetables constitute the major dietary sources with yellow-orange
vegetables and fruits providing most of the ß-carotene and α-carotene, orange fruits providing
α-cryptoxanthin, dark green vegetables providing lutein and tomatoes and tomato products
lycopene [2, 10].

Table 1. Examples of major contributors of carotenoids in North American diet
Carotenoid Food source Amount

ß-Carotene Apricot, dried 17600
Carrots, cooked 9771
Spinach, cooked 5300
Green Collard 5400
Cantaloupe 3000
Beet Green 2560
Broccoli, cooked 1300
Tomato, raw 520
α-Carotene Carrots, cooked 3723
Lycopene Tomatoes, raw 3100
Tomato juice 10000
Tomato paste 36500
Tomato ketchup 12390
Tomato sauce 13060
ß-Cryptoxanthin Tangerine 1060
Papaya 470
Lutein Spinach, cooked 12475
Green collard 16300
Beet, green 7700
Broccoli, cooked 1839
Green peas, cooked 1690
Table 1 shows typical food sources and amounts of carotenoids present. In the case of
lutein and zeaxanthin, they are also present in high concentrations in egg yolks [3]. Due to the
unsaturated nature of the carotenoids they are subject to changes due mainly to oxidation.
However, other factors such as temperature, light and pH can also produce alterations that can
influence the color of foods as well as their nutritional value [11]. In general carotenoid
content of foods is not altered to a great extent by common household cooking methods such
as microwave cooking, steaming and boiling but extreme heat can result in oxidative
destruction of carotenoids [12]. Although several nutrient data basis provide estimations of
the daily intake of carotenoids by humans these values vary considerably due to the
sensitivity and specificity of different analytical methods that are used in the detection of
these phytochemicals. Also consideration is often times not given to seasonal variations and
methods of processing of foods containing carotenoids [4]. In spite of the recognition of the
beneficial role of carotenoids in human health they are not considered as essential nutrients
and as such do not have a dietary reference intake (DRI) value assigned to them.
ß-Carotene
ß-carotene is the most widely studied carotenoid and is one of the major carotenoids in
our diet and in human blood and tissues [13, 14]. Major sources of dietary ß-carotene include
green leafy vegetables as well as orange and yellow fruits and vegetables (Table 2).

Table 2. ß-Carotene content of foods [20]
Food Content (mg/100 g wet wt)a

Carrots, raw 18.3
Mangos, canned 13.1
Sweet potato, cooked 9.5
Carrots, cooked 8.0
Pumpkin, canned 6.9
Kale, cooked 6.2
Spinach, raw 5.6
Spinach, cooked 5.2
Winter butternut squash 4.6
Swiss chard, raw 3.9
Apricots, raw 2.6
Pepper, red, raw 2.4
Pepper, red, cooked 2.2
Cantaloupe, raw 1.6
Lettuce, romaine, raw 1.3
Tomato paste 1.2
a
Edible portion.
However, the bioavailability of ß-carotene from green leafy vegetables such as spinach is
thought to be low [15]. Factors other than the food vehicle are thought to be important in the
bioavailability of ß-carotene.
These include, cooking, chopping and the presence of dietary fat, all of which improve
the bioavailability [16, 17]. Of the 50 different carotenoids that can be metabolized into
vitamin A, ß-carotene has the highest provitamin A activity. However, this bioconversion is
highly variable among individuals and perhaps food sources. Tang et al. reported that spinach
ß-carotene conversion to retinal averaged 21 to 1 (range: 10–47 to 1) and carrot ß-carotene
conversion to retinol was 15 to 1 (range: 8–25 to 1) [18]. The National Health and Nutrition
Examination Survey, 1999–2000 for the US population reported dietary intakes of ß-carotene
to be 5.4 ± 0.3 (mean ± SE, n = 8604) [19]. However, intakes much higher than this are
possible through over-the-counter supplements that are commonly available in health food
stores in doses of 3–20 mg/capsule.
Lycopene
Dietary lycopene is derived predominately from tomatoes and tomato products. In the
United States, more than 85% of lycopene consumed is from tomato products although other
dietary sources include dried apricots, guava, watermelon, papaya, and pink grapefruit (Table
3) [20]. Similar to the effect on ß-carotene bioavailability, heating tomatoes in oil was found
to be associated with an increase in lycopene absorption when compared to the absorption for
unprocessed tomato juice [21].
Also, the lycopene bioavailability was greater from a single dose of tomato paste than it
was from an equal lycopene dose from fresh tomatoes [22].

Table 3. Lycopene content of foods [20]
Food Content (mg/100 g wet wt)a

Tomato paste 29.3
Catsup 17.0
Tomato puree 16.7
Pasta sauce 16.0
Tomato sauce 15.9
Tomato soup 10.9
Tomato, canned, whole 9.7
Tomato juice 9.3
Watermelon, raw 4.9
Tomato, cooked 4.4
Tomato, raw 3.0
a
Edible portion.
Interestingly, these studies support the findings of Giovannucci et al., that the association
between consumption of various tomato products and the risk of prostate cancer depends on
the bioavailability of lycopene [23]. That is, an association was found with the consumption
of tomato paste or sauce and not with consumption of minimally processed tomato juice.
Typical dietary intakes of lycopene in the United States are about 2–5 mg/d, which probably
reflects a diet high in tomato and tomato products e.g., pizza [24-26]. Supplemental lycopene
is also available, usually in amounts of 5–10 mg/capsule.
Lycopene absorption from dietary sources is influenced by several factors including the
breakup of the food matrix containing lycopene, cooking temperatures and the presence of
lipids and other lipid soluble compounds including other carotenoids. Absorption of lycopene
is similar to other lipid soluble compounds and is absorbed across the gastrointestinal tract via
a chylomicron mediated mechanism [27]. In general, 10–30% of the dietary lycopene is
absorbed by humans [22, 28]. It is absorbed equally efficiently from different sources of
lycopene including tomato sauce, tomato juice and tomato oleoresin capsules [22, 29]. Other
studies have shown that lycopene is absorbed more efficiently from processed tomato
products compared to raw tomatoes. The increased absorption of lycopene from processed
tomato products is attributed to the presence of cis-isomers of lycopene [21].
Absorbed lycopene is distributed throughout the body via the circulatory system.
Lycopene is the most predominant carotenoid in human plasma with a half-life of about 2–3
days [30]. Although the most prominent geometric isomers of lycopene in plants are the all-
trans, in human plasma, lycopene is present as an isomeric mixture containing 50% of the
total lycopene as cis isomer [31]. When animals were fed lycopene containing predominantly
the all trans isomeric form, serum and tissue lycopene showed the presence of cis lycopene
[32]. Similar results were also observed in human serum. Testes, adrenal glands, prostate,
breast and liver were shown to have the highest levels of lycopene in humans [32, 33].
Lycopene in the tissues undergoes oxidation and metabolism. Several oxidized form of
lycopene and polar metabolites have recently been isolated and identified [34]. The biological
significance of these findings is not clear at present and is the subject of many studies.

Lutein and Zeaxanthin
The two foods that were found to have the highest amount of lutein and zeaxanthin are
spinach and kale (Table 4) [20, 35]. Other major sources include broccoli, peas, and brussel
sprouts. Another good source of lutein and zeaxanthin to consider is egg yolk. Though the
values are relatively low in eggs, recent data suggest that lutein and zeaxanthin from this food
source are highly bioavailable [36, 37, 38]. Data on the lutein content of foods include
zeaxanthin, i.e. lutein + zeaxanthin, making examination of specific effects of dietary lutein
difficult.
However, in terms of food sources, human metabolism, and tissue storage, lutein and
zeaxanthin are similar. Intakes of lutein and zeaxanthin in the US are generally lower than
that of ß-carotene or lycopene, but levels of ~3 mg/d can be easily achieved with a high fruit
and vegetable diet [26].
Although lutein and zeaxanthin are considered to be major carotenoids in the US diet,
data from the 1987 and 1992 National Health Interview Surveys suggest that there was a
decline in lutein intake (from dark green leafy vegetables) [39]. Currently, there are a variety
of supplement products available in health food stores that contain lutein in amounts of 6–25
mg/capsule. At this point, lutein is found in many multi-vitamin products in much smaller
amounts (0.25 mg/capsule).
Table 4. Lutein and zeaxanthin content of foods [20]
Food Content (mg/100g wet wt)a

Kale, cooked 15.8
Spinach, raw 11.9
Spinach, cooked 7
Lettuce, romaine, raw 2.6
Broccoli, raw 2.4
Broccoli, cooked 2.2
Summer squash, zucchini 2.1
Corn, sweet, cooked 1.8
Peas, green, canned 1.4
Brussels sprouts, cooked 1.3
Corn, sweet, canned 0.9
Beans, green, cooked 0.7
Beans, green, canned 0.7
Beans, green, raw 0.6
Okra, cooked 0.4
Cabbage, white, raw 0.3
Egg yolk, medium b 0.3
Celery, raw 0.2
Orange, raw 0.2
Tomato, past 0.2
a
Edible portion.
b
[36, 37].

Absorption
Carotenoids, being fat-soluble, follow the same intestinal absorption path as dietary fat.
Release from the food matrix and dissolution in the lipid phase appears to be important initial
steps in the absorption process. Carotenoids are thought to be absorbed by the small intestinal
mucosa via a passive, diffusion process, although recent studies in Caco-2 cell monolayers
indicate that intestinal absorption is a facilitate process [35, 40]. Fatty acid esters of
xanthophylls are cleaved in the lumen of the small intestine prior to uptake by the mucosa.
Carotenoids are taken up by the mucosa of the small intestine and packaged into
triacylglycerol-rich chylomicrons. ß-Carotene and other provitamin A carotenoids are partly
converted to vitamin A, primarily retinyl esters, in the intestinal mucosa, and both carotenoids
and retinyl esters are incorporated into chylomicrons and secreted into lymph for transport to
the liver [27].
Transport
In fasting serum, hydrocarbon carotenes are found primarily in low-density lipoprotein

(LDL), while xanthophylls (containing one or more polar functional groups) are more evenly
distributed between LDL and high-density lipoprotein (HDL) [41]. The carotenes, being
lipophilic, are located in the core of lipoprotein, which may explain why they do not transfer
between lipoproteins at an appreciable rate [42]. The xanthophylls, being more polar, are
probably located on the surface of lipoproteins, and are likely to undergo more rapid surface
transfer, resulting in the observed apparent equilibration between LDL and HDL.
Prevention of Chronic Diseases
Carotenoids and Cancer Lycopene
The evidence in support of lycopene in the prevention of chronic diseases comes from
epidemiological studies [23, 43-47] as well as tissue culture studies using human cancer cell
lines [48–50], animal studies [32, 51-54] and also human clinical trials [55-59].
Of all the cancers, the role of lycopene in the prevention of prostate cancer has been
studied the most. An inverse relationship between the consumption of tomatoes and the risk
of prostate cancer was first demonstrated in a 1995 publication [60]. Lycopene was suggested
as being the beneficial compound present in tomatoes. A follow up met analysis of 72
different studies in 1999 showed that lycopene intake as well as serum lycopene levels were
inversely related to several cancers including prostate, breast, cervical, ovarian, liver and
other organ sites [44]. Several other studies since then demonstrated that with increased
intake of lycopene and serum levels of lycopene the risk of cancers were reduced significantly
[43, 61-63]. To study the status of oxidative stress and antioxidants in prostate cancer patients
a study was undertaken [55]. Results showed significant differences in levels of serum
carotenoids, biomarkers of oxidation and prostate specific antigen (PSA) levels in these
subjects. Although there were no differences in the levels of ß-carotene, lutein, cryptoxanthin,

vitamin E and A between the cancer patients and their controls, levels of lycopene were
significantly lower in the cancer patients. As expected the PSA levels were significantly
elevated in the cancer patients who also had higher levels of lipid and protein oxidation
indicating higher levels of oxidative stress in cancer patients. In the same study, serum PSA
levels were shown to be inversely related to the serum lycopene [55]. Other carotenoids did
not show similar inverse relationship. In more recently reported studies, lycopene was shown
to decrease the levels of PSA as well as the growth of prostate cancer in newly diagnose
prostate cancer patients receiving 15 mg of lycopene daily for 3 weeks prior to radical
prostactomy [57, 63]. In another study when tomato sauce was used as a source of lycopene,
providing 30 mg lycopene/day for three weeks preceding prostatectomy in men diagnosed
with prostate cancer, serum and prostate lycopene levels were elevated significantly [59].
Oxidative damage to DNA was reduced and serum PSA levels declined significantly by 20%
with lycopene treatment. Although small in number, these observations raise the possibility
that lycopene may be involved not only in the prevention of cancers but may play a role in the
treatment of the disease.
Other than prostate cancer there is now growing evidence in support of the protective role
of lycopene in cancers of other sights including breast, lung, gastrointestinal, cervical, ovarian
and pancreatic cancers [44]. Tissue culture studies using human cancer cell lines have shown
that their growth is inhibited significantly in the presence of lycopene in the growth media
[54, 64, 65]. Similarly, several animal studies have also confirmed the inverse association
between dietary lycopene and the growth of both the spontaneous and transplanted tumors
[66, 67]. Human dietary intervention studies are now beginning to be undertaken to study the
role of lycopene in breast, ovarian and cervical cancers.
Overall, epidemiological studies, in vitro tissue culture studies, animal studies and now
some human intervention studies are showing that increased intake of lycopene will result in
increased circulatory and tissue levels of lycopene. In vivo lycopene can act as a potent
antioxidant and protect cells against oxidative damage and there by prevent or reduce the risk
of several cancers.
Further studies are needed to get further proof and to gain better understanding of the
mechanisms involved.
One of the earliest case-control studies examining the relationship between lycopene-rich
foods and cancer risk reported that weekly tomato consumption associated with a 40%
reduction in risk for esophageal cancer [68].
Several studies have reported protective effects for diets rich in tomato products against
gastric cancer [46, 69-72] whereas others have found no relationship [73, 74]. A study of
prospectively collected serum samples from a cohort was used to compare serum
micronutrients from those developing pancreatic cancer and matched controls [75]. The
greatest difference was observed for lycopene. However, because lycopene uptake and serum
concentrations may be related to the digestibility of dietary lipids, diseases of the pancreas
could significantly reduce absorption and perhaps confound such studies. In a single study
examine the role of tomatoes, lycopene and bladder cancer risk [76], serum micronutrient
profiles were quantitated, and only lycopene and selenium concentrations were found to be
inversely related to risk. However, others have found no such relationship [77-79]. Less
consistent results have been observed for a relationship between estimated lycopene intake or
serum lycopene concentrations and breast cancer risk [80, 81]. A recent study of dietary
intake interview of 4697 women in Finland showed no relationship between estimate intake

of tomato products and breast cancer risk [82]. However, a case-control study from Boston
examined the relationship between breast adipose tissue lycopene concentrations and breast
cancer risk, and an inverse association between lycopene and breast cancer risk was observed
[83].
In a study conducted in a cohort of Seventh Day Adventists, dietary and lifestyle factors
were evaluated and related to subsequent risk of prostate cancer in a follow up of 6 years.
Multivariate analysis indicated that increasing age and previous prostate problem were among
the risk factors, whereas, unexpectedly, smoking and alcohol consumption were not. Among
dietary factors, high tomato consumption of five or more per week was associated with a
significantly decreased risk [84]. An inverse association between high intake of tomato
products and prostate cancer risk was confirmed in an analysis of the US Health Professionals
Follow-up Study [50]. A dietary frequency questionnaire was applied at baseline and related
to the incidence of prostate cancer that was assessed biennially. It was shown that lycopene
intake but not the overall intake of fruits and vegetables was inversely related to prostate
cancer risk. Among tomato-derived products, this association was strongest for tomato
sauces, followed by tomatoes and pizza. No relationship was found for tomato juice. This
may be explained by the low bioavailability of lycopene from unprocessed juice [21]. A
summary of the epidemiologic studies evaluating lycopene and cancer risk was reported
recently [85].
The reports of lycopene concentrations being relatively high in the prostate compared to
other tissue [86] suggests that this carotenoid may exert unique biological effects at this site
which may be related to cancer prevention. Clinton et al. have observed 14–18 different
isomers of lycopene in prostate [87]. Approximately 80% of total lycopene is found as cis
isomers in prostate tissue, compared to 50% in blood [26] and 5–10% in foods [87]. The
biological significance of this is not known. cis isomers differ in molecular shape from the
all-trans form and, therefore, may differ in metabolism (as has been reported for ß-carotene
[88]. A chemoprevention effect of lycopene remains to be determined.
Observational Epidemiology
Although epidemiological studies cannot provide evidence of causal relationship, they

have proven useful in evaluating the possible protective effects of foods or food components
in disease prevention. Observational epidemiologic studies have been very consistent in
showing that people who consume higher dietary levels of fruits and vegetables have a lower
risk of certain types of cancer. The results of 10 of 17 case-control studies show that a high
intake of fruits and vegetables that are rich in carotenoids has been associated with decreased
risk of cancer at a number of common sites. This association appears to be most consistent for
lung and stomach cancer [89, 90]. Given the variability in study design and statistical analysis
among the epidemiologic studies, the consistency of the relationship between fruit and
vegetable intake and lung cancer risk strongly suggests a real effect of fruit and vegetables on
cancer prevention. It has been suggested that carotenoids are the chemopreventive agents in
fruits and vegetables. There are several mechanisms by which a carotenoid can function in
cancer prevention. As a provitamin A, a carotenoid would have an effect on cellular
differentiation and proliferation [91-93]. Moreover, the antioxidant function could prevent
free radical-induced damage to cellular DNA and other molecules [94]. Immunomodulatory

effects could enhance immune surveillance in tumorigenesis [95] and enhanced cell–cell
communication would restrict clonal expansion of initiated cells [96].
ß-Carotene
The epidemiologic observations, along with what is known about carotenoid function,
have led to further study of the effect of ß-carotene on cancer risk. Although other types of
cancers have been evaluated, e.g. breast cancer [97] with a reduced risk with increased dietary
intake, the relationship between ß-carotene and lung cancer has been most studied and the
data have been more consistent [98]. Evidence for ß-carotene as the chemoprotective agent in
fruits and vegetables comes from prospective epidemiologic studies in which 11 of 15 have
demonstrated a significant inverse relation of ß-carotene intake and/or plasma level and risk
of lung cancer [85, 99].
Intervention Trials
Based on the epidemiological evidence, long-term large randomized intervention trials

were designed to test the efficacy of high doses of ß-carotene (20–30 mg/d) in the prevention
of cancer. Surprisingly, the results from two trials provide possible evidence of harm from ß-
carotene supplements in relation to cancer among high-risk individuals such as smokers and
asbestos workers [100, 101] and no effect (neither beneficial nor detrimental) in a generally
well-nourished population [102]. However, in the Linxian (Chinese) Cancer Prevention
Study, it was found that supplementation with ß-carotene, vitamin E and selenium led to a
significant reduction in total mortality (9%), especially from cancer (13%), particularly
stomach (21%) [103]. The positive results of the Chinese study may reflect the correction of a
nutrient deficiency in this study population.
Therefore, the usefulness of high doses of ß-carotene in high-risk individuals needs to be
re-evaluated. It should be noted that, like the earlier epidemiologic studies that lead to these
intervention studies, there are recent studies that report a similar relationship between diet
and/or blood ß-carotene levels and cancer prevention [90,104-108]. It is probable that ß-
carotene is serving as a marker of increase fruit and vegetable intake and therefore of all
components that have cancer-prevention potential, e.g. vitamin C, folic acid, other
carotenoids.
Carotenoids and Ocular Diseases
Lutein and zeaxanthin have been suggested to be protective against certain eye diseases.
Given that lutein and zeaxanthin, and their metabolites, are the only carotenoids found in the
retina [109, 110] and lens [111] and that the retina and lens suffer oxidant damage, these two
antioxidant nutrients may serve a unique role in the protection against eye disease.

Age-Related Macular Degeneration
Lutein and zeaxanthin are concentrated in the retina at the macula lutea and are
responsible for the yellow colour that gives the macula its name. Although the term macula
lutea, or macula, originally applied to the yellow pigment, it is now commonly used to refer
to the corresponding region of the retina. This region includes the fovea, which is the region
that is responsible for our highest visual acuity and which contains the highest density of cone
photoreceptors. Toward the periphery of the retina, the concentration of zeaxanthin declines
rapidly whereas the concentration of lutein declines gradually [112-114]. By preventing light
initiated oxidative damage to the retina and retinal pigment epithelium, the macular pigment
(lutein and zeaxanthin) may protect against age-related deterioration.
Lutein and zeaxanthin are thought to protect the eye in two ways. One hypothesis is that
the macular pigment filters blue light, which is particularly damaging to photoreceptors and to
the retinal pigment epithelium [115, 116], and lutein and zeaxanthin absorb blue light. The
second hypothesis is that these carotenoids act as antioxidants to limit the oxidant stress of the
tissue that results from metabolism and light [115, 117, 118]. It has been shown that one of
the ways light damages the retina is by generation of free radicals that lead to peroxidation of
membrane lipids [115,116]. Carotenoids have long been known as powerful antioxidants
[117, 119, 120].
Evidence from human studies suggest that dietary intake of carotenoids can lead to their
accumulation in the retina and, therefore, may provide protection against retinal degeneration.
In a recent prospective study [121], eleven subjects modified their usual daily diets by adding
60 g/d of spinach (containing 11 mg lutein and 0.3 mg zeaxanthin) for 15 weeks. Eight
subjects had increases in serum lutein and macular pigment density, two subjects showed
substantial increases in serum lutein but not macular pigment, and one subject showed no
changes in serum lutein or macular pigment density. Although the results were varied,
augmentation of macular pigment through dietary modification appears to be possible for
many people. Similar to this study, Landrum et al. have found that supplementation with
lutein (30 mg/d for 140 days) resulted in increased serum levels of lutein and corresponding
increases in the concentration of lutein in the macula on the human eye [122].
Population studies provide evidence to suggest that protection from AMD can be
obtained from lutein [85]. Investigators from the Eye Disease Case-Control study [123]
reported that patients in the group with the highest level of plasma lutein/zeaxanthin had a
decreased for AMD. In a subsequent study [124], the investigators found that protection from
AMD was associated with dietary intake of specific carotenoids. In this case-control study,
AMD patients and matched control subjects (who had other eye problems) were divided into
five groups on the basis on their intake of various nutrients from foods. The nutrient class that
was found to have the strongest protective effect against AMD was carotenoids. Those in the
highest quintile of carotenoid intake had a 43% lower risk of developing AMD compared to
those in the lowest quintile. The authors then investigated which specific carotenoids were
responsible for this effect. The strongest association with protection from AMD was found for
lutein/zeaxanthin. Subjects who were in the highest quintile for their intake of lutein
/zeaxanthin had a 57% lower risk of advanced AMD compared to those in the lowest quintile.
This odds ratio was calculated from a multivariate node, indicating that the reduction in odds
associated with consumption of lutein/zeaxanthin was independent of effects from other
carotenoids. A final analysis was performed by arranging the subjects on the basis of

consumption of specific foods. In a multivariate model that included consumption of broccoli,

cabbage-related vegetables, carrots, spinach or collard greens, sweet potatoes, and winter
squash, only consumption of spinach was associated with protection from AMD. Subjects in
the highest quintile for consumption of spinach had an 86% lower odds ratio of advanced
AMD.
It should be noted that not all studies have found an association between serum
carotenoids and protection from AMD. For example, a case-control study that used the
population for the Beaver Dam Eye Study found no such association for lutein or zeaxanthin,
but did find a weak protective effect of serum lycopene [85, 125, 126]. However, this study
included many fewer subjects than did the Eye Disease Case-Control study.
Recent studies suggest that lutein supplementation may improve visual function in AMD
patients. Falsini et al., evaluated the influence of short-term antioxidant supplementation on
retinal function in age-related maculopathy patients and control subjects (54–84 years) by
recording focal electroretinograms (FERG) [127]. The supplementation regimen included 15
mg/d lutein for 180 days. They reported a significant increase in amplitude change of FERG
in patients and controls with antioxidant supplementation. However, the supplementation
regimen included vitamin E and nicotinamide. Therefore, the specific effects of any one
component could not be assessed. It was concluded that increasing the level of retinal
antioxidants may influence macular function early in the disease process, as well as in normal
aging. In another study, it was reported that AMD patients (n=59) supplemented with various
antioxidants including 10 mg lutein resulted in positive effects in visual function, including
improved contrast sensitivity, glare recovery, and Snellen acuity [128].
Cataracts
Lutein and zeaxanthin are the only two carotenoids that have been identified in the
human crystalline lens [111]. Like the antioxidant enzymes found within the lens, the lipid-
based lutein and zeaxanthin, are primarily found in the metabolically active epithelium/outer
cortex of the lens [129], and therefore, may have a preferential role in protecting against UV-
induced oxidative damage. This function would be similar to the role that lutein and
zeaxanthin play in the retina, where they are optimally located to reduce risk from blue light
[109].
Few studies have specifically examined the relationship between lutein and zeaxanthin
with cataract risk. In a recent report, Chasan-Taber et al. (1999) [130] observed in women that
those with the highest intake of lutein and zeaxanthin had a 22% decreased risk of cataract
extraction compared with those in the lowest quintile. Brown et al. (1999) [131] also observed
that there was a lower risk of cataract extraction in men with higher intakes of lutein and
zeaxanthin but not other carotenoids. In this study, men in the highest fifth of lutein and
zeaxanthin intake have a 19% lower risk of cataract relative to men in the lowest fifth. Mares-
Perlman and coworkers observed in women a significant inverse trend across quintiles of
lutein intake [125, 126]. Women in the highest quintile of lutein intake (median 0.95 mg/d)
had a 27% lower prevalence of nuclear cataract than women in the lowest lutein intake
quintile (median 0.28 g/d). The trend was in the same direction in men, but did not reach
significance. Hankinson et al. (1992) [132] reported that the rate of cataract surgery was

associated with lower intakes of lutein rich foods such as spinach and other green vegetables.
These studies suggest that dietary lutein and zeaxanthin play a role in cataract prevention.
In a double blind study involving dietary supplementation with lutein (15 mg/d, 3
times/wk for up to 2 years, n = 5), α-tocopherol (100 mg/d, 3 times/wk (n = 6) or placebo (n =
6) in patients with cataracts visual performance (visual acuity and glare sensitivity) improved
in the lutein supplemented group only [133].
In a recent study, it was reported that a high dose combination of antioxidants (vitamins
C and E, ß-carotene, and zinc) had no significant effect in the development or progression of
cataract [134]. The Linxian trial [135] examined the role of antioxidants in prevention of
cataract, and the effect was not clear. The intervention was a combination dose of 14 vitamins
and 12 minerals. Therefore, a specific role of any one nutrient could not be accurately
evaluated. The multivitamin component demonstrated that nutrition can modify the risk of
nuclear cataract, but specific nutrients were not evaluated.
Also, the population examined had suboptimal nutritional intakes at the study start and
the effect may have been due to a correction of certain nutrient deficiencies.
The Roche European–American Anticataract Trial (REACT) was carried out to examine
if a mixture of oral antioxidant micronutrients (ß-carotene, 18 mg/d; vitamin C, 750 mg/d;
vitamin E, 600 mg/d) would modify the progression of age-related cataract [136]. This was a
multi-center prospective double masked randomized placebo-controlled 3 yr trial in 445
patients with early age-related cataract. REACT demonstrated a statistically significant
positive treatment effect after 2 years for US patients and for both subgroups (US, UK) after 3
years, but no effect for the UK patients alone. The conclusion from this study was that daily
supplementation with these nutrients for 3 years produced a small deceleration in progression
of age-related cataract.
In recent studies of the AREDS Group [134, 137], it was reported that a high dose
combination of antioxidants (including ß-carotene and also vitamins C and E, and zinc) had
no significant effect on the development or progression of cataract but significantly reduce
the risk of age-related macular degeneration (AMD). It was found that people at high risk for
developing advanced stages of AMD (people with intermediate AMD or advanced AMD in
one eye but not the other eye) lowered their risk by about 25% when treated with the high
dose combination.
In the same high-risk group the nutrients reduced the risk of vision loss caused by
advanced AMD by about 19%. For those subjects who had either no AMD or early AMD, the
nutrients did not provide a measured benefit. Because single nutrients were not evaluated,
specific effects could not be determined. However, it should be noted that ß-carotene is not
found in the lens [111] or macula [109] of the eye and, therefore, the result may be due to the
other nutrients tested.
Cardiovascular Disease
Several reports have now appeared in the literature in support of the role of lycopene in
the prevention of CVD [138–140]. The strongest population-based evidence comes from a
multicenter case-control study (EURAMIC) that evaluated the relationship between adipose
tissue antioxidant status and acute myocardial infraction [141, 142]. Subjects that included
662 cases and 717 controls were recruited from 10 different European countries. Results of

this study showed a dose-response relationship between adipose tissue lycopene and the risk
of myocardial infraction. Another study that compared the Lithuanian and Swedish
populations showed lower lycopene levels to be associated with increased risk and mortality
from coronary heart disease (CHD) [143]. Serum cholesterol levels have traditionally been
used as a biomarker for the risk of CHD. Oxidation of the circulating low density lipoprotein
(LDL), that carries cholesterol into the blood stream, to oxidized LDL (LDLox) is also
thought to play a key role in the pathogenesis of arteriosclerosis which is the underlying
disorder leading to heart attack and ischemic strokes [144–146]. Lycopene was also shown to
significantly reduce the levels of oxidized LDL (LDLox) in subjects consuming tomato sauce,
tomato juice and lycopene oleoresin capsules as sources of lycopene [147]. In another small
study, lycopene was shown to reduce serum total cholesterol levels and thereby lowering the
risk of CVD [148].
Although epidemiological studies have provided convincing evidence in support of the
protective role of lycopene in CVD, these observations need to be validated by conducting
well controlled human intervention studies in the future. Important aspects of such studies
will be to use well-defined subject populations, standardized outcome measures of oxidative
stress and the disease, and lycopene ingestion that is representative of normal healthy dietary
intakes.
Osteoporosis
Oxidative stress and antioxidants may contribute to the pathogenesis of the skeletal
system including osteoporosis, the most prevalent metabolic bone disease [149]. Oxidative
stress control the functions of both osteoclasts [150, 151] and osteoblasts [152].
Endogenous [151, 153] and synthetic [48] antioxidants counteract the effects of oxidative
stress in these cells. Recent studies reported that antioxidants from natural sources, such as
the lycopene from tomatoes, can also counteract the damaging effects of oxidative stress. The
findings that lycopene has a stimulatory effect on the cell proliferation [154] and the
differentiation marker alkaline phosphatase of osteoblasts [154, 155], as well as its inhibitory
effects on osteoclasts formation and resorption [156, 157] are evidence of the involvement of
lycopene in bone health and warranted further investigation in clinical studies.
Epidemiological studies have shown that oxidative stress is associated with osteoporosis
and that antioxidants may counteract this effect. Certain antioxidants including vitamin C, E
and ß-carotene may reduce the risk of osteoporosis [158–161] and counteract the adverse
effects of oxidative stress on bone that are produced during strenuous exercise [160] and
among smokers [158]. Osteoporotic women have been shown to have reduced levels of
antioxidant vitamins and enzymes indicating a decrease in their antioxidant defences [162].
A recently published clinical study showed a direct correlation between serum lycopene
and decrease in the risk of osteoporosis among postmenopausal women for the first time
[163]. The relationship between serum lycopene, oxidative stress parameters and bone
turnover markers in postmenopausal women were investigated. Study participants were asked
to complete a seven-day food intake record prior to giving fasting blood samples. Oxidative
stress parameters, total antioxidant capacity, serum lycopene and the bone turnover markers
including bone alkaline phosphatase (bone formation) and cross-linked N-telopeptides of type

I collagen (NTx) (bone resorption) were measured in the serum samples. Results showed a
direct correlation between lycopene intake and serum lycopene levels.
Increase in serum lycopene levels resulted in significant decreases in protein oxidation
and NTx values [163]. Based on these results an important role for lycopene mediated via its
antioxidant property in reducing the risk of osteoporosis is suggested. Dietary intervention
studies with varying levels of lycopene are currently being conducted with the objective of
demonstrating the beneficial effects of lycopene in the prevention and management of
osteoporosis.
Other Human Diseases
Since the recognition of lycopene as a potent antioxidant, and its preventive role in
oxidative stress mediated chronic diseases, researchers are beginning to investigate its role in
other human diseases. Hypertension is commonly referred to as the ‘silent killer’ since
symptoms of this disorder are not observed until a more advanced and fatal stage of the
diseases is reached. A causal relationship between oxidative stress and the incidence of
hypertension is now recognized. The antioxidant property of lycopene has attracted scientific
research into its protective role in hypertension.
A recent study showed lycopene supplementation at the rate of 15 mg per day for 8
weeks to significantly decrease systolic blood pressures from the baseline value of 144mmHg
to 134mmHg in mildly hypertensive subjects [164, 165]. In another study a significant
reduction in plasma lycopene was observed in the hypertensive patients compared to normal
subjects [166]. When patients with liver cirrhosis, a condition closely associated with
hypertension and disorders of the lymphatic circulation, were compared with matched
controls a significant reduction in serum lycopene was observed along with other carotenoid
antioxidants, retinol and vitamin E in the cirrhotic group [33, 164]. Recognizing the
importance of antioxidants in the management of hypertension a ‘dietary approach to control
hypertension (DASH)’ diet is recommended that contains substantially higher levels of
lycopene along with other carotenoids, polyphenols, flavanols, flavanones and flavan-3-ols
[167].
Male infertility, a common reproductive disorder, is now being associated with oxidative
damage of the sperm leading to loss of its quality and functionality. Significant levels of ROS
are detectable in the semen of up to 25% of infertile men, whereas fertile men do not produce
detectable levels of ROS in their semen [168, 169]. A number of studies have reported the
beneficial effects of vitamins C and E. and other antioxidants, including taurine [170], l-
carnitine [171], coenzyme Q10 [172, 173], and glutathione [174] on sperm quality.
Researchers are beginning to investigate the role lycopene in protecting sperm from oxidative
damage leading to infertility. Men with antibody-mediated infertility were found to have
lower semen lycopene levels than fertile controls [175]. In another study infertile men
consumed a daily dose of 8mg lycopene in capsule form. After consuming lycopene for 12
months significant increases in serum lycopene concentration and improvements in sperm
motility, sperm motility index, sperm morphology and functional sperm concentration were
observed. Lycopene treatment resulted in a 36% successful pregnancies. Other studies are
now in progress and their results will further advance our knowledge of the beneficial role of
lycopene in male infertility.

