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HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS AND
FRUITS AND ITS TRADITIONAL IMPLICATIONS IN THERAPY INCLUDING

ALTERNATIVE MEDICINES
Dr. Subha Ganguly (Editor-in-Chief)
ISBN: 978 – 978 – 52231 – 8 - 7
SCIENCE AND EDUCATION DEVELOPMENT INSTITUTE, NIGERIA

HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS AND
FRUITS AND ITS TRADITIONAL IMPLICATIONS IN THERAPY INCLUDING
ALTERNATIVE MEDICINES
Dr. Subha Ganguly (Editor-in-Chief)

AICRP on Post Harvest Technology (ICAR), Faculty of Fishery Sciences, West Bengal
University of Animal and Fishery Sciences, 5, Budherhat Road, P.O. Panchasayar,
Chakgaria, Kolkata - 700 094, WB, India
Knowlegde for Global Development

© All rights reserved. No reproduction, copy or transmission of this publication may be
made without written permission.
This first edition published 2014 by

2 Church Avenue, Oke Eri Quarters
Oba Ile
P.O.Box 214, Akure
Ondo State
Nigeria
+2348122469297
ISBN: 978 – 978 – 52231 – 8 - 7
ii

Vision
To Become a Centre of Excellence Recognized Worldwide in Skill Development and
Research
Mission
To Be a Role Model of Academic Excellence in Science and Education
iii

BOARD
Abulude, F.O. (Nigeria) - President/CEO
LIST OF ADVISORY BOARD MEMBERS
Mr Balogun G. A. Sanni
Saag Chemical (Nig.) Ltd, 4 Sanni Way, Off Godwin Omonua, Off Banks Way, Isolo
Illasamaja, Lagos, Lagos State, Nigeria.
Prof. Mohammad S. Mubarak

Chemistry Department, University of Jordan, Amman-11942, JORDAN
Prof. T. T. Adebolu
Department of Microbiology, Federal University of Technology, Ondo State, Akure,
Nigeria
Prof. Francisco Torrens

Universitat de València, InstitutUniversitari de Ciència Molecular, Universitat de
València, Edificid'Instituts de Paterna, València, Spain
Hon. Niyi Jones Akinyugha

30B, Olufumilayo Str., Dideolu Estate, P.O.Box 4822K, Ikeja, Lagos, Nigeria
Prof. V. A. Aletor
Elizade University, Ilara Mokin, Ondo State, Nigeria
Prof. E. A. Aderinola
Department of Agricultural Economics, Federal University of Technology, Akure,
Ondo State, Nigeria
Mr. Sola Akitimehin

Akinrinaye Street, Ilesha Garage, Akure, Ondo State, Nigeria
iv

CONTENTS
CHAPTER ONE 1–5
HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS

AND FRUITS AND ITS TRADITIONAL IMPLICATIONS IN THERAPY
INCLUDING ALTERNATIVE MEDICINE : AN INTRODUCTION TO READERS
CHAPTER TWO 6 - 20
COMPARTMENT OF PLANT CELL - Md. Rageeb Md. Usman
CHAPTER THREE 21 - 26
DEVELOPMENT OF MOSQUITO REPELLENT FINISHES IN

KNITTED FABRICS USING Rosmarinus officinalis LEAVES
- Banupriya.J and Maheshwari.
CHAPTER FOUR 27 – 38
IN VITRO ANTIBACTERIAL ACTIVITY OF METHANOLIC – AQUA

EXTRACT OF Tetradenia riparia LEAVES - Anthoney Swamy T and
Ngule Chrispus Mutuku
CHAPTER FIVE 39 – 79
MEDICINAL PROPERTIES OF INDIGENOUS PLANTS USED

IN THE PREPARATION OF TRADITIONAL RICE BEVERAGE
“HANDIA” AMONG THE TRIBALS OF MAYURBHANJ DISTRICTS,
ODISHA, INDIA - Sujogya Kumar Panda, Laxmipriya Padhi and
Akshaya Kumar Bastia
CHAPTER SIX 80 – 125
USE OF PLANT EXTRACTS IN PLANT DISEASE MANAGEMENT:

ROLE AND SIGNIFICANCE OF CHARACTERIZATION OF
SECONDARY METABOLITES FROM HIGHER PLANTS IN
PHYTO-DISEASE MANAGEMENT - Enyiukwu, D N., Awurum,
A. N., Ononju, C. C and Nwaneri, J.

CHAPTER SEVEN 126 – 177
COMPREHENSION ON USE OF MEDICINAL PLANTS TO

CURE DIARRHEA AMONG THE TRIBALS OF MAYURBHANJ
DISTRICTS, ODISHA, INDIA
- Laxmipriya Padhi and Sujogya Kumar Panda
CHAPTER EIGHT 178 - 201
MAXIMUM LIKELIHOOD ESTIMATES IN RELIABILITY OF

TYPHOID FEVER DRUG IN THE RECOVERY OF TYPHOID FEVER
PATIENTS AT AKUSE (A CASE STUDY AT THE AKUSE
GOVERNMENT HOSPITAL) - Eric Boahen
vi

ACKNOWLEDGMENTS
The President/CEO wishes to thank members of staff of Science and Education
Development Institute, Nigeria for their selfless service in making this publication a
reality.
vii

PREFACE
The book aims towards providing the basic and fundamental information to the
researchers and scientists worldwide on the vast herbal and natural medicinal treasure
available to us derived from plants, herbs and fruits obtained from traditional
agricultural practices. This book is dedicated to the professionals of Agriculture,
Horticulture and Forestry Sciences and has been composed exclusively for providing
first-hand knowledge on the related issues for the development of science and
education.
SUBHA GANGULY
Editor-in-Chief
viii

CHAPTER ONE
HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS AND FRUITS AND ITS
TRADITIONAL IMPLICATIONS IN THERAPY INCLUDING ALTERNATIVE MEDICINE: AN
INTRODUCTION TO READERS
Subha Ganguly, Editor-in-Chief (FSEDInst)

AICRP on Post Harvest Technology (ICAR), Department of Fish Processing
Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery
Sciences, 5, Budherhat Road, P.O. Panchasayar, Chakgaria, Kolkata - 700 094, WB, India
ABSTRACT
A herbal immunomodulator is a substance which stimulates or suppresses the
components of immune system including both innate and adaptive immune
responses (Agarwal and Singh 1969). The modulation of immune system by
various medicinal plant products has become a subject for scientific
investigations currently worldwide.
KEY WORDS: Herbal immunomodulator, Medicinal plants
INTRODUCTION
Under Indian scenario, poultry industry has become a means for earning livelihood for
the economically distressed farmers in India due to its promising results in productivity
and National economy. Poultry rearing is currently the fastest growing industry in our
National livestock sector which is benefiting us from production and advantages in
prices along with provision of proteinacous food.
Mode of action in immunostimulation of different herbal extracts

Many herbal plant preparations are prescribed to strengthen host resistance (Thatte and
Dahanukar 1986). Many useful plants fall under this category. They exhibit
immunomodulatory activities. One such plant, Tinospora cordifolia, commonly called
„Guduchi‟ has been examined for its immunomodulatory properties. Guduchi means to
rejuvenate dead cells. It is widely used in veterinary folk medicine and has also been
claimed to be beneficial according to „Ayurveda‟ for the cure of jaundice, skin diseases,
diabetes, anemia, emaciations and various infections for its anti-spasmodic, anti-
inflammatory, anti-arthritic and anti-allergic properties (Chopra et al. 1982). It has also
been reported that it improves the phagocytic and bactericidal activities in patients
suffering from polymorphism in surgical jaundice (Thatte at al. 1989). Kolte et al. (2007)
studied the effect of feeding T. cordifolia in broiler birds which were immunosuppressed
with cyclophosphamide. They had found a significant rise in antibody titer against ND
virus with augmentation of inflammatory reaction to skin contact sensitivity test. Rege
Knowlegde for Global Development 1

et al. (1989) and Bishavi et al. (2002) have proved the hepato-protective effect of T.
cordifolia. Manjrekar et al. (1999) also found that aqueous extract of T. cordifolia is capable
of increasing leukocyte count in mice.
Also, Ocimum sanctum, commonly known as „tulsi‟ is also used in Ayurveda for various
ailments including treatment of allergies. The plant has been reported to evince
significant anti-stress properties. The beneficial effects of O. sanctum could therefore be
due to its direct or indirect effect on the immune system. O. sanctum has been reported
to modulate humoral immune response by releasing mediators for hypersensitivity
reactions (Kujur 2001; Krishnamohan et al. 1997; Kumar 2003).
Withania somnifera also fall in this category with many other useful plants. They exhibit
immunomodulatory activities. Withania somnifera (commonly called „Ashwagandha‟)
root extracts possess anti-estrogenic, adaptogenic, anti-cancer and anabolic activities
having beneficial effects in the treatment of arthritis, geriatric problems and stress. The
root of Asparagus racemosus (commonly called „Satavar‟) possess anti-diarrheal, anti-
ulcerative, anti-spasmodic, aphrodisiac, galactogogue and other properties and has
therefore gained its importance in Ayurveda, Siddha and Unani systems of medicine
(Nadkarni, 1954). It has been observed that feeding W. somnifera and A. racemosus dried
root powder significantly stimulates both humoral and cell mediated immune
responses in swiss albino mice by Kuttan and Kuttan (1992). W. somnifera and A.
racemosus extracts increase phagocytic activities of macrophages in vitro (Rege and
Dahanukar 1993). There have been studies on the immunomodulatory activities of W.
somnifera and A. racemosus in mice with myelo-suppression induced by
cyclophosphamide, azathioprim or prednisolone. Extracts of W. somnifera and A.
racemosus have also shown immunopotentiating effects in cyclophasphamide treated
mouse with ascitic sarcoma (Diwanay et al. 2004). Kalita and Dutta (1999) reported that
maternal antibody was persistently found in sera samples tested against ND virus
during the first week of age in broilers. This was attributed to transfer of natural passive
immunity in young chicks as demonstrated by Hellar (1975). Muruganandan et al.
(2001) reported the effects of ethanolic extracts of W. somnifera and A. racemosus on
humoral immune system which was assessed by humoral immune response and cell
mediated immune response in mice.
CONCLUSION
The use of various plant extracts and herbal fed additives in a specific dose during the
scheduled vaccination regimen may be helpful in obtaining higher protective antibody
against different infections including production and development of more effective cell
mediate immune response for protection against various bacterial, viral and other
diseases. Herbal formulation may be therefore recommended for use as positive
immunomodulator in normal and immunocompromized susceptible animals and birds.

REFERENCES
Agarwal S S, Singh V K. 1969. Immnomodulators: a review of studies on Indian
Medicinal Plants and Synthetic Peptides. PINSA. 65 (3-4): 179-204.
Bishavi B, Roychowdhury S, Ghosh S and Sengupta M. 2002. Hepatoprotective and

immunomodulatory properties of Tinospora cordifolia in CCl4 intoxicated mature albino
rats J. Toxicol. Sci. 27(3): 139-146.
Chopra R N, Chopra L C, Handa K D and Kapur L D. 1982. Indigenous Drugs of India. 2nd
edn. Dhur & Sons Pvt Ltd, Calcutta, India.
Diwanay S, Chitre D and Patwardhan 2004. Immunoprotection by botanical drugs in

cancer chemotherapy. J. Ethnopharmacol. 90(1): 49-55.
Hellar E.D. 1975. Res. Vet. Sci.18: 117 (Cited by Rao et al. 1987). Resistance of maternal
antibodies against Newcastle disease virus in chicks from immune parents and its effect
on vaccination. Indian J. Comp. Microbiol. Immunol. Inf. Dis. 8(3): 106-110.
Kalita D N and Dutta G N. 1999. Immunomodulatory effect of levamisole upon

Newcastle disease, pigeon pox and Mark‟s disease vaccination in broiler chicks. Indian
Vet. J. 76: 490-492.
Karnataka B C, Shukla S K, Kumar M and Dixit V P. 1993. Immunomodulatory effect of

levamisole on the antibody response to RD vaccination. Indian J. Vet. Med. 13(2): 48-51.
Kolte A Y, Siddiqui M F and Mode S G. 2007. Immunomodulating effect of Withania

somnifera and Tinospora cordifolia in broiler birds.
Krishnamohan A V, Reddy D B, Sarma B and John Kirubharan J. 1997. Studies on the

effects of levamisole against Newcastle disease virus in chicken. Indian J. Comp.
Microbiol. Immunol. Infict. Dis. 8: 1-6.
Kujur R T. 2001. Evaluation of certain immunomodulatory agents in countering

immunosuppressive effects of vaccine strain of infectious bursal disease virus in chicks.
M.V.Sc. thesis. Rajendra Agricultural Univ., Bihar, India.
Kumar P. 2003. Studies on comparative immunomodulatory effect of herbal preparation

and Vitamin E-Se in comparison to Levamisole in broiler chicks. M.V.Sc. thesis. Birsa
Agricultural Univ., Ranchi, India.

Kuttan G and Kuttan R. 1992. Immunomodulatory activity of a peptide isolated from
Viscum album extract. Immunol. Invest. 21: 285-296.
Manjrekar P N, Jolly C I and Narayan S. 1999. Comparative studies of the

immunomodulatory acivities of Tinospora cordifolia and Tinospora sinensis. Fitoterapia. 71:
254-257.
Muruganandan S, Garg H, Lal J, Chandra S and Kumar D. 2001. Studies on the

immunostimulant and anti-hepatotoxic activities of Asparagus recemosus root extract.
J. Med. Arom. Pl. Sci. 22: 49-52.
Nadkarni A.K. 1954. Indian Materia Medica, Bombay, Popular book Depot, 3rd edn., 1:
153-155.
Panda S K and Rao A T. 1994. Effect of levamisole on chicken infected with infectious
bursal disease (IBD) virus. Indian Vet. J. 71: 427-439.
Rege N.N and Dahanukar S.A. 1993. Quantitation of microbicidal activity of

mononuclear phagocytes : an in vitro technique. J. Postgrad. Med. 39(1): 22-25.
Rege N N, Nazareth H M, Bapat R D and Dhanukar S A. 1989. Modulation of

immunosuppression in obstructive jaundice by Tinospora cordifolia. Ind. J. Med. Res. 90:
478-483.
Renoux and Renoux. 1971. Mechanism of action of some immunomodulatory drugs

used in Veterinary Medicine. Adv. Vet. Sci. Comp. Med. 35: 43-49. 293
Soppi E, Lassila O, Vilijanen M K, Lehtonon O P and Eskole J. 1979. Clin. Exp. Immunol.
38: 609. (Cited by Chakraborty D and Chatterjee A. 1998. Studies on
immunomodulatory effect of Levamisole in Newcastle disease vaccinated chicks. Indian
J. Comp. Microbiol. Immunol. Infect. Dis. 19: 85-87).
Thatte U M and Dahanukar S A. 1986. Ayurveda and contemporary scientific thought.

Trends in Pharmacol. Sci. 17: 248-257.
Thatte U M and Dahanukar S A. 1989. Immunotherapeutic modification of experimental

infection by Indian medicinal plants. Phytothe. Res. 3: 43-49.

Vyas G P, Dholakia P M and Kathiria L G. 1987. Studies on immunomodulation by
levamisole along with vaccination in chicks against Ranikhet disease. Indian Vet. J. 64:
456-462.
______________________________________________________________________________
Suggested Readings:
Ganguly, S. and Prasad A. (2010) Role of plant extracts and cow urine distillate as
immunomodulator in comparison to levamisole – a Review. J. Immunol. Immunopathol.,
12(2): 91-94.
Prasad, Arun and Ganguly, Subha (2012) Herbal Immunomodulators. AV

Akademikerverlag GmbH & Co. KG, Saarbrücken, Germany with trademark LAP
LAMBERT Academic Publishing. ISBN 978-3-659-30401-9.
Ganguly, Subha (2013) A Handbook on Traditional Medicinal Plants, Herbs and Fruits
in Indian Agriculture and Forestry. 1st Edition. International E-Publication. ISBN 978-81-
927544-5-1. Official E-Book (Section: Agriculture & Forestry Sciences). Publication of the
International Science Congress Association, Indore, UP, India.

CHAPTER TWO
COMPARTMENT OF PLANT CELL
Md. Rageeb Md. Usman

Department of Pharmacognosy, Smt. S. S. Patil College of Pharmacy, Chopda,
Maharashtra, 2/899 SURMAZ Villa, KGN Colony, Chopda, Maharashtra, India.
A. PLANT TISSUE
Types of plant tissue
Meristematic tissue
Apical meristems
Lateral meristems
Intercalary meristems
Permanent tissue
Simple permanent tissue
Parenchyma
Collenchyma
Sclerenchyma
Complex permanent tissue

Xylem
Phloem
Protective tissue
Epidermis
Cork (phellem)
Meristematic tissue
Apical meristems
These are situated at the growing tip of the stems & roots, i.e., at shoot apex & root
apex. Apical meristems are also found at apices of the leaves. It brings about the
elongation of the root & stem. It results in increase in the height of the plant, which is
called primary growth.
Lateral meristems
These are found beneath the bark (cork cambium) & in vascular bundles of dicot roots
& stems (cambium).They occurs in thin layers. Cambium is the region which is
responsible for growth in thickness. It causes the organ (stem or root) to increase in
diameter & girth. This is called secondary growth.

Intercalary meristems
They are located at the base of leaves or internodes, e.g., Stem of grasses & other
monocots.
It produces an increase of length of organ.
Meristematic Tissue
Nature-cells of meristems divide continuously & help in increasing the length

girth of the plant. These cells show the following characteristics:
1. The cells of meristematic tissue are similar in structure & have thin cellulose
cell walls.
2. The meristematic cells may be spherical, oval, polygonal or rectangular in
shape.
3. The meristematic cells are compactly arranged & do not contain any
intercellular space between them.
4. Each meristematic cell contains dense or abundant cytoplasm & a single large
nucleus.
5. The meristematic cells contain few vacuoles or no vacuoles at all.
6. Occurrence-Meristematic tissues are growth tissues & are found in those
regions of the plant that grow. According to their position in the plant,
meristems are apical, lateral & intercalary.
7. Function-the main function of meristematic tissue is to continuously form a
number of new cells.
Permanent tissue
These tissues derived from the meristematic tissues but their cells have lost the power
of division & have attained their definite forms. Permanent tissues are classified into
two-simple & complex.

These tissues are composed of cells which are structurally & functionally similar. Thus,
these tissues are all made of one type of cells.

They are of three types
I. Parenchyma
II. Collenchyma
III. Sclerenchyma

The complex tissues consist of more than one type of cells. All this co-ordinate to
perform a common function. Complex tissues transport water, mineral salts (nutrients)
& food material to various parts of plant body. Complex tissues are of following two
types:
I. Xylem or wood
II. Phloem or bast
Xylem & phloem are both conducting tissues & also known as vascular tissues; together
both of them constitute vascular bundles.

1. Parenchyma
Living & possess the power of their division. The cells are rounded or isodiametric, i.e.,
equally expanded on all sides. The parenchymatous cells are oval, round, polygonal or
elongated in shape. The cell wall is thin & encloses a dense cytoplasm which contains a
small nucleus & surrounds a large central vacuole. In other words, parenchyma cells
have living protoplasm. Intercellular spaces are abundant
Occurrence
The parenchyma is widely distributed in plant body such as stem, roots. Leaves,
flowers & fruits. Thus, the parenchyma tissue is found in the soft parts of the plant such
as cortex of roots, ground tissues in stems & mesophyll of leaves. It is also distributed in
pith, medullary rays & packing tissue in xylem & phloem.
Functions
1. Parenchyma serves as a packing tissue to fill the spaces between other tissues &
maintain the shape & firmness of the plant due to its turgid cells.
2. Due to turgidity property, parenchyma forms the main means of support to the
stem of herbaceous plants.
3. The main function of parenchyma is to store & assimilate food. Parenchyma
serves as food storage tissue.
4. Transport of materials occurs through cells or cell walls of parenchyma cells.
5. Parenchyma cells are metabolically active their intercellular air spaces allow
gaseous exchange.

Parenchyma
2. Collenchyma
Nature-collenchyma tissue consists of living cells. It shows many of the features of
parenchyma but is characterised by the deposition of extra cellulose at the corners of the
cells. In collenchyma, intercellular spaces are generally absent. Collenchymas cells are
elongated in shape. They often contain a few chloroplasts.
Occurrence
The cells of collenchyma are located below the epidermis of dicotyledons stem &
petiole. These cells also occur in midribs of dicot leaves. Collenchyma is absent in
monocot stems, roots & leaves.
Functions
Collenchyma is a mechanical tissue; it provides mechanical support & elasticity. Thus,
collenchyma provides tensile strength with flexibility to those organs in which it is
found. It allows easy bending in various parts of a plant without actually breaking it.
When cells of collenchyma contain some chloroplasts, they manufacture sugar & starch.
Collenchyma

Sclerenchyma
Nature-sclerenchyma cells are dead cells & they are devoid of protoplasm. The walls of
cells of sclerenchyma are greatly thickened with deposition of lignin. Such cells are
called lignified. Due to excessive thickening of the wall of a sclerenchyma cells, its cell
cavity or lumen becomes nearly absent. The cells of sclerenchyma are closely packed
without intercellular spaces.
Occurrence
The sclerenchyma occurs in abundance either in patches or definite layers. They are
found in stems, roots, veins of leaves, hard covering of seeds & nuts. Sclereids form the
gritty part of the most of the ripe fruits & contribute hardness to the seed coat &
nutshells.
Functions
The sclerenchyma is mainly mechanical & protective in function. It gives strength,
rigidity, exibility & elasticity to the plant body &, thus, enables it to withstand various
strains.
Sclerenchyma

Xylem
Nature-xylem is a vascular & mechanical tissue. In other words, it is a conducting
tissue. Xylem is composed of cells of four different types like, tracheids; vessels
ortracheae, xylem parenchyma; xylem sclerenchyma. Except xylem parenchyma, all
other xylem elements are dead & bounded by thick lignified walls. Of these four types
of cells of xylem are most important cells are vessels. Vessels are very long tube-like
structures formed by a row of cells placed end to end. Tracheids are elongated cells
with tapering ends. They conduct water.
Functions

The main function of xylem is to carry water & minerals salts upward from the root to
different parts of shoots. Since walls of tracheids, vessels & sclerenchyma of xylem are
lignified, they give mechanical strength to the plant body.
Xylem
Phloem
Nature-like xylem, it contains tubes but has no mechanical function. Phloem is
composed of following four elements or cells like, sieve tubes, companion cells, and
phloem parenchyma& phloem fibres. Except for phloem fibres, phloem cells are living
cells.
Functions
Phloem transport photosynthetically prepared food materials from the leaves to the
storage organs & later from storage organs to the growing regions of the plant body.
Phloem Vascular Bundles with Xylem & Phloem
Protective tissue
Epidermis
It is usually present in the outermost layer of the plant body such as leaves, flowers,
stem & roots. Epidermis is one cell thick & is covered with cuticle. Cuticle is a water
proof layer of a waxy substance called cutin which is secreted by epidermal cells.
Cuticles possess variable thickness in plants such as it is thicker in xerophytic plants.

Cells of epidermis are elongated & flattened & do not contain any intercellular space
between them. Their inner contents are similar to parenchyma cells.
The main function of epidermis is to protect the plant from desiccation & infection. In
fact, cuticle of epidermis helps to reduce water loss by evaporation from the plant
surface as well as helping in preventing the entry of pathogens
Epidermis
Cork
As plants grow older, the outer protective tissue undergoes certain changes. A strip of
secondary meristems, called phellogen or cork cambium replaces epidermis of stem.
Cork cambium is a simple tissue having only one type of cells. The cells of cork
cambium are rectangular & their protoplasts are vacuolated & contain tannins &
chloroplasts. Cork cambium gives off new cells on its both sides, thus, forming cork on
the outer side & the secondary cortex or Phelloderm on the inner side. The layer of cells
which is cut by cork cambium on the outer side ultimately becomes several layered
thick cork or the bark of trees. Cells of cork are dead & compactly arranged without
intercellular spaces. The walls of cork cells are heavily thickened by deposition of an
organic substance, called suberin. Suberin makes these cells impermeable to water &
gases. The cork cells do not contain protoplasm but are filled with resin or tannins. In
case of onion bulb too, in the skin of onion the cell walls become thick & water proof
due to addition of suberin. Cork is protective in function. Cork cells prevent desiccation,
infection & mechanical
Cork

B. THE CELL
The elementary organs from which the body of the plant is constructed are the cells.
Most plants (all the more highly organized ones) consist of numerous cells. Among the
lower plants there are, however, many which arc formed of but single cells, some of
which assume the most manifold forms, branch abundantly, and, indeed, without being
in any manner divided by lateral walls, imitate a stem, leaf, and root.
Contains of the Cell

Cell-contents with the distinction already made the cell contents may be grouped into
two classes: (1) Protoplasmic, or those in which the life-processes of the plant, or cell,
are manifested, and (2) non-protoplasmic, or those which are the direct or indirect
products of the protoplast. The first class includes the protoplasm with its various
differentiated parts, and the second, the various carbohydrates (starches and sugars),
calcium, oxalate, aleurone, tannin, oil, and a number of other substances.
Cell Wall
It is rigid wall made up of cellulose, proteins, and carbohydrates
Function: boundary around the plant cell outside of the cell membrane that provides
structure and support
Protoplasm

Protoplasm occurs as a more or less semi fluid, slimy, granular, or foam-like substance,
which lies close to the walls of the cell as a relatively thin layer and surrounding a large
central cavity or vacuole filled with cell-sap. or it may be distributed in the form of
threads or bands forming a kind of network enclosing smaller vacuoles. Protoplasm
consists of two comparatively well differentiated portions: (1) Certain more or less
distinct bodies which appear to have particular functions and to which a great deal of
study has been given, as the nucleus and plastids, and (2) a less dense portion which
may be looked upon as the ground substance of the protoplast and which is now
commonly referred to as the cytoplasm. These differentiated bodies and the cytoplasm
are intimately associated and interdependent. The nucleus and cytoplasm are present in
all living cells and it is through their special activities that cell division takes place.
When in addition plastids are present, constructive metabolism takes place, whereby
complex substances are formed from simpler ones. Besides the nucleus and plastids
other protoplasmic structures are sometimes found embedded in the cytoplasm
Centrospheres
Small spherical bodies are associated with the nucleus and appear to be concerned in
cell division. There are in fact quite a number of minute bodies in the cytoplasm which
may be always present or only under certain conditions, and which are grouped under
the general name of microsomes or microsomata.
Chemically protoplasm is an extremely complex substance, but does not appear to have
a definite molecular structure of its own, being composed in large measure of proteins,
a class of organic compounds which always contain nitrogen, and frequently
phosphorus and sulphur. The molecule of the proteins is large and more or less
unstable, and hence subject to rapid changes and a variety of combinations, and it is to
these interactions that the vital activities of the plant are attributed Nucleus. The
nucleus consists of a ground substance in which is embedded a network composed of
threads containing a granular material known as chromatin, and generally one or more
spherical bodies called nucleoles, the while being enclosed by a delicate membrane. The
chromatin threads are readily stained by some of the aniline dyes, and are mainly
composed of nucleins (proteins) rich in phosphorus, which by some writers are
supposed to be essential constituents of the nucleus and necessary to the life of the
protoplast. Chromatin is constant in the nucleus and prior to cell division the threads
become organized into bodies of a definite number and shape known as chromosomes.
Plastids
The plastids or chromatoid hores form a group of differentiated protoplasmic bodies
found in the cytoplasm (Frontispiece) and are associated with it in the building up of
complex organic compounds, as starch, oil and proteins. The term chromatophore

means colour-bearer, but applies also to those plastids which may be colourless at one
stage and pigmented at another.
Hence we may speak of colourless chromatophores. According to the position of the

cells in which these bodies occur and the functions they perform, they vary in colour
three distinct kinds being recognized. (1) In the egg-cell and in the cells of roots,
rhizomes and seeds the plastids are colourless and are called leucoplastids. (2) When
they occur in cells which are more or less exposed to light and produce the green
pigment called chlorophyll, they are known as chloroplastid or chloroplasts. (3) In other
cases, independently of the position of the cells as to light or darkness, the plastids
develop a yellowish or orange-colored principle, which may be termed chlorophyll, and
are known as chromoplastids.
Chromo plastids.
Chloroplastid is found in all plants except Fungi and non-chlorophyllous flowering
plants, and chromoplastids in all plants except Fungi. Plastids vary in form from more
or less spherical to polygonal or irregular-shaped bodies, and they increase in number
by simple fission. They suffer decomposition much more readily than the nucleus, and
are found in dried material in a more or less altered condition.
Leucoplastids. The chief function of the leucoplastids is that of building up reserve

starches or those stored by the plant for food, and they may be best studied in the
common potato tuber, rhizome of iris, and the over ground tubers. The reserve starches
are formed by the leucoplastids from sugar and other soluble carbohydrates. The
chloroplastid occurs in all the green parts of plants. They vary from 3 to 11 /x in
diameter and are more or less spherical or lenticular in shape, except in the Algae,
where they are large and in the shape of bands or disks, and generally spoken of as
chromatophores. Chloroplastid are found in greater abundance in the cells near the
upper surface of the leaf than upon the under surface, the proportion being about five
to one. These grains upon close examination are found to consist of (1) a colourless
stroma, or liquid, in which are embedded (2) green granules; (3) colourless granules; (4)
protein masses; (5) starch grains; and (6) a membrane which surrounds the whole. The
green granules are looked upon as the CO2 assimilation bodies, the colourless grains are
supposed to assist in the storing of starch or in the production of diastase, the
conditions for these processes being directly opposite, i.e., when CO2 assimilation is
active, starch is stored, and when this process is not going on. As at night, diastase is
produced and the starch is dissolved. The protein grains may be in the nature of a
reserve material of the plastid and are also probably formed as a result of CO2 as
simulation. While the protoplasm has been termed by Huxley " The physical basis of
life," the chloroplastid has been spoken of as the mill which supplies the world with its
food, for it is by the process of photosynthesis that the energy of the sun is converted

into vital energy, and starch and other products formed, which become not only the
source of food for the plant itself, but also the source of the food-supply of the animals
which feed upon plants. It other words, horse-power is derived from the energy of the
sun which is stored by the chloroplastid in the plant.
Chromoplastids. In many cases, as in roots, like those of carrot, or flowers and fruits,
which are yellowish or orange colored, there is present a corresponding yellow
pigment, and to this class of pigments the name chlorophyll may be applied. Some of
these pigments, as the carotin in carrot, have lien isolated in a crystalline condition
Chromoplastids usually contain, as first pointed out by Schimper and Meyer, protein
substances in the form of crystal-like bodies; starch-grains may also be present. The
chromo plastids are variable in shape and in other ways are markedly different from the
chloroplastid. They are more unstable than the chloroplastid, and are formed in
underground parts of the plant, as in roots, as well as in parts exposed to the light, as in
the flower. Their formation frequently follows that of the chloroplastid, as in the
ripening of certain yellow fruits, such as apples, oranges, persimmons, etc.
Chromo Plastids
Plastid pigments
They are distinguished from all other colour substances in the plant by the fact that they
are insoluble in water and soluble in ether, chloroform and similar solvents. In fact they
are but little affected by the usual chemical reagents under ordinary conditions. Apart
from the difference in colour, the yellow pigment (chlorophyll) is distinguished from
the green (chlorophyll) by the fact that the latter is said to contain nitrogen, and also by
their difference in behaviour when examined spectroscopically, chlorophyll giving
several distinct bands in the yellow and orange portion of the spectrum, which arc
wanting in the spectrum of the yellow principle.
Non-protoplasmic cell-contents
The non-protoplasmic constituents of plants may be said to differ from the
protoplasmic cell-contents in two important particulars, namely, structure and function.

For convenience in considering them here, they may be grouped as follows : (1) Those
of definite form including (a) those which are colloidal or crystalloid, as starch and
inulin; (b) those which are crystalline, as the sugars, alkaloids, glucosides, calcium
oxalate (c) composite bodies, as aleurone grains, which are made up of a number of
different substances. (2) Those of more or less indefinite form, including tannin, gums
and mucilages, fixed and volatile oils, resins, gum-resins, oleo-resins, balsams, and also
silica and calcium carbonate.
Substances definite in form. Colloidal or crystalloid.

Starch is the first visible product of photosynthesis although it is probable that simpler
intermediate products are first formed. This substance is formed in the chloroplastid
and is known as assimilation starch. Starch grains are usually found in the interior of
the chloroplastid, but may attain such a size that they burst through the boundary wall
of the plastid, which latter in the final stage of the growth of the starch grain forms a
crescent-shaped disk attached to one end of grain, as in Pellionia. Starch is changed into
soluble carbohydrates by the aid of ferments and probably other substances, and in this
form is transported to those portions of the plant requiring food. The starch in the
medullary rays and in other cells of the wood and bark of plants is distinguished by
being in the form of rather small and nearly spherical grains. In rhizomes, tubers, bulbs
and seeds the grains are, as a rule, quite large, and possess more or less distinct
characteristics for the plant in which they are found.
Crystalline substances.
The sugars constitute a group of crystalline principles of wide distribution. They occur
in the cell-sap, from which by evaporation or on treatment with alcohol they may be
crystallized out. Quite a large number of distinct principles belonging to this class have
been recognized, of which the following may be mentioned
Dextrose
(grape-sugar or dextro-glucose) is found in sweet fruits, the nectarines of the flowers,
and stems and leaves of various plants. It crystallizes in needles and varies in amount
from i to 2 per cent, (in peaches), to 30 per cent
Sucrose
(saccharine or cane-sugar) is found rather widely distributed, as in the stems of corn,
sorghum and the sugar-cane ; in roots, as the sugar-beet ; in the sap of certain trees, as
sugarmaple and some of the palms
Maltose
It is found in the germinating grains of cereals (see malt); it forms colourless, needle-
shaped crystals resembling those of dextrose, and forms compounds with calcium,
strontium, barium and acetic acid.

Cell-sap
The majority of the other colour-substances found in the higher plants besides the green
and yellow principles previously mentioned occur in solution in the cell-sap, and may
be in the nature of secondary substances derived from the plastid pigments, or they
may be produced directly by the protoplasm. Upon making sections of the tissues
containing cell sap colour substances, not infrequently strikingly contrasting colours are
observed in contiguous cells; as in the petals of the poppy and petals of certain lilies,
where we find some cells of a deep purple colour, others of a deep red and still others of
intermediate shades. Calcium oxalate is found in many of the higher plants, and in the
algs and fungi as well; while in the mosses, ferns, grasses and sedges it is seldom found.
It occurs in plants in crystals of either the monoclinic or tetragonal system. The crystals
dissolve in any of the mineral acids without effervescence and their identity is usually
confirmed by the use of dilute hydrochloric acid. The crystals of the monoclinic system
are rather widely distributed, while those of the tetragonal system are less frequent in
their occurrence, being found in species of Allium, crystals belonging to the monoclinic
system include a number of forms, as follows: (1) Rosette aggregates, or what are
commonly termed rosette-shaped crystals; (2) prisms, pyramids and elongated or
irregular polygonal-shaped crystals (3) crystal-fibers (4) raphides; (5) sphenoid micro-
crystals.
Rosette aggregates of calcium oxalate

It consists of numerous small prisms and pyramids, or hemihedral crystals more or less
regularly arranged around a central axis, have the appearance of a rosette or star. The
development of these aggregates may is readily observed in the stem of Datura.
Crystals of this class are more widely distributed than any of the others, and are found
in a number of drugs. Monoclinic prisms and pyramids are also widely distributed and
are frequently so in form that they are of an elongated or irregular polygonal shape. The
crystals of this group are sometimes mistaken for silica, owing to the fact that in some
instances the lumen of the cell is completely filled by the crystal, and the inner wall
having the contour of the crystal, it is impossible to determine whether the crystal is
affected by the use of hydrochloric acid. It should be stated in this connection that silica
never occurs as a cell-content in sharp, angular crystals, but either in more or less
ellipsoidal or irregular hollow masses, or in somewhat solid, irregularly branching
masses.
Crystal Fibers.
In quite a number of drugs a single monoclinic prism occurs in each of the parenchyma
cells adjoining the sclerenchymatous fibers, and to this single longitudinal row of
superimposed cells the name crystal fiber has been applied

Raphides
Raphides are groups of needle-shaped crystals which are found in various plants .These
have been mistaken by several observers for calcium phosphate. Calcium phosphate,
however, occurs in plants either in solution or in combination with protein substance.
The cells containing raphides are long, thin-walled and contain sooner or later a
mucilage, which arises from the cell-sap and behaves with reagents much like cherry
gum.
Amorphous substances
Cystoliths.
Occasionally cells are found among the parenchyma or in the inner row of the
epidermal cells on the upper side of the leaf, the walls of which form an inward
protrusion in cell and impregnated with and encrusted by calcium Carl ovate, giving
rise to more or less stalked bodies known as Cystoliths
The calcium carbonate

Calcium carbonate dissolves on the application of acetic acid, leaving a core which
responds to the tests for cellulose. Cystoliths are not of common occurrence, being
found with but few exceptions in the two families Acanthaceae and Moraceae, and in a
few species of the Cucurbitacese. In the leaves of the cultivated ruber plant the
Cystoliths have long stalks, whereas in cannabis indica, they are sessile.
Tannin and Tannoids

Tannins are astringent principles which belong to the class of phenol acids and give
blue or green precipitates with iron salts. The Tannoids, in addition, precipitate
aluminous compounds, and when applied to animal hides convert them into leather.
These principles are widely distributed, occurring dissolved in the cell-sap, in
parenchyma cells or in distinct reservoirs or vessels, and vary in amount from T per
cent, or less to as high as 70 per cent, in Chinese galls. Tannin may be precipitated in the
plant cells by copper acetate.
Mucilages and Gums

By the terms mucilages and gums are meant those substances which are soluble in
water, or swell very perceptibly in it, and which, upon the addition of alcohol, are
precipitated in the form of a more or less amorphous or granular mass.
REFERENCES
Botanic drugs Their materia medica, pharmacology And therapeuticspublished by The
therapeutic digest publishing co. Cincinnati, ohio 1917.
Herbal Pharmacognosy 312,Practical manual Coordinators: Miss M. Hess and Miss S.

Nyati.

Medicinal plants, kerala agricultural university Aromatic and medicinal plants research
station, 1998.
The Ayurvedic pharmacopoeia of India part - ii (formulations) Volume – I. First edition

Government.
The Principles of Pharmacognosy An introduction to the study of the crude substances

of the vegetable kingdom, New York William Wood & Company 1887.
The Ayurvedic Pharmacopoeia of India Part- I, Volume – i, ii, iii, iv, government of
India Ministry of Health and Family Welfare Department of Ayush.

