An investigation on the Pharmacognosy, Phytochemistry and ...
Post on 05-Mar-2023
1 Views
Preview:
Transcript
1
An investigation on the Pharmacognosy, Phytochemistry and Pharmacology of
Adenia wightiana (Wall. ex Wight & Arn.) Engl. (Passifloraceae)
THESIS
submitted to the Pondicherry University
for the award of the degree of
DOCTOR OF PHILOSOPHY in
BOTANY
submitted by
J. PRESENA
under the guidance of Dr. A. PRAGASAM
DEPARTMENT OF BOTANY
KANCHI MAMUNIVAR CENTRE FOR POST GRADUATE STUDIES LAWSPET, PUDUCHERRY
INDIA
DECEMBER-2016
2
Dr. A. PRAGASAM Research Supervisor Department of Botany Kanchi Mamunivar Centre for Post Graduate Studies Lawspet, Puducherry – 605 008 India.
CERTIFICATE
I hereby certify that this thesis entitled “An investigation on the
Pharmacognosy, Phytochemistry and Pharmacology of Adenia wightiana (Wall.
ex Wight & Arn.) Engl. (Passifloraceae)” submitted for the award of the Degree of
Doctor of Philosophy in Botany is a record of research work done by the candidate
Mr. J. PRESENA, during the period of his study under my guidance at Kanchi
Mamunivar Centre for Post Graduate Studies, Puducherry and the thesis has not
formed the basis for the award to the candidate of any Degree, Diploma, Associate
ship, Fellowship or any other similar titles. I further certify that the thesis represents
the independent work of the candidate.
Place: Puducherry Dr. A. PRAGASAM
Date:
3
J.PRESENA Ph.D. Research Scholar Department of Botany Kanchi Mamunivar Centre for Post Graduate Studies Lawspet, Puducherry – 605 008 India.
DECLARATION
I hereby declare that the thesis entitled “An investigation on the
Pharmacognosy, Phytochemistry and Pharmacology of Adenia wightiana (Wall.
ex Wight & Arn.) Engl. (Passifloraceae)” submitted for the award of the Degree of
Doctor of Philosophy in Botany is my original work and has not formed the basis for
the award of any Degree, Diploma, Associateship, Fellowship or any other similar
titles.
Place: Puducherry J.PRESENA
Date:
4
ACKNOWLEDGEMENT
My heart with great pleasure bows with immense gratitude and indebtedness
to Dr. A. PRAGASAM, Research Supervisor, Department of Botany, Kanchi
Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry in guiding me
from the start of the title to the thesis without whom my research would not have seen
the light of the day.
I thank Dr. Thamizharasi Tamizhmani, the Director, Kanchi Mamunivar
Centre for Post-Graduate studies, Puducherry, for her Co-operation in carrying out the
research successfully.
My heartfelt thanks to Dr. T. Ganesan, Professor and Head of the Department
of Botany, Kanchi Mamunivar Centre for Post-graduate studies, Puducherry for
having provided me the necessary laboratory facilities and valuable suggestions for
my research work.
I am searching words to express my indebtness to my Doctoral Committee
members Dr. V.S. Negi, Professor and Head, Department of Clinical Immunology,
JIPMER and Dr. Bijaya Kumar Nayak, Assistant Professor, Department of Botany,
Kanchi Mamunivar Centre for Post Graduate Studies, Lawspet who had given
valuable suggestions to come over the stumbling blocks in my research.
Words are inadequate to express my special thanks to Dr. B. Kumaran,
Principal, Indira Gandhi College of Arts and Science, Kathirkamam, Puducherry for
permitting me to carry out research work along with my profession.
I am searching words to express my indebtness to Dr. P. Jayaraman,
Director, Plant Anatomy Research Centre, Chennai for his suggestions and help
throughout the course of investigation.
I thank Dr. V. Gopal, Principal, Department of Pharmacognosy and Dr. V.