A recent review article elaborated on the possible role of lycopene in neurodegenerative

diseases including Alzheimer’s disease [176]. Due to high levels of oxygen uptake and
utilization, high lipid content and low antioxidant capacity, human brain represents a
vulnerable organ for oxidative damage.
Although the role of antioxidant vitamins in neurodegenerative diseases have been
reported in the literature, only a small number of studies have been reported for lycopene.
Lycopene was shown to cross the blood brain barrier and be present in the central nervous
system in low concentrations. Significant reduction in the levels of lycopene was reported in
Parkinson’s disease and vascular dementia patients [177]. In the Austrian Stroke Prevention
study, lower serum lycopene and α-tocopherol levels were associated with increased risk of
microangiopathy [178].
Lycopene was also suggested as providing protection against amyotrophic lateral
sclerosis (ALS) disorder in humans [179]. When a population of elderly subjects were tested
for functional capacity including the ability to perform self-care tasks, a significant positive
correlation was observed between blood lycopene levels and functional capacity [180]. On
the basis of the relationship between oxidative stress and neurodegenerative diseases and the
potent antioxidant properties of lycopene it is logical to expect further intervention studies to
be carried out in the future to address this important area of human health.
Incidence of emphysema, a disorder of the lungs is reported to be high in certain
countries of the world. A recent study showed protective role of lycopene in the prevention of
emphysema in a mouse model. At a recent conference on the role of processed tomatoes in
human health, data was provided for the protective role of lycopene in the prevention of
emphysema in a Japanese population. Undoubtedly, future research will also explore the role
of lycopene in other human diseases including diabetes, ocular and skin disorders, rheumatoid
arthritis, periodontal diseases and inflammatory disorders [61]. The antioxidant property of
lycopene is also opening up new applications in pharmaceutical, nutraceutical and
cosmoceutical products [181].
The scientific interest to explore innovative strategies for the prevention of human
diseases underlines the common etiological and mechanistic nature of these diseases. The
hypothesis that oxidation of cellular components as an initial event eventually leading to the
incidence of several diseases brings the focus to the use of antioxidants. Examples of this
hypothesis include oxidation of LDL leading to increases risk of CVD; oxidation of DNA as
an early step in the progression of cancers; and protein oxidation resulting in possible
alterations in the activity of several metabolic enzymes and influencing many disease
conditions. Lycopene by acting as an antioxidant can prevent the progression of many human
diseases at an early stage and improve the quality of life.
Daily Intake Level Estimations

and Suggested Levels of Intake
Reports about the daily intake levels of lycopene have varied significantly due to the
methods of estimation used. In general they range from 3.7 to 16.2 mg in the United States of
America, 25.2 mg in Canada, 1.3 mg in Germany, 1.1 mg in United Kingdom and 0.7 mg in
Finland [56]. However, it is important to note that close to half of the population in North

America are estimated to consume less than 2 mg of lycopene per day. It is evident that the
average intake levels of lycopene are lower than required to provide its beneficial effects.
Although the beneficial effects of lycopene in the prevention of human diseases has been well
documented it is not yet recognized as an essential nutrient. As a result there is no official
recommended nutrient intake (RNI) level set by health professionals and government
regulatory agencies. However, based on reported studies a daily intake level of 5–7 mg in
normal healthy human beings may be sufficient to maintain circulating levels of lycopene at
levels sufficient to combat oxidative stress and prevent chronic diseases [182]. Under the
condition of disease such as cancer and cardiovascular diseases, higher levels of lycopene
ranging from 35 to 75 mg per day may be required [58].
Conclusion
Carotenoid pigments are a group of bioactive compounds that are of interest to the food
scientists, nutritionists and food industries due to their positive impact on human health and
their economic benefits. Carotenoids are responsible for the attractive colour of most fruit and
vegetables, having diverse biological functions and activities. So, if we want to take
advantage of all of these benefits we must consider the bioaccessibility event as a necessary
stage in the complex process of converting a visual attraction into biological actions. As we
await a better scientific understanding of carotenoid metabolism and the mechanisms of
action, a prudent strategy to reduce the risk of cancer incidence and mortality would include
increased consumption of vegetables and fruits as a part of a healthy, balanced diet. This
would include eating between five to nine servings of fruits and vegetables every day. There
is currently no evidence of any dangers associated with high levels of dietary β-carotene from
natural food sources, aside from the occasional appearance of carotenodermia, an
accumulation of β-carotene in the skin that gives it a yellow or orange tint. At present,
supplemental doses of β-carotene taken to meet vitamin A needs beyond the recommended
dietary intake dose are not advisable for the general population. Smokers and alcohol drinkers
are especially encouraged to avoid high doses of supplemental β-carotene.
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Chapter XI
Health Benefits of Carotenoids

for Human Skin
Maxim E. Darvin* and Juergen Lademann

Charité - Universitätsmedizin Berlin, Department of Dermatology,
Venerology and Allergology, Center of Experimental and Applied Cutaneous Physiology
(CCP), Berlin, Germany
Abstract
Human skin is rich in different antioxidants. Amongst a variety of antioxidants, fat-
soluble carotenoids play an important role in the protection of human skin against the
action of most reactive free radicals, such as singlet oxygen and other reactive oxygen
species. Recent studies performed with electron paramagnetic spectroscopy show that
epidermal carotenoids could serve as marker substances for the whole antioxidant
capacity of human skin. This means that the individual concentration of carotenoids in
human skin reflects the current antioxidative status of human skin. Non-invasive optical
methods are well suited for in vivo investigation of kinetics of carotenoids in human skin
and irreplaceable for this task. Results obtained on a large group of volunteers with
resonance Raman spectroscopy and reflection spectroscopy show that the concentration
of cutaneous carotenoids reflects the current lifestyle of volunteers. A high concentration
of carotenoids in human skin is always associated with a carotenoid-rich supplementation
and lifestyle with reduced stress. A low carotenoid concentration is attributed to an
unhealthy lifestyle and increased stress. Smoking, alcohol abuse, inflammation, illness,
sun radiation, environmental conditions, heavy sport loads, insomnia, fatigue, etc. are
stress conditions giving rise to the generation of free radicals in the skin and subsequent
reduction of the antioxidative status. Volunteers with a higher concentration of cutaneous
lycopene were shown to have a reduced skin roughness value, which is determined by the
depth and density of furrows and wrinkles. Thus, a high concentration of carotenoid
lycopene in the skin is associated with a young-looking visual appearance of the skin.
* Corresponding Author address Email: [email protected].

230 Maxim E. Darvin and Juergen Lademann
Keywords: Antioxidants, oxidation, epidermis, stress, lifestyle, supplementation, protection
Introduction
Free radicals are highly reactive molecules formed in human skin as a result of metabolic
processes [1]. They play an important role for signalling between cells [2, 3] and for action
against viruses and bacteria [4]. Increase of metabolic intensity or exposure to external stress
factors, such as sun irradiation or environmental pollutants, give rise to a substantial increase
in the concentration of free radicals in human skin [5-10]. To avoid the development of
oxidative stress and subsequent injuries at cellular level, human skin contains different
antioxidant substances, which act synergistically [11-13] and neutralize generated free
radicals [14, 15].
The most powerful of these substances include the carotenoids, which neutralize singlet
oxygen and other reactive oxygen species (ROS) that exhibit an enhanced oxidative activity
compared to other free radicals [16, 17]. Among the variety of cutaneous carotenoids,
lycopene shows the highest antioxidative activity in neutralizing ROS [18]. Carotenoids
cannot be synthesized by the human organism itself and must be taken up with food or
supplements rich in these substances, for example with fruit and vegetables [19]. After
digestion, the carotenoids accumulate at high concentrations in the adipose tissue, liver and
blood [20]. The further accumulation in the epidermis occurs via two different pathways,
which are characterized by different velocities. The first one, the ―slow‖ pathway, is the
diffusion of fat-soluble carotenoids from the subcutaneous fat tissue, blood and lymph flows
into the epidermis; whereas the second one, the ―quick‖ pathway, is the spread of carotenoids
with the sweat and/or sebum onto the skin surface and their subsequent penetration into the
stratum corneum [21].
Healthy volunteers with skin types II and III according to the Fitzpatrick classification
[22] without skin diseases, visible skin disorders and allergies were included into the studies on
non-invasive determination of carotenoid antioxidants in human skin. The studies were
approved by the Ethics Committee of the Charité – Universitätsmedizin Berlin and conducted
in accordance with the Declaration of Helsinki Principles.
Non-Invasive Measurement of Carotenoids

in the Skin
For measurement of kinetics of carotenoids in human skin, non-invasive methods are
irreplaceable and should be performed. Optical methods are ideally suited for determination
of carotenoids in human skin in vivo as they are both non-invasive and quick. The resonance
Raman spectroscopy [23], reflection spectroscopy [24], Raman microscopy [25] and skin
color measurements [26] could be mentioned in this regard. Fluorescence-based methods
cannot be performed for carotenoid analyses due to the very low fluorescence effectiveness of
carotenoids [27]. The metrological and bio-physical principles of these optical methods were
previously described and compared in detail in the literature [28]. The results presented in this

Health Benefits of Carotenoids for Human Skin 231
chapter were obtained in vivo with the use of resonance Raman spectroscopic, confocal
Raman microscopic and reflection spectroscopic methods.
Distribution of Carotenoids in Human Skin

The stratum corneum, as a first defence line of the epidermis is rich in different
antioxidants including carotenoids [15, 21, 29]. The typical distribution of carotenoids in the
epidermis measured by confocal Raman microscopy shows the highest carotenoid
concentration in the superficial stratum corneum layers. Moreover, in comparison to different
body sites, the highest carotenoid concentration was found in the palmar and plantar areas and
on the forehead [30], where the concentration of sweat glands is maximal [31, 32]. Such
distribution confirms the quick pathway of carotenoid accumulation in the stratum corneum
including the spread of carotenoids with the sweat and/or sebum onto the skin surface and
their subsequent penetration into the lipid layers of the stratum corneum. This pathway was
confirmed by the kinetics of cutaneous carotenoids measured after the influence of exogenous
stress factors [33]. The recovery of carotenoids started relatively quick in the superficial layer
of the stratum corneum developing from the outside to the inside of the skin. Thus, sweat
and/or sebum secretion provides an additional antioxidant protection against negative
environmental influences, and the stratum corneum‘s biologically dead corneocytes remain
strongly protected by an increased concentration of antioxidants, including carotenoids.
Factors Influencing the Carotenoid Concentration

in Human Skin
Stress factors, such as smoking [34], alcohol abuse [35], inflammation [36], illness [37,
38], sun irradiation [5, 7, 8, 39], environmental conditions [9], heavy sport loads [40], etc. are
always associated with the generation of free radicals and especially ROS in human skin.
Using resonance Raman spectroscopy, the subsequent reduction of the carotenoid
concentration in human skin was clearly observed after UV [41] and IR irradiation [42],
alcohol abuse [43], heavy sport loads [10], during inflammation [44], illness [45] and fatigue
[44]. Carotenoids cannot continuously neutralize ROS generated in the skin. After
neutralization of a few ROS attacks, carotenoids are destroyed and lose their antioxidative
activity [46, 47]. The amount of destroyed cutaneous carotenoids can be well correlated with
the amount of generated free radicals in the skin, which was confirmed by electron
paramagnetic resonance measurements, showing that carotenoids could serve as marker
substances for the whole antioxidative status of human skin [48].
The process of accumulation of carotenoids in human skin is an important part of
cutaneous physiology. Many research studies show that the intake of carotenoid-containing
food or supplements increase the carotenoid concentration in the human skin [44, 49-57]. The
subsequent increase could be measured already a few days after the start of supplementation
and the decay time lasted up to 5 weeks after discontinuing the application [55].

Cutaneous carotenoids show seasonal variations. During the summer and autumn months
the concentration of carotenoids in the skin of ten healthy volunteers, which had not taken any
supplements or special diet, was found to be significantly higher in comparison to winter and
spring months. The average ―seasonal increase‖ of the carotenoid concentration in human
skin was found to be 1.26 fold [44]. The obtained seasonal variations are explained by the
increased consumption of ripe fruit and vegetables, which are naturally rich in carotenoids,
and by increased sweat secretion, which could deliver carotenoids to the skin surface.
The carotenoid concentration in the skin is immediately increased if a carotenoid-
containing formulation is topically applied. Contrary to systemic application, topically
applied carotenoids are stored in the stratum corneum only for a short time due to their
depletion by desquamation, textile contact, washing and environmental stress [55] saturating
only a superficial layer of stratum corneum [25]. It was shown that combined topical and
systemic application of carotenoids represent an optimal form of protection against stress
induced by environmental influences [55]. Immediate and prolonged protective action could
be achieved. The carotenoid concentration of volunteers showed drastic individual variations
depending first of all on their lifestyle. Dietary supplementation rich in carotenoids on the one
hand and the influence of stress factors on the other, determine the current concentration of
carotenoids in human skin.
Lycopene and Skin Aging

Visual manifestation of skin aging is usually associated with an increased amount of
furrows and wrinkles. This subjective observation was objectively investigated using optical
skin surface topography [58]. The value of cutaneous roughness characterized by the depth
and density of furrows and wrinkles was determined and used as an objective criterion of the
skin aging extent. Skin roughness and concentration of cutaneous carotenoids were measured
non-invasively on the light-exposed forehead area of twenty healthy volunteers aged between
40 and 50 years. The strong negative correlation (R=-0.84) was found between the skin
rougness and concentration of cutaneous lycopene providing an evidence that volunteers with
a high concentration of carotenoid lycopene in their skin are more protected against
development of premature skin aging. Thus, a healthy lifestyle including an antioxidant rich
nourishment and reduced stress, could be associated with a young-looking visual appearance
of the skin exhibiting an reduced amount and depth of furrows and wrinkles. The obtained
results are in accordance with the results obtained by Heinrich et al. [59] showing that the
skin roughness value can be reduced by supplementation of antioxidants including carotenoid
lycopene.
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Chapter XII
Nanoparticles of Chitin Nanofibril-

Hyaluronan Block Polymer Entrapping
Lutein as UVA Protective Compound
P. Morganti1, H. D. Chen2, X. H. Gao3, P. Del Ciotto4,

F. Carezzi4 and G. Morganti4
1
Dermatology Depart, 2nd University of Naples, Italy,
China Medical University, Shenyang, China,
Centre of Nanoscience Mavi Sud S.r.l, Aprilia, Italy
2
Key Lab of Immunodermatology, Ministry of Health,
No 1 Hospital of China Medical University, Shenyang – China
3
Head Depart Dermatology No 1 Hospital of China Medical University Shenyang, China
4
Centre of Nanoscience Mavi Sud S.r.l, Aprilia, Italy
Abstract
As the exterior barrier of the human body, the skin in direct contact with the
environment is exposed to oxygen and light generating reactive oxygen species (ROS)
and consequently experiences photoaging and photocarcinogenesis. Thus, the mainstay of
skin protective strategies is UV photoprotection, inside and outside, and the use of
antioxidant compounds, such as carotenoids. These natural compounds are a class of
molecules found in plants, algae and in some fungi, used as food, that protect the human
body against excess light. Besides their specific activity, carotenoids are used as natural
photo-protectant compounds, being involved in the dissipation of excess light energy
through the xanthophyl cycle, quenching excited triplet state molecules, and singlet,
oxygen. The use of these natural molecules as antioxidant compounds may prevent the
oxidative damage of proteins, lipids and DNA, also modulating the cell signaling-
initiating mechanism of the oxidative stress. Essential for the effectiveness and safety of
carotenoids and chitin derivatives, is their transport to the target site. Promising
candidates considered safe innovative carriers for both endogenous and topical
photoprotection are Chitin nanofibril-hyaluronan block polymers (CN-HA). Both Chitin

238 P. Morganti, H. D. Chen, X. H. Gao et al.
and Hyaluronan are, in fact, natural biocompatible ingredients and environmentally

friendly. The activity of CN-HA, as carrier, and carotenoids, such as skin
photoprotectants, are reported in this chapter.
Introduction
Skin Aging
Chronoaging, fundamentally characterized by a loss of cells and interstitial matrix, is

affected by a genetic program as well as by cumulative environmental and endogenous insults
that take place throughout the organism's life-span [1, 2]. In the skin, the aging process
involves characteristic changes of tissue architecture, clinically manifested as wrinkles, tissue
slackening, and irregular pigmentation, increased by photoaging as a result of cumulative
damage from UVB and UVA radiations [3, 4]. Although the energy per photon delivered as
UVA is approximately 1,000-fold less than UVB, it penetrates the skin to reach deeper
dermal layers, causing oxidative damage especially by the activity of Reactive Oxygen
Species (ROS) [5-7]. Excessive amounts of ROS, in fact, may be a primary cause of
biomolecular oxidation and may result in significant damage to cell structure, contributing to
various diseases.
To counteract this oxidative damage, human skin is equipped with a network of
enzymatic and non-enzymatic antioxidant systems [8]. An imbalance between ROS, natural
bioproducts of the normal metabolism of oxygen, and these protection mechanisms leads to
the so-called oxidative stress, involving oxidation of DNA, lipids and proteins.
The result is a loss of structural and functional integrity of the epidermal barrier's key
components and dermis' macromolecules that can lead to cell cycle arrest, and premature
aging [9]. Thus the increasing use of sunscreens for the skin's daily protection, designed as
cosmetic products containing both UVB and UVA filters. These topically applied products
protect against UV-induced skin lesions and prevent aging and sunburns. However, being
they are not sufficient to give 100% photoprotection, current strategies are geared not only
toward the use of sunscreens adsorbing UV radiation but also toward cosmetic formulations
that, enriched with antioxidant and immune-protective compounds, should reduce skin
damage, modulating or inhibiting oxidation processes in human body, and thus monitoring
the ROS activity [10]. These active ingredients have the capacity to prevent some of the
biochemical and molecular consequences which occur in the skin after UV radiation has been
absorbed, significantly reducing the adverse effects of ROS, RNS (Reactive Nitrogen
Species) and RIS (Reactive Iron Species) on human physiological functioning [11, 12].
To enhance the activity of topical sunscreens, and also because the population is living
longer thus increasing its sun exposure, the use of endogenous photoprotective ingredients
have became a complementary necessity. It implies systemic delivery of these protective
agents and their transport to the target site, providing a desirable basic UV shield for the
entire body surface. For all these reasons, enhancement in the delivery and absorption of
active ingredients, via a specific route of administration, becomes essential in the design of
novel topical and systemic photoprotective agents and therapies [13]. Thus the necessity of
effective biomaterials and formulations based on the use of the right carriers are necessary to
obtain the best results for their specific cellular effectiveness and compatibility [14-16]. In

Nanoparticles of Chitin Nanofibril-Hyaluronan Block Polymer … 239
any way, both topical and systemic photo protectants need to be safe in the recommended
dose range and over a longer period of time.
Promising candidates as safe innovative carriers for both endogenous and topical
photoprotection are found in Chitin Nanofibrils (CN) [17], while antioxidant compounds,
such as tocopherols, flavonoids, polyphenols and carotenoids, and immune stimulant
ingredients such as beta-glucan and ectoin, are considered effective active ingredients to
prevent the skin aging processes [18-21]. What it is important to underline is that carotenoids
seem to play a protective role in humans similar to that in plants
Carotenoids
Over 600 carotenoids are known. They are split into two classes: hydrocarbon
carotenoids without oxygen, and xanthophylls containing oxygen (Fig. 1).
Figure 1. Major Carotenoids in human diet.
All these natural compounds, produced from 8 isoprene molecules containing 40 carbon
atoms, are powerful singlet-oxygen quenchers and exhibit additional antioxidant properties
[22, 23].
Peroxyl radicals and singlet-oxygen, due to the specific physical chemical properties they
possess, are, in fact, the oxygen species efficiently scavenged by carotenoids, acting as
antioxidant compounds synergistically with tocopherol and tocotrienols. Their conjugated
isoprene backbone has the ability to delocalize a charge or an unpaired electron along the
different chains. Thus, carotenoids play an important role in protecting skin and eyes against
excess light and premature aging being involved in the dissipation of light energy through the
xanthophyll cycle, capable of quenching excited triplet state molecules and single oxygen [23,

24]. They can be grouped into several subclasses, with the most familiar being the provitamin
A carotenoids (partially converted in vitamin A), and the non-provitamin A carotenoids.
Lutein and zeaxanthin belong to the subclass of non-provitamin A carotenoids that are
classified as xanthophylls. Although xanthophylls have structural similarities to other
carotenoid compounds, they possess free hydroxyl groups at each end of the molecule,
allowing lutein and zeaxanthin to orient within the cell lipid membranes and lipoproteins in
ways other carotenoids cannot (Fig. 2) [25-28]. However, oxidative stress and lipid
peroxidation are the cause of a number of chronic diseases, such as cancer, stroke, diabetes,
and atherosclerosis, but also of degenerative processes associated with skin aging and age-
related macular degeneration (AMD) [29, 30].
Figure 2. Xanthophylls structure and Lutein orientation into the Cell membrane.

The carotenoids‘ antioxidant system helps reduce potential cell damage at the level of
skin and the eye macula of the retina by neutralizing the free radicals produced especially by
UVA1 (the lutein and zeaxanthin activity) and UVA2 (the beta-carotene and lycopene
activity) [11, 28-31]. In normal health there is, in fact, a balance between the formation of
chemical species and their effective removal by protective antioxidants through the so-called
redox system. Such control offers two major advantages, (a) the ability to remove toxic levels
of oxidants before they damage critical biological molecules and (b) the ability to manipulate
changes of molecules that can function as signal, trigger or messenger carriers [32-36].
Normally the resting cell has a redox potential with a reduced state that is progressively
activated as lipid oxidation increases at the level of the cell membrane [37]. Thus, a normal
build-up of lipid peroxides triggers new cell growth [38], while too much oxidation deposes
all functions until apoptosis or necrosis is triggered [39]. As a general rule low levels of ROS
activate cellular processes, whilst higher levels turn them off. This is the reason for the use of
the antioxidant therapy to reduce the ROS' excessive production provoked by the
environment.
Therefore the antioxidant activity of carotenoids may prevent many degenerative
diseases, modulating the oxidative damage of proteins, lipids and DNA, thus affecting the
signaling mechanisms which initiate the oxidative stress.
Among the carotenoids, lutein seems to exhibit the highest activity as an antioxidant to
protect the eyes' photoreceptor cells from potential damage, caused by free radicals generated
in these cells by the high oxygen tension and excessive exposure to light. In addition to
antioxidant activity lutein is believed to be responsible for filtering the high-energy
wavelengths of UVA1 and blue light (Fig. 3), reaching both eye-macula and the skin, where it
appears to have interesting moisturizing and anti-aging properties [40, 41].
Figure 3. Lutein Spectrum.

For these reasons, the strategic placement of topical or dietary lutein into specific body
tissues makes this xanthophyll particularly useful, also because epidemiologic studies have
shown an inverse relationship between several diseases states and the level of carotenoid
content in blood serum. [41-47]. Recording the baseline carotenoid concentration in the blood
serum of all the patients was necessary before starting the clinical study, to obtain realistic
and comparable results of effectiveness. However, it is important to remember the difficulty
of maintaining stability, since this molecule is extremely rich in unsaturated double bonds and
is therefore easily oxidizable. It must also remain stable for penetration and adsorption,
respectively, through the skin barrier and epithelial mucous membranes. Therefore, saving the
activity of lutein and all carotenoids by using boosting polymers protects them from the
oxidative power of oxygen and increases their antioxidant effectiveness, acting as vehicles
capable of improving skin penetration [41, 46, 47].
Chitin Nanofibrils
Chitin, a copolymer of N-acetyl-D-glucosamine and D-glucosamine and natural
polysaccharide, is widely and abundantly distributed among living organisms on the Earth. It
is extracted from crab and shrimp shells as a byproduct of the seafood industry. Chitin
Nanofibrils (CN), which represents the purest crystalline form of chitin with a dimension of
240x7x5 nanometers, has the same backbone of hyaluronic acid (Fig. 4) and is, therefore,
capable of easily binding a great quantity of water and many different active ingredients
through ionic charges. Moreover, CN has shown to be an interesting natural compound of
great industrial interest and probably, because of its geometrical and topographical
architecture mimicking the native state of Extra Cellular Matrix (ECM) in living tissue,
possesses interesting biological activities (Fig. 5) [48].
Figure 4. Chitin and Hyaluronic acid Backbone.

Figure 5. Some of the Chitin Nanofibrils' Characteristics.
What seems interesting is that this polysaccharide, industrially produced from fishery
waste, may be considered a safe natural polymer, biocompatible and environmentally friendly
[49-52]. For this reason our research group designed these chitin nano-crystals to use as an
active nano-carrier for delivering the entrapped active compounds at different skin layers, and
also because of their capacity to release their contents extracellulary or intracellulary.
The properties that make nano-materials more useful for cosmetic and biomedical
applications are, in fact, their enhanced mechanical, optical, magnetic and electrical
characteristics, which must be controlled in advance. The physicochemical properties of the
nanoparticle size, shape, surface charge, hydrophobicity or hydrophilicity, and eventual
surface functional added groups, seems to allow the ability to determine the nanoparticle cell
interaction and ultimate cellular response [53, 54]. Moreover, the mode by which they enter
cells is relevant because it will dictate the initial cellular microenvironment to which
nanoparticles will be exposed [55]. Cationic lipophilic moieties, for example, are favorable
for mitochondrial targeting (when a redox activity has to be designed and obtained) due in
part to the negative membrane potential of mitochondria [56].
For this reason we produced, using the gelation method and a refining process,
nanostructured block-co-polymers mixing the polycation CN with the polyanion Hyaluronic
acid (HA) to obtain nanoparticles, positively or negatively charged, according to the
productive process selected (Fig. 6) [57-59]. By building detailed models and computerized
simulations, it was possible to design its structure in advance, also predicting its properties.
The obtained nanoparticles, entrapping lutein as an active ingredient, were characterized
controlling their size, release times, release rates, surface charges, and bioavailability (Fig. 7)
[59, 60].

Figure 6. The Gelation method to obtain the Chitin Nanofibril- Hyaluronan Block polymers.
Figure 7. Diversified Skin release of Chitin-Hyaluronan Nanoparticles.
Moreover, it has been shown that CN-HA may serve as a vehicle for delivering a wide
range of active ingredients, which can be controlled for optimal transepidermal and/or
transdermal delivery because of the specific physicochemical properties and morphology of
the nanoparticles obtainable, according to the designed productive methodology [57-60]. Of
course, the stability of the nanoemulsion used to include these nanoparticles into the final
cosmetic formula, together with product effectiveness and safety were also monitored.

Compared with traditional cosmetic carriers CN-HA nanoparticles, included into the right
micro/nanoemulsions, combine the advantage of polymeric nanoparticles and multiple
emulsions for cosmetic delivery, having not only a very low toxicity, good biocompatibility,
and targeting ability, but also a high effectiveness [58-66].
Polymeric nanoparticles are preferred, in fact, over other colloidal carrier systems, like
micelles, owing to their higher stability and flexibility in tailoring the active ingredient load
and release rate. Their physicochemical properties, in fact, can be engineered at the molecular
level [67, 68], their shape, size and charger can be controlled, and the surface density of the
targeting entrapped or ligand compound can be optimized for specific applications [69-72].
Thus, in order to achieve a specific effect such as, for example, effective antioxidant activity,
some active ingredients have to target specific cell organelles, such as mitochondria or
lisosomes [73].
Figure 8. Anti-aging activity of CN-HA Block polymers entrapping antioxidant compounds applied topically
and /or taken by oral route.
The size of nanoparticles and its surface chemistry can, in fact, enhance the cellular
uptake. The smaller the particle, especially when positively charged, the easier it can be
delivered to cells, also acting as cell signaling [74]. These nanoparticles can, therefore, be
designed as carriers to deliver active molecules and to target specific skin cells and tissues,
being engineered as nano-platforms for effective and targeted delivery of active cosmetic
ingredients, overcoming the skin barrier. With the obtainable size smaller than human cells,
these nanoparticles can offer unprecedented interactions with biomolecule-signals both on the
surface and inside the cells, which may innovate, for example, the skin anti-aging treatment,
as reported on Figure 8 [61, 75].

Table I. SPF and UVA PF of Chitin Nanofibril-Hyaluronan entrapping carotenoids

and ZnO and TiO2
It must be remembered that biological signals, coordinating all physiological responses,

play an important role during the normal tissue development process, mediating the tissue
remodeling during injury.
Cell signaling is part of a complex system of communication, which governs and
coordinates the basic cellular activities, acting locally or systematically depending on their
diffusion limit, eventual access to the circulatory system, and stability in the body [76-79].
The ability of cells to perceive and correctly respond to the interior and exterior
environment represent, in fact, the basis of tissue development, repair, and immunity, as well
as normal tissue homeostasis. Errors in cellular information processing are, in fact,
responsible for diseases such as cancer, autoimmunity, and diabetes.
For all these reasons cosmetics, often considered as products capable of preventing and
protecting the skin from negative environmental conditions, must be designed for modulating
activities of the cells, even at the level of all the signals arriving at their surface membranes
[79].
To obtain real effectiveness and safety from cosmetic products, it is necessary to select
the active ingredients to direct the promise they carry, using the right vehicle to deliver them
to the designed site of action. Thus, our interest in using the CN-HA nanoparticles as

innovative cosmetic carriers, entrapping lutein, lycopene and/or beta-carotene as antioxidant

and UV protective ingredients, would be especially useful in prevent photoaging of the skin.
For this reason, we began our study to entrap lutein into CN-HA block polymers to verify
the stability over time of the obtained nanoparticles, and the bioavailability and release in
vivo of this natural ingredient (Fig. 9). At the moment we are verifying in vivo the
photoprotective activity versus both UVB and UVA of these nanoparticles entrapping
lycopene, betacarotene and other active ingredients for cosmetic use, as well as their delivery
and release at different skin layers, as shown for lutein. From the first obtained data shown in
Table I (Unpublished and in progress data), it is interesting to see the capacity CN-HA has to
increase the UV-screening capacity of both the inorganic and organic filters, trapped into a
block polymeric structure. What seems interesting is that lutein and lycopene have been
shown to increase the sunscreen activity of ZnO and TiO2, versus both UVA and UVB, much
more than betacarotene. Also, the CN-HA block polymer acts not only as a carrier but also as
an interesting boosting agent.
Figure 9. Lutein release from skin treated for one month by both CN-HA nanoparticles (electropositive on the
surface) and HA-CN nanoparticles (electronegative on the surface) entrapping lutein.
As previously reported and shown in Figure 9, it was possible to obtain a release at levels
of the different corneocytes layers, depending on the size of the designed and obtained
nanoparticles, as well as on the electrical charges characterizing their surface [57-66].
Conclusion
As reported, both skin aging and photoaging is the result of an imperfect repair of
cumulative damage caused by external and internal factors, such as environment-pollutants,
UV light, allergies, and overactive immune responses. While the first visible consequences of

skin aging are the development of wrinkles and sagging, characterized by the decrease of
keratinocytes differentiation and turnover, and alteration of skin barrier, at a microscopic
level the damaged cells present an altered redox system and cell proliferation [35-39], and an
unphysiologic skin microbiota [80]. When a redox imbalance occurs there are two major
implications: (a) the production of toxic levels of ROS, RNS and RIS, damaging key
molecules in cells, and/or (b) the production at sub-toxic levels changing the cell signal
responses to proliferation and apoptosis [35-39], often altering the equilibrium between
saprophyte and pathogen bacteria and fungi [80]. The use of antioxidant compounds as
carotenoids seems useful to prevent both singlet oxygen- and free radical-mediated damage
and to increase the expression of a gene encoding connexin-43- a gap junction protein [81,
82].
Figure 10. The cellular activity according to the NICE-TCM approach.
Among the carotenoids, lycopene exhibits the highest physical quenching rate constant
with singlet oxygen, being at least three times more effective than Beta-carotene in reducing
NOO• radicals [81, 82]. By the combination of different carotenoids, together with other
antioxidant and immune stimulant ingredients, it seems possible not only to repair the altered
redox system and the disrupted skin barrier, but also to neutralize the errors in cellular
information processing. For this reason, the systemic biology research and the NICE-TCM
approach to designing cosmetic products and diet supplements, involving billions of Nervous,
Immune, Cutaneous and Endocrine cell signals, may help to understand the underlying
structure of cell signaling networks and how these changes may affect the transmission and
flow of information at skin level (Fig. 10) [83-86].

Naturally to obtain a real effect on cells, the active ingredients should be transported until
the cells‘ membrane by a carrier capable of accumulating them in the target tissue owing to its
physicochemical properties, as much as necessary to select an effective active ingredient load,
based on the distribution properties of the carrier. CN-HA nanoparticles seem to provide this
new approach for delivery to specific tissues and cells, different active compounds, at the
designed dose, and in a controlled release [57-59]. However, while the intrinsic
biocompatibility, effectiveness and safety of these natural nanoparticles have been supported
by the results of many studies [57-66], their large surface area rich of different functionalities
should be verified, being ideal for conjugating various biochemical entities, useful for specific
cosmetic activities as well as drug therapies.
Table II. Entrapment of lutein into the block polymer Chitin-Hyaluronan
For these reasons, the CN-HA suitability as a carrier was controlled for its loading
capacity (Tab. II), as well as for the ability that some components, entrapped into the block-
polymer, have to protect the skin and retain the inherent moisturizing and anti-inflammatory
activity (Fig. 11) [87], or for the anti-aging effects shown entrapping, for example,
phosphatidylcholine (Fig. 12) [61, 75, 88, 89].

Figure 11. Anti-inflammatory activity of CN-HA nanoparticles compared to corticosteroids on an erythema

area after 12 hours of treatment (A clinical double-blind study).
Figure 12. Anti-aging efficacy of CN-HA entrapping phosphatidylcholine. A clinical study.
By the high loading capacity, obtained with lutein, as shown in Tab. II [57-60], it was
possible to allow a higher concentration of the payload to be delivered, using a lower quantity

of the delivery agent normally necessary to obtain the same results. In parallel to the loading
capacity, the regular and continuous release of lutein from the carrier was also obtained,
according to the necessity of formulating an effective cosmetic product (Fig. 13).
Figure 13. Release profile of lutein from Chitin-Hyaluronan nanoparticles.
As clearly shown on Figure 11, this innovative block polymer has the capacity to mitigate
the inflammatory cascade of cytokines reducing the intensity of the erythema, similar to a
corticosteroid compound without having side effects. It may also ameliorate the appearance
of aged skin by an increase of collagen I production (Fig. 14). Naturally, these two different
results were obtained by the use of different active ingredients entrapped into the CN-HA
nanoparticles.
The feasibility of being able to tune the release of cargo from the carrier is advantageous
in developing novel applications of innovative delivery agents and tissue-engineering
scaffolds.
For this reason, the delivery system of CN-HA nanoparticles, engineered at the molecular
level, has shown the potential to significantly impact skin treatments probably affecting the
physico-chemical properties of all the active ingredients used, such as their solubility,
molecular size, stability and the distribution targeting at different levels of skin layers and
cells as previously shown on Figure 9. Thus, these polymeric nanoparticles may be
considered both as vehicles for delivering a wide range of active ingredients throughout the
skin layers, and contemporary active compounds, being easily metabolized in glucosamine
and acetyl glucosamine from the human chitinases [90], or in glucose to be used as cellular
energy ingredient [91, 92]. With the use of CN-HA nanoparticles, the skin permeability may
be temporarily improved by the probable formation of micro pores, caused when the positive

charges recover their external surface, so that the nanovector and active ingredients can
diffuse across the skin with less difficulty (Fig. 15).
Figure 14. Increase of collagen produced by fibroblasts' ex vivo cultures added with the CN-HA block
polymer entrapping phosphatidylcholine.
Figure 15. Micropores of penetration created by Chitin-Hyaluronan nanoparticles positively charged on their
surface.