CHAPTER THREE
DEVELOPMENT OF MOSQUITO REPELLENT FINISHES IN KNITTED FABRICS

USING Rosmarinus officinalis LEAVES
Banupriya.J and Maheshwari.V

Costume Design and Fashion, PSG College of Arts and Science/ Bharathiar University,
India.
ABSTRACT
The textile materials were considered primarily for economic and functional
point of view some end users in particular demands on the safety of textiles for
the health. An insect-repellent helped to prevent and control the outbreak of
insect-home disease such as malaria, dengue fever. The most herbal plants
contain compounds that are preventing attack from phytophagous regulators.
Insect repellent textiles are also a part of protective textiles which help in
protection from the species that are prone to cause damage in some or the other
manner. These textile products find their application over a wide range. The
knitted fabrics were finished with mosquito repellent test by excite chamber.
The treated fabrics show 100% mosquito repellent efficiency. The finished
fabrics were tested for laundering process they retained their activity until 16
washes. So these types of plant based mosquito repellents has been used for
generations in practice as a personal protection against mosquito.
KEYWORDS: Eco-friendly fabrics, herbal plants, laundering properties.
INTRODUCTION
Protective textiles are among one such smart application of smart technology in textiles.
Protective textiles refer to those textile products which have a functionality of giving
protection from something in some or the other sense. Mosquitoes have a complex
method of detecting hosts and different types of mosquitoes react to different stimuli.
At present, there are very few durable repellents that can be applied to clothing and
almost all the repellents are designed to be applied directly on the skin. This poses a
great risk to the individuals using them and hence, with a view to reducing this risk and
at the same time meeting the needs of industry. Basically mosquito repelling textiles are
the ones which have a character of repelling mosquitoes. This feature was developed as
a need in sense of protection from the mosquitoes in the areas which are habitats of the
mosquitoes and are prone to disease like malaria. Global warming has resulted in the
spread of mosquitoes from tropical regions to rest of the world resulting in spread of
viral infection to different parts of the world. Anti- mosquito finishing on textile
products can suppress mosquito-transmitted diseases such as West Nile fever; malaria.

Herbs are available in a variety of forms, including fresh, dried, in tablets or capsules, or
bottled in liquid form. Buy them individually or in mixtures formulated for specific
conditions. Finishing is the general term for a multitude of processes and treatments
which a fabric may undergo after it has been knitted. Finishes are also categorized by
their degree of performance.
Cotton is the natural vegetable fiber of great economic importance as a raw material for
cloth. Bamboo is grown using methods and materials that have low impact on the
environment.
MATERIALS AND METHODS

Fabric selection
The fabric used as the textile substrates in the present research work was 50‟s combed
bamboo/cotton blended knitted fabrics.
Pretreatment
The material is treated with soap at 50 0C for 20 minutes to remove the dirt on the
untreated fabric with water. The soap solution is added into water in the proportion of
3: 1.Then the material is given hot wash and cold wash. The M: L is 1:30.
Selection of the Medicinal Valuable Herbs

The herbal plant were identified and collected from the natural resources in a pure
form. The following plant was chosen for the study Rosemary. The procedure begins
with the selection of natural herb, which was screened and identified. The extract was
tested for its Mosquito Repellent which was done by Excito chamber method.
Rosemary has been around for a long time, and therefore has a long list of claims
regarding its medicinal uses, including use as a tonic, a digestive aid, to treat
depression, headache, and muscle spam, and as an expectorant, promoter of menstrual
flow, and stimulant for production of bile.
Extraction of Rosemary leaves

Methanol Extraction
For extraction, 6g of dry powder from each herb (Rosemary) was taken and mixed into
50ml of 80% Methanol. The container was closed and kept for over night. After over
night incubation, the extract was filtered through filter paper and evaporated to
concentrate the extract. The extract was finished on the fabric by dip dry method and
tested for its mosquito repellency activity.

Collection of mosquitoes
Anopheles mosquitoes were identified based on morphologic keys and they were
collected during the evening time. All the mosquitoes were starved of blood and sugar
for 4 hours.
Mosquito Repellency Behavioral Test

Specially designed two excito repellency test chambers were used to evaluate the
efficiency of repellency activity. The wooden outer chamber of excito-repellency testing
device measures 34 cm × 32 cm × 32 cm and faces the front panel with the single escape
portal. The box is composed of a rear door cover, an inner Plexiglas glass panel with a
rubber latex-sealed door, a Plexiglas holding frame, a screened inner chamber, an outer
chamber, a front door, and an exit portal slot. Mosquitoes were deprived of all nutrition
and water for a minimum of 4 hours before exposure. Laboratory tests were performed
during daylight hours only and each test was replicated four times. Observations were
taken at one-minute interval for 30 minutes. After each test was completed, the number
of Escaped specimens and those remaining inside the chamber was recorded separately
for each exposure chamber, external holding cage, and paired control chamber. Escaped
specimens and those remaining inside the chamber, for the treated samples, were held
separately in small holding containers with food and water. The repellency were
calculated by below formula:
Fig-1 shows the Excito chamber

Wash durability test
The rosemary extract finished samples was then subjected for 16 machine washes and
the washed fabric was tested for its mosquito repellency efficiency using excite chamber
method.
RESULTS AND DISCUSSION

The results shows that the mosquito repellent activity was the highest with Rosemary
extract finished samples. The mosquito repellent activity for the fabrics was determined
by examining the mortality rates of the mosquitoes after exposure to the leaf extracts.
Percentage of mosquito repellency
Table-1 shows the percentage of mosquito repellency.
Mosquito
S.No. methods samples Repellency
in %
1 Untreated Controlled 0
2 herbal Rosemary 88
Mosquito Repellency in %
100
50
Mosquito Repellency in
0
%
Controlled Rosemary
Untreated herbal
1 2
Fig-2 represents the herbal extracts treated fabrics percentage.
Wash durability
The laundering durability of the treated fabrics were tested with excite
chamber after each and every washes it was calculated using formula. The
coated fabrics were with stand nearly up to 16 washes.

Table-2 shows the wash durability test for treated f abrics.
S.No Samples Mosquito

Repellency %
Herbal
1. Untreated 0
2. Before laundering 88
3. After launderings 4 88
Mosquito Repellency % Herbal

100
80
60
40
20 Mosquito Repellency
0 % Herbal
Before…
After…
After…
After…
After…
After…
Untreated
1 2 3 4 5 6 7
Fig-3 represents the percentage of washing property.
CONCLUSION
From the study it was concluded that rosemary herbal extracts treated fabrics eco-
friendly, bio-degradable and non-toxic to the skin. Apart from the industrial use,
mosquito repellent finish on textiles has become essential in our day today life to live in

free diseases and hygienic atmosphere. The finish has excellent potential in various
textile uses baby care products and Night wears etc.
REFERENCE
Butter Worth, (1964), Review of Textile Process, Butter Worth And Co. Publishers
Ltd, Bangalore, P-330
Carey AF, Wang G, Su CY, Zwiebel LJ, Carlson JR. Odorant reception in the malaria
mosquito Anopheles gambiae Nature. 2010; 464:66–71.
Elementary idea of textile dyeing, printing and finishing, kanwar varinder pal singh,
Kalyani Publishers, 2004, pg.no-5, 29
Hallem EA, Dahanukar A, Carlson JR., (2006). “Insect odour and taste
receptors” Annu Rev Entomol. Vol.51. P.113–135
Shell ER. Resurgence of a deadly disease. Atlantic Monthly. August 1997:45-60.

Malaria. Fact sheet. No. 94. Geneva: World Health Organization, 1999. (Accessed May
3, 2002,
The Journal of Textile Association.
Taubes G. A mosquito bites back. New York Times Magazine. August 24, 997:40-6.

CHAPTER FOUR
IN VITRO ANTIBACTERIAL ACTIVITY OF METHANOLIC – AQUA EXTRACT OF

Tetradenia riparia LEAVES
Anthoney Swamy T and Ngule Chrispus Mutuku

Department of Chemistry, University of Eastern Africa Baraton, P.O. Box 2500, Eldoret
-30100, Kenya.
ABSTRACT
Ethnobotanical medicine has attracted great interest due to the belief that it is
safe, cheap and more dependable than aliphatic drugs, which have adverse side
effects. The current study was conducted to analyze the antibacterial activity of
Tetradenia riparia leaves hydroalcoholic extract. The plant sample was
extracted using methanol and water in the ratio of 9:1. From the study the
plant Tetradenia riparia was found to inhibit the growth of Escherichia coli
with a zone of inhibition of 11.67 ± 0.882, Serratia liquefaciens zone of
inhibition of 12.67 ± 0.667, Bacillus cereus 28.00 ± 1.154, Enterobacter
aerogenes 8.67±0.882 and Proteus vulgaris 11.00 ± 0.577. The data collected
and documented in this paper is a scientific justification that the plant
Tetradenia riparia can be used to treat against various diseases caused by
Escherichia coli, Serratia liquefaciens, Bacillus cereus, Enterobacter aerogenes
and Proteus vulgaris. However, further research is needed to isolate the active
compounds identify their structure, their mode of action to the microorganisms
and their effect in the in vivo environment.
KEYWORDS: Tetradenia riparia, Antibacterial, Medicinal herbs, Leaves.
INTRODUCTION
Nature is a paradise of medicinal solutions to all ailments affecting human beings
through medicinal plants. Medicinal plants are being used widely to treat against the
currently widespread strains of drug resistant bacteria. Scientists all over the world are
working hard to provide scientific justification on the traditional use of medicinal plants
to treat against the ailments affecting human beings
Pharmacological studies have reported appealing results showing the importance of

using plant extract to treat diseases. The reports have shown that plants can be used as
antitumor, anti-inflammatory, antibacterial activity, anti-hyperlipidemic, anti-
hyperglycemic and hypoglycemic activity (Kaoli and Kauli, 2011). The antibacterial
activity of plants has been associated with the presence of certain compounds referred

to as phytochemicals such as tannins, saponins, terpenoids, flavonoids, phenols and
alkaloids (Ngule et al., 2013; Nyaberi et al., 2008).
All chemicals found in plants are potential drugs. Certain tree barks produce chemicals
that discourage caterpillars from feeding on them, a good example being the Indian
neem tree, which keeps off desert locusts. The twigs are chewed by people in Serengeti
national park in east Africa to prevent tooth decay. Plants produce more than 10,000
different compounds to prevent themselves against animals who feed on them. Almost
half of all prescribed drugs contain chemicals produced by plants, fungi and bacteria.
Aliphatic drugs also contain synthesized compounds in the laboratory that have been
modeled after plants originating compounds (Moore et al., 1995).
The use of medicinal plants to treat diseases is as old as man. Medicinal plants have
been used since ancient times to treat many illnesses (Mir et al., 2013). Research has
shown that the concentration of these compounds in plants is directly related to their
capability to treat certain illness. Many of these non-nutritive secondary metabolites are
found in plants which are even used for food. Over 80% of the plants in Nigeria used
for treatment of malaria and other sicknesses are also used as food (Cousins and
Huffman, 2002); there seem to be not much distinction between medicinal benefits of
plants and their nutritive value.
Over the past few years much research has been done and is still going on to prove
scientifically the plants nutritional value and medicinal value. A good number of
chemical compounds have been discovered from plants and found to have
pharmacological value; this has led to the development of over 25% of all the artificial
medicines used today. Many of the traditional plants species used all over the world
have been found to have great pharmacological value. Studies carried out throughout
Africa confirm that indigenous plants are the main constituents of traditional
medicines. The published WHO traditional strategy addressed the issues and provided
a framework for countries to develop policies to govern medicinal plants use. The
strategy put forward by WHO advocates the formulation of a policy by states as the
first component of developing traditional medicine. India is one of the few countries
which have started to develop such policies (Prajapati and Purohit, 2003).
Over 80% of the people in developing countries use medicinal plants to treat the
illnesses which affect them from time to time (Ganga et al., 2012). This can be attributed
to poverty in these countries which has led to inefficient health care system in hospitals
and inadequate resources to access these facilities. People in these countries look for
cheap and available medicines which are known traditionally to cure the illnesses. The
use of herbal medicines in the western world is steadily growing with 40% of the
population using plants to treat illnesses; while in Kenya 90% of the population has one

time in their life used medicinal plants (Adongo et al., 2012). The use of these plants in
treatment of ailments is mainly based on the type of flora in that region.
Our environment is very rich with a great range of medicinal plants and this mainly
explains the reason why our grand‟s lived for quite some time. They could stay in the
bush during war and even could use plants to treat ailments and wounds affecting
soldiers in the battle ground. People all over the world should go back to these basics of
treatment. Many communities in Africa still consider the use of medicinal plants as an
important part of their culture, just to mention, the Maasai community in Kenya still
value their culture very much, the Kalenjn community and their medicinal fermented
milk which is prepared mainly from medicinal plants such as Senna didymobotrya stem
which previous studies have shown this plant to have a great potential in treatment of
diseases such as typhoid, diarrhea and food poisoning caused by Salmonella typhi, E.coli
and Bacillus cereus (Ngule et al., 2013). The reason why herbal medicine still remains
controversial is because of some greedy practitioners who want to become wealthy by
pretending to know much about the treatment of every disease that clients complain
about. This has led to administration of wrong drugs which do not cure a patient
leading to death of the individual. Proper scientific evidence needs to be provided in
order to create confidence in medicinal herbs. The increase of multi-resistant strains of
bacteria calls for new discoveries of new classes of antibiotics that can clearly inhibit
these resistant strains. This is the reason why much research should be turned to plants
which have been used since ancient times to treat many diseases (Cousins and
Huffman, 2002).
The non-nutritive plant components are referred to as phytochemicals, which can be

divided in two major categories primary and secondary, with the primary constituting
of carbohydrates, proteins and chlorophyll and the secondary consisting of tannins,
alkaloids, saponins, steroids, flavonoids, terpenoids and anthroquinones (Maobe et al.,
2013). The secondary metabolites help the plant survive in the environment by
protecting them against predators but research has shown that these metabolites can be
used to treat diseases in both animals and humans (Kokwaro, 2009). The antibacterial
activity of plants has been closely associated with the presence of these important
compounds in the plant. Plants antibacterial activity against various bacteria such as
B.cereus, Klebsiella sp., Streptococcus pyogenes and Proteus vulgaris has been closely
associated to the presence of phytochemicals in the plants (Swamy et al., 2013).
Physiological activities of phytochemicals have been found to include cancer
prevention, antibacterial, antifungal, anti-oxidative, hormone action and enzyme
stimulation.
Natural bioactive compounds have been investigated in plants and their

pharmacological effects analyzed. Secondary metabolites functions on growth,

photosynthesis and other important plant activities have not been discovered but their
medicinal values have been identified in most of them (Ghasemzadeh and
Ghasemzadeh, 2011). Phytochemicals have been used to a greater extend in Asia for
various purposes such as treatment of diseases (Bodeker, 2000).
The lack of scientific knowledge on the phytochemical constituents, antibacterial,

antioxidants and toxicological properties limits the use of traditional herbal medicine
(Nyaberi et al., 2008). Phytochemicals can really improve the activity of the currently
used drugs by acting as efflux of existing pump inhibitors. Many drug resistant
microbes are emerging from time to time and causing the need to such for new
antibiotics to kill and inhibit their growth. Phytochemicals have been associated with
reduction of drug resistant forms of bacteria (Stauri et al., 2007).
A big percentage of plants in the savanna and semi-arid areas of east Africa where
Kenya is located contains alkaloids which have been associated with increase in renal
secretion when ingested, hence used as a diuretics and in the treatment of dropsy
(Kokwaro, 2009). The use of alkaloids, saponins and tannins as antibiotics has been
scientifically justified (Mir et al., 2013). Majority of the pharmacologically active
chemical compounds were found mainly in ethanol extracts which is contrary to
previous researches which had affirmed the traditional way of extracting these
compounds using water (Iqbal, 2012).
According to Coopoosamy and Naidoo (2011), the plant Tetradenia riparia has great
effect in the treatment against skin infections. The plant has also shown great potency in
the treatment of chest diseases. According to Ndamane et al., (2013), the plant showed
antibacterial activity against all the gram positive and gram negative bacteria it was
tested against. In Kenya, the plant has a wide variety of uses depending on the region.
The plant is used traditionally in the rift valley and western region to treat against
stomach problems, wound infections, throat infections and also it is believed to treat
have anti-cancer activity. The plant is grown by the local Nandi community in Kenya
along farm edges and around their homes. The plant is greatly used by traditional
practioners to treat various diseases; however, some of the practioners find it difficult to
transfer this information to their clients even upon request for the information about the
plant. This calls for scientific documentation to enable the transfer of knowledge about
the plants use. The literature available is also contradicting and therefore the current
study was not only done to give a scientific justification of the plants traditional use but
also to compare the data obtained in this study with that in literature and provide a
scientific view on the same.

Sample Collection and Preparation
The herb was randomly collected in the natural forest around University of Eastern
Africa, Baraton. The plant samples were collected and identified by a taxonomist in the
University of Eastern Africa, Baraton. The samples were thoroughly mixed and spread
to dry at room temperature in the chemistry laboratory for about three weeks. They
were then ground into fine powder and put in transparent polythene bags.
Extraction procedure
Using electric analytical beam balance fifty grams of the powdered leaves of the
Tetradenia riparia was placed in 1000 ml conical flask, methanol and water were then
added in the ratio of 9:1 respectively until the leaves were completely submerged in the
solvent. The mixture was then agitated for thorough mixing. The mixture was kept for
24 hours on a shaker for effective extraction of the plant components. The extract was
filtered using Butchner funnel; Whatman no.1 filter paper and a vacuum and pressure
pump. The filtrate was re-filtered again using the same apparatus. The solvent was
evaporated using rotary vacuum evaporator (R-11) with a water bath at 40oC. The
extract was brought to dryness using vacuum and pressure pump at room temperature.
The residue was then obtained and used for the experiment.
BIOASSAY STUDY
Preparation of the Bacterial Suspension:
The turbidity of each of the bacterial suspension was prepared to match to a 0.5
McFarland standard, a procedure similar to that used by Biruhalem et al., (2011) and
Donay et al., (2007). The McFarland standard was prepared by dissolving 0.5 g of BaCl2
in 50 ml of water to obtain a 1% solution of Barium chloride (w/v). This was mixed
with 99.5 ml of 1% sulphuric acid solution. Three – five identical colonies of each
bacterium was taken from a blood agar plate (Himedia) culture and dropped in Mueller
Hinton broth (Himedia). The broth culture was incubated at 370C for 2 - 6 hours until it
achieved turbidity similar to the 0.5 McFarland standards. The culture that exceeded
the 0.5 McFarland standard were each adjusted with the aid of a UV spectrophotometer
to 0.132A0 at a wavelength of 600 nm in order to obtain an approximate cell density of
1x108 CFU/ml.
Preparation of the Extract Concentrations and Antibiotic:

Stock solutions for the extracts were prepared by dissolving 500 mg in 1 ml of
dimethylsulfoxide (DMSO). An antibiotic control was made by dissolving 500 mg of
Penicillin in 1 ml of sterile distilled water. DMSO served as a negative control.
Determination of bioactivity of the Extract:

Mueller Hinton agar plates were prepared by the manufacturer‟s instruction. The media
was sterilized in an autoclave at 1210C for 15 minutes. The plates were also sterilized at

the same temperature. The media was then poured on to the plates and air bubbles
removed from the surface of the plates using non luminous Bunsen burner flame. The
bacterial suspension was smeared on the surface of the plates using a sterile swab. Five
wells were then drilled in each agar plate. Three of the wells were filled with the plant
extract. The other wells were filled with penicillin and DMSO control respectively.
Three plates were made for each bacterial organism and extract giving a triplicate
reading for each microorganism and extract. The wells were labeled on the underside
of the plate and incubated at 370C for between 24 to 48 hours and the zones of inhibition
were measured in millimeters with the aid of a ruler.
RESULTS AND DISCUSION
Table 1: Zones of Inhibition (mean ± S.E.) of 500 mg/ml of Tetradenia riparia Extract
against Selected Microorganisms.
Microorganisms Mean ± S.E. Penicillin DMSO control
Escherichia coli 11.67±0.882 40.00±0.000 0.00±0.000
Salmonella typhi 0.00±0.000 31.00±0.000 0.00±0.000
Serratia liquefaciens 12.67±0.667 36.00±0.000 0.00±0.000
Enterobacter aerogenes 8.67±0.882 40.00±0.000 0.00±0.000
Bacillus cereus 28.00±1.154 45.00±0.000 0.00±0.000
Proteus vulgaris 11.00±0.577 46.00±0.000 0.00±0.000

dimethylsulfoxide (DMSO)
The average mean zone of inhibition (±S.E.) was calculated for each of the microbial
organism. The zones of inhibition of the microorganisms were also analyzed by
analysis of variance (ANOVA) and it was shown that there were significant differences
in the zones of inhibition among the microbial organisms (p<0.05). The biggest zone of
inhibition was against B. cereus (28.00±1.154) followed by S. liquefaciens (12.67±0.667),
Escherichia coli (11.67±0.882), Proteus vulgaris (11.00±0.577) and Enterobacter aerogenes
(8.67±0.882). Penicillin which was used as the positive control inhibited the growth of
all the organisms while DMSO did not have any inhibition against the organisms tested.

Table 2: Turkey’s Honestly Significant Difference among Microorganisms.
(Using 500mg/ml of Tetradenia riparia Extract)
Comparison P Value Significance
E. coli vs Salmonella typhi 0.001 S

E. coli vs S. liquifaciens 0.995 NS
E. coli vs E. aerogenes 0.084 NS
E. coli vs Bacillus cereus 0.000 S
E. coli vs Proteus vulgaris 0.999 NS
Salmonella typhi vs S.liquefacines 0.000 S
Salmonella typhi vs E. aerogenes 0.111 NS
Salmonella typhi vs B. cereus 0.000 S
Salmonella typhi vs P. vulgaris 0.001 S
S. liquefaciens vs E. aerogenes 0.036 S
S. liquefaciens vs B. cereus 0.000 S
S. liquefaciens vs P. vulgaris 0.953 NS
E. aerogenes vs B. cereus 0.000 S
E. aerogenes vs P. vulgaris 0.145 NS
B. cereus vs P. vulgaris 0.000 S
The study shows that the plant Tetradenia riparia can inhibit the growth of five
microorganisms out of the six it was tested against. The plant show clear zones of
inhibition against Escherichia coli with a zone of inhibition of 11.67 ± 0.882, S. liquefaciens
12.667 ± 0.667, Bacillus cereus, 28.00 ± 1.154, Enterobacter aerogenes, 8.67±0.882 and Proteus
vulgaris, 11.00 ± 0.577. Comparing for the mean zones of inhibition of 500 mg/ml of
Tetradenia riparia showed that there was significant difference in the zones of inhibition
among the organisms (p<0.001). Further comparison using the Tukey‟s pairwise
comparison showed that the zones of inhibition for E. coli were significantly higher than
those of Salmonella sp. and P. vulgaris (p<0.05). Zones of inhibition of Salmonella typhi
were significantly lower than all of the other organisms (p<0.05) except for E. aerogenes.
Significant difference were also observed between B. cereus and all the organisms
(p<0.05). The study is in conformity with previous studies in which the plants
methanol and water extracts were found to inhibit the growth of all the gram positive
and gram negative bacteria (Ndamane et al., 2013), The study is completely in
agreement with the same study in which the plant inhibited B. cereus most. The current
study use a system of solvent which constituted methanol and water in the ratio of 9:1
unlike in previous studies the two were used separately. The data obtained in this study
in conformity with previous studies which shows the two solvents to have great
antibacterial activity, however, contradicts with the results obtained by Erasto et al.,

(2005), the plant did not show antibacterial activity against E. coli and B. cereus. The
antibacterial activity of the plant can be attributed to the presence of the
phytochemicals. The phytochemicals found in the plant have been investigated and
found to have antibacterial activity therefore justifying traditional plant use to treat
against various diseases caused by bacteria (Gazim et al., 2010).
The plant extract can be used to treat infections caused by Bacillus cereus viz
posttraumatic wounds, self-limited gastroenteritis, burns, surgical wounds infections,
and ocular infections such as endophthalmitis, corneal abscess and panophthalmitis
(Garcia-Arribas et al., 1988) & Sankararaman and Velayuthan, 2013). The plant extract
can also be used to treat immunologically compromised patients including AIDS and
malignant disease victims (Cotton et al., 1987 & Tuazon et al. 1979). The plant‟s ability
to inhibit the growth of E. coli is a scientific justification that the plant can be used to
treat against enteric infections caused by the bacteria. The plants extract can also be
used to treat against gastro-intestinal diseases, ear infections, urinary tract infections
and wounds infections caused by Proteus vulgaris (Goodwin et al., 1971 & Neter and
Farrar, 1943).
Indigofera arrecta can be a good source of active compounds for a variety of diseases
affecting human beings in the world today. The plants ability to inhibit the growth of
Serratia liquefaciens shows how the plant can be important to treat against the bacteria
which according to Okunda et al., (1984) cause nosocomial urinary tract infections. The
inhibition of the plant against these bacteria is, therefore, note worthy since the
microorganisms have been found to have resistance against most of the currently used
antibiotics. Enterobacter aerogenes is a major cause of a wide variety of nosocomial
infections viz, pneumonia, urinary tract infections, meningitis, wound infections and
intravascular and prosthetic devices infections (Santos et al., 1990, Blot et al., 2003,
Donnenberg, 2005).
CONCLUSION
The data provided in this study is a scientific justification that the plant Tetradenia
riparia can be used to treat against diseases such as abdominal cramps and diarrhea
caused by Bacillus cereus due to its ability to cause food poisoning, treat against
Escherichia coli which causes serious and even life threatening effects such as hemolytic-
uremic syndrome (HUS), diarrhea and neonatal meningitis. It can also be used to treat
against throat problems caused by Serratia liquefaciens, opportunist pathogen Proteus
vulgaris which causes wound infections. From the study the plant Tetradenia riparia has
shown to have great medicinal value and therefore justifying its traditional use to treat
against various diseases. More research needs to be done to identify the mode of action
of the active compounds in the plant.

ACKNOWLEDGEMENTS
The authors of this paper are very much thankful to the Department of Chemistry and
Medical Laboratory Science, University of Eastern Africa, Baraton. Authors are also
thankful to taxonomist Mr. Joel Ochieng Ondiek, University of Eastern Africa, Baraton
for his great assistance in the identification of the plant.
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Fig 1. Picture showing the zones of inhibition of the plant extract and positive and
negative action against Proteus vulgaris
Fig 2. Tetradenia riparia plant leaves

CHAPTER FIVE
MEDICINAL PROPERTIES OF INDIGENOUS PLANTS USED IN THE

PREPARATION OF TRADITIONAL RICE BEVERAGE “HANDIA” AMONG THE
TRIBALS OF MAYURBHANJ DISTRICTS, ODISHA, INDIA
Sujogya Kumar Panda1, Laxmipriya Padhi1 and Akshaya Kumar Bastia2

1Department of Zoology; North Orissa University; Baripada, Odisha, India-757003
2Department of Botany; North Orissa University; Baripada, Odisha, India-757003
ABSTRACT
The district Mayurbhanj is charecterised by a diverse tribal population of
people with different ethinic background. Similipal Biosphere Reserve is
present at the center of Mayurbhanj District; its rich biodiversity is
acknowledged internationally. The district as a whole and SBR in particular,
offers unique opportunities to study the indigenous knowledge and their uses
prevalent among the local tribes. The native tribals depend on wild as well as
cultivated plants for their various food and medicinal purposes. As a part of
the socio-cultural life, all tribes prepare rice beverage using their own unique
starter culture. In the preparation of starter they use some of the wild plants as
antimicrobials without knowing the actual role of these plants in fermentation.
They say that yeast is formed from these plants, responsible for yeast action
during the fermentation. This chapter deals with the available plants used for
starter preparation of Handia by the tribes of Mayubhanj, their ethnomedicinal
uses. All these plants were evaluated for the phytochemical constituents and
antibacterial activity against enteric pathogens. Results found that alcoholic
extracts of the plants contain abundant alkaloids, flavonoids, carbohydrate,
protein and amino acids, saponins, tannin and phenolic compounds. With the
exception of Aspargus racemosus (root), Cissampelos pareira (leaf), Dioscorea
sp. (tuber), Rauwolfia serpentina (leaf) extracts, all test plant parts exhibited
antibacterial activity by agar cup method. The zone of inhibition was found
maximum against Staphylococcus aureus followed by Shigella sonnei and S.
flexneri. The MIC result ranged from 125 to 1000 µg/ml (w/v) with the lowest
against S. aureus (125, 156, 250 µg/ml) followed by S. sonnei (156, 250, 312,
500, 625 µg/ml). MBC test validate that in between 1000-2500 µg/ml (w/v)
concentrations, most of test bacteria were killed due to broad spectrum activity.
This chapter also provides information on different types of starter used for
preparation of rice beverage through out India.
KEYWORDS: Bakhar, Alcoholic beverage, Traditional knowledge, Process

technology, Ethnomedicine, Enteric pathogens

INTRODUCTION
A wide range of cereal based fermented foods exist throughout the Asian and African
countries. Since rice is the major cereal in these areas, a global interest in rice and its
fermented product is increasing due to their caloric value, unique quality characteristics
and high acceptability (Steinkraus, 1994). In most of the countries, rice is fermented
either by using mixed culture(s) into alcoholic beverages, or by natural fermentation
into leavened batter formed dough breads which are usually baked or steamed
(Yokotsuka, 1991). Rice beer is an integral part of life of several aboriginal communities
throughout the world. In India, from time immemorial, both fermented and distilled
beverages have been prepared by fermenting different varieties of rice. These beverages
are primarily prepared and used by different tribal communities of the northern and
eastern part of India.
In Odisha, from time immemorial, both fermented and distilled beverages have been
prepared by fermenting different varieties of rice. These beverages are primarily
prepared and used by different tribal communities all over the state. About 62 ethnic
tribal communities are reported from the state (Naik, 1998) and they mostly inhabit
forest villages. They meet most of their requirements, including food and primary
healthcare, from the forest resources. Out of 62, 30 communities (48%), several
aboriginals, are found in the district of Mayurbhanj, (largest district of the state; area -
10, 418 sq km forest cover – 4, 392 sq km. population – 25, 13, 895/2011 census).
Similipal Biosphere Reserve (SBR, 5569 sq. km.) is located at the centre of the district
and its rich biodiversity is acknowledged internationally. The district as a whole and
SBR in particular, offers unique opportunities to study the indigenous knowledge and
their uses prevalent among the local tribes. Santal, Kolha, Bathudi, Bhumij, Munda and
Gond are major tribes whereas Mankidia, Lodha, Kisan and Baiga are minor tribal groups
that inhabit the area. Santals constitute the largest tribal group of the district and are
scattered throughout. The social, cultural and religious life of aboriginal people is
influenced by the nature and natural resources available in and around their habitat
that provide food, fodder, medicine, shelter and various other material and cultural
needs.
The fermented food, locally known as handia, is an inseparable food item in the life of
tribals of Mayurbhanj and most other districts of the state. The word handia finds its
origin from the word Handi in Odia (local language), means large earthen pot. Handia
occupies a key position in the social, cultural and economic life of Santals and accepted
as a traditional drink (Sahu, 1996). Handia is generally prepared throughout the year,
but most common during the summer months (March to June). Women and children
are also fond of these beverages but consume in small quantity and preferably during
festivals, ceremonies and on Sundays. Tribals get 5-10% of their daily nutrient
requirements that plays a supplementary role in the nutrition of the people (Roy, 1978).

It is prepared from rice along with some of the locally available plant parts through
some indigenous method. Otherwise known as country liquor or poor man‟s whiskey,
it is relished by one and all and in most occasions. The process of starter (locally known
as Bakhara/Ranu) preparation of each tribe is almost similar with slight differences.
The main ingredients of starter preparation are rice along with some of the locally
available plant parts through some indigenous method. So, a survey of the use of
medicinal plants in preparation of Bakhara/Ranu by the tribes in the District
Mayurbhanj was carried out. The tribals do not know the authentic role of these plants
in the fermentation. According their knowledge, either yeast is formed from these
plants or these plants are responsible for the yeast‟s action in fermentation. The present
paper deals with the description and ethnomedicinal uses of plants for the starter
preparation by the tribes of Odisha. For scientific validation all these plants were
subjected for screened of antimicrobial activity and phytochemical screening.
LITERATURE REVIEW
Traditional rice-based alcoholic beverages
Traditional rice beverages have different compositions according to the formulation and
processes used. The principle of their manufacture can be characterized as a
biochemical modification of cereal starches brought about by microorganisms. Moulds
produce the amylases that degrade the starch into dextrins and sugars and yeasts
convert these sugars to alcohol (Lim, 1991; Motarjemi and Nout, 1996; Nout and Aidoo
2002).
The preparation and the use of fermentation starters as a source of inoculum are
important for preparation of rice beverage. The choice of starter tablets influences the
yield and quality of the rice beer. Even in certain region the local processors claim that
using a combination of two or three different starters yields better quality with a
stronger sweet alcoholic taste and more attractive flavor than is obtained with a single
starter. The dried starters normally include yeasts, moulds and bacteria and convert
starchy materials to fermentable sugars and subsequently to alcohol and organic acids
(Hesseltine et al., 1988; Nwosu and Ojimelukwe, 1993; Luong, 1998; Nout and Aidoo,
2002). A variety of starter cultures is available in the through out India especially in the
North East region (Table-1).

Table-1: Starters for alcoholic beverages used by different tribes of India
Product Plants used during preparation Functional moulds, yeasts
and bacteria
Angkur Xanthium strumarium, Scoparia dulcis L., Un known
Clerodendrum viscosum L.
Aopo Achyranthes aspera L., Cinnamomum bejolghata, Un known
pitha Adhatoda Vasica Nees., Ageratum conyzoides L.,
Ananas comosus (L.) Merr., Artocarpus
heterophyllus, Asparagus racemosus Willd.,
Cinnamomum tamala Nees., Capcicum annum L.,
Centella asiatica L., Clerodendrum viscosum L.,
Costus speciosus (Koen. ex. Retz.) J.E. Smith,
Drymeria cordata L., Gomphostemma parviflora
Wall., Ipomoea aquatica Forsk, Ipomea mauritiana
Jacq., Kaempferia rotunda L., Leucas plukenetii
(Roth) Spreng., Lygodium flexuosum, Melothrea
heterophylla (Lour) Cogn, Microsorum punctatum
(L.) Copel, Musa balbisiana Colla, Naravelia
feylavica (D.C), Oldenlandia corymbosa L., Piper
longum L., P. nigrum L., Phlogacanthus
thyrsiformis
(Hardw.) Mabb., Psidium guajava L., Pueraria
tuberose (Roxb. ex. Willd.) DC, Ptridium
aquilinum Kuhn, Saccharum officinarum L.,
Scoparia dulcis L., Selaginella species, Swernia
chirata (Buch-Hem), Vitex negundo L.,
Zanthoxylum nitidum (Roxb.) (Gogoi et al., 2013;
Das et al., 2012)
Bakhar Heteropogon contortus (L.) Beauv. ex R. & S., Mucor sp., Rhizopus sp.,
Bambusa vulgaris Schrad, Amylomyces sp., Yeast
Epop Scoparia dulcis (Linn.); Pteridium sp; Adhatoda Un known
vasica (Nees); Cyclosorus sp., Adhatoda zeylenica,
Costus speciosus, Naravelia zeylanica, Melothrea
heterophyla; Zanthoxylum hemiltonianum (Wall);
Phogocanthus thyrsiflorus (Nees); Hydrocotyl
rotundifolia (Roxb); Centella asiatica; Swertia
chirata (Buch-Hem); Actinodaphne obovata
(Blume); Piper nigrum (Linn); Piper longum
(Linn); Selaginella sps., Lygodium japonicum (L);
Naravelia feylavica (D.C.); Melothrea heterophylla
(Lour) Cogn

Hamei Albizia myriophylla Benth. Mucor sp. And Rhizopus sp.;
Saccharomyces cerevisiae,
Pichia anomala, P.
guilliermondii, P. f abianii,
Trichosporon sp., Candida
tropicalis, C. parapsilosis, C.
43ontana, and Torulaspora
delbrueckii; Pediococcus
pentosaceus and Lactobacillus
brevis (Tamang et al. 2007a;
Jeyaram et al. 2008b; Singh
and Singh, 2006).
Humao/ Glycyrrhiza glabra L. (Chakrabarty et al., 2009) Saccharomyces cerevisiae
Umhu Acacia pinnata (Das et al., 2012)
Khekhrii Lagenaria siceraria [Molina] Standley; Elscholtzia Saccharomyces cerevisiae
blanda Benth., Elscholtzia blanda, Justicia adhatoda (Teramoto et al., 2002;
Nees. (Tamang, 2010) Sekar and Mariappan, 2007)
Marcha Plumbago zeylanica, Buddleja asiatica, Vernonia Rhizopus sp., Mucor sp.,
cinerea, Polygala arillata, Clematis grewiaeflora, Mucor circinelloides, M.
Polygala sp., Polygala arillata, Piper chaba, P. hiemalis, Rhizopus chinensis,
longum, Christella appendiculata, Elephantopus and R. stolonifer var.
scaber, Inula sp., and Scoparia dulcis (Tamang, lyococcus;
2010) Endomycopsis fibuliger.
Saccharomycopsis fibuligera,
Saccharomycopsis capsularis,
Pichia anomala, P. burtonii,
Saccharomyces cerevisiae, S.
bayanus, and Candida
glabrata; Pediococcus
pentosaceus, Lactobacillus
bifermentans, and Lb. brevis
(Tamang 1992; Tamang and
Sarkar 1995; Thapa 2001;
Tsuyoshi et al. 2005;
Tamang, 2010).
Mod Allium sativum L., Artocarpus heterophyllus Un known
pitha Lamk., Ananas comosus (L.) Merr. Alpinia
malaccensis Rosc., Alternanthera sessilis (L.) R. Br.
ex DC., Capsicum annuum L., Cinnamomum
bejolghota (Buch.-Ham) Sw., Centella asiatica (L.)
Urban, Coffea bengalensis Roxb., Costus speciosus

J. E. Sm., Desmodium sp., Cyprus sp., Desmodium
pulchellum (L.) Benth. Equisetum sp., Lygodium
flexuosum (L.) Sw., Melastoma malabathricum L.
Mussaenda roxburghii Hook.f., Myxopyrum
smilacifolium (Wall.) Bl., Naravelia zeylanica (L.)
DC., Oryza sativa L. Psidium guajava L., Pothos
scandens L., Pteridium aquilinum (L.) Kuhn.,
Pycnarrhena pleniflora Miers., Rubus sp.,
Saccharum officinarum L., Selaginella semicordata
(Wall) Spreng, Scoparia dulcis L., Solanum
torvum Sw., Thunbergia grandiflora Roxb.,
Zanthoxylum oxyphyllum Edgw., Zingiber
officinale Rosc. (Deori et al., 2007)
Perok Jasminum sambac; Cinnamomum byolghata; Un known
Kushi Zanthoxylum hamiltonianum; Lygodium flexuosum;
Acanthus leucostychys; Cyclosorus exlensa; Alstonia
scholaris; Alpinia malaccensis; Costus speciosus
(Das et al., 2012)
Ranu Oryza sativa L., Coccinia grandis L., Vernonia Several mycelia fungus and
dabai cinerea L., Clerodendrum viscosum Ventenat,, Yeast
Plumbago zeylanica L.,, Stephania japonica
(Thunb.)Miers,, Stephania glabra (Roxb.) Miers,,
Oroxylum indicum L.,, Mussaenda roxburghii
Hook.f.,, Scoparia dulcis L.,, Rauvolfia serpentina
L.,, Artocarpus heterophyllus Lam., Wattakaka
volubilis (L.f) Benth
Ranu Argyreia bella (C.B.Clarke) Raizada, Bombax ceiba Several mycelia fungus and
goti/ L., Buchanania lanzan Spreng., Casearia graveolens Yeast
tablets Dalz., Cassine glauca (Rottb.) O. Ktze,
Catunaregam spinosa (Thunb.), Cissampelos pareira
L., Crotalaria albida Heyne ex. Roth, Cryptolepis
buchanani Roem. & Schult., Datura metal L.,
Elephantopus scaber L., Euphorbia prolifera Buch.-
Ham. ex D. Don, Hemidesmus indicus (L.) R. Br.,
Holarrhena pubescens Wall. ex Don, Knoxia
sumatrensis (Retz.) DC., Pueraria tuberosa
(Willd.) DC., Scoparia dulcis L., Senecio nudicaulis
Buch.-Ham. ex D. Don, Symplocos racemosa
Roxb., Tylophora rotundifolia Buch.- Ham. ex.
Wt., Wattakaka volubilis (L.f.) Stapf (Kumar and
Rao, 2007)

Siiyeh/ Clerodendrum viscosum L., Veronia sp. (Das et al., Un known
Siye/Op 2012)
op/Ipof
Thiat Amomum aromaticum Roxb. Yeast
Thap Croton joufra, Artocarpus heterophyllus, Un known

Phlogocanthus thysiflorus, Solanum viarum and
Acacia pennata (Das et al., 2012)
Vekur Lygodium flaxuosum Linn., Leucas aspera Spreng, Saccharomyces cerevisiae
pitha Cissampelos pereira, Scoparia dulsis Linn., Centella
asiatica; Cinamommum glanduliferum Meissn.,
Piper betle L., Piper nigrum L., Hydrocotyle
sibthorpioides (Das et al., 2012)
Starter preparation and Alcoholic fermentation

The raw ingredients for the preparation of starter tablets can be either rice flour or
cassava flour or combinations of rice flour and cassava flour. Also mixed flours are
preferred in certain region. These mixtures are ground and thoroughly mixed with
spices and herbs that are believed to play a role in preventing the growth of undesirable
microorganisms. The spices and herbs used include mixtures of garlic, pepper, onion,
rhizomes and root of oriental herbs and producers jealously guard their own secret
recipes. The ratio of ground rice to mixed spices is different about 14:1 by weight. Water
is added to form a dough-like mass with moisture content of 55-60% which is
inoculated with dry powdered starter from previous batches, followed by thorough
mixing. The inoculated dough is shaped into small flattened or ball-shaped cakes about
4 cm in diameter and 1cm thick. The cakes are placed on a bamboo tray and are then
covered with a thin layer of rice husks. According to producers this reduces
overheating and facilitates aeration. The tray is covered with a cloth and incubated in a
ventilated place at ambient temperature (approx. 28-32°C) for 2-5 days during which
time the dough rises slightly and becomes covered with fungal mycelium. The cakes are
air or sun-dried and then have a shelf life of several months.
Throughout the world three different kinds of starters are used e.g. starters without
oriental medicinal herbs; starters supplemented with oriental medicinal herbs; and
starters supplemented with plant containing aromatic essential oils. In the most cases,
starters supplemented with medicinal plants are predominating. It was suggested by
some authors that a number of plants have a stimulatory effect on biomass and on yeast
count. Few authors believe that plants are used in starter because of their antibacterial
properties and their fragrant flavour.