Kavimani, Professor, Department of Pharmacology of Mother Theresa Post Graduate
5
and Research Institute of Health Sciences, Puducherry for giving complete shape to
my research.
My thanks to Dr. R. Ponnusamy, Mr. C. Krishnakumar, Mr. N.
Balachander for selection, collection, identification of my plant of interest for my
research.
I thank Dr. Dhamodharan, PDF, South Korea, who helped me a lot to give a
final shape to the thesis.
I thank Mr. J. Srinivasan and Dr. B. Sundarakrishnan, Research Associate,
Pondicherry University for their valuable suggestions in helping me to finish this
research work.
I thank Dr. A. Moushumi Priya, Mrs. P. Gunavathy, Mrs. R. Sharmila,
Mrs. Devika, Mr. M. Gandhi Pragash, Mr. M. Manikandan for helping me to
finish the project.
I wish to thank my family members for their constant help and encouragement
in finishing my thesis.
I do thank all other innumerable sources and persons who guided me for the
thesis to be in its right shape.
J. PRESENA
6
CONTENTS
Page No.
INDEX
LIST OF TABLES
LIST OF FIGURES
LISTS OF PLATES
ABBREVIATIONS
Chapter I. INTRODUCTION Chapter 2. REVIEW OF LITERATURE Chapter 3. MATERIALS AND METHODS Chapter 4. RESULTS Chapter 5. DISCUSSION Chapter 6. SUMMARY AND CONCLUSION REFERENCES PUBLICATION
7
INDEX
Chapter Title Page No. I. INTRODUCTION 1.1. Pharmacognosy 1.2. Phytochemistry 1.3. Pharmacology 1.3.1. Anti-oxidation 1.3.2. Anti-microbial study 1.3.3. Cytotoxicity study 1.3.4. Anti-inflammation 1.3.5. Anti-Ulcer 2. REVIEW OF LITERATURE 2.1. Family Passifloraceae 2.1.1. Passiflora 2.1.2. Ethnopharmacology of Passiflora 2.1.3. Phytochemical constituent of Passiflora 2.2. Objectives of the Present Study 3. MATERIALS AND METHODS 3.1. Collection of plant materials 3.2. Taxonomy of the plant species 3.3. Morphological features 3.4. Pharmacological Studies 3.4.1. Anatomical studies 3.4.2. Histo-chemical colour reaction 3.4.4. Fluorescence analysis 3.5. Phytochemistry 3.5.1. Physico-chemicalconstants 3.5.2. Prepartation of the Extract 3.5.3. pH Determination of Powdered Drug 3.5.4. Preliminary Phytochemical Screening 3.5.5. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis 3.6. Pharmacology 3.6.1. In-vitro antimicrobial study 3.6.2. In-vitro Antioxidant Activity 3.6.2.1. DPPH 3.6.2.2. Ferric Reducing Antioxidant Power (FRAP) Assay 3.6.2.3. Phosphomolybdenum assay 3.6.3. Cytotoxicity (MTT assay) 3.6.4. Invitro anti-inflammatory activity 3.6.5. Acute Toxicity Study 3.6.5.1. Invivo anti-inflammatory activity 3.6.5.2. Invivo Anti-Ulcerous Study
8
4. RESULTS 4.1. Pharmacognosy 4.1.1. Anatomy 4.1.1.1. Leaf epidermis in surface view 4.1.1.2. Quantitative values of foliar epidermis 4.1.1.3. Venation 4.1.1.4. Powder microscopy 4.1.1.5. Transverese section of Leaf 4.1.1.6. Transverse section of Leaf- midrib 4.1.1.7. Transverse section of Petiole 4.1.1.8. Transverse section of Stem 4.1.1.9. Transverse section of Root 4.1.1.10. Transverse section of Root tuber 4.1.1.11. Distribution of crystals and starch grains 4.1.2. Histochemicalcolour reaction 4.1.3. Fluorescence analysis 4.2. Phytochemistry 4.2.1. Physico-chemicalanalysis 4.2.2. Extractive values 4.2.3. pH determination of powdered drug 4.2.4. Preliminary phytochemical screening 4.2.5. GC-MS analysis 4.2.5.1. GC-MS analysis of leaf 4.2.5.2. GC-MS analysis of stem 4.2.5.3. GC-MS analysis of root 4.3. Pharmacology 4.3.1. In-vitro antioxidant activity 4.3.1.1. DPPH scavenging activity 4.3.1.2. FRAP method 4.3.1.3. Phosphomolybdenum