This is because charged particles may create electrostatic interactions with charged
elements at the level of lipid lamellae and/or in the interstitial ECM, where positively charged
collagen or negatively charged glycosaminoglycans are embedded [93, 94]. At this purpose,
according to scientific-legal considerations reported elsewhere from our group [95], it is
necessary to remember that the entire epidermis, and not only the stratum corneum, plays an
important role in the barrier function of the skin, representing the borderline for the activity of
both dermatologic and cosmetic products. While a drug must have a pharmacological activity,
the cosmetic product must possess a biological efficacy. Either way, all the ingredients must
show their effectiveness at skin level: dermatologicals must solve pathological conditions of
the skin, and cosmetics must maintain skin in good condition, ameliorating the weakened
condition of the cells. Innovative cosmetic products should be effective not only at the level
of corneocytes (dead cells) but also at level of both keratinocytes, melanocytes, fibroblasts
(living cells), different ECM components and all the cell signals [96-99]. This effectiveness
must be controlled and regulated by the use of the right active ingredients delivered at the
desired level of the skin by the right vehicle. In this way, according to EU rules on cosmetics,
the skin may be protected and maintained in good condition, explaining a biological activity
by use of these innovative products. However, innovation in cosmetic products will be
possible only by the modernization of regulatory science and policy, adopting new scientific
approaches and advanced technologies, such as nanotechnology. This is the reason why the
market of nanotechnology-based products reached $254 billion in 2009 and is expected to rise
to $1 trillion by 2015 [100].
The multi-functionality represents the key approach of these CN-HA nanoparticles over
the traditional cosmetic emulsions. Their capacity to entrap many different molecules,
provide, in fact, enormous sensitivity and flexibility, having the potential to profoundly
impact cosmetic/drug production and customer management in the near future. Nanoparticles-
based delivery systems are likely to be below toxicity and side effects (via targeted delivery),
to increase the effectiveness of the active ingredients used, to reduce cost and even time of
treatments, with respect to alternative drug/cosmetic therapies/treatments. Regarding lutein
and the other carotenoids in the study, it was shown that these particular ingredients seem to
represent one of the more interesting groups among all the naturally recovered compounds,
not only for their antioxidant effectiveness [22-33], but also for their probable capacity to
modulate the signals molecules by the NICE-TCM approach, where Nervous, Immune,
Cutaneous and Endocrine Systems (i.e. NICE) all work together towards the health of body,
beauty and wellness [83-86, 101-103]. In conclusion while carotenoids represent an
interesting group of natural ingredients, skin-friendly and environmentally friendly, useful to
prevent the premature skin aging, CN-HA nanoparticles must be considered an innovative
technology solution to increase the activity and effectiveness of many active ingredients at
skin level. These nanoparticles represent an essential requirement for success in the medical
and beauty marketplace of both cosmetic and pharmaceutical fields [104].
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Chapter XIII
Bixin and Norbixin: Chemistry,

Production and Health Benefits
Miguel Roehrs1 and Rafael Roehrs2

1
Post-graduate Program of Pharmacology, Center of Healthy Sciences,
Santa Maria, Brazil, Clinical Laboratory, University Hospital of Santa Maria,
Federal University of Santa Maria, Santa Maria, Brazil
2
Natural Products and Physico-chemical Studies Laboratory,
Post-graduate Program of Biochemistry, Federal University of Pampa,
Uruguaiana, Brazil
Abstract
Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts
of plants and some other photosynthetic organisms like algae, bacteria, and fungi. There
are over 600 known carotenoids; they are divided into two classes, xanthophylls (which
contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen).
All carotenoids are tetraterpenoids, meaning that they are produced from 8 isoprene (2-
methyl-1,3-butadiene) molecules and contain 40 carbon atoms. In humans, four
carotenoids (beta-carotene, alpha-carotene, gamma-carotene, and beta-crypoxanthin)
have vitamin A activity (meaning they can be converted to retinal). Bixin
((2E,4E,6E,8E,10E,12E,14E,16Z,18E)-20-methoxy-4,8,13,17-tetramethyl-20-oxoicosa
2,4,6,8,10,12,14, 16,18-nonaenoic acid) is an polyunsaturated, norcarotenoid, red dye
from the main fruit of annatto, its seeds reduced to powder are widely used to color food
and sunscreen. The annatto seeds contain about 5% pigment, which consist of 70-80% of
bixin. The bixin is soluble in fats but insoluble in water. When exposed to alkalis, the
methyl ester is hydrolyzed and produces the norbixin dicarboxylic acid, a water soluble
derivative. It is a chemically unstable compound when isolated and is converted, via
isomerization, in bixin trans-(β-bixin), the cis-trans isomer of bixin. Carotenoids bixin
and norbixin have two stereo configurations, i.e., cis and trans. In extracts, under normal
conditions, the cis-bixin or cis-norbixin are more unstable. The cis-bixin or cis-norbixin
solution under heating are partially converted into the trans configuration, which is more
stable and known as isobixin and isonorbixin. Brazil is one of the major producers of

262 Miguel Roehrs and Rafael Roehrs
annatto and its planting occurs primarily in the North and Northeast regions, although its
cultivation in the last decades expanded to other regions, in the Southeast, especially Sao
Paulo and Rio de Janeiro. The international market growing demand is justified by the
prohibition of synthetic dyes due to studies that have proven their toxicity. Like many
carotenoids, bixin and norbixin also present significant antioxidant properties. Both
carotenoids can reduce levels of malondialdehyde (biomarker of lipid peroxidation) when
induced by cyclophosphamide (immunosuppressant), as well as protect the DNA from
oxidative damage in vitro. Bixin inhibited in vitro generation of superoxide and the
generation of reactive oxygen species such as hydrogen peroxide and hydroxyl radicals
and with protective action against mutagenicity in human lymphocytes. Besides the
antioxidant property, these carotenoids also present important metabolic actions in lipids
and sugars. Bixin acts as PPAR-γ agonist (receptor activated by peroxisome proliferators
range), a nuclear receptor that acts in the metabolism of lipids and carbohydrates and has
anti-inflammatory, immunomodulatory and anti-atherosclerotic action. This interaction of
BIX and the PPAR gamma receptor may increase the sensitivity to insulin in adipocytes
so that there is a greater glucose uptake. Furthermore, the interaction with the receptor
regulates lipid metabolism improving metabolic syndrome present in diabetic patients.
Keywords: Carotenoids, bixin, norbixin, oxidative stress, Bixa orellana, Annatto
Carotenoids: Chemistry and Dietary Sources

Color is essential to the full enjoyment of our food. Yellow, orange and red colored
carotenoids are much more than pigments. For this reason the multidisciplinary area of
carotenoid anti- and pro-oxidant chemistry is not only important, but also attractive.
Carotenoids are a family of pigmented compounds that are synthesized by plants and
microorganisms but not by animals. In plants, they contribute to the photosynthetic
machinery and protect them against photo-damage. Fruits and vegetables constitute the major
sources of carotenoid in human diet. All carotenoids possess a polyisoprenoid structure, a
long conjugated chain of double bond and a near bilateral symmetry around the central double
bond, as common chemical features [1]. Different carotenoids are derived essentially from
modifications in the basic structure by cyclization of the end groups and by introduction of
oxygen functions giving them their characteristic colors and antioxidant properties. Due to the
presence of the conjugated double bonds, carotenoids can undergo isomerization to cis-trans
isomers. Although the trans isomers are more common in foods and are more stable, very
little is known about the biological significance of carotenoid isomerization in human health
[1]. Although carotenoids are present in many common human foods, deeply pigmented
fruits, juices and vegetables constitute the major dietary sources with yellow-orange
vegetables and fruits providing most of the β-carotein and α-carotein, orange fruits providing
α-cryptoxanthin, dark green vegetables providing lutein and tomatoes and tomato products
lycopene as well [1]. Examples of food that show carotenoids are: Apricot, carrots, spinachs,
anteloupe, green beet, broccoli, tomato, tangerine, papaya and green peas. Due to the
unsaturated nature of carotenoids they are subject to changes due mainly to oxidation.
However, other factors such as temperature, light and pH can also produce alterations that can
influence the color of foods as well as their nutritional value. In general carotenoid content of
foods is not altered to a great extent by common household cooking methods such as

Bixin and Norbixin 263
microwave cooking, steaming and boiling but extreme heat can result in oxidative destruction
of carotenoids. A diet with total vitamin E intake and total carotenoids was associated with a
reduced risk of type 2 diabetes, whereas vitamin C intake was not. Considering the individual
tocopherols and tocotrienols, the intake of α-tocopherol, γ-tocopherol, δ-tocopherol, and β-
tocotrienol was inversely associated with diabetes risk. Among the carotenoids considered, β-
cryptoxanthin intake showed the strongest inverse association with diabetes risk. In general,
the results were similar among men and women with the exception of α-carotene, which
showed a difference between genders [2]. Beyond β-carotein and lycopene, the absorption of
other major carotenoids is not well known. Several factors influence the absorption of
carotenoids. Food processing and cooking that cause mechanical breakdown of the tissue,
releasing the carotenoids, improve their absorption. They are absorbed into the
gastrointestinal mucosal cells and appear unchanged in the circulation and tissues. In the
intestine the carotenoids are absorbed by passive diffusion after being incorporated into the
micelles, which are formed by dietary fat and bile acids. The micellular carotenoids are then
incorporated into the chylomicrons and released into the lymphatic system. They are then
incorporated into the lipoprotein at the site of the liver and released into the blood stream.
Carotenoids are absorbed differently by different tissues. Little is known about the
mechanisms of tissue absorption of carotenoids at this time. The major site of tissue storage
of carotenoids is the adipose tissue [1].
Chemistry and Dietary Sources

In developing countries, people of native communities use Bixa orellana L., commonly
known as ‗achiote/ annatto‘ (Family: Bixaceae)‘, in folk medicine in the form of decoctions,
teas and juices for the treatment of common infections. In Philippines, the leaf decoction is
used to cure skin diseases and burns. The leaves are a popular febrifuge in Cambodia. The
infusion of leaves is prescribed as a purgative and used in the treatment of dysentery. In
Central America, the oil derived from seeds is used to cure leprosy and decoction is given to
treat jaundice. A colored compound obtained from the pulp of the seeds called ‗bixin‘ is used
all over the world as a red-orange dye, for coloring rice, cheese, soft drinks, oil, butter and
soup. The dye is also used in some regions in textile industry and the seeds as a condiment.
Ayurveda practitioners in India use it as an astringent and mild purgative and it is also
considered a good remedy for treating dysentery and kidney diseases. The root bark is anti-
parasitic and antipyretic. The traditional healers claim that Bixa sp are more efficient to treat
infectious diseases than synthetic antibiotics [3]. Bixa orallana L. (B. orallana), belonging to
the family Bixaceae, is used for the treatment of liver disorders. It is a small evergreen tree,
leaves are cordate, acuminate, flowers are white or pink in terminal panicle, fruits reddish
brown, and seeds are trigonous covered with a red pulp. The roots bark and seeds of B.
orellana are antiperiodic, antipyretic and astringent. Seeds and leaves of the annatto tree were
used by the Aztecs to prepare remedies for a variety of illnesses such as tonsillitis, asthma,
pleurisy, rectal disorders, headache, jaundice, sunstroke, and burns [4]. A study with
methanolic leaf extract of B. orellana showed that, it is 50% more efficacious than the seed
extract, on average. The inhibitory activity of seed extract of B. orellana could be attributed
to the presence of flavonoids. Flavonoids have the ability to complex with extra

cellular/soluble proteins and with bacterial cell walls. Bixa orellana L. (annatto) is a unique
plant, having massive storage of bixin, a carotenoid, in the vacuoles of aril layer of seeds. In
mature dried seeds, the stored pigments can be extracted as crude powder of carotenoids [5].
Figure 1. Chemical structures of the carotenoids bixin (BIX, CAS 6983-79-5) (a) and norbixin (NOR, CAS
542-40-5) (b).
Bixa orellana L. is a native shrub from South America; its seeds, which are known as
annatto, contain an orange-red pigment (Fig. 1). Anatto is used for the industrial production
of a variety of foods such as butter, margarine, cheese and ice cream; more than 70% of the
annual production of anatto is commercialized at domestic markets and used for home
cooking (for rice dishes, manioc flour, sauces, steaks, and white meat), particularly in the
north and northeastern regions of Brazil [6].
The bixin is the natural pigment annatto seed, representing 80% of all carotenoids. From
it, the other pigments are obtained as the normal bixin (lipid-soluble), the salt of norbixin
(water) and products of thermal degradation, which are characterized by lipid solubility and a
more stable yellow color, suitable for use in pastas [7].
Bixin (cis-bixin, cas 6983-79-5) is the main oil-soluble pigment of Bixa orellana seeds. It
is a carotenoid devoid of provitamin A activity with two carboxylic groups, one of which is a
methyl ester. Hydrolysis of this methyl ester group yields the corresponding dicarboxylic
acid, norbixin, which is an annatto pigment soluble in aqueous alkaline solutions [8]. Bixin is
one of the few naturally occurring carotenoids in the cis configuration. Small amounts of
norbixin are also found in annatto seeds making the carotenoid annatto soluble in aqueous
extracts. Cis isomers, both of bixin as the norbixin are naturally present in the plant, being
converted into trans form, more stable, when subjected to high temperatures. The structural
differences between cis-trans isomers result in physical characteristic particularities. The
bixin, while soluble in solutions of low polarity, when presented as the isomer cis it does not
demonstrate extensive solubility in vegetable oils due to the polarity provided by groups
located on the same side of the structure. Moreover, the trans-bixin is soluble in oils and
provides red color, differing from the cis isomer, orange-colored [9].
Currently, annatto is extensively used as a colorant in Western foods including
margarine, candies, and cheeses, with>8,000 tons produced annually for use as a food
colorant, and an additional 3,000 tons used in the cosmetics industry. The Joint Federal
Agricultural Organization – World Health Organization Committee on Food Additives (
JECFA) – has established an acceptable daily intake (ADI) of annatto extracts as 0–12 mg=kg

body weight, with the average North American consuming approximately 0.38–0.63 mg of
annatto extract per day. The intake of annatto is much higher in some Latin American
countries, where annatto is commonly used as a condiment [10].
Extraction and Obtaining

Nowadays, several methods are used for the extraction of antioxidant compounds in
plants, since traditional techniques, using organic solvents, as well as more advanced
processes such as supercritical fluid extraction and use of micro-waves. The FAO describes
six different annatto extracts characterized by the final concentrations of each carotenoid and
the methods used to obtain. Extractions performed by means of organic solvents reach
concentrations around 92% and the proportion of carotenoids at the end of each pigment is
affected by the acidity extractor [8].
The bixin can be extracted by traditional Soxhlet method, resulting in a yield of
approximately 1.3% of bixin. However, innovations in this method aiming at the total
depletion of pigments, provided concentrations at 3.6% of this carotenoid. In this method, the
seeds were dried, fragmented and subjected to extraction for 6 hours using chloroform as
solvent. The conventional percolation system was replaced with a perforated Teflon plate and
covered with cotton. From this process it was obtained a concentrated intense red-purple
color, characterized as bixin by means of nuclear magnetic resonance of hydrogen and carbon
(¹H NMR and ¹³C). The easier form of extraction is: the seeds not crushed, dried in an oven at
45 oC, are extracted with hexane in soxhlet for a period of 8 hours after evaporation of the
solvent in rotavapor obtaining an oily product called dark red oily fraction. Then, the seeds
are extracted with chloroform, also for a period of 8 hours, after evaporation of the solvent
yields a solid residue, reddish, called concentrate bixin. The pure bixin was obtained as
follows: the residue chloroform was solubilized in a mixture chloroform-acetone (1:1) and
left in the -20oC for 24 hours. The next day the precipitate was filtered and recrystallized from
CHCl3, thus providing the pure bixin, in the form of purple crystals with melting point 195-
196 oC [10].
The supercritical extraction is a technique guided in a modern and efficient way which
uses pressure for liquefaction of extractor solvent and its subsequent evaporation, allowing
obtaining a final product free from solvent. This method can be further improved by the use
of co-solvents such as methanol and ethanol, aiming to improve the extraction yield. The use
of CO2 as a supercritical fluid offers advantages because it is an inert, nontoxic,
nonflammable, low cost gas and gaseous under normal conditions, facilitating the separation
of solutes. The process is carried out at relatively low temperatures, favoring obtaining
thermolabile compounds and better preserving the extracts. Moreover, this technology has
high operational cost, preventing, sometimes, the industrial application [8].
The microwave extraction is a method of obtainment characterized by simplicity and
effectiveness, and it optimizes the process performance while requiring less time and less use
of solvents. The presence of water is a fundamental requirement in the microwave extraction,
since it is the radiation absorber medium and generator of heat responsible for promoting the
breakdown of plant cells releasing their phytochemicals. The extraction of bixin from annatto
seeds using the method of extraction by microwaves and traditional method, based on heating

the seed in ethyl acetate solution were compared. The microwave extraction was performed
for 18 minutes, again in a medium containing water and ethyl acetate and it showed twice the
efficiency, demonstrating the efficiency and rapidity of this technique. Analyzes carried out
with antioxidants the bixin obtained by the two techniques were similar, indicating no change
in the chemical properties of carotenoids obtained by extraction assisted by microwaves [9].
Norbixin can be obtained with a chemical synthesis of bixin by a saponification reaction
which was used to convert cis-bixin to norbixin. In brief, cis-bixin was added to aqueous
sodium hydroxide, and the reaction is agitated until the solution turned from cloudy to clear.
Complete conversion to norbixin is confirmed with HPLC [11].
Health Benefits
Previous pharmacological studies have revealed that Bixa orellana extracts, which has as
main components carotenoids bixin and norbixin, possess antiprotozoal, anthelmintic and
platelet antiaggregant activity. Root´s extracts have been reported to have spasmolytic
activity. Extracts from the leaves and the branches have shown to be effective in neutralizing
the effects of snake venoms. Extracts from different parts (leaves and seeds in particular)
have displayed in vitro antimicrobial activity. The seed extracts have been reported to exhibit
chemopreventive and antioxidant activity. Bixin has also been found to have anticlastogenic
activity [12]. Furthermore, there has been increasing interest in the study of these carotenoids
in the metabolism of carbohydrates and lipids. Also, there is interest in deepening the study
on antioxidant activity, characteristic common to all carotenoids.
Action in Lipid and Carbohydrates Metabolism

Bixin, which is the major carotenoid in the seeds of Bixa orellana, has been studied in
detail regarding its effects onlipid and carbohydrate metabolism, as some researchers realized
that after ingestion of glucose some abnormalities occurred in animals. There are reports in
literature showing that normal animals presented hyperglycemia after ingesting this
carotenoid. On the other hand, animals with induced diabetes, where glucose levels were
high, this same carotenoid decreased glycemia. It is also known that the action on
carbohydrate metabolism is also dependent on the type of extraction. The oil suspension
depicts hypoglycemic activity; the methanol solution shows hyperglycemic action [13]. The
hypoglycemic activity may be occurring for two reasons: the first and most accepted theory is
due to relation to modulation of PPAR γ receptor; the second would be its action directly on
pancreatic beta cells by stimulating the production of insulin. There are theories that both
mechanisms occur synergistically, improving the effect of the carotenoid, especially in heart
disease and diabetes.
The most accepted theory for hypoglycemic activity of bixin is based on the action at the
nuclear receptor PPAR γ. The receptor peroxisome proliferator-activated (PPARs) are
transcription factors belonging to the family of nuclear receptors that regulate glucose
homeostasis, lipid metabolism and inflammation, which are activated by small hydrophobic
ligands. PPAR γ is necessary and sufficient for adipocyte differentiation. In adipocytes,

PPAR gamma regulates the expression of numerous genes involved in lipid metabolism,
included aP2, acyl-CoA synthetase, and lipoprotein lipase (LPL). Since PPAR γ is expressed
predominantly in adipose tissue, the prevailing hypothesis on bixin mechanim of action is the
side effects on insulin-responsive tissues such as skeletal muscle and liver. PPAR γ agonists
as bixin, with beneficial effects in distant tissues (muscle and liver) are probably because the
accentuate uptake mediated by insulin in adipose tissue, with combined effects of stock and
catabolism of free fatty acids. This induces the production of factors derived from adipocytes
with the potential for insulin sensitizing action, suppressing circulating levels or actions of
factors derived from adipose tissue, such as tumor necrosis factor alpha (TNF alpha) or
resisitin that cause insulin resistance. Then, activation of the PPAR gamma by bixin, induced
an increase in the clearance of fatty acids by adipose tissue, with a consequent decrease in the
plasma concentration and transport into the muscle. This decrease in fatty acids in the muscle
increases sensitivity to insulin [14].
Another mechanism by which bixin may be acting in the metabolism of lipids and
carbohydrates is via PPAR α. This PPAR isoforms mostly expressed in tissues with high rates
of fatty acid (FA) oxidation and peroxisomal metabolism. In these tissues, PPAR α regulates
the expression of genes involved in FA oxidation. PPAR α agonists are, therefore, commonly
used to treat hyperlipidemia and other dyslipidemic states. Besides their hypolipidemic
effects, PPAR α activators also ameliorate obesity-induced dysfunctions of carbohydrate
metabolism, including insulin resistance, hyperglycemia, and hyperinsulinemia, in animal
models of obesity and type-2 diabetes mellitus. Moreover, PPAR α activators induce the
mRNA expression of adiponectin and its receptors, which enhance insulin sensitivity and
suppress inflammation in adipose tissues. Therefore, appropriate spatial and temporal controls
of ligand-dependent PPAR α activation are important for the treatment of obesity-induced
metabolic diseases [15]. Although there are few studies using norbixin cause it is obtained via
synthetic pathway. Despite the fact that norbixin has a similar structure to bixin, it has a
hydrophilic character. Due to this feature, the modulating PPAR receptors α and γ, which are
primarily responsible for reduction of blood glucose with bixin, does not occur. Also, due to
the hydrophilic characteristic of norbixin, an interaction occurs with the plasma membrane
which results in alteration in membrane permeability to glucose affecting the intrinsic activity
of GLUT receptor (receptor responsible for the entry of glucose into the cell). This causes a
low absorption of glucose, thereby leading to hyperglycemia. Another theory on the
hyperglycaemic activity of norbixin may be by the interference of insulin response due to
changes in membrane fluidity [16].
Bixin and Norbixin: Oxidative Stress X

Antioxidants
Carotenoids have a wide range of actions in many pathologies, as well as lycopene has
beneficial actions in prostate cancer and heart disease, lutein and zeaxanthin for macular
diseases. These beneficial actions occur mainly for its antioxidant activity. This feature occurs
because antioxidant carotenoids have an extensive system of conjugated double bonds,
usually containing 40 carbon atoms, typically having internal isomerism, and always having
one or two cyclic structures terminating in conjugated bonds. This peculiar chemical structure

of carotenoids presents important features, such as singlet oxygen quenching, which is much
more reactive than the triplet oxygen present in air and can interact with many cellular
components producing oxidative products inactivated. There is also interaction of carotenoids
with oxygen to produce singlet oxygen and a carotenoid triplet, which makes their energy
harmlessly. These highly reactive molecules can interact with other radicals such as peroxide
and hydroxide radicals, generating non radicals, or they may interact with other molecules to
restore the carotenoid to the ground state while producing some free radicals. Removal of free
radicals cells is generally considered beneficial, and generation of free radicals in cells is
generally harmful [17, 18]. Under physiological conditions the antioxidant action of
carotenoids shows up prevailing. Addition reaction type mediator mentioned above, may be
oxidized carotenoids, both chemical and biologically by strong oxidants, generating a variety
of products. The extract of Bixa orellana, which largely contains the carotenoid bixin, also
has antioxidant properties. It can reduce the levels of malondialdehyde (a marker of lipid
peroxidation) and still modulate the action of antioxidant enzymes. It has been hypothesized
that the extract of annatto exerts a direct scavenger of reactive species produced mainly by
neutrophils as well as nitric oxide. One of the mechanisms the extract is acting through by
increasing the production of the antioxidant enzyme superoxide dismutase. The action of
bixin to inhibit the generation of superoxide anion as well as the generation of reactive
oxygen species was comparable to the action of lycopene, a powerful and well established
antioxidant. This leads to the conclusion that bixin the same manner as lycopene, can help
modulate oxidative stress encountered in cancer therapy with cisplatin [19, 20].
Conclusion
Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts of
plants and some other photosynthetic organisms like algae, bacteria, and fungi. Carotenoids
are derived essentially by modifications in their basic structure by cyclization of the end
groups and by introduction of oxygen functions giving them their antioxidant properties. Due
to the presence of the conjugated double bonds, carotenoids can undergo isomerization to cis-
trans isomers. Although the trans isomers are more common in foods and are more stable
very little is known about the biological significance of carotenoid isomerization in human
health. The carotenoids bixin and norbixin, main carotenoids extracted from the plant Bixa
orellana and synthesized from bixin, respectively, are included in the diet of the world
population usually as dyes in most foods because they are very safe involving toxicity. The
benefits provided by these carotenoids are very large, besides being considered antioxidants,
peculiar characteristics have been attributed to them, such as their action in the metabolism of
carbohydrates and lipids.
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[2] Montonen, J; Knekt, P. Jarvinen, R; Reunanen, J. Dietary Antioxidant intake and risk of
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[6] Barcelos, Grm; Grotto, D; Serpeloni, Jm; Aissa, Af; Antunes, Lmg; Knasmüler, S;
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[14] Tavares, V; Hirata, Mh; Hirata, Rc. Peroxissome proliferator-activated receptor gamma:
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[17] Olson Ja, Krinsky Ni. Introduction: the colorful, fascinating world of the carotenoids
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Chapter XIV
Biology of Carotenoids and their

Potential Cardiovascular Health
Benefits
Assunta Pandolfi*
Department of Experimental and Clinical Sciences, ―G. d‘Annunzio‖ University;
Aging Research Center, Ce.S.I., ―Gabriele d‘Annunzio‖ University Foundation,
Chieti-Pescara, Italy
Abstract
More than 700 carotenoids have been identified, but a few of them are considered of
nutritional relevance and circulate at micromolar levels, therefore they receive the most
attention from health researchers. Among them, β-carotene, α-carotene and β-
cryptoxanthin are the major carotenoids having significant pro-vitamin A activity, while
lutein, lycopene, and zeaxanthin are not converted into active retinoids by humans. Their
biological activity is therefore independent of retinoid-associated pathways. Given their
chemical structure, carotenoids have been conjectured to act as free-radical scavengers,
even though they can act as pro-oxidant molecules, at least at high oxygen concentration.
However, more recently, several biologically beneficial activities relating to their ability
to regulate various cellular functions have been proposed. Of note, a number of
epidemiological studies have shown a correlation between elevated dietary carotenoid
intake and circulating levels and decreased risk of cardiovascular disease (CVD). It has
recently been demonstrated that circulating serum carotenoids are associated, beneficially
it would seem, with markers of inflammation, oxidative stress, and endothelial
dysfunction, which are known to be associated with CVD.
At present, it is widely accepted that one of the earliest detectable pathogenic events
in both human and experimental atherosclerosis is vascular inflammation associated with
activation of NF-θB pathway, in turn triggering up-regulation of the expression of the
vascular cell adhesion molecules (VCAM-1), intercellular cell adhesion molecules
(ICAM-1) and E-Selectin.
*
Tel and fax ##39-0871-541425, e-mail: [email protected].

272 Assunta Pandolfi
Nitric Oxide (NO), constitutively generated by the endothelial cells, plays an

important role in maintaining vascular homeostasis and the pro-inflammatory response
that characterizes the early stages of atherosclerosis. It is known that NO inhibits the
vascular inflammatory response, down-regulating NF-θB-dependent expression of
adhesion molecules. The maintenance of endothelial NO bioavailability is therefore
considered beneficial to endothelial functions and more in general to vascular health.
However, in the dysfunctional endothelium, NO may rapidly react with superoxide anion
(O2-) to form a stable potent oxidant peroxynitrite (ONOO-) resulting in decreased
vascular relaxation, and contributing to the up-regulation of NF-θB dependent cellular
response. Thus, the general effect of anti-oxidant molecules on the biological function of
NO is likely to be due, at least in part, to a direct removal of O2-. Within this scenario,
carotenoids may be considered potential anti-oxidant modulators of endothelial response
to pro-oxidant/inflammatory stimuli.
Even though in vitro and in vivo experiments have recently demonstrated that
carotenoids are able to reduce inflammation, while epidemiological studies indicate a
strong correlation between dietary carotenoid consumption and decreased risk of CVD,
the mechanism underlying the carotenoid‘s cardiovascular protective activities, is still
little known.
Keywords: Carotenoids, Endothelial dysfunction, Inflammation, Nitric Oxide, Reactive

Oxygen Species, Cardiovascular health
Introduction
Carotenoids are tetraterpenoids that are synthesized by plants and microorganism but not
by animals. Various fruits and vegetables form the main source of carotenoids in the human
diet [1, 2]. They are a class of pigmented compounds which have long been mooted as
cardiovascular disease-preventive food constituents.
More than 700 carotenoid compounds have been characterized; about 50 of them
consumed in the human diet [3, 4]. About 10-15 carotenoids represent most of the dietary
intake, and these are found in measurable concentrations in human blood and tissues [5, 6] the
most common being lycopene, lutein, β-carotene, α-carotene, β-cryptoxanthin, and zeaxanthin
[7].
Various biological effects have been ascribed to carotenoids. One possible action
mechanism of carotenoids is via their antioxidant and scavenging capacity [8]. There is, in
fact, reason to think that carotenoids act as modulators of intracellular redox status [9]. The
conjugated double bond structure is primarily responsible for the chemical reactivity of
carotenoids with free radicals such as the peroxyl, hydroxyl, and superoxide radicals. Of note,
carotenoids have proved able to prevent or decrease oxidative damage to DNA, lipid and
proteins [3, 10].
Conversely, a number of reports claim that carotenoids can act as pro-oxidant molecules
and increasing the total radical yield in a system [11, 12]. The key factor to determine the
switch of carotenoids from antioxidant to pro-oxidant is the oxygen partial pressure (pO2)
and the carotenoid concentration [13, 14]. At higher pO2 a carotenoid radical can react with
molecular oxygen to generate a carotenoid-peroxyl radical which can act as a pro-oxidant by
promoting oxidation of unsaturated lipids.

Biology of Carotenoids and their Potential Cardiovascular Health Benefits 273
Although the antioxidant properties of carotenoids have been proposed as the main
mechanism behind their beneficial effects, recent studies are also beginning to show that these
compounds may mediate their effects via other mechanisms such as cell growth relulation,
gap-junction communication, modulating gene expression, etc [7]. In addition, carotenoids
are a sizable dietary source of vitamin A, dividing into provitamin A (β-carotene, α-carotene
and β-cryptoxanthin) and non-provitamin A compounds [15].
Interestingly, a number of epidemiological reports have shown a correlation between
elevated dietary carotenoid intake and circulating levels and prevention of CVD [16, 17]. For
example a relationship has recently been demonstrated between circulating carotenoid
concentrations and several markers of inflammation, oxidative stress, and endothelial
dysfunction [18], which are known to be associated with CVD [19-21].
At present it is well recognized that atherosclerosis is an inflammatory disease [22], and
there is some evidence to suggest that the beneficial effects of carotenoids may result from
modulation of the inflammatory responses.
NF-θB inflammatory pathway activation, in turn triggering up-regulation of the
expression of the VCAM-1, ICAM-1 and E-Selectin [23], has been shown to be partially
regulated by Radical Oxygen Species (ROS) and has been implicated in various forms of
CVD [24, 25]. Moreover, NO, constitutively generated by endothelial cells, plays an
important role in the maintenance of vascular homeostasis and in the pro-inflammatory
response that characterizes the early stages of atherosclerosis [26]. An imbalance entailing
reduced production of NO and increased production of ROS may be involved in impaired
endothelium-dependent vasodilation in patients with cardiovascular risk factors and diseases.
It is known, in fact, that NO inhibits the vascular inflammatory response by quenching NF-θB
nuclear transfer thanks to its regulatory activity on IθBα synthesis [27] and also by directly
inhibiting NF-θB binding to DNA [28]. These events, in turn, down-regulate NF-θB-
dependent expression of adhesion molecules [29].
Carotenoids and vitamins could have an antioxidant-mediated tempering influence on
endothelial function and inflammation, thereby reducing the risk of atherosclerosis [30]. We
recently demonstrated that β-Carotene or lycopene treatment reduce the inflammatory
response in tumor necrosis factor-α (TNF-α)-treated human umbilical vein endothelial cells
(HUVECs). This is due to redox balance protection and to the maintenance of NO
bioavailability [31]. Lee and colleagues demonstrated that Akt-specific inhibitor reverses the
inhibitory effect of carotenoids on tissue factor activity, indicating that carotenoids enhance
phosphorylation of Akt and suppress tissue factor activity in endothelial cells potentially
through a NO-mediated mechanism [32]. This would support the idea that maintenance of
endothelial NO bioavailability may be beneficial to endothelial function and more in general
to vascular health. In line withthis hypothesis, an astaxanthin-enriched diet reduces
endothelium-mediated blood pressure and improves cardiovascular parameters in in vivo
experiments performed on spontaneously hypertensive rats [33]. In a human study, George
and colleagues [34] recently demonstrated significant effects of chronic and acute
consumption of fruit- and vegetable-puree-based drinks on endothelium-mediated
vasodilation, known to be due to regular NO availability.
However, although the studies mentioned support the idea that carotenoids may exert
their cardiovascular protective action by increasing NO bioavailability, the majority of papers
published to date indicate that such benefits stem from carotenoids‘ anti-inflammatory action,
though the mechanism/s underlying this vascular activity is/are still largely unknown.

Martin et al. [35], showed that preincubation of lycopene using human aortic endothelial
cell cultures resulted in a 13% decreased expression of the vascular cell adhesion molecule.
Further study confirmed that lycopene also inhibited LPS-induced IθB phosphorylation, IθB
degradation, and NF-θB translocation. Moreover, lycopene blocked the phosphorylation of
ERK1/2 and p38 MAP kinase but not c-Jun NH2-terminal kinase [36].
Notably, Riso et al. [37] have reported that concentrations of the proinflammatory
cytokine TNF-α in the blood of healthy volunteers were decreased after dietary
supplementation with a tomato-based drink.
However, because carotenoids are a complex group of chemicals, and studies of the
health effects of carotenoids are very heterogeneous, it is difficult to perform a meta-analysis
or even a detailed systematic review of the health effects of carotenoids.
Thus, although plasma concentrations of carotenoids are considered useful biomarkers of
total dietary intake of vegetables and fruit [38] while epidemiological studies have provided
convincing evidence in support of the protective role of carotenoids in CVD [39], these
observations need to be validated on the one hand by carrying out in vitro studies on the
molecular mechanisms and, on the other hand by conducting well controlled human
intervention studies in the future.
The present chapter will outline the current situation of relations between the carotenoids
and prevention of CVD, examining epidemiological studies, clinical trials and in vitro
experiments as well as in vivo animal studies. As a final point, a conclusion as to the
importance of these compounds in cardiovascular health will be drawn.
Epidemiological Studies
In the scientific research community there has been keen interest in the relationship
between diet and health outcomes. Numerous epidemiological studies over the past decades
have shown that protection against many chronic diseases, including CVD, is due to high
consumption of fruit and vegetables. Recently, Donaldson analyzed sixty-two studies of
plasma carotenoids in relation to health outcomes, mostly prospective cohort studies or
population-based case-control studies. Based on these data, a carotenoid health index has
been proposed with risk categories as follows: very high risk: <1 κM, high risk: 1–1.5 κM,
moderate risk: 1.5–2.5 κM, low risk: 2.5–4 κM, and very low risk: >4 κM [40]. Notably, over
95 percent of the USA population falls into the moderate or high risk category of the carotenoid
health index [41].
Today, CVD is the primary cause of death in Western cultures and accounts for up to a
third of all deaths worldwide. In comparison to the Northern European or other Western
countries, the Mediterranean area has lower rates of mortality from cardiovascular diseases
and cancer, and this is ascribed, at least in part, to the so-called Mediterranean diet, which is
rich in plant-derived bioactive phytochemicals [42]. Recognition of the active constituents of
the Mediterranean diet is therefore fundamental to any formulation of correct dietary
guidelines. Several articles have appeared in support of the role of carotenoids in the
prevention of CVD, mostly based on epidemiological studies showing a relationship between
carotenoid plasma levels and CVD. A less well-defined and more multifaceted picture
emerges from interventional trials, where a number of works have reported contradictory

results (see below). Although many features of carotenoids‘ in vivo metabolism, functions and
clinical indications remain to be clarified, supplementation of low doses of these compounds
has been already suggested as a preventive measure for combating and improving many
aspects of CVD.
In recent times, 139 Cretan (Greek) men aged 79 years and over were compared to men
from Zutphen (The Netherlands). The Cretan men had approximately fourfold higher mean
levels of lycopene as well as a lower level of oxidative stress and higher levels of antioxidants
in plasma than men of the same age from Zutphen [43].
Lately, Karppi et al. [44] assessed relations between the concentrations of serum
carotenoids and CVD mortality among Eastern Finnish men, demonstrating that low
concentrations of serum β-carotene concentrations may increase the risk for CVD mortality
among Eastern Finnish men. In addition, the same authors had previously evaluated serum
samples from 349 subjects in relation to concentrations of conjugated dienes in low-density
lipoprotein (LDL), this being one marker of lipid oxidation. The lycopene content in plasma
correlated significantly and negatively with the content of conjugated dienes. Thus, dietary
carotenoids proved significantly to lower LDL oxidative modification in vivo [45]. To
examine whether serum concentrations of carotenoids are related to the risk of sudden cardiac
death (SCD) in middle-aged men, Karppi et al. [46] studied a population consisting of 1031
Finnish men aged 46-65 years enllisted in the Kuopio Ischemic Heart Disease Risk Factor
(KIHD) cohort. Their results suggest that low serum β-carotene concentrations may enhance
the risk of SCD in middle-aged Finnish men. Additionally, low serum-carotene
concentrations may be associated with the risk of CVD and total mortality. Early
observational studies reported an association between a high dietary intake of β-carotene and
reduced incidence of CVD [47, 48]. In a case-control study, the risk of nonfatal acute
myocardial infarction (MI) in women was inversely associated with daily intake of 𝛽-
carotene-containing diet [49]. In the Rotterdam study, a population-based cohort study
targeting the elderly, the dietary intake of 𝛽-carotene was inversely associated with the risk of
MI [50].
Interestingly, in the American Health Professional‘s Study conducted on 39,910 US
males, the carotene intake was associated with a lower risk of Coronary heart disease (CHD)
among current smokers but not nonsmokers [51].
To search for associations between serum carotenoids and risk factors for development of
atherosclerosis, Xu et al. [52] studied 40 early atherosclerosis patients without clinical
cardiovascular events, and comparable healthy controls. The results suggested that early
atherosclerosis patients had lower serum concentrations of lutein and zeaxanthin than healthy
subjects. Serum carotenoids were associated with reduced risk of atherosclerosis. This
association was further supported by the study of Dwyer et al. [53] on 573 middle-aged
women and men who were free of symptomatic cardiovascular disease at baseline. The
findings suggest that higher levels of plasma oxygenated carotenoids (lutein, zeaxanthin,
beta-cryptoxanthin) and α-carotene may be defensive against early atherosclerosis.
Supporting this observation, Marin et al. [54] demonstrated that the Mediterranean diet
induces a decrease in endothelial injury and dysfunction, which is associated with an
improvement in the regenerative capacity of the endothelium in elderly subjects. Notably,
recent findings show that the favourable effects of fruit and vegetable intake on markers of
inflammation and oxidative stress are already present by early puberty [55]. In addition,
Azzini et al. [56] showed that the Mediterranean dietary pattern is associated with significant