Preparation of Angkur by the Bodos tribes of Assam (Das et al., 2012)
Jou Bishi is the local rice beer prepared by the Bodos using the starter cakes known as
angkur. For preparing angkur, different plant materials are said to be used based on their
availability in different regions. The most common species are leaves of Xanthium
strumarium, Scoparia dulcis and either roots or leaves of Clerodendrum viscosum. These
plants are first washed properly and allowed to dry in the air. Rice grains are soaked for
about 5 hours in normal temperature water and allowed to soften. This is then mixed
with the plants and grinded together in a wooden mortar with a pestle and this set of
apparatus is called wayal. Dough is made by adding a little water to the mixture. They
are then made into round cakes with different size and covered with powder of the
mixture to which water is not added. This is followed by covering with gigab (paddy
straw) and allowed to dry for a period of 3-4 days. These can be stored in moisture free
places for more than a year.
Preparation of Apop Pitha by the Mising tribes of Upper Assam (Gogoi et al., 2013)
For preparing apop pitha a mixture of plants (table) collected, cleaned and dried by
placing on a bamboo mat. Soaked rice and the leaves are grinded separately in a
wooden grinder and they are mixed together in a vessel with a little of water. From the
dough, oval shaped balls of about (6 cm x3cm) are made and dried in the sun. Apop
pitha is used for preparation of Apong-rice beer. The earthen pot is use as a fermentor
and before starting the fermentation process, it is fumigated by placing it on a bamboo
frame constructed over the fire place until the pot turns blackish. There after the boiled
rice are spread over a large banana leaf and allowed to cool. To this powdered apop pitha
is added (1 apop pitha for 1 kg of rice) and the whole mixture is kept inside the fermentor
and the mouth of the pot is covered with banana leaves or leaves of bhilongoni
(Cyclosorus exlensa). The fermentor is left for a period of at least 5 days. A little water is
added to the fermented product and is filtered to get the apong (Das et al., 2012). Similar
type of starter is also prepared by the Khampti tribe of Arunachal Pradesh locally
known as Khamtip for preparation of Apong (Srivastava et al., 2012).
Preparation of Bakhor, Surachi/Phap by the Rabha tribes, Assam (Deka and Sharma,
2010)
Rice-beer cake is popularly known as bakhor, surachi or phap among the Rabhas of
Goalpara district while rice-beer is known as choko or jongamod. Paste of 2 kg wet
seeds of rice (Oryza sativa L.) is prepared after mixing several plants. A considerable
amount of old rice-beer cake is mixed along with these plant materials for the
preparation of fresh rice-beer cakes. Some round and flat globules (each of around 50
gm) are prepared from the grinded mixture. The globules are placed on straw. Different
parts of ten plant species are used in particular amount to prepare rice-beer cake.
Heteropogon contortus is kept scattered on a broad sieve made of bamboo (Bambusa

vulgaris) and then sun dried. To prepare rice-beer choko or phap, tightly cooked fresh
rice (using less amount of water) is used. After cooking, Rabhas scatter rice on a broad
mat made of bamboo and cooled. Then a particular amount of rice beer cake (generally
2 pieces for boiled rice prepared from 2 kg of fresh rice) is powdered and mixed with
the cooked rice. A special type of cylinder made of bamboo net, known among Rabhas
as janthi, is placed inside an earthen pitcher, known as jonga. Now, already prepared
mixture of rice and rice-beer cake is kept inside jonga and outside janthi. At last, open
mouth of jonga is tightly sealed with banana (Musa balbisiana) leaf warmed in fire and
placed in a dark place. Rabhas place Ricinus communis L. leaf and one piece of wood
charcoal over the lid of jonga made of banana leaf ward off the effect of evil sprit.
During summer, (after 4-5 days) and during winter (after 7-8 days), choko or rice-beer
attains the actual stage for drinking. A hole is made at the venter of dried shell of
matured fruit of Lagenaria siceraria Standl. and used to collect the local alcoholic drink
stored inside janthi placed inside jonga. The rice-beer prepared through the above
process of fermentation is again fermented adding particular amount of water and rice-
beer cake. After 3-4 days, rice beer is collected and distilled through a local process
using 3 earthen or metalic pitcher-like pots (hadi / luduki), placing one over another;
2nd and 3rd pots having a hole at the bottom. The whole equipment is made air-tight
using jute (Corchorus capsularis L.) fibre and mud at the junctions. The resulting drink is
strong liquor, known as fotika and Rabhas believe that it has curative effect on
psychiatric patients.
Preparation of epop (starter culture) by Mishing tribe of Assam (Kardong et al., 2012)
The epop is prepared from rice, preferably glutinous, soaked in water for about 2 hrs.,
ground together and mixed properly with semidried powder of leaves or the whole
plant/plant parts which is then kneaded to specific shape (usually oval) followed by the
transfer (inoculation) of microbial consortium with desired quality from the old stock of
previous batch culture. The preparation of epop and the poro apong is somewhat
similar with babud preparation reported from Phillipines and the Ipoh preparation by
Adi tribe of Arunachal Pradesh, India (Tiwari and Mohanta, 2007). Mishing tribe
generally uses at least 16 different herbs with different interpretations (Table-1). This
number may vary from place to place, persons to persons within the tribe itself.
Preparation of Hamei as a starter by Meitei tribes, Manipur (Singh and Singh, 2006)
Yu is a distilled product of the fermented local rice of Meitei communities of Manipur.
For quality and more alcohol production of Yu, Hamei (a fermented product) is added
generally, because of its action as a starter/catalyst. Moreover, the preparation of Hamei
is a popular domestic business for the people of scheduled caste and tribes, as it is also
used as an ingredient of cattle foods. The traditional practitioners believe that, the
quality of Hamei fermentation will be responsible for the quality and quantity of ethyl
alcohol.

Hamei has different name viz. Andro, Sekmai, Phayeng, Jiribam, Bishenpur, Tengnoupal etc
and prepared using similar ingredients and methods except with the slight differences
in shape, size and coverings during the process of fermentation. White rice of about 3
kg was pre-soaked for about half an hour and dried for 15 min to remove excess water.
The white rice is prepared traditionally by pounding in a wooden mortar (Shumban)
with a wooden mallet (Shuk) and the powder mass thus obtained is called Yam. Finely
chopped or powdered about 250-300 gm dried bark of Yanglee (Albizia myriophylla
Benth.) plant is mixed with required amount of water and filtered. The filtrate obtained
appears brownish in colour. A homogenous mixture paste is prepared by mixing Yam
and Yanglee filtrate. From this paste mass, a cake like structure in the form of elliptical
or rounded flattened mass is prepared known as Hamei. Pressing a small portion of
paste mass in between the palms does the preparation of Hamei cake in the form of a
flattened mass. The shape, sizes and forms are changed according to the convenience of
the practitioner.
Preparation of Humao as a starter by Dimasa tribes, Assam (Das and Deka, 2012)
Dimasa tribe of Assam prepared rice beer popularly known as Judima. The starter
culture needed for Judima preparation is humao. For preparing humao, brown rice is
soaked in water for 10-12 hours at room temperature. It is then crushed with the barks
of Glycyrrhiza glabra L. The mixture is then made into paste by adding water and flat
cakes are prepared from this mixture and sundried (Chakrabarty et al., 2009). For
preparing judima, rice is first cleaned and washed. This is then cooked and dewatered.
After cooling it is mixed properly with humao in appropriate quantity. This mixture is
then spread on a banana leaf for overnight and then transferred to an earthen pot and
made partially air tight. Fermentation is allowed to take place at ambient temperature
for 3-4 days during summer and 6-7 days during summer. The resultant juices are
known as judima (Chakrabarty et al., 2009). In similar manner the North East tribes used
Acacia pennata bark, are cut into small pieces and dried in the sun. Rice is soaked in
water until it is softened. It is then grinded in a wooden or metallic mortal pestle called
rimin along with the barks of Acacia pennata. A little water is added in order to make a
paste. They are then made into cakes of appropriate sizes and allowed to dry for a
period of one week. They can be stored for many months (Das et al., 2012).
Preparation of Siiyeh/ Siye/Opop/Ipof/Ipoh by tribes of Arunachal Pradesh (Das and

Deka, 2012; Das et al., 2012)
Apong and Ennog/ Sai Modis are alcoholic rice beverage prepared by the tribes of
Arunachal Pradesh and the Mising tribes of Assam (Tiwari and Mahanta, 2007; Das and
Deka, 2012). Both bears very important place in the tradition of the people of their
region. The starter culture used for their preparation is called Siiyeh/
Siye/Opop/Ipof/Ipoh which contains the yeast to carry out the fermentation. For
preparation of ipoh rice is first dried and grinded into fine powder. This is then mixed

with powder of seeds and barks of the locally available plants Veronia cinerea Less and
Clerodendron viscosum Vent. This mixture is taken into a vessel called Dekchi and made
into a paste using water of previously prepared apong. This paste is poured and spread
on bamboo mats and made into disc shaped small cakes. They are then carefully dried
over the fireplace or left in a cool place for 3 to 4 days. After drying they can be stored
for up to a year (Tiwari and Mahanta, 2007). Similarly for preparation of Ennog/Sai
Mod (black beer) rice is first boiled and spread on a bamboo mat to cool.
Simultaneously, paddy husk is filled into a large tin sheet or drum and allowed to burn
slowly and evenly till they become black. The burnt husk, while still hot is mixed with
the boiled rice and allowed to cool. After cooling, the mixture is again mixed with
crumbled ipoh cakes and transferred to bamboo basket for fermentation.
Preparation of Khekhrii by the Mao tribes of Nagaland (Mao, 1998)

Khekhrii is a unique starter culture prepared from fermented germinated rice in
Nagaland by Mao tribes (Mao 1998). The ethnic alcoholic drink prepared using
Khekhrii is called zutho or zhuchu. Water used for making khekhrii is traditionally
brought in a gourd (Lagenaria siceraria [Molina] Standley) (family Cucurbitaceae) shell
from a spring (Mao, 1998). Mao tribes believe that if water is brought in any other jar it
may spoil the starter culture. Rice is collected and cleaned and put into an earthen jar
that is filled with water brought in the gourd shell. Two pieces of charcoal and two
fresh twigs of Elscholtzia blanda Benth. are also put into the jar. Khekhrii makers believe
that the addition of charcoal pieces and Elscholtzia blanda are important, acting as
antimicrobial regulators to keep fermenting rice from contamination. The mouth of the
jar is closed tightly with fresh leaves of Justicia adhatoda Nees. and the jar is kept in a
warm place for 7-14 days, depending on the room temperature. In general, it takes
about a week in summer and two weeks in winter. At the end of fermentation, a typical
flavor comes out when the mouth of the jar is opened. After fermentation, the contents
of the jar are poured into a sieve and the water is discarded, and the fermented paddy is
kept inside the basket. The basket is opened after germination of the paddy has taken
place. The germinated paddy is then dried in the sun and stored in a dry container for
use as a starter, called khekhrii which is pounded into powder and used in the
preparation of zutho or zhuchu, a traditional alcoholic beverage of Nagaland.
Preparation of Mod pitha (natural starter) by Deori tribe of Assam (Deori et al., 2007)
Saol (rice grains), plant species (Table 1), Kula (a round bamboo utensil), Saloni (round
bamboo utensil or sieving), Dheki (wooden grinder), Dhua sang (a rectangular frame
made of bamboo), Soriya (aluminium utensil) and Kher (straw) are required for the
preparation of Mod pitha (Figs. 1-3). A handful each of cleaned leaves, fronds, barks,
roots and bulb of the plant parts are put in a Saloni and kept for a day for sun drying. 3-
5 kg of Saol is soaked in water for about 2 hrs, mixed with the dried plant materials and
grounded in a Dheki. The grounded powder is taken out, sieved in a Saloni and the

coarse part is returned to the Dheki for grinding. The process is continued until a fine
powder is obtained. 2-3 old Mod pitha are added to the mixture while grinding, which
acts as an inoculant. Grounded powder is put into a Soriya, water is added to make a
sticky paste and small round cakes (2-3 cm in diameter and ca.1 cm in thickness) are
prepared. Cakes are then kept on clean, dry paddy straws spread on a Kula (a round
bamboo utensil) and again covered with straws. Kula is then kept on a Dhua sang tied
about 1 m above the fireplace in the kitchen for drying. This procedure of baking
continues for a couple of weeks until the Mod pitha becomes hard. Pitha is then ready for
use in Sujen brewing. Unused Mod Pihta is stored in Tekele (small earthen pot), mouth of
which is covered with a bunch of straws. It can be stored for 2-3 months and can be
used as and when required.
Preparation of Marcha by the tribals of Darjeling and Sikkim (Tamang, 2010)

Marcha are dry round to flattened, creamy, white to dusty white, solid ball which is
used as an amylolytic starter to produce ethinic alcoholic beverage in the Himalaya
Regions (Darjelling hills, Sikkim) of India, Nepal, Bhutan and Tibet in China (Tamang,
2010). It has various vernacular names among the Himalayan tribes viz. Phab by
Tibetians and Bhutia; khesung by Limboo; Bharama by Tamang; Bopkha or Khabed by
Rai, Buth/Thanbum by Lepcha; Poo by Drukpa, Manapu and Mana by Newar
(Tamang, 2010).
For marcha preparation, glutinous rice (Oryza sativa) is soaked in water for 8–10 h at
ambient temperature and unheated soaked rice is crushed in a foot-driven, heavy
wooden mortar and pestle. In 1 kg of ground rice, ingredients added include roots of
guliyo jara or chitu (Plumbago zeylanica), 2.5 g; leaves of bheemsen paate (Buddleja asiatica),
1.2 g; flowers of sengreknna (Vernonia cinerea), 1.2 g; ginger, 5.0 g; red dry chilli, 1.2 g;
and previously prepared marcha as mother culture, 10.0 g. The mixture is then made
into a paste by adding water and kneaded into flat cakes of varying sizes and shapes.
These are then placed individually on the ceiling–floor, above the kitchen, made up of
bamboo strips inlaid with fresh fronds of ferns (Glaphylopteriolopsis erubescens), covered
with dry ferns and jute bags and are left to ferment for 1–3 days, depending upon the
temperature. Completion of fermentation is indicated by a distinct alcoholic and ester
aroma and puffy/swollen appearance of the marcha (Tamang, 2010).
Preparation of Perok Kushi by the Deoris tribals of Assam (Das et al., 2012)
The indigenous rice beer of the Deoris is known as Sujen, prepared using starter material
perok kushi. The plant materials used for preparing perok kushi are leaves of Jasminum
sambac, Cinnamomum byolghata, Zanthoxylum hamiltonianum, Lygodium flexuosum,
Acanthus leucostychys, Cyclosorus exlensa, Alstonia scholaris and roots of Alpinia malaccensis
and the stem and rhizome of Costus speciosus. All these are washed and cut into small
pieces, dried and grinded using a specialized wooden grinder called as dheki. The

mixture is then soaked in water in a vessel until the water becomes coloured. The whole
mixture is added to grinded rice in a vessel in order to make dough. Round balls of
about 4 cm diameter is made out of this and dried either in the sunlight or over the fire
hearth by placing in a bamboo mat called as aaphey. After getting dried they are placed
in a bamboo container called as kula the inside of which is laid with kher (paddy straw).
Its mouth is again covered with kher and is kept over the hearth for storage. They can be
kept in this manner for many months and can be used as and when required.
Preparation of starter (Ranu dabai) by the tribals of West Bengal (Ghosh and Das,
2004)
Ranu dabai is the starter used for preparation of Jhara/Harhia similar to Hnadia
preparation by the tribes of West Bengal. Ranu dabai are the mixture of roots, barks,
rhizomes, leaves of about 10-12 plant species (Table-1) and binded with the rice flour. 10
Kg of rice grains are taken on a flat traditional utensil generally made of sliced bamboo
(„Soop‟) washed properly. Clean water is poured in it, stirred and decanted. The
decanted wash-water is preserved in a bucket for future use. The most common plants
viz. tuberous roots of Coccinia grandis (500 gm); leaves of Clerodendrum viscosum (300
gm); whole plant of Vernonia cinerea (350 gm) and leaves of Plumbago zeylanica (250 gm)
are taken and chopped and ground properly on Soop. All rest plant (50-100 gm only)
depending on the availability are taken and added for preparation which will improve
the quality of the starter. In some cases 300 gm of Rauvolfia serpentina roots can replaces
Coccinia grandis roots. Rice grains are then put in the pit of wooden husking machine
(Dhiki) and where partially powdered a few (3–4 large tablets for 10 kg of rice) old Ranu
Dabai are added. After some time, the plant paste is also added to it and allowed to mix
properly. When the rice grains are properly powdered and mixed with plant paste, it is
then taken out on a sieve (Chakni) and the coarse part is returned to the wooden
husking machine. After completing sieving, woody and fibrous materials are rejected.
The powdered material (Gunda) is now taken in a large vessel (Dikchi) and made into
paste using the previously stored rice wash water. The paste becomes slightly greenish-
white and emits the smell of mixed herbage. Clean gunny bags are then spread on the
floor under shade or inside the rooms. Different size such as small, medium and large
(standard size is 4.5-7 cm in diameter), are prepared by pressing with hand and
arranged in rows on the gunny bags, where these are kept for 40-60 minutes. Tablets
loose some amount of water and become little tough. All the materials are taken in a
large basket (Dhakiya) made of sliced bamboo. Clean and dry straw is spread on the
bottom of the basket and some tablets are kept on it. These are then covered with straw
and another layer of tablets is kept on it. The entire set is covered with polythene sheet
and/or gunny bags and stored in a dark and warm place. The incubation period varies
from 2-3 days in warm season and 4–6 days in winter. The inside temperature rises
considerably (fever) and the set starts emitting pungent Harhia-like smell. During this, a
layer of cottony mycelia develops on the tablets. The fungal mycelia produce a mat of

black sporangia in damp weather or if stored for a slightly longer period. The tablets are
taken out of the basket and are kept in single layer on large sized circular flat bamboo
basket called „Dagra‟ and get dried under the sun for 7–8 days. Now, the Ranu Dabai is
ready for storing and for use.
Preparation of fermentation cake (Ranu goti/ Ranu tablets) by the tribals of Central
India (Kumar and Rao, 2007)
In the preparation of Handia and Mahua, Ranu tablets play an important role, act as
yeast starter or fermentor, and help in fermentation of both beverages. Ranu tablets or
Ranu goti are the mixture of roots, barks, rhizomes, leaves of about 20-25 plant species
(Table-1) and binded with the rice flour. The preparation is almost same with the
preparation of Ranu dabai with only difference in plant species used and their ratio. For
preparation of tablets, rice is soaked in water, pounded, and kept in shady place for
drying. The plant species used in preparation of Ranu goti are collected mostly from
forests in wild condition. The roots, leaves, bark, seeds of the plants are sun dried and
pounded, powdered and dried in sun. The powder is mixed with flour thoroughly in
the ratio of 1:2, and rolled in small pieces in the form of small cakes. These tablets are
kept in closed room for drying. After drying, these Ranu tablets or Ranu goti are used for
preparing local beverages Handia.
Preparation of Thiat by the tribes of Mahalaya (Samati and Begum, 2007)

Leaves of Amomum aromaticum Roxb. are cleaned, sun dried and ground into powder in
a mortar made of hard wood of Schima wallichii (DC.) Korth (Thlong) by a pestle made
of hard wood of Docynia indica (Wall.) Decne (Surai). Then, 1-2 kg of Oryza sativa L.
soaked in water is also ground in a mortar made of hard wood of Schima wallichii by a
pestle made of hard wood of Docynia indica (Wall.) Decne (Surai). The process is
continued until a fine powder is obtained. Thiat (natural yeast) cakes are made from the
ground rice powder mixed with Amomum aromaticum Roxb (Khaw-iang/Haw-iang)
leaves powder, which are put in a cone-shaped basket (Khrie) and spring water (Um-
pohliew) is added to make a sticky paste and small round cakes are prepared with
standard size of 4-5 cm in diameter and 0.8-1cm in thickness. These cakes are then kept
in a round basket (Malieng) and covered by leaves of banana. A round basket (Malieng)
is hanged on a bamboo made a rectangular frame, which is exposed to sunlight or tied
about 1.20-1.50m above the fireplace/hearth for drying until the cakes get harden and
then are used for rice brewing as natural yeast.
Preparation of Vekur pitha by the Ahoms tribes of Assam (Saikia et al., 2007)
Vekur Pitha is the starter prepared by the Ahoms tribes of Assam for preparation of rice
beer xaj-pani (Saikia et al., 2007). For preparing vekur pitha, a mixture of plant leaves viz.
Lygodium flaxuosum Linn., Leucas aspera Spreng, Cissampelos pereira, Scoparia dulsis Linn.,
Cinamommum glanduliferum Meissn. and Piper betle Linn. are dried in the sunlight for 1-2

days. These are then grounded to powder and mixed with powdered rice in a vessel
with some amount of water. To this powder of previously prepared pitha called ghai
pitha is added which serves as a source of yeast. The mixture is then made into disc
shaped cakes and wrapped with banana leaves (Musa paradisiacal Linn.) and kept in air
locked condition above the fire heart for 4 to 5 days. After getting dried, the cakes are
known as vekur pitha which serves as the source of Saccharomyces cerevisae and can be
stored for future use.

Survey and documentation of preparation of Handia: A survey was conducted in 10
selected localities (Palbani, Takatpur, Roxy Road, Chhaupadia, Baghra Road,
Tulasichoura, Darogadahi, Station Bazar, Hospital Road and Baripada Hat) of Baripada
city in the district of Mayurbhanj where preparation and large scale selling of handia is a
normal practice. Information was collected on methods of preparation, raw materials
used and frequency and timing of consumption through informal personal interview (N
= 5 at each sampling point) among various tribal communities. Accuracy of the
information was ensured through cross verification.
Survey and documentation of plants in Bakhar preparation: Survey was conducted in

different villages of Mayurbhanj district of Odisha and gathered information through
questionnaires and personal interaction with native tribal people regarding the use of
plants and plant parts in the preparation of starter culture and its detail method of
preparation is recorded. As most of the tribals obtain the principal constituents in form
of the powder plant parts from the market, so a survey was conducted by showing the
plant parts along with their dried form. Accuracy of the information was ensured
through cross verification. The plant specimens used in Bakhar preparation were
collected, identified and deposited as voucher specimen in Department of Botany,
North Orissa University. Medicinal uses of the same plants were obtained by
interviewing (once only) traditional healers of 24 villages of the district (Figure-2). All of
them were males with an average age of 46 years. Prior informed consent was not
taken from the informants as most of them were professionals and reputed in their
respective areas and prescribed plant preparations for different ailments.

Figure-2: MAP of Mayurbhanj district with sampling site (*)

Collection and processing of plants: Bark, flower, leaves, roots, rhizome and young
shoot of plants have separately been collected during field trips to different places of
Mayurbhanj. The roots are dug out from the soil and the adhering soils were removed
by shaking and washing. The leaves were plucked from the trees, washed properly and
infected leaves were discarded. After collection, the healthy leaves were dried at room
temperature to maintain their green color and volatile oils, if present. The material is
completely shed dried so long it does not allow for the growth of any type of fungi,
molds, bacteria and other microorganisms. The dried bark, flower, leaves, roots and
rhizome are powdered separately by using mortar and pestle.
Extraction of plants: Hundred grams of each powdered samples were dissolved in

200ml of ethanol separately in wide mouth bottle. The suspension was then filtered
(Whatman No. 40) separately and utilized for studying antimicrobial properties and
phytochemicals. Ethanol extract was dried in rotary evaporator (Sonax, India) at 40 °C
and store in refrigerator for further study.
Phytochemical Analysis:
Qualitative phytochemical analysis was carried out using methods described by Trease
and Evans (1989). Each extract was screened for presence of alkaloids (using Mayer‟s,
Wagner‟s, Hager‟s and Dragend dorff‟s reagents); flavonoids (NaCl and HCl);
carbohydrates (using Molisch‟s reagent); glycosides (using Keller Killiani and
Borntrager‟s reagents); protein and amino acids (using Biuret, Xanthoproteic,
Ninhydrin and Millon‟s reagent); tannin and phenolic compounds (FeCl3 and Gelatin);
triterpenoids (thionyl chloride solution); steroid and sterols (using Liebermann
Burchard and Salkowski‟s reagents), fat and fixed oils with alcoholic KOH reagents.
Antimicrobial activity
Enteric pathogens viz. enteropathogenic and enterotoxigenic Escherichia coli,
Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, S. sonnei, Staphylococcus
aureus and Vibrio cholerae were used in the present study as discussed earlier by Panda
et al., (2010). The antibiogram was carried out by adopting disc diffusion method using
several antibiotics. The result of the antibiogram was published in our earlier report
(Panda et al., 2010). The agar cup method and MIC were used to study the antibacterial
activity. Broth microdilution technique adopted using 96-well microtiter plate and
tetrazolium salt, 2,3,5-triphenyltetrazolium chloride (TTC), was carried out to determine
the MIC following the method as described by Eloff, (1998). Selected extracts were
serially diluted in the 96-well plate with an overnight culture of microorganisms (0.5
McFarland) grown at 37°C to obtain the final concentration of extracts ranging from 78
to 2500 µg/ml. The microplate was sealed and incubated at 37°C and observed for the
growth of the microorganism. 10µl of the broth from each well of 96 microtiter plate
(≤MIC) and control wells were taken aseptically and plated on one day old MH agar

plate as a point inoculum and allowed to dry for 10 min. under the laminar air hood.
These plates were then sealed and incubated at 37°C for 24 hours and observed for
growth of the bacteria. Absence of growth of the bacteria showed the MBC result of the
respective bacteria.
RESULTS
Handia preparation includes two distinct phases: preparation of ranu or bakhar tablets
and making of handia.
Ethnomedicinal uses, Preparation of Bakhar or Ranu tablets

Ranu or bakhar tablets act as starter for fermentation. Ranu tablets are mixtures of
various plant parts (50%) and powdered un-boiled rice (50%). The plant species and
parts thereof used for the purpose along with local names, family, parts used are listed
in Table-2 along with its medicinal properties throughout India. Survey was carried out
and plants are collected after documenting its ethnomedicinal uses are listed in Table-3
and Figure-3. Some species viz. Asparagus racemosus (Willd.), Cissampelos pareira L. var.
hirsuta (DC) Forman, Clerodendrum serratum (L.) Moon, Coccinia grandis (L.) Voigt,
Holarrhena antidysenterica Wall ex. A. DC., Woodfordia fruticosa (L.) Kurz. and Benth. are
commonly used by the tribal of all localities while plants such as Madhuca longifolia
(Koenig), Smilax macrophylla (Roxb.), Rauwolfia serpentina (L.), Elephantopus scaber L.,
Gardenia gummifera L.f. and Dioscorea sp. are rarely use. Depending on the season and
availability in a particular locality, plant parts of one or more species are used. The
accurate ratio of different plants used for ranu preparation could not be ascertained as
the informants were reluctant to disclose the same. However, C. pareira forms the major
part in most of the preparations (70%) followed by other plants in combination (1-30%).
R. serpentina and G. gummifera are used in very small proportion. According to one
informant (Sama Singh, Male, Age-62) the ratio of the plant (root) is 6:2:1:1 (C. pareira:
W. fruticosa: A. racemosus: H. antidysenterica). Preferred parts and plants varied at
different places. Dried root, stem and other parts used for the purpose, both as such and
powdered, are abundantly and openly sold in the local markets (Figure-4. a & b).
Powdered plant ingredients are mixed with equal amount of rice (Oryza sativa L.)
powder. A suitable amount of water is added to make dough. Ranu is prepared in the
form of rounded tablets and spread over straw beds in layer over layer with a final thin
layer of straw cover. After 3 days, the ranu tablets are picked up from straw beds and
dried under sun for about 2 days and stored for use in fermentation of rice beverages
(Figure- 3. e). These tablets are not only used for fermenting rice beverage but also used
for treatment of various ailments.

Figure-3: Plant parts used for preparation of Bakhar
a. Asparagus racemosus root
b. Cissampelos pareira root
c. Clerodendrum serratum root
d. Coccinia grandis rhizome
e. Dioscorea sp. tuber
f. Elephantopus scaber root
g. Holarrhena antidysenterica bark
h. Madhuca longifolia flower
i. Woodfordia fruticosa root

Table-2: Phytotherapeutic uses of plants that are used in preparation of ranu tablets
Sl. Name of the plant Local name Family Parts Medicinal uses References
No. used
1. Asparagus Gaisiro Liliaceae Root Nutritive tonic and Thatoi et al.,
racemosus Willd. demulcent, cures 2008; Wani
fever, sexual debility, et al., 2011
leprosy, bronchitis
and cough.
2. Cissampelos pareira Andiakidula Menisper- Root, Leaves used for Rout &
var. hirsuta (Buch.- maceae Leaf cough, urinary Panda,
Ham. ex DC.) troubles, diarrhea, 2010; Thatoi
Forman inflammation and et al., 2008
colic pain.
3. Clerodendrum Samarkand Verbenaceae Root, Roots used in fever, Kirtikar et
serratum ( L.) Leaf snakebite, asthma al., 2005
Moon and cough.
4. Coccinia grandis Banokunduri Cucurbitaceae Root Ear pain, jaundice, Panda et al.,
(L.) Voigt tuber blood dysentery and 2011;
diabetes. Tamilselva
et al., 2011
5. Dioscorea sp. Sanga Dioscoreaceae Root Not known.
tuber
6. Dipteracanthus Chaulia Acanthaceae Root Renal problems. Nayar et al.,
suffruticosus 1956
(Roxb.) Voigt
7. Elephantopus scaber Tatmuli Asteraceae Root Diarrhea, dysentery, Panda et al.,
L. colic, vomiting, 2011
headache and tooth
ache.