assay 4.3.1.4. Antimicrobial activity of Adeniawightiana 4.3.1.5. In-vitro anti-inflammatory study 4.3.1.6. In-vitro cytotoxicity study 4.3.2. Invivo Pharmacological Study 4.3.2.1. Acute toxicity & Gross behavioural study 4.3.2.2. In-vivo anti-inflammatory study 4.3.2.3. In-vivo anti-ulcerous study 5. DISCUSSION 5.1. Pharmacognosy 5.2. Phytochemistry 5.2.1. Phytochemical analysis 5.2.2. GC-MS analysis 5.2.2.1. Comparative analysis of compounds present in leaf, stem and root 5.2.2.2. Chemical compound groups present in GC-MS analysis 5.2.2.3. Chemical compounds and their biological 5.3. Pharmacology 5.3.1. Antioxidant activity
9
5.3.2. Anti-microbial activity 5.3.3. In-vitro cytotoxicity study 5.3.4. In-vivo acute toxicity study 5.3.5. In-vivo, anti-inflammatory study 5.3.6. In-vivo, anti-ulcerous study 6. SUMMARY AND CONCLUSION REFERENCES PUBLICATION
10
LIST OF TABLES
Table No. Title
Table 1 : Quantitative values of foliar epidermis of Adeniawightiana
Table 2 : Histo-chemical colour reactions of leaf, stem and root of Adeniawightiana
Table 3 : Fluorescence analysis of leaf powder of Adeniawightiana
Table 4 : Fluorescence Analysis of stem powder of Adeniawightiana
Table 5 : Fluorescence Analysis of root powder of Adeniawightiana
Table 6 : Physico-chemicalanalysis of leaf, stem and root of Adenia wightiana
Table 7 : Extractive values of leaf, stem and root of Adeniawightiana by batch process
Table 8 : pHDetermination of leaf, stem and root of Adenia wightiana
Table-9 : Phytochemical screening of various solvent extracts of leaf of Adeniawightiana
Table-10 : Phytochemical screening of various solvent extracts of stem of Adeniawightiana
Table-11 : Phytochemical screening of various solvent extracts of root ofAdeniawightiana
Table- 12 : GC-MS analysis of methanolic extract of leaf of Adenia wightiana
Table-13 : GC-MS analysis of methanolic extract of stem of Adenia wightiana
Table-14 : GC-MS analysis of methanolic extract of root of Adenia wightiana
Table-15 : A comparative analysis of compounds in GC-MS analysis of methanolic extract of leaf, stem and root of Adenia wightiana
Table-16 : Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of leaf of Adeniawightiana
11
Table-17 : Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of stem of Adeniawightiana
Table-18 : Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of root of Adeniawightiana
Table-19 : A comparative analysis total number of chemical compounds groups present in the GC-MS analysis of Adeniawightiana
Table-20 : Biological activities possessed by the chemical compounds present in the GC-MS analysis of Adeniawightiana
Table21 : Antioxidant activity of leaf extract of Adeniawightiana - DPPH Assay
Table 22 : Antioxidant activity of stem extract of Adeniawightiana - DPPH Assay
Table 23 : Antioxidant activity of root extract of Adeniawightiana - DPPH Assay
Table 24 : Antioxidant activity of leaf extract of Adeniawightiana - FRAP method
Table 25 : Antioxidant activity ofstem extract of Adeniawightiana - FRAP method
Table 26 : Antioxidant activity ofroot extract of Adeniawightiana - FRAP method
Table-27 : Antioxidant activity of leaf extract of Adeniawightiana - Phospho molybdenum assay
Table-28 : Antioxidant activity of stem extract of Adeniawightiana - Phospho molybdenum assay
Table 29 : Antioxidant activity of root extract of Adeniawightiana - Phospho molybdenum assay
Table 30 : Antimicrobial activity of methanolicleafextract ofAdenia wightiana