amelioration of multiple risk factors, including a better cardiovascular risk profile, reduced
oxidative stress and modulation of inflammation.
However, evidence regarding the health benefits of carotenoids is controversial. In recent
times, the effects of serum carotenoids and their interactions on mortality have been examined
in a representative sample of US adults. The study consisted of adults aged ≥20 years enrolled
in the National Health and Nutrition Examination Survey (NHANES) III, 1988–1994, with
measured serum carotenoids and mortality (CVD and cancer) follow-up through 2006
(N=13,293). Analyses with continuous carotenoids confirmed associations of serum total
carotenoids, α-carotene, and lycopene with all-cause mortality (P<0.001). In a random
survival forest analysis, very low lycopene was the carotenoid most strongly predictive of all-
cause mortality, followed by very low total carotenoids. α-carotene/β-cryptoxanthin, α-
carotene/lutein+zeaxanthin and lycopene/lutein+zeaxanthin interactions were significantly
related to all-cause mortality (P<0.05). Interestingly, low α-carotene was the only carotenoid
associated with CVD mortality (P=0.002). Very low serum total carotenoid, α-carotene, and
lycopene concentrations may be risk factors for mortality, but carotenoids show interaction
effects on mortality. Studies of balanced carotenoid combinations are necessary for
confirmation [57].
Moreover, several years ago the EURAMIC study already suggested that lycopene, or
some highly correlated substance which is found in a common food source, may contribute to
the protective effect of vegetable consumption on myocardial infarction risk [16].
Subsequently, De Waart et al. [58] suggested that serum levels of individual carotenoids,
particularly the oxygenated species, are inversely associated with all-cause mortality.
Recently, epidemiological data concerning lycopene and its potential cardiovascular
health benefits have been extensively reviewed by Bohm [59]; the fundamental studies are
summarized in this chapter and shown below.
A total of 264 serum samples obtained from healthy Korean women were analysed for
their lycopene content [60]. In addition, arterial stiffness - a possible marker involved in the
pathophysiology of CVD - was assessed by brachial-ankle pulse wave velocity (baPWV).
Serum lipid profile, high-sensitivity C-reactive Protein (hs-CRP) and contents of oxidized
low density lipoprotein (oxLDL) were also analysed. A negative correlation was found
between lycopene and oxLDL and also between lycopene and baPWV. Thus, lycopene may
be responsible for a reduced oxidative modification of LDL, this possibly being one
mechanism by which lycopene could reduce arterial stiffness and the risk of CVD.
Again, a total of 299 Korean men were investigated as to the interrelationship between
arterial stiffness, antioxidant status and the risk of metabolic syndrome. The authors analysed,
among other parameters, baPWV, lycopene content, lipid profile and oxLDL. baPWV
inversely correlated with lycopene content in serum. A negative correlation was also seen
between lycopene and oxLDL. Thus, an interrelationship was shown between circulating
lycopene, baPWV and metabolic syndrome [61].
In another study, 3061 participants were invited to fill in a questionnaire and to give
serum samples. Lycopene contents in serum tended to be lower for those who died due to
CVD than for those who survived [62]. A case-control study with 760 cases and 682 controls
showed a decreased risk of acute myocardial infarction with increasing intake of α-carotene,
β-carotene and β-cryptoxanthin but no association for lycopene [63]. The CARDIA Study
(Coronary Artery Risk Development in Young Adults) with 4580 participants showed that

those people with higher lycopene contents tended to have less healthy lifestyles. Serum total
and individual carotenoids, with the exception of lycopene, were inversely associated with
markers of inflammation, oxidative stress and endothelial dysfunction [18]. The Minnesota
Heart Survey Study with 5369 men and 6070 women used a 24-h dietary recall. The authors
developed a Heart Disease Prevention Index. This index improved between 1980/1982 and
2000/2002: for men by 2.58 points (8.3%) from 31.14 to 33.72 and for women by 2.44 points
(7.9%) from 30.97 to 33.41. Thus, overall diet quality has moderately improved. However,
improvement plateaued and levelled off during the last 5-year period that was studied.
Regarding the carotenoids, only uptake of β-carotene, lutein, zeaxanthin and β-cryptoxanthin
significantly increased from 1980/1982 to 2000/2002, but not consumption of lycopene [64].
So, controversial epidemiological data exist regarding the CVD-preventive effects of
lycopene.
In Vitro Experiments
As mentioned above, carotenoid-rich diets have been associated with decreased risk of
CVD, but the underlying mechanism is still unknown.
In the last ten years some in vitro studies have suggested that carotenoids significantly
inhibit TNF-α-induced ICAM-1 and VCAM-1 expression in both vein and arterial endothelial
cells [65] and have barrier integrity activity, as well as inhibitory activity on cell adhesion and
migration to endothelium by blocking the activation of NF-θB, CD14 and TLR4 expression
and production of TNF-α [66].
We recently demonstrated that in HUVECs, both β-carotene and lycopene significantly
affected TNF-α-induced inflammation. Notably, this was associated with a significant
decrease in the generation of ROS and nitrotyrosine (an index of ONOO-), increased
NO/cGMP (cyclic guanosine monophosphate) levels, and down-regulation of NF-kB-
dependent adhesion molecule expression and monocyte–HUVEC interaction. Thus, our
results indicate for the first time that treatment with β-carotene or lycopene reduces the
inflammatory response in TNF-α-treated HUVECs through redox balance protection and the
maintenance of NO bioavailability [31].
The maintenance of endothelial NO bioavailability is therefore considered beneficial to
endothelial function and more in general to vascular health. However, in TNF-α-stimulated
endothelium NO rapidly reacts with superoxide anion (O2-) to form a stable potent oxidant
ONOO- resulting in decreased vascular relaxation, and contributing to the up-regulation of
NF-kB-dependent cellular response. Thus, the general effect of anti-oxidant molecules on the
biological function of NO is likely to be due, at least in part, to direct removal of O 2- [67].
Within this scenario, carotenoids may be considered potential anti-oxidant modulators of
endothelial response to pro-oxidant/inflammatory stimuli.
Again, the effects of lycopene on oxidative injury and apoptosis in endothelial cells
following exposure to H2O2 were investigated by Tang et al. [68] using human vascular
endothelial cells (ECV304 cells). Pre-treatment with lycopene dose-dependently decreased
malondialdehyde (MDA) contents in H2O2-treated cells. Lycopene also significantly reduced
the number of cells undergoing apoptosis in response to H2O2 inhibiting the upregulation of
p53 messenger ribonucleic acid (mRNA) and caspase3 mRNA. These results tend to suggest

that protecting endothelial cells from oxidative injury may be one of the mechanisms
underlying the cardiovascular-related beneficial effects of lycopene. In agreement with the
hypothesis that carotenoids might exert their protective role through oxidative stress
reduction, Rossoni-Junior et al. [69] recently showed that neutrophils from diabetic animals
produce significantly more reactive oxygen species and NO than their respective controls and
that supplementation with beta carotene and annatto (which has been identified as a
carotenoid having antioxidative effects) is able to modulate the production of these species.
Interestingly, annatto extract may have therapeutic potential for modulation of the reactive
oxygen species/NO balance induced by diabetes. The study published by Bai et al. [70] in
2005 already supported the idea that carotenoids may protect against oxidative stress. β-
carotene, in fact, directly blocked the intracellular accumulation of reactive oxygen species in
RAW264.7 cells stimulated with LPS, just as both the NADPH oxidase inhibitor diphenylene
iodonium and antioxidant pyrrolidine dithiocarbamate did. The inhibition of NADPH oxidase
also inhibited NO production, inducible Nitric Oxide (iNOS) expression, and iNOS promoter
activity. Moreover, carotene inhibited the expression and production of these inflammatory
mediators in both LPS stimulated RAW264.7 cells and primary macrophages in a dose-
dependent fashion, as well as in LPS-administrated mice. Furthermore, this compound
suppressed NF-θB activation and iNOS promoter activity in RAW264.7 cells stimulated with
LPS. These results suggest that β-carotene possesses anti-inflammatory activity by
functioning as a potential inhibitor of redox-based NF-θB activation, probably due to its
antioxidant activity.
Astaxanthin, a xanthophyll carotenoid, is a nutrient with unique cell membrane actions.
This molecule neutralizes free radicals or other oxidants by either accepting or donating
electrons, and without being destroyed or becoming a pro-oxidant in the process. Its linear,
polar-nonpolar-polar molecular layout equips it to precisely insert itself into the membrane
and span its entire width.
In this position, astaxanthin can intercept reactive molecular species within the
membrane‘s hydrophobic interior and along its hydrophilic boundaries. In cultured cells,
astaxanthin protected the mitochondria against endogenous oxygen radicals, conserved their
redox (antioxidant) capacity, and enhanced their energy production efficiency. The
concentrations used in these cells would be attainable in humans by modest dietary
intakes [71].
Thus, increasing evidence suggests that carotenoids may protect against atherosclerosis,
although, the exact mechanism(s) is still unknown. Because carotenoids may be considered
efficient antioxidants, it has long been proposed that this property may be responsible for its
beneficial effects. However, recently other mechanisms such as modulation of lipid
metabolism through control of cholesterol synthesis and oxysterol toxic activities have been
evoked as relevant effects [72].
Consistent with this hypothesis, Palozza et al. recently demonstrated that lycopene (0.5–2
mM) dose-dependently reduced the intracellular content of total cholesterol in THP-1 cells.
This effect was due to a reduction in expression of 3-hydroxy- 3-methylglutaryl-coenzym-A
(HMG-CoA) reductase, an enzyme promoting the deacylation of HMG-CoA to mevalonate
[73]. As hypercholesterolaemia is one of the most important risk factors for atherosclerosis,
these results imply a potential role of lycopene in attenuating foam cell formation and thus in
CVD risk reduction. Lycopene (0.5–2 mM) also dose-dependently reduced 7-ketocholesterol-
induced ROS production and 8-hydroxydeoxyguanosine formation in human THP-1

macrophages. In addition, lycopene was able to counteract 7-ketocholesterol-induced

apoptosis by limiting caspase-3 activation [74]. Moreover, in the same cellular model
lycopene prevented oxysterol-induced increase in pro-inflammatory cytokine secretion and
expression. That effect was accompanied by inhibition of oxysterol-induced ROS production,
mitogen-activated protein kinase phosphorylation and NF-θB activation. In additon, the
carotenoid increased peroxisome proliferator-activated receptor γ levels in THP-1
macrophages. Taken all together, these data bring new information on the anti-atherogenic
properties of lycopene, and on its action mechanisms in atherosclerosis prevention [75].
Among the mechanisms proposed as potentially responsible for the beneficial effects of
carotenoids we may consider inhibition of Vascular Smooth Muscle Cells (SMCs)
proliferation through regulation of the molecular pathways involved in cell proliferation and
apoptosis. In fact, several growth factors, among them platelet-derived growth factor (PDGF),
and increased SMCs proliferation, play an important role in the development and progression
of CVDs. Lycopene inhibited PDGF-BB induced signalling in SMCs of rats via binding to
PDGF-BB and inhibiting of the PDGFBB-SMC interaction as well as PDGFBB-induced
SMC proliferation [76].
Another key factor in atherogenesis is intravascular thrombosis [77]. Lycopene inhibited
both aggregation in human platelets in a dose-dependent manner and the ATP-release reaction
stimulated by agonists such as collagen or arachidonic acid. These results may suggest that
tomato-based foods might be especially beneficial in the prevention of platelet aggregation
and thrombosis [78].
The majority of the discussed in vitro studies [65, 68, 76, 78] used very high lycopene
concentrations up to 20 mM, which is an undue amount physiologically. When using lower
concentrations between 0.5 and 2 mM, an unphysiologically long treatment of up to 24 h [73]
was used.
In this connection, our recent results on HUVECs treated with high carotenoid
concentrations (2.5 mmol/L to 1 mmol/L), strongly indicate that in these experimental
conditions all the carotenoids tested are active in suppressing cell proliferation and decreasing
cell viability [31], thus demonstrating that unphysiological doses of carotenoids may bring
about their in vitro effects not specifically, but through a general cytotoxic action.
This evidence also corroborates the hypothesis that in vivo supplementation of
carotenoids at pharmacological levels may have adverse effects, possibly through their pro-
oxidant activity or, in the case of pro-vitamin A molecules, by overactivating retinoid acid-
related signalling [7, 79]. However, at lower doses (below 2.5 mmol/L) we found that both
carotenoids reduced the U937–endothelium interaction, confirming their potential beneficial
effects in reducing vascular inflammation.
In order to elucidate the mechanism/s potentially underlying the effects of carotenoids,
we considered their role in modulating NO bioavailability, according to the evidence that an
increased release of this molecule leads to down-regulation of the expression of NF-kB-
dependent adhesion molecules in endothelial cells [29, 80].
In our study, we demonstrate that in a model of vascular inflammation, the presence of
‗‗physiological‘‘ concentrations of β-carotene and lycopene is associated with a significant
increase in NO level and its bioavailability (as indicated by the increase in cGMP levels).
As expected, TNF-α treatment led to a fall in NO availability and release due to the
reduction of eNOS phosphorylation and to an increase in ROS generation, inducing a
situation of endothelial nitro-oxidative stress [81]. The inactivation of NO by ROS, in

particular O2-, is hence recognized to be a crucial factor in reducing NO bioavailability [67].

In this respect, we demonstrated that in cultured HUVECs, either β-carotene or lycopene (2.5
mmol/L, 2 hours) suppressed the increase in ROS generation and the intracellular levels of
NT (an index of ONOO- formation) due to TNF-α treatment. This activity significantly
quenched the oxidative stress generated by an inflammatory condition, allowing NO to exert
its biological effects, as documented by increased cGMP levels. Note that the increase in NO
levels associated with β-carotene and lycopene treatment was not affected by the presence of
the eNOS inhibitor L-NAME, indicating that their effect was not due to any specific
enzymatic activation of eNOS.
We propose that the candidate mechanism/s potentially responsible for the positive
modulation of NO bioavailability by β-carotene and lycopene is associated with their
reducing potential. Carotenoids are known to be able to directly interact with several free-
radical species in vitro [7], and this may account for their radical quenching or scavenging
properties. In pro-oxidant conditions, such as in the presence of TNF-α, reducing molecules
might contribute to maintaining NO availability by directly interacting with O2- and therefore
minimizing its reaction with intracellular NO and the formation of the potent oxidant-
nitrosylating agent, peroxinitrite.
In conclusion, our results, obtained in human endothelial cells exposed to a physiological
concentration of carotenoids, proved similar to those occurring in the vessels of subjects
consuming a ‗‗normal‘‘ diet, which provides robust evidence that these molecules may act on
an inflammatory vascular state by increasing vascular NO bioavailability thanks to their
reducing activity. This looks like another interesting mechanism to further elucidate why
carotenoids can prevent and/or delay cardiovascular disease.
Although most of the effects shown in in vitro studies cannot be directly transferred to
the in vivo situation, these findings offer an opportunity to understand the mechanisms
underlying the beneficial cardiovascular effects of carotenoids observed in vivo.
In Vivo Studies
Animal Studies
In the last few years, several studies have been conducted on a variety of animal models
in order to better understand whether diet supplementation with carotenoids, administered
alone or in combination with other molecules of nutritional interest, could exert the
hypothesized protective cardiovascular effects in animals.
Bansal et al. [82] fed adult male Wistar rats with lycopene dissolved in olive oil for 31
days. Lycopene reduced levels of lipid peroxides and augmented glutathione levels as well as
glutathione peroxidase (GSHPx) activity. The study is thus an example where lycopene
reduced oxidative stress in rats, in contrast to other studies where it failed.
In another experiment with female Wistar rats, lycopene was given for 2 week (0.001 and
0.1 g/kg body weight/day). The activity of glutathione reductase, GSH-Px and super oxid
dismutase (SOD) was significantly induced by various different doses of lycopene. In
contrast, that of catalase (CAT) was not modified [83].

Gitenay et al. [84] explored the potential role of yellow tomato, red tomato or lycopene
beadlets in a rat model with mild oxidative stress induced by a diet low in vitamin E. Six
week of feeding with 16% freeze-dried yellow tomato, 16% freeze-dried red tomato or 0.05%
lycopene beadlets did not affect the cholesterol concentration in plasma. Red tomato
intervention decreased triacylglycerol levels compared to controls, yellow tomato and
lycopene beadlets. Moreover, thiobarbituric acid-reactive substances levels in the heart were
lower after feeding on red tomatoes and yellow tomatoes, as compared to controls and
beadlets. Thus, tomatoes had a higher potential than lycopene to affect oxidative stress-related
parameters, possibly due to the synergy of all the phytochemicals in tomatoes.
Hsu et al. characterized the lipid-lowering effects and antioxidant mechanisms of tomato
paste (t. p., containing approx. 0.1% lycopene) in another animal experiment, this time
hamsters. Following 8 weeks of feeding, the authors observed significantly reduced contents
of total cholesterol and LDL cholesterol in serum due to feed containing 9% tomato paste.
Conversely, High Density Lypoproteins (HDL) cholesterol increased by 19.4% (3% t. p.) or
by 28.8% (9% t. p.). In addition, MDA in plasma was reduced by 80.2% (3% t. p.) or by
89.3% (9% t. p.). Regarding the antioxidant enzymes, the activities of CAT, SOD and GSH-
Px turned out to have significantly increased after 8 weeks feeding on 9% t. p. [85].
Astaxanthin is a carotenoid with antioxidant, anti-cancer and anti-inflammatory
properties. After the intravenous (5, 10 and 20 mg/kg) and oral (100 and 200 mg/kg)
administration of astaxanthin, its pharmacokinetic parameters proved dose-dependent and
dose-independent, respectively, in rats. The absorption of astaxanthin after oral administration
followed the flip-flop model. The hepatic and gastrointestinal first-pass extraction ratios were
approximately 0·490 and 0·901, respectively. Astaxanthin was unstable up to a 4 h incubation
in the rat gastric juices and 24 h incubation in various buffer solutions having a pH of 1–13
[86].
Recently several studies have suggested a cardiovascular protective role by this
compound. Khan SK et al. [87] studied the effect of a proprietary astaxanthin prodrug (CDX-
085) on thrombus formation, using a mouse model of arterial thrombosis. The influence of
free astaxanthin, the active drug in CDX-085, on human endothelial cells and rat platelets was
evaluated to investigate its potential action mechanisms. When compared to control mice, the
CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant
delays in occlusive thrombus formation following the onset of vascular endothelial injury.
Primary human umbilical vein endothelial cells and platelets isolated from Wistar-Kyoto rats
treated with free astaxanthin demonstrated significantly increased levels of released NO and
significantly decreased peroxynitrite levels. Thus, this study supports the potential of CDX-
085 and its metabolite astaxanthin to treat or prevent thrombotic cardiovascular
complications. Interestingly, astaxanthin-enriched diet reduces blood pressure and improves
cardiovascular parameters in spontaneously hypertensive rats. These effects are accompanied
by a decrease in oxidative stress and improvements in NO bioavailability [33]. The
ameliorative effect of astaxanthin was again recently demonstrated on endothelial dysfunction
in streptozotocin-induced diabetes in rats where it inhibited the ox-LDL-LOX-1-eNOS
pathway. This indicates that treatment with astaxanthin might be clinically useful for diabetic
complications associated with endothelial dysfunction [88]. Conversely, Jacobsson et al. [89]
evaluated the influence of alpha-tocopherol and astaxanthin on LDLoxidation lag time and
atherosclerotic lesion formation in Watanabe heritable hyperlipidemic (WHHL) rabbits. They
concluded that alpha-tocopherol but not astaxanthin prolonged the LDL oxidation lag time.

The two antioxidative substances did not prevent atherogenesis in WHHL rabbits in this
setting.
Verghese et al. [90] recently demonstrated that dietary lycopene has a protective effect on
cardiovascular disease in New Zealand male rabbits. Animals were fed for 12 weeks on a
normal diet, a high cholesterol (5 g/kg) diet and a high cholesterol diet containing various
amounts of lycopene. The highest lycopene dose reduced serum cholesterol by 42.8%,
increased HDL cholesterol levels and reduced HMG-CoA reductase activity as well as acyl-
CoA-cholesterolacyltransferase activity. Of note, the highest dose of lycopene significantly
reduced a plaque area in the aorta by 64.3%. Recently, Lee et al. [91] showed the inhibitory
effects of lycopene on HMGB1-mediated pro-inflammatory responses in both cellular
(HUVECs) and mouse animal models, thereby suggesting its usefulness for vascular
inflammatory disease.
The comparison of lycopene and fluvastatin effects on atherosclerosis induced by a high-
fat diet in 40 adult male New Zealand white rabbits was lately published by Hu and collegues
[92]. The high-fat diet led to increased levels of total cholesterol, total triacylglycerol, LDL
cholesterol and IL-1. Lycopene (8 wk) was better than fluvastatin in reducing the changes in
these parameters. Lycopene and fluvastatin also markedly reduced the formation of
atherosclerotic plaques in the aorta compared to the situation in rabbits on a high-fat diet
alone [92].
In contrast, Frederiksen et al. [93] did not show any effects from an intervention (16 wk)
with extract of lycopene rich tomatoes when investigating 65 male Watanabe heritable
hyperlipidemic rabbits. They fed on a control diet, a control diet supplemented with a mixture
of plant oils or a control diet supplemented with tomato extract (0.25 g tomato extract
(containing 6% lycopene)/100 g:15mg lycopene/ 100 g diet). The tomato extract had no effect
on cholesterol and triacylglycerol levels in plasma, on cholesterol in lipoprotein fractions and
on aortic atherosclerosis (cholesterol in tissue, microscopy). Oxidation of plasma lipids was
also unaffected by the intake of tomato extract. These results were recently confirmed by
Lorenz M et al. [94]. In fact, although they found that lycopene supplementation for 4 weeks
increased its plasma levels and strongly reduced total and LDL cholesterol serum levels as
well as significantly lowering amounts of cholesteryl ester in the aortae in lycopene-treated
New Zealand white rabbits, no significant differences in initial lesions to the aorta were
detected.
One notable recent study demonstrates that a dietary mix of fish oil, resveratrol,
lycopene, catechin, d-α-tocopherol, and vitamin C, which was shown to be well tolerated in
humans, improves lipid and inflammatory risk factors for CVD in humanized models of
disease [95]. These findings support the concept of combination strategies with several
bioactive nutrients and a systems-based, multi-target approach for complex multifactorial
diseases, such as type 2 diabetes. In this regard, Zhu J et al. [96] demonstrated that chronic
lycopene treatment could attenuate endothelial dysfunction by reducing oxidative stress in
streptozotocin-induced diabetic rats, indicating that chronic lycopene treatment might be
useful in preventing the diabetic vascular complications associated with endothelial
dysfunction.
In conclusion, taking into account that most such animal experiments [82-85, 90, 92, 93]
were carried out to evaluate the possible beneficial effects of dietary lycopene in humans, we
can assume, first, that the intervention periods, lasting between 2 and 16 weeks, are also
relevant to the human situation. Secondly, the lycopene dosage may be a critical parameter.

The authors used lycopene dosages between 0.001 mg/kg b.w. [83] and 127.8 mg/kg
diet [90].
As the animals partly had free access to the feed (e.g. [90]) and otherwise got a restricted
amount (e.g. 100 g: [93]), the studies are not comparable. Experiments with a lycopene dose
of approx. 10 mg/kg b.w. cannot be transferred to the human situation. In addition, it must be
considered that experiments with rats [82-84], hamsters [92] and rabbits [90, 92, 93] cannot
be directly converted into the conditions found in the human organism.
Human Intervention Studies
In recent times, Yang et al. [97] reviewed the major publications relating to the potential
effects on cardiovascular risk factors and outcomes of some common dietary constituents:
carotenoids, flavonoid-rich cocoa, tea, red wine and grapes, coffee, omega-3 fatty acids, and
garlic. Increased intake of some of these has been associated with reduced all-cause mortality
or reduced incidence of myocardial infarction, stroke, and hypertension. However, although
the evidence from observational studies, in vitro studies and animal studies showed linkages
between carotenoids and prevention of cardiovascular disease and is supportive of beneficial
effects for most of these foodstuffs taken as part of the diet, the potential benefits of using
supplements derived from these natural products remain largely inconclusive.
The fact is that although many epidemiological studies have reported an association
between β-carotene and the risk of CVD, several large randomized trials failed to reveal any
reduction in CVD with β-carotene consumption. For instance, the MRC/BHF Heart
Protection Study showed no benefit from β-carotene 20 mg daily, in combination with
vitamin E 600 mg and vitamin C 250 mg, on morbidity or mortality in high-risk individuals
[98]. In the α-tocopherol and β-carotene (ATBC) study conducted on 1,862 male smokers
who had had a previous myocardial infarction, there were no significant differences in the
number of major coronary events between any supplementation group and the placebo group.
Moreover, the risk of fatal CHD was increased in the β-carotene and combined α-tocopherol
and β-carotene groups as compared to the placebo group [99]. Likewise, the Women‘s
Antioxidant Cardiovascular Study (WACS) found no CVD risk reduction in women at high
risk, whether using β-carotene 50 mg every other day, or vitamin C 500 mg daily or vitamin E
600 IU every other day [100]. The prospective evaluation of the relation between vegetable
intake and CHD risk in the Physicians‘ Health Study concluded that the consumption of
vegetables rich in carotenoids was associated with a reduced risk of CHD [101], but after 12
years of follow-up there was no impact from supplementation of alternate day β-carotene 50
mg on CVD, cancer, or overall mortality among primarily non-smokers [102].
Moreover, in the Physicians‘ Health Study, no association between increasing
concentrations of plasma lycopene and the risk of CVD was found [103]. A recent review of
the controlled clinical studies with lycopene in well-defined subject populations found no
definite evidence for CVD prevention [104].
To compare the effect of lutein- and zeaxanthin-rich foods and supplements on macular
pigment levels (MPL) and serological markers of endothelial activation, inflammation and
oxidation in healthy volunteers, Graydon R et al. [105] conducted two 8-week intervention
studies concluding that this 8-week supplementation with lutein and zeaxanthin, whether as
foods or as supplements, had no significant effect on MPL or serological markers of

endothelial activation, inflammation and oxidation in healthy volunteers, but may improve
MPL in the highest serum responders and in those with initially low MPL.
The effects of lycopene (from cooked tomatoes) on serum antioxidant enzymes, lipid
peroxidation rate and lipid profile were evaluated in a case-control study involving 20
coronary heart disease patients. They were asked to eat 200 g cooked tomatoes every day for
60 days. Supplementation with tomatoes significantly reduced MDA levels, indicating a
lower rate of lipid peroxidation. and conversely increased levels of antioxidant enzymes
(SOD, glutathione reductase, GSH-Px), while lipid status parameters were not affected [106].
The same authors studied the effect of lycopene from cooked tomatoes on plasma antioxidant
enzymes, lipid peroxidation rate and lipid profile in grade-I hypertension, and concluded that
tomato lycopene may have considerable natural therapeutic potential as an antioxidant but
may not be used as a hypolipidemic agent in hypertension [107]; they demonstrated that a
relatively high daily consumption of tomato-based products (equivalent to 32-50 mg
lycopene/d) or lycopene supplements (10 mg/d) is ineffective in reducing conventional CVD
risk markers in moderately overweight, healthy, middle-aged individuals.
The effects of a tomato-based drink on markers of inflammation, immunomodulation,
and oxidative stress were studied in a randomised, placebo-controlled, double-blind,
crossover study. 26 healthy men and women were supplemented with placebo and tomato-
based drink (ingesting 5.7 mg lycopene per day) for 26 days per period. The tomato-based
drink significantly reduced TNF-α production in challenged whole blood. By contrast, DNA
damage and urinary 8-iso-PGF2α concentration were not affected by tomato drink
consumption [37]. Effects of lycopene supplementation on oxidative stress and markers of
endothelial function in healthy men were recently published in another randomised, placebo-
controlled, double-blind study [108]. Supplementation of 126 healthy men (22–57 years),
with 6 or 15 mg lycopene daily for 8 weeks led to significantly increased lycopene contents in
serum. As was shown by decreased DNA damage as well as by increased SOD activity,
oxidative stress was reduced by lycopene supplementation. Endothelial function, evaluated by
the reactive hyperemia peripheral arterial tonometry index, was significantly improved by
lycopene (15 mg per day). This dosage also significantly decreased the hs-CRP content in
serum, a marker of inflammatory status. Plasma levels of adhesion proteins sICAM- 1 and
sVCAM-1 were significantly decreased by lycopene. Thus, supplementation with 15 mg
lycopene per day over 8 weeks was able to reduce oxidative stress as well as to improve
endothelial function. This study specially focused on middle-aged Korean men; hence the
results cannot be generalised to women. However, the results demonstrated the antioxidative
and anti-inflammatory effects of lycopene [108].
By way of contrast, one recent intervention with 70 g tomato purèe per day (46 mg
lycopene per day) in a group of 31 non-smoking healthy postmenopausal women did not
affect endothelial function. Though the concentration of lycopene in plasma significantly
increased, flow-mediated dilation did not change during the intervention period [109].
A meta-analysis, employing human intervention trials between 1955 and September
2010, investigated the effect of lycopene on blood lipids and on blood pressure [110]. Ried
and Fakler‘s meta-analyses indicated that 25 mg daily of lycopene effectively reduced total
cholesterol and LDL cholesterol in serum. Regarding the potential role of lycopene in the
regulation of blood pressure, the clinical trails published to date are too low in number (54) to
offer any firm evidence of this. Although they do suggest lycopene has a lowering effect on

systolic blood pressure, in particular in hypertensive subjects, further studies are necessary to
prove these results.
Astaxanthin, a xanthophyll carotenoid, is a nutrient with unique cell membrane actions
and diverse clinical benefits, with excellent safety and tolerability. Significant antioxidant
powers have been ascribed to astaxanthin, based primarily on experimental findings. The real
breakthrough with this nutrient, however, is that it produces clinically significant antioxidant
benefits in human subjects, including groups especially vulnerable to oxidative stress, such as
smokers, the obese, and the overweight.
In a Korean prospective, randomized, double-blind study, astaxanthin ―normalized‖
oxidative stress in individuals with weight challenges [111]. In this three-week study,
twenty‐three adults with Body Mass Index (BMI)> 25.0 kg/m2 enrolled in this study and
were randomly assigned to two dose groups: astaxanthin 5 mg and 20 mg once daily for 3
weeks, and compared to a control group (n=10) with normal body weight (BMI <25.0 kg/m2)
who received no intervention. Malondialdehyde, isoprostane, superoxide dismutase and total
antioxidant capacity, as oxidative stress biomarkers, were measured at baseline and 1, 2 and 3
weeks after astaxanthin administration. Compared with baseline, the malondialdehyde (by
34.6% and 35.2%) and isoprostane (by 64.9% and 64.7%) levels were significantly lowered,
whereas superoxide dismutase (by 193% and 194%) and total antioxidant capacity (by 121%
and 125%) levels were significantly increased in two dose groups after the 3 week
intervention. This study revealed that supplements of astaxanthin for 3 weeks improved
oxidative stress biomarkers by suppressing lipid peroxidation and stimulating the activity of
the antioxidant defense system.
Another double-blind, randomized controlled trial was conducted by the same group
[112]. Thirty-nine heavy smokers (≥20 cigarettes/day) and 39 non-smokers were enrolled in
this study. Smokers were randomly allocated to receive astaxanthin at 5-, 20-, or 40 mg/day
for three weeks. Oxidative stress biomarkers such as malondialdehyde, isoprostane,
superoxide dismutase, and total antioxidant capacity, and astaxanthin levels in plasma were
measured at baseline and after 1, 2, and 3 weeks of treatment. Compared with baseline, the
plasma malondialdehyde and isoprostane levels decreased, whereas superoxide dismutase
levels and total antioxidant capacity increased in all astaxanthin intervention groups over the
3-week period. In particular, isoprostane levels showed a significant dose-dependent decrease
after astaxanthin intake. The results suggest that astaxanthin supplementation might prevent
oxidative damage in smokers by suppressing lipid peroxidation and stimulating the activity of
the antioxidant system in smokers.
In the Park double-blind, randomized controlled trial [113], astaxanthin also significantly
lowered C-reactive protein, a biomarker of systemic inflammation [114].
Astaxanthin improved certain blood lipids in subjects with moderately elevated serum
triglycerides. Healthy non-obese subjects (BMI <28 kg/m2), aged 20-65 years (n=61) with
fasting triglycerides in the range 120-200 mg/dL, were recruited into a double-blind
randomized controlled trial [115]. They were randomly allocated to receive astaxanthin at 6,
12, or18 mg/day, or a placebo for 12 weeks. Astaxanthin, as compared to placebo,
significantly elevated HDL-cholesterol at the doses of 6 mg/day (p<0.05) and 12 mg/day
(p<0.01). It also significantly lowered triglycerides at doses of 12 mg/day and 18 mg/day
(p<0.05 for both) as compared to placebo. There was no effect on LDL-cholesterol at any
dose.

Astaxanthin also significantly increased blood adiponectin levels (p<0.01 at 12 mg/day;

p<0.05 at 18 mg/day). Adiponectin is a hormone produced by adipose tissue, cardiac and
skeletal muscle, and vessel endothelia. Serum levels of adiponectin tend to be reduced in
obese and/or diabetic subjects, smokers, patients with coronary heart disease, and individuals
with metabolic syndrome [116]. Although the results of this study suggest a normalization of
adiponectin levels, 12 weeks of supplementation had no effect on BMI. Further investigation
is required under better controlled conditions in order to clarify astaxanthin‘s utility for this
condition.
As reviewed by Fasset and Coombes [117], the safety, bioavailability and effects of
astaxanthin on oxidative stress and inflammation that have relevance to the pathophysiology
of atherosclerotic cardiovascular disease have been assessed in a small number of clinical
studies. No adverse events have been reported and there is evidence of a reduction in
biomarkers of oxidative stress and inflammation with astaxanthin administration.
Experimental studies in several species using an ischaemia-reperfusion myocardial model
demonstrated that astaxanthin protects the myocardium when administered both orally or
intravenously prior to induction of the ischaemic event. At this stage we do not know whether
astaxanthin is of benefit when administered after a cardiovascular event and no clinical
cardiovascular studies in humans have been completed and/or reported. Cardiovascular
clinical trials are recommended and warranted, judging by the physicochemical and
antioxidant properties, the safety profile and preliminary experimental cardiovascular studies
on astaxanthin.
As widely reviewed by Bohm [59], most intervention studies published, especially those
conducted using lycopene [37, 106, 108, 109], were perfomed on healthy subjects. Thus, the
authors of these studies investigated the possible primary preventive effect of lycopene
contained in tomatoes or their products. Some studies investigated only men [108] or women
[109] while others used men and women as volunteers [37, 106]. This renders a comparison
of the investigations complex. In addition, the duration of intervention trials also varied
between 7 days [109] and 8 weeks [108] and the authors used lycopene dosages from 5.7 [37]
up to 46.2 mg per day [109]. Another factor affecting the results could be the matrix of the
intervention products, such as, tomato-based drink, tomato oleoresin capsules, raw tomatoes
and tomato purée. Thus, no direct comparison among the studies is conceivable.
Conclusion
Increasing evidence indicates that an appropriate redox balance may be implicated in
preserving health and longevity. Altering this equilibrium in favour of oxidants may result in
pathological responses causing functional disorders and disease. Promising epidemiological
studies on nutrition, associating high levels of carotenoids with low levels of oxidative stress
(oxLDL, conjugated dienes, etc.) establishes that higher fruit and vegetable intakes tend to be
associated with lower rates of heart and vascular diseases, including coronary heart disease
and stroke. Notably, short-term dietary intervention trials suggest that those who consume
higher amounts of fruits and vegetables tend to have improvements in coronary risk factors
and reduced cardiovascular mortality.

Carotenoids are a large family of pigmented compounds that are synthesized by plants
and micro-organisms but not animals. In human diet fruit and vegetables constitute the main
sources of carotenoids, fat-soluble pigments that can function as antioxidants. Although more
than 600 carotenoids have been identified, most research in nutrition has focused on the five
most common carotenoids: α-carotene, β-carotene, lycopene, lutein/zeaxanthin, and β-
cryptoxanthin. In addition, astaxanthin is a xanthophyll carotenoid present in micro-algae,
fungi, complex plants, seafood, flamingos and quail. It is an antioxidant with anti-
inflammatory properties and as such has potential as a therapeutic agent in atherosclerotic
cardiovascular disease.
As discussed earlier, carotenoids may prevent cardiovascular disease in a number of
ways. For these reasons, carotenoids from plants may represent one possible mechanism by
which fruit and vegetables reduce the risk of heart and vascular disease.
Previously, carotenoids were known to exert relevant beneficial properties on human
health. Their biological role in the prevention and possibly the treatment of cardiac and
vascular diseases is now being studied and partially understood.
Epidemiological and human intervention studies have verified the significance of these
natural molecules in the prevention of human diseases. In vitro and animal investigations
have tested the hypotheses generated from the epidemiological reports. Although some
human clinical trials are beginning to be undertaken, there is a great need for well-designed
human intervention studies designed to define subject selection, end point measurements and
the levels of carotenoids being tested. It is only through such studies that our knowledge of
the crucial role played by carotenoids will improve and enable us to develop complementary
approaches to the prevention, cure and management of cardiovascular diseases.
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Chapter XV
Effects of Carotenoids on Mammalian

DNA Polymerase Inhibition
and Anti-Inflammatory Activity
Yoshiyuki Mizushina1,2,, Isoko Kuriyama1, Toshifumi Takeuchi3,

Fumio Sugawara3 and Hiromi Yoshida1
1
Laboratory of Food & Nutritional Sciences, Faculty of Nutrition, Kobe Gakuin
University, Nishi-ku, Kobe, Hyogo, Japan
2
Cooperative Research Center of Life Sciences, Kobe Gakuin University,
Chuo-ku, Kobe, Hyogo, Japan
3
Department of Applied Biological Science, Tokyo University of Science,
Noda, Chiba, Japan
Abstract
We screened for selective inhibitors of mammalian DNA polymerase (pol) species
from food components, focusing on carotenoids in this review. Ten major carotenoids in
food, including α-carotene (1), β-carotene (2), lycopene (3), β-cryptoxanthin (4),
zeaxanthin (5), lutein (6), canthaxanthin (7), astaxanthin (8), capsanthin (9), and
fucoxanthin (10), were investigated for their inhibitory effects on in vitro mammalian pol
activities. Compounds 5, 6, and 9 strongly inhibited the activities of mammalian pol ι,
which is a DNA repair- and/or recombination-related X-family pol. In contrast, all
carotenoids tested had no influence on the activities of mammalian pols α, γ, and θ,
which are members of the B-, A-, and Y-families, respectively. Lutein (6) was the
strongest inhibitor of mammalian pol ι of the 10 carotenoids that were tested, and lutein
showed noncompetitive inhibition with both the DNA template-primer substrate and the
dNTP substrate. The tendency for pol ι inhibition by these carotenoids showed a positive
correlation with suppression of tumor necrosis factor (TNF)-α production induced by

Corresponding author: Yoshiyuki Mizushina, PhD. E-mail: [email protected]; Tel.: +81-78-974-
1551 (ext. 3232); Fax: +81-78-974-5689.