8. Gardenia gummifera Bhurudu Rubiaceae Young Used as a digestive Kirtikar et
L.f. shoot tonic and antiseptic. al., 2006
9. Holarrhena Kuruchi Apocyanaceae Bark, Bark is Kirtikar et

pubescens (Buch.- Seed antihelminthic, al., 2006;
Ham.) Wall. ex. G. antipyretic and used Panda et al.,
Don in dysentery. Seeds 2011; Thatoi
are astringent, used et al., 2008
in cough, cold, fever,
scabies, leprosy and
malaria.
10. Homalium nepalense Danmari Flacourtiaceae Bark Stomach disorder. Nayar et al.,
(Wall.) Benth. 1956
11. Lygodium Mahajal Lygodiaceae Root Fresh roots used as Panda et al.,
flexuosum (L.) Sw. expectorants and 2011
healing wounds.
12. Madhuca longifolia Matkam Sapotaceae Seed, Seed oil used in Kirtikar et
(Koenig) MacBride Leaf, rheumatism. al., 2006
var. latifolia Roxb. Bark Leaf and bark used Nayar et al.,
for diabetes. 1956
13. Ochna obtusata DC. Otchampa Ochnaceae Root Used as antidote for Kirtikar et
var. obtusata snake bite, menstrual al., 200517
complaints and
asthma.
14. Orthosiphon Chandua Lamiaceae Root Used to cure stomach Nayar et al.,
rubicundus tuber disorder. 1956
(D.Don) Benth.
15. Polygala Lilkathi Polygalaceae Bark Cold decoction of Nayar et al.,
crotalarioides bark for cough. Used 1956

Buch.-Ham. ex. as antidote against Kirtikar et
DC snake bite. al., 2005
16. Phoenix acaulis Khajuri Arecaceae Root Used as laxative. Nayar et al.,
Roxb. ex. Buch.- 1956
Ham.
17. Rauwolfia Patal Garuda Apocynaceae Root Used as antidote for Nayar et al.,
serpentina (L.) snake bite, malaria. 1956
Benth. Rout &
Panda, 2010
18. Smilax macrophylla Ramadantani Smilaceae Root, Roots used for Panda et al.,
Roxb. Stem urinary complaints 2011
and dysentery. Stem
used in tooth ache.
19. Woodfordia fruticosa Icheba Lythraceae Flower Dried flowers used Panda et al.,
(L.) Kurz as astringent, 2011; Thatoi
leprosy, burning et al., 2008
sensation and liver
disorders.
20. Xantolis tomentosa Ghurmur Sapotaceae Fruit Used as an antiseptic Rout &
(Roxb.) Raf. and digestive tonic. Panda, 2010

A paste of ranu tablets with saliva is applied on mumps by the tribes before sleeping to
get relief. Santal tribals also disinfect silk worm (tasar) eggs during indigenous rearing.
From the study conducted in the various villages, it is evident that all tribes use some
plants for starter preparation; however the type of plant varies from tribe to tribe and
village to village. The tribal peoples of Sukruli and Karanjia block use the same plant
viz. C. pareira, C. serratum, C. grandis, Dioscorea sp. for starter preparation. Similarly
tribes of Bijatola and Rairangpur use the
Figure-4: Preparation of Bakhar

a. Clerodendrum serratum root powder
b. Cissampelos pareira root powder
c. Asparagus racemosus root powder
d. Holarrhena antidysenterica bark powder
e. Drying of Bakhar in mats
f. Bakhar in large quantity for sale at Baripada hata
g. Bakhar prepared by a tribal (male) at Gorumahisiani, Mayurbhanj

same plant in addition to A. racemosus instead of Dioscorea sp. However, the tribes of
Samakhunta use different plants A. racemosus, C. pareira, H. antidysenterica, M. longifolia,
S. macrophylla and W. fruticosa for the same purpose. This is an age old practice in these
communities which is followed generation after generation.
Preparation of Handia
Mechanically de-husked rice is soaked and boiled in water. The cooked rice is dried on
a bamboo mat under sun (Fig. 1d). After drying, the rice is mixed with required amount
of powdered ranu tablets (approximately 10 tablet per kg rice), kept in a large earthen
pot or handi (hence the name of the product) followed by addition of required amount
of water.
Table-3: Ethnomedicinal uses of plants used for preparation of Bakhar

Sl. Name of the plant Ethno-medicinal uses
No.
1. Asparagus racemosus Willd. Root paste in water is administered (10 g) two
times a day in gonorrhea and jaundice. Root is
taken as tonic.
2. Cissampelos pareira L. var. Filtered root juice is taken with water to cure colic.
hirsuta (DC) Forman
3. Clerodendrum serratum ( L.) Paste of leaves applied locally for treatment of
Moon septicemia, worms and foot diseases
4. Coccinia grandis (L.) Voigt Fresh leaves along with leaves of Kalanchoe pinnata
and sugar are ground with water and taken twice a
day for four to five days to cure jaundice.
5. Dioscorea sp. Tuberculous rhizome are cooked and eaten as
famine food.
6. Elephantopus scaber L. The entire plant is cooked with rice and eaten to
cure migraine. An entire root is tied over forehead
to get relief from headache.
7. Gardenia gummifera L.f. The leaves are used in stomach pain and stems
against teeth pain.
8. Holarrhena antidysenterica Stem bark infusion with honey (3:1) is taken once a
Wall ex. A. DC. day in empty stomach for cure of dysentery. Bark
of the plant and black pepper are powered together
and orally taken against malarial fever.
9. Madhuca longifolia Koenig Flowers are boiled in water with a pinch of salt and
the decoction is given with honey, thrice a day for
seven days for cure of piles and fistula.
10. Rauwolfia serpentina (L.) Juice extracted from leaves mixed with the juice of
Benth. Andrographis paniculata and Azadirachta indica and

given it with honey to drink for seven day
continuously to cure malaria.
11. Smilax macrophylla Roxb. Roots are boiled in water, is given orally with
honey to cure gastric problems.
12. Woodfordia fruticosa (L.) Juice is good for treating dysentery. Leaves are
Kurz used for edible purpose.
The mixture is kept untouched for 3-4 days for fermentation. After proper fermentation
a white supernatant was present at the upper layer containing 8-10% alcohol called
rashi, which fetches higher price. After 2-3 days the fermented liquid is allowed to
trickle down through a bamboo sieve, collected in earthen pots and is ready for
consumption (Fig. 1e &f). The taste of handia depends on the plants used for ranu/bakhar
preparation. About 8-10 bakhar tablets are used for 1 kg of rice, which together produce
about 10 L of handia. The quality gets lowered on dilution. On an average, some 30% of
families prepare handia for their own consumption. Per capita consumption amounts to
be about 1 L/day. Consumption is much higher during summer and hence, it is
essentially a summer drink (Figs. 1g & h). It keeps the stomach cool, protects from
extreme heat and is also intoxicating. Handia preparation and selling is a secondary
source of livelihood for tribals and some accept it as a primary occupation. It is used in
all social, cultural and religious purposes and no social occasion is considered complete
without it. The Santals believe it to possess medicinal value, i.e. use it in the cure of
jaundice, colic disorders and dysentery.
Screening of phytochemicals and antimicrobial activity of plants parts used in starter

The qualitative phytochemical analysis of ethanol extracts indicated the presence of
alkaloid, flavonoid, carbohydrate, protein and amino acid, tannin and phenolic
compounds and saponin in most of the plants (Table-3). However, glycoside, steroid &
sterols, gum & mucilages, oil & fats and triterpenoids were found in less number of test
plants. Alkaloid, carbohydrates, and saponin are universally present in all test plant
extracts. Preliminary screening of antimicrobial activity was evaluated by using agar
cup method against eight human pathogenic bacteria are given in Table-4. The zone of
inhibition was found maximum against S. aureus followed by S. sonnei and S. flexneri.
Organism such as S. typhimurium and V. cholerae show moderate zone of inhibition
while enteropathogenic and enterotoxigenic E. coli and P. aeruginosa show least zone of

Table-4: Phytochemical screening of plant parts used for preparation of Bakhar
Name of the Qualitative test Ar Cp Cs Cg Ds Es Gg Ha Ml Rs Sm Wf

phytochemicals
Alkaloid MR - + + + + + + + - + + +
DD + + - + - + - + - - + +
HR - + + + + + - + - + + +
WR - - - + - - + - - - - +
Carbohydrates MT + + + + - + + + + + + +
FT + + + + + + + + + + + +
BT + + - + + + + + + - + +
Tannin and Phenolic WFC - + + + - - + + + + - +
compounds WLA - - + - - - - + - + - -
WGS - - - - - - - - - - - -
Glycoside KKT + - + + - - - + + - - +
LT + - - - - - - - - - - -
BRT - - - - - - - - - - - -
Proteins and amino BIT - + + + + + + + + - - +
acids NT - + + + + + + + + + - +
XT - + + + - + + + + + - +
MLT - + + + - - - - - - - +
Flavonoids WN - + + + - + - + - + + +
WS - + + + - + - + - + + +
Saponins HCT + + + - - + + + + + + +
FT + + + + + + + + + + + +
Steroids and sterol ST - - + + - - - - - - - +
LBT - - + + - - - - - - - +
Triterpenoids TCT - - + + - - - + - - - -
Oil and fats WFC + - + - - - - + - - - -

Ar- Asparagus racemosus; Cp- Cissampelos pareira; Cs- Clerodendrum serratum; Cg- Coccinia
grandis; Ds- Dioscorea sp.; Es- Elephantopus scaber; Gg- Gardenia gummifera; Ha-
Holarrhena antidysenterica; Ml- Madhuca longifolia; Rs- Rauwolfia serpentina; Sm- Smilax
macrophylla; Wf- Woodfordia fruticosa; MR- Mayer‟s reagent; DD- Dragend droff‟s test;
HR- Hager‟s reagent; WR- Wagner's reagent; MT- Molisch‟s test; FT- Fehling‟s test; BT-
Benedict‟s test; WFC-With Ferric chloride; WLA-With lead acetate; WGS-With gelatin
solution; KLT-Keller-Killiani test; LT-Legal Test; BRT-Borntrager's test; BIT-Biuret test;
NT-Ninhydrin test; XT-Xanthoproteic test; MLT-Millon‟s test; WN-With NaOH; WS-
With H2SO4; HCT-Honeycomb test; FT-Foam test; ST-Salkowski‟s test; LBT-Liberman
Burchard ; TCT-Thionylchloride test; WFP-With filter paper; (-) Absence; (+) Presence
inhibition. No test organisms were inhibited by A. racemosus (root), C. pareira (leaf),
Dioscorea (tuber), R. serpentina (leaf) extract. So these plant extracts are not subjected to
further study on MIC and MBC. The MIC results among all test bacteria are
summarized (Table-5) and showed that extracts were able to prevent the growth of
most of the test strains with selective activities. The growth inhibition of the test bacteria
ranged from 125 µg/ml (w/v) to 1000 µg/ml (w/v) with the lowest MIC value against
S. aureus (125, 156, 250 µg/ml) followed by S. sonnei (156, 250, 312, 500, 625 µg/ml).
MBC test showed that in between 1000-2500 µg/ml (w/v) concentrations, most of test
bacteria were killed.
DISCUSSION
Preparation and consumption of cereal based beverages is a common practice in many
Mid-Asian, Middle East and African countries (Hesseltine, 1979). These are known by
different names at different places: sake in Japan, Shaosingiju and lao-chao in China,
Chongju and Takju in Korea, Brem bali, Tapuy and Tape ketan in Indonesia, khao-mak in
Thailand, Tapai pulal in Malaysia and jhara, daru, kali, pachwai, apong, bunkchung, chi,
laopani, jumai, suze, morpo, jou, zu, mod, harhia and handia in India (Lee, 2009).
Table-5: Screening of antibacterial activity of ethanol extracts of plants

Name of the plant Parts Zone of inhibition in mm (extract volume 40 µl
tested from 20 mg/ml)
EP ET Pa Sa Sf Ss St Vc
Asparagus racemosus Root - - - - - - - -
Cissampelos pareira Leaf - - - - - - - -

Root - - 12 16 14 16 - -
Clerodendrum serratum Leaf 12 - - - 10 - 14 -
Root* 15 12 12 14 17 16 15 14
Coccinia grandis Rhizome* - - - 14 15 12 14 12

Dioscorea sp. Rhizome - - - - - - - -
Elephantopus scaber Root - - - 12 10 - 14 -
Gardenia gummifera Young - - - 12 - - - -
shoot
Holarrhena antidysenterica Bark* 12 - 11 14 12 14 10 12
Madhuca longifolia Flower* - - - 16 14 12 10 -

Rauwolfia serpentina Leaf - - - - - - - -
Root - - - 16 12 14 - 10
Smilax macrophylla Root - 12 - 14 - 12 - -

Woodfordia fruticosa Leaf - 14 13 15 - 15 12 12
Root* 13 18 12 18 12 14 16 14
Ciprofloxacin 15 20 17 24 18 26 24 22
Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic E. coli (ETEC), Pseudomonas

aeruginosa (Pa), Salmonella typhimurium (St), Shigella flexneri (Sf), S. sonnei (Ss),
Staphylococcus aureus (Sa) and Vibrio cholerae (Vc), C-Ciprofloxacin, *Stock concentration
(25mg/ml)

Figure-5: Selling of Bakhar at different localities
a. A women selling bakhar, rice and plant powder at Station Bajara hata,
Baripada
b. A seller selling bakhar and plant powder at Dhenkikote hata, Mayurbhanj
c. A seller selling bakhar at Kaptipada hata, Mayurbhanj
These products have many advantages like superior digestibility and nutritive value
compared to their unfermented counterparts. The consumption of rice beer prepared
from rice is a common practice among many tribal communities residing among the
tribals of Odisha and many of them have been preparing it since time immemorial.
Among the Santal tribes of Mayurbahnj, it is prepared in almost every third house.
More or less all tribals are fond of drinks and consume during every ceremony,
festivals, marriages, funeral feasts, and offer it to their, guests, gods and deities 7. In
marriages, the amount of handia to be given to the girl side and is decided well in
advance.

Table-6: Results of screening of selected extracts for MIC and MBC by 96 well
microtiter plate
Plant parts Plant extracts in µg/ml
used Minimum Inhibitory Minimum Bactericidal
Concentration in µg/ml Concentration in µg/ml
Sa Sf Ss St Sa Sf Ss St
CpR 125 500 250 1000 1000 >2000 1000 >2000
CsR 125 250 250 500 1000 1000 1000 2500
CgRh 156 312 625 312 1250 2500 1250 2500
EsR 125 250 250 250 1000 1000 1000 2000
HaB 156 156 156 312 1250 2500 1250 >2500
MlF 156 312 312 625 1250 2500 >2500 >2500
RsR 250 250 500 1000 1000 1000 >2000 >2000
SmR 250 1000 250 1000 1000 1000 1000 >2000
WfL 250 1000 250 250 2000 2000 2000 2000
WfR 156 156 312 625 1250 2500 1250 >2500
Sa- Staphylococcus aureus; Sf- Shigella flexneri; Ss- S. sonnei; St- Salmonella typhimurium;
Cpr- Cissampelos pareira root; Csr- Clerodendrum serratum root; CgRh- Coccinia grandis
rhizome; Esr- Elephantopus scaber root; Hab- Holarrhena antidysenterica bark; MlF-
Madhuca longifolia flower; RsR- Rauwolfia serpentina root; SmR- Smilax macrophylla root;
WfL- Woodfordia fruticosa flower; WfR- Woodfordia fruticosa root
The consumption of rice beverage emerged mainly due to the climatic conditions and
discovering the use of surrounding natural resources (Roy et al., 2004). There are also
reports of rice beer being used as a drug from North east region of India (Singh and
Singh, 2006). It works effective against insomnia, headache, body ache, inflammation of
body parts, diarrhoea and urinary problems, expelling worms and as a treatment of
cholera (Samati and Begum, 2007; Deka and Sarma, 2010). It cures jaundice, colic, and
dysentery. It protects from sun stroke and maintains the motility and tone of the gastro-

intestinal system. It is taken as a light tranquillizer by Maria tribe of Baster (Sahu, 1996).
It is also given to treat fever, dysentery, diarrhea and gynecological complaints (Usha,
1999). Mahua is given to treat dysentery by Baiga, Gond, and Kol tribes of Surguja district
(Kumar and Rao, 2001). Ranu tablets are also used in treating cholera by Gond tribe of
Surguja district (Kumar and Rao, 2001). The tribes of Mayurbahnj use handia in the cure
of jaundice, colic disorders and dysentery (Panda et al., 2014).
Plant ingredients are used in preparation of starter culture (ranu or bakhar) universally
that are essential for fermentation of rice to prepare beverages. After surveying 12
districts of Odisha including Mayurbhanj (Dhal et al., 2010) recorded the use of bark and
root of six plant species in the preparation of bakhar by the tribals.
Singh, 1996 has reported the use of several plant species viz. mature leaves of Allophylus
cobbe (L.) Blume, Antidesma roxburghii Wall. ex Tul. and tender leaves of Artocarpus
heterophyllus (Lam.), in equal proportions together with little chilli, are used in the
preparation of Choarak, a local wine of Tripura state, India. Tribal inhabitants of tea
gardens in Terai region of West Bengal use 12 plants, specific parts for particular
purpose, for preparation of rice beer jhara or haria (Ghosh and Das, 2004). According to
Ghosh and Das, 2004, Sweetness of the liquor is developed by the use of tuberous roots
of Coccinia grandis L. (Voigt), whole plant including fleshy roots of Veronia cinerea L.
(Lessing) and leafy twigs of Scoparia dulcis L.. Likewise, young and soft leaves of
Clerodendrum viscosum Ventinat, bark of Oroxylum indicum (L.) Benth. Ex. Kurz, root
bark of Rauvolfia serpentina (L.) Benth. Ex. Kurz, and bark of Wattakaka volubilis (L.f.)
Staph. Produce a bitter taste. Leafy branches of Plumbago zeylanica (L.) act as a process
enhancer. Roots of Stephania japonica (Thumb.) Miers and Stephania glabra (Roxb.) Miers,
are used for long storing. Roots of Mussaenda roxburghii (Hook.f.) and leaves of
Artocarpus heterophyllus (Lam.), impart sweetness and yellowish tint to the liquor. The
tribals of Central India use a number of roots, bark, rhizomes, leaves and seeds of some
21 plants for making ranu (Kumar and Rao, 2007). The tribals get the phytotherapeutic
value of these plants through the drink. The use of specific plants and parts thereof is a
tradition and passed through generations. However, due to the loss of biodiversity and
fragmentation of habitat, all the plants are not available in the vicinity of a particular
village.
Wani et al., 2011 screened presence of phytochemicals in A. racemosus root from

Himalaya region, India. According their findings, both alcoholic and aqueous extracts
confirm presence of carbohydrate, glycoside, mucilage and saponin. In a study
conducted by Mandal et al., 2010 have shown antibacterial property of methanol extract
of A. racemosus root against Escherichia coli, Shigella dysentrae, S. sonnei, S. flexneri,
Salmonella typhimurium, Pseudomonas putida and Staphylococcus aureus. However, in the

present investigation antibacterial activity was not recorded against any test pathogens
with the ethanolic extracts of A. racemosus.
Jhuma et al., 2012 studied phytoconstituents from methanolic extracts of Cissampelos

pareira flower and result revealed presence of alkaloid, carbohydrate, flavonoid, protein,
tannin and terpenoids. In the present study similar results were obtained for presence
of phytochemicals in the ethanol extracts except triterpenoids. On the other hand, leaf
extract don‟t show antibacterial activity against any strain while root extract show good
inhibitory property against S. aureus and S. sonnei. This may be due to presence of
certain additional phytochemicals in root comare to leaf.
Prasad et al., 2012 reported presence of alkaloid, anthroquinoles, glycoside, phenol,

saponin, tannin and terpenoids in Clerodendrum serratum. The same authors also
evaluate the antibacterial activity of various extracts and result found that isoamyl
alcohol had better antibacterial property against Bacillus subtilis, S. aureus, S. typhi and
Proteus species. Similar type of results was also recorded by Vidya et al., 2010 during
their study on the same plant. Khatun et al., 2012 studied the antibacterial activity of
various extracts of Coccinia grandis and result found that methanol extracts showed
antibacterial activity against S. aureus, S. dysentrae, E. coli and S. typhi. These authors
also investigated presence of phytochemicals such as flavonoids, phenols, saponin,
tannin and terpenoids. Similar study conducted by Umamaheswari and Chattargee,
2008 shown presence of phytoconstituents viz. alkaloid, glycoside, flavonoid, phenols,
saponin and tannin. Presence of triterpenoids reported by Syed et al., 2009 whereas
proteins and aminoacid by Umamaheswari and Chattargee., 2009 while presence of
both these phytochemicals in the ethanol extract right through the present study.
Povendran et al., 2011 reported antibacterial activity of leaf extracts of C. grandis against
Heliobacter pylori. Nevertheless, in the present study the rhizome extract exhibited very
good antibacterial activity against S. aureus, S. flexneri and S. typhimurium.
Poli et al., 1992 evaluated pharmacological properties of Elephantus scaber and observed
the presence of alkaloid, anthocyanin, chalcones, flavonoid, lactone and triterpenoids.
In another study conducted by Kamalkannan et al., 2012 showed presence of alkaloid,
carbohydrate, proteins and saponin while absence of volatile oil, gum, mucilage and
steroid. The same authors studied the antibacterial property and conclude that the
methanol extract of Elephantus scaber has potential antimicrobial activity against
Streptococcus pyogene while no activity against E. coil, P. aeruginosa, S. aureus and S. typhi.
In contrary to this work the study conducted by Kumar et al., 2004 and Avani and
Neeta, 2005 reported promising antibacterial activity against pathogens such as B.
subtillis, E. coil, P. aeruginosa and S. aureus. At this juncture, antibacterial activity was
confirmed against S. aureus, S. typhimurium and S. flexneri whereas absence of activity
against E. coli, P. aeruginosa, S. sonnei and V. cholerae. Tambekar and Khante[23]

evaluated antibacterial activity of Gardenia gummifera and result originated that this
plant exhibit activity against Enterococcus aerogenes, Klebesiella pneumonae, S. aureus
whereas no activity was evidenced against E. coli, P. aeruginosa, S. typhi, Proteus vulgaris
and S. typhimurium. These authors also reported presence of phytochemicals viz.
alkaloid, flavonoid, glycoside, steroid, tannin and phenolic compounds whilst absence
of carbohydrate, protein and aminoacid. On the other hand, in the present study
antibacterial activity was not observed by ethanol extract of G. gummifera except S.
aureus (zone of inhibition 12 mm).
Ballal et al., 2011 studied antibacterial activity of Holarrhena antidysenterica against

enteric pathogens and outcome confirmed antibacterial activity against EPEC, EIEC, P.
aeruginosa, Shigella boydii, S. flexneri, V. cholerae, S. aureus and S. typhimurium. Study
conducted by Preethi et al., 2010 validated antibacterial activity of H. antidysenterica
against B. subtillis, E. coil, S. aureus and S. typhimurium. Chakraborty and Brantner, 1999
reported antibacterial activity of steroid alkaloid from stem bark of Holarrhena pubescens
against B. subtillis, P. aeruginosa, S. aureus, S. epidermidis, Streptococcus faecalis and S.
typhimurium. The present findings correlate with the study conducted by all these
authors, relating to the antibacterial activity. Chakma, 2011 studied antimicrobial
activity of Madhuca longifolia fruit and conclude that the plant act as a potential agent
against B. subtillis, E. coli, P. aeruginosa and S. aureus. Later, Gopalkrishnan and Shimpi,
2012 carried out pharmacological studied on stem bark of Madhuca longifolia and result
evaluated the presence of starch, protein, terpenoid, glycoside, saponin, tannin while
absence of alkaloid, flavonoid and steroid. In the present investigation, antibacterial
activity was recorded against S. aureus while E. coli and P. aeruginosa was not inhibited
by M. longifolia extract. Nevertheless, the extract revealed presence of phytochemicals
such as carbohydrate, protein, terpenoid, glycoside, saponin and tannin, and these
finding are correlate with the study conducted by Gopalkrishnan and Shimpi, 2012.
Harisaranraj et al., 2009 examine phytochemicals such as alkaloid, flavonoid, phenolic

compounds and tannin in the root extracts of Rauwolfia serpentina. Deshmukh et al., 2012
studied on antimicrobial activity of indole alkaloids from R. serpentina and conclude
that root extracts illustrate better antimicrobial activity in compare to leaf extracts
against B. subtillis, E. coli, S. aureus and S. typhimurium. The same authors at preliminary
level investigated presence of phytochemicals viz. alkaloid, tannin, saponin, flavonoid
and starch. Deshwal and Vig, 2012 studied antibacterial activity of R. serpentina and
proved that ethanol extract showed higher zone of inhibition compare to norofloxacin
against S. aureus. In the present experiment, ethanol extract of root exhibited
antibacterial activity against S. aureus, S. sonnei, S. flexneri and V. cholerae while leaf
extract do not inhibit any test strain. Furthermore, the phytochemical study correlate
with the study carried out by Deshmukh et al., 2012.

Hooda et al., 2011 reported presence of phytochemicals such as carbohydrate, protein,
saponin, flavonoid, alkaloid and tannin in the root extracts of Smilax zeylanica. The
present study finds presence of phytochemicals such as carbohydrate, saponin,
flavonoid and alkaloid. Nonetheless, antibacterial activity is the primary information
reported on ethanol extract of this plant.
Parekh and Chanda, 2007 studied antibacterial activity of W. fructicosa flowers and
result found that methanol extract exhibited potential activity against B. cereus, S.
aureus, S. epidermidis, P. vulgaris, P. seudoalcaligens and S. typhimurium. Later, similar
experiment carried out by Kumarswamy et al., 2008 showed promising antibacterial
activity against E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis, S. typhi, S. boydii, S.
flexneri, S. sonnei and S. aureus. Chougale et al., 2009 also studied antibacterial activity of
fraction of leaf extracts of W. fructicosa with promising antibacterial activity against B.
subtillis, E. coli, S. aureus and P. aeruginosa. Further, Bhattarai and Bhuju, 2011 tested both
leaf and flower extracts of W. fructicosa and concluded that methanol extracts have
antibacterial activity against B. cereus, E. coli, P. aeruginosa, P. mirabilis, S. dysentriae, S.
typhimurium and S. aureus. Finose and Devaki, 2011 investigated presence of
phytochemicals viz. carbohydtate, aminoacid, glycoside, saponin, flavonoid, alkaloid
and tannin in W. fructicosa. Later, Gyawali et al., 2012 establish presence of same
phytoconstituents in addition with terpenoids.
The detail study and uses of these plants clearly indicate that the presence of these plant
materials and their bactericidal activity are mainly responsible for protecting and
preserving starter cultures since traditional system of fermentation normally operates in
unhygienic condition which sometimes contaminates the system and cause toxication of
drinks. But due to the presence of antimicrobial chemical principles of these plants or
plant parts, they are able to continue such practice for generations without much
decline in the characteristics of microorganisms involved in fermentation.
CONCLUSIONS
The study establishes the deep knowledge of tribal people in terms of selection of plants
for preparation of handia and the processing technique. The native skill of rice beer
preparation using unique starter culture technique is well recognized. Both the Bakhar
tablets and handia are used in the treatment of several ailments. Probably, this is central
theme in the preparation of rice beverages by the tribals across the world.
RECOMMENDATION
It is possible that the traditional knowledge of beverage preparation along with various
parts of different plant species used will be a useful lead for phytochemists and
pharmacologists for further study. Once the efficacy of these indigenous drinks is
scientifically established, the popularization of these remedies can be recommended in

conventional healthcare systems for wider applications. Further work is required to
fully characterize the predominant microorganisms and to establish their technological
roles and contribution to product quality and safety.
ACKNOWLEDGEMENTS
The present research has been funded by the Department of Science and Technology,
Government of Odisha (Grant No. ST-BIO-70/2010/ST). We wish to express our
profound gratitude to Dr. A. K. Biswal (Department of Botany, North Orissa University)
for identification of the plant samples. We are also grateful to the authorities of North
Orissa University for providing necessary facilities to carry out this research. Finally,
we wish to thank to all the informants and sellers; they allowed us to take photographs
of plant materials. We also thank to Dr. Abhijeet Das for photographic documentation.
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CHAPTER SIX
USE OF PLANT EXTRACTS IN PLANT DISEASE MANAGEMENT: ROLE AND

SIGNIFICANCE OF CHARACTERIZATION OF SECONDARY METABOLITES
FROM HIGHER PLANTS IN PHYTO-DISEASE MANAGEMENT
Enyiukwu, D N., Awurum, A. N., Ononju, C. C and Nwaneri, J.

Department of Plant Health Management, Michael Okpara University of Agriculture,
Umudike, P.M.B 7269 Umuahia, Abia State Nigeria.
ABSTRACT
Several sources reported the advantages of synthetic fungicides in boosting
food production. However, there are serious concerns and negative
consequences associated with their use on ecological and human health in
recent years. Besides, pathogens’ resistances to some of the most effective
fungicides have been reported. These amongst others prompted search for
alternatives. Plants offer an expanse of exploitable chemical space in this
regard which is unparalleled in nature and not beaten by combinatorial
chemistry as yet.Extracts of higher plants have demonstrated a wide range of
activity against plant pathogenic organisms. These plants extracts have been
found to contain broad spectra of phytochemicals (secondary metabolites) such
as alkaloids, flavonoids, tannins, saponins, phenols, glycosides, terpenoids,
phlobatannins, polyphenols and steroids. Secondary metabolites constitute
plants’ weaponry against pest and pathogen invasion. These groups of
phytochemicals possess wide ranging chemical functional groups; by which
they establish and bind to sites on target pathogens to ineffectuate them.
Complexes of these chemical compounds occur and incrude extracts. Need
therefore arises in the present to identify, isolate and purify the active
principle(s) in the extracts and to determine theone(s) effecting the reported
kills and fungitoxity of the extracts. With the exception of neem assertive
reports on their modes of action are unavailable. We have to understand the
functional groups of the isolates, their position on the carbon skeleton of the
principles, binding sites on target pathogenic species; and the metabolic
process(es) which they affect and ineffectuate. This will aid in either
synergizing or synthesizing them so as to combat the enormous and obvious
challenges of fungicide resistance threating agricultural production and food
security. This work reviews available literature in these regards.
KEYWORDS: Plant extracts, secondary metabolites, Plant disease, chemical

characterization, MOA

INTRODUCTION
The National Bureau of Statistics (NBS) of the Government of Nigeria listed the
important field crops grown in Nigeria. These crops include yam, cocoyam, cassava,
cowpea, groundnut, sorghum, millet, maize, rice, melon and cotton. According to Abate
et al. (2011), FAOSTAT also included soybean as an emerging important field cropalso
grown inNigeria. Statistics show that Nigeria is the world‟s largest producer of yam
accounting for 70-76% of the world production. In 2008 for instance, Nigeria produced
35.017 million MT of yam valued at US$ 654 billion,on 3.045 million ha of farmlands
(Kleih et al., 2012; Wikipedia, 2012). FAO (2006) on the other hand reported that Nigeria
is the largest producer of cocoyam in the world, with 5.49 million tonnes of
cormsproduced annually which accounts for 37-40% of the world‟s total. Similarly,
statistics reveal furtherthat out of the 29.5 million MT of groundnut produced
worldwideper annum, Nigeria alone contributes 1.51-2.57million MTproduced on 2.09
million ha; similarly,0.53 million MT of soybean werereported produced from0.582
million ha of arable farmlands respectively (Abate et al. 2011, Soytech, 2011; Awurum
and Uwajimgba, 2013). She is also the world‟s largest producer of the grain legume,
cowpea. Furthermore, Singh et al. (2002) noted that Nigeria produced 2.0 million MT of
cowpea grains on 5 million ha per annum.Many socio-economic factors, pests and
disease pressures however, threaten the sustainableproduction, storage and
preservation of these staple foods as well as several leafy vegetables, fruits and
spicesamongst others in the country (Awurum et al 2000; Abate et al., 2012); and these
threats are projected to increase in the face of climate change challenges (Sadiku and
Sadiku, 2011).
Fungi represent the greatest number of pathogens responsible for these plant diseases
and deteriorations (Ajibade and Amusa, 2001). Many species of fungi decimate
agricultural crops or products in field, transit or store. It has been reported that up to
8000 – 750,000 crop diseases are caused by fungi with between 50 – 200 species, races
and/or biotypes attacking a single crop (Ragsdale, 1994; Madden et al., 2008). Infection
by fungi interferes with normal physiological function(s) of the host plant such as
photosynthesis, biosynthesis, nutrient and water uptake among others, leading to great
reductions in crop yield and product quality. Some pathogenic fungi such as Aspergillus
spp. and Fusarium moniliformeproduce toxins – mycotoxins – which cause cancer or may
be nerve or organ poisons, making affected produce dangerous for human and
livestock consumption (Ragsdale, 1994). The incidence of these plant diseases upon a
susceptible host is dependent on the presence of specific conducive conditions of
moistures in the atmosphere, in soil and/or on plant surfaces; in addition to optimum
temperatures for infection and disease development (Ragsdale, 1994; Amadioha, 2012).
Field phyto-fungal diseases may be nematode-assisted. Nematodes of the order
Xiphenima, Trichoderma, Longidorus and Para-longidorus have been implicated in this
regard. These stylet-possessing organisms puncture and burrow into tender, succulent

plant roots sucking out vital growth factors from the infested crop-plants, creating
portals of entry for pathogenic fungi and bacteria; reducing plant anchorage and
vigour, and predisposing the affected crop to damaging infections. For example the
yam nematode Scutellonema bradys and species of Melodogyne have been noted to attack
yam in Southeast Nigeria, cowpea isseriously galled by members of the genera
Meloidogynewhile Practylenchus spp. attack plantains and bananas in the rainforest zone
of the country (Wikipedia, 2013).
Emechebe and Shoyinka (1985) reported that pathogenic fungi are capable of causing
up to 100% yield loss in crops. Worldwide, it is projected that 20 – 40% of potential yield
of crops are lost annually to plant diseases (Sygenta, 2012). This may aggravate to 50%,
particularly in Africa due to climate change impacts, noted Sadiku and Sadiku (2011).It
is estimated that globally these yield losses amounts tobetween 60 – 525 billion US
dollars annually (Agrois, 2004; Sygenta, 2012). According to Amadioha (2012), in
Nigeria alone, Nkama et al. (1994) estimated that 1.5 – 20 million tonnes of cereals,
tubers and legumes amounting to 0.3 – 0.4 billion Naira are lost annually in storage due
to fungal attacks. In a bid to control these devastating attacks, synthetic fungicides are
employed.Without controversy, fungicides contribute to yield increases in crop
production. Adipala and Edema (1994) reported that the application of Mancozeb and
Dithane M-45 significantly improved the yield of cowpeas in Uganda.In the USA noted
Bernnett (2005), 1.5 billion pounds of onion was harvested from onion plants cared for
with 0.8 million pounds of fungicides. Economic use of fungicides in agriculture
translates to improved returns on farm investment. According to this source, in
California apple growers invested 70 million dollars on various fungicides, and reaped
an attractive 1.2 billion dollars while grape growers got 2.6 billion dollars as return on
investment (ROI) of 128 million dollars in the same period. A total of 880 million dollar-
investment in fungicides by growers in the USA all together reaped the sum of 12.8
billion dollars ROI translating to US$1 fungicides investment to US$14.6financial
returns for them. Fungicides play a very active role in production of high value crops
with uniform appearances and quality (Biobank, 2009). Highly intensive and developed
crop farming as practiced in the USA and Europe, involves use of highly-bred crop
varieties to maintain uniform crop height, canopy, fruit size and shape as well as overall
appearance and quality of produce in mechanized farms. Without fungicides and other
pesticides it will be difficult to grow such crops ofhigh horticultural characteristics in
large monocultures given serious potential pathogenic challenges in the environment.
In the USA, concluded Croplife (2012) many crops would not be produced
commercially without fungicidal agents.
However, the high intensity of chemical pesticide applications and/or their

inappropriate applications in agriculture have become a serious cause of concern in
recent years reported Biopesticides (2012). Several demerits are obviouslyassociated

with use of these synthetic fungicides in agriculture and pest controlhave been reported
such as pathogen resistance, pathogen resurgence, effects on non-target species,
ecological and human health concerns among others. Resistance to chemical agents is a
very serious matter. Recently FAO according to Par and Rajul (1994) noted 150 fungal
pathogens to exhibit resistance to fungicides. Some authorities assert that evolution of
races and biotypes of pathogens to previously effective chemical agents occur 5-10 years
after introduction of the agent (Oreason, 2010; Pallant, 2010).Besides, synthetic
pesticides leave undesirable residues in the treated food materials and the environment.
Some of these residues retain their toxic properties for a long time in the food chain,
impairing metabolic processes when consumed by non-target species. These and many
other factors gave impetus for alternatives to be sought (Awurum et al., 2005; Okwu et
al., 2007; Amadioha, 2012).Some of the preferred alternatives are:
 Use of natural enemies of the pathogen such as Trichoderma sp., Gliocladium sp.,
Bacillus thurengiensis and Baculoviruses in managing challenges from the pathogen
(Carson‟s Silent Spring, 1963).
 Use of Intercropping to forestall and manage pests and pathogens problems in
smallholder farms (Awurum et al., 2001).
 Farming systems re-design, through adopting crop rotation practices, proper
field sanitation, good crop density, improved field aeration, and furrow instead
of overhead sprinkler irrigation systems etc., so as to reduce predisposing crops
to fungal attacks (Abawi and Hunter, 1979; Anonymous, 2012).
 Use of botanical pesticides such as neem (Azatin, Bioneem, Tomco and
Mangosan)and extracts of other higher plants to combat challenges from phyto-
pathogenic organisms (Amadioha and Obi, 1998; Amadioha, 2002; 2003; 2004;
Awurum et al., 2005)
This review focuses on use of botanical pesticides in plant health management. Many
higher plants of the rain forest are being screened for fungicidal properties (Amadioha.
2001; 2002; 2003). Several workers have conclusively asserted the fungitoxic properties
of these plant-based extracts for management of phyto-fungal diseases (Olufolaji, 1999;
Awurum et al., 2005; Opara and Obana, 2010). These extracts have been reported to
have the merits of being readily available in faming localities of the tropics, cheap, eco-
compatible, less harmful to non-target organisms and useable in Integrated Disease
Management (IDM) programmes for smallholder resurce-poor farmers. They are also
reported to provide sustainable disease management solutions especially in organic
farming where synthetic pesticides are non-tolerable. Above all, they are systemic and
contain multiple bioactive metabolites which make pathogens‟ resistance to them less
likely (Adjaye-Gbewonyo et al., 2010; Opara and Obana, 2010; Gurjar et al., 2012).