Table 31 : Antimicrobial activity of methanolicstemextract ofAdenia wightiana
12
Table 32 : Antimicrobial activity of methanolicrootextract ofAdenia wightiana
Table 33 : Minimum Inhibitory Concentration of antimicrobial activity of methanolic leaf, stem and root extract of Adenia wightiana
Table 34 : In-vitro anti-inflammatory study of methanolic leaf, stem and root extract of Adeniawightiana
Table 35 : In-vitro cytotoxicity study of methanolic leaf, stem and root extract of Adeniawightiana
Table 36 : Animal gross behavioural study methanolic leaf, stem and root extract of Adeniawightiana
Table 37 : In-vivo anti-inflammatory study of methanolic leaf, stem and root extract of Adeniawightiana
Table 38 : In-vivo anti-ulcerous study of methanolic leaf, stem and root extract of Adeniawightiana
13
LIST OF FIGURES
Plate No. TITLE
1 : GC-MS chromatogram of methanolic leaf extract of Adeniawightiana
2 : GC-MS chromatogram of methanolic stem extract of Adeniawightiana
3 : GC-MS chromatogram of methanolic root extract of Adeniawightiana
4 : Structure of chemical compounds present in the GC-MS analysis of methanolic extract of leaf of Adeniawightiana
5 : Structure of chemical compounds present in the GC-MS analysis of methanolic extract of stem of Adeniawightiana
6 : Structure of chemical compounds present in the GC-MS analysis of methanolic extract of root of Adeniawightiana
7 : Anti-oxidant activity– DPPH Scavening assay
8 : Anti-oxidant activity – FRAP assay
9 : Anti-oxidant activity - Phosphomolybdenum assay
14
LIST OF PLATES
Plate No. TITLE
1 : Adenia wightiana– Morphology
2 : Adenia wightiana– Anatomy
3 : Adenia wightiana– Anatomy
4 : Adenia wightiana– Anatomy
5 : Antimicrobial activity of methanolic leaf extract
6 : Antimicrobial activity of methanolic leaf extract
7 : Antimicrobial activity of methanolic leaf extract
8 : Regeneration of Adeniawightiana
15
ABBREVIATIONS
IPNI - International Plant Names Index
RIP - Ribosomal Inactivating Protein
TDEF - Tropical Dry Evergreen Forests
FAA - Formalin Acetic Acid
TBA - Tertiary-Butyl Alcohol
DPX - DibutylPhathalate Xylene
UV - Ultra Violet
GC-MS - Gas Chromatography-Mass Spectrometry
pH - Potential of Hydrogen
HCl - Hydrochloric acid
GC-MS/EI - Gas Chromatography-Mass Spectrometry/Mass Spectrometry Electron Ionization NIST - National Institute Standard and Technology
MTCC - Microbial Type Culture Collection
PDA - Potato dextrose agar
DPPH - 1,1-diphenil-2- picrylhydrazyl
DMSO - Dimethyl sulfoxide
PRP - Phosphomolybdenum reduction potential
MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
HRBC - Human Red Blood Cell
OECD - Organization for Economic Co-operation and Development
PL - Pylorus Ligated
UI - Ulcer index
ROS - Reactive oxygen species
p.o. - per oral
17
CHAPTER 1
INTRODUCTION
Since olden times, medicines are being obtained from the plants for the
treatment of various diseases. Eventhough many synthetic drugs are being used for
various diseases, plants play a vital role being the integral part of health care in
majority of the developing countries. According to World Health Organisation
(WHO) a majority of the World’s population depends on plant based therapies to
address the needs of the Primary Health Care (WHO, 1999). Abu-Dahab et al., 2007
stated that a big share in the drug market has been contributed by plant based
products, neutraceuticals and food supplement comprising the complementary and
alternative therapies towards the end of 20th century.