298 Yoshiyuki Mizushina, Isoko Kuriyama, Toshifumi Takeuchi et al.
lipopolysaccharide (LPS) in cultured mouse macrophage RAW264.7 cells and TPA (12-
O-tetradecanoylphorbol-13-acetate)-induced inflammation in mouse ear in vivo. We
discuss the relationship between the structures and bioactivities of carotenoids.
Carotenoids containing lutein may be particularly useful due to their anti-inflammatory
properties based on the ability to inhibit mammalian pol ι activity.
Keywords: Carotenoids, lutein, DNA polymerase (pol), enzyme inhibitor, anti-inflammation,

structure and bioactivity relationship
Abbreviations
BER base excision repair
Clog P calculated log P
DMSO dimethyl sulfoxide
dNTP 2′-deoxyribonucleoside 5′-triphosphate
DS Discovery Studio
dsDNA double-stranded DNA
dTTP 2′-deoxythymidine 5′-triphosphate
ELISA enzyme-linked immunosorbent assay
HIV human immunodeficiency virus
IC50 50% inhibitory concentration
Ki inhibition constant
Km Michaelis constant
LPS lipopolysaccharide
MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide
Pol DNA-dependent DNA polymerase (EC 2.7.7.7)
TLS translesion synthesis
Tm melting temperature
TNF tumor necrosis factor
TPA 12-O-tetradecanoylphorbol-13-acetate
Vmax maximum velocity
XPV Xeroderma pigmentosum variant
1. Introduction
Accumulating scientific evidence supports an association between diet and chronic
diseases. Based on this evidence, dietary guidelines have been developed worldwide for
preventing chronic diseases such as cancer, cardiovascular disease, diabetes, inflammation,
and osteoporosis. One of the main recommendations of these dietary guidelines involves
increasing the consumption of plant-based foods, including fruits and vegetables, which are
good sources of carotenoids and other biologically active phytochemicals. Fruits and
vegetables exert their beneficial effects via several mechanisms involving metabolism,
immune modulation, and hormonal induction.

Effects of Carotenoids on Mammalian DNA Polymerase Inhibition … 299
Carotenoids have attracted interest in diverse fields, including chemistry, biochemistry,

biology, food science and technology, medicine, pharmacy, and nutrition for more than a
century, and these fascinating compounds continue to be intensely investigated. Carotenoids
are a family of pigmented compounds that are synthesized by plants and microorganisms, but
not by animals. In plants, they assist the photosynthetic machinery by protecting them from
photo-damage. Fruits and vegetables are the major sources of carotenoids in human diet [1–
3]. These micro-components are responsible for the yellow, orange, and red colors of fruits
and vegetables. Carotenoids are thought to be responsible for the beneficial properties of
fruits and vegetables, such as preventing human diseases including inflammation, cancer, and
other chronic diseases [4, 5].
This review will focus on the novel bioactivity of carotenoids such as the in vitro
inhibition of DNA polymerase (pol) of different species; this novel bioactivity of carotenoids
has recently been extensively investigated for its potential use in treating diseases.
2. Chemistry and Dietary Sources of Carotenoids

The basic structure of carotenoids is a symmetrical, linear, 40-carbon tetraterpene built
from eight 5-carbon isoprenoid units joined such that the order is reversed at the center [6].
This basic skeleton may be modified by hydrogenation, dehydrogenation, cyclization, double
bond migration, chain shortening or extension, rearrangement, isomerization, introduction of
oxygen functions, or by combinations of these processes, thus generating significant
structural diversity.
Structures of 10 major carotenoids present in food are shown in Table 1. Due to the
presence of conjugated double bonds, carotenoids can undergo isomerization to cis-trans
isomers. Although trans isomers are more common in foods and are more stable, very little is
known about the biological significance of carotenoid isomerization in human health.
Hydrocarbon carotenoids are collectively known as carotenes (compounds 1–3 in Table 1);
those containing oxygen are referred to as xanthophylls (compounds 4–10 in Table 1). The
most common oxygen-containing groups include hydroxy (OH) and epoxy (5,6- or 5,8-
epoxides) groups. In addition, aldehyde (CHO), keto (C=O), carboxy (COOH), carbomethoxy
(COOMe), and methoxy (OMe) groups are also found in carotenoids.
More than 600 carotenoids have been identified in nature. However, only approximately
40 are present in a typical human diet. Of these, approximately 20 carotenoids have been
identified in human blood and tissues. Nearly 90% of the carotenoids in the diet and human
body are represented by α-carotene (1), β-carotene (2), lycopene (3), β-cryptoxanthin (4), and
lutein (6) [7].
The typical food sources of carotenoids are listed in Table 1. Although carotenoids are
present in many common human foods, deeply pigmented fruits, juices, and vegetables are
the major dietary sources. Yellow-orange vegetables and fruits contain most of the α-carotene
(1) and β-carotene (2), citrus fruits such as orange contain β-cryptoxanthin (4), dark green
vegetables contain lutein (6), and tomatoes and tomato products contain lycopene (3) [1, 8].
Zeaxanthin (5) and lutein (6) are also present in high concentrations in egg yolks [2]. Because
carotenoids are unsaturated, they are subject to changes primarily due to oxidation. However,

other factors such as temperature, light, and pH can also alter carotenoid structure and
influence the color of foods as well as their nutritional values [9].
Generally, the carotenoid content of foods is not significantly altered by common
household cooking methods such as microwave cooking, steaming, and boiling, but extreme
heat can result in oxidative destruction of carotenoids [10]. Although nutrient data can be
used to estimate daily carotenoid intake by humans, these values vary considerably, due to the
sensitivity and specificity of different analytical methods used for detecting these
phytochemicals. In addition, seasonal variations and methods of processing food containing
carotenoids are often not considered when determining the nutritional content of carotenoids
[3]. Despite recognizing the beneficial roles of carotenoids in human health, they are not
considered essential nutrients and as such have not been assigned dietary reference intake
(DRI) values.
Table 1. Major carotenoids (1–10) in food
Carotenoid Structure Food source

Carrot, Sweet potato,
α-Carotene (1) Citrus fruits, Chicken
oil, Egg yolk
Carrot, Coconut oil,
β-Carotene (2) Citrus fruits, Green
leaves, Chicken oil
Tomato, Watermelon,
Lycopene (3)
Carrot, Persimmon
Persimmon, Corn, Citrus

β-Cryptoxanthin (4)
fruits, Egg yolk
Zaxanthin (5) Corn, Spinach, Egg yolk
Egg yolk, Sweet potato,

Lutein (6) Pumpkin, Cauliflower,
Citrus fruits
Salmon, Trout,
Canthaxanthin (7)
Mushroom
Astaxanthin (8) Salmon, Prawn, Crab
Capsanthin (9) Red pepper, Paprika
Fucoxanthin (10) Seaweed, Algae

3. Eukaryotic Pol Species and Their Inhibitor

Screening
Pol (DNA-dependent DNA polymerase, E.C. 2.7.7.7) catalyzes the polymerization of
deoxyribonucleotides along a DNA strand that is ―read‖ and used as a template [11]. The
newly polymerized molecule is complementary to the template strand and identical to the
non-template strand. Pol can only add free nucleotides to the 3′ end of a newly formed strand,
resulting in the elongation of the new strand in the 5′ to 3′ direction.
The human genome encodes at least 14 pols that conduct cellular DNA synthesis [12–
14]. Eukaryotic cells contain 3 replicative pols (α, δ, and ε), mitochondrial pol γ, and at least
12 non-replicative pols (β, δ, ε, ζ, η, θ, ι, µ, λ, and REV1) [12–14]. Pol structure is highly
conserved, with little variation among their overall catalytic subunits among species.
Structure conservation typically indicates that the molecule has an important and often
irreplaceable function, and maintaining this function provides evolutionary advantages. Based
on sequence homology, eukaryotic pols can be divided into 4 main different families: A, B,
X, and Y [15]. Family A includes mitochondrial pol γ and pols ζ and λ, and family B includes
3 replicative pols (α, δ, and ε) and pol δ. Family X contains pols β, ι, and µ, and family Y
includes pols ε, η, θ, and REV1.
Because pols play important roles in maintaining key eukaryotic systems, such as DNA
replication, DNA recombination, and DNA repair [16], pol inhibitors can be employed as
anticancer chemotherapy agents to inhibit cell proliferation. Based on the strategic effects pol
inhibitors, we established an assay for determining pol inhibitors [17] and screened
mammalian pol inhibitors in natural products, including food materials and chemically
synthesized materials for more than 15 years. More than 100 compounds were identified as
inhibitors [18, 19], including vitamin A, vitamin B6, vitamins D2 and D3, vitamin E, vitamin
K3, long-chain fatty acids, fatty acid derivatives, bile acid derivatives, steroid derivatives,
triterpenoids, cerebrosides, alkaloids, flavonoids, anthocyanins, glycolipids, catechins,
coenzyme Qs, isosteviols, dipeptide alcohols, and nucleotide analogs. Through pol inhibitor
studies, we found that pol ι-selective inhibitors, such as curcumin derivatives [20–22],
display 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anti-inflammatory activity [23–
25]. These results led to the hypothesis that selective inhibitors of mammalian pol species
could be utilized as chemotherapeutic agents not only due to their anticancer effects but also
due to their anti-inflammatory activities.
In this review, we focused on the mammalian pol inhibitory effect of carotenoids and
investigated the chemical constituents responsible for inhibiting pol activities and their anti-
inflammatory activities.
4. Effects of Carotenoids on Pol Activity
4.1. Preparation of Pols
Pol α was purified from calf thymus by using immunoaffinity column chromatography as
described by Tamai et al. [26]. Recombinant rat pol β was purified from E. coli JMpβ5 as

described by Date et al. [27]. The human pol γ catalytic gene was cloned into the pFastBac
vector. Histidine-tagged enzyme was expressed using the BAC-TO-BAC HT Baculovirus
Expression System, according to the supplier‘s instructions (Life Technologies, Inc.,
Gaithersburg, MD, USA) and purified using ProBoundresin (Invitrogen, Carlsbad, CA, USA)
[28]. Human pols δ and ε were purified by nuclear fractionation of human peripheral blood
cancer cells (Molt-4) by performing the affinity column chromatography of the second
subunits of pols δ and ε, respectively [29].
A truncated form of human pol ε (residues 1–511) tagged with His6 at its C-terminus was
expressed in E. coli cells and was purified by the method of Kusumoto et al. [30]. A
recombinant mouse pol η tagged with His6 at its C-terminus was expressed and purified by
performing Ni-NTA column chromatography as per a previously described method [Masutani
et al., in preparation]. A truncated form of pol θ (residues 1–560) with His6-tags attached at
the C-terminus was overproduced in E. coli and was purified by the method of Ohashi et al.
[31]. Recombinant human His-pol ι was overexpressed and purified according to the method
of Shimazaki et al. [32]. Fish pol δ was purified from the testis of cherry salmon
(Oncorhynchus masou) [33]. Fruit fly pols α, δ, and ε were purified from early embryos of
Drosophila melanogaster, as per the method of Aoyagi et al. [34, 35]. Pol α from a higher
plant, cauliflower inflorescence, was purified according to the method of Sakaguchi et al.
[36]. The Klenow fragment of pol I from E. coli was purchased from Worthington
Biochemical Corp. (Lakewood, NJ, USA). Taq pol and T4 pol were purchased from Takara
Bio, Inc. (Shiga, Japan).
4.2. Pol Assays
The reaction mixtures for calf pol α, rat pol β, plant pol α, and prokaryotic pols have been
described previously [37, 38]. Those for human pol γ and for human pols δ and ε were as
described by Umeda et al. [28] and Ogawa et al. [39], respectively. The reaction mixtures for
mammalian pols ε, η, and θ were the same as that for calf pol α, and the reaction mixture for
human pol ι was the same as that for rat pol β. Poly(dA)/oligo(dT)18 (A/T = 2/1) and tritium-
labeled 2′-deoxythymidine 5′-triphosphate ([3H]-dTTP) were used as the DNA template-
primer substrate and nucleotide (i.e., 2′-deoxyribonucleoside 5′-triphosphate [dNTP])
substrate, respectively.
Carotenoids were dissolved in distilled dimethyl sulfoxide (DMSO) at various
concentrations and sonicated for 30 s. Aliquots of 4 µL of sonicated samples were mixed with
16 µL of each enzyme (final amount, 0.05 U) in 50 mM Tris-HCl (pH 7.5), containing 1 mM
dithiothreitol, 50% glycerol, and 0.1 mM EDTA, and incubated at 0°C for 10 min. These
inhibitor-enzyme mixtures (8 µL) were added to 16 µL of each of the enzyme standard
reaction mixtures and incubated at 37°C for 60 min, except for Taq pol, which was incubated
at 74°C for 60 min. Activity without the inhibitor was considered to be 100%, and the
remaining activity at each inhibitor concentration was determined relative to this value. One
unit of pol activity was defined as the amount of enzyme that catalyzed the incorporation of 1
nmol of dNTP (i.e., dTTP) into synthetic DNA template-primers in 60 min at 37°C under the
normal reaction conditions adopted for each enzyme [37, 38].

4.3. Pol Inhibition by Carotenoids
Ten major carotenoids in foods (i.e., compounds 1 to 10 in Table 1) were purchased from
Sigma-Aldrich Inc. (St. Louis, MO, USA), and the purity of these carotenoids was ~98%.
Inhibition of 4 mammalian pols, including calf pol α, human pol γ, human pol θ, and human
pol ι, by 100 µM of each compound was investigated (Figure 1). For mammalian pols, pols
α, γ, θ, and ι were used as representatives of the B, A, Y, and X pol families, respectively
[12–15]. As shown in Figure 1, carotenes, such as hydrocarbon carotenoids of compounds 1–
3, had no influence on the 4 mammalian pols that were tested. Xanthophylls (compounds 4–
10 in Table 1)only minimally affected the activities of pols α, γ, and θ, but zeaxanthin (5),
lutein (6), and capsanthin (9) potently inhibited pol ι activity. Lutein (6) showed the strongest
inhibitory effect on pol ι of all the tested carotenoids, and the effect of the 3 compounds were
as follows: lutein (6) > zeaxanthin (5) > capsanthin (9). When activated DNA (DNA with
gaps digested by bovine deoxyribonuclease I) and dNTP were used as the DNA template-
primer substrate and nucleotide substrate pair in place of synthesized DNA
(poly[dA]/oligo[dT]18 [A/T = 2/1]) and dTTP, respectively, the inhibitory effects of these
compounds did not change (data not shown).
Calf pol 
-Carotene (1)
Human pol 
Human pol 
-Carotene (2) Human pol 
Lycipene (3)
-Cryptoxanthin (4)
Zeaxanthin (5)
Lutein (6)
Canthaxanthin (7)
Astaxanthin (8)
Capsanthin (9)
Fucoxanthin (10)
Mammalian pol relative activitiy (%)
Figure 1. Inhibitory effect of major carotenoids (compounds 1–10) in food on the activities of mammalian
pols. Each compound (100 µM) was incubated with calf pol α (B-family pol), human pol γ (A-family pol),
human pol θ (Y-family pol), and human pol ι (X-family pol) (0.05 U each). Percent relative activity is
shown. Pol activity in the absence of the compound was considered to be 100 %. Data are shown as mean ±
SD (n = 3).

5. Effects of Lutein on the Activities

of Various Pols
5.1. Inhibition of Various Pols by Lutein
Among the 10 major carotenoids investigated, lutein displayed the strongest inhibitory
effect on human pol ι (Figure 1) and is therefore the focus of this section. Briefly, we
succeeded in obtaining 9 mammalian pol species including pols α, β, γ, δ, ε, ε, η, θ, and ι.
Table 2 shows the inhibitory effect (IC50 value) of lutein against various pol species,
including the 9 mammalian pols obtained.
Table 2. IC50 values of lutein (6) for the activities of various pols and other DNA
metabolic enzymes
Enzymes IC50 (µM)

Mammalian pols
[A-Family]
Human pol γ >200
[B-Family]
Calf pol α >200
Human pol δ >200
Human pol ε >200
[X-Family]
Rat pol β 45.0 ± 2.3
Human pol ι 12.8 ± 0.7
[Y-Family]
Human pol ε >200
Mouse pol η >200
Human pol θ >200
Fish pol
Cherry salmon pol δ >200
Insect pols
Fruit fly pol α >200
Fruit fly pol δ >200
Fruit fly pol ε >200
Other pols
Cauliflower pol α >200
E. coli pol I >200
Taq pol >200
T4 pol >200
Other DNA metabolic enzymes
HIV-1 Reverse transcriptase >200
T7 RNA polymerase >200
T4 polynucleotide kinase >200
Bovine deoxyribonuclease I >200
Lutein (6) was incubated with each pol (0.05 U) and other DNA metabolic enzymes. One unit of pol
activity was defined as the amount of enzyme catalyzing the incorporation of 1 nmol of dNTP
substrate (i.e., dTTP) into the synthetic DNA template-primer substrate (i.e., poly(dA)/oligo(dT)18,
A/T = 2/1) in 60 min at 37°C under normal reaction conditions for each enzyme. Enzyme activity
in the absence of the compound was considered to be 100%. Data are shown as mean ± SD of 3
independent experiments.

Lutein inhibited the activities of both rat pol β and human pol ι, which are from the X-
family of pols. Pol activities were suppressed in a dose-dependent manner to the same extent,
with IC50 values of 45.0 and 12.8 µM, respectively; therefore, the inhibitory effect of lutein
on pol ι was 3.5-fold stronger than that on pol β.
In contrast, lutein showed no inhibitory effect on fish (i.e., cherry salmon) pol δ, insect
(i.e., fruit fly) pols α, δ, and ε, plant (i.e., cauliflower) pol α, or prokaryotic pols, such as the
Klenow fragment of E. coli pol I, Taq pol, and T4 pol. These results suggest that lutein
probably selectively inhibit mammalian pols, particularly the X-family of pols β and ι.
5.2. Mode of Inhibition of Mammalian Pols β and λ by Lutein
Next, to elucidate lutein‘s mechanism of selective inhibition of mammalian pols, we

investigated the mode of inhibition of the compound against pols β and ι. For kinetic
analysis, poly(dA)/oligo(dT)18 and dTTP were used as the synthetic DNA template-primer
substrate and nucleotide (dNTP) substrate, respectively. The extent of inhibition as a function
of DNA template-primer substrate or nucleotide substrate concentration was measured
(Table 3).
Double reciprocal plots (i.e., Lineweaver–Burk plots) showed that lutein-induced
inhibition of rat pol β activity was noncompetitive with respect to both the DNA template-
primer substrate and nucleotide substrate. For the DNA template-primer substrate, the
apparent Michaelis constant (Km) was unchanged at 2.17 µM, whereas decreases of 111, 71.4,
52.6, and 41.7 pmol/h in maximum velocity (Vmax) were observed in the presence of 0, 10, 20,
and 30 µM lutein, respectively. Km for the nucleotide substrate was unchanged at 3.05 µM,
and Vmax for the nucleotide substrate decreased from 62.5 to 27.8 pmol/h in the presence of 30
µM lutein. Inhibition constant (Ki) values, obtained based on Dixon plots, were found to be
17.5 and 27.8 µM for the DNA template-primer substrate and nucleotide substrate,
respectively.
The data were expressed as double reciprocal plots, which showed that lutein inhibited
pol ι activity in a noncompetitive manner with respect to both the DNA template-primer
substrate and the nucleotide substrate. For the DNA template-primer substrate, the apparent
Km was unchanged at a concentration of 2.38 µM lutein, whereas 29.4, 45.4, and 55.5%
decreases in Vmax were observed in the presence of lutein at 3, 6, and 9 µM, respectively. Km
for the nucleotide substrate was unchanged at 1.18 µMm, and Vmax for the nucleotide
substrate decreased from 52.6 to 26.0 pmol/h in the presence of 0–9 µM lutein. Ki values,
obtained from Dixon plots, were found to be 4.8 µM for the DNA template-primer substrate
and 7.6 µM for the nucleotide substrate. Because Ki value for the DNA template-primer
substrate was ~1.6-fold lower than that for the nucleotide substrate, the affinity of lutein was
greater for the enzyme-DNA template-primer binary complex than for the enzyme-nucleotide
substrate complex.
When activated DNA and 4 dNTPs were used as DNA template-primer substrate and
nucleotide substrates, respectively, the mode of inhibition of mammalian pols β and ι by
lutein was the same as that observed for the synthetic DNA template-primer (data not shown).
These results suggest that lutein binds to or interacts with a site distinct from both the
DNA template-primer substrate-binding site and the nucleotide substrate-binding site of each
pol.

Table 3. Kinetic analysis of lutein (6) inhibitory effects on the activity of rat pol β or
human pol λ as a function of DNA template-primer substrate dose and nucleotide
substrate concentration
Lutein (6) Km a) Vmax a) Ki b)

Pol Substrate Inhibitory mode
conc. (µM) (µM) (pmol/h) (µM)
0 111
10 71.4
DNA template-primer c) 2.17 18.5 Noncompetitive
20 52.6
30 41.7
β
0 62.5
10 44.1
dNTP d) 3.05 27.8 Noncompetitive
20 34.1
30 27.8
0 83.3
3 60.6
DNA template-primer c) 2.38 4.8 Noncompetitive
6 47.6
9 39.2

0 52.6
3 39.2
dNTP d) 1.18 7.6 Noncompetitive
6 31.3
9 26.0
a) b)
Data obtained from a Lineweaver Burk plot. Data obtained from a Dixon plot.
c)
Poly(dA)/oligo(dT)18.d)dTTP.
6. Effects of Lutein on the Activities of Other DNA

Metabolic Enzymes
6.1. Other DNA Metabolic Enzyme Assays
Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, T7 RNA

polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I were prepared from
commercial sources, and the activities of these enzymes were determined using standard
assays, as per the manufacturer‘s specifications and as per the method of Mizushina et al. [35,
36], Nakayama and Saneyoshi [40], Soltis and Uhlenbeck [41], and Lu and Sakaguchi [42],
respectively.
6.2. Inhibition of Other DNA Metabolic Enzymes by Lutein
As shown in Table 2, lutein only minimally influenced the activities of other DNA-
metabolic enzymes such as HIV-1 reverse transcriptase, T7 RNA polymerase, T4
polynucleotide kinase, and bovine deoxyribonuclease I. These results suggest that lutein

selectively inhibits the activities of mammalian X-family pols among the DNA metabolic
enzymes tested. Interestingly, some carotenoids, particularly lutein, are inhibitors of
mammalian pols.
We next performed specific assays to determine whether lutein-induced inhibition
resulted from the ability of the compound to bind to DNA or the enzyme. The interaction of
lutein with double-stranded DNA (dsDNA) was investigated by examining its thermal
transition. For this, the melting temperature (Tm) of dsDNA in the presence of excess lutein
(200 µM) was observed using a spectrophotometer (260 nm) equipped with a thermoelectric
cell holder. As shown in Figure 2, thermal transition (i.e., Tm) from 75 to 90°C was not
observed within the concentration range used in the assay; the typical intercalating
compound, ethidium bromide, (15 µM) was used as a positive control, and a clear thermal
transition was observed.
Whether the inhibitory effect of lutein resulted from nonspecific adhesion to mammalian
pol molecules or from its selective binding to specific sites was investigated by determining if
an excessive amount of nucleic acid (poly[rC]) or protein (bovine serum albumin, BSA) could
prevent the inhibitory effect of lutein. Poly(rC) and BSA had little or no influence on pol
inhibition by lutein (data not shown), suggesting that this compound selectively binds to
mammalian pol molecules. These observations indicate that lutein does not act as a DNA-
intercalating agent or as a DNA template-primer substrate but that this compound binds
directly to the enzyme to inhibit its activity.
Collectively, these results suggest that lutein is a potent and specific inhibitor of
mammalian pols β and ι. We investigated in detail whether pol inhibition by major
carotenoids in foods was effective for inducing an anti-inflammatory response.
0.9
□ Control
● Lutein (6) (200 M)
△ EtBr (15 M)
0.85
Absorbance (260 nm)
0.8
0.75
0.7
75 80 85 90
Temperature ( oC)
Figure 2. Effect of lutein (6) on the thermal transition of dsDNA. Control (open-square), lutein (200 µM,
closed-circle), and ethidium bromide (EtBr, 15 µM, open-triangle) were incubated with 6 µg/mL of calf
thymus dsDNA in 0.1 M Na-phosphate buffer (pH 7.0). Data are shown as mean ± SD (n = 3).

7. Effects of Carotenoids on Anti-Inflammatory

Activity
7.1. Measurement of Tumor Necrosis Factor (TNF)-α Level in Mouse

Macrophages
The mouse macrophage cell line RAW264.7 was obtained from American Type Culture
Collection (ATCC) (Manassas, VA, USA). The cells were cultured in Eagle‘s minimum
essential medium (MEM) supplemented with 4.5 g of glucose per liter containing 10% fetal
calf serum, 5 mM L-glutamine, 50 U/mL penicillin, and 50 U/mL streptomycin. The cells
were cultured at 37°C in standard medium in a humidified atmosphere of 5% CO2 and 95%
air. RAW264.7 cells were placed in a 12-well plate at 5 × 104 cells/well and incubated for 24
h. The cells were pretreated with the 10 carotenoids (compounds 1–10 in Table 1) (final
concentrations of 10 or 100 M) for 30 min before adding 100 ng/mL of lipopolysaccharide
(LPS). After stimulation with LPS for 24 h, the cell culture medium was collected to measure
the amount of secreted TNF-α. TNF-α concentration in the culture medium was quantified by
using a commercially available enzyme-linked immunosorbent assay (ELISA) development
system (Bay Bioscience Co., Ltd., Kobe, Japan), in accordance with the manufacturer‘s
instructions.
7.2. Suppression of TNF- Production by Carotenoids
Next, we investigated whether carotenoids can inhibit TNF-α production induced by LPS
stimulation in cultured mouse macrophage RAW264.7 cells. The inflammatory cytokine
TNF-α activates the major inflammation signaling pathway by binding to the TNF-α receptor
(TNFR), resulting in various inflammatory diseases [43]. In RAW264.7 cells, no compound
showed cytotoxicity at 100 µM because the LD50 values of these 10 carotenoids were greater
than 200 µM. Among the xanthophylls (compounds 4–10 in Table 1), each at a concentration
of 100 µM, zeaxanthin (5), lutein (6), and capsanthin (9) significantly suppressed LPS-
stimulated production of TNF-α, while -cryptoxanthin (4), canthaxanthin (7), astaxanthin
(8), and fucoxanthin (10) slightly influenced TNF-α production. The inhibitory effects of
lutein (6) and zeaxanthin (5) were the strongest and second strongest among these
compounds, respectively. In contrast, hydrocarbon carotenoids at a concentration of 100 µM,
including carotenes (compounds 1–3 in Table 1), displayed no inhibitory effect on TNF-α
production. The effect of carotenoids on the suppression of LPS-evoked TNF-α production
showed nearly the same tendency as that observed for their inhibitory effect on mammalian
pols β and ι. These results suggest that some carotenoids, such as lutein, inhibit mammalian
pol activities to prevent TNF-α production in LPS-induced macrophages, but they do not
affect cell growth.

7.3. Anti-Inflammatory Assay in Mice
A mouse inflammatory test was performed according to the method of Gschwendt‘s [44].
Briefly, an acetone solution containing 1 of the 10 carotenoids (compounds 1–10 in Table 1)
(250 or 500 µg in 20 µL) or 20 µL of acetone as a vehicle control was applied to the inner
part of the mouse ear.
Thirty minutes after applying the test compound, TPA solution (0.5 µg/20 µL of acetone)
was applied to the same site in the ear. This TPA solution was also applied as a control to the
other ear of the same mouse. After 7 h, a punch biopsy disk (6-mm diameter) was obtained
from each ear and weighed.
The inhibitory effect (IE) of the test solution was expressed as the ratio of the test
solution-prevented increase in weight to that of the response induced by TPA alone observed
in the ear disks:
IE = ([TPA only] – [tested compound plus TPA]/[TPA only] – [vehicle]) × 100.
This animal study was performed according to the guidelines outlined in the ―Care and
Use of Laboratory Animals‖ directory of the Kobe Gakuin University. The animals were
anesthetized with pentobarbital before undergoing cervical dislocation. Female 8-week-old
ICR mice bred in-house were used for all the experiments. All the mice were maintained
under a 12-h light/dark cycle and housed with food and water ad libitum at a room
temperature of 25°C.
7.4. Anti-Inflammatory Effect of Carotenoids
In a previous pol inhibitor study, we observed a relationship between pol ι inhibitors and
TPA-induced anti-inflammatory activity [22–25].
Thus, using the mouse ear inflammatory test in vivo, we examined the anti-inflammatory
activities of the 10 carotenoids. Application of TPA (0.5 µg) to the mouse ear induced edema,
resulting in a 241% increase in ear disk weight 7 h after application. As shown in Figure 4,
lutein (6) showed the strongest reduction in TPA-induced inflammation among the
compounds tested, with inhibition effects of 31.4 and 54.0% for 250 and 500 µg/ear,
respectively.
The 2nd and 3rd compounds, zeaxanthin (5) and capsanthin (9), respectively, showed
inhibitory effects, but the other compounds had little or no effect on inflammation. Therefore,
the mouse ear anti-inflammatory effects of these compounds were correlated with their
inhibitory effects on both mammalian pols β and ι (Figure 1) and TNF-α production in
macrophages (Figure 3).
Lutein (6) showed the strongest effect among the 10 major carotenoids in foods. These
results suggest that the inhibition of pol β and pol ι activities has a positive correlation with
anti-inflammatory activity.

-Carotene (1) 10 M
uM
100 M
uM
-Carotene (2)
Lycipene (3)
Zeaxanthin (5)
Lutein (6)
Canthaxanthin (7)
Astaxanthin (8)
Capsanthin (9)
Fucoxanthin (10)
0 20 40 60 80 100
Rate of TNF- production (%)
Figure 3. Inhibitory effects of major carotenoids (compounds 1–10) in food on lipopolysaccharide (LPS)-
induced production of TNF-α in the mouse macrophage cell line RAW264.7. These cells were pretreated
with 10 µM (gray bar) and 100 µM (black bar) of each compound and were considered as a vehicle control
(TNF-α level, 43 pg/mL) for 30 min and then treated with 100 ng/mL LPS for 24 h (LPS-evoked TNF-α
level, 538 pg/mL). The TNF-α concentration in the cell medium was measured by performing ELISA. The
relative effect in the absence of the compound was considered to be 100%. Data are shown as the mean ± SD
(n = 4).
-Carotene (1) 250g/ear

250 ug/ear
500g/ear
500 ug/ear
-Carotene (2)
Lycipene (3)
Zeaxanthin (5)
Lutein (6)
Canthaxanthin (7)
Astaxanthin (8)
Capsanthin (9)
Fucoxanthin (10)
0 10 20 30 40 50 60
Anti-inflammatory effect (%)
Figure 4. Anti-inflammatory activity of major carotenoids (compounds 1–10) in food toward TPA-induced
edema on mouse ear. A total of 250 µg (gray bar) and 500 µg (black bar) of each compound were applied
individually to one ear of each mouse, and after 30 min, TPA (0.5 µg) was applied to both ears. Edema was
evaluated after 7 h. The inhibitory effect is expressed as the percentage of edema. Data are shown as mean ±
SD (n = 6).

8. Three-Dimensional Structures of Carotenoids
8.1. Molecular Modeling
The energy-minimized 3-dimensional molecular structures of compounds 1–10 (Table 1)

were determined by molecular dynamics using Minimization and Dynamics protocols within
the Discovery Studio (DS) 3.5 modeling software (Accerlys Inc., San Diego, CA, USA). The
calculations involved a CHARMm (Chemistry at HARvard Macromolecular Mechanics)
force field. Both the calculated log P (Clog P) values and pKa values of compounds 1–10
(Table 1) were obtained from the calculated properties in SciFinder Scholar, which were
originally calculated using the Advanced Chemistry Development (ACD/Lab) Software
V8.14 for Solaris (ACD/Labs).
8.2. Structure and Bioactivity Relationship of Carotenoids
To obtain information regarding the molecular basis of the inhibitory properties of

carotenoids, computational analyses were performed using molecular simulation. We focused
on the Clog P value (partition coefficients for octanol/water) and pKa (acid dissociation
constant) of the 10 carotenoids (Table 4). Values of Clog P, which indicate hydrophobicity,
for carotenes (compounds 1–3 in Table 1) were larger than that for xanthophylls (compounds
4–10 in Table 1); therefore, the presence of an oxygen atom determines hydrophobicity. Clog
P values of the inhibitory and anti-inflammatory activities of mammalian pols β and ι on
carotenoids such as zaxanthin (5), lutein (6), and capsanthin (9), were in the range of 10.50 to
11.50; a value within this range is probably important for bioactivity. In contrast, pKa values
of compounds 4–6 and 9–10 (Table 1) were in the same range (14.5–14.9); therefore, the
acidity of carotenoids is very similar and has no influence on their bioactivity. The molecular
length and width of the 3-dimensional structure of the compounds were nearly the same (29
to 37 Å and 4.2 to 5.0 Å, respectively); thus, the molecular sizes of the carotenoids were
similar, indicating that size does not affect the bioactivities of pol inhibition and anti-
inflammation.
Lutein (6), a member of the xanthophyll family of carotenoid, is composed of chains
possessing 8 conjugated double bonds (polyene chain) containing closed rings at each end of
the chain. Zeaxanthin (5) is a stereoisomer of lutein, differing only in the location of a double
bond relative to 1 of the hydroxyl groups. Figure 5A indicates groups essential for carotenoid
bioactivity that were determined using lutein. For the bioactive carotenoids (i.e., lutein [6],
zeaxanthin [5], and capsanthin [9]), 2 hydroxyl groups (i.e., red circles in Figure 5A), 1 on
each side of the carotenoid molecule, is thought to be important for inhibiting pols β and ι
and for its anti-inflammatory activity. Although astaxanthin (8) possesses 2 hydroxyl groups
on both side rings, this compound showed no prominent inhibitory activities against these
pols and no anti-inflammatory effects either; therefore, the 2 ketone groups on both sides of
the carotenoid molecule markedly prevented these activities.
Lutein was the strongest inhibitor of mammalian pols β and ι and showed the strongest
anti-inflammation activity among the carotenoids tested. To obtain information regarding the
molecular basis of differences in inhibition between lutein and its 9 related carotenoids,

computational analysis was performed using molecular simulation and surface analysis
software.
Table 4. Clog P values, pKa values, and molecular length and width of the 3-
dimensional structure of carotenoids
Carotenoid Clog P pKa Length (Å) Width (Å)

α-Carotene (1) 15.14 – a) 30 5.0
β-Carotene (2) 14.80 – a) 30 5.0
Lycopene (3) 14.50 – a) 37 4.2
β-Cryptoxanthin (4) 13.00 14.9 30 5.0
Zaxanthin (5) 10.90 14.6 30 5.0
Lutein (6) 11.50 14.6 30 5.0
Canthaxanthin (7) 9.50 – a) 30 5.0
Astaxanthin (8) 8.20 12.3 30 5.0
Capsanthin (9) 10.50 14.9 32 5.0
Fucoxanthin (10) 7.30 14.5 29 5.0
a)
Not calculated.
Unless otherwise noted, both the Clog P values and pKa values of compounds 1–10 were obtained from
the calculated properties in SciFinder Scholar, which were originally calculated using the
Advanced Chemistry Development (ACD/Lab) Software V8.14 of Solaris (ACD/Labs). The length
and width of the energy-minimized 3-dimensional compounds 1–10 were measured using
Discovery Studio (Accelrys, San Diego, CA, USA).
These hydroxyl groups at both ends of the compound are essential

for bioactivity.
(The ketone groups at both ends of the compound prevent bioactivity.)
A
B
Figure 5. Structures of lutein (6) as a bioactive carotenoid. (A) Chemical structures of lutein. Hydroxyl
groups are highlighted in red circles. (B) Stick model of energy-minimized 3-dimensional structure of
carotenoids was built using DS 3.5 modeling software (Accelrys Inc., San Diego, CA, USA). Carbon,
oxygen, and hydrogen are indicated in gray, red, and white, respectively. Electostatic potential over
commonly surfaces was also analyzed using DS. Blue areas are positively charged, red areas are negatively
charged, and white areas are neutral.