USE OF PLANT EXTRACTS IN PLANT DISEASE CONTROL
According to Cowan (1999), initial screening of plants for antimicrobial activities; begin
with their crude aqueous or alcoholic extracts.Literatureavailable to us indicated
thatextracts of wild Ipomea carnea and isolates of Penicillin sp. had fungitoxic effects
against Heminsthoporium oryzae, H. sativum and Colletotrichum capsici which affect paddy
rice, wheat and chilli respectively (Narian, 1970). Many plant extracts as pesticides have
been reported to inhibit spore germination and mycelial growth of pathogenic fungi
(Amadioha and Obi, 1998; Olufolaji, 1999).These crude extracts were evaluated in vitro
for activities against germination of the spores and mycelial growth of the pathogens,
and later, for in vivo inhibition of the development and spread of the organisms in
actual field conditions.Gurjar et al. (2012) however argued that majority of these works
on extract evaluations against pathogenic organisms were conductedin vitro. The use of
plant extracts in plant protection is summarized in Table 1. They have been found
effective against seed-inhabiting pathogens, soil dwelling, nutrient and water uptake
impairing organisms such as the wilt-inducing Fusariumoxysporium of egg plants and
rootknot nematode (Meloidogyne spp.) of okra as well as rots and other phyto-fungal
pathogens ofAmaranthus, legumes, tomato, yam, and avocado etc. (Amadioha and Obi,
1998; Olufolaji, 1999; Amadioha, 2001; 2003). They have also proven effective for
arresting the development and spread of bacteria-induced diseases of vegetables and
tuber crops. Several plant extracts have been evaluated and found efficacious against
pre- and post-emergence damping-off, post-harvest rot and transit-decay inciting
pathogens of great implications in threating post-harvest storage and preservation of
produce in agriculture such as species of Diplodia, Aspergillus, Botrytis, Botrydiplodia,
pythium, Fusarium, mucor, Rhizopus, Penicillin, Sclerotinia, Alternaria ,Rhizoctonia and
phytophtora (Okigbo and Ogbonnaya 2006; Sarpeleh et al., 2009; Gupta et al., 2012; Islam
and Faraq, 2012). For example, recently phytochemicals from some tropical plants
(Carica papaya and Piper guineense) strongly retarded the germination of spore
ofColletotrichum destructivum (Enyiukwu and Awurum,2012).Okigbo et al. (2012)
reported the isolation ofBotrydiplodia theobromae as the most virulent amongst other
pathogenic rot fungi from cocoyam corms in Southeast Nigeria and showed that
extracts fromAllium sativumandAzadirachta indica were the most fungitoxic against them.
Similarly, Amienyo and Ataga (2007)indicated also that 30% strength of extracts of
Alchornea cordifolialeaves reduced development of rot in mechanically injured and
artificially inoculated sweet potato by the same organism to the tune of 46%. In an
evaluation in sorghum, Cymbopogoncitratus(30% strength) completely inhibited the
growth of Colletotrichum graminicola and Phoma sorghomi causing seed and seedling rot
in the plant (Somda and Sereme, 2007). Gupta etal. (2012) reported that extracts from
Eucalyptus terticornis and A. indica improved seed germination and seedling vigour by
decreasing the pre and post-emergence mortality and number of seedlings showing
symptoms of black mould in attacked onion.Greenhouse studies conducted in
Southeast Nigeria revealed that Piper guineense and Carica papaya extracts inhibited the

development and spread of anthracnose caused by C. destructivum and the results
compared well with a placebo. In field trials, Awurum and Nwaneri(2011) and
Awurum and Ogbonna (2013) reported extracts from Dennettia tripetala and Spondias
mombin comparable in fungitoxic effects to benomyl in combating Choanephora
cucurbitarium-induced wet rot of Amaranthus vegetable.On-farm research depicted that
while in Cameroun, Ambang et al. (2011) found methanoic extracts of Thevetiaperuviana
effective in controlling cercospora leaf spot (CLS) in groundnut, Awurum and
Uwajimgba also found Dennettia tripetala fungicidal against Fusarium wilt of the same
crop; and both reports compared well with benomyl treated plants. In Kenya,
experiments on-farm on Common bean (Phaseolus vulgaris L.), indicated that neem
inhibited Fusarium spp. in amended soils better than tobacco, Mexican marigold and
periwinkle (Obongoya et al., 2010). There is something to note here however that in soil
amendment trials, combinations of extracts involving 500 extracts from different
families/genera showed antagonistic, synergistic and neutral effects. Glove and black
pepper (1% concentration) combination for example, was found synergistic against
mycelial growth and sclerotialformation of the pathogen Sclerotium cepivorium while
antagonism was observed in all extract combination involving allspice, and clove and
cinnamon at 0-3% against the organism (Montes-Belmont and Prados-Ligero,
2006).Theseevaluations in general clearly demonstrated that the plant extracts from a
wide range of families are potently efficacious in cultures, glasshouse studies and in
field trials in inhibiting mycelial growth of diverse organisms,arresting their spore
germinationanddevelopment as well as spread of pathogenic diseases from both
artificially and naturally infected plants in actual field conditions.
Table 1: Some important phyto-diseases controlled with extracts of higher plants

DISEASE CROP PATHOGEN EXTRACT USED SOURCE
Bacterial Egg plant Xanthomonas Piper guineense, Opara and

blight campestris pv Obana, 2010
Zingiber sp.,
vesicatoris
Opara and
Allium sp.
Wokocha, 2008.
Anthracnose Cowpea Colletotrichum Piper guineense seed, Enyiukwu and

destructivum Awurum (2011;
Carica papaya root, seed
2012; 2013)
Wet rot Amaranthus Choanephora Azadirachta indica bark. Olufolaji, 1999.

cucurbitarium
Awurum and

Dennetia tripetala leaf, Nwaneri, 2011.
Spondias mombin leaf
Sclerotium Cowpea Sclerotium Afromonium meleguata Okwu and

stem rot rolfsii seed Njoku, 2009
Monodora myristica seed
Stem rot Cowpea Rhizoctonia Piper guineense leaf, Amadioha

solani (2001; 2012)
Carica citratus leaf,
Ocimum sp. Leaf.
Ocimum sanctum.
Blast Rice Pyricularia Azadirachta indica seed Amadioha

oryzae oil. (2000; 2012)
Brown spot Rice Cochlobolus Azadirachta indica Amadioha, 2002

miyabeanus (ethanolic extract).
Black pod Cocoa Phytophtora Carica papaya seed Wokocha and

palmivora Nwogu, 2008.
Garcinia kola seed.
Mould Mung bean Aspergillus Vernonia sp. Leaf Onuegbu, 1996.

niger
(Black) Onion Eucalyptus terticornia Gupta et al.,

Aspergillus 2012
Azadirachta indica
niger
Basal stem Tomato, Sclerotium Garcinia kola, Wokocha and

rot rolfsii Okereke, 2005.
Hyptis spp.
Okwu et al; 2007

Cowpea Citrus peel.
Curvularia Datura stramonium root,

lunata Colotropis procera stem,
Ocimum sanctum Leaf
Stem rot Vanilla Fusarium Eugenia aromatica leaf, Suprita and

oxysporium f. Khalim, 2009.
Piper bettle leaf,
sp. vanilla
Alpinia galangal
rhizome,
Sphaeranthus indica leaf.
Rot Cashew-nut Aspergillus Anacardium occidentalis Suleman and

spp., leaf Ogundana,
2010.
Trichodrema Vernonia amygdalina
spp., leaf.
Cephalosporiu
Musk bean, m sp.
Sarpeleh et al.,
Cucumber 2009.
Perganum hamala
Phytophtora
(shoot, flower, seed)
drechsleri
Verticillium
diahlae
Scleretina
scleroiorium
Aternaria sp.
Botrytis
cinerea
Macrophomina
phaseolus
Root rot Cowpea Pythium Aloe vera leaf, Suleman and

alphanidermat Emma, 2009.

um Garcinia kola seed,
Azadirachta indica,
Zzingiber officinale
Suleiman and
Emua, 2009.
Cowpea Garcinia kola
Pythium Ziginber officinale

aphanidermatu
Maize/tom m
ato Sangrikar and
Hermidesmus indicus
Wadje, 2012.
Withania sominifera
Alternaria
solani Rauwolfia tetraphylla.
Fusarium
moniliforme
Post-harvest Yam Ocimum gratissimum Okigbo and

rot leaf, Ogbonna, 2006.
Afromonium meleguata.
Okigbo and
Aspergillus
Nmeka, 2005.
niger Xylopia aethiopica
Aspergillus Zingiber officinale

flavus
Fusarium Okigbo et al.,

oxysporium 2009.
Cassava
Afromonium meleguata
seeds
Botrydiplodia
Azadirachta indica
theobromae
Fusarium
Ugwuoke et al.,

Cocoyam solani 2008.
Penicillin Ocimum basilium

oxalicum
Vernonia amygdalina
Azadirachta indica
Geotrichum
candida
Cortichum
rolfsii
Sclerotium
rolfsii
Aspergillus
niger
B. theobromae
F, oxysporium
Cercospora Cowpea Cercospora Dennettia tripetala Nwachukwu,

leafspot spp. 2010
Curvularia
Maize Phyllanthus amarus
leaf spot
Curvularia Akinbode, 2010
Tithonia diversifolia
lunata
Morinda lucida
Gliricidia sepium
Damping off Cowpea Scleretium Annona municata Chukwu, 2010.

rolfsii
Eggplant, Azadirachta indica

Chilli Rhizoctonia
Allium sativum Islam and
pepper,

Tomato solani Zingiber officinale Faraq, 2012.
Fusarium Allamonde leaf

oxysporium
Sclerotium
rolfsii
Rice blast Rice Pyricularia Chloranthus japonica Choi et al., 2004.

oryzae roots,
Paulonia coreana stem
Rootknot Okra Meloidogyne Azadirachta indica Asaawalam and

nematode spp. Adesanya, 2001.
Dry rot Yam Fusarium Aloe babadensis leaf, Taiga, 2009

oxysporium,
Nicotinia tabacum leaf,
Aspergillus
Azadirachta indica leaf.
niger.
Seed rot Melon Ocimum gratissimum Chuku et al.,

(dry) 2010.
Azadirachta indica
Rhizopus
stolonifer
Penicillium
italicum
Sorghum Cymbopogon citratus
Aspergillus
Eucalyptus Somda and
niger
camaldulensis Serene, 2009.
Colletotrichum
gramminis
Phoma
sorghumi

Fusarium
moniliforme
Potato/ Rhizoctonia Aloe vera Gurjar and

bataticola Telwankar, 2012
Vegetables Azadiractha indica
Rhizoctonia
Ocimum sanctum
solani
Aspergillus
flavus
Aspergillus
niger
Brown spot Rice Bipolaris Nerium oleander leaf Harishet al.,

oryzae 2008.
Callistemon citrinus
Nguefack et al.,
Ocimum gratissimum.
2007
Fruit rot Pawpaw Aspergillus Carica papaya Ilondu, 2011

niger
Chromolaena odorantum
Botrydiplodia
Acalypha ciliate
theobromae
Fusarium
White rot Onion solani Pimenta dioica Montes-
Penicillin sp. Belmont and
Syzygium aromaticum
Prados-Ligero,
Sclrerotium Piper nigrum 2006
ceporium
Wilt Brijal Fusarium Azadirachta indica Babu et al., 2008.

oysporium f.
Artemisia annua
sp. Melongae
Eucalyptus glabulus

Ocimum sanctum
Rheum emodi
Tomato Azadirachta indica Agbenin and

kernel, stem, leaf Marley, 2006.
Fusarium
oxysporium f.
sp. lycopersici
Brown blight Tea Glomerella Pongamia pinnata Kuberan et al.,

cingulate 2012
Syzigium aromatia
Alcorous calamus
Ageratum conyzoides
Allium sativum
Abutilon indicus
Blight Wheat Bipolaris Adhatoda vasicola Nagis et al., 2012

sorokiniana
Zingiber officinale
Seed-borne Maize Fusarium Allium sativum Debnath et al.,

diseases(rot, oxysporium 2012
Azadirachta indica
seedling
Fusarium
blight,
moniliforme
Bipolaris leaf
spot, Penicillin spp
curvularia leaf
spot) Aspergillus
spp.
Bipolaris
maydis
Curvularia

lunata
Rhizoctonia
stolonifer
Seed/seedln Soybean Cephalosporiu Acacia nilotica Rathod and

g rot m acromonium Pawar, 2012.
Azadirachta indica
Rhizopus
Datura stramonium
leguminosa
Polyathia longifolia
Colletotrichum
dermatium Annna squamosal
Macrophomina Allium sativum
phaseolina
Phoma sp.
Sclerotium
rolfsii
Curvularia
lunata
Fusarium
oxysporium
Penicillin
chrysogenum
Mucor mucedo
Alternaria
alternate
Aspergillus
flavus
Aspergillus
fumigatus
Aspergillus

niger
Plants extracts as seen from Table 1, are hence suitable for exploitation as potent sources
of pesticides to reduce losses arising from pathogenic attacks on crops and stored
products (Amadioha, 2012). The use of these natural products for pathogenic disease
management is particularly important and necessary in the developing economies of
the world like Nigeria where synthetic fungicides are not only unavailable but farmers
who produce about 98% of food in the country are poorly equipped to handle them
making their use uneconomic for resource-poor farmers.
Phytochemical evaluation of extracts

It has been noted that, despite the use of plant extracts in ethno-medicine, African
cuisines and recently in plant protection, that their phytochemical composition and
active ingredients have notbeen fully documented (Okwu and Njoku, 2009).
Corroborative studies estimated that only 4% -10% of the 250,000 plant species
constituting the biodiversity of the world‟s flora have been examined chemically for
antimicrobial activity (Earnsworth, 1990; Pallant, 2010). Ahuge potential therefore exists
in this regard; especially in the tropical areas like Nigeria with vast untapped rainforest,
for these fungitoxic plant species to be extensively examined chemically. Scientific
analysis of plants it has been observed follows a logical pathway beginning with a lead
from the natives. Hence, Cowan (1999) reported further, that initial aqueous and
alcoholic screening of plants extracts for antimicrobial activities are followed by other
organic extraction methods for determination of their phytochemical compositions.
According to Wessells and Hopsons (1988), marvelous assortments of chemicals which
are noxious to pathogens and even pests, have been found to be present in plants.
These antimicrobial constituents (phytochemicals) include alkaloids, flavonoids,
saponins, tannins, phenols, terpenoids, glycosides, anthraquinones, coumarins,
polyphenols, Phlobatannin and steroids (Wessells and Hopsons, 1988; Edeoga et al.,
2005;Okwu and Njoku, 2009; Soladoye and Chukwuma, 2012). The phytochemical
constituents of some plant materials used in plant protection are presented in Table 2.
They may be qualitatively represented or quantitatively documented (Edeoga et al.,
2005; Jeruto et al., 2011). In a qualitative evaluation, Ayodele et al. (2009) reported the
absence of alkaloids and anthraquinones from the leaves, stem bark and root of Ficus
exaspeata. Quantitative documentation of phytochemicals in plant materials are
presented in mg/100g of dry weight of specimen, g/100g of weight of the specimen
(Aliyu et al., 2008; Harisaranraj et al., 2009; Okwu and Iroabuchi, 2009; Senthilkumar et
al., 2011) or as percentages of weight per volume (w/v) of extract (Edeoga et al., 2005;
Okwu et al., 2007; Uchegbu and Okwu, 2012; Enyiukwu and Awurum, 2013). This
review will only present results expressed as percentages. Studies show that sometimes
there may be disparity in the reported yield values of these phytochemicals by different

investigators. For instance,Enyiukwu and Awurum (2013) reported 1.63% alkaloids
present in the seeds of Piper guineense which figure differed hugely from the value 5-8%
noted in Purseglove (1976). This difference in values the authors attributed to influences
from time of harvesting of the plant materials, extracting solvent, and method of
extraction. The latter two factors seem most important to the reviewers. Variation in
extracting methods are usually dependent on the length of the extraction period,
solvent used, solvent pH, temperature, particle size of plant material and the solvent-to-
sample ratio. According to Gurjar et al. (2012), the finer the particles size the higher and
better the rate of extraction. Furthermore, in a study by (Elloff, 1998) it was found that 5
minutes extraction of very fine particles of diameter 10 nm gave higher quantities of
phytochemicals than values obtained after 24 h in a shaking machine with less finely
ground materials. Later investigations revealed that solvent-to-sample (solvent to dry
weight) ratio of 10:1 had proved ideal for extraction of phytochemicals (Green, 2004;
Gurjar et al., 2012).The observed differences in the yield of phytochemicals amongst
parts (leaf, stem, bark, flower, seed) of the sameplant, the authors further added may be
occasioned by factors of age of the plant, plant part, sex and cultivar used in the
investigation. Enyiukwu and Awurum (2013) noted that in a study at Cornell
University, that male Carica plants yielded more phytochemicals than female ones and
older plant parts yielded more than the young ones. Similarly, it was also noted in the
samestudy that the potency of activity of the yielded phytochemicals were age of plant
or plant part dependent. For instance, phytochemicals from young plants or plant parts
were observed to be more active than those from older plants or plant parts.Variations
in yield values of phytochemicals may also be occasioned by the climatic and edaphic
variations in the geographic locations of growth of the plant (Pallant, 2010).
Phytochemicals of the group alkaloids have complex structure. They are bitter tasting,
colourless, basic, and toxic,and contain nitrogen in a heterocyclic ring.At room
temperature alkaloids may be liquids or crystalline solids (Okigbo etal., 2009). They are
the most physiologically active compounds of medical importance found in plants.
Alkaloids and their derivatives are used as basic starting points for drugs. They possess
antifungal and bactericidal properties (Okwu and Uchendu, 2009). Karlovsky (2008)
reported that alkaloids can inactivate enzymes, block ionchannels, interfere
withneurotransmission and cause loss of electrical coordination (ataxia) in organisms.
Flavonoids are polyphenolic compounds possessing 15 carbon atoms made up of two

benzene rings joined by linear carbon chain. They represent the most common and
widely distributed class of plant phenolics. Flavonoids are a class of secondary
metabolites known most commonly for their antioxidant and free radicals scavenging
activities. Aside of prevention oxidative cell damage, flavonoids also play roles in
combating allergies and microbes (Okigbo et al., 2009). Some flavonoids of the sub-class
isoflavonoids, exhibit high pesticidal activity by anaesthetic-like action related to

electron transport blockade in the mitochondria brought about by inhibiting oxidation
linked to NADH2. (Freidli, 2008; Okwu and Njoku, 2009).

Table 2: Phytochemical composition of some plants materials used in plant protection.
Phytochemical constituents *(%)
Plant Materials Alka Flav Tan Sap Phe Ter Gly Ste Source/References
Ricimus communis leaf - + + + + - + + Yadav and Agarwala, 2011.
Xanthium strumarium leaf + + + + + - + - “ “ “
Tinospora cordifolia + + + + + + + + “ “ “
Hyptis sauveolens + - + + - + + - Pachkore et al., 2011
Clutiaabyssinica - + - + + + - -
Euphobia hirta + + + + + + + - Ibrahim et al., 2012
Eruca sativum 11.27 24.43 4.15 6.20 26.90 - 2.76 - Mohammed et al., 2011.
Alchornea cordifolia leaf 5.90 4.20 6.80 3.20 Adeshina et al., 2012
Cleome rutidosperma 0.34 0.34 15.25 2.00 0.20 - + - Edeoga et al., 2005
Emillia coccinea 0.92 0.96 11.85 2.30 0.81 + + + “ “
Euphobia heterophylla 0.86 0.74 12.46 0.00 0.10 + + - “ “
Physalis angulate 0.40 0.15 13.15 3.92 0.80 + + + “ “
Richadis brasilienss 0.45 0.56 12.13 1.12 0.14 + + + “ “

Scorpania dulchis 0.81 0.88 6.23 0.00 0.04 + + - “ “
Sida acuta 1.04 0.98 6.08 0.00 0.08 - + - “ “
Spigella anthemia 0.84 0.77 15.05 2.26 0.10 - + + “ “
Stachytaphyta cayennensis 0.68 0.00 9.98 3.10 0.13 - + - “ “
Tridax procumbens 0.58 0.61 7.45 1.70 0.06 - - - “ “
Carica papaya seed 0.62 0.34 0.22 0.68 0.08 - + - Enyiukwu and Awurm, 2013
Carica papaya root 0.75 0.57 0.34 1.40 0.05 + + + “ “
Piper guineense seed 1.63 1.23 0.88 2.64 0.66 + - + “ “
Monodora myristica leaf 4.28 8.29 0.34 0.02 0.03 Okwu and Njoku, 2009
Monodora myristica seed 0.41 0.12 0.03 0.87 0.02 “ “
Bryophyllum pinnatum 1.48 1.72 0.51 1.74 0.06 Okwu and Uchenna, 2009
Cissis populnea root 2.79 4.13 1.18 1.11 - 0.36 0.53 0.13 Soladoye and Chukwuma,
2012
C. populnea stem 4.70 1.46 1.10 1.21 - 0.27 0.41 0.15 Okwu and Uchenna, 2009
Cassia alata seed 3.24 0.50 2.46 6.44 0.95 “ “
Nauclea latifolia leaf 4.32 0.36 0.01 0.98 0.06 “ “

Nauclea latifolia seed 0.59 0.56 0.06 1.34 0.05 “ “
Nauclea latifolia fruit 0.28 0.81 0.01 0.42 0.02 “ “
Azadirachta indica leaf 0.52 0.62 9.10 2.10 0.02 Khrishnaiah et al., 2009
Molinger oleifera 0.36 0.51 9.20 2.30 0.08 “ “
Cleredendon sp. Leaf 5.41 0.70 3.60 2.10 0.08 Okwu and Uchenna, 2009.
Spondias mombin leaf 6.00 3.00 3.80 7.60 1.00 Njoku and Amaefula, 2007.
Afromonum meleguata leaf 0.29 2.15 0.16 0.14 0.10 Okwu and Njoku, 2009
Afromonum meleguata seed 5.64 5.78 0.39 1.24 0.11 “ “
Chromolaena odorantum - - 1.98 0.38 - 0.13 Igboh et al., 2009.
Citrus limonum 0.54 0.64 1.31 0.34 0.25 Okwu et al., 2007.
Detarium Senegalese 0.72 5.68 0.79 4.60 0.25 Uchegbu and Okwu, 2012.
Uvaria chamae 0.81 5.70 0.40 0.38 0.10 Okwu and Iroabuchi, 2009.
*= qualitative representation. + indicate presence of constituent; - indicate absence of constituent.

Tannins comprised of a large assemblage of natural products which have unpleasant
taste and are employed in tanning leather (Okigbo et al., 2009). They have great natural
diversity and generally are made up hydrolysable and condensed tannins (Okwu and
Njoku, 2009). They have pain killing properties. Tannic acids are used for prevention of
loss of plasma and limiting secondary infection in burn wounds. Tannins-rich plants
extracts are used by Asian natives for the treatment of ulcers. Tannins have uniquely
high affinity for precipitating proteins and complexing with all kinds of biomolecules
(Peru, 2001; Dharmananda, 2007). Phenols or carbolic acids are aromatic alcohols
consisting of a benzene ring bonded directly to a hydroxyl group (OH) (De-Reuter,
2005). They are weakly acidic, and have long history of roles in antisepsis and
disinfection (Okigbo et al., 2009). They are used as the starting ingredients in the
industrial production of drugs, herbicides. synthetic resins and additives to inhibit
microbial growth in various ranges of pesicides (Greener Industry, 2009). Phenolics
slow growth, block microbial cell division and enzyme activity. According to Okwu et
al. (2007) they caused swelling of fungal hyphae tips, plasma seeping and leaking
around hyphae tips, cell wall distortions, abnormal branching or fusion of hyphae
surface.
Saponins are glycosides of both triterpenes and steroids known for the soap-like
foaming they produce in aqueous solutions. Saponins have a characteristic property of
being bitter or astringent (Okigbo et al., 2009). Their soap-like nature makes them
useable as surfactants and adjuvants for vaccines to enhance macromolecule
penetration (Wikipedia, 2008). Saponins can ward off microbes and this makes them
good candidates for treating yeast, viral and fungal infections. They are known to play a
role in cytolysisby complexing with cell membrane bilayers (Okwu and Njoku, 2009)
sometimes creating pores on them (Rongai et al., 2012).
In recent years noted a study from Italy, plants of the family Brassicacaea have attracted
scientific attention due to their high contents of glucosinolates. These glycosidic
compounds have no biocidal activity in their native forms but are converted through
enzymatic hydrolysis within living systems to their active forms (isothiocyanates)
which have strong cytotoxic activity (Rongai et al., 2012). Methyl isothiocyanate (MITC)
and benzyl isothiocyanate (BITC) were reported present in Carica papaya (Morton, 987;
Cornell University, 2012). BITC occurs in the range of 1.37-1.96% in the Family Caricaceae
(Tang et al., 2001). According to Reulas et al. (2003) BITC isolated from Papaya seeds is
known to be a strong antifungal compound. At 0.5mg/ml the compoundinhibited the
fungus Alternaria alternata causing post-harvest rot of tomato. Later evaluations
indicated that aqueous seed extract from C. papaya was superior to metalaxyl (ridomil)
in inhibition effects against Phytophtora palmivora inciting black pod disease of cocoain
vitro. Investigators believed BITC was responsible for this reported activity(Wokocha
and Nwaogu, 2008; Enyiukwu and Awurum, 2013).Furthemore, for instance, the

efficacy of Afromonium meleguata and Monodora myristicaseeds and leaves extract against
Sclerotium rolfsii, the incitant of basal stem rot of cowpea, according to Okwu and Njoku
(2009)were due to their high contents of alkaloids. Enyiukwu and Awurum (2011; 2012),
also attributed the potency of Piper guineense seed extracts in the inhibition of spore
germination, mycelial elongation, development and spread of the causal organism of
anthracnose (Colletotrichum destructivum) of cowpea in culture and glasshouse, largely
to its high contents of alkaloids and perhaps, terpenoids in the test seeds extracts.
Similarly, alkaloids are also reported responsible for the fungitoxic activities of extracts
of Raecimus communis and Datura stramonium against several phytopathogenic fungi,
while Cucumina longa and Azadirachta indica contain bioactive terpenoids which have
been implicated for activities against a broad array of plant pathogenic fungi and
bacteria (Gurja et al., 2012).Amadioha and Obi (1998) in a study with spices suspected
and attributed the potency of extracts of Xylopia aethiopica againt C. lindemuthianium the
incitant of antheacnose of cowpea to perhaps the presence of xylopic acids in the plant
materials.
Functional chemical groups

In a study on cocoyam in Nigeria, it was inferred that rot causing fungi produce
pectinolytic and cellulolytic enzymes which degrade cell wall polymers and as such
make available carbon sources for the invading pathogens. Ocimum basilium extract
which inhibited the rot-causing fungi in the study was seen as possessing active
principle(s) that arrested the ravages of the pathogens or their enzymes or both
(Ugwuoke et al., 2008).Tests with infra-red reveal that these phytochemicals, according
to several workers, contain many chemically potent functional groups. Functional
groups are specific groups of atoms or bonds within molecules that are responsible for
the characteristic chemical reactions of the molecules. Same functional groups are
known to undergo same or similar chemical reaction regardless of the size of the
molecule it is a part of. However its relative reactivity is modified by nearby functional
groups. Functional groups include amides, carbonyls, esters, aldehydes, phenyls,
hydroxyls, ethyls, methyls etc. With these functional groups extracts (isolates) establish
bonds with target enzymes, hormones, organelles or processes of pathogens to their
harm (Brown, 2006; Okwu and Ukanwa, 2010).Echeverrigaray et al (2010) reported that
hydroxyl and carbonyl groups or substituents on carbon skeletons of various
monoterpenes are responsible for their inhibitory activity against soil inhabiting
nematodes. The report further suggested that in addition, the position of these
functional groups also influence the observed activity. Malheiros et al. (2005) in
agreement from a parallel study, reported that dramane sesquiterpenes fromDrimys
brasilliensis inhibited a variety of human myco-pathogens including the stubborn
Epidermophyton floccosum; and showed that their activity decreased 8 times (from 3
mg/ml to 25 mg/ml) when a bulky substituent (p- methoxy or p-hydroxycinnamoyl) is

present in carbon position 1. Some of the essential functional groups of a phytochemical
are listed in Table 3.
Table 3: Infra-red analysis of isolates from Afromonium meleguata and Monodora
myristica for functional groups.
Plant Isolate Frequency Functional Compound Reference
group type
Afromoniun meleguata 3413.07 OH Hydroxyl Okwu and

seed phenol Njoku, 2009.
3108.08 C-N Amine
2925.88 C-H Aliphatic

stretching

stretching
1709.15 C=O Carbonyl ester
1654.99 C=O Carbonyl

ketone
1604.88 C=C Aromatic
1121.00 C-O Ether
3418.90 O-H Hydroxyl

phenol

stretching

stretching

substitution
1164.45 C-O Aromatic

substitution
Afromonium meleguata 3401 O-H Hydroxyl

leaf phenol
3008 C-N Amine
2926 C-H Aliphatic

stretching
2864 C-H Aliphatic

stretching
1464 C=C Aromatic
1378 C-O Ester
1244 C-O Ether
1177 C-N Amine
1098 C-O Ether
1050 =C-H Aromatic

substitution
801 =C-H Aromatic

substitution
723 =C-H Aromatic

substitution
Monodoramyristicaseed 3442.25 O-H Hydroxyl

phenol

hydrocarbon

1634.29 C-=O Carbonyl
668.13 =C-H Aromatic

substitution
Monodora myristica leaf 3421.22 O-H Hydroxyl

stretching

stretching

ketone

ketone

ketone
1375.28 C-O Ether flavonoid
799.60 =CH Aromatic

substitution
According to Okwu and Uchenna (2009), many other functional groups exist in these
phytochemicals besides the ones shown above in Table 3, such as pyrones,double and
triple bonds etc. These chemically reactive groups have been postulated to be isolates‟

arsenals of attack against metabolic sites and enzymes of pathogens (Okwu and
Ukanwa, 2010) and even host plants in the case of phytotoxic chemicals (Echeverrigaray
et al., 2010).
Characterizedisolates of some extracts

The effectiveness of the activity of phytochemicals against pathogenic organisms has
been reportedly suggested to depend on the type and concentration of bioactive
principles they contain (Owolade and Osinkalu, 1999). In India, Stripathi and
Poongothai (2010) reported that from bioassay-guided fractionation of Pisonia glandis a
topical medicinal plant, isolated fraction A inhibited the fungus Monascus purpureus
better than clotrimazole a standard drug against its infections in humans.Of all the
plant materials listed in Table 1 above, reportedly used in many researches on toxicity
to phyto-parasites, only neem (Azadirachta indica) has been most extensively studied,
characterized; and asserted to contain the terpenoid azadirachtin. Scott et al., (2002; 2005)
also documented that Piper guineense contain the alkaloids piperine and piperidine which
have strong fungitoxic and pesticidal attributes. Allicin isolatedfrom Allium spp.
effectively controlled seed-borne Alternaria spp. in carrot, Phytophtora leaf blight of
tomato and tuber blight of potato. Guie et al. (2003) indicated the isolation of
flavanolglycoside isorhamnetin fromAlchornea cordifolia. Later investigation revealed the
presence of anthocyanidine glycoside in Alchornea cordifolia, andfingered this compound
to underpin its antibacterial, antiviral and antimicrobial properties (Okwu and Ukanwa,
2010). Polygodial (sesquiterpenes) has been isolated from Drimys brasilliensis
(Malheiros, 2005). The compound has been found to exhibit insect antifeedant,
antimicrobial activities as well as fungicidal activities to yeast, filamentous fungi and
the medically difficult to eradicate endomycotic Epidermophyton fluccosum (Taniguchi et
al., 1998; Lunde and Kubo, 2000; Malheiroset al, 2005). Recently a few other plant
materials have been examined chemically and their active principles isolated,
characterized and documented Table 3.
Table 4: Some characterized plant materials with antimicrobial activities

PLANTS/PLANT CHARACTERISED SOURCE
MATERIALS ISOLATE(S)
Alchornea cordifolia Isopenteneyl guanidine Lamikanra et al., 1990
Aspilia Africana leaf Inisitol Ita et al., 2010
Limonene,

Alpha-pinene
Ethyl 3-(3,4-dihydroxyphenyl) Faleye and Ogundaini, 2012

acrylate
3-(4-dihydroxyphenyl)-oxo-
2H-chromene-6-carbaldehyde
Ocimum basilicum Linalool Klimankova, 2008
Methyl chavicol
Eugenol
Bergamotene
Methyl cinnamate
Geranyl acetate Ozcan and Chalchat, 2002.

Methyl eugeno
Rosmarinic acid Jamal et al., 2002
Stachyterpheta jamaicensis Lanostance glycoside Okwu and Offiong, 2009.

linn vahl
S. jamaicensis Steroidal glycoside Okwu and Ohenhen, 2010.
Nerium oleander Oxoocyl-1-2- Sharma et al., 2010.

hydroxyundecanoate
Heptacosane-3enyl-5-
hydroxyhexanoate
Butelin
Butelinic acid
Stigmasterol

Gupta andMittal, 2010
Neridienone A
Neridienone B
Syzygium aromaticum 4-allyl-2-methoxyphenol Rahimi et al., 2012
Eugenia carophyllata Eugenol Singh et al., 2012
Hyptis suaveolus Stigmasterol Jasani et al., 2012.
Beta-myrcene Vijay et al, 2011
Sabenene
(2E)-1-(2-hydroxy Jayakumar and Ganesh,

pheyl)penta-2-en-1-one 2012
1-{(3-hydroxy-5, 5-
dimethylcyclohex-3-en-
1yl)oxy}hexan-3-one
Datarium senegalense gmelin Tetrahydroxyl anthonyanides Okwu and Uchegbu, 2009.
Dataru metel linn Alkaloid sitosterol 1 Okwu and Igara, 2009.
Vernonia amygdalina Stigmasterol Luo et al., 2008
Chondrillasterol
n-Hexadecanoic acid
Beta-Sitosterol Onuegbu, 1996
Melanthera scandens Beta-caryophyllene Affia et al., 2011
Limonene

Beta-phellandrene
Alpha-bisabolol
Alpha-humulene
Afromonium meleguata Monoterpene indole alcohol Okwu and Ojukwu, 2010.
Bridelia ferruginea berth Flavonoid chalcones, Okwu and Ukanwa, 2010a
Anthocyanidines
Garcinia kola seed Flavone glycoside Okwu, 2007.
Datura metel linn Beta-Carboline alkaloid Okwu and Igara, 2011.
Ficus exaasperta Ficusamide Dongfack et al., 2012
Furanocoumarines
Bergapten
Alpha-terpineol Oladosu, et al., 2009.
Alpha-pinene
Peperomia pellucida Phytol Wei et al., 2011
Hexadeconoic acid
Naphthaleno
Octadecanoic acidl
Bryophyllum pinnetum Flavonoid glycoside, Okwu and Uchenna, 2009.
Rutin,
Kaemoforol-3-glycoside,
Beta-Sitosterol

Cleredendon splendens Flavonone-diglucoside, Okwu and Uchenna, 2009.
Hispudilin
Ageratum conyzoides Coumarin Wildodo et al., 2008
Cassia alata Chrysanthemic acid, Okwu and Uchenna, 2009.
Luteoline
Nauclea latifolia Nauclechine Okwu and uchenna, 2009.
Azadirachta indica (Neem) Azadirachtin Brown, 2006
Alchornea cordifolia Anthocyanidine glycosides Okwu and Ukanwa, 2010b
Piper guineense Piperine, Scott et al., 2002.
Piperidine.
Carica papaya Carpaine, Njoku and Obi, 2009.
Pseudo-carpaine,
Carica papaya Xanthine, Violaxanthine Njoku and obi, 2009.
Allium cepa L. Allicin Gurjar et al., 2012.
Allium satvum L. Allicin Gurjar et al., 2012.
Thymus vulgaris L. Caffeic acid ““
Ricinus cumminis L. Ricinine, ““
Ricininoleic acid
Plants contain a marvelous array of potent and bioactive chemical compounds which
play roles in warding off microbial, pest and herbivoral attacks. A source noted that 12,
000 of such chemical compounds have been isolated from the plant kingdom. In the
thoughts of Pallat (2010), these are still infinitesimal compared to the enormous
chemical space provided by the 250,000 species constituting the world‟s flora.
Identification, isolation, characterization and purification of novel potent compounds of
plant origin will pave way to sustainable food production; reduceeco-contamination
while delaying or reversing pathogen resistance to plant health management chemicals.

Mode of action of isolates from biopesticides
Mechanism of action (MOA) of a chemical substance refers to the specific biochemical
interaction through which a chemical substance exerts or produces its effects. MOA
includes a crystal clear mention of specific molecular targets to which the chemical
binds such as enzymes or receptors. For example, in a related field of pharmacology, it
is known that salicylic acid, acts by irreversible inhibition of the enzyme cyclo-oxygenase
and suppressing the production of the hormones prostaglandins and thromboxanes
leading to pain relief. The demand for bio-pesticides especially botanicals however, are
seriously constrained by dearth of knowledge of their modes of action, in particular
their target specificity, and slow action. These have put them in a serious disadvantage
vis-a-viz the synthetic chemicals noted for their fast action. Despite the glorious
potentials of plant extracts for crop disease management, they are also demerited in
being photo and themo unstable, and like their predecessors the pyrethrins have short
half-life, shelf storageability and are rapidly degraded by ultra-violent radiations
(Kumar, 1986; Eno, 2011; Gurjar et al., 2012). Aside of the pyrethrins (sodium channel
modulators), nicotine (acetylcholine agonist) and rotenone (electron transport inhibitor
at cytochrome A) whose modes of action have long been determined; of all the plant
materials above, only azadirachtin isolatedfrom neem has been clearly reported to act
by antagonizing prothoracicotropichormone (PTTH) of target organisms (Brown,
2006).From available literature, many reports only offer postulations on the modes of
action of these bioactive metabolites. For example, in a parallel study, Okwu and
Ukanwa (2010) suggested that anthocyanidine glycoside from Alchornea cordifolia
inhibited Klesbiella sp and Staphyllococcus aureaus probably by the mechanism of
membrane disruption or enzymes inactivation. Binding to adhesins, proteins, substrate
deprivation, and intercalation into cell walls and DNA have been advanced as well by
many workers as possible mechanisms for the observed inhibitions (Echeverrigaray et
al., 2010; Gurjar et al., 2012).Allicin (Alliaceae) is thought to be readily cell-membrane
permeable and undergoes thiodisulphide exchange reaction with amino acids and
proteins and its fungitoxic property is assumed pivoted on this attribute (Shusarenko et
al., 2008). Besides it is also thought that the mode of action of allicin may also be by
mediation of lipoperoxide production in fungal plasmamembrane leading to increased
permeability (Rongai et al., 2012). With regard to saponins, remarked the fore-going
source, their mechanism of antifungal action is not well understood but it is believed to
complex with sterols in the cell membrane leading to pores formations and consequent
loss of cell membrane integrity.Furthermore, according to Brown (2006),
cinnamaldehyde (Cinnacure, Cinnamite) a botanical pesticide is believed to impair
energy production of target organisms. He however reported that its exact mode of
action is not well understood though interference with glucose uptake is assumed.
Concrete studies to ascertain these claims and postulations seem lacking. However,
experts maintain that unless something is done drastically to improve the effectiveness
of biopesticides, the growth in their popularity will remain only gradual (Biopesticides,

2012). We, as a matter of necessity must understand how these active isolates interfere
with the physiology of the target organisms before thinking of improvements. To this
end therefore, the challenges facing us in this area of plant disease management now
are to determine:
 What constituent(s) make up the test extracts and what functional groups does
the extract possess?
 What are their binding sites on the target pathogen?
 What metabolic processes or pathways do they affect by so doing?
 How do theytherefore effect the observed inhibitions and/oreffect kills on the
target pathogen?
 In most cases listed in Table 1, these are notthoroughly understood yet.
CONCLUSION
Reports show that majority of the evidences of plant-based chemical activity against
plant pathogenic fungi and other micro-organisms of agricultural importance were
provided from in vitro evaluations. Crude extract evaluations have become very
rudimentary. Numerous phyto-chemicals which have demonstrated in vitro effects
should be evaluated on-farm under natural or induced infections, to further determine
and establish their efficacy in controlling the incidences of pathogen-borne diseases in
crops in a multi-factor environment. Information on the active ingredients of plant
extracts is sparingly available while that ofthe mechanism of action of isolates is almost
non-existent.These are high profile, high-tech areas of scientific endeavours, and multi-
disciplinary in nature.Therefore further focused and articulated researches to improve
the effectiveness, target specificity and shelf storageability of botanicals are pressingly
imperative. To achieve this, we must liaise and collaborate effectively with scientists in
biochemistry, plant physiology, biotechnology, pharmacology and natural products
chemistry. It is in the light of this kind of collaboration and purpose-guided assays that
the much anticipated outcome of identifying, isolating, characterizing and
understanding isolate-pathogen interaction which will lead us to the knowledge of the
mechanisms of action (MOA) of the active principles of plant-based natural products
will be achieved in the near future.
SUGGESTIONS AND FUTURE TRENDS

1. Investigators should be encouraged to conduct on-farm evaluations of crude plant
extracts against a wide range of pathogens of various high value field and vegetable
crops. In vitro trials have become too narrow for us to base our conclusion of bioefficacy
of crude extracts against pathogenic fungi upon.
2. Given proven phyto-fungal toxicity of the plant materials and assertions on their
effectiveness especially from actual field trials in the management of plant health
challenges;many concerted and directed efforts and thrusts should hence be geared

toward chemical examinations of the plant materialsin all future investigations with a
view to:
 Determining their phytochemical compositions,
 Determining their chemical functional groups and their relative positions on the
carbon skeletons,
 Isolating their active ingredients,
 Elucidating and characterizing the structure of isolates so as to enhance:
 Studies on their modes of action on pathogens,
 Phytotoxicity of the principles on host plants and,
 Possible means of improving their effectiveness and synthesis
3. Departmental collaborations should be sought with highly equipped and established

laboratories in the USA, UK, China and India to enable investigators overcome complex
issues of chemical structure elucidation of isolates and isolate-pathogen interaction.
4. Cost-benefit evaluations should be incorporated in our trials to scientifically establish

the cost effectiveness of the plant extracts vis-a-viz synthetic chemical product rather
than base this on guess works. Appropriate tests on the mammalian toxicity of isolates
should thoroughly and speedily conducted to overcome the challenges bans after
introduction of products
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CHAPTER SEVEN
COMPREHENSION ON USE OF MEDICINAL PLANTS TO CURE DIARRHEA

AMONG THE TRIBALS OF MAYURBHANJ DISTRICTS, ODISHA, INDIA
Laxmipriya Padhi and Sujogya Kumar Panda

Department of Zoology; North Orissa University; Baripada, Odisha, India-757003
ABSTRACT
This chapter presents a comprehension on ethnomedicinal uses of medicinal plants by the
indigenous tribals of District Mayurbhanj, Odisha, India. In addition to this the chapters also
scientifically validated how many of these plants are active to kill bacteria responsible to cause
infectious diarrhea. Aqueous and methanol extracts of 150 plants were recorded as medicinal
value, of which 136 plants were tested for antibacterial activity using agar well diffusion against
eight pathogenic bacteria involved in diarrhea and dysentery. The results indicated that out of
136 plants species, 100 species exhibited antibacterial activity against one or more test
organisms. In total 288 (144 methanolic+144 aqueous) extracts, 107 methanolic and 74 aqueous
extracts expressed antibacterial properties. From the screening results plant family’s viz.
Fabaceae (08), Rutaceae (07), Combretaceae (06), Caesalpinaceae (06), Anacardiaceae (05) and
Apocynaceae (04) showed highest antibacterial activity in terms of test plant numbers. However,
families viz. Burseraceae, Crassulaceae, Hypoxidaceae, Liliaceae, Lygodiaceae, Musaceae,
oxalideceae, Pedaliaceae, Strychnaceae, Trapaceae, Ulmaceae did not showed activity against any
test organism. Moreover, Euphorbiaceae, Rubiaceae are the two largest families from which
plants did not show result significant activity against diarrheal causing bacteria. Among the
plants viz. Acacia leucophloea, Adhatoda vasica, Andrographis paniculata, Annona reticulate,
Anogeissus latifolia, Anthocephalus chinensis, Bombax ceiba, Buchanania lanzan, Butea
monosperma, Careya arborea, Cassia fistula, Centella asiatica, Cissampelos pareira , Coccinia
grandis, Croton roxburghii, Curculigo orchioides, Diospyros melanoxylon, Eleutherine bulbosa,
Erycibe paniculata, Ficus racemosa, Flemingia nana, Helicteres isora, Holarrhena pubescens,
Helicteres isora, Lannea coromandelica, Litsea glutinosa, Mesua ferrea, Mimusops elengi,
Moringa oleifera, Nyctanthes arbortristis, Phyllanthus emblica, Piper longum, Pterocarpus
marsupium, Pterospermum acerifolium, Punica granatum, Quisqualis indica, Semecarpus
anacardium, Smilax zeylanica, Terminalia arjun, Terminalia bellirica, Tinospora cordifolia and
Vitex negundo experimentally proved to inhibit most of the diarrheal causing bacteria.
KEYWORDS: Antidiarhheal, Similipal Biosphere Reserve, Preliminary screening, Plant

extracts, Ethnomedicinal uses
INTRODUCTION
Diarrhea, particularly infectious diarrhea is the second leading cause of mortality and
morbidity throughout the world in children less than 5 yrs of age. This is especially true
in developing countries like India where there is poor sanitation and overcrowding.