Since the inception of mankind, herbs were used to treat to cure various
diseases and hence herbal medicine is considered to be the oldest medicine. Plants are
capable of synthesizing myriads of organic compounds which encompasses almost all
the possible combinations of organic compounds yet to be explored and hence they
act as chemical industries. The different types of plants act as the factories of food,
dye and colorant, perfumes and cosmetics, gum, sugar, vegetable oil, insecticide,
textile fibers, drugs etc. Plants are unique in creating the specific chemical compounds
in them, by sucking the nutrients from the soil and these chemical compounds are
responsible for various biological acitivities and to act as a drug to cure various
diseases. In the Primary Health care Declaration of Alma Ata, WHO recognised the
importance of traditional medicine as the source of Primary Health Care and has been
globally addressed since 1976. The traditional medicinal program of WHO has
defined traditional medicine as the sum total of knowledge and practice, whether
18
explicable or not, used in diagnosis, prevention and elimination of physical, mental or
social imbalance and relying exclusively on practical experience and observation
handed down from generation to generation either verbally or in writing (Rukangira,
2004).
According to WHO about 80% of the people in the developing countries of the
world depend upon the traditional medicine for their primary helath care needs and
85% of them makes use of plant extracts in their formulations. This percentage
attributes to nearly 3.5 billion to 4 billion people of the World depend upon plant as a
source of drug (Farnsworth et al., 1985). In the present scenario India stands seventh
in its position in species richness out of all the fourteen megabiodiverse nations of the
world. India has got innumerable medicinal plants being used for various ailments
since ages. Humans slowly lost their direct access to the plants because of the reason
that they got settled in different parts of the world where the plants cannot grow due
to their specificity towards environment. The gap between plants and humans were
being bridged by the World’s largest industry, Pharmaceutical industry, by isolating
the pure chemical compounds from the plant source using modern methods adopted in
the field of pharmacognosy, phytochemistry and pharmacology. Even though, the
pharmaceutical industry has unraveled some of the plants of ethno-medicinal
importance, many of them remain untapped so far. Hence, this study has been planned
to bring out the medicinal efficacy of an ethno-medically important plant of local
availability under the canopy of pharamcognosy, phytochemistry and pharmacology.
1.1. Pharmacognosy
Pharmacognosy is the study of medicines derived from natural sources, mainly
from plants. It basically deals with standardization, authentication and study of natural
19
drugs. Most of the research in pharmacognosy has been done in identifying
controversial species of plants, authentication of commonly used traditional medicinal
plants through morphological, phytochemical and physicochemical analysis. The
importance of pharmacognosy has been widely felt in recent times. Unlike taxonomic
identification, pharmacognostic study includes parameters which help in identifying
adulteration in powder form also. This is again necessary because once the plant is
dried and made into powder form, it loses its morphological identity and easily prone
to adulteration. Pharmacognostic study ensures plant identity, lays down
standardization parameters which will help and prevent adulterations. Such studies
will help in authentication of the plants and ensures reproducible quality of herbal
products which will lead to safety and efficacy of natural products. The
pharmacognostic standardization includes five major methods of evaluation of crude
drug namely organoleptic or morphological, microscopical or histological, physical,
chemical and biological (Chanda, 2014).