As shown in Figure 5B, the energy-minimized 3-dimensional structure of lutein is stick-

shaped, with both sides of the compound showing a ball-like conformation. The length and
width of this molecule were 30 and 5.0 Å, respectively, which may fit into the active site
pocket of mammalian pol β and pol ι molecules. Lutein consists of both hydrophilic ends and
a hydrophobic center, and the distribution of positively and negatively charged regions in the
hydrophilic areas of both sides are probably important for lutein‘s bioactivity.
9. Discussion
Lutein is one out of 600 known naturally occurring carotenoids. Found in green leafy
vegetables such as spinach and kale, this compound acts as an antioxidant in organisms and is
involved in light absorption in plants. Lutein is sometimes present in plants as a fatty-acid
ester, with 1 or 2 fatty acids bound to 2 hydroxyl groups. Saponification of lutein esters yields
lutein in an approximately 1:2 molar ratio. This compound is also found in egg yolks, animal
fats, and the corpus luteum. Lutein is a lipophilic molecule that is generally insoluble in
water. The presence of the long chromophore of conjugated double bonds provides the
distinctive light-absorbing properties. The polyene chain is susceptible to oxidative
degradation by light or heat and is chemically unstable in acids. Lutein is widely known as
the primary nutrient used for protecting ocular function. No toxicities or adverse reactions
have been reported in the scientific literature for lutein administered at doses of up to 40 mg
daily for 2 months [45]. It has long been thought that carotenoid intake also reduces the risk
of certain forms of cardiovascular disease [46–48], stroke [49, 50], and cancer [51, 52]. On
the basis of our results, we can conclude that some carotenoids, such as lutein, show anti-
inflammatory activity.
Eukaryotic cells reportedly contain 14 pol types belonging to 4 families, including family
A (pols γ, ζ, and λ), family B (pols α, δ, ε, and δ), family X (pols β, ι, and µ), and family Y
(pols ε, η, and θ, and REV1) [12–14]. Among the X family of pols, pol ι appears to show
activity similar to that of pol β [53]. Pol β is involved in the short-patch base excision repair
(BER) pathway [54–57] and plays an essential role in neural development [58]. Recently, pol
ι was found to possess 5′-deoxyribose-5-phosphate (dRP) lyase activity, but not
apurinic/apyrimidinic (AP) lyase activity [59]. Pol ι was effectively substituted for pol β
during in vitro BER, suggesting that pol ι also participates in BER. Northern blot analysis
indicated that transcripts of pol β are abundantly expressed in the testis, thymus, and brain in
rats [60], whereas pol ι is efficiently transcribed primarily in the testis [61]. Bertocci et al.
reported that mice in which pol ι expression is knocked out are not only viable and fertile, but
also display a normal hyper-mutation pattern [62].
In addition to causing inflammation, TPA influences cell proliferation and has
physiological effects on cells due to its tumor promoter activity [63]. Therefore, anti-
inflammatory agents are thought to suppress DNA replication/repair/recombination in nuclei
to block the action of TPA. Because pol ι is a repair/recombination-related pol [53], our
finding that the molecular target of carotenoids such as lutein is pol ι is in good agreement
with this expected mechanism of anti-inflammatory agents. Thus, the pol ι inhibitor may
inhibit inflammation. A positive relationship between anti-inflammatory and pol ι-inhibitory
activities were found in this review, which may be useful as a convenient in vitro assay for

screening and identifying novel anti-inflammatory compounds. To clarify the exact

mechanism of the anti-inflammatory effect of lutein, further studies are necessary.
Conclusion
We showed that some major carotenoids in foods potently and selectively inhibit
mammalian pol activity, particularly pols β and ι, and that these compounds prevent the
inflammatory response both in vitro and in vivo. Particularly, lutein showed the strongest of
these effects among the 10 carotenoids that were tested. As reported previously, the phenolic
compound curcumin, an anti-inflammatory agent, is a pol ι-specific inhibitor [22–25].
Interestingly, the principle molecular target of carotenoids, such as lutein, is also pol ι. The
inhibitory effects on repair/recombination-related pols showed the same tendency as that
observed for TPA-induced inflammation, suggesting that the inhibitory activity of pol ι may
be important for the anti-inflammatory effect. Carotenoids, particularly lutein, may provide
valuable structural information that can be used in designing drugs for developing new anti-
inflammatory agents.
Acknowledgments
This study was supported in part by MEXT (Ministry of Education, Culture, Sports,
Science and Technology, Japan)-Supported Program for the Strategic Research Foundation at
Private Universities, 2012–2016. Y.M. acknowledges Grants-in-Aid for Scientific Research
(C) (No. 24580205) from MEXT, the Takeda Science Foundation (Japan), and the Hyogo
Science and Technology Association (Japan). I. K. acknowledges a Grant-in-Aid for Young
Scientists (B) (No. 23710262) from MEXT.
Conflict of Interest
None of the authors have any financial interest in any of the compounds reviewed in this
article.
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Chapter XVI
Dietary β-Cryptoxanthin Stimulates

Osteogenesis: Role in Prevention
and Treatment of Osteoporosis
Masayoshi Yamaguchi
Department of Hematology and Medical Oncology,
Emory University School of Medicine
C Clifton Road, NE, Atlanta, US
Abstract
Bone homeostasis is maintained through a balance between osteoblastic bone
formation and osteoclastic bone resorption. Aging induces bone loss due to decreased
osteoblastic bone formation and increased osteoclastic bone resorption.
Osteoporosis with its accompanying decrease in bone mass is widely recognized as a
major public heath problem. Food functional factors may play a role in prevention of
bone loss with aging.
Among various carotenoids (carotene and xanthophylls including β-cryptoxanthin,
lutein, lycopene, β-carotene, astaxanthin, and rutin), β-cryptoxanthin, which is abundant
in Satsuma mandarin orange (Citrus unshiu MARC.), has been found to uniquely have a
stimulatory effect on osteoblastic bone formation and an inhibitory effects on osteoclastic
bone resorption in vitro, thereby increasing bone mass.
β-Cryptoxanthin has an effect on the gene expression of various proteins that are
related to osteoblastic bone formation and osteoclastic bone resororption in vitro. Intake
of β-cryptoxanthin has been found to have preventive effects on bone loss in animal
models for osteoporosis and healthy human or postmenopausal women. Epidemiological
studies supported a potent role of β-cryptoxanthin to improving bone health of human
subjects. β-Cryptoxanthin may be important as osteogenic factor in preventing
osteoporosis in human subjects.

E-mail: [email protected].

320 Masayoshi Yamaguchi
Keywords: Carotenoid, β-cryptoxanthin, osteoblastic bone formation, osteoclastic bone

resorption, osteoporosis
Introduction
Bone is a dynamic tissue that preserves skeletal size, shape, and structural integrity and to
regulate mineral homeostasis. Bone homeostasis is maintained through a balance between
osteoblastic bone formation and osteoclastic bone resorption. Aging and numerous
pathological processes induce decrease in bone formation and increase in bone resorption,
leading to osteoporosis, a devastating bone disease [1]. Osteoporosis, which is induced with
decrease in bone mass, is widely recognized as a major public health problem. The most
dramatic expression of the disease is represented by fractures of the proximal femur for which
the number increases as the population ages [2].
Nutritional and functional food factors may have potent effects to prevent bone loss with
increasing age. There is growing evidence that supplementation of nutritional and food
factors may have preventive effects on bone loss that is induced in animal model of
osteoporosis and in human subjects [3-6]. Chemical compounds in food and plants, which
regulate on bone homeostasis, have been to be worthy of notice in maintaining of bone health
and prevention of bone loss with increasing age [7-13].
Carotenoids (carotene and xanthophyll) are present in fruit and vegetables. Carotenoids,
which are a provitamin A, may have an anabolic effect on bone metabolism. Vitamin A
(retinol, retinal, and retinoic acid), which is formed from carotenoids in animal and human,
has been shown to have a role in regulation of bone cells and it may have an anabolic effect
on bone [14-16]. However, vitamin A is also known to have a detrimental effect on bone at
high doses [17-20]. In laboratory animals, high levels of vitamin A lead to accelerated bone
resorption, bone fractures, and osteoporotic bone lesions [17].
β-Cryptoxanthin, a kind of xanthophyll, is abundant in Satsuma mandarin orange (Citrus
unshiu MARC.). Of various carotenoids, β-cryptoxanthin has been found to have a potent-
anabolic effect on bone due to stimulating osteoblastic bone formation and inhibiting
osteoclastic bone resorption [21, 22]. This chapter has been written to outline the recent
advances concerning the role ofβ-cryptoxanthin in bone homeostasis and in prevention of
osteoporosis.
Role of β-Cryptoxanthin in Bone Homeostasis

Bone is a dynamic tissue that undergoes continual adaptation during vertebrate life to
attain and preserve skeletal size, shape, and structural integrity and to regulate mineral
homeostasis. Bone homeostasis is regulated by the functions of osteoblasts, osteoclasts, and
osteocytes that are major cells in bone tissues [23, 24]. Osteoclasts, which develop from
hematopoietic progenitors, are recruited to the site and excavate the calcified matrix.
Osteoblasts arise from local mesenchymal stem cells assemble at the bottom of the cavity and
bone formation begins. Bone acts as major storage site for growth factors [25]. Growth
factors, which are produced by osteoblasts, diffuse into newly deposited osteoid and are

Dietary β-Cryptoxanthin Stimulates Osteogenesis 321
stored in the bone matrx including isulin-like growth factors (IGF- I and II), transforming
growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), and bone morphologic
protein (BMP). These bone-derived factors, which can be liberated during subsequent periods
of bone resorption, act in an autocrine, paracrine, or delayed paracrine fashion in the local
microenvironment of the bone surface. Thus, bone homeostasis is regulated through complex
mechanism. There is growing evidence that functional food factors can regulate bone
homeostasis [4-13].
The effects of carotenes, canthaxanthin, fucoxanthin, antheraxanthin, and violaxanthin on
bone homeostasis have been not shown so far. Among various carotenes and xanthophylls
(including β-carotene, lycopene, lutein, astaxanthin, and β-cryptoxanthin), β-cryptoxanthin
has been found to have a potent-anabolic effect on bone calcification in vitro [21, 22].
Myricetin, kaempferol, isorhamnetin, curcumin, hesperidin, and rutin (quercetin-3-rutinoside)
of various flavonoids did not have an effect on bone formation and calcification in vitro [22].
Vitamin A (retinol, retinal, and retinoic acid) is formed from carotenoids, which are
provitamin A, in the animals and humans. The retinoic acid receptors may be required for
skeletal growth, matrix homeostasis and growth plate function in postnatal mouse, suggesting
a role of retinoic acid in bone growth [14]. However, vitamin A has also been known to have
a detrimental effect on bone at high doses [17-20]. α-Carotene, canthaxanthin, and lycopene
have also been shown to inhibit MC3T3-E1 cell proliferation and to increase alkaline
phosphatase activity and osteopontin mRNA expression [15].
β-Cryptoxanthin Stimulates Bone

Formation and Suppresses Bone Resorption
in Bone Tissue Culture In Vitro
Xanthophyll β-cryptoxanthin, which is a kind of carotenoid, is abundant in Satsuma
mandarin orange (Citrus unshiu MARC). Molecular weight of β-cryptoxanthin is 552.
Biological function of β-cryptoxanthin in animal and human, however, was not determined.
Yamaguchi et al. firstly found that β-cryptoxanthin has been shown to have unique anabolic
effects on bone calcification; such effect was not seen in lutein, lycopene, or astaxanthin,
which is other carotenoids and flavonoid rutin (quercetin-3-rutinoside) [21, 22].
Culture with β-cryptoxanthin (10-7 or 10-6 M) has been found to cause an increase in
calcium content and alkaline phosphatase activity in the femoral-diaphyseal (cortical bone)
and -metaphyseal (trabecular bone) tissues in vitro [21]. Lutein, lycopene, and rutin (10-8 to
10-6 M) did not have anabolic effects on alkaline phosphatase activity and calcium contents in
rat femoral tissues [21]. Astaxanthin and β-carotene did not have an effect on the femoral
calcium contents [22]. Myricetin, kaempferol, isorhamnetin, curcumin, or hesperidin (10-7 to
10-5 M) had no effect on bone calcium content in tissue cultures in vitro [22]. Quercetin
significantly increased calcium content in femoral diaphyseal tissues but not metaphyseal
tissues. Thus, β-Cryptoxanthin has unique anabolic effects on bone calcification in vitro. The
effect of β-cryptoxanthin increasing bone components was completely depressed in presence
of cycloheximide, an inhibitor of protein synthesis, suggesting that this effect is needed newly
protein synthesis [22].

β-Cryptoxanthin has also been shown to inhibit bone resorption in bone tissue cultures in
vitro [22]. Parathyroid hormone (PTH) or prostaglandin E2 (PGE2), which is a bone-resorbing
factor, can stimulate osteoclastic bone resorption in vitro [30-32]. Culture with PTH or PGE2
caused a decrease in calcium content in the diaphyseal and metaphyseal tissues [22]. This
decrease was completely inhibited in presence of β-cryptoxanthin (10-8 to 10-6 M). Likewise,
culture with β-cryptoxanthin completely suppressed the PTH- or PGE2-induced increase in
medium glucose consumption and lactic acid production by bone tissues [22]. β-
Cryptoxanthin has suppressive effects on bone resorption in tissue culture in vitro.
As described above, β-cryptoxanthin has been shown to have a stimulatory effect on
osteoblastic bone formation and a suppressive effect on osteoclastic bone resorption in bone
tissue culture in vitro. Serum concentration of β-cryptoxanthin due to consumption of
vegetable juice in women is shown to be in the range of 1.3×10-7 to 5.3×10-7 M [33]. β-
Cryptoxanthin in the range of 10-8 to 10-6 M has an anabolic effect on biochemical
components in rat femoral tissues in vitro, suggesting a physiologic role in the regulation of
bone metabolism.
β-Cryptoxanthin Stimulates Osteoblastogenesis

The cellular and molecular mechanisms by which β-cryptoxanthin stimulates bone
formation in bone tissues has been examined using osteoblastic MC3T3-E1 cells in vitro. β-
Cryptoxanthin has been found to stimulate proliferation of osteoblastic cells in subconfluent
monolayers in a medium containing 10% fetal bovine serum [34]. Culture with β-
cryptoxanthin also caused an increase in biochemical components (alkaline phosphatase
activity, protein, and DNA contents) of osteoblastic cells [34]. This effect was depressed in
presence of staurosporine, an inhibitor of protein kinase C, or PD98059, an inhibitor of
mitosis activated protein kinase (MAP) kinase, although the effect of β-cryptoxanthin in
increasing cellular biochemical components was not suppressed in culture with dibucain, an
inhibitor of Ca2+/calmodulin-dependent protein kinase [34]. Stimulatory effects of β-
cryptoxanthin on osteoblastic cell components seem to be partly mediated through signaling
factors of protein kinase C or MAP kinase in the cells. The effects of β-cryptoxanthin in
increasing the biochemical components in osteoblastic cells were completely suppressed in
the presence of 5,6-dichloro-1- β-D-ribofuranosylbenzimidazole (DRB), an inhibitor of RNA
polymerase II, suggesting that effect of carotenoid results from a stimulatory effect on
transcriptional activity in osteoblastic cells. The mineralization in osteoblastic cells has been
shown to stimulate in prolonged culture with β-cryptoxanthin [35]. Stimulatory effects of β-
cryptoxanthin on mineralization may result from the carotenoid-induced proliferation and
differentiation of osteoblastic cells.
β-Cryptoxanthin may stimulate gene expression for proteins that are involved in bone
formation and mineralization in osteoblastic cells. The effect of β-cryptoxanthin on gene
expression in osteoblastic cells using reverse transcription-polymerase chain reaction (RT-
PCR) has been examined. Culture with β-cryptoxanthin was found to stimulate the mRNA
expression of IGF-I or TGF- β1 in osteoblastic cells [34]. Finding may support the view that
β-cryptoxanthin has the stimulatory effect on transcriptional activity in osteoblastic cells.
IGF-I or TGF-β1 is a bone growth factor produced from osteoblasts [36-38]. Stimulatory

effects of β-cryptoxanthin on the proliferation of osteoblastic cells may be partly mediated

through the action of IGF-I or TGF-β1 produced from the cells.
TGF-1 has a potent activity on osteoblast-lineage commitment an event that is partly
mediated through Smad transcription factors [38]. It has been reported that NF-θB signaling
represses basal osteoblast differentiation and mineralization in MC3T3 cells and antagonizes
TGF-1 and BMP-2 mediated MC3T3 mineralization by downregulating Smad activation
[39]. Interestingly, -cryptoxanthin has been shown to have the capacity to suppress the
receptor activator of nuclear factor-kappa B (NF-θB) activity in MC3T3 preosteoblastic cells
[40], suggesting that one mechanism by which -cryptoxanthin may stimulate osteoblast
differentiation may be due to promoting Smad activation. Such finding also suggests that -
cryptoxanthin may lead to enhanced Smad signaling. While, -cryptoxanthin failed to directly
stimulate Smad activity, it did however amplify TGF-1-induced Smad signaling [41].
Furthermore, whether -cryptoxanthin directly stimulates Smad activation or regulates
TGF-1-induced Smad activation in osteoblastic cells in vitro has been examined [41]. -
Cryptoxanthin was found to potentiate TGF-1-induced, but not Bmp-2-induced, Smad
activation in MC3T3 preosteoblastic cells, suggesting that the mechanism by which -
cryptoxanthin stimulates bone formation enhances the TGF-1-mediated commitment of
preosteoblasts to differentiate along the osteoblastic pathway [41].
Runx2 (Cbfa1) is a member of the runt domain family of transcription factors and a
master regulator of osteoblast differentiation [42]. α1 (I) Collagen is a matrix protein that is
related to bone formation and mineralization in osteoblast lineage cells [43]. Alkaline
phosphatase participates in the mineralization process in osteoblastic cells [44]. β-
Cryptoxanthin (10-7 or 10-6 M) was found to increase the mRNA expression of Runx2, α1 (I)
collagen, and alkaline phosphatase in osteoblastic MC3T3-E1 cells [35].β-Cryptoxanthin had
the stimulatory effect on the gene expression of for various proteins involved in osteoblasic
bone formation [35]. Such effect of β-cryptoxanthin were blocked in the presence of DRB
[34], supporting the view that the carotenoid stimulates transcriptional activity in osteoblastic
MC3T3-E1 cells.
Vitamin A (retinol) may be able to bind to nuclear receptors in cells. Retinol and β-
carotene has been shown to inhibit the proliferation of osteoblastic MC3T3-E1 cells as well as
DNA synthesis of the cells, due to increasing alkaline phosphatase activity dose dependently
(10-9 to 10-7 M) [15]. Vitamin A (10-7 to 10-6 M) increases alkaline phosphatase activity in
osteoblastic cells [34]. β-Cryptoxanthin (10-7 or 10-6 M) caused an increase in alkaline
phosphatase activity and protein content in osteoblastic cells. This effect was also seen in the
presence of vitamin A (10-6 M) [34].
Moreover, the stimulatory effect of β-cryptoxanthin on the expression of Runx2 type 1
and α1 (I) collagen mRNA was observed in presence of vitamin A [34]. Vitamin A did not
have a significant effect on Runx2 type 1 mRNA expression in osteoblastic MC3T3-E1 cells
[34]. Thus, the mode of action of β-cryptoxanthin on gene expression in osteoblastic cells
may differ from that of vitamin A, which is mediated through the RXR receptor in the
nucleus of the cells [34]. β-Cryptoxanthin may be able to bind other receptors (including
orphan receptors that ligands has been unknown), and that the carotenoid may stimulate
transcriptional activity in osteoblastic cells.

β-Cryptoxanthin Suppresses Osteoclastogenesis

NF-θB ligand (RANKL) plays a pivotal role in osteoclastogenesis from bone marrow
cells. RANKL expression is induced in osteoblastic cells and bone marrow stromal cells in
response to osteoporotic factors, such as PTH, PGE2, and 1,25-dihydroxyvitamin D3 (VD3),
and combined treatment of hematopoietic cells with macrophage colony-stimulating factor
(M-CSF), and the soluble form of RANKL (sRANKL) induce osteoclast differentiation in
vitro [45, 46]. The receptor protein RANK (receptor activator of NF-θB) is expressed on the
surface of osteoclast progenitors.
β-Cryptoxanthin (10-8 to 10-6 M) has been shown to have a potent-inhibitory effect on
osteoclast-like cell formation in mouse marrow culture in vitro [47]. Inhibitory effect of β-
cryptoxanthin on osteoclast-like cell formation was seen at the later stage of osteoclast
differentiation in bone marrow cultures [47]. Culture with β-cryptoxanthin caused a marked
inhibition of osteoblast-like cell formation induced in presence of PTH, PGE2, VD3,
lipopolysaccharide, or tumor necrosis factor-α (TNF-α). β-Cryptoxanthin also had the
suppressive effect on osteoclast-like cell formation induced by RANKL [47]. Suppressive
effects of β-cryptoxanthin were equal to that of 17 β-estradiol (E2), calcitonin, genistein, and
zinc sulfate, which can inhibit osteoclast-like cell formation induced by bone-resorbing
factors [47].
Interaction of RANKL with its receptor RANK leads to the recruitment of the signaling
adaptor molecules TRAFs (TNF receptor-associated factors) to the receptor complex and the
activation of NF-θB and c-Jun N-terminal kinase (JNK) [48, 49]. Protein kinase C family
enzyme has a role in regulation of osteoclast formation and function potently due to
participating in the extracellular signaling-regulated kinase (ERK) signaling pathway of M-
CSF and RANKL [49].
Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated
osteoclast-like cell formation in mouse marrow cultures, and PMA-induced
osteoclastogenesis was found to inhibit in presence of β-cryptoxanthin [47]. Moreover, β-
cryptoxanthin had the suppressive effect on dibutyryl cyclic adenosine monophosphate
(DcAMP)-induced osteoclast-like cell formation in mouse marrow cultures [47]. Activation
of protein kinase C and protein kinase A pathways may lead to increased RANKL expression,
and that β-cryptoxanthin can inhibit protein kinase C- or protein kinase A-related RANKL
expression in osteoclastogenesis.
The effect of β-cryptoxanthin on mature osteoclasts has been demonstrated [50]. M-CSF-
dependent bone marrow macrophages were cultured in presence of M-CSF and RANKL for 4
days [50]. Osteoclastic cells formed were further cultured in medium containing β-
cryptoxanthin with or without M-CSF and RANKL for 24-72 hours. The number of
osteoclastic cells has been found to decrease in culture with β-cryptoxanthin (10-7 or 10-6 M)
in presence or absence of M-CSF and RANKL for 72 hours. The β-cryptoxanthin-induced
decrease in osteoclastic cells was depressed in the presence of caspase-3 inhibitor. Results of
agarose gel electrophoresis showed the presence of low-molecular-weight DNA fragments of
adherent cells cultured with β-cryptoxanthin. These findings indicate that the carotenoid
induces apoptotic cell death.
Apoptosis-related gene expression has been determined using RT-PCR [50]. The
expression of caspase-3 mRNA or Apaf-2, which involves apoptosis, in osteoclastic cells was

found to stimulate when cultured with β-cryptoxanthin in prsence or absence of M-CSF and
RANKL [50]. β-Cryptoxanthin-induced apoptotic cell death may be partly mediated through
caspase-3 expression in osteoclastic cells.
The expression of Bcl-2 mRNA, which is involved in the rescue of apoptosis, was
decreased after culture with β-cryptoxanthin in the presence or absence of M-CSF and
RANKL [50]. However, Akt-1 mRNA expression was not significantly changed after culture
with β-cryptoxanthin. Decrease in Bcl-2 mRNA expression may partly contribute to effect of
β-cryptoxanthin in stimulating the apoptotic cell death of osteoclastic cells.
Culture with β-cryptoxanthin has been found to have suppressive effects on tartrate-
resistant acid phosphatase (TRACP) activity, and it decreases TRACP and cathepsin K
mRNA expressions in osteoclastic cells in presence or absence of M-CSF and RANKL [50].
These findings suggest that β-cryptoxanthin can inhibit enhancement of bone-resorbing
activity in osteoclasts. β-Cryptoxanthin inhibitd various bone-resorbing factors-induced
decrease in bone calcium content and increase in lactic acid production in rat femoral tisuue
culture system in vitro. Presumably, β-cryptoxanthin has an inhibitory effect on activation of
mature osteoclasts.
As mentioned above,β-cryptoxanthin has stimulatory effects on apoptotic cell death due
to activating gene expression of its related proteins. Carotenoid also has the suppressive
effects on TRACP activity and gene expression of enzymes that involve in bone-resorbing
activity in osteoclastic cells.
β-Cryptoxanthin Prevents Bone Loss in Vivo

As mentioned above, β-cryptoxanthin has been shown to have stimulatory effects on
osteoblastic bone formation and suppressive effects on osteoclastic bone resorption in vitro.
Furthermore, preventive effects of β-cryptoxanthin on osteoporosis have been demonstrated
using animal models in vivo.
Anabolic effects of β-cryptoxanthin on bone components in young and aged rats have
been examined. β-Cryptoxanthin (100, 250, or 500 κg/kg body weight) was orally
administered once daily for 7 days to young male rats [51]. Administration of β-cryptoxanthin
(250, or 500 κg/kg) caused an increase in alkaline phosphatase activity, DNA and calcium
contents in the femoral-diaphyseal and -metaphyseal tissues [52]. Such an effect was also
observed in the femoral tissues of aged (50-week-old) female rats [62]. β-Cryptoxanthin has
been shown to have anabolic effects on bone components in rats in vivo.
β-Cryptoxanthin has been shown to have preventive effects on bone loss in the
pathophysiologic state. Bone loss is induced in streoptozotocin (STZ)-diabetic rats [53].
Young rats received a single subcutaneous administration of STZ (60 mg/kg body weight),
and then the animals received oral administration of β-cryptoxanthin (50 or 100 κg/kg) once
daily for 7 or 14 days. Administration of STZ caused a decrease in body weight and a
significant increase in serum glucose, triglyceride, and calcium levels, indicating a diabetic
state [53]. These alterations were restored after administration of β-cryptoxanthin (50 or 100
κg/kg) for 14 days [53]. Alkaline phosphatase activity, DNA and calcium contents in the
femoral-diaphyseal and -metaphyseal tissues were decreased in STZ-diabetic rats [62]. These
decreases were prevented after administration of β-cryptoxanthin (50 or 100 κg/kg) for 14

days [53]. Thus, intake of β-cryptoxanthin was found to have restorative effects on STZ-
diabetic state and bone loss in STZ-diabetic rats.
Bone loss is induced after ovriectomy (OVX), which is a model of postmenopausal
osteoporosis. β-Cryptoxanthin (50 or 100 κg/kg body weight) was orally administered once
daily for 3 months to OVX rats. Analysis using peripheral quantitative computed tomography
shows that OVX induced a significant decrease in mineral content and mineral density in the
femoral-diaphyseal and -metaphyseal tissues [54]. Such decreases were protected after
administration of β-cryptoxanthin (50 or 100 κg/kg). Moreover, OVX caused bone loss. This
was completely prevented after administration of β-cryptoxanthin (50 or 100 κg/kg). β-
Cryptoxanthin had preventive effects on OVX-induced bone loss in vivo [54].
Intake of Dietary β-Cryptoxanthin Reveals

Preventive Effect in Menopausal Women
Serum bone-specific alkaline phosphatase and γ-carboxylated osteocalcin are bone
metabolic markers of osteoblastic bone formation [55, 56]. Serum bone TRACP and N-
telopeptides of type I collagen are metabolic markers of osteoclastic bone resorption [57, 58].
The effect of prolonged intake of juice prepared from Satsuma mandarin (Citrus unshiu
MARC) containing β-cryptoxanthin has been found using circulating biochemical markers of
bone metabolism in subjects including menopausal woman [59 - 61]. Twenty-one volunteers
(10 males and 11 females) were divided into two groups of ten volunteers (5 males and 5
females) and eleven volunteers (5 males and 6 females). Each group was given sequentially
juice (192 ml) containing two different contents of β-cryptoxanthin once a day for 28 or 56
days either a regular juice with naturally occurring 802 κg β-cryptoxanthin/100 ml or a
reinforced juice containing 1500 κg β-cryptoxanthin/100 ml [59].
Intake of regular juice for 28 or 56 days in healthy subjects caused a significant increase
in serum γ-carboxylated osteocalcin concentration, and intake for 56 days produces a
decrease in serum bone TRACP activity [59]. Moreover, intake of the β-cyptoxanthin
reinforced juice for 28 or 56 days caused an increase in serum γ-carboxylated osteocalcin
concentration and a corresponding decrease in serum bone TRACP activity and N-telopeptide
of type I collagen [59]. Such findings suggest that intake of β-cyptoxanthin reinforced juice
has stimulatory effects on osteoblastic bone formation and suppressive effects on osteoclastic
bone resorption in normal individuals [59].
Serum β-cyptoxanthin concentration was increased after intake of regular juice for 56
days [60]. This increase was enhanced after intake of β-cyptoxanthin-reinforced juice. Intake
of regular juice or of β-cyptoxanthin-reinforced juice for 56 days caused an increase in serum
γ-carboxylated osteocalcin and a decrease in serum bone TRACP activity [60]. A relationship
between serum β-cyptoxanthin and circulating γ-carboxylated osteocalcin concentrations was
found using the value obtained from all groups for before intake and with the intake of regular
juice and β-cyptoxanthin-reinforced juice.
A negative relationship between serum β-cyptoxanthin concentation and circulating
TRACP activity was observed [60]. This study shows that a relationship between serum β-
cyptoxanthin and circulating bone metabolic markers is found in healthy individuals with
intake of juice containing β-cyptoxanthin.

Ninety volunteers, aged 27-65 years (19 men and 71 women), were enrolled in this study
[61]. The seventy-one females included 35 premenopausal women (ages, 27-50 years) and 36
menopausal women (ages, 46-65 years). Volunteers were divided into four groups; placebo
juice without β-cyptoxanthin (5 men and 19 women), juice containing β-cyptoxanthin at 1.5
mg/200 ml of juice/day (4 men and 17 women), 3.0 mg/day (5 men and 17 women), and 6.0
mg/day (5 men and 18 women). Placebo or juice (200 ml) was ingested once a day for 28 or
56 days.
Serum β-cryptoxanthin concentrations were increased after intake of juice containing β-
cryptoxanthin (1.5, 3.0, or 6.0 mg/day) for 28 or 56 days, and the increases were dose-
dependent [56].
An increase in serum β-cryptoxanthin concentration was also observed at 28 days at the
end of intake, indicating that the carotenoid is stable in the serum. Serum β-cryptoxanthin
concentration was in the range of 4.20 x 10-7 M to 4.89 x 10-7 M in the placebo groups. The
intake of juice reinforced with β-cryptoxanthin concentration at doses of 1.5, 3.0, or 6.0
mg/day increased the serum concentration to 2.43 x 10-6, 4.06 x 10-6, or 5.38 x 10-6 M,
respectively [61]. These increases were about 5 or 10 fold as compared with the value
obtained before intake or after placebo intake. It has been shown that the serum concentration
of β-cryptoxanthin increased due to consumption of vegetable juice in women from 1.3 x 10-7
to 5.3 x 10-7 M [33].
In ninety volunteers (aged 27-65 years), serum bone-specific alkaline phosphatase
activity was increased after intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for
56 days as compared with the value obtained before intake [61]. γ-Carboxylated osteocalcin
concentration was increased after intake of juice containing β-cryptoxanthin (3.0 or 6.0
mg/day) for 28 or 56 days as compared with the value obtained before intake or after intake
of placebo juice [61].
Serum TRACP activity and type I collagen N-telopeptide concentration were decreased
after intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as
compared with the value obtained before intake or after intake of placebo juice, and
significant decreases were also seen after intake of 1.5 mg/day β-cryptoxanthin as compared
with the value obtained before intake [61].
In menopausal women (36 volunteers), bone-specific alkaline phosphatase activity and γ-
carboxylated osteocalcin concentration were increased after intake of juice containing β-
cryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained after
placebo intake [61].
Also, this intake caused a decrease in bone TRACP activity and type I collagen N-
telopeptide concentration. Thus, prolonged intake of β-cryptoxanthin-reinforced juice has
been demonstrated to have stimulatory effects on osteoblastic bone formation and inhibitory
effects on osteoclastic bone resorption in menopausal women.
Meanwhile, serum calcium, inorganic phosphorous, and parathyroid hormone (intact)
were not changed after intake of β-cryptoxanthin-containing juice for 28 or 56 days. Other
serum biochemical findings were not changed after intake of juice containing β-cryptoxanthin
(3.0 or 6.0 mg/day) for 56 days. Safety of β-cryptoxanthin in human is confirmed [61].
As mentioned above, intake of juice reinforced with β-cryptoxanthin (3.0 or 6.0 mg/day)
has been found to have an effect on circulating bone metabolic markers in men,
premenopausal women, and menopausal women [61]. This indicates that effects of β-

cryptoxanthin in stimulating bone formation and inhibiting bone resorption are present in
both sexes.
Interstingly, intake of juice reinforced with β-cryptoxanthin (3.0 or 6.0 mg/day) has been
found to have effects on circulating bone metabolic markers in menopausal women,
indicating that supplementation of β-cryptoxanthin has preventive effects on bone loss due to
osteoporosis in menopausal women. Such preventive effect was obvious at a dose of β-
cryptoxanthin of 3.0 mg/day in menopausal women. This dose may be suitable in prevention
of osteoporosis in human subjects. Thus, intake of reinforced juice, which contains more β-
cryptoxanthin than regular juice, was demonstrated to have the preventive effect on bone loss
that accompanies an increase in age.
β-Cryptoxanthin and Bone Health:

Epidemiological Evidence
On the based on our findings, epidemiological studies support the view that the intakes of
fruit and vegetables containing β-cryptoxanthin may reduce risk of osteoporosis [62-64]. The
effect of dietary antioxidants on knee structure in a cohort of healthy, middle-aged subjects
with no clinical knee osteoarthritis is reported [62]. Two hundred and ninety-three healthy
adults (mean age = 58.0 years) without knee pain or knee injury were selected from an
existing community-based cohort. Intake of antioxidant vitamins and food sources by these
individuals was estimated from a food frequency questionnaire at baseline. The cartilage
volume, bone area, cartilage defects and bone marrow lesions were assessed approximately
10 years later using magnetic resonance imaging. Higher vitamin C intake was associated
with a reduced risk of bone marrow lesions and with a reduction in the tibial plateau bone
area. There was an inverse association between fruit intake and the tibial plateau bone area
and between fruit intake and the risk of bone marrow lesions. Neither fruit intake nor vitamin
C intake was significantly associated with the cartilage volume or cartilage defects.
Lutein and zeaxanthin intake was associated with a decreased risk of cartilage defects,
and vitamin E intake tended top be positively associated with the tibial plateau bone were
only after adjusting for vitamin C intake. Intake ofβ-cryptoxanthin was inversely associated
with the tibial plateau bone area after adjusting for vitamin E intake. These observations
suggest beneficial effects of fruit consumption and vitamin C intake as they are associated
with a reduction in bone size and the number of bone marrow lesions, both of which are
important in the pathogenesis of knee osteoarthritis [62].
Bone mineral density (BMD) in post-menopausal female subjects has been shown to
associate with serum antioxidant carotenoids. A total of six hundred ninety-nine subjects (222
males and 477 females) who had received health examinations in the town of Mikkabi,
Shizuoka Prefecture, Japan, participated in the study [63]. Radial BMD was measured by
using dual-energy X-ray absorptiometry. Associations of serum carotenoid concentrations
with the dadial BMD were evaluated cross-sectionally. In male and pre-menopausal female
subjects, the six serum carotenoids were not associated with the radial BMD. On the other
hand, in post-menopausal female subjects, serum β-cryptoxanthin and β-carotene were
weakly but positively correlated with the radial BMD. After adjustment for confounders, the
odds ratio (OR) for the lowest quartile of BMD in the high groups of serum β-cryptoxanthin

against the lowest quartile was 0.45 in post-menopausal female subjects. However, this
association was not significant after further adjusting for intakes of minerals and vitamins.
Antioxidant carotenoids, especially β-cryptoxanthin, significantly but partly associated with
the radial BMD in post-menopausal female subjects [63].
Moreover, epidemiological studies showed that high intakes of carotenoids might be
useful to maintain bone health [64]. A follow-up on 146 male and 99 pre- and 212 post-
menopausal female subjects from the Mikkabi study, Shizuoka, Japan, was conducted [64].
Those who participated in previous BMD surveys and completed four years of follow-up
were examined longitudinally. During a 4-year follow-up, 15 of the post-menopausal female
subjects developed new-onset osteoporosis. In contrast, none of the male and pre-menopausal
female subjects did. In male and pre-menopausal female subjects, the six serum carotenoids at
the baseline were not associated with bone loss. On the other hand, in post-menopausal
female subjects, the 4-year bone loss of radius was inversely associated with the serum
carotenoid concentrations, especially in β-carotene. After adjustments for confounders, the
odds ratios (OR) for osteoporosis in the highest tertiles of serum β-carotene and β-
cryptoxanthin against the lowest tertiles were 0.24 (95% confidence interval 0.05-1.21) and
0.07 (CI: 0.01-0.88), respectively. Serum β-cryptoxanthin was also inversely associated with
risk for osteopenia and/or osteoporosis (P for trend, 0.037). In addition, this retrospective
analysis revealed that subjects who developed osteoporosis and/or osteopenia during the
survey period had significantly lower serum concentrations of β-cryptoxanthin and β-carotene
at the baseline than those in normal group. Thus, antioxidant carotenoids, especially β-
cryptoxanthin and β-carotene, have been found to be inversely associated with change of
radial BMD in post-menopausal female subjects.
Figure 1. Change in bone mass with increasing age. Bone mass increases with growing and reaches to peak in
the range of age between twenty and twenty-five years. Then, bone mass is gradually decreased with aging.
In postomenopausal women, bone mass decreases dramatically and it induces osteoporosis. It is important to
prevent decrease in bone mass with increasing age.