Estimation of crude death rate due to diarrhea in India was 9.3 per 1000 population
(WHO, 2005). If this continues, then the predicted case of burden will rise to 126.35
cores during 2016 (WHO, 2005). Among the leading causes of infectious diarrhea,
Salmonella and Shigella contributes highest number. The current the chemotherapeutical
treatment of Salmonellosis and Shigellosis is complicated as a result of drug resistance.
Moreover, majority of the people in these developing countries have no access for
modern health care facilities. This necessitated the search for alternative therapies such
as, the use of medicinal plants. Shigellosis is an important cause of diarrheal deaths. It
has been reported that not less than 140 million cases of Shigellosis occur worldwide
with 6,00,000 deaths annually, 60% of such deaths are of under 5 years old children
(Peirano et al., 2006; Sur et al., 2004). Among the different species of Shigella, S.
dysentriae is known for its fatality, and life threatening situation. The emergence of
multiple drug resistant strains of diarrheagenic pathogens has made the treatment of
dysentery more difficult hence there is increasing interest in plants as source of
antimicrobial agents for the treatment of such diseases (Munshi et al., 1987; Monroe and
Polk, 2000). Salmonellosis, another type of diarrheal diseases is caused by a group of
bacteria called Salmonella. It is primarily transmitted through ingestion of contaminated
food by infected faeces from man or animal, through fecal oral route. Active cases of
Salmonella in man are source of contamination and transmission to other human beings
and to lower animals. Strains of Salmonella species with resistance to antimicrobial
drugs are now widespread in both developed and developing countries. In developed
countries, it is now increasingly accepted that for the most part such strains are zoonotic
in origin and acquire their resistance in the food-animal host before onward
transmission to humans through the food chain. On the other hand, indiscriminate use
of commercial antimicrobial drugs commonly employed in the treatment of infectious
diseases lead to high resistance among the strains. This has forced researchers to search
for new antimicrobial substances from various sources like the medicinal plants. Search
for new antibacterial agents should be continued by screening many plant families.
Several studies have identified that the compounds within herbal plants are effective
antibiotics (Basile et al., 2000). Traditional healing systems around the world that utilize
herbal remedies are an important source for the discovery of new antibiotics (Okpekon
et al. 2004); some traditional remedies have already been produced compounds that are
effective against antibiotic-resistant strains of bacteria (Kone et al., 2004).
Diarrheal disease continues to be a major cause of morbidity and mortality throughout

the world, particularly among children in developing countries, often as a result of
infection by bacteria, viruses, and protozoal parasites. Given the increasing resistance in
many common pathogens to currently used chemotherapeutic agents, there is renewed
interest in the discovery of novel compounds that can be used to fight infectious
diseases. There have been numerous studies that have served to validate the traditional
use of medicinal plants used to treat or prevent diarrhea. Several methods can be used

for selecting plants of potential therapeutic interest (Vlietinck and Vanden-Berghe, 1991;
Farnsworth, 1993). The search can follow three main routes: random, ethno (including
ethnobotanical, ethnomedical and ethnopharmacological) and ecological search
(Fabricant and Farnsworth, 2001). The ethnobotanical and ethnopharmacological
approach uses information obtained from traditional medical practitioners and other
people such as village elders and local women who are traditional users of medicinal
plants. Ethnomedicinal reports are available on the use of plants for the treatment of
diarrhea and dysentery by the tribals from district Mayurbhanj (Rout and Pandey, 2002;
Pandey and Rout, 2002; 2006; Rout and Panda 2010, Panda et al., 2011a,b,c; Panda et al.,
2012; Panda, 2014). There is limited information on the safety of traditional plant
extracts, although some clinical trials have evaluated the safety and tolerability of
herbal medicine preparations used to treat diarrhea and generally indicate that minimal
side effects are observed. Based on earlier reports and data collected from the present
ethnomedicinal uses, plants were selected to find out the experimental validation of
tribal knowledge for the curing of infectious diarrhea.
LITERATURE REVIEW
Diarrhea poses a significant economic and societal burden throughout the world both in
developing and developed countries. Annually, more than five million people, 80% of
whom are less than one year of age, die from acute infectious diarrhea (Sood and
Pacheco, 2002). Additional agents of infectious diarrhea for which clinical diagnostic
testing is not routinely available include enterotoxigenic, enteropathogenic,
enteroaggregative, and enteroinvasive strains of E. coli, toxin-producing Clostridium
perfringens, Staphylococcus aureus, Bacillus cereus and rotaviruses (Guerrant, 1998).The
emergence of multiple drug resistant strains of diarrheagenic pathogens has made the
treatment of dysentery more difficult. In developing countries, the majority of people
living in rural areas almost exclusively use traditional medicines in treating all sorts of
diseases, including diarrhea. A range of medicinal plants with anti-diarrheal properties
have been widely used by the tribes of Mayurbhanj. The effectiveness of many of these
plants has not been scientifically evaluated.
Diarrhea: Definition and related terms

Diarrhea is loosely defined as an alteration in the normal bowel movement
characterized by an increase in the volume, frequency and water content of stool (Baldi
et al., 2009). The patho-physiology of diarrhea include microbial and parasitic infections
(Hodges and Gill, 2010), stress (oxidative or physical) (Soderholm and Perdue, 2006),
dysfunctional immunity (Schulzke et al., 2009), disrupt GIT integrity and neurohumoral
mechanisms (Vitali et al., 2006; Spiller, 2004) . Diarrhea can also be a symptom of other
diseases such as cholera, irritable bowel syndrome (IBS), gastroenteritis (intestinal
inflammation and ulcerative colitis) (Schiller, 1999; Baldi et al., 2009), malaria (Gale et
al., 2007) and diabetes mellitus (Forgacs and Patel, 2011) etc.

Classification
Diarrheal disease can be either infectious or non-infectious in nature. In infectious
diarrhea, the potential causative pathogens include bacterial agents (Mathabe et al.,
2006), rarely fungal (Robert et al., 2001), viral and parasite pathogens (Brijesh et al.,
2006). Non-infectious diarrhea can be caused by adverse reactions to drugs, toxins,
allergy to food, poisons and acute inflammation which promote the release of
secretagogues and some enteric nervous system (ENS) receptors (prostaglandin,
serotonin, substance P, vasoactive intestinal peptides, and hormone) in the GIT (Wynn
and Fougere, 2007) . Diarrhea can be classified in several ways viz. according to the
duration of the symptoms (Acute, persistant, chronic); according to stool
characteristics or pathological mechanisms (watery, osmotic, altered motility or
inflammatory diarrhea) (Ravikumara, 2008); according to age group (infantile,
weanling, childhood, adult); based on epidemiological pattern (sporadic, endemic,
epidemic, pandemic); based on seasonal (summer, winter, monsoon) etc.
Plant metabolites as potential therapeutic agents

Since ancient time, plants have been used as medicines by peoples. Today, the majority
of people in the world use traditional medicines for their primary course of treatment
because (i) biomedical healthcare systems and pharmaceuticals are not available in most
places, (ii) Synthetic drugs are not safe. Thus, to improve health and to instill pride in
traditional knowledge systems, several governments (e.g., China, India, and South
Africa) are incorporating traditional healthcare practices into their national regimes
(WHO, 2003). Medicinal plants have therapeutic properties due to biosynthesis of
various complex phytochemical substances grouped broadly as phenolic compounds,
quinones, stilbinoids, flavonoids, tannins, coumaranis, alkaloids, terpenoids, lectins and
polypeptides. Pharmacological and clinical studies of phytochemicals in plants have
shown that they exhibit various medicinal uses (Van Wyk and Wink, 2004). Synergistic
interaction among the multiple phytochemicals may be responsible for the overall
bioactivity of a given medicinal plant. Several plant extracts, formulations, or pure
natural compounds are used in controlling diverse diseases incluiding diarrhea to
parasitic infection in both human and veterinary medicine.
Plant-based remedies in improving diarrhea

Ethnobotanical theories support the effectiveness of plants for therapeutics, incliding all
type of gastrointestinal diseases. All animals including humans have develop senses to
select appropriate plants for ingestion, digestive enzymes to acquire nutrients from
plants, and behaviors or detoxifying enzymes to neutralize harmful plant chemicals
(Johns, 1996). Due to the widespread occurrence of diarrhea as a disease together with
the prevalence coinciding with human development, plants have featured widely in the
management of the disease both in human and veterinary medicines. Johns (1999)
postulates that “fundamental forms of medicine involve the gastrointestinal tract”

because of the immediate cognitive link between ingesting effective medicinal plants
and relief of gastrointestinal ailments. This cognizant association could be one reason
why all known pharmacopeias of the world possess remedies for gastrointestinal
illness. Another reason is that gastrointestinal ailments are ubiquitous. Therefore, the
need for treatments is universal.
Possible mechanisms of action for anti-diarrheal plants

Plants may act in various ways in alleviating diarrhea. It also providing nutrients and
generally increasing gastrointestinal health. However, for treatment purpose plants are
known as anti-infectious agents due to their secondary metabolites to fight against
diarrheal pathogens.
Antimicrobial: Many plant metabolites are known to exhibit some level of toxicity
toward microorganisms. Numerous mechanisms of actions have been hypothesized to
explain their antimicrobial activity such as microbial enzymes inhibition, deprivation of
essential growth substrates, cell membrane disruption (Cowan, 1999) or direct
interference with metabolic pathways.
Antiadhesion: Adhesion of some enteric pathogen to the mucosa epithelium of the host
cells is the first important step in intestinal infections that may lead to the development
of diseases (Ofek and Sharon, 1990). Application of antiadhesives chemotherapy can be
effective only against microorganisms that depend on the surface contact with host cells
as prerequisite for survival, multiplication and virulence (Lengsfeld et al., 2007).
Antitoxin: Since enteric pathogens may induce diarrhea through the production of toxin
(endotoxin or cytotoxin) the neutralization with plant antidiarrheal compounds may
beneficial in the management of diarrhea. Activated charcoals processed from plants
are also used as toxin binders.
Immunomodulatory: With immune suppression being a pre-disposing, drugs or

medicinal plant preparations with immune stimulating activities may help in
attenuating many infectious diseases.
Phytochemicals responsible for anti-diarrheal properties

Much plant-based anti-diarrheal research has analyzed the effects of phytochemicals on
intestinal tissues (Brijesh, et al., 2006; Grover, et al., 2002; Sagar, et al., 2005; Shaphiullah,
et al., 2003; Shilpi, et al., 2006; Teke, et al., 2007; Gutierrez et al., 2007). Using rodent
models, extracts are evaluated for antispasmodic effects, gut motility suppression, or
water and electrolyte reabsorption (Akindele and Adeyemi, 2006; Mbagwu and
Adeyemi, 2008; Sairam, et al., 2003; Thakurta, et al., 2007) with tannins and flavonoids
exhibiting promising results for water and electrolyte retention (Palombo, 2006).

Astringent and pectin-rich plants often are used to treat diarrheal disease, as are opiates
that slow smooth muscle contractions of the intestines (Lewis and Elvin-Lewis, 2003).
However, these remedies that suppress intestinal function block the symptoms of
diarrhea and not the causes.
Bacteria, viruses and parasites are the major causes of infectious diarrhea, with bacteria
leading highest (2-4 billion cases of infection with 3-5 million deaths per year (Sanchez
and Holmgren, 2005). Phytochemicals inhibit the growth and virulence of diarrhea-
causing bacteria in numerous ways. When bacteria invade the intestines, they follow
similar etiologies. The sequence, known as the five stages of pathogenicity (Mitchell,
1998), includes: 1) adherence to host tissue, 2) invasion or control of host tissues, 3)
multiplication in host tissues or with nutrients from host tissues, 4) evasion of host
defenses, and 5) damage and spread.
Phytochemicals can inhibit bacterial growth or virulence at any of these five stages of
pathogenicity. For example, mucilaginous, astringent and fibrous properties of some
plants can mechanically prevent bacterial adhesion to host intestinal cells by direct
competition between plant-derived lectins and bacterial membrane glycosides (Coutião
Rodriguez et al., 2001; Rabbani et al., 2004).
Agents involved in causing diarrhea

Bacterial causes of diarrhea
Escherichia coli
E. coli is a gram-negative rod shaped bacteria that shares a symbiotic relationship with
animal host as part of normal digestive intestinal flora. Under certain define conditions
these organisms or pathogenic strains of these organisms are known to induce diarrhea
(Clarke, 2001; Le Bouguenec, 2005). There are six main types of pathogenic E. coli
associated with diarrhea, namely enterotoxigenic E. coli (ETEC), enteroinvasive E .coli
(EIEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC),
enteroaggregative E. coli (EAEC) and diffusively adherent E. coli (DAEC) (Clarke, 2001).
The characteristics and mode of actions of each type of the pathological strains in
diarrhea diseases are listed in Table-1.
Table-1: Charecterstice of E. coli starins causing diarrheal diseases

Pathogens Epidemiology Symptoms
Enterotoxigenic E. Most common cause of Watery diarrhea ranging from
coli (ETEC) traveler‟s diarrhea. Most of mild, self-limiting disease to
the children are affected in severe purging.
developing countries.
Enteroinvasieve E. Important cause of diarrhea Bloody, mucoid diarrhea and
coli (EIEC) in the areas of poor hygiene. dysentery.

Enterohaemorrhagic Most important in human Bloody diarrhea, fever, vomiting,
E. coli (EHEC) infection. Out breaks and haemorrhagic colitis, haemolytic
sporadic cases all over the uremic syndrome (HUB), acute
world. renal failure, haemolytic anaemia
Enteropathogenic E. Sporadic case and mostly Self-limiting watery diarrhea
coli (EPEC) occurs in baby with fever and vomiting.
Staphylococcus aureus
S. aureus is a gram-positive coccus present in normal intestinal and skin flora of human
and homoeothermic animal. Under define conditions, the pathogenic strains produces
heat stable staphylococcal enterotoxins (SETs) and toxic shock syndrome toxins (TSST-
1) (de Oliveira, 2010) both of which are known to induce diarrhea. Upon ingestion of
the contaminated food with the SETs, results diarrhea associated with fever, nausea and
vomiting (Rosengren et al, 2010; Perez-Bosque and Moreto, 2009). In contrast, TSST-1 is
characterized by sudden onset of fever, vomiting, diarrhea, erythematous rash with
skin peels, hypotensive shock, impairment of renal and hepatic functions. Toxicity
results by way of the production of pro-inflammatory cytokines and chemokines.
Toxicity is usually aggravated by further interaction between the activated immune
system and inflammatory mediators (Krakauer et al., 2001).
Campylobacter jejuni
C. jejuni is an invasive Gram-negative, spiral-shaped rod bacterium present in the GIT
of mammals, birds and primates (Lengsfeld et al., 2007). The clinical signs of
campylobacter infections include pyrexia, abdominal pains, watery diarrhea and
dysentery (Podewils et al, 2004). The characteristic mechanisms Campylobacter
infection involves invasion and translocation of the epithelium with a concomitant
induction of inflammation (Hu et al., 2008).
Shigella species
Four species of Shigella (S. flexneri, S. dysenteriae, S. sonnei and S. boydii) invades the
colon with resulting inflammation and diarrhea (Podewils et al., 2004). S. flexneri is
responsible for dysenteric symptoms and persistent illness while S. dysenteriae type-1
produces Shiga-toxin causes bloody diarrhea (Podewils et al, 2004), S. sonnei causes
bacterial gastroenteritis and bacillary dysentery and S. boydii causes fever, chills,
abdominal pain and diarrhea.
Vibrio cholerae
V. cholerae is a motile, facultative anaerobic Gram-negative rod associated with
potentially fatal diarrhea (Granum, 2006) that results from the ingestion of the cholera
enterotoxins (CT) (Podewils et al, 2004). Watery, colour less mucous-flecked stool and
vomiting are the main clinical signs associated with cholera which in severe cases can

result in a life-threatening fluid and electrolyte imbalance (Podewils et al, 2004).
Pathophysiologically, toxicity results from the CT induction of intestinal hypersecretion
through the activation of the mucosal epithelium cAMP-adenylate cyclase system in the
mucosal epithelium (Casburn-Jones and Farthing, 2004). Other species of Vibrio such as
V. parahaemolyticus and V. vulnificus also caused watery diarrhea, abdominal cramps,
nausea and vomiting.
Bacillus species
B. cereus is a sporulating bacterium that causes diarrhea due to food poisoning resulted
by enterotoxins such as haemolysin BL (HBL), non-haemolytic enterotoxin (NHE) and
cytotoxin K (CytK) (Lund et al., 2000). Other species of Bacillus such as B. subtilis, B.
licheniformis, B. pumilus and B. megaterium can also produce enterotoxins and emetic
toxins involved in food borne illness but are usually considered relatively safe (From et
al, 2007).
Yersinia species
Yersinia species are Gram-negative facultative anaerobic nonsporing rods bacteria
belonging to Enterobacteriaceae family. Two pathogenic species Y. enterocolitica and Y.
pseudotuberculosis are responsible for yersiniosis with clinical signs such as diarrhea,
vomiting, fever and abdominal pain (Linscott, 2011).
Listeria monocytogenes
L. monocytogenes is the only single species which causes life-threatening invasive
diseases referred to listeriosis in human and animals (Chaturongakul et al., 2008; Todd
and Notermans, 2011). Upon ingestion of the bacteria through contaminated foods,
causes diarrhea by colonizing the intestinal tract (Chaturongakul et al., 2008).
Clostridium species
Three species of Clostridium viz. C. difficile, C. botulinum, C. perfringens are responsible
for diarrheal diseases. C. difficile causes a spectrum of diseases ranging from benign
diarrhea to fatal colitis and most often as a consequent of antibiotics treatment. Most
antibiotics predispose C. difficile overgrowth leading to the production and
accumulation of and diarrhea are Toxins A (enterotoxin) and B (cytotoxin) in the
intestine. Both toxins A and B inactivate intracellular Rhoproteins by glycosylation,
leading to desorption of the cytoskeleton, production of inflammatory cytokines and
damage to tight junctions. In contrast, C. perfringens is an important food poisoning
bacterium with clinical sign as diarrhea, abdominal cramping and nausea. C. botulinum
play a role in diarrheal diseases due to botulinum toxin resulting abdominal cramping,
nausea, vomiting, diarrhea, double vision, long term nerve damage and possible even
death from paralysis (Linscott, 2011).
Salmonella typhimurium

S. typhimurium is a bacterium that may be associated with mild gastroenteritis to enteric
(typhoid) fever, bacteraemia and septicaemia commonly referred to as salmonellosis
(Mastroeni and Maskell, 2006). The clinical signs of salmonellosis include diarrhea,
fever and abdominal cramps.
Enterococcus faecalis
E. faecalis is a gram-positive bacterium that survives symbiotically in the intestinal tract.
However, under stress conditions such as the disruption delicate host-commensal
relationship due to overuse of antibiotic use, abdominal surgery or changes in host
immunity, the enterococci becomes invaders of the intestinal wall (Butler, 2008)
through the production of adhesin, aggregating and binding substances (Butler, 2008).
E. faecalis is known to produce superoxide (O2-) that can results in hydroxyl radical
formation which contributes to oxidative stress in the intestine and membrane lipid
peroxidation (Huycke and Moore, 2002; Sun et al, 2010) resulting diarrhea.
Other bacterial species

Bacteria such as Klebsiellae species, Aeromonas species, Enterobacter species, Citrobacter
species, Proteus species, Pseudomonas aeruginosa and Plesiomonas shigelloides were also
important bacteria involved in causing diarrheal diseases.
Table-2: Clinical feuture of bacterial diarrheal disese other than E. coli

Pathogens Incubation Symptoms
period
Bacillus cereus, Staphyloccus aureus 1-8 hr Diarrhea, vomiting
Clostridium perfringens 8-24 hr Diarrhea, abdominal
cramps
Vibrio cholerae, Klebesiella pneumoniae 8-72 hr Diarrhea, vomiting,
abdominal cramps, fever
C. difficile 24-72 hr Diarrhea, abdominal
cramps
Salmonella, Campylobacter, Aeromonas, 12hr-11 day Diarrhea, vomiting,
Yersania species and V. abdominal cramps, fever
parahaemolyticus
Shigella sp. 1-4 days Diarrhea, abdominal
cramps, fever
Giardia sp. 1-8 days Diarrhea, fever
Rotavirus, Norvirus 24-72 hr Diarrhea, fever
Viral agents
Several viruses belonging to Rotavirus, Norwalk viruses, Adeno viruses, Calciviruses,
Coronaviruses, Astroviruses and Enteroviruses are responsible for causing diarrhea
throughout the world.

Rotavirus
Rotavirus is a major cause of severe diarrhea due to the production of enterotoxin NSP4
which inducesd Na-glucose dependent malabsorption and destruction of enterocytes
(cytotoxicity). The toxin also has a direct effect on the intestinal barriers by blocking TJs
formation with resultant diarrhea through a „leak flux‟ mechanism in which water is
secreted into the lumen of the intestine (Dickman et al., 2000).
Norovirus
Norovirus is considered as major global causes of gastroenteritis resulting diarrhea
(Mattison, 2010). The disease is opportunistic with clinical signs viz. nausea, vomiting,
diarrhea and abdominal pains (Koopmans, 2008).
Hepatitis A virus
Hepatitis A is a small, non-enveloped spherical with cubic symmetry, thermostable and
acid resistant entero virus. It multiply in the intestinal epithelium and reaches the liver.
The clinical signs are dark urine, jaundice, malaise, weakness, fever, anorexia, nausea
and vomiting, abdominal pains, and diarrhea (Koff, 1992).
Human immunodeficiency virus (HIV)

Chronic diarrhea is one of the complications associated with HIV infection and acquired
immune deficiency syndrome (AIDS) due to multiple enteric opportunistic microbes
(DuPont and Marshall, 1995). While HIV is important in secondary enteric diseases as a
result of immune suppression (CD4+ T-lymphocytes destruction), the virus can result in
diarrhea directly by altering the mucosa structural arrangement referred as
HIVenteropathy (Epple et al., 2009). The diarrhea resulting from HIV appears to be
caused by the releases of cytokines from the infected immune cells (Schmitz et al., 2002).
Protozoa and Parasitic agents

Sevral protozoans and parasites such as Entemoeba histolytica, Giardia lamblia, Balantidium
coli, Cryptosporidium sp., isospora belli, Fasciola hepatica, Taenia saginata, T. solium,
Hymenolepis nana, Schistosoma mansoni, S. japonica, Trichuris trichiura, Ancyclostoma
duodenale, Necator americanus etc. are involved in diarrheal diseases.
Giardia intestinalis
G. intestinalis (syn. G. doudenalis, G. lamblia) is a flagellate protozoa parasite colonizes
the small intestinal lumen and induces non-inflammatory and malabsorptive diarrhea
(Schulzke et al., 2009). The pathophysiology of Giardiasis involves Na+ dependent D-
glucose absorption impairment, active electrogenic anion secretion activation, mucosal
inflammation and leak flux (Buret, 2007; Troeger et al., 2007). Clinical signs of Giardia
infection include bloating, steatorrhea and nausea.
Entamoeba histolytica

E. histolytica is a protozoa parasite which infects the large intestine with resultant
intestinal dysfunction characterized by invasive illness and severe dehydration
commonly referred to as amoebiasis (Ralston and Petri, 2011). The pathophysiology of
amoebiasis involves villus structural destruction and increase in epithelial permeability
(Lauwaet et al., 2004). The clinical signs are usually similar to S. dysenteriae or
enteroinvasive E. coli with blood and pus contaminated stool. Other related infectious
species include E. dispar and E. moshkovskii (Ralston and Petri, 2011).
Cryptosporidium parvum
C. parvum is an intracellular protozoa parasite that infects epithelia causing
cryptosporidiosis which manifest clinically as profuse watery diarrhea containing
mucus and some times blood or leukocytes (Kenny and Kelly, 2009; O‟Hara and Chen,
2011).
Cyclospora cayetanensis
C. cayetanensis is a protozoan parasite which invades the epithelial cells of the small
intestine (Chacin-Bonilla, 2010; Manfield and Gajadhar, 2004). The clinical signs of the
infection include watery diarrhea, loss of appetite, weight loss, abdominal bloating and
cramping, increased flatulence, nausea, fatigue, and low-grade fever (Linscott, 2011).
Trichinella spiralis
T. spiralis is a food-borne zoonotic parasite induced changes in intestinal function by
hypersensitivity mechanism resulting in an increased intestinal chloride and fluid
secretion (Cui et al., 2011). The clinical intestinal symptoms are nausea, abdominal
pain, vomiting and diarrhea (Linscott, 2011).
Fungal induced diarrhea by Candida albicans

C. albicans is the only fungal originate organism is responsible for diarrhea and it exist
as a member of normal flora in the GIT and mucocutaneous membrane. Due to overuse
of antibiotic therapy that results in sterilization of the GIT flora, C. albicans can
overgrowth to take the place of removed organisms with end result of diarrheal
symptoms (Henry-Stanley et al., 2003). Other predisposing factors include altered
intestinal permeability and diminished host immunity response. It has been postulated
that this organism produces virulence factors which increases fungal adherence to host
cells and secretion of proteolytic enzymes (Henry-Stanley et al., 2003). Clinical signs
associated with enteric candidiasis are abdominal pain, cramping, rectal irritation and
absence of nausea, vomiting, bloody and mucus stool, and pyrexia (Levine et al., 1995).

The phytogeography of Similiapl Biosphere Reserve, Mayurbhanj, Odisha
The Similipal Biosphere Reserve (Figure 1), one of the mega biodiversity zones of the
country, is situated in the central part of the Mayurbhanj district of Odisha (20 o17‟ -

22o34‟N and 85o40‟ - 87o10‟E) and covers an area of 5569 km2. The reserve has been
divided into three zones i.e. Core Zone, Buffer Zone and Transition Zone. The core zone
(845Km2) is reserved for wildlife habitat development and no exploitation activities are
entertained in this area. The buffer zone (around 2,129 Km2) is partially prohibited and
is allowed for some activities like research, education and tourism. On the other hand
the transition zone, which lies in the peripheral region covering 2,595 Km 2, is allowed
for research, tribal settlement, tourism and other environment friendly activities.
Health care in the tribal villages of Mayurbhanj

In Similipal Biosphere Reserve there are 4 villages inside the core area, 65 villages in the
buffer zone and 1200 villages in the peripheral zone. The human population in all being
more than 4.5 lakhs, where tribals occupy about 52 % of its population and 53
communities, both aboriginal and migrated, inhabit the district glorifying the rich
heritage of tribal culture. Among the tribal communities, the chief ones are Santal, Kol,
Bhomij, Bhuyan, Bathuri, Kharia, Gonds, Mankdias, Pauri-Bhuyan, Saharias, Mahalis
and Sounti. Some of these tribes namely Kharias, Mankdias and Saharas are still in
primitive state of living. Most of the villages are situated in the mountainous terrain of
the district i.e. not well communicated with the district head quarter hospital. Till now
the health care system are still sparse in the core areas. The local tribal baidays and
tribal old man usually are the first to prescribe medicines for various remedies when
illness strikes. Regarding the common diseases the respondents informed about the
frequent occurrence of skin infection with other diseases like dysentery, diarrhea and
snakebite.

Figure-1: MAP OF Mayurbhanj District showing Similipal Biosphere Reserve
Knowledge on types of diarrheal disease among the tribes of Mayurbahanj

The most common type of diarrhea among the villagers was the green color and rice
water diarrhea. All these diarrheal diseases mostly are induced by bacteria. The green
color occurs because of un-processed green bile secretions from the upper small
intestines which normally turn brown during transit. The black color results from
blood that is acidified, as in the acidic environment of the upper small intestine

(Navaneethan and Giannella, 2011). The tribals also have also knowledge on bloody
diarrhea. Most cases of bacillary dysentery, caused by Shigella species, lead to blood in
the stool as bacteria lyse and kill the epithelial lining of the intestines (Fernandez and
Sansonetti, 2003). Watery diarrhea can be caused by too many salts or fats in the colon.
Watery diarrhea is caused by bacterial infection releasing toxin by E. coli, Vibrio cholerae,
V. parahaemolyticus, or Campylobacter jejuni.
Ethnomedicinal documentation
In Mayurbhanj district, phytotherapy (treatment with medicines from plant and their
derived products) forms an integral part of the local culture, and the information about
plants and their uses are passed from generation to generation through oral folk-lore,
primarily amongst the elderly; the natural retainers of traditional knowledge in their
respective communities. The field study was carried out from September 2006 to
November 2008, and the information on the use of medicinal plants for treatment of
diarrhea was obtained through structured questionnaires, complemented by free
interviews and informal conversations (Huntington, 2000). The interviews were
individually carried out during the first contacts with the local population, native
specialists were identified, i.e. people considered by the community as having
exceptional knowledge about the use of plants.
Collection of medicinal plants

The present work is based on the explorations made in Similipal Biosphere Reserve
during 2006-10. Field trips to Similipal Biosphere Reserve were undertaken for
collection of medicinal plants. Identification of these medicinal plants was done at the
Post Graduate Department of Botany, North Orissa University, Baripada.
Processing
Stems, leaves, bulb, barks, roots, rhizomes, seeds etc. of plants have separately been
collected during field trips to different parts of Similipal Biosphere Reserve. The roots
are dug out from the soil and the adhering soils were removed by shaking and washing.
The leaves were plucked from the trees, washed properly and infected leaves were
discarded. After collection, the healthy leaves were dried at low temperature to
maintain their green color and volatile oils, if present. The materials were completely
shed dried so long it does not allow for the growth of any type of fungi, molds, bacteria
and other microorganisms. The dried leaves, roots and stems were powdered separately
using mortar and pestle and then were passed through sieve to get the uniform
powdered sample.
Preparation of plant extracts

Twenty grams of each powdered samples were dissolved in 100ml of sterile distilled
water and 80% methanol separately in wide mouth bottles. The aqueous samples were

prepared by adding distilled water (steam for 30 minutes) and stored overnight.
Similarly, methanol samples were incubated at room temperature for 48 hrs. The
suspension was then filtered (Whatman No. 40) separately and made up to 100ml with
respective solvent and utilized for studying their antimicrobial properties.
Test bacteria
Pathogenic bacteria under study were Escherchia coli, Escherichia coli O157:H7, Salmonella
typhimurium, Salmonella typhi, Shigella dysentriae, Vibrio alginolyticus, Vibrio cholerae and
Vibrio cholerae O139.
Maintenance of bacteria
The bacterial cultures were maintained on nutrient agar (NA) slants and stored at 4 оC.
Activation of the bacterial was carried out by streaking culture from the slants on to a
Muller Hinton agar (MHA) plate and incubating overnight at 37 оC. Single colony was
picked up from each plate and transferred to nutrient broth, incubated for 1 day at 37 оC
prior to the test.
Antibiotics
The following antibiotics sensitivity test disc (Hi media Pvt. Ltd., Mumbai, India) at the
given concentration were used to determine antibiotic sensitivity profile of reference
bacteria: Amikacin-Ak (30μg); Amoxicillin-Aug (10μg); Amphotericin-Ap (100unit);
Ampicillin-A (10μg); Bacitracin-B (10units); Cefoxitin-Ctn (10μg); Ceftriaxone-Cez
(10μg); Cephotaxime-Ce (30μg); Chloroamphinecol-Ch (10μg); Ciprofloxacin-C (10μg);
Erythromycin-E (15μg); Gatifloxacin-Gf (30μg); Gentamycin-G (10μg); Levofloxacin-Lvx
(5μg) ; Naladixic acid-Nal (30μg); Ofloxacin-Ofl (5μg); Polymyxin-B-Pb (300 unit);
Streptomycin-St (10μg); Tetracycline-Te (10μg) and Vancomycin-Vn (30 μg).
Sensitivity test
Antibiogram was done by disc diffusion method (Bauer et al., 1966) with commonly
used antibiotics. Antibiotic sensitivity was tested in MHA plates. The test microbes
were removed from the slant aseptically with inoculating loops and transferred to
separate test tubes containing 5.0ml of sterile distilled water. Inoculums added until the
turbidity equaled 0.5 McFarland (108 CFU/ml). For each of the bacteria, one milliliter of
the test tube suspension was added to 15-20ml of nutrient agar and transferred to the
agar plate (90 mm diameter). After cooling the inoculated agars at room temperature for
25 min, antibiotic sensitivity test discs were placed on the surface of solid agar. The
plates were incubated at 37°C. The plates were examined thereafter, clear zones of
inhibition formed around the discs were measured and antibiotic sensitivity was
assayed from the diameter of the clear inhibition zones (in mm) (Figure-2).

Figure-2: Antibiogram among strains
Screening of antimicrobial properties by agar cup method
The agar cup method was followed to study antibacterial activity of the extracts.
Overnight Muller Hinton Broth culture of the test organisms were firmly seeded over
the MHA plates. Wells of 6 mm diameter was punched over the agar plates using a
sterile borer. The bottoms of the wells were sealed by pouring 50-100 µl of molten MHA
into the scooped out wells. 100 µl of extracts were poured into the wells. The water was
allowed to evaporate and the plates were incubated at 37 °C for 18-24 hours. Presence of
zone of clearance around the wells, confirmed the antibacterial activity of the extracts.
RESULTS
The antidiarrheal screening of 134 plants collected from Similipal Biosphere Reserve,
Orissa, India was carried out by agar cup method. The selection of plants was based on
ethnomedicinal uses reported earlier as well as freshly recorded data (Table-3). Two
different solvents viz. methanolic (80%) and aqueous used for the preparation of crude
extracts (Table-2). A total of 288 plants extract (Table-4) belonging to 66 families were
tested for anti-diarrheal activity. Among the selected 136 plants, 100 species showed
anti-bacterial activity against at least two or more test organisms. Two families with
higher number of plants didn‟t show any activity belonged to Rubiaceae and
Euphorbiaceae. Acanthaceae, Anacardiaceae, Apocynaceae, Ceasalpiniaceae,
Clusiaceae, Combretaceae, Fabaceae, Moringaceae, Oleaceae, Rutaceae, Punicaceae and
Verbenaceae were some of important families which showed anti-diarrheal activity
against pathogens. Screening results showed antibacterial activity with 107 methanol
and 74 aqueous extracts against one or more test strains.
Some of the active species have already been reported earlier to have antibacterial
activity. The effects of few medicinal plants viz. Justicia adhatoda, Achyranthes aspera,
Andrographis paniculata, Holarrhena antidysenterica, Cassia fistula, Carica papaya, Punica
granatum, Moringa oleafera, Vitex negundo, Hemidesmus Spondias pinnata were previously
described (Valsaraj et al. 1997) and was confirmed in this work. Similar results were

described by several authors by adding plants such as Achyranthes aspera, Annona
reticulate, Annona squamosa, Cassia fistula, Diospyros melanoxylon, Holarrhena pubescens,
Moringa oleifera, Mesua ferrea, Punica grantum, Semecarpus anacardium, Tamarindus indica,
Terminalia arjuna etc. (Ahmad et al., 1998; PerumalSamy et al., 1998, 1999; Jeevan Ram et
al., 2004; Prashantkumar et al., 2006; Parekh and Chanda 2008).
Figure 3: Agar cup method showing zone of inhibition
DISCUSSION
Diarrhea is known to be caused by several factors including the non infectious type of
diarrhea such as metabolic diseases, food allergy and other organic causes. On the
contrary, infectious diarrhea is a type of diarrhea which is caused by an infectious agent
(bacteria, fungus, parasites and viruses) due to the invasion and colonization of the host
tissue. Infectious diarrhea is characterized by an alteration in a normal bowel
movement, an increase in the volume of water content, or frequency of stools, nausea,
vomiting, cramps and/or abdominal discomfort (Sood and Pacheco 2002). Studies on
pathogens responsible for acute diarrhea in developing countries, revealed that the
contribution of Rotavirus (15-25%), entrerotoxigenic E. coli (10-20%), Shigella species (5-
15%) Salmonella species (1-5%), C. jejuni (10-15%) and entropathogenic E. coli (1-5%)
(EHNRI 2002). E. coli (EIEC) entroinvassive and enterohemorragic (EHEC), Salmonella,
Shigella, V. cholerae are the major bacterial pathogens most often responsible for causing
pandemic and epidemic infectious diarrheal disease in developing countries (Black et
al. 1984).