1.2. Phytochemistry
Phytochemicals are generally the secondary metaboliteswhich are biologically
active and are originated from plants. These secondary metabolites are of very little
importance for the plants because fo the reason they are required by them in very
trace quantity. They are synthesized naturally by all parts of the plant viz. leaf, stem,
root, flower, fruit, seed and bark (Tiwari et al., 2011).The phytochemical compounds
of plants have been used as therapeutic agent, new synthetic compound for drug
formulations and as taxonomic markers for discovery of new compounds. Base
compositions of more complex semisynthetic chemical compounds are also derived
from phytochemical compounds of plants (Akerele, 1992).Plants are the reservoirs of
20
potentially useful biologically active chemical compounds which could be new leads
and clues for the synthesis of modern drugs. Of all the chemical compounds naturally
synthesised by the plants flavonoids, alkaloids, phenolic compounds and tannins are
conosideered to be the most important (Doss, 2009). There is always a very close
correlation between the phytoconstituents and the bioactivity of plant from which the
specific biologically active chemical compounds can be synthesised to treat various
chronic ailments (Pandey et al., 2013).Owing to the significance in the above context,
such preliminary phytochemical screening of plants is the need of the hour in order to
discover and develop novel therapeutic agents with improved efficacy. In the last few
years, Gas Chromatography Mass Spectrometry (GC-MS) has become firmly
established as a key technological platform for secondary metabolite profiling in both
plant and non-plant species (Fernie et al., 2004).
1.3. Pharmacology
Pharmacology is the branch of medicine and biology concerned with the study
of drug action where a drug can be broadly defined as any man-made, natural, or
endogenous molecule which exerts a biochemical and physiological effect on the cell,
tissue, organ or organism. More specifically, it is the study of the interactions that
occur between a living organism and chemicals that affect normal or abnormal
biochemical function. If substances have medicinal properties, they are
considered pharmaceuticals.
1.3.1. Anti-oxidation
In many countries of the world, especially in the developing countries herbal
plants are still being the source of phytochemicals to cure vaious ailments such as
urinary infections, vaginitis, cervicitis, blood infections, skin infections and gastro
21
intestinal tract disorder. Medicinal plants are still the backbone for the traditional
medicine and hence they are the good source of biologically active compounds called
phytochemicals. Phytochemicals derived from plants are found to be very good
antioxidants to scavenge the free radicals for many of the tissue damaging diseases
(Hausladen et al., 1999; Lee, et al., 2000). It is very candid that the free radicals are
the major cause of various chronic tissue damage and degenerative diseases such as
inflammation, cancer, coronary heart disease, stroke and diabetes milletus (Scalbert et
al., 2005). Hence there is a need to assess the potentiality of medicinal plants for their
anti-oxidant and the corresponding anti-inflammatory activity and to know the
mechanism of action against inflammation phramcologically.
In recent years there has been great interest in screening various plant extracts
for natural antioxidants because of their good antioxidant properties. Since ancient
time, spices added to different food preparations to improve flavor and taste are also
well known for their antioxidant capacities. Exploring the anti-oxidation activities of
the plants and identification of their crude drugs is gaining importance throughout the
world. In the present work Adenia wightiana is considered for evaluating its
medicinal potential.
1.3.2. Anti-microbial study
In developing countries, infectious diseases are the vital cause for morbidity
and mortality among the general population. Hence, the pharmaceutical industries are
in the urge to develop new anti-microbial drugs particularly because of the constant
emergence of new drug resistance microbes for the conventional drugs. It is because
of the ability of the microorganism to acquire genetic change for the drug resistance
against the currently available anti-microbial drugs. Many recent studies from various
22
parts of the world corroborates the same stating the multi-resistant activity shown by
the microorganisms to many of the drugs available in the market (Nascimento et al.,
2000, Sakagami et al., 2002). Once again he human community relies on the plant
community in search for the new drug for the microbes which are multiresistant to the
traditional one. India being one among the fourteen richest nations of the mega-
biodiversity is in search of novel drugs from the plant resource to cull out more
efficient drug to battle against the multi-resistant microbes.