Interestingly, seasonal variation of serum α- and β-cryptoxanthin and 25-OH-vitamin D3

in women with osteoporosis has been showed [65]. In six hundred fourty-four women with
osteoporosis, serum β-cryptoxanthin and 25-OH-vitamin D3 showed a weak but significant
correlation and exhibited a complementary seasonal distribution [65]. Dietary intake and
serum levels of β-cryptoxanthin have been inversely related to different bone and joint
disorders and in vitro and animal studies have shown that β-cryptoxanthin displays a unique
anabolic effect on bone calcification.
Due to the emerging role of β-cryptoxanthin in bone biology, this study was aimed to
assess serum distribution and variability of β-cryptoxanthin and their potential relation to 25-
OH-vitamin D3 in women with osteoporosis [65]. Overall, significant seasonal variations
were found for the three analyses and inter-individual variation was also high (60-73%). β-
Cryptoxanthin and 25-OH- vitamin D3 exhibited a marked complementary seasonal
distribution in serum, with vitamin D displaying the highest values in summer and β-
cryptoxanthin in winter. Given anabolic effects of β-cryptoxanthin on bone calcification and
its complementary seasonal distribution with respect to 25-OH- vitamin D3, potential role of
β-cryptoxanthin as a sustainable nutritional approach to improving bone health deserves to be
further evaluated [65].
Conclusion
Bone mass is changed with increasing ages as shown in Figure 1. Bone loss is
dramatically induced in postmenopausal women. Nutritional and food factors may have a
preventive role in the decrease in bone mass with aging and pathophysiological conditions.
Among various carotenoids, β-cryptoxanthin has been found to have unique anabolic effects
on bone mass due to stimulating osteoblastic bone formation and inhibiting osteoclastic bone
resorption. β-Cryptoxanthin modulates the gene expression of various proteins that involve in
osteoblastic bone formation and osteoclastic bone resorption.
Intake of dietary β-cryptoxanthin has been found to have preventive effects on bone loss
in animal models for osteoporosis and in menopausal women, suggesting the possibility of
pharmacological use ofβ-cryptoxanthin in prevention and therapy of osteoporosis and other
bone diseases. Moreover, role of β-cryptoxanthin in bone health has also been shown in
human subjects with epidemiological studies. Supplemental intake of β-cryptoxanthin with
higher dose may have a pharmacologic role in therapy of osteoporosis with clinical studies.
Potent effects withβ-cryptoxanthin derivatives will be expected in development of new drug
for treatment of bone diseases.
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Index
advancements, 156
A adverse effects, 42, 213, 238, 279
adverse event, 286
absorption spectra, 105
adverse weather, 148
abstraction, 100, 121
aetiology, 234
abuse, 231
aflatoxin, 141
access, 177, 200, 246, 283
age, 10, 11, 12, 28, 29, 40, 45, 46, 47, 94, 111, 112,
acetaminophen, 269
115, 116, 118, 119, 120, 121, 122, 123, 143, 152,
acetone, 15, 62, 97, 98, 118, 119, 265, 309
161, 166, 190, 197, 208, 210, 211, 212, 223, 224,
acetonitrile, 19
225, 240, 275, 320, 328, 329
acid, 8, 13, 57, 58, 64, 69, 70, 71, 75, 76, 97, 99,
age-related diseases, 11
104, 108, 109, 111, 117, 150, 153, 157, 159, 182,
aggregation, 93, 103, 104, 108, 152, 279
192, 206, 226, 242, 243, 256, 257, 261, 264, 267,
aging process, 238, 239
279, 281, 301, 311, 313, 317, 320, 321, 331, 333,
agonist, xi, 262
334
AIBN, 104
acidity, 265, 311
Alaska, 81, 90, 91
activation energy, 101
albumin, 96
active compound, 243, 249, 251
alcohol abuse, xi, 229, 231
active oxygen, 39, 117
alcohol consumption, 208
active site, 313
alcohols, 65, 301
adaptation, 170, 180, 233, 320
algae, xi, 35, 38, 44, 130, 131, 137, 144, 145, 147,
additives, 2, 7, 19, 166
149, 156, 157, 200, 237, 261, 268, 287, 331
adenine, 80
alkaloids, 96, 301
adenocarcinoma, 108, 122
almonds, 16
adenosine, 324
alpha-tocopherol, 117, 124, 224, 233, 234, 281, 288,
adhesion, xii, 40, 257, 271, 272, 273, 277, 279, 284,
291, 293, 294
288, 289, 291, 307
ALS, 151, 160, 215
adipocyte, 266
alternative hypothesis, 178
adiponectin, 267, 286, 295
alters, 139, 141
adipose tissue, 13, 30, 156, 208, 212, 221, 230, 263,
aluminum oxide, 17
267, 286, 290
alveoli, 108
adiposity, 162
amines, 96
adjustment, 328
amino acid(s), 96, 100
adolescents, 291
amplitude, 99, 211
adrenal gland(s), 130, 204
amyotrophic lateral sclerosis, 151, 160, 215, 227
adsorption, 242
anatomy, 193
adulthood, 190
ancestors, 80, 175
adults, 234, 276, 285, 291, 295, 328
ancient forms, 90

338 Index
androgen(s), 153, 176, 185

angiogenesis, 162
B
annatto, xi, 7, 261, 263, 264, 265, 268, 269, 278
BAC, 302
ANOVA, 167
bacteria, x, 35, 44, 45, 173, 174, 230, 248, 261, 268
anthocyanin, 81
bacterial infection, 137
anti-/pro-oxidant, ix, 94, 102, 103
bacterial pathogens, 269
antibody, 141, 214
bacterium, 46, 121
anti-cancer, 281
barriers, 258
antigen, 70, 71, 141
basal cell carcinoma, 110
anti-inflammatory, vii, xii, xiii, 145, 249, 262, 273,
base, 201, 254, 298, 313, 318
278, 281, 284, 287, 298, 301, 307, 309, 311, 313,
BD, 225
314, 316
beams, 89
anti-inflammatory agents, 313, 314
beer, 159
antioxidative activity, 230, 231
behaviors, 18
antioxidative potential, 234, 236
Beijing, 166
antipyretic, 263
beneficial effect, viii, 2, 7, 10, 11, 22, 39, 93, 114,
antitumor, 136
155, 181, 214, 216, 267, 273, 278, 279, 282, 283,
aorta, 282
298, 328
apoptosis, 77, 122, 145, 152, 219, 241, 248, 277,
benefits, vii, ix, x, 1, 2, 7, 9, 10, 39, 42, 43, 45, 131,
279, 292, 324, 325
135, 143, 144, 145, 154, 180, 199, 216, 268, 273,
appetite, 8
276, 285, 292
aquaculture, ix, 145, 154, 165, 166, 171
benign, 221
aquarium, 167
benzene, 15, 65, 104
arrest, 238, 292, 333
beta-carotene, vii, 1, 30, 35, 45, 47, 116, 118, 124,
arteriosclerosis, 213
139, 140, 141, 142, 170, 218, 234, 235, 241, 247,
artery, 225
261, 270, 288, 290, 292, 294
asbestos, 41, 209
BI, 218
ascorbic acid, 71, 77, 96, 109, 126, 151
Bible, 269
Asia, 24
bile, 11, 131, 263, 301
asparagus, 37
bile acids, 11, 131, 263
assessment, 157, 224, 234, 236, 287
bilirubin, 96
assimilation, 13, 153
bioavailability, vii, xii, 1, 2, 3, 8, 10, 12, 13, 14, 17,
asthma, 263
24, 26, 31, 47, 140, 152, 180, 191, 203, 204, 208,
astringent, 263
218, 227, 243, 247, 272, 273, 277, 279, 280, 281,
asymmetry, 181
286, 287
atherogenesis, 9, 27, 279, 282
biochemical processes, 182
atherosclerosis, ix, xii, 3, 11, 47, 109, 127, 136, 160,
biochemistry, 81, 119, 182, 192, 299
225, 240, 271, 272, 273, 275, 278, 282, 291, 292,
biocompatibility, 245, 249
293, 294
bioconversion, vii, 1, 2, 8, 13, 23, 153, 162, 203
atherosclerotic plaque, 9, 282
biodiesel, 151
athletes, 235
biodiversity, 158
atmosphere, 15, 308
bio-efficacy studies, ix, 128
atmospheric pressure, 20, 133
biological activity(s), viii, x, 3, 7, 12, 13, 16, 40, 49,
atoms, 3, 57, 98
128, 133, 199, 242, 253, 271
ATP, 279
biological functions, vii, x, 2, 3, 15, 22, 38, 39, 133,
attachment, 257
138, 144, 199, 216
autoimmunity, 246
biological markers, 220
autooxidation, ix, 94
biological systems, 98, 105, 287
avian, 174, 175, 176, 177, 182, 192, 196, 197
biomarkers, 109, 206, 219, 274, 285, 286, 290, 293
axons, 111
biomass, 16, 147, 149, 150, 151, 154, 155, 158, 170
biomaterials, 238
biomedical applications, 243
biomolecules, 95, 181

Index 339
biopsy, 309 calcium, 160, 227, 321, 322, 325, 327, 331, 332
biosensors, 94 calibration, 20
biosynthesis, viii, ix, 24, 29, 49, 50, 57, 71, 73, 74, Cambodia, 263
144, 145, 147, 155, 157, 163 cancer cells, 42, 71, 77, 113, 122, 140, 145, 155,
biotechnology, 16, 158 219, 220, 302
birds, vii, x, 2, 10, 35, 173, 174, 175, 176, 177, 179, cancer death, 219
180, 182, 185, 187, 188, 189, 191, 192, 194, 197, cancer therapy, 268
200 candidates, viii, xi, 79, 98, 147, 237, 239
bladder cancer, 109, 207, 220, 221 capsanthin, viii, xii, 7, 10, 17, 49, 50, 51, 52, 53, 54,
bleaching, 7, 99, 103, 104 57, 59, 61, 64, 65, 68, 69, 70, 71, 73, 76, 127,
blindness, 2, 8, 111, 135, 152 130, 131, 136, 139, 141, 297, 303, 308, 309, 311
block polymers, xi, 237, 247 capsule, 203, 204, 205, 214
blood flow, 234, 281 carbohydrate(s), xii, 81, 95, 262, 266, 267, 268, 269
blood plasma, 112, 200 carbohydrate metabolism, 266, 267
blood pressure, 226, 273, 281, 284, 289, 295 carbon, 3, 22, 36, 38, 44, 47, 48, 57, 80, 81, 100,
blood stream, x, 131, 153, 174, 176, 213, 263 128, 129, 144, 146, 157, 159, 239, 261, 265, 267,
blood vessels, 153 299
bloodstream, 145, 176 carbon atoms, 3, 57, 128, 239, 261, 267
BMI, 285, 286 carbon dioxide, 80, 81, 159
body fat, 153 carbonyl groups, 65, 69
body size, 184 carboxylic groups, 264
body weight, 265, 280, 285, 325, 326 carcinogen, 9, 41, 42, 109
bonds, 5, 22, 93, 97, 101, 105, 153, 267 carcinogenesis, 9, 70, 115, 140, 153, 219, 221
bone, xiii, 8, 40, 42, 44, 213, 219, 225, 226, 319, carcinoma, 102
320, 321, 322, 323, 324, 325, 326, 327, 328, 329, cardiovascular disease(s), vii, ix, xii, 1, 2, 8, 9, 10,
330, 331, 332, 333, 334 23, 40, 41, 43, 45, 46, 47, 114, 127, 128, 136,
bone biology, 330 141, 160, 199, 200, 201, 216, 219, 222, 225, 271,
bone cells, 320 272, 274, 275, 280, 282, 283, 286, 287, 290, 292,
bone form, xiii, 213, 319, 320, 321, 322, 323, 325, 293, 294, 295, 298, 313
326, 327, 328, 330, 332, 333 cardiovascular disorders, 136
bone growth, 8, 321, 322 cardiovascular risk, 125, 273, 276, 283, 288
bone loss, xiii, 319, 320, 325, 326, 328, 329, 330, carotenes, vii, 4, 30, 35, 39, 44, 94, 103, 106, 108,
331, 334 112, 118, 127, 128, 144, 151, 153, 175, 176, 182,
bone marrow, 226, 324, 328, 331, 332 194, 206, 261, 299, 303, 308, 311, 321
bone mass, xiii, 319, 320, 329, 330, 333 cartilage, 328, 334
bone resorption, xiii, 214, 219, 226, 319, 320, 321, cascades, 111
322, 325, 326, 327, 328, 330, 332, 333, 334 case studies, 134
brain, 102, 215, 313 catabolism, 267
Brazil, xi, 1, 35, 37, 43, 44, 261, 264 cataract, 2, 8, 29, 40, 46, 135, 140, 143, 200, 211,
breakdown, 123, 131, 263, 265 212, 224
breast cancer, 11, 29, 141, 207, 209, 220, 221, 222 cataract extraction, 135, 140, 211, 224
breeding, 185, 189, 192 cation, 100, 117, 119
budding, 82, 89 causal relationship, 208, 214
butadiene, 261 CDC, 288, 290
butyl ether, 19 cell culture, 121, 137, 148, 159, 160, 274, 292, 308,
by-products, 22, 60, 145, 181 332
cell cycle, 136, 140, 220, 238, 292
cell death, 324, 325, 334
C cell differentiation, 9, 39, 40, 153, 221, 333
cell line(s), 113, 136, 206, 207, 308, 310
Ca2+, 322
cell membranes, 95, 108, 112, 182
cabbage, 130, 211
cell metabolism, 181
calcification, 321, 330, 331
cell organelles, 245
calcitonin, 324

340 Index
cell signaling, 237, 245, 248 coenzyme, 214, 226, 301

cell surface, 289 coffee, 283
cellular energy, 251 collagen, 110, 152, 196, 214, 251, 252, 253, 279,
central nervous system, 215 323, 326, 327, 332, 334
ceruloplasmin, 96, 110 collisions, 96
cervical cancer, 207 colon, 10, 11, 29, 43, 102, 220
challenges, 188, 190, 285 colon cancer, 10, 11, 43
cheese, 263, 264 colon carcinogenesis, 11, 29
chelates, 91 color, vii, viii, ix, x, xi, 2, 3, 5, 6, 7, 12, 35, 36, 43,
chemical(s), vii, viii, ix, xiii, 1, 2, 3, 7, 12, 16, 17, 79, 86, 99, 127, 130, 140, 165, 166, 167, 169,
20, 22, 34, 38, 49, 53, 59, 94, 95, 96, 97, 98, 107, 170, 174, 176, 184, 185, 189, 192, 202, 230, 234,
111, 114, 118, 126, 129, 133, 144, 149, 150, 200, 236, 261, 262, 264, 265, 300
201, 221, 239, 241, 251, 261, 262, 266, 267, 271, combined effect, 151, 267
272, 274, 301 commercial, 17, 19, 20, 25, 138, 147, 148, 150, 151,
chemical properties, 12, 239, 251, 266 154, 155, 158, 306
chemical reactions, 97 communication, x, 9, 11, 39, 40, 42, 113, 128, 133,
chemical reactivity, 95, 118, 272 136, 140, 173, 174, 177, 209, 222, 223, 246, 273
chemical structures, vii, 2, 3, 22, 95 community(s), 263, 274, 328
chemoprevention, 29, 122, 208 compatibility, 238
chemopreventive agents, 208 competition, ix, 143, 145
chemotherapeutic agent, 301 competitive advantage, 154
chemotherapy, 71, 301 complex interactions, 180
chicken, 191 complexity, 103
children, 2, 7, 26 complications, 281, 282
China, 145, 165, 166, 169, 170, 222, 237, 256 composition, vii, 1, 2, 3, 5, 6, 11, 15, 22, 25, 35, 46,
chirality, 69, 76 47, 50, 52, 53, 54, 73, 74, 103, 107, 121, 123,
chitin, xi, 39, 237, 242, 243 175, 184, 221, 257
Chitosan, 257, 258, 259 comprehension, 180, 181, 190
chloroform, 15, 104, 265 computed tomography, 326
chlorophyll, viii, 5, 16, 36, 72, 79, 81, 82, 86, 87, 89, computer, 135, 161
90, 150 conceptualization, 182
chloroplast, viii, 6, 13, 79, 81, 89, 144 conditionally essential, 28
cholesterol, 9, 39, 99, 136, 139, 141, 151, 213, 225, conditioning, 149
226, 278, 281, 282, 284, 285, 292, 295 conference, 215
chopping, 203 configuration, 57, 60, 200, 261, 264
chromatographic technique, 132 confounders, 328, 329
chromatography, 17, 19, 20, 49, 53, 151, 159, 301, conjugated dienes, 103, 104, 275, 286, 290
302, 316 conservation, 145, 301
chronic diseases, viii, ix, 11, 40, 42, 47, 93, 108, 114, constituents, 41, 44, 272, 274, 283, 294, 301
128, 138, 196, 201, 206, 214, 216, 218, 219, 240, construction, 150
274, 298, 299, 314 consumers, x, 199, 200
cigarette smoke, 109, 138, 142 consumption, ix, xii, 1, 2, 3, 8, 10, 11, 14, 16, 24, 37,
circulation, 13, 114, 186, 214, 263 41, 42, 43, 44, 102, 128, 133, 135, 140, 145, 204,
City, 79 206, 207, 208, 210, 216, 226, 232, 235, 236, 272,
classes, vii, 18, 35, 44, 239, 261 273, 274, 276, 277, 283, 284, 289, 291, 298, 317,
classification, 128, 129, 144, 230 322, 327, 328, 332
cleavage, 7, 8, 9, 12, 57, 65, 98, 124, 153 consumption frequency, 1
climate(s), 5, 7, 91 contamination, 148
clinical trials, 206, 274, 286, 287 Continental, 91
cloning, 74 contrast sensitivity, 211
CO2, 16, 31, 33, 147, 150, 159, 265, 308 control group, ix, 165, 285
coatings, 30 controlled exposure, 235
cocoa, 283 controversial, 95, 180, 190, 276, 277

Index 341
COOH, 299
cooking, 14, 30, 36, 131, 202, 203, 204, 262, 264,
D
300
damages, 36, 156, 210
cooling, 99
dandruff, 257
cooperation, 51, 64, 133
database, 47
copolymer, 242
deaths, 274
coronary artery disease, 43, 47, 114, 120, 141, 288
decay, 231
coronary heart disease, 47, 136, 141, 213, 225, 284,
decomposition, viii, 81, 89, 94, 100, 101, 103, 105
286, 291, 294, 295, 317
defects, 328
corpus luteum, 313
defence, 134, 231
correlation, xii, 10, 22, 40, 43, 213, 232, 271, 272,
defense mechanisms, 10, 95
273, 276, 330
deficiency(s), 8, 209, 212, 330, 331
cortex, 211
degradation, 3, 6, 7, 13, 14, 16, 17, 36, 51, 97, 98,
cortical bone, 321
103, 235, 274, 313
corticosteroids, 250
dehydration, 12, 14, 31
cosmetic(s), 7, 16, 158, 233, 238, 243, 245, 246, 247,
denaturation, 14
248, 249, 251, 253, 254, 264
deoxyribose, 313, 318
cost, x, 17, 19, 20, 22, 97, 147, 150, 156, 157, 165,
deposition, 175, 176, 179, 186, 192
166, 169, 175, 177, 178, 181, 253, 265
deprivation, 146, 147, 162, 235
cotton, 265
depth, xi, 229, 232
covering, 37, 177, 182
derivatives, xi, 5, 38, 44, 56, 76, 91, 94, 140, 155,
critical period, 81
237, 301, 316, 330
crop(s), 43, 81, 90
dermatology, 161
cross sectional study, 141, 225, 226, 334
dermis, 185, 238
CRP, 276, 284
destruction, 36, 89, 93, 95, 108
Cryptoxanthin, vi, xiii, 50, 131, 202, 300, 312, 319,
detectable, xii, 214, 269, 271
320, 321, 322, 323, 324, 325, 326, 328, 330, 332,
detection, 15, 31, 32, 34, 76, 99, 202, 236
333, 334
detoxification, 89, 115, 194
crystal structure, 73
developed countries, 94, 111, 153
crystalline, 67, 211, 242
developing countries, 2, 8, 263
crystallization, 15, 49
diabetes, 215, 240, 246, 263, 266, 267, 278, 281,
crystals, 265
293, 298
CSF, 324, 325
diabetic patients, xii, 262
cultivars, 43
diatoms, 144
cultivation, xi, 145, 147, 149, 150, 155, 157, 158,
dienes, 275
262
dietary fat, 131, 145, 203, 206, 263
cultivation conditions, 145
dietary intake, viii, 41, 93, 128, 137, 153, 200, 203,
culture, 117, 136, 147, 148, 150, 151, 154, 158, 159,
204, 207, 209, 210, 213, 216, 272, 274, 275, 287
206, 207, 219, 308, 322, 324, 325, 331, 332
dietary supplementation, 13, 154, 212, 218, 235, 274
culture conditions, 150
differential scanning calorimetry, 119
culture medium, 151, 308
diffusion, 16, 104, 107, 124, 131, 175, 206, 230, 246,
curcumin, 301, 314, 316, 321
263
cure, ix, 127, 263, 287
diffusion process, 16, 206
CVD, xii, 212, 213, 215, 271, 272, 273, 274, 275,
digestibility, 207
276, 277, 278, 282, 283, 284
digestion, 16, 230
cycles, 147, 183
digestive enzymes, 39
cyclophosphamide, xi, 262
dilation, 284
cysteine, 109
dimorphism, 184
cytochrome, 47, 110
direct action, 108
cytokines, 153, 251, 289
direct measure, 22
cytoplasm, 144
disability, 330
cytotoxicity, 308
discrimination, 152, 176
discrimination tasks, 152

342 Index
discs, viii, 79, 82, 87, 88 electron(s), x, 3, 5, 20, 65, 81, 93, 94, 95, 96, 100,
diseases, vii, ix, 2, 3, 8, 9, 11, 40, 41, 42, 69, 93, 112, 102, 104, 117, 123, 129, 229, 231, 235, 239, 278
127, 128, 133, 135, 143, 151, 153, 181, 199, 200, electron paramagnetic resonance, 231, 235
201, 207, 209, 214, 215, 216, 238, 241, 242, 246, electrophoresis, 324
255, 263, 267, 273, 282, 287, 298, 299, 330 ELISA, 298, 308, 310
dislocation, 309 elongation, 301
disorder, 107, 133, 213, 214, 215 elucidation(s), vii, 49, 56, 75
dispersion, 12 e-mail, 271
dissociation, 311 emission, 81, 89
distress, 151 emphysema, 215
distribution, 3, 5, 12, 15, 45, 46, 107, 116, 120, 123, EMS, 147
144, 171, 183, 184, 218, 223, 231, 234, 249, 251, emulsions, 119, 245, 253
313, 330 encoding, 153, 155, 248
diversity, 17, 192, 299 encouragement, 138
DNA breakage, 270 endocrine, 176
DNA damage, 9, 10, 24, 27, 28, 42, 108, 109, 110, endothelial cells, xii, 117, 272, 273, 277, 279, 280,
113, 115, 117, 119, 123, 141, 151, 284 281, 289, 292
DNA polymerase, xii, 297, 298, 299, 301, 315, 316, endothelial dysfunction, xii, 271, 273, 277, 281, 282,
317, 318 288, 293, 294
DNA repair, xii, 297, 301, 318 endothelium, xii, 226, 272, 273, 275, 277, 279, 291
DNA strand breaks, 160 endurance, 233, 235
domain structure, 112 energy, xi, 5, 43, 81, 89, 94, 96, 97, 98, 104, 111,
domestic markets, 264 144, 175, 177, 178, 237, 238, 239, 241, 268, 278,
dominance, 154 311, 312, 313, 328
donors, 96 energy input, 104
dosage, 152, 282, 284 energy transfer, 96
dose-response relationship, 213 engineering, 251
dosing, 188 England, 139, 140, 193
double blind study, 212 environment(s), xi, 10, 41, 95, 97, 105, 114, 116,
double bonds, 5, 6, 20, 36, 89, 97, 99, 101, 102, 108, 151, 167, 182, 237, 241, 246, 247
111, 129, 153, 200, 201, 242, 262, 267, 268, 299, environmental conditions, xi, 147, 154, 158, 180,
311, 313 229, 231, 246
down-regulation, 277, 279 environmental factors, 190
Drosophila, 302, 316, 317 environmental influences, 231, 232
drugs, 7, 314 environmental stimuli, 144
drying, 7, 14, 30, 150, 154 environmental stress, 180, 232
dyes, xi, 5, 7, 262, 268, 269 enzymatic activity, 81
dynamism, 185 enzyme(s), viii, 3, 6, 7, 9, 12, 16, 32, 40, 41, 42, 47,
dyspepsia, 142 49, 57, 60, 61, 74, 96, 109, 110, 134, 136, 137,
142, 152, 153, 154, 177, 191, 211, 213, 215, 268,
278, 281, 284, 288, 293, 294, 298, 302, 304, 305,
E 306, 307, 308, 315, 316, 318, 324, 325
enzyme-linked immunosorbent assay, 298, 308
earthquakes, 81
epidemic, 317
Eastern Europe, 317
epidemiologic, 27, 45, 125, 141, 208, 209, 219, 242,
ECM, 242, 253
288
ecology, 158, 196, 197
epidemiologic studies, 27, 208, 209, 242, 288
edema, 309, 310, 317
epidemiology, 115, 290
edible mushroom, 130
epidermis, 94, 110, 184, 188, 230, 231, 253
editors, 219
epigenetic modification, 155
egg, 10, 37, 43, 118, 127, 130, 139, 183, 196, 202,
epinephrine, 226
205, 218, 299, 313
epithelial cells, 9
elderly population, 219
epithelium, 10, 210, 211
electric field, 7, 26

Index 343
epoxy groups, 6, 57 fermentation, 38

EPR, 99, 119, 122, 123, 124 fetal development, 8
Epstein-Barr virus, 70, 77 fiber(s), 9, 13, 111, 116
equilibrium, 248, 286 fiber content, 9
equipment, 150 fiber membranes, 116
erythropoietic protoporphyria, 137, 152 fibroblasts, 110, 142, 161, 252, 253
esophageal cancer, 207 fidelity, 315, 316
esophagus, 9, 42 filters, 43, 94, 210, 238, 247
ester, 12, 13, 64, 65, 70, 133, 261, 264, 282, 313 filtration, 154
estrogen, 141, 153, 220 financial, 90, 114, 156, 314
ethanol, 15, 32, 81, 150, 265 financial support, 90, 114, 156
ethers, 19 Finland, 207, 215
ethyl acetate, 15, 19, 266 fish, ix, 2, 130, 154, 165, 166, 167, 169, 170, 282,
EU, 253, 258 294, 305
eukaryotic, 301, 315, 317 fish oil, 282, 294
Europe, 256, 288, 290 fitness, 178, 179
evaporation, 265 flank, 184
evolution, 157, 174, 177, 190, 196 flavonoids, 2, 11, 96, 107, 161, 239, 263, 301, 318,
examinations, 328 321
excision, 298, 313, 318 flavor, 7
excitation, 5 flexibility, 180, 245, 253
exciton, 76 flight, viii, 79, 81, 89
excitotoxicity, 160 flocculation, 154
exclusion, 7 flour, 264
excretion, 14 flowers, 2, 54, 74, 128, 130, 263
exercise, 213, 226, 233, 235 fluid, 16, 33, 106, 112, 150, 159, 226, 265
exonuclease, 317 fluid extract, 16, 33, 150, 159, 265
experimental condition, 101, 279 fluorescence, 230
experimental design, 182, 190 folate, 222, 318
exploitation, 150 folic acid, 209, 317
exposure, 3, 6, 7, 10, 11, 12, 15, 17, 29, 110, 126, follicle, 176
127, 137, 144, 147, 149, 152, 154, 161, 191, 230, food additive(s), vii, 5, 22, 130
238, 241, 277 Food and Drug Administration (FDA), 29, 154
external environment, 151, 166 food chain, 25
extinction, 133 food industry, 7, 17, 23
extraction, 14, 15, 16, 17, 22, 31, 32, 43, 87, 89, 132, food intake, 213
150, 151, 154, 156, 159, 211, 265, 266, 281 food production, ix, 143, 145
extracts, 7, 33, 38, 46, 49, 71, 131, 139, 150, 152, food products, 2, 3, 7, 22, 47, 130, 131, 200
261, 264, 265, 266, 269 force, 178, 311
Ford, 288
formula, 132, 154, 245
F fovea, 111, 210
fractures, 320, 330, 331
families, xii, 297, 301, 303, 313
fragments, 324
family functioning, 75
France, 183
fasting, 206, 213, 285
free radicals, ix, x, 3, 9, 11, 39, 40, 43, 93, 95, 96,
fat, x, 8, 12, 16, 127, 131, 145, 176, 184, 185, 206,
99, 100, 104, 106, 107, 109, 116, 119, 126, 128,
229, 230, 282, 287, 293
151, 156, 210, 225, 229, 230, 231, 233, 235, 241,
fat soluble, 127
268, 272, 278
fatty acids, viii, 11, 16, 44, 49, 70, 79, 81, 89, 90,
freedom, 106
104, 159, 267, 283, 301, 313, 317
freezing, 16, 80, 150
feces, 14
freshwater, 125, 147
female rat, 269, 325, 331
friction, 16
femur, 320

344 Index
fucoxanthin, xii, 10, 38, 113, 144, 145, 152, 158,

161, 297, 308, 321
H
functional food, 30, 320, 321
H. pylori, 137
fungi, x, xi, 35, 38, 39, 44, 173, 174, 237, 248, 261,
habitat, 197
268, 287
hair, 40, 259
fungus, viii, 36, 82
half-life, 97
harmful effects, 95, 111, 116
G harvesting, 5, 144, 154, 156
Hawaii, 221
GAO, 256, 257 hazards, 223
gastrointestinal tract, 204 headache, 263
gel, 17, 324 healing, 39
gelation, 243 health condition, 143
gene expression, xiii, 40, 111, 113, 133, 136, 139, health effects, ix, 100, 128, 274
141, 144, 155, 222, 258, 273, 289, 292, 319, 322, health researchers, 271
323, 324, 325, 330, 333, 334 health status, 186, 189, 190
gene promoter, 162 heart attack, 136, 213
genes, 90, 111, 153, 155, 162, 166, 197, 267 heart disease, 93, 108, 218, 266, 267, 275, 290
genetic disease, 178 height, 83, 86, 150
genetic engineering, 145 Helicobacter pylori, 137, 142
genetic factors, 8, 12 helium, 64
genetics, 123 heme, 152
genomic stability, 27, 116 heme oxygenase, 152
genomics, 158 hemisphere, 187
genus, 38 hepatocarcinogenesis, 11, 29
Germany, 166, 215, 229 hepatocytes, 12
global warming, 81 hepatotoxicity, 77
glucocorticoids, 180 herbal medicine, 128, 133, 134, 138
glucose, xii, 96, 251, 262, 266, 267, 269, 270, 308, heterogeneous systems, 104
322, 325 hexane, 15, 22, 104, 117, 150, 265
glucoside, 121 high density lipoprotein, 12, 136
GLUT, 267 history, 71, 80, 81, 90, 192, 194, 217
glutamate, 152, 160 HIV-1, 304, 306
glutamine, 308 homeostasis, xii, xiii, 160, 180, 266, 269, 272, 273,
glutathione, 96, 110, 112, 115, 214, 227, 280, 284 319, 320, 321, 331
glycerol, 38, 47, 48, 149, 302 homocysteine, 226
glycine, 149 honesty, 174, 177, 180, 181, 183, 189, 191
glycol, 315 hormone(s), x, 42, 58, 141, 166, 174, 179, 180, 181,
glycosaminoglycans, 253 189, 190, 193, 219, 286, 322
gravity, 151 hormone levels, 181, 190
green alga, 57, 130, 142, 158 host, 125, 178, 181, 183, 188, 193, 194
growth, viii, 8, 9, 10, 40, 44, 54, 79, 85, 86, 113, hue, 185
122, 128, 133, 136, 145, 147, 148, 149, 150, 153, human body, xi, 12, 37, 108, 130, 135, 237, 238, 299
154, 155, 157, 158, 162, 170, 171, 207, 220, 241, human brain, 215
259, 273, 279, 308, 320, 321, 331, 333 human genome, 301
growth factor, 153, 220, 279, 320, 333 human health, x, 1, 2, 8, 9, 10, 22, 36, 42, 44, 45, 46,
growth hormone, 153 47, 114, 116, 123, 128, 138, 139, 141, 156, 157,
growth rate, 149 196, 199, 200, 201, 202, 215, 216, 218, 219, 220,
Guangdong, 169 222, 225, 226, 227, 262, 268, 287, 288, 299, 300,
Guangzhou, 165, 166 314, 315
guidance, 11 human immunodeficiency virus (HIV), 41, 109, 136,
guidelines, 155, 274, 298, 309 298, 304, 306
human neutrophils, 107

Index 345
human skin, x, 116, 145, 160, 229, 230, 231, 232, immunoglobulin, 136
233, 234, 235, 236, 238, 254 immunomodulation, 40, 284, 290
human subjects, xiii, 285, 295, 319, 320, 328, 330 immunomodulator, 128
Hunter, 221, 222 immunomodulatory, xii, 113, 262
hydrazine, 29 immunostimulant, 178, 182
hydrides, 65, 66, 76 immunosuppression, 110, 137, 181, 194, 195
hydrocarbons, vii, 4, 94, 129, 144, 201, 261 improvements, 150, 154, 214, 281, 286
hydrogen, xi, 44, 81, 89, 100, 101, 104, 120, 129, in vivo, viii, xi, xii, xiii, 8, 24, 39, 41, 42, 70, 94, 95,
175, 262, 265, 312 108, 109, 110, 113, 116, 122, 123, 141, 160, 196,
hydrogen peroxide, xi, 81, 89, 101, 120, 262 229, 230, 234, 235, 236, 247, 269, 272, 273, 274,
hydrogenation, 3, 36, 299 275, 279, 280, 288, 292, 298, 309, 314, 325, 326,
hydrolysis, 7, 12, 60 331, 334
hydroperoxides, viii, 69, 94, 101, 105, 226, 232 incidence, 2, 10, 41, 45, 152, 159, 208, 214, 215,
hydrophilicity, 243 216, 222, 275, 283, 294
hydrophobicity, 106, 243, 311 independence, 176
hydroxide, 268 India, 127, 138, 145, 263
hydroxyl, xi, 57, 65, 69, 70, 94, 95, 105, 106, 240, individuals, x, 8, 10, 41, 151, 174, 177, 178, 179,
262, 272, 311, 313 180, 186, 189, 190, 203, 209, 283, 284, 285, 286,
hydroxyl groups, 69, 70, 105, 106, 240, 311, 313 294, 326, 328, 334
hypercholesterolemia, 289 induction, 9, 88, 89, 122, 286, 289, 298
hyperemia, 284 industrial processing, 43
hyperglycemia, 266, 267 industry(s), x, 7, 16, 41, 154, 155, 159, 199, 216,
hyperinsulinemia, 267 242, 263, 264
hyperlipidemia, 267, 295 infarction, 124, 225, 317
hyperplasia, 110 infection, x, 137, 142, 174, 178, 180, 181, 186, 188,
hypertension, 214, 226, 283, 284, 294 189, 191, 194, 196, 235
hypocotyl, 269 infertility, 214
hypothesis, 42, 112, 114, 153, 177, 180, 181, 185, inflammation, xi, xii, xiii, 111, 119, 137, 141, 152,
186, 189, 190, 191, 193, 195, 210, 215, 225, 267, 229, 231, 235, 266, 267, 271, 272, 273, 275, 277,
273, 278, 279, 301 279, 283, 284, 285, 286, 288, 289, 290, 291, 294,
295, 298, 299, 308, 309, 311, 313, 314
inflammatory disease, 273, 282, 308
I inflammatory mediators, 278
inflammatory responses, 273, 316, 318
ICAM, xii, 271, 273, 277, 291
information processing, 246, 248
ideal, 249
infrared spectroscopy, 22, 51
identification, 5, 14, 16, 17, 19, 20, 22, 98, 116, 120,
ingest, 30, 36
133, 154
ingestion, vii, 12, 22, 107, 145, 152, 213, 218, 266,
identity, 20, 33, 177
269
illumination, 152
ingredients, xi, 156, 238, 239, 242, 244, 245, 246,
images, 183, 187
247, 248, 249, 251, 253
immersion, 14
inheritance, 170
immune activation, 191, 196
inhibition, xii, 9, 40, 70, 75, 89, 95, 115, 122, 133,
immune defense, 178, 180
136, 140, 141, 151, 153, 160, 219, 220, 259, 278,
immune function, 178, 191, 194, 195
279, 292, 297, 298, 299, 305, 307, 309, 311, 316,
immune modulation, 42, 298
324
immune response, x, 8, 11, 39, 174, 178, 179, 181,
inhibitor, xii, 120, 135, 141, 149, 273, 278, 280, 297,
186, 188, 189, 190, 191, 194, 247, 295
298, 301, 302, 307, 309, 311, 313, 314, 315, 318,
immune system, ix, x, 2, 8, 10, 40, 128, 136, 178,
321, 322, 324
181, 190, 199
initial state, 293
immunity, 39, 41, 109, 133, 138, 174, 178, 180, 181,
initiation, 29, 94, 103, 113, 217, 315
186, 189, 195, 246
injury(s), 137, 151, 230, 246, 275, 277, 281, 292,
immunization, 26
293, 328
immunodeficiency, 41, 306