Table-3: Ethnomedicinal uses of medicinal plants from district Mayurbhanj, Orissa, India
Botanical name Family Parts Cure for References
used diseases
Acacia catechu (L.f.) Willd Mimosaceae Bk Diarrhea Kar et al., 2013
Acacia leucophloea Mimosaceae Bk Diarrhea Panda et al., 2011a
(Roxb.)Willd. Panda et al., 2014
Kar et al., 2013
Acacia nilotica (L.) Delile Mimosaceae Lf Diarrhea Kar et al., 2013
Dysentery
Achyranthes aspera L. Amaranthaceae Wp Dysentery Panda et al., 2011a
Rt Rout and Panda 2010
Mohanta et al. 2006
Kar et al., 2013
Panda et al., 2014
Acorus calamus L. Araceae Rh Diarrhea Panda et al. 2011a
Panda et al., 2014
Aegle marmelos L. Rutaceae Lf, Fr Diarrhea Rout and Panda 2010
Kar et al., 2013
Ageratum conyzoides L. Asteraceae Rt, Lf Diarrhea, Kar et al., 2013
Dysentery
Alangium salviifolium (L.f) Alangiaceae Bk Dysentery Kar et al., 2013
Wang.
Albizia lebbeck Benth. Mimosaceae Bk Dysentery Kar et al., 2013
Allium cepa L. Liliaceae Bl Diarrhea, Rout and Panda 2010
Dysentery Kar et al., 2013
Allophylus serratus (Roxb.) Sapindaceae Wp Diarrhea Kar et al., 2013
Kurz
Alstonia scholaris (L.) R. Br. Apocynaceae Bk Diarrhea, Panda et al., 2014

Andrographis paniculata Acanthaceae Lf, St Dysentery Rout and Panda 2010
(Burm. f.) Wall. ex Nees Kar et al., 2013
Angiopteris evecta Forst. Angiopteridaceae Lf Dysentery Panda et al. 2011a
Hoff
Annona reticulata L. Annonaceae Lf Diarrhea Padhi et al. 2011
Annona squamosa L. Annonaceae Lf Diarrhea Padhi et al. 2011
Anogeissus latifolia (Roxb. Combretaceae Lf Diarrhea Panda et al. 2011a
ex DC.) Wall. ex Bedd Bk Mohanta et al. 2006
Panda et al., 2014
Kar et al., 2013
Anthocephalus chinensis Rubiaceae Bk Diarrhea Kar et al., 2013
(Lam.)A.Rich.ex.Walp.
Ardisia solanacea Roxb. Myrsinaceae Rt Diarrhea Kar et al., 2013
Asparagus racemosus Willd. Liliaceae Rt Diarrhea Kar et al., 2013
Dysentery
Azadirachta indica A.Juss. Meliaceae Lf Diarrhea Kar et al., 2013
Dysentery
Barringtonia acutangula Barringtoniaceae Lf Diarrhea Kar et al., 2013
(L.) Dysentery
Gaertn.
Bauhinia purpuria L. Caesalpiniaceae Bk Diarrhea Kar et al., 2013
Bauhinia racemosa Lam. Caesalpiniaceae Bk Dysentery Kar et al., 2013
Bauhinia vahlii W. & A. Caesalpiniaceae Bk Dysentery Panda et al. 2011a
Kar et al., 2013
Bauhinia variegata L. Caesalpiniaceae Bk Dysentery Kar et al., 2013
Boerhavia diffusa L. Nyctaginaceae Rt Diarrhea Kar et al., 2013
Dysentery
Bombax ceiba L. Bombacaceae Gum Diarrhea Panda et al., 2012

Boswella serrata Roxb. Burseraceae Bk Diarrhea Kar et al., 2013
Bryophyllum calycinum Crassulaceae Lf Dysentery Panda et al., 2012
Salis.
Buchanania lanzan Spreng. Anacardiaceae Lf Diarrhea Panda et al., 2012
Butea monosperma (Lam.) Fabaceae Rt, Sd Dysentery Rout and Panda 2010
Taub. Kar et al., 2013
Butea superba Roxb. Fabaceae Rt Dysentery Panda et al., 2012
Calotropis gigantea R. Br. Asclepiadaceae Rt Dysentery Panda et al., 2012
Kar et al., 2013
Calotropis procera (Ait.) R. Asclepiadaceae Rt Cholera Kar et al., 2013
Br.
Canthium dicoccum Rubiaceae Rt Diarrhea Kar et al., 2013
(Gaertn.) Teijsm. & Lf
Binnend.
Careya arborea Roxb. Lecythidaceae Bk Blood Mohanta et al. 2006
Dysentery Rout and Panda 2010
Kar et al., 2013
Cassia fistula L. Caesalpinaceae Lf Dysentery Panda et al. 2011c
Catharanthus roseus (L.) G. Apocynaceae Lf Dysentery Rout and Panda 2010
Don
Catunaregam spinosa Rutaceae Bk Diarrhea, Kar et al., 2013
(Thunb.) Tirven Dysentery
Centella asiatica (L) Urban Apiaceae Lf Diarrhea, Panda et al., 2014
Dysentery
Cissampelos pareira L. Menispermaceae Rt Diarrhea Rout and Panda 2010
Citrus limon (L.) Burm. f. Rutaceae Fr Diarrhea Panda et al., 2014
Kar et al., 2013
Citrus medica L. Rutaceae Fr Diarrhea Panda et al. 2011a;
Rout and Panda 2010

Clausena excavata Burm. f. Rutaceae Rt, Lf Diarrhea, Panda et al. 2011a
Dysentery Mohanta et al. 2006
Panda et al., 2014
Kar et al., 2013
Cleome viscosa L. Capparaceae Wp Diarrhea Kar et al., 2013
Commelina benghalensis L. Commelinaceae Wp Dysentery Kar et al., 2013
Coccinia grandis (L.) Voigt Cucurbitaceae Lf Diarrhea, Panda et al., 2012
Dysentery
Crotalaria spectabilis Roth. Fabaceae Rt Dysentery Rout and Thatoi,
2009
Panda et al., 2014
Croton roxburghii Balak. Euphorbiaceae Lf Dysentery Panda et al. 2010
Panda et al., 2014
Curculigo orchioides Hypoxidaceae RT Dysentery Panda et al., 2012
Gaertn.
Curcuma angustifolia Roxb. Zingiberaceae Rh Dysentery Panda et al. 2011a
Mohanta et al. 2006
Panda et al., 2014
Kar et al., 2013
Curcuma aromatica Salisb. Zingiberaceae Rh Dysentery Panda et al., 2014
Cynodon dactylon (L.) Pers. Poaceae Wp Diarrhea Panda et al. 2011a
Kar et al., 2013
Panda et al., 2014
Cyperus rotundus L. Cyperaceae Rh Diarrhea Kar et al., 2013
Desmodium gangeticum (L.) Fabaceae Rt Dysentery Panda et al., 2014
DC.
Diospyros malabarica Ebenaceae Bk Diarrhea Panda et al. 2011a
(Desr.) Kostel Kar et al., 2013
Diospyros melanoxylon Ebenaceae Lf, Bk Diarrhea, Panda et al. 2009

Roxb. Dysentery Kar et al., 2013
Elephantopus scaber L. Asteraceae Rt, Lf Diarrhea Kar et al., 2013
Eleutherine bulbosa (Miller) Iridaceae Bl Dysentery Panda et al., 2012
Urban
Emilia sonchifolia (L.) DC Asteraceae Wp Diarrhea Kar et al., 2013
Erycibe paniculata Roxb. Convolvulaceae Bk Diarrhea, Panda et al., 2014
Eryngium foetidum L. Apiaceae Lf Diarrhea Panda et al., 2014
Ficus benghalensis L. Moraceae Bk Dysentery Kar et al., 2013
Ficus racemosa L. Moraceae Bk Dysentery Panda et al., 2012
Diarrhea Kar et al., 2013
Flemingia nana Roxb. Fabaceae Rt Diarrhea, Panda et al., 2012
Dysentery Panda et al., 2014
Gardenia gummifera L. f. Rubiaceae Rs Diarrhea, Kar et al., 2013
Dysentery
Gardenia resinifera Roth. Rubiaceae Rs Diarrhea, Kar et al., 2013
Dysentery
Glycyrrhiza glabra L. Fabaceae Bk Diarrhea Rout and Panda 2010
Grewia abutilifolia Vent.ex Tiliaceae Lf Dysentery Panda et al., 2012
A.L.Juss Bk Kar et al., 2013
Grewia helicterifolia Tiliaceae Fr Diarrhea, Kar et al., 2013
Wall.ex.G.Don Dysentery
Haldinia cordifolia (Roxb.) Rubiaceae Rt Diarrhea, Kar et al., 2013
Ridsd. Dysentery
Helicteres isora L. Sterculiaceae Rt Diarrhea, Panda et al., 2012
Hibiscus rosasinensis L. Malvaceae Tw Dysentery Panda et al. 2011a
Mohanta et al. 2006
Kar et al., 2013

Panda et al., 2014
Holarrhena pubescens Apocyanaceae Lf Dysentery Panda et al. 2011a
(Buch.-Ham.) Wall ex. G. Mohanta et al. 2006
Kar et al., 2013
Panda et al., 2014
Hyptis suaveolens (L.) Poit. Lamiaceae Wp Dysentery Panda et al., 2014
Indigofera cassioides Rottl. Fabaceae Rt, Fl Dysentery Panda et al. 2011a
ex DC. Mohanta et al. 2006
Kar et al., 2013
Panda et al., 2014
Ixora pavetta Andr. Rubiaceae Bk Dysentery Panda et al., 2014
Kar et al., 2013
Justicia adhatoda L. Acanthaceae Lf Diarrhea Rout and Panda 2010
Kar et al., 2013
Kalanchoe pinnata (Lam.) Crassulaceae Lf Dysentery Panda et al. 2011a
Pers. Mohanta et al. 2006
Panda et al., 2014
Kar et al., 2013
Lannea coromandelica Anacardiaceae Lf Diarrhea, Panda et al., 2012
Houtt. Dysentery Kar et al., 2013
Lawsonia inermis L. Lythraceae Rt, Lf Diarrhea, Kar et al., 2013
Dysentery
Leea indica (Burm.f.) Merr. Rutaceae Fr Diarrhea, Kar et al., 2013
Dysentery
Limonia acidissima L. Rutaceae Fr Diarrhea, Kar et al., 2013
Dysentery
Litsea glutinosa (Lour.) Louraceae Bk Diarrhea, Kar et al., 2013
Robin Dysentery
Lygodium flexuosum L. Lygodiaceae Wp Blood Panda et al. 2011a

Dysentery
Lygodium microphyllum Lygodiaceae Lf Dysentery Panda et al. 2011a
(cav.)R.Br.
Mangifera indica L. Anacardiaceae Bk Blood Panda et al. 2011a
Dysentery Rout and Panda 2010
Kar et al., 2013
Panda et al., 2014
Melastoma malabathricum Melastomataceae Lf Diarrhea Kar et al., 2013
L. Dysentery
Mesua ferrea L. Clusiaceae Lf Diarrhea Panda et al., 2012
Dysentery
Mimusops elengi L Sapotaceae Bk Diarrhea, Kar et al., 2013
Dysentery
Momordica charantia L. Dipterocarpaceae Bk Diarrhea, Mohanta et al. 2006
Dysentery
Morinda citrifolia L. Rubiaceae Rt Dysentery Panda et al. 2011a
Panda et al., 2014
Kar et al., 2013
Moringa oleifera Lam. Moringaceae Lf Diarrhea Mohanta et al. 2006
Kar et al., 2013
Murraya koenigii (L.) Rutaceae Lf Diarrhea, Kar et al., 2013
Spreng Dysentery
Musa paradisica L. Musaceae Fr Dysentery Mohanta et al. 2006
Panda, 2010
Nicotiana tabacum L. Solanaceae Wp Diarrhea Rout and Panda 2010
Kar et al., 2013
Nyctanthes arbortristis L. Oleaceae Bk Dysentery Panda et al. 2011b
Rout and Panda 2010
Panda et al., 2014

Ocimum canum Sims Lamiaceae Lf Dysentery Kar et al., 2013
Ocimum sactum L. Lamiaceae Lf Diarrhea Kar et al., 2013
Oroxylum indicum (L.) Bignoniaceae Lf, Bk Dysentery Kar et al., 2013
Vent.
Oxalis corniculata L. Oxalidaceae Wp Diarrhea, Panda et al., 2012
Dysentery
Paederia foetida L. Rubiaceae Lf Diarrhea Panda et al. 2011b
Panda et al., 2014
Phyllanthus emblica L. Euphorbiaceae Lf Diarrhea, Kar et al., 2013
Dysentery
Phyllanthus fraternus Euphorbiaceae Wp Diarrhea Panda et al., 2012
Webster Kar et al., 2013
Piper longum L. Piperaceae Wp Cholera Kar et al., 2013
Piper nigrum L. Piperaceae Sd, Fr Diarrhea Rout and Panda 2010
Plumbago zeylanica L. Plumbaginaceae Rt Diarrhea Rout and Panda 2010
Kar et al., 2013
Pogostemon benghalensis Lamiaceae Lf Dysentery Kar et al., 2013
(Burm.f.) Kuntze
Psidium guava L. Myrtaceae Lf, Fr Diarrhea Rout and Panda 2010
Kar et al., 2013
Pterocarpus marsupium Dipterocarpaceae Bk Blood Panda et al. 2011
Roxb. Dysentery Mohanta et al. 2006
Panda et al., 2014
Kar et al., 2013
Rout and Panda 2010
Pterocarpus santalinus L. f. Fabaceae Fr Dysentery Kar et al., 2013
Pterospermum acerifolium Sterculiaceae Bk Diarrhea Panda and Dutta,
(L.) Willd. Dysentery 2011
Panda et al., 2014

Punica granatum L. Punicaceae Lf Dysentery Pandey and Rout
2002
Kar et al., 2013
Quisqualis indica L. Combretaceae Sd Diarrhea Kar et al., 2013

Randia uliginosa (Retz.) DC Rubiaceae Fr Dysentery Panda et al., 2012
Rauvolfia serpentina (L.) Apocynaceae Rt Diarrhea Kar et al., 2013
Benth.ex.Kurz.
Rubia cordifolia L. Rubiaceae Rt Diarrhea Kar et al., 2013
Dysentery
Saraca asoca (Roxb.) de Caesalpiniaceae Bk, Lf Dysentery Kar et al., 2013
Wilde
Schleichera oleosa (Lour.) Sapendaceae Wp Diarrhea Panda et al. 2011a
Securinega virosa (Roxb. ex Euphorbiaceae Bk Diarrhea Kar et al., 2013
Willd). Baill
Semecarpus anacardium Anacardiaceae Fr Diarrhea Panda et al., 2012
L.f.
Sesamum orientali L. Pedaliaceae Lf Cholera, Kar et al., 2013
Dysentery
Shorea robusta Gaertn. F. Dipterocarpaceae Bk Dysentery Mohanta et al. 2006
Kar et al., 2013
Sida acuta Burm.f. Malvaceae Lf Dysentery Kar et al., 2013
Smilax zeylanica L. Smilacaceae Rt Diarrhea Panda et al., 2012
Soymida febrifuga (Roxb.) Melastomataceae Bk Dysentery Rout and Panda 2010
A. Juss.
Spilanthes calva DC. Asteraceae Fl Dysentery Kar et al., 2013
Spondias pinnata (L.f.) Anacardiaceae Lf, Fr, Dysentery Panda et al. 2011a
Kurz Bk Mohanta et al. 2006
Kar et al., 2013

Panda et al., 2014
Streblus aspera Lour. Moraceae Bk Dysentery Kar et al., 2013
Strychnos nux-vomica L. Strychnaceae Rt Cholera Kar et al., 2013
Syzygium cumuni L. Skeels Myrtaceae Bk Dysentery Mohanta et al. 2006
Diarrhea Kar et al., 2013
Tamarindus indica L. Caesalpiniaceae Lf, Sd Diarrhea Rout and Panda 2010
Tamilnadia uliginosa (Retz.) Rubiaceae Rt Diarrhea Kar et al., 2013
Tirveng. Dysentery
Terminalia alata Heyne Combretaceae Bk Diarrhea Mohanta et al. 2006
ex. Roth Kar et al., 2013
Terminalia arjuna (Roxb. ex Combretaceae Bk Chronic Pandey and Rout
DC.) W. & A. Dysentery 2002
Kar et al., 2013
Rout and Panda 2010
Terminalia bellirica (Gartn.) Combretaceae Bk Dysentery Kar et al., 2013
Roxb.
Terminalia chebula Retz. Combretaceae Bk Diarrhea Rout and Panda 2010
Thalictrum foliolosum DC. Ranunculaceae Rh Dysentery Kar et al., 2013
Tinospora cordifolia (Willd.) Menispermaceae Rt, St Diarrhea Kar et al., 2013
Hook.f.& Thoms. Dysentery
Tragia involucrate L. Euphorbiaceae Wp Diarrhea Rout et al. 2009
Trapa natans L. Trapaceae Fr Diarrhea Panda et al., 2014
Trema orientalis (L.) Bl. Ulmaceae Rt Diarrhea Kar et al., 2013
Trewia nudiflora L. Euphorbiaceae Lf Dysentery Kar et al., 2013
Tridax procumbens L. Asteraceae Rt Diarrhea Kar et al., 2013
Vitex negundo L. Verbenaceae Lf Diarrhea Mohanta et al. 2006
Kar et al., 2013
Woodfordia fruticosa (L.) Lythraceae Rt Dysentery Panda et al. 2011a
Kurz Rout and Thatoi 2009

Kar et al., 2013
Panda et al., 2014
Wrightia tinctoria (Roxb.) Apocynaceae Bk Diarrhea Kar et al., 2013
R. Br.
Zingiber officinale Rosc. Zingiberaceae Rh Diarrhea Kar et al., 2013
Ziziphus mauritiana Lam. Rhamnaceae Bk Dysentery Kar et al., 2013
Bl-bulb; Bk-bark; Fl-flower; Fr-fruit; Lf-leaf; Rt-root; Rh-rhizome; Rs-Resin; Sd-seed; Tw-twig; Wp-whole plant

A total of 288 plants extracts (Table-2) belonging to 66 families were tested for anti-
diarrheal activity. Among the selected 136 plants, 100 species showed anti-bacterial
activity against at least two or more test organisms. Some of important families with
anti-diarrheal activity against pathogens are Acanthaceae, Anacardiaceae,
Apocynaceae, Ceasalpiniaceae, Clusiaceae, Combretaceae, Fabaceae, Moringaceae,
Oleaceae, Rutaceae, Punicaceae and Verbenaceae. Screening results showed
antibacterial activity with 107 methanol and 74 aqueous extracts against one or more
test strains. Solvent or the extraction agent used in the preparation of
phytopharmaceuticals must be suitable for dissolving the important therapeutic drug
constituents. In addition, solvents used should be easy to remove, inert, nontoxic, and
not easily flammable. The aqueous extracts have commonly been used in preliminary
studies. Methanol efficiently penetrates cell membranes, permitting the extraction of
high amounts of endocellular components in contrast to the solvents with lower
polarity. Such solvents are limited for extracting mostly extracellular material. Hence,
methanol chiefly dissolves polar constituents together with medium and low polarity
compounds extracted by co-solubilazation. So, the aqueous and methanolic (80%)
extracts of different plant belonging to a wide range of families are selected for
screening based on random sampling and observed ethnomedicinal uses.
Table-4: Preliminary screening of plants against diarrhea causing bacteria
Plant description PU E S S S S S S S S
1 2 3 4 5 6 7 8
Acanthaceae
Adhatoda vasica Nees Lf A 12 - 11 - - 12 12 12
M 11 12 10 12 - 11 10 10
Andrographis paniculata Lf A 11 - - 11 12 12 12 10
(Burm. f.) Wall. ex Nees M 14 - - - - - - 13
St A 12 12 - - - - - 12
M 13 12 10 12 12 12 10 -
Alangiaceae
Alangium salviifolium (L.f) Lf A - - - - - - 11 12
Wang. M 12 - - - - - 10 13
Amaranthaceae
Achyranthes aspera L. Wp A - - 9 - - - - 11
M - 10 8 - - - - 12
Anacardiaceae
Buchanania lanzan Lf A - - - - - - - -
Spreng. M 10 - - 12 12 14 - 13
Lannea coromandelica Lf A 12 - - 10 - - 10 9

154
(Houtt.) Merr. M 14 12 - 14 - 12 10 14
Mangifera indica L. Lf A - - - - - - - -
M - - 12 - - - - 10
Semecarpus anacardium Fr A - 13 - - - 12 - 12
L.f. M 12 11 - - 14 12 - 13
Spondias pinnata Lf A - - - - - - - -
(L.f.) Kurz M 12 - 13 - - - - 12
Fr A - - - - - - - -
M - - - - - - - -
Angiopteridaceae
Angiopteris evecta Forst. Lf A - - 11 - - - - -
Hoff M - - 14 - - - - -
Annonaceae
Annona reticulata L. Lf A - - 12 - - - - -
M 12 - 12 - - - 12 -
Annona squamosa L. Lf A 12 - - - - - - -
M 11 - - - - 11 12 -
Apiaceae
Centella asiatica (L) Urban Wp A 11 - 10 - - - 11 -
M 12 - 14 12 - - 12 12
Eryngium foetidum L. Lf A 10 - - 11 12 - - 13
M 11 - - 11 12 - - 13
Apocynaceae
Alstonia scholaris (L.) R. Lf A - - - - - - 11 -
Br. M 12 - 10 - - - 11 14
Catharanthus roseus Lf A - - - - - - - -
(L.) G. Don M - - - - - - - -
Holarrhena pubescens Lf A - 11 - 11 12 11 - 14
(Buch.-Ham.) Wall ex. G. M - 10 12 12 - - - 14
Rauvolfia serpentina (L.) Lf A - - - - - - - -
Benth. ex. Kurz. M - - - - - - - -
Wrightia tinctoria (Roxb.) ND
R. Br.
Araceae
Acorus calamus L. Rh A - - 09 - - - 12 -
M - - 12 12 - - 14 11
Asclepiadaceae
Calotropis gigantea L. Lf A - - - - - - - -
M - - - - - - - -
Calotropis procera (Ait.) Lt A - - 12 - - - 11 -

155
R. Br. M - - 12 - - - 10 -
Asteraceae
Ageratum conyzoides L. Wp A 12 12 - - - - - 11
M - 12 - - - - - -
Elephantopus scaber L. Lf A - - - - 8 - - 11
M - - 10 - 10 - - 14
Emilia sonchifolia (L.) DC ND
Spilanthes calva DC. ND
Tridax procumbens L. Lf A 10 - 13 - - - - -
M 12 - 12 - - - - -
Barringtoniaceae
Barringtonia acutangula ND
(L.) Gaertn.
Bombacaceae
Bombax ceiba L. Gu A - - - - - - - -
m
M 10 - - - - - 12 -
Bignoniaceae
Oroxylum indicum (L.) Bk A 10 - - - - - - -
Vent.
M 10 12 - - - - - 13
Burseraceae
Boswella serrata Roxb. Lf A - - - - - - - -
M - - - - - - - -
Caesalpiniaceae
Bauhinia purpuria L. Lf A - - - - - - - -
M - - 11 - - - - -
Bauhinia racemosa Lam. Lf A - - - - - - - -
M - - - - - - - -
Bauhinia variegata L. Lf A - 10 - - - - - -
M - 12 12 - - - - -
Bauhinia vahlii W. & A. Lf A - - - - - - - -
M - 12 10 - - - - -
Cassia fistula L. Lf A 9 12 12 11 12 12 11 9
M 13 12 13 12 12 12 10 12
Saraca asoca (Roxb.) de Lf A - - - - - - - -
Wilde M - - 13 - - - - -
Tamarindus indica L. Lf A - - 12 - - - - 10

156
M 12 - 14 - - - - 12
Capparaceae
Cleome viscosa L. Lf A 10 - - - - - - 10
M - - - - - - - 11
Clusiaceae
Mesua ferrea L. Lf A 12 10 - - 12 - 12 12
M 10 12 - - 14 - 12 10
Combretaceae
Anogeissus latifolia (Roxb. Lf A 10 - - - 12 12 12 12
ex DC.) Wall. ex Bedd M 10 14 11 12 15 14 12 12
Quisqualis indica L. Lf A 10 12 - - 12 - 12 12
M 12 15 14 - 12 12 15 12
Terminalia alata Lf A - - - - - - - -
Heyne ex. Roth M - - 12 - - - - -
Terminalia arjuna Bk A 10 12 12 12 12 - 14 12
(Roxb. ex DC.) W. & A. M 12 15 14 12 12 12 15 12
Terminalia bellirica (Gartn.) Bk A 11 13 - - - - - -
Roxb. M 14 12 - - 10 11 12 -
Terminalia chebula Retz. Bk A - - - 14 12 - - -
M - - - 12 11 - - -
Commelinaceae
Commelina benghalensis L. Lf A - - - - - - - -
M - - - - - - - -
Erycibe paniculata Roxb. Lf A 10 - 10 - - - - 12
M 10 12 14 - 14 - - 17
Crassulaceae
Bryophyllum calycinum Lf A - - - - - - - -
Salis. M - - - - - - - -
Kalanchoe pinnata Lf A - - - - - - - -
(Lam.) Pers. M - - - - - - - -
Cucurbitaceae
Coccinia grandis Lf A 12 11 - - - - - 11
(L.) Voigt M - 14 - - - - - 12
Momordica charantia L. Lf A - 12 - - - - 12 -
M - 14 - - - - - -
Cyperaceae
Cyperus rotundus L. Lf A - 10 - - - - - -
M - 12 - - - - - 10
Dipterocarpaceae

157
Shorea robusta Gaertn. f. Lf A - - 11 - 12 - - -
M 11 - 13 - 9 - - 12
Ebenaceae
Diospyros malabarica Bk A - - - - - - - -
(Desr.) Kostel M 10 - - - - - - 12
Diospyros melanoxylon Lf A - - - - - - - 11
Roxb. M 14 - - - - - - 12
Bk A - - 11 11 10 - - 12
M 12 15 10 10 10 - - 16
Euphorbiaceae
Croton roxburghii Lf A 17 - 13 - - - 12 -
Balak. M 14 10 10 12 14 - 15 15
Bk A - - - 10 - - - 14
M - - - - - - - 15
Phyllanthus emblica L. Fr A 12 10 - - - - - 12
M 12 - - 12 10 - - 15
Phyllanthus fraternus Lf A - - 11 - - - - -
Webster M - - - - - - - -
Securinega virosa (Roxb. ex ND
Willd). Baill
Tragia involucrate L. Wp A - - - - - - - -
M - - - - - - - -
Trewia nudiflora L. Lf A - - - - - - - -
M - - - - - - - -
Fabaceae
Butea monosperma Lf A - - - - - - - -
(Lam.) Taub. M 12 - 12 - - - - 12
Fl A 10 - 11 - - - - 12
M 12 - 12 10 - - - 14
Butea superba Roxb. Lf A 10 10 - - - - - 10
M 10 12 - - - - - -
Crotalaria spectabilis Roth Lf A - - - - - - - -
M - - - - - - - -
Desmodium gangeticum (L.) Lf A 8 - - - - - - 10
DC. M 10 10 - - - - - 12
Flemingia nana Roxb. Rt A - - 10 - - - - -
M 10 - 12 - - - - 10
Glycyrrhiza glabra (L.) Bk A - - 10 - - - - -
M - - 18 - - - - -
Indigofera cassioides Lf A - - - - - - - -

158
Rottl. ex DC. M - 10 10 8 - - - 10
Pterocarpus marsupium Bk A - 10 - - - - - 10
Roxb. M 10 12 - - 9 - - 12
Pterocarpus santalinus L. f. Bk A - 10 - - - - - -
M 10 12 - - 9 - - -
Hypoxidaceae
Curculigo orchioides Rt A - - - - - - - -
Gaertn. M - - - - - - - -
Iridaceae
Eleutherine bulbosa Bl A - 14 - - - - - 17
(Miller) Urban M 12 15 - - - - 11 15
Lamiaceae
Hyptis suaveolens (L.) Poit. Lf A - - - - - - - -
M - - 10 - - - - -
Ocimum canum Sims Lf A - - - - - - - 10
M 12 - - - - - - 12
Ocimum sactum L. Lf A - 10 - - - - - 10
M 10 10 - - - - - 10
Pogostemon benghalensis ND
(Burm.f.) Kuntze
Lauraceae
Litsea glutinosa (Lour.) Lf A - - 10 - - - - -
Robin M 10 11 12 - - - - -
Lecythidaceae
Careya arborea Roxb. Lf A - - - 10 - - - -
M 12 - 12 12 - 15 - 14
Liliaceae
Allium cepa L. Bl A - - - - - - - -
M - - - - - - - -
Asparagus racemosus Rt A - - - - - - - -
Willd. M - - - - - - - -
Lygodiaceae
Lygodium flexuosum L. Wp A - - - - - - - -
M - - - - - - - -
Lygodium microphyllum Lf A - - - - - - - -

159
(cav.) R. Br. M - - - - - - - -
Lythraceae
Lawsonia inermis L. Lf A - - - - - - - -
M - - 12 - - - - -
Woodfordia fruticosa Lf A - - - - - - - 12
(L.) Kurz M - - 11 - - - - 14
Malvaceae
Hibiscus rosasinensis L. Lf A - - - - - - - -
M - - - - - - - -
Sida acuta Burm.f. Lf A - - 10 - - - - -
M - - 13 - - - - -
Melastomataceae
Melastoma malabathricum L Bk A - - 10 - - - - -
M - - 20 - - 12 - 16
Soymida febrifuga Lf A - - - - - - - -
(Roxb.) A. Juss. M - - - - - - - -
Meliaceae
Azadirachta indica A. Juss. Bk A 11 - 13 - - - - 12
M 10 - - - - - - 12
Menispermaceae
Cissampelos pareira L. Rt A - - - - - - - 12
M 12 - - 14 - 10 10 14
Tinospora cordifolia (Willd.) Wp A - - - - - - - 12
Hook.f.& Thoms. M 10 - 13 11 - 10 10 14
Mimosaceae
Acacia catechu (L.f.) Willd ND
Acacia leucophloea Lf A - 10 - - - - - 9
(Roxb.)Willd. M 10 12 10 - - - 12 14
Acacia nilotica (L.) Delile ND
Albizia lebbeck Benth. Lf A - - - - - - - -

M - - - - - - - -
Moraceae
Ficus benghalensis L. Lf A - - - - - - - -

160
M - - - - - - - -
Ficus racemosa L. Lf A 12 - - - - - - -
M - - 14 - - - - 12
Streblus aspera Lour. ND
Moringaceae
Moringa oleifera Lam. Lf A 12 10 8 - - - - -
M 14 11 12 - - - 10 12
Musaceae
Musa paradisiaca L. Fl A - - - - - - - -
M - - - - - - - -
Myrsinaceae
Ardisia solanacea Roxb. Lf A - - - - - - - -
M 10 10 - - - - - -
Myrtaceae
Psidium guajava L. Lf A - - - - 10 - - 12
M - - - - 13 - - 14
Syzygium cumunis L. Bk A - - - - - - - -
M - - - - - - - -
Nyctaginaceae
Boerhavia diffusa L. Lf A - - - - - - - -
M - - 9 - - - - -
Oleaceae
Nyctanthes arbortristis L. Lf A 12 - 10 - - 10 - 12
M 20 - 13 10 - 12 - 10
Bk A 12 10 10 12 - 12 - 14
M 23 10 18 12 12 11
Oxalidaceae
Oxalis corniculata L. Wp A - - - - - - - -
M - - - - - - - -
Pedaliaceae
Sesamum orientali L. Wp A - - - - - - - -
M - - - - - - - -
Piperaceae
Piper longum L. Sd A 10 10 - 12 - 10 10 10
M 13 13 10 12 - 12 13 14
Piper nigrum L. Lf A - - - - - - - -
M - - - - - - - -
Plumbaginaceae
Plumbago zeylanica L. Lf A - - - - - - - -

161
M - - - - - - - -
Cynodon dactylon Wp A - - - - - - - -
(L.) Pers. M - - - - - - - -
Punicaceae
Punica granatum L. Lf A 10 10 14 - 10 - - 12
M - - 12 - 12 - - 10
Ranunculaceae
Thalictrum foliolosum DC. ND
Ziziphus mauritiana Lam. Lf A - - - - - - - -

M - - 9 - - - - 10
Rubiaceae
Anthocephalus chinensis Lf A 12 - - - - - 10 -
(Lam.)A. Rich. ex. Walp. M 12 - 14 - 12 - - -
Canthium dicoccum Lf A - - - - - - - -
(Gaertn.) Teijsm. & M - - - - - - - -
Binnend.
Gardenia gummifera L. f. Lf A - - - - - - - -
M - 10 10 - - - - -
Gardenia resinifera Roth. ND
Haldinia cordifolia (Roxb.) Lf A - - - - - - - -

Ridsd M 10 10 12 - - - - 12
Ixora pavetta Andr. Lf A 10 - - - - - - -
M 12 - - - - - - -
Morinda citrifolia L. Lf A - - - - - - - -
M - - - - - - - -
Paederia foetida L. Lf A - - - - - - - -
M - - - - - - - -
Randia uliginosa Lf A - - - - - - - -
(Retz.) DC M - - - - - - - -
Rubia cordifolia L. Lf A - - - - - - - 10
M 10 - - - - - - 12
Tamilnadia uliginosa (Retz.) ND
Tirveng.
Rutaceae
Aegle marmelos L. Lf A - - - - - - - -
M - - - - - - - -

162
Fr A - - - - - - - -
M - - 12 - 10 - - 12
Catunaregam spinosa Lf A - - - - - - - -
(Thunb.) Tirven M 12 - 12 - - - - -
Citrus limon L. Fr A - - 10 - - - - -
M 10 - 12 - - - - 11
Citrus medica L. Lf A - - - - - - - -
M - - 9 - - - - -
Clausena excavata Burm. Lf A - - - - - - - 14
M 14 - - - - - - 12
Leea indica (Burm.f.) Merr. Lf A - - - - - - - -
M - - 12 - - - - -
Limonia acidissima L. ND
Murraya koenigii (L.) Lf A - - - - - - - -

Spreng M 12 - - - - - - 10
Sapendaceae
Allophylus serratus (Roxb.) ND
Kurz
Schleichera oleosa (Lour.) Wp A - - - - - - - -
Oken M - - - - - - - 10
Sapotaceae
Mimusops elengi L Bk A - - 10 12 - - - -
M - - 14 12 12 - 12 -
Smilacaceae
Smilax zeylanica L. Lf A - - - - - - - -
M - 12 - - 11 - 10 12
Solanaceae
Nicotiana tabacum L. Lf A - - - - - - - -
M - - - - - - - -
Sterculiaceae
Helicteres isora L. Lf A 11 - - 10 - - - 12
M - - - 8 - - - 10
Fr A - - - - - - - -
M 11 - - - - - - 12
Rt A 12 12 - 12 - - - 14
M 14 10 - 10 - 10 - 12
Pterospermum acerifolium Lf A 10 10 - - - 10 - 12

163
(L.) Willd. M 10 12 - 19 - 18 - 15
Strychnaceae
Strychnos nux-vomica L. Sd A - - - - - - - -
M - - - - - - - -
Tiliaceae
Grewia hirsute Vahl. Lf A - - - - - - - -
M 10 - - - - - - -
Grewia helicterifolia Lf A - - - - - - - -
Wall. ex. G. Don M - - - - - - - -
Trapaceae
Trapa natans L. Lf A - - - - - - - -
M - - - - - - - -
Ulmaceae
Trema orientalis (L.) Bl. Lf A - - - - - - - -
M - - - - - - - -
Verbenaceae
Vitex negundo L. Lf A 10 10 - - - - - -
M 12 10 14 10 - - - 10
Bk A 10 - 10 - - - - 12
M 18 - 16 - - - - 17
Zingiberaceae
Curcuma angustifolia Lf A - - - - - - - -
Roxb. M - - 8 - - - - 10
Curcuma aromatica Salisb. Rh A - - - - - - - -
M - - 12 - - - - 12
Zingiber officinale Rosc. Rh A - - - - - - - -
M 10 - 12 - 12 - - 14
PU-Parts used, E-Extract, A-Aqueous, M-Methanol, Bl-bulb; Bk-bark; Fl-flower; Fr-fruit;

Lf-leaf; Rt-root; Rh-rhizome; Rs-Resin; Sd-seeds; Tw-twig; Wp-whole plant, S1-
Escherichia coli, S2- Salmonella typhi, S3- Vibrio cholerae, S4- Vibrio alginolyticus, S5- Vibrio
cholerae 0139, S6- Escherichia coli O157:H7, S7- Shigella dysentriae, S8- Salmonella
typhimurium
Among the test strains the most sensitive strains were recorded in decreasing order
were S. typhi followed by E. coli, Vibrio cholerae, S. typhimurium, V. alginolyticus, E. coli

164
O157:H7 and V. cholerae O139. However, the strain S. dysentriae showed least activity in
compared to above referred strains.
Nigella satavia (Ranunculaceae), used to treat numerous ailments including diarrhea and
its essential oil has been shown to exhibit activity against Staphylococcus aureus,
Salmonella, Shigella, V. cholerae, and E. coli (Ali et al. 2003). Swertia corymbose shows
antibacterial activity against a wide range of microorganisms, including (E. coli,
Salmonella sp., V. cholerae and Staphylococcus aureus) that cause diarrhea. Vijaya et al.
1997 reported 10 Indian traditional plants (Allium sativum, Bauhinia racemosa, Camellia
sinensis, Euphorbia hirta, Cissampelos pareira, Acorus calamus, Psidium guajava and
Sphaeranthus indicus) used to treat dysentery and diarrhea showed high antibacterial
activity. Methanol and water extracts of a number of medicinal plants used to treat
dysentery and diarrhea in the Democratic Republic of Congo showed activity against
one or more enteropathogens, including Shigella, Salmonella, E. coli, Vibrio and
Campylobacter (Longanga-Otshudi et al. 1999). Diehl et al. 2004 evaluated 60
traditionally used plants in human or veterinary medicine to treat worm infections
(worms in general, round worms, Guinea worms, or flatworms), diarrhea, dysentery
and abdominal pain.
Modern scientific evaluation of medicinal plants and herbs is concerned with validating
the traditional use of plants as well as identifying the active components of extracts and
preparations. With respect to traditional medicines used to treat diarrheal diseases, such
medicines will continue to be used as long as there are communities with limited access
to modern therapies. In future, it may be possible to supplement conventional ORS
treatment with plant extracts resulting in complementary treatments that may lead to a
reduction in the length of disease symptoms.
CONCLUSIONS
This study proves that use of plants for treatment of diarrhea and dysentery among the
tribes of Similipal is still a major part of life and culture. The data collected show that
majority of remedies are taken orally in fresh form. The study concludes that the tribes
are depending on traditional medicinal uses and modern health system is far away
from them. It is necessary to acquire and proper documentation of the knowledge of the
tribes of Similipal, Mayurbahnj. The result obtained in the present study point out that
in their crude form bout 75 % have scientifically proved as medicinal uses with respect
to the antibacterial properties against diarrhea causing bacteria.
RECOMMENDATION
Surely, the evidence provided by this study will encourages further investigation in the
expectation that alternative treatments for diarrheal diseases will be developed. Further

165
study is required to isolate compounds from plants which have not been reported as
antimicrobial potential.
ACKNOWLEDGEMENT
The authors wish to thank Prof. S. K. Dutta and Dr. A. K. Bastia (North Orissa
University) for their guidance during my Ph. D. and providing facilities for this work.
We wish to express our profound gratitude to Dr. A. K. Biswal (North Orissa
University) for identification of the plant samples. Thanks are also to S. K. Jena, B.
Behera, D. Dubey, N. Patra and Y. K. Mohanta (North Orissa University), Dr. Sanat
(Ayurvedic doctor, Badasahi, Mayurbahnj) for collection of plant samples and expert
technical help during experiment. SKP is also grateful for financial support provided by
DST, Govt. of Odisha. Finally, we wish to thank to all the tribal informants as they
travelled with me and help in collection, identification and provided traditional uses of
plants. We also thankful to all other informants those provide me information on
medicinal uses and helps in communicating in vernacular languages. Hope that the
information produced from these studies will be of use to them in future.
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CHAPTER EIGHT
MAXIMUM LIKELIHOOD ESTIMATES IN RELIABILITY OF TYPHOID FEVER DRUG

IN THE RECOVERY OF TYPHOID FEVER PATIENTS AT AKUSE
(A CASE STUDY AT THE AKUSE GOVERNMENT HOSPITAL)
Eric Boahen
Department of Statistics, Faculty of Mathematical Sciences, University For Development
Studies –Tamale.Box 24 uds Navrongo- Ghana.
ABSTRACT
Typhoid fever is one of the top ten outpatient diseases that is recorded by the
Akuse Government Hospital. Akuse is a big town located in the eastern region
of Ghana. The Government Hospital located in the area recorded a continuous
substantial increase in the number of typhoid fever cases. This substantial
increase was partly attributed to the fact that the probability of an individual
to be healed of the disease was relatively low. As a result of this, a five staged
clinical trial was conducted by the hospital to assess the probability of being
cured after receiving treatments. This five staged clinical trial was used in the
research to assess the survivability growth and reliability of the typhoid fever
drugs administered. The research was carried out by first, obtaining the
observed survival probabilities at each stage and then the maximum likelihood
estimates and least squares estimates were found. The Newton-Raphson
procedure was used in the calculation of the maximum likelihood estimates. An
appropriate (1-α) 100 percent lower confidence limit was then constructed for
each stage of the clinical trial. In clinical trials, patients response to treatment
is classified as success or failure, therefore, the overall probability of success in
the clinical trial was then assessed with the aid of smoothen constants. The
study identified that the drugs administered had huge effect on the survival of
an individual and it was therefore recommended that the Hospital creates an
awareness program for the people of Akuse to know that treating typhoid the
drugs used in the clinical trial helps in curing typhoid. Also, the Akuse
Government Hospital in their endeavor to the increase the curing rate of
typhoid fever in the township should encourage patients to take drugs
prescribed to the patients well.
INTRODUCTION
Introduction
The purpose of the study was to find out the reliability and growth in the healing
process of typhoid fever at Akuse in the Lower Manya District. This chapter consists of

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background of the study, problem statement, purpose of the study, objectives, research
questions, scope of the study and Limitations.
Background of the study

Akuse is a town located in the Eastern Region of Ghana off the Akosombo – Tema
highway. Akuse is under the Lower Manya Constituency and is situated close to the
Volta River. Akuse Government Hospital is located at the entrance of the town,
opposite the Ghana Commercial Bank. The hospital serves people from other
surrounding towns such as Somanya, Atua, Kpong among other smaller towns located
closes to Akuse.
Typhoid Fever also known as Enteric Fever can be a life – threatening febrile disease,
caused by the bacterium Salmonella Typhi. According to the Centre for Disease and
Control (CDC) an estimated 22 million cases of typhoid fever and 200 000 related deaths
occur worldwide every year. Typhoid is common in most parts of the developing
world. In Ghana, a study conducted in 2010 has shown that typhoid fever ranks among
the leading causes of outpatient illness accounting for 0.92% of hospital admissions.
The disease is most common in developing countries of which Ghana is no exception

contributing factors being poor sanitary system and lack of antibiotics putting travelers
to Asia America and Africa in high risk group.
Typhoid fever belongs to the family Enterobacteriaceae and has been associated with
gastroenteritis and food borne diseases. The salmonella species is the etiological agents
of most food borne disease and gastroenteritis.
In the year 2011, three hundred and twenty two cases (322) of typhoid fever were
recorded in the Akuse Government Hospital with seven hundred and seventy two (772)
cases and one thousand (1000) cases in 2012 and 2013 respectively.Domestically
acquired typhoid fever, however, is often transmitted by chronic carrier of salmonella
typhi. After recovering from typoid fever, some individuals carry the bacterium
asymptomatically for long periods; others are asymptomatic carriers without ever
having the disease. The silent carriers contribute to continued episodes of infection.
Most of the disease is acquired by people travelling overseas (Prescott, 1999).
The disease, typhoid fever, is caused by Salmonella typhi which is acquired by

ingestion of food or water contaminated by faeces of infected humans. The pathogen
colonizes the small intestines penetrating the lymph node spleen and other lymphoid
tissues within 6-14 days after exposure, to headache and fever developments. The latter
can continue for weeks and rise about 40⁰ Celsius. In most cases, salmonella typhi is

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shed in faeces for several weeks. However, approximately 3% of those who recover
continue to shed salmonella typhi for extended periods but show no symptoms of the
disease. In such individuals, termed carriers, the bacterium grows in the gall bladder
and finds its way to the intestines through the bile duct.
Typhoid fever is usually confirmed by identifying Salmonella Typhi in a culture of the

blood or other body fluid (urine and stools). For the culture, a small sample of the
patient‟s blood, urine and stool is placed in a container to growth the bacteria. This
takes between 48 to 72 hours, for the culture to be checked under a microscope for the
presence of the bacteria.
According to CDC, one can get typhoid fever if food or drink beverages that have been
handled by a person who is shedding Salmonella Typhi are ingested or if sewage
contaminated with Salmonella Typhi bacteria gets into the water used for drinking or
washing food. Therefore, typhoid fever is more common in areas of the world
where hand washing is less frequent and water is likely to be contaminated with
sewage. World Health Organization (WHO) also mentioned the mode of transmission
of the disease as through the ingestion of food or drink contaminated by the faeces or
urine of infected persons.
Typhoid fever in Akuse is mostly attributed to the works of witchcraft and other
demonic powers. The people of Akuse believe that the disease can be transmitted onto a
person by spiritual means. In a blog posted on March 10th, 2012 by one researcher
named BiorKwerBiorJr (Borglobe), he mentions that the people of South Sudan and
other neighboring countries have a myth that typhoid fever is attributed to the sun.
Case-fatality rates of 10% can be reduced to less than 1% with appropriate antibiotic
therapy. However, strains resistant to chloramphenicol and other recommended
antibiotics (ampicillin, cotrimoxazole and even ciprofloxacin) have become prevalent in
several areas of the world. It usually takes two weeks for patients treating typhoid fever
to be fully cured.
Typhoid fever can also be treated herbally using the roots, bark and leaves of plants.
The use of mauringa is highly recognized in Akuse for the treatment of typhoid fever
and other fever related diseases.
CDC described measures of preventing the disease in four ways; boil it, cook it, peel it
or forget it. The Centre for Disease Control listed these measures of preventing or
avoiding typhoid;

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 If you drink water, buy it bottled or bring it to a rolling boil for 1 minute before
you drink it. Bottled carbonated water is safer than uncarbonated water.
 Ask for drinks without ice unless the ice is made from bottled or boiled water.
Avoid popsicles and flavored ices that may have been made with contaminated
water.
 Eat foods that have been thoroughly cooked and that are still hot and steaming.
 Avoid raw vegetables and fruits that cannot be peeled. Vegetables like lettuce are
easily contaminated and are very hard to wash well.
 When you eat raw fruit or vegetables that can be peeled, peel them yourself.
(Wash your hands with soap first). Do not eat the peelings.
The following problems are faced in the treatment process of typhoid fever;
 Patients do not take medications as prescribed by the doctor or health
practitioner.
 Patients do not heed to the doctor‟s advice by not returning for a checkup after
two weeks when the bacteria and the symptoms are expected to have gone.
 Some patients feel reluctant to report signs and symptoms early but rather wait
till the symptoms become severe before seeking medical attention
Problem Statement
Typhoid fever is one of the leading causes of death among children and adults in
Ghana.
Study has shown that, EasternRegion has high cases of typhoid. Our study has also
shown that, Eastern Region is one of the regions with high typhoid cases due to the
open defecation practice, dirty surroundings and unhygienic practices.Typhoid fever is
one of the top 10 out-patient diseases in Ghana according to the Ghana health service. It
is rated among the top killer diseases in Ghana.
Typhoid fever cases in Akuse are on the rise. The typhoid fever cases at Akuse in the
eastern region for the year 2011, 2012 and 2013 are three hundred and twenty two (322),
seven hundred and seventy two (772) and one thousand (1000) respectively and the
recovery rate for the patients have been low. There is a situation at Akuse where
infected persons who report to the hospital, diagnosed with the disease, given
medication to follow and are asked to come back for review end up, taking the
medication until when the infected person feels his situation is better but not when the
dosage of the medication has been fully completed.
The infected persons also ignore the review instruction set by the doctor or health
practitioner. Therefore, the fact that, an infected person is fully cured of the disease and

181
the bacteria have been removed from the system is in doubt. These patients will still
harbor the bacteria in their bodies.When these infected persons have a bowel
movement, they may pass stools (faeces) that contain the Salmonella typhi bacteria.
There is a low level of sanitation at Akuse and hygienic conditions are also poor.
Infected persons, who do not wash their hands before handling food and water,
contaminate the food. When another person consumes tainted food or water, he picks
up the typhoid bacteria and become infected.
Some people, known as chronic carriers, still harbor typhoid bacteria (and can still
contaminate food and water supplies) even after receiving antibiotic treatment and
proving to be free of symptoms.
All these conditions add up to the increasing rate of typhoid cases at Akuse. In this
regard, the research was interested in studying the survivability growth and the
reliability of typhoid fever drugs at Akuse in the Eastern Region of Ghana.
Purpose of the study

The purpose of the study is to:
 Assess the survivability growth rate of the healing process
 Assess the reliability of the drug
Objectives of the study

The main objectives of the study are to assess the reliability of the drug and growth in
the healing process of typhoid fever and also to determine the overall probability of
success or failure in each stage of the clinical trial.
Research Questions
These research questions have been outlined for the project.
 How reliable is the drug?
 Is the healing process improving?
 Is the overall survival probability at each stage of the clinical trial good?
Scope of the study

The study is confined to the Lower Manya District of the Eastern Region, Akuse to be
precise. The data used for the project was collected in five stages with ten patients in
each stage. The conclusion of the study was however generalized and made applicable
to all typhoid cases treated with cefuroxime and ciprofloaxin throughout the country.
The study was not made applicable to other drugs used in the treatment of typhoid
fever.

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Limitations of the Study
These constraints among others were encountered during the study;
 Those in charge of the data were reluctant to give out the data for analysis.
 It was difficult for those at the records department to really understand the kind
of data needed for the research.
 It was not easy moving from Navrongo to Akuse to collect the data as far as
monetary issues are concerned.
METHODOLOGY
Introduction
This chapter introduces the methodology adopted in the study. It discusses the study
area, the design of the research, sample size selection, population of study, technique
used in the data collection, sampling techniques, statistical analysis, and procedures
and describes the theory of the model used, formulations and methods of analyzing the
available data to satisfy the objectives of the study.
Study area
The study was conducted among typhoid fever patients in the Akuse Government
Hospital. The hospital is one of the biggest in the ManyaKrobo District and also one of
the oldest in the country. The Akuse Government hospital is located at the entrance of
the Akuse town, opposite the Ghana Commercial Bank. It serves people from
neighboring towns and villages. The people of Akuse are mainly farmers and petty
traders. Some of the indigenes are employed with the anana farm plantation located
close to the town. The Kpong Generation Station, with four power turbines, which
produces power for the national grid is also located at Akuse. Therefore, Akuse also
inhabits the Volta River Authority (VRA) workers, who work in the generation station.
Population and sample

The population of the study was made up of typhoid fever patients in the ManyaKrobo
district. However, the accessible population of the research consisted of all typhoid
fever patients who were diagnosed with the disease in the month of October. A total
number of 63 cases was recorded by the hospital for that month.
Sources of data collected

Secondary data was our main source of collecting data for the study. The secondary
data was collected from the hospital in a clinical trial conducted in five stages. The trial
was to test the survivability growth of patients‟ remission from typhoid fever. The trial
had each in each stage ten patients who were receiving treatment from typhoid. Each
patient had a healing period of two weeks since the typhoid fever drugs used are
supposed to cure a patient with the disease within that period. Each stage of the clinical
trial has a new group of patients thereby making the trial carried out, independent.

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Statistical analysis, procedures and models used
The data collected was a research conducted in 5 (five) stages such that at the ithstage
nipatients enter the study each with a probability of surviving the ith stage. At each
stage a new group of patients is entered; thus stages are assumed to be independent as
well as identical in their time periods. After the ith stage, the probability there are
xisurvivors (number of patients healed) is
* + . / ( ) …(1)
Some general parametric growth models is assumed by for measuring survivability

growth from stage to stage. Examples of these growth models are the hyperbolic
growth model, exponential growth model and the logistic growth model.
The proportion of survivors from the data, increases from stage to stage. is a function
of two parameters as well as the stage number i. The exponential model is given by
( ) …(2)
where are the parameters of the model, . Two

methods commonly used to estimate and are least squares and maximum
likelihood. Maximum likelihood estimators are generally preferred to least squares
estimators because of the desirability of large sample properties of maximum likelihood
estimator. On the other hand least square estimators are often obtainable in closed form
and are a good first approximation to the maximum likelihood estimator.
Least Squares
Let us define ( ) as
( ) ∑ 0 ( )1 …(3)
The least squares estimator and are the values of the parameters and ,
respectively, that simultaneously minimize ( ). Assuming that ( ) is
differentiable in and , , and that the minimum is obtained by
differentiation, we find and as the solution of
( )
∑ 0 ( )1 …(4)
and
( )
∑ 0 ( )1 …(5)
where

184
( ) ( )
| …(6)
That is, and are the least squares estimators of and , respectively.
Maximum Likelihood
Given the probability that patients of on trial at stage survive, Thus
the stages being statistically independent, the likelihood function for all k stages is
given by
( ) ∏ . /, ( )- , ( )- (7)
Assuming that the parameter vector ( ) can be maximized with respect to the
observations by maximum likelihood procedures, the maximum likelihood estimator ̂
and ̂ are the values of the parameters and , respectively, that simultaneously
maximize ( ) or equivalently ( ) The vector ( ̂ ̂ ) is then the
simultaneous solution of
(̂ ̂ ) (̂ ̂ )
∑ ∑ (8)
(̂ ̂ ) ̂ (̂ ̂ ) ̂
and
(̂ ̂ ) (̂ ̂ )
∑ ∑ (9)
(̂ ̂ ) ̂ (̂ ̂ ) ̂
that maximizes ( ̂ ̂ ), where
(̂ ̂ ) ( )
̂
| ̂
. (10)
̂

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Estimation for the Exponential Growth Model
The exponential model was chosen for this research after plotting the survival
probabilities at each stage of the trial.
7
theoritical growyh in survival
4
Y-Values
3
0
0 1 2 3 4 5 6
stages
Fig 1: Plot of survival probabilities against stages
This model depicts a slower growth in the early stages which sustains itself in the later
stages.
Let ( ) be defined by the exponential growth model. That is
( ) ( ) (11)
Since for this model

( ) ( ) (12)
the least squares estimator and are obtained in the following ways. First
. / ( ) (13)
Where
As a result of this a least squares fit on the logarithm is used to obtain and . The
least squares equation is then
( ) ∑ 0 1 (14)

186
Let us set *( )⁄( )+. Differentiating ( ) with respect to and
and setting the resulting equations equal to zero we obtain
( )
∑ (15)
and
( ) ( )( )
∑ (16)
Solving in terms of and we find

, ( )- {( )∑ ∑ } (17)
and
∑
, ( )- {∑ } (18)
These are used as the least squares estimators of the exponential growth model. The
maximum likelihood function for the exponential growth model. Let ( ) be the
likelihood function for all k stages of the trials.
Then we have
( ) ∏ . /( ( )) ( ( ))
(19)
Taking logarithms and then differentiating with respect to and , the maximum
likelihood estimators ̂ and ̂ are obtained as the unique solution to
∑ ( )
∑ (20)
, (̂ ) ̂ - ̂
And
̂ ∑ , ( ) ̂ -
∑ ( ) (21)
Therefore taking the logarithm and differentiating with respect to and , we have
0∑ ∑ 1 (22)
, ( ) -
( )
∑ (23)
, ( ) -
and
( )
∑ (24)
, ( ) -
Confidence regions and intervals for the exponential growth model

An approximate (1- )100 percent elliptical confidence region for the parameter vector
. / is

187
(̂ ) (̂ )) ( (̂ ) )( ̂
…(25)
Where ( ) is the (1- )100 percentage point of the chi-square distribution with 2
degree of freedom and
[ ] (26)
For this model

∑ (27)
( )
∑ (28)
( )
and
∑ (29)
( )
We obtain an appropriate ( ) percent lower confidence limit ( ̂ ̂ ) for the

exponential growth model. We have
(̂ ̂ ) ( ̂ ( ̂ )) ( )√ ̂ ( ̂ ( ̂ ))
(30)
Where
i. Var( ̂ ( ̂ )) is given as
( ̂ ( ̂ )) ( )[ ̂ ̂ ̂ ̂ ]
(31)
ii. The values ̂ ̂ and ̂ ̂ are elements of the matrix
̂ ̂ ̂
* + 0 1 (32)
̂ ̂ ̂
Assessment model for the overall probability of success in the clinical trial
Patients‟ response to treatment in clinical trials is classified as “success” or “failure”.
Models developed have been concerned in estimating the probability of success (failure)
at each stage for clinical trials conducted in stages. We are to assess the overall
probability of success at the end of each stage of the clinical trial conducted in stages.
At the first stage of the trial, ̃ the estimate for patient response is given by
̃ (33)
Where, = patients under stage ὶ, = patients are those who respond for stage ὶ,
The overall assessed probability of patient response after each stage the model is

188
̂ ̂ . ̂ /̂ (34)
Where, proportion of patient respond at the
̂ is the smoothing constants of each stage
Using the empirical approach or method discussed by Gross [1971b] in determining the
smoothing constant, we make the following assumptions,
 ̂ is an unbiased estimator of , where is the theoretical assessment of the
overall probability of success after the trial,
 ( ), thus is the probability that a patient on study during the stage
responds.
 All patients within a stage have the same independent probability of response.
Furthermore, the stages are independent.
The criterion for the choice of each smoothing constant ̂ is to minimise the variance
̂ of its associated value ̂ by assumption 1 through 3.
̂
̂ (35)
∑
( )
̂ (36)
Hence
̂ ( )
(37)
̂ = inverse estimated variance for each stage
̂ = smoothing constant for each stage
RESULTS AND DISCUSSION

Introduction
This chapter presents the details of the analysis and results of the research. This consists
of presentation of data collected, numerical analysis (least square estimates and
maximum likelihood estimates), confidence region and interval at each five stages of the
of the clinical trial, assessment of the overall probability of success in the clinical trial
and discussion of the results.
Presentation of data collected

Analysis on this study began with the data collected from the Akuse Government
Hospital. The data collected was a clinical trial which was conducted in five (5) stages.
The clinical trial was conducted with the aim of improving the probability that patients
suffering from typhoid fever will be cured after receiving treatment. The data, which
was made up of ten patients in each stage of the clinical trial adding up to fifty (50)

189
patients. Out of the fifty patients, twenty two (22) were male and twenty eight (28) were
females.
The five staged clinical trial, has in each stage a total of ten patients who underwent the
trials. In each stage of the clinical trial some of patients were unable to be cured of the
disease, others did. The patients, who after two weeks, are supposed to be cured of the
disease, were grouped into healed and not healed. That is, those patients that were
cured of the disease were grouped under healed and those that were not cured, placed
under not healed. Stage one of the clinical trial had six patients healed and four patients
not healed. Stage two and stage three had seven patients healed and three patients not
healed each. Stage four had eight patients being healed and two patients not healed.
Stage five had nine patients out of ten healed.
The proportion of patients being cured at each stage of the clinical trial is derived. This
is done by dividing the total number of patients that were healed in the stage by the
total number of patients in that stage of the clinical trial. This proportion of patients also
depicts the survival probabilities of each stage. The table below shows the details of the
data collected.
Table 1: Stage by stage clinical trial with survival probability of each stage
Stages Total Number of Number of Number of Number of Survival
number of males females patients patients not probability
patients healed healed
1 10 4 6 6 4 0.6
2 10 3 7 7 3 0.7
3 10 5 5 7 3 0.7
4 10 6 4 8 2 0.8
10 4 6 9 1 0.9
Table 1 shows an improvement in the number of patients that are healed from stage-to-
stage. Plotting the survival probabilities of each stage aids in determining the
exponential model used in the analysis of the data. The graph in Fig 2 shows the
graphical display of the survival probabilities.

190
1.2
1
Survival Probabilities
0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6
Stage Number
Fig 2: Graph of Survival probabilities as a function of the stages (i) for typhoid fever
remission
X-axis = stages, Y-axis = survival probabilities
Fig 2 shows the survival probabilities having a slower growth in the early stages which
sustains itself in the later stages. This determines the use of the exponential model in the
analysis of the data. The exponential model is used when at the early stages of the
clinical trial the survival probabilities show a slow growth and later sustains itself.
Numerical analysis
Two methods are commonly used in the estimation of and . These are the
maximum likelihood and least squares. Maximum likelihood estimators are generally
preferred to least squares estimators because of the desirability of the large sample
properties of maximum likelihood estimators. On the other hand, least square
estimators are often obtainable in closed form and are good first approximation.
Least square estimates

The exponential model as given in equation (19), computes ̂ and ̂ , the maximum
likelihood estimates of and respectively, where
= the growth in the healing process (survivability growth)
= the drug intervention (reliability of the typhoid drugs)
To this end, we obtained the initial estimates ̂ and ̂ by least square equations (17)
and (18). The estimates ̂ and ̂ were obtained from the layout in Table 2.

191
Table 2: Least square computation layout
Stages Number of cured
patients
1 6 4/11 -1.01160 -1.01160
2 7 3/11 -1.29928 -2.59856
3 7 3/11 -1.29928 -3.89784
4 8 2/11 -1.70475 -6.81899
5 9 1/11 -2.39790 -11.98948
Totals -7.71281 -26.31647
From Table 2 and equations (17) and (18), it follows that our initial estimates ̂ was
0.55480 and ̂ was 0.317805. These initial estimates are our least square estimates.
Maximum likelihood estimates

The initial estimates were used in the computation for the Newton-Raphson procedure.
This procedure was used to obtain the maximum likelihood estimates of ̂ and ̂ . By
the Newton-Raphson procedure, the maximum likelihood estimates was obtained by
going through series of iterations for the Newton-Raphson procedure to converge at an
estimate.
To facilitate the computation of the Newton-Raphson procedure, Table 3 is given. In

this table we let ̂ , ̂ , ̂ , ̂ and ̂ be, respectively,
∑ ( )
, (̂ ) ̂ - ̂
̂
( )
, (̂ ) ̂ -
( )
, (̂ ) ̂ - ̂
(̂ )
, (̂ ) ̂ -
and

192
(̂ )
̂
, (̂ ) ̂ -
For the first iteration we used the initial estimates in the computation.
Table 3: First iteration

Stages ̂ ̂ ̂ ̂ ̂
1 -0.1134454 0.06293951 -21.933657 12.28224 -6.814186
2 0.1575125 -0.17477582 -13.683755 14.86847 -16.498054
3 1.9755692 -3.28813741 -11.428942 13.09562 -21.796356
4 0.9474480 -2.10257666 -7.380420 12.58884 -27.937144
5 -0.2692907 0.74701253 -3.725733 11.68164 -32.404857
Total 2.697794 -4.755538 -58.15251 64.5168 -105.4506
The values ̂ and ̂ were then found by solving the matrix equation
̂
( ) ( ) 0 1 ( )
̂
Solving the matrix ̂ and ̂ . ̂ and ̂ were used in the second

iteration. The iteration is continued since the Newton-raphson procedure did not
converge to maximum likelihood estimates.

193
Table 4: Second iteration
Stages ̂ ̂ ̂ ̂ ̂
1 -0.5249250 0.2852968 -23.902550 13.51596 -7.345925
2 -0.5243284 0.5699450 -15.374736 17.76099 -19.306201
3 1.3438211 -2.1911004 -12.647220 16.58983 -27.049715
4 0.2786689 -0.6058263 -8.216665 16.74835 -36.410921
5 -0.9432078 2.5631673 -4.245978 16.25448 -44.171559
total -0.3699712 0.6214824 -64.38715 80.86962 -134.2843
The values ̂ and ̂ were then found by solving the matrix equation
. ̂̂ / ( ) 0 1 ( )
Solving the matrix ̂ and ̂ . ̂ and ̂ were used in the third

iteration. The iteration was continued since there was no convergence of the maximum
likelihood estimates.
Table 5: Third iteration

Stages ̂ ̂ ̂ ̂ ̂
1 -0.4675435 0.2542502 -23.726761 13.37016 -7.270691
2 -0.4444299 0.4833620 -15.221282 17.44353 -18.971579
3 1.4187522 -2.3145523 -12.543850 16.20778 -26.441373
4 0.3555019 -0.7732877 -8.142917 16.29047 -35.435021
5 -0.8676804 2.3592230 -4.195555 15.74612 -42.813689
Total -0.005399756 0.008995103 -63.83036 79.05804 -130.9324
From Table 5 ̂ and ̂ were found by solving the matrix

194
̂
( ) ( ) 0 1 ( )
̂
Solving the matrix, ̂ and ̂ and were then used in the fourth
iteration to verify the convergence of the maximum likelihood estimates.
Table 6: Fourth iteration

Stages ̂ ̂ ̂ ̂ ̂
1 -0.4662067 0.2535232 -23.723275 13.36692 -7.268933
2 -0.4426859 0.4814652 -15.218312 17.43681 -18.964272
3 1.4203726 -2.3171958 -12.541953 16.19982 -26.428394
4 0.3571305 -0.7768302 -8.141565 16.28101 -35.414452
5 -0.8661063 2.3549430 -4.194608 15.73567 -42.785292
total 0.002504157 -0.004094581 -63.81971 79.02024 -130.8613
From Table 6, ̂ and ̂ was found by solving the matrix
̂
( ) ( ) 0 1 ( )
̂
Solving the matrix, ̂ and ̂ . The iteration was stopped since the
Newton-Raphson procedure converged to the maximum likelihood estimates. Table 7
shows the results at each step of the iteration. The third iteration was chosen since
further iterations gave the requisite maximum likelihood estimates and does not change
the result.

195
Table 7: Values of ̂ and ̂ at each iteration of the Newton-Raphson procedure
Iteration ̂ ̂
Table 7 shows clearly that after the second iteration, the estimates begin to become the
same, that is, they converge.
It is intrusive to compare the observed and expected relief probabilities at each stage
using both least squares and maximum likelihood estimates. These comparisons appear
in Table 4.10.
Table 8: Expected and observed relief probabilities for typhoid fever patients at each
stage when the clinical trial is conducted
Stages Observed Expected Maximum Expected Least Squares
Likelihood Estimate likelihood Estimate
Probability probability probability
1 0.6 0.5851647 0.5962472
2 0.7 0.6835448 0.7061711
3 0.7 0.7585937 0.7861676
4 0.8 0.8158443 0.8443846
5 0.9 0.8595177 0.8867517
Table 8 indicates the growth in survivability at each stage of the clinical trial. The table
also indicates that the least square estimates tend to overestimate and the maximum

196
likelihood estimates tend to underestimate the observed probabilities of relief at each
stage. Thus the least square estimates are somewhat optimistic whereas the maximum
likelihood estimates are somewhat conservative.
Confidence region and interval for the probability of relief

We obtained an appropriate (1-α) 100 percent lower confidence limit ( ̂ ̂ ) for the
model by means of (25). Thus the values of ̂ ̂ and ̂ ̂ was obtained by inverting
the matrix of as by equations (27), (28) and (29), respectively. Thus,
using the maximum likelihood estimates, we found , and
. Thus we see that
̂ ̂ ̂
* + 0 1 0 1
̂ ̂ ̂
By through equations (30) through to (32), we found that
(̂ ̂ ) ( ( ))
( )√ ( ), -
is a 95 percent lower confidence limit for ( ̂ ̂ ), , noting that 1.645 is the

upper 95 percentage point of the standard normal distribution.
th
Table 9: Ninty-five (95) percent lower confidence limits for the probability of relief
from typhoid at each of the five clinical trial, plus predicted sixth stage lower limit
Stages (̂ ̂ )
1 0.3340049
2 0.5299582
3 0.6604890
4 0.7432562
5 0.7957258
6 0.8324348

197
Table 9 indicates the values of ( ̂ ̂ ) for the exponential model at each of the five
stages of the clinical trial and in addition, the predicted value ( ̂ ̂ ) with
confidence 0.95.
Assessing the overall probability of success in the clinical trials

Assessing the overall probability of success at each stage of the clinical trial, the values
of ̂ , and ̂ was found.
Table 10: Assessed overall survival probabilities at each stage of the clinical trial
Stage ̂ ̂ ̂
1 0.6 37.5 - 0.600
2 0.7 42.86 0.533 0.653
3 0.7 42.86 0.348 0.669
4 0.8 56.25 0.313 0.710
5 0.9 100.00 0.358 0.778
Table 10 contains the overall assessed probability of patients‟ response after each stage
as well as the inverse estimated variance and smoothing constant for each stage. The
estimated variances were found to be 37.5, 42.86, 42.86, 56.25 and 100.00. Our smoothing
constants were found to minimize the variances of its associated value ̂ . The
smoothing constant were 0.533, 0.348, 0.313 and 0.358. ̂ was our overall remission
probabilities from typhoid fever at each stage.
SUMMARY, CONCLUSION AND RECOMMENDATION

Summary
Typhoid fever is one of the top 10 out-patient diseases in Ghana according to the Ghana
health service.It is rated among the top killer diseases in Ghana. The research began
with information gathered from the Akuse Government Hospital depicting a rise in the
number of typhoid fever cases recorded from 2011, 2012 to 2013. The figures recorded
by the hospital were 322, 772 and 1000. These indicate an increase from year to year.
The increase was amounted to the fact that the residents in the Akuse Township had
bad hygienic practices which has contributed to the spread of the disease in the vicinity.

198
In this regard, the research was interested in assessing the survivability growth and
reliability of typhoid fever drugs in Akuse.
The main objectives of the research was to assess the survivability growth and
reliability of typhoid fever drugs and also the overall probability of survival in each
stage of the clinical trial conducted by the hospital. The methodology employed to
assess the survivability growth was to find least squares and maximum likelihood
estimates of the exponential model chosen to analyze the data collected from the
hospital. The exponential model was chosen after the data collected from the hospital
was plotted on a graph. The graph showed a slow survivability growth in the early
stages which sustained itself in the later stages of the clinical trial.
The maximum likelihood estimates and the least squares estimates was used to find
expected maximum likelihood estimate probabilities and expected maximum likelihood
probability estimates, respectively, which was compared with the observed probability
estimates. The confidence interval and region for the probability of relief at each stage
was also calculated. The overall probability of success at each stage of the clinical trial
was calculated for by finding the inverse estimated variance and smoothing constants
for each stage. These in turn aid in assessing the overall probability of success in each
stage of the clinical trial.
The Least squares estimates were found to be the initial estimates of the maximum
likelihood estimates. These initialestimates ̂ was 0.55480 and ̂ was 0.317805. The
initial estimates was used in the computation for the Newton-Raphson procedure for
the calculation of the maximum likelihood estimates. By the Newton-Raphson
procedure, the maximum likelihood estimates was obtained by going through series of
iterations for the Newton-Raphson procedure to converge at an estimate. After the
second iteration, the Newton-Raphson procedure converged at ̂ and
̂ . The third iteration was chosen since further iterations gave the requisite
maximum likelihood estimates and does not change the result. The observed and
expected relief probabilities at each stage using both least squares and maximum
likelihood estimates was compared.
A 95 percent lower confidence limit for ( ̂ ̂ ), , noting that 1.645 is the

upper 95 percentage point of the standard normal distribution was constructed. Each
th
stage with its lower limit was found to be 1=0.3340049, 2=0.5299582, 3=0.6604890,
4=0.7432562, 5=0.7957258 and 6=0.8324348. A predicted value ( ̂ ̂ ) with
confidence 0.95 was also calculated for.

199
Finally, in finding the assessed overall probability of success, the smoothing constants
were calculated for and was used in assessing the overall probability of success. The
overall remission probabilities for stage 1 through to five were 0.600, 0.653, 0.669, 0.710
and 0.778, respectively.
CONCLUSION
From the analysis made and estimates derived in the Newton-Raphson procedure, we
conclude that since ̂ , the drug is reliable and very useful in the healing process of
typhoid fever.
On whether the healing process is improving, conclusion was made on the observed
survival probabilities derived from the data collected from the hospital. The observed
probabilities showed an increase in the number of survivors at each stage of the clinical
trial. This depicts improvement in the healing process. This is further confirmed by the
expected least squares estimates probabilities and maximum likelihood estimates
probabilities derived for each stage which show increasing probabilities from stage to
stage. Noting that is a function of and , and also since is increasing that is, the
drug reliability increase from stage to stage, this in turn affects the survivability growth
and hence we conclude that there is improvement in the healing process.
Considering the probability values derived in the assessed overall survival

probabilities, we conclude that a patient that faces treatment in stage one will have a 0.6
chance of survival, whereas stages two and three have 0.653 and 0.669 chances of
survival respectively. Stages four and five had their overall survival probabilities to be
above 0.7 and hence a patient chance of being cured of typhoid fever in that stage is 0.7
which is a very high chance of success.
RECOMMENDATION
From the conclusions theseare the recommendations we came out with.
 It is obvious that typhoid fever has very high chances of being cured and hence
people affected with the disease are advised to visit the hospital to seek
treatment.
 The Akuse Government Hospital in their endeavor to increase the curing rate of
typhoid fever in the township should encourage patients to take drugs
prescribed to them.
 The Ghana Health Service should engage in more clinical trials to improve upon
the overall survival probabilities of patients
 Future assessments and or researchers are encouraged to collect primary data
for their research.

200
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Alan J. Gross, Virginia A. Clarke, (1975), Survival Distribution and Reliability Applications
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Balley and Scott‟s (1973) Diagnostic Microbiology, 9th Ed. Pasadena City College.
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Giannella R.A. (1996) “Salmonella” Baron’s Medical Microbiology 4th Ed., University of
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HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS AND

FRUITS AND ITS TRADITIONAL IMPLICATIONS IN THERAPY INCLUDING

Dr. Subha Ganguly (Editor-in-Chief)

ISBN: 978 – 978 – 52231 – 8 - 7

SCIENCE AND EDUCATION DEVELOPMENT INSTITUTE, NIGERIA

Dr. Subha Ganguly (Editor-in-Chief)

SCIENCE AND EDUCATION DEVELOPMENT INSTITUTE, NIGERIA

Knowlegde for Global Development

This first edition published 2014 by

ISBN: 978 – 978 – 52231 – 8 - 7

Knowlegde for Global Development

Knowlegde for Global Development

Abulude, F.O. (Nigeria) - President/CEO

LIST OF ADVISORY BOARD MEMBERS

Prof. Mohammad S. Mubarak

Prof. Francisco Torrens

Hon. Niyi Jones Akinyugha

Mr. Sola Akitimehin

Knowlegde for Global Development

CHAPTER ONE 1–5

HERBAL IMMUNE ENHANCERS AND INDIGENOUS HERBS, PLANTS

COMPARTMENT OF PLANT CELL - Md. Rageeb Md. Usman

DEVELOPMENT OF MOSQUITO REPELLENT FINISHES IN

IN VITRO ANTIBACTERIAL ACTIVITY OF METHANOLIC – AQUA

MEDICINAL PROPERTIES OF INDIGENOUS PLANTS USED

CHAPTER SIX 80 – 125

USE OF PLANT EXTRACTS IN PLANT DISEASE MANAGEMENT:

Knowlegde for Global Development

COMPREHENSION ON USE OF MEDICINAL PLANTS TO

CHAPTER EIGHT 178 - 201

MAXIMUM LIKELIHOOD ESTIMATES IN RELIABILITY OF

Knowlegde for Global Development

Knowlegde for Global Development

Knowlegde for Global Development

Subha Ganguly, Editor-in-Chief (FSEDInst)

KEY WORDS: Herbal immunomodulator, Medicinal plants

Mode of action in immunostimulation of different herbal extracts

Knowlegde for Global Development 1

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Bishavi B, Roychowdhury S, Ghosh S and Sengupta M. 2002. Hepatoprotective and

Diwanay S, Chitre D and Patwardhan 2004. Immunoprotection by botanical drugs in

Kalita D N and Dutta G N. 1999. Immunomodulatory effect of levamisole upon

Karnataka B C, Shukla S K, Kumar M and Dixit V P. 1993. Immunomodulatory effect of

Kolte A Y, Siddiqui M F and Mode S G. 2007. Immunomodulating effect of Withania

Krishnamohan A V, Reddy D B, Sarma B and John Kirubharan J. 1997. Studies on the

Kujur R T. 2001. Evaluation of certain immunomodulatory agents in countering

Kumar P. 2003. Studies on comparative immunomodulatory effect of herbal preparation

Knowlegde for Global Development 3

Manjrekar P N, Jolly C I and Narayan S. 1999. Comparative studies of the

Muruganandan S, Garg H, Lal J, Chandra S and Kumar D. 2001. Studies on the

Rege N.N and Dahanukar S.A. 1993. Quantitation of microbicidal activity of

Rege N N, Nazareth H M, Bapat R D and Dhanukar S A. 1989. Modulation of

Renoux and Renoux. 1971. Mechanism of action of some immunomodulatory drugs

Thatte U M and Dahanukar S A. 1986. Ayurveda and contemporary scientific thought.

Thatte U M and Dahanukar S A. 1989. Immunotherapeutic modification of experimental

Knowlegde for Global Development 4

Prasad, Arun and Ganguly, Subha (2012) Herbal Immunomodulators. AV

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COMPARTMENT OF PLANT CELL