346 Index
insects, 2, 183 kidney, 13, 41, 102, 110, 217, 263, 270
insomnia, xi, 229 kinetic studies, 14
insulin, xii, 220, 262, 266, 267, 333, 334 kinetics, xi, 31, 103, 104, 121, 229, 230, 231, 236
insulin resistance, 267 KOH, 150
insulin sensitivity, 267
integration, 159
integrity, 238, 277, 320 L
integument, 176, 177, 185, 192, 196
lactic acid, 322, 325
interaction effect(s), 276
lactose, 38
intercellular adhesion molecule, 289
landscapes, ix, 143
interface, 106, 121
larvae, 188
interference, 22, 267
lateral sclerosis, 160
intermediaries, 3
Latin America, 265
internalization, 257
LDL, 9, 11, 12, 107, 114, 116, 117, 123, 133, 136,
intervention, 2, 11, 27, 41, 44, 45, 115, 141, 152,
145, 206, 213, 215, 225, 275, 276, 281, 282, 284,
207, 209, 212, 213, 214, 215, 222, 224, 225, 274,
285, 290, 293
281, 282, 283, 284, 285, 286, 287, 295
lead, 42, 81, 89, 93, 95, 100, 106, 108, 109, 110,
intestine, 41, 131, 151, 175, 206, 263
111, 145, 152, 155, 177, 209, 210, 238, 320, 323,
intima, 225, 291
324
intraocular, 140
lecithin, 104, 117
intravenously, 286
legs, 183, 184, 185, 189
investment, 191, 196
lens, 140, 209, 211, 212, 224
ionization, 20, 133
leprosy, 263
ions, 107, 157
leptin, 295
Iran, 139, 220
lesions, 223, 238, 282, 320, 328
iron, 8, 147, 150, 157, 290
leukemia, 153, 162
irradiation, 90, 104, 142, 151, 230, 231, 233
leukocytes, 69
irrigation, 86
Lewis acids, 65
islands, 183
liberation, 16
isolation, 22, 53, 55, 56, 74, 154, 316
life cycle, 5
isomerization, 3, 6, 7, 12, 15, 16, 17, 201, 261, 262,
lifetime, 89
268, 299
ligand, 153, 245, 257, 267, 316, 324, 333
isomers, 3, 7, 12, 15, 16, 18, 19, 20, 24, 32, 33, 50,
light-emitting diodes, 157
106, 125, 200, 201, 204, 208, 217, 221, 233, 262,
linear model, 167
264, 268, 299
lipid metabolism, xii, 262, 266, 269, 278
isoprene, 128, 239, 261
lipid oxidation, 83, 124, 241, 275
isotope, 14
lipid peroxidation, ix, xi, 24, 70, 91, 93, 95, 98, 99,
Israel, 145
101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
Italy, 183, 220, 237, 271, 291
115, 117, 121, 123, 124, 125, 127, 136, 138, 141,
142, 186, 223, 226, 240, 262, 268, 284, 285, 294
J lipid peroxides, 96, 100, 241, 280
lipids, ix, xi, 5, 7, 11, 13, 16, 49, 83, 93, 94, 95, 96,
Japan, 75, 79, 81, 160, 220, 297, 302, 308, 314, 328, 100, 101, 104, 105, 111, 112, 114, 121, 144, 176,
329 181, 182, 204, 207, 210, 234, 237, 238, 241, 262,
jaundice, 263 266, 267, 268, 272, 282, 284, 285
Jordan, 138 lipopolysaccharide, xiii, 292, 298, 308, 310, 324
lipoproteins, 11, 12, 13, 14, 94, 109, 113, 122, 125,
153, 161, 176, 206, 219, 225, 226, 240
K liposomes, 103, 107, 113, 115, 118, 121, 123, 124,
125, 126, 197
kaempferol, 321
liquid chromatography, 17, 20, 22, 32, 33, 34
keratinocytes, 152, 160, 232, 248, 253
Lithuania, 136
ketones, 65, 72, 76

Index 347
liver, 12, 41, 42, 102, 107, 108, 130, 131, 134, 136, matrix metalloproteinase, 152
137, 141, 142, 176, 184, 185, 192, 204, 206, 214, measles, 41
217, 230, 263, 267, 269, 270, 293, 315 measurement(s), 20, 34, 39, 99, 86, 87, 194, 197,
liver cirrhosis, 214 220, 230, 231, 287
localization, 107, 123, 125 meat, 264, 269
loci, 161 mechanical properties, 120
longevity, 286 media, 136, 207, 225, 291
Louisiana, 220 median, 211
low risk, 40, 274 medical, 181, 182, 253
low temperatures, 7, 15, 86, 265 medicine, 193, 263, 299
low-density lipoprotein, 107, 116, 118, 120, 140, Mediterranean, 183, 219, 274, 275, 290, 291
151, 206, 275 melanin, 183
lumen, 206 melanoma, 110, 126, 152, 153, 162
lung cancer, 9, 41, 45, 46, 71, 138, 151, 159, 160, melody, 258
208, 209, 221, 222, 318 melting, 200, 265, 298, 307
Luo, vi, 162, 165, 170, 171 melting temperature, 298, 307
lymph, 12, 14, 206, 230 membrane damage, ix, 98, 127
lymphatic system, 131, 176, 263 membrane permeability, 115, 267
lymphocytes, xi, 107, 108, 109, 156, 262 membranes, 7, 13, 39, 93, 98, 99, 105, 106, 107, 108,
lymphoid, 108 111, 112, 116, 117, 118, 121, 124, 125, 142, 240,
246, 255
memory, 199
M mercury, 102
mesenchymal stem cells, 320
machinery, 201, 262, 299
messenger ribonucleic acid, 277
macromolecules, 238
meta-analysis, 182, 197, 274, 284
macrophages, 278, 279, 290, 292, 308, 309, 324
Metabolic, 142, 306, 333
macular degeneration, 1, 2, 8, 10, 13, 23, 29, 43, 45,
metabolic pathways, 42
46, 47, 94, 98, 111, 115, 118, 119, 120, 123, 133,
metabolic syndrome, xii, 262, 276, 286, 291, 295
143, 152, 161, 200, 212, 223, 224, 225, 240
metabolism, xii, 3, 9, 14, 40, 42, 46, 95, 108, 141,
magnesium, 227
151, 153, 155, 170, 177, 181, 190, 194, 195, 204,
magnetic resonance, 20, 328
205, 208, 210, 216, 218, 232, 238, 262, 266, 267,
magnetic resonance imaging, 328
268, 275, 298, 320, 322, 326, 331
magnetic resonance spectroscopy, 20
metabolites, 145, 151, 154, 204, 209, 223
magnitude, 254
metabolized, x, 5, 14, 135, 174, 176, 203, 251
majority, 20, 70, 107, 273, 279
metabolizing, 40, 42, 133, 136, 137, 142, 293
malignancy, 332
metal ion, 101
malonedialdehyde, viii
metalloproteinase, 161
mammal(s), 182
metals, 7
man, 29, 217
metastasis, 113
management, 193, 214, 253, 287
methanol, 15, 18, 19, 97, 265, 266
manipulation, 156
methodology, 22, 150, 244
marine environment, 130
methyl group(s), 3
marine species, 149
mice, 11, 12, 41, 102, 134, 136, 137, 142, 152, 162,
marketing, 154
220, 269, 270, 278, 281, 294, 309, 313, 318, 332
marketplace, 253
microbiota, 248
marrow, 324, 328, 333
micrograms, 3
Marx, 30
micronutrients, 3, 207, 212, 220
mass, xiii, 14, 16, 20, 31, 32, 34, 38, 98, 104, 133,
microorganism(s), vii, viii, 2, 16, 17, 35, 36, 39, 74,
140, 150, 191, 329, 330
128, 130, 133, 200, 262, 272, 299, 315
mass spectrometry, 20, 32, 34, 98, 140
microscopy, 230, 231, 282
materials, 154, 243, 257, 301
Microsoft, 167
matrix(s), 2, 3, 7, 8, 12, 13, 14, 15, 16, 20, 22, 152,
microsomes, 102, 107
161, 204, 206, 238, 286, 320, 321, 323, 331, 333

348 Index
microstructure, 176
microwaves, 14, 265
N
migration, 277, 299
NADH, 80, 81
mineralization, 322, 323, 333
nano-crystals, 243
Ministry of Education, 114, 314
nanofibril-hyaluronan, xi, 237
misuse, 108
nanomedicine, 257, 258
mitochondria, 98, 243, 245, 278
nanometers, 242
mitogen, 279
nanoparticles, 243, 244, 245, 246, 247, 249, 250,
mitosis, 322
251, 252, 253, 256, 257
mixing, 147, 150, 154, 243
Nanostructures, 257
MMA, 23
nanotechnology, 253
MMP, 152
National Academy of Sciences, 123, 191
model system, ix, 94, 95, 103, 160
National Health and Nutrition Examination Survey
models, xiii, 93, 103, 108, 112, 117, 137, 182, 243,
(NHANES), 203, 276, 291
267, 280, 282, 293, 319, 325, 330
natural compound, xi, 55, 153, 237, 239, 242
moderate activity, 70
natural food, 200, 216
modernization, 253
natural gas, 81
modifications, 4, 114, 201, 262, 268
near infrared spectroscopy, 22
molasses, 38, 149, 157
necrosis, 241
molecular dynamics, 311
negative effects, 14, 188
molecular oxygen, 5, 57, 121, 272
negative relation, 326
molecular structure, 144, 311, 316
nematode, 188, 189, 195
molecular weight, 7
nerve fibers, 111
monomers, 112
Netherlands, 275
Montenegro, 95, 96, 104, 109, 121
neural development, 313
morbidity, 283
neurodegenerative diseases, 215, 227
Morocco, 199
neurogenesis, 318
morphology, 214, 244
neutral, 100, 104, 312
morphometric, 234
neutrophils, 107, 268, 270, 278, 292
mortality, 2, 9, 41, 42, 138, 141, 209, 213, 216, 222,
New England, 159, 160
225, 274, 275, 276, 283, 286, 288, 290, 291
New Zealand, 282, 293
mortality risk, 291
NH2, 274
Moscow, 235
nicotinamide, 80, 211
motor neurons, 160
nicotine, 234
MR, 216, 218, 220
NIR, 22, 32
mRNA, 161, 267, 277, 321, 322, 323, 324, 325
nitric oxide, xii, 69, 77, 268, 270, 289, 292
mtDNA, 316
nitrogen, 96, 100, 101, 146, 147, 148, 149, 163, 288
mucosa, 176, 186, 206
nitrogen compounds, 96
mucous membrane(s), 242
nitrogen dioxide, 100, 101
mucus, 40
Nitzschia, 149, 159
multiple factors, 10
NMR, 20, 98, 118, 132, 133, 265
multivariate analysis, 22
non-arable land, ix, 143
muscular dystrophy, 143
non-polar, 15, 18, 101, 106
mussels, 61
nonsmokers, 142, 160, 275
mutagenicity, xi, 262
non-smokers, 283, 285
mutant, viii, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
normal aging, 211
90, 147, 149, 157, 158, 160, 194, 317
normal development, 40
mutation(s), 81, 85, 88, 89, 90, 117, 160, 233, 313
North America, 9, 37, 202, 216, 265
mycelium, 38
Northern blot, 313
myocardial infarction, 43, 109, 114, 124, 275, 276,
nuclear magnetic resonance, 133, 265
283, 288, 290, 291, 294, 317
nuclear receptors, 13, 153, 266, 323
myocardial ischemia, 293
nuclei, 71, 313
myocardium, 286
nucleic acid, 307

Index 349
nucleotides, 301
nucleus, 323
P
null, 9
p53, 277
nutraceutical, 215
pachytene, 318
nutrient(s), 1, 8, 28, 138, 145, 147, 151, 160, 199,
Pacific, 81, 139, 269
202, 209, 210, 212, 216, 224, 278, 282, 285, 292,
pain, 328
300, 313, 315
pairing, 95
nutrient concentrations, 224
palm oil, 16, 113, 118, 131
nutrition, vii, 1, 27, 39, 47, 121, 140, 154, 156, 157,
pancreas, 207
158, 181, 197, 212, 224, 226, 236, 286, 287, 299
pancreatic cancer, 207, 220
nutritional status, 2, 14, 177, 186, 190
paprika, viii, 7, 49, 50, 52, 55, 57, 58, 60, 70, 72, 73,
74, 75, 76, 77, 130, 139, 141
O parallel, 105, 107, 178, 251
parasite(s), x, 174, 175, 178, 179, 180, 181, 186,
obesity, 11, 44, 145, 267, 269, 293 188, 189, 190, 193, 194, 195, 196
OCD, 17 parathyroid, 225, 327, 331, 332
OH, 95, 299, 330, 335 parathyroid hormone, 225, 327, 331, 332
oil, ix, 3, 12, 30, 32, 119, 127, 128, 130, 151, 154, parkinsonism, 316
157, 203, 263, 264, 266, 300 participants, 41, 101, 213, 276, 331
old age, 191 partition, 311
olive oil, 3, 12, 24, 151, 280 pasteurization, 7
omega-3, 283 pathogenesis, 114, 115, 213, 225, 233, 328
operations, 3, 6, 7, 151 pathogens, x, 174, 178, 181
optic nerve, 10 pathophysiological, 330
optical density, 28, 161 pathophysiology, 276, 286
optimization, 32 pathways, ix, 115, 117, 144, 145, 175, 179, 182, 189,
organ(s), 12, 13, 42, 72, 94, 108, 127, 130, 176, 206, 190, 195, 230, 257, 271, 279, 317, 324
215, 221, 332 PCR, 322, 324
organic solvents, 15, 16, 265 penicillin, 308
organism, x, 43, 95, 109, 174, 175, 181, 182, 190, peptic ulcer, 137
230, 238, 283 peptide, 332
osteoarthritis, 328 percolation, 265
osteoblastic, xiii, 225, 319, 320, 322, 323, 324, 325, periodontal disease, 215
326, 327, 330, 331, 333, 334 peripheral blood, 302
osteoclastogenesis, 324, 331 permeability, 16, 108, 151, 251
osteogenic factor, xiii, 319 permit, 22
osteoporosis, vii, xiii, 93, 108, 213, 214, 225, 298, peroxidation, ix, 11, 94, 98, 100, 102, 103, 104, 105,
319, 320, 325, 326, 328, 329, 330, 331, 332, 334, 110, 112, 120, 121, 122, 123, 125, 134, 140, 210,
335 235, 284
ovarian cancer, 162 peroxide, 80, 99, 104, 268
ovaries, 176 peroxynitrite, xii, 122, 272, 281
overweight, 284, 285, 295 petroleum, 81
ox, viii, 4, 35, 94, 120, 127, 128, 130, 136, 141, 144, pH, 36, 131, 149, 154, 202, 262, 281, 300, 302, 307
275, 276, 281 pharmaceutical, viii, 16, 36, 145, 159, 215, 253
oxidation products, 57, 97, 98, 103, 108, 116, 123, phenolic compounds, 96
141, 153, 155, 223 phenotype(s), 81, 88, 90
oxidative damage, ix, xi, 11, 106, 109, 110, 111, 112, Philippines, 263
114, 115, 120, 121, 124, 128, 130, 133, 140, 181, phosphate, 96, 225, 307, 313, 318
182, 186, 188, 196, 197, 207, 210, 211, 214, 215, phosphatidylcholine, 118, 124, 126, 249, 250, 252
237, 238, 241, 255, 262, 272, 285 phospholipids, 11, 107, 108, 112, 126, 134
oxidative destruction, 36, 202, 263, 300 phosphorous, 327
phosphorus, 147
phosphorylation, 273, 274, 279, 289

350 Index
photocarcinogenesis, xi, 237 pools, 13, 147

photodegradation, 97 population, 2, 6, 9, 22, 37, 43, 120, 122, 136, 180,
photodynamic therapy (PDT), 89 185, 190, 195, 196, 203, 209, 211, 212, 215, 216,
photolysis, 104, 122 220, 222, 224, 238, 268, 274, 275, 288, 290, 291,
photons, 104, 110, 111 292, 320
photooxidation, 91, 119, 137 population control, 120
photosensitivity, 137, 234 population density, 180, 190
photosensitizers, 104, 111, 121 Porifera, 75
photosynthesis, 36, 81, 89, 118, 144, 162 portal vein, 12
phototoxicity, 160 Portugal, 32, 173
physical properties, 100, 118, 200 positive correlation, viii, xiii, 93, 109, 215, 297, 309
physicians, 118 positive relationship, 40, 313
physicochemical properties, 33, 94, 243, 244, 245, potato, vii, 32, 35, 130, 203, 300
249 potential benefits, 283
Physiological, 161, 191, 192, 194, 195, 196 poultry, 154
physiological factors, 166, 190 predators, 180
physiology, 158, 170, 231 preparation, 8, 15, 16, 68, 89, 132, 302
phytochemicals, x, 28, 43, 127, 128, 199, 200, 202, preservation, 6
265, 274, 281, 298, 300 primate, 120
pigmentation, x, 110, 152, 165, 166, 169, 170, 171, principles, 32, 230
178, 180, 182, 185, 191, 193, 194, 238 probability, 11, 168
pilot study, 135, 140, 161, 224, 317 producers, xi, 147, 261
placebo, 142, 161, 212, 224, 283, 284, 285, 294, 295, professionals, 216
327 pro-inflammatory, xii, 272, 273, 279, 282, 293
plant growth, 91 project, 114
plaque, 282 proliferation, 8, 9, 11, 40, 42, 113, 119, 120, 133,
plasma levels, 120, 141, 274, 282 135, 141, 151, 208, 213, 219, 220, 225, 248, 279,
plasma membrane, 81, 267 295, 301, 313, 321, 322, 323, 333
platelet aggregation, 279 promoter, 70, 77, 278, 313, 317, 318
platelets, 279, 281 pro-oxidant/inflammatory, xii, 272, 277
playing, 102 propagation, 104
pleurisy, 263 prostate cancer, 27, 42, 45, 46, 109, 113, 119, 136,
point mutation, 90 141, 152, 153, 162, 204, 206, 207, 208, 218, 219,
Poland, 93 220, 221, 267
polar, 15, 17, 18, 65, 98, 103, 105, 106, 107, 121, prostate specific antigen, 136, 206
124, 125, 144, 204, 206, 278 prostatectomy, 46, 136, 141, 207, 220
polar groups, 105, 106 protection, x, 7, 10, 11, 27, 39, 40, 47, 94, 97, 108,
polarity, 11, 15, 17, 19, 98, 103, 104, 264 109, 113, 120, 126, 134, 135, 137, 138, 144, 145,
policy, 253 151, 152, 160, 161, 209, 210, 211, 215, 223, 229,
pollen, 51, 55, 331 230, 231, 232, 233, 235, 238, 273, 274, 277
pollutants, 230, 247 protective mechanisms, 145
pollution, 233 protective role, 26, 46, 111, 121, 135, 207, 213, 214,
polymer(s), 242, 243, 244, 245, 247, 249, 251, 252, 215, 226, 239, 274, 278, 281, 287
257 protein folding, 144
polymerase, 304, 306, 316, 317, 322 protein kinase C, 322, 324
polymerase chain reaction, 322 protein oxidation, 207, 214, 215
polymerization, 301 protein synthesis, 321
polymorphisms, 153 proteins, xi, xiii, 3, 13, 39, 70, 93, 95, 96, 100, 109,
polynucleotide kinase, 304, 306, 317 111, 112, 115, 125, 144, 153, 155, 181, 237, 238,
polyphenols, 107, 214, 239 241, 264, 272, 284, 293, 319, 322, 323, 325, 330,
polypropylene, 150 333
polysaccharide, 242, 243
ponds, 148, 158

Index 351
provitamin A, x, 8, 9, 12, 13, 27, 30, 32, 113, 135, reconditioning, 19

142, 144, 153, 199, 200, 203, 206, 208, 240, 264, recovery, 19, 151, 211, 231
273, 320, 321 rectum, 220
psoriasis, 235 red wine, 283
puberty, 275 regeneration, 101
public health, 8, 200, 320 regenerative capacity, 275, 291
pulp, vii, 35, 263 regulatory agencies, 216
purification, 20, 150, 317 rejection, 7
purity, 75, 303 relatives, 24
relaxation, xii, 272, 277
relevance, 271, 286
Q reliability, 162, 235
repair, 11, 28, 39, 117, 246, 247, 248, 298, 313, 314,
quality control, 159
318
quality of life, 22, 199, 215
replication, 301, 313, 315
quantification, 5, 15, 16, 17, 20, 22, 31, 34
reproduction, 40, 176, 186, 188, 197, 227
quantum dot(s), 257
requirements, ix, 13, 165, 166
quartile, 328
researchers, 2, 17, 103, 106, 150, 214, 266
Queensland, 143
reserves, 175
quercetin, 321
residues, 16, 302, 318
questionnaire, 208, 217, 276, 328
resistance, 6, 16, 70, 71, 77, 91, 104, 178, 179, 191,
quinone, 123
196, 219
resolution, 17, 19, 180, 181
R resource availability, 190
resources, 190
radiation, xi, 110, 111, 115, 117, 145, 149, 151, 152, respiration, 98, 124
156, 157, 158, 159, 160, 229, 233, 235, 238, 254, response, x, xii, 14, 42, 136, 153, 163, 174, 179, 180,
265 181, 186, 188, 191, 218, 221, 234, 243, 267, 272,
radical formation, 100, 235 273, 277, 289, 293, 307, 309, 314, 324
radical mechanism, 95 responsiveness, 178, 186, 195
radical reactions, 105, 121, 233 restrictions, 182
radicals, viii, xi, 3, 47, 94, 95, 96, 98, 99, 100, 101, resveratrol, 282, 294
102, 103, 104, 106, 109, 113, 114, 117, 119, 121, retail, 154
122, 126, 145, 193, 225, 229, 230, 231, 233, 235, retina, 10, 13, 28, 43, 46, 94, 98, 111, 112, 113, 120,
239, 248, 262, 268, 272, 278 135, 152, 161, 209, 210, 211, 223, 241
radio, 190 retinitis, 28
radius, 329 retinitis pigmentosa, 28
Ramadan, 318 retinol, 8, 12, 13, 27, 40, 41, 96, 128, 135, 141, 144,
Raman spectroscopy, xi, 22, 125, 161, 229, 230, 231, 162, 203, 214, 218, 220, 221, 291, 294, 318, 320,
234 321, 323, 331, 332
reaction mechanism, 60, 153 reverse transcriptase, 306
reactions, ix, 3, 6, 10, 15, 16, 57, 59, 94, 95, 98, 99, RH, 28, 101, 255
100, 104, 109, 110, 120, 136, 182, 313 rheumatoid arthritis, 3, 143, 215
reactive oxygen, x, xi, 42, 93, 95, 152, 225, 226, 229, riboflavin, 96
230, 237, 262, 268, 270, 278, 288, 292, 332 rings, 5, 6, 51, 57, 59, 61, 69, 179, 183, 184, 185,
reactive sites, 95, 96 186, 189, 311
reactivity, 95, 96, 98, 99, 102, 104, 126 risk factors, 11, 124, 208, 275, 276, 278, 282, 286,
real time, 121 290, 291, 295, 317
recall, 277 RNA, 3, 304, 306, 317, 322
receptors, 13, 267, 321, 323, 331, 334 rodents, 110
recognition, 41, 202, 214 room temperature, 309
recombination, xii, 297, 301, 313, 314 root(s), vii, 35, 263, 269
recommendations, 41, 298 roughness, xi, 152, 160, 229, 232

352 Index
routes, 58 silicon, 150

Royal Society, 119, 191, 192, 193, 195, 196, 197 silver, 113, 124
rubber, 150 simulation(s), 135, 243, 311, 312
rules, 253 Singapore, 294
runoff, 91 skeletal muscle, 267, 286
skeleton, 3, 36, 98, 129, 299
skin cancer, 110, 115, 117, 118, 122, 137, 152
S skin diseases, 230, 263
small intestine, 159, 206
safety, 237, 245, 246, 249, 285, 286
smoking, 11, 160, 208, 231, 234, 284
salinity, 145, 149
smooth muscle, 151, 292
salmon, 10, 36, 127, 130, 131, 154, 170, 200, 302,
smooth muscle cells, 292
304, 305, 317
snake venom, 266
salts, 65, 154
social behaviour, 190
saprophyte, 248
social environment, 180
saturation, 11, 145, 185
sodium, ix, 147, 165, 166, 167, 168, 169, 266
scaling, 154
sodium hydroxide, 266
scattering, 196
sodium taurocholate, ix, 165, 166, 167, 168, 169
scavengers, vii, xii, 93, 100, 105, 271
software, 311, 312
science, 25, 44, 253, 288, 299
soil type, 5
scientific understanding, 216
solid tumors, 258
SEA, 257
solubility, 93, 104, 112, 200, 251, 264
seafood, 242, 287
solution, 15, 99, 100, 103, 104, 114, 117, 118, 126,
sebum, 230, 231
200, 253, 261, 266, 309
secretion, 14, 40, 218, 231, 232, 279
solvents, 15, 16, 17, 19, 265
sedimentation, 154
South America, 264
seed, 263, 264, 266
Spain, 173, 183, 220
seedlings, 79
specifications, 150, 306
selectivity, 17, 18, 19, 218
spectrophotometry, 22, 236
selenium, 152, 160, 207, 209
spectroscopic techniques, 133
self-assembly, 76, 257
spectroscopy, x, 22, 69, 104, 229, 230, 233, 234,
semen, 214, 226
235, 236
sensitivity, xii, 140, 152, 162, 202, 212, 235, 253,
speculation, 144
262, 267, 276, 300
sperm, 214, 226
Serbia, 93, 114
spermatocyte, 318
serum albumin, 118, 307
spermatogenesis, 8
sex, 190, 197
spin, 99, 122, 124
sexual dimorphism, 185
spin labeling, 99
shape, 20, 125, 129, 208, 243, 245, 257, 320
sponge, 62, 64, 75
shellfish, 2, 127, 154
squamous cell carcinoma, 110
showing, vii, viii, 22, 79, 81, 85, 88, 104, 137, 180,
stability, vii, 3, 6, 7, 22, 24, 26, 29, 70, 96, 99, 104,
183, 184, 185, 207, 208, 231, 232, 266, 274, 313
121, 145, 193, 200, 233, 242, 245, 246, 247, 251,
shrimp, 139, 242
287, 289
side effects, 110, 123, 251, 253, 267
stabilization, 5, 96, 112, 185
signal transduction, 91, 111
stabilizers, 16
signaling pathway, 113, 308, 324
standard deviation, 167
signalling, 122, 144, 178, 189, 191, 193, 195, 230,
starvation, 146
279
state(s), xi, 5, 12, 50, 80, 81, 89, 95, 96, 97, 111,
signals, 40, 46, 123, 174, 175, 176, 177, 178, 179,
119, 121, 122, 141, 144, 157, 197, 225, 237, 239,
181, 182, 189, 190, 191, 193, 194, 195, 196, 197,
241, 242, 267, 268, 280, 325
233, 245, 246, 248, 253
steel, 150
significance level, 167
stimulant, 188, 239, 248
silanol groups, 18
stimulation, 9, 40, 151, 308
silica, 17, 33

Index 353
stock, 267 territorial, 183, 195

stomach, 43, 137, 208, 209, 220 test efficiency, 41
storage, 5, 7, 12, 13, 15, 19, 26, 36, 45, 205, 263, testing, 88
264, 320 testis, 302, 313
stress factors, 230, 231, 232, 235 testosterone, 179, 180, 181, 188, 190, 192, 194, 195,
stress response, 181 196
stressors, 180 tetrahydrofuran, 15, 19, 65
stretching, 22, 94 texture, 31
stroke, 114, 227, 240, 283, 286, 313, 317, 318 TGF, 321, 322, 323
stromal cells, 324 T-helper cell, 141
strong interaction, 105 therapeutics, 333
subgroups, 212 therapy, 91, 227, 241, 330
substitution, 104 thermal degradation, 116, 264
substrate(s), xii, 7, 38, 95, 153, 297, 302, 303, 304, thermal treatment, 7, 12
305, 306, 307 thrombosis, 279, 281, 292, 293, 295
sugar cane, viii, 38, 44 thrombus, 281, 292
sugarcane, 88 thymine, 315
sulfur, 91 thymus, 301, 307, 313, 316, 318
Sun, 141, 157, 222, 257, 258 tissue, viii, 3, 5, 6, 8, 16, 39, 70, 93, 98, 108, 111,
supplier, 302 113, 153, 155, 176, 185, 191, 204, 205, 206, 207,
suppression, xiii, 70, 117, 225, 297, 308 208, 210, 213, 217, 218, 219, 230, 233, 238, 242,
surface area, 3, 6, 166, 249 246, 249, 251, 263, 267, 273, 282, 289, 320, 321,
surface chemistry, 245 322, 331, 332
surveillance, 209 tissue homeostasis, 246
survival, 29, 144, 171, 188, 196, 276 TLR4, 277
susceptibility, 11, 15, 117, 148, 180, 182 TNF, 267, 273, 274, 277, 279, 280, 284, 289, 291,
sweat, 230, 231, 232, 234 293, 297, 298, 308, 309, 310, 317, 324, 333
Sweden, 136 TNF-alpha, 289, 291
swelling, 186 TNF-α, 273, 274, 277, 279, 280, 284, 308, 309, 310,
symmetry, 201, 262 324
symptoms, 214 tobacco, 137
syndrome, 28, 219, 276 tocopherols, 33, 109, 119, 140, 223, 224, 239, 263
synergistic effect, 11, 106, 124, 155, 197 toluene, 119
synthesis, viii, 2, 38, 39, 40, 49, 51, 64, 65, 73, 74, tonometry, 284
76, 146, 153, 225, 266, 273, 278, 292, 298, 301, tonsillitis, 263
316, 318, 321, 323 total cholesterol, 213, 278, 281, 282, 284
systolic blood pressure, 214, 285 toxicity, xi, 19, 245, 253, 262, 268
toxicology, 233
toxin, 316
T TPA, 298, 301, 309, 310, 313, 314
trade-off, 178, 179, 180, 181, 190, 191, 192, 194
tar, 138
traits, x, 174, 175, 176, 177, 178, 179, 180, 181, 184,
target, xi, 101, 219, 237, 238, 245, 249, 282, 313,
185, 186, 189, 190, 191, 193
314
transcription, 153, 162, 266, 322, 323
tartrate-resistant acid phosphatase, 325
transcription factors, 162, 266, 323
taxa, x, 173, 182, 183, 185
transcripts, 313
techniques, 2, 12, 14, 15, 17, 20, 21, 98, 104, 132,
transduction, 292
133, 265, 266
transformation(s), viii, 4, 49, 58, 68, 160, 163, 175,
technology(s), 2, 7, 20, 147, 148, 151, 157, 158, 253,
176, 177, 190
265, 299
transforming growth factor, 321, 333
telomere, 316
translocation, 122, 162, 274, 289
temperature, 6, 7, 18, 19, 33, 36, 80, 81, 99, 106,
transmission, 248
147, 149, 150, 158, 167, 170, 202, 262, 300
tension, 241

354 Index
transport, xi, 13, 39, 106, 139, 152, 175, 195, 200,
206, 218, 237, 238, 267
V
transportation, 11, 12, 101
vacuum, 15
treatment, xiii, 3, 9, 11, 16, 22, 80, 82, 141, 142, 147,
variables, 12, 25, 31, 190
149, 150, 153, 159, 167, 196, 199, 207, 212, 214,
variations, 108, 129, 149, 155, 156, 176, 184, 185,
219, 245, 250, 258, 263, 267, 273, 277, 279, 281,
197, 202, 232, 300, 330
282, 285, 287, 316, 324, 330
varieties, 12, 34
trial, 46, 118, 141, 160, 162, 212, 218, 220, 224, 285,
vascular cell adhesion molecule, xii, 271, 274, 289
294
vascular dementia, 215, 227
triggers, 241
vascular diseases, 114, 286, 287
triglycerides, 12, 14, 30, 285
vascular inflammation, xii, 271, 279
tumor(s), xiii, 10, 70, 71, 77, 113, 114, 133, 136,
vasodilation, 273, 289
137, 162, 207, 220, 258, 267, 273, 289, 297, 298,
VCAM, xii, 271, 273, 277
313, 317, 318, 324
vector, 302
tumor cells, 71, 136
vegetable oil, 16, 31, 151, 159, 264
tumor growth, 114
vehicles, 242, 251
tumor necrosis factor, xiii, 267, 273, 289, 297, 298,
vein, 108, 273, 277, 281
324
velocity, 276, 291, 298, 305
tumorigenesis, 209
vertebrates, 175, 197
tumours, 153
very low density lipoprotein, 12
turnover, 213, 248
vessels, 12, 280
type 2 diabetes, 263, 269, 282
virus infection, 193
tyrosine, 109
viruses, 230
viscosity, 19
U vision, ix, 8, 40, 43, 94, 127, 133, 135, 140, 153,
212, 224, 225
ultrasound, 16, 32, 150 visual acuity, 126, 135, 210, 212
ultraviolet irradiation, 235 vitamin B1, 96
United Kingdom (UK), 34, 197, 212, 215, 254, 255, vitamin B6, 301
256 vitamin C, 2, 11, 96, 109, 116, 142, 151, 160, 182,
United States (USA), 74, 82, 123, 133, 140, 191, 209, 212, 213, 226, 263, 282, 283, 317, 318, 328
203, 204, 215, 217, 218, 226, 259, 274, 290, 302, vitamin D, 330, 335
303, 308, 311, 312, 317 vitamin E, 2, 11, 45, 96, 107, 109, 117, 125, 140,
urban, 37 142, 152, 159, 207, 209, 211, 212, 214, 221, 222,
urea, 149 263, 281, 283, 293, 301, 317, 328
uric acid, 96, 182 vitamin K, 301
Uruguay, 222 vitamin supplementation, 294
US Department of Health and Human Services, 217 vitamins, 11, 31, 47, 96, 108, 109, 114, 116, 119,
USDA, 217 160, 212, 213, 214, 215, 217, 222, 223, 224, 226,
UV, xi, 20, 22, 27, 31, 39, 53, 65, 94, 103, 104, 110, 273, 289, 290, 292, 294, 301, 328, 329
111, 115, 116, 117, 119, 121, 122, 125, 132, 133, VLDL, 12, 145
137, 139, 145, 146, 149, 152, 157, 159, 160, 161, volatility, 19
185, 188, 196, 211, 231, 235, 237, 238, 247, 254, Volunteers, xi, 229, 327
255 vulnerability, 160
UV irradiation, 125
UV light, 39, 116, 137, 247
UV radiation, 27, 39, 110, 125, 235, 238
W
UV-irradiation, 103, 104, 117
Washington, 23, 26, 30, 138, 193, 226
UV-radiation, 94, 110
waste, 243
wastewater, 145

Index 355
water, xi, 6, 14, 18, 22, 40, 82, 86, 103, 105, 106,
113, 119, 125, 140, 147, 152, 167, 170, 242, 261,
Y
264, 265, 309, 311, 313
yeast(s), viii, 2, 36, 38, 44, 46, 170
wavelengths, 5, 65, 241
yield, viii, 38, 90, 94, 95, 148, 151, 154, 158, 265,
wellness, 158, 253
272
Western Australia, 148
yolk, 10, 127, 130, 205, 218, 300
Western countries, 274
wild type, 87
workers, 26, 41, 200, 209 Z
World Health Organization, 264
worldwide, 1, 8, 274, 298, 330 zinc, 212, 225, 324, 331
zinc sulfate, 324
ZnO, 246, 247
X
zooplankton, 130
xanthophyl cycle, xi, 237
xerophthalmia, 8
X-ray diffraction, 121
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Carotenoids in Herbal Medicine.

Chapter · January 2013

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BIOCHEMISTRY RESEARCH TRENDS

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Chapter VIII Sodium Taurocholate Optimum Amount of Blood Parrots

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Carotenoids as Bioactive Compounds

Aline Inácio Alves1, Afonso Mota Ramos1, Paulo César Stringheta1

Keywords: Bioactive compounds, pro-vitamin A, antioxidant, natural pigments

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antioxidant properties of carotenoids by deactivating free radicals and sequestrating singlet

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polymeric chain shortening or combinations thereof, resulting in a wide variety of structures

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Main Sources and Applications in Food Industry

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carotenoids, such as α-carotene, α- or β-cryptoxanthin, zeaxanthin, antheraxanthin and lutein-

Table 1. Structures, characteristics and main sources of carotenoids in food

Common name Characteristics Main sources

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The isomerization of trans-carotenoid compounds, the most abundant arrangement in

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Carotenoids and Health Benefits

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Figure 2. Symmetric and asymmetric cleavage of -carotene.

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a coordinated or synergistic manner, reinforcing the importance of variety in meal

Absorption and Bioavailability

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for micellization efficiency. In this way, techniques such as mechanical homogenization,

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