1.3.3. Cytotoxicity study
Cancer is considered as a disease which is characterised by uncontrolled cell
division or the disease where the tissue growth regulation fails. Alteration of gene
which regulates the cell growth and differentiation is the major cause for the
transformation of normal cell to a cancer cell (Priya et al., 2013). Enenthough, several
classes of anti-cancer drugs are available in the modern medicine their adverse side-
effects are very severe distorting the basic body structural plan of the person who
undergoes such medication. Hence there is always a need to develop herbal medicine
drug for cancer prevention and treatement without any side-effects. Various plant
extracts are being under research by various researcheres all over the world to bring
out a potential drug for cancer because of their low toxicity and side-effects.
1.3.4. Anti-inflammation
Whenever we encounter damage of tissuedue to injurythe tissue gets inflamed.
In general, a short course of anti-inflammatory drug is administered for all sorts of
painful conditions. This may also lead to severe side-effects and adverse effects in
many of the people. Many herbs can act as an anti-inflammatory drug without causing
any side effects (Burke et al., 2005).Eventhough the modern medicines has become
23
very popular in many parts of the World, till 70% of the people from developing
country still rely on complementary and alternative systems of medicine, which is
also called as traditional medicne for their complete cure (Shaikh et al., 2005).
According to the literature, many of the medicinal plants possess anti-inflammatory
properties by reducing the swelling both internally and externally. Herbal drugs had
gained its momentum again among the public because of their safety, potentiality to
cure the disease completely and being cost effective (Apu et al., 2012).
1.3.5. Anti-Ulcer
Ulcer is a very common gastrointestinal disorder among the people all over the
world which causes break in the inflamed skin or in the mucus membrane lining the
digestive tract. Ulceration is caused by loss of homeostasis either by increased
aggression or decreased mucosal resistance. This loss of homeostasis of digestive tract
may be due to the continuous usage of drugs, stress, changed food habits, etc. An
array of synthetic drugs are available in the market to treat ulcer but being expensive,
in-addition-to, minimal antiulcer effect shown by the drug it also cause many side-
effects. The literature survey unveiled that there are many herbal palnts and poly-
herbal formulations being used for the cure of ulcer by many ayurvedic doctors and
traditional medicinal practioners till date. The first and foremost objective of
treatment of peptic ulcer is to subside pain, curing of ulcer and postpone the
occurrence of ulcer again. Many attempts have been made by various researchers all
over the world to know the plants that are being used in Ayurvedic medicine which
could be used in modern medicine to cure the disease namely ulcer (Vimala et al.,
2014).
24
The present study is an effort to fulfill the gap in the field of medicinal plants
availability and to enlist existing wild medicinal plants as potent drug for various
ailments like anti-inflammation, anti-ulcer, anti-microbial and cytotoxicity through
pharmacognostical, phytochemical and pharmacological tools. Hence, Adenia
wightiana of Passifloraceae is selected to unravel the natural chemical compounds
which may lead to the discovery of new drug to treat diseases.
25
CHAPTER 2
REVIEW OF LITERATURE
Plants are said to be the one of the major group of organisms to trap the solar
energy to prepare food for their survival. The primary metabolites namely,
carbohydrates, proteins, lipids and nucleic acids by subjecting themselves under
various metabolic pathway produce secondary metabolites like alkaloids, tannins,
gums, resins, aromatic oils, terpenoids etc. These phytochemicals have been attracting
much interest as natural alternatives to synthetic compounds. Many higher plants
accumulate extractable organic substances which are sufficient for economic
management of disease. Plants have been a rich source of medicines because they
produce wide array of bioactive molecules, most of which probably evolved as
chemical defense against predation or infection. Recently, the phytochemicals of a
large number of plants and their effects on human health have been intensively
studied. Particularly, a search for antioxidants, antimicrobial, hypoglycemic and
anticancer agents of plants is in progress throughout the world.
A number of plants have been studied intensively to reveal their medicinal
potential. The studies of Santhaet al., (1988), Ahmad (1994), Nambiaret al., (1996),
Seetharamet al., (1999), Annamalaiet al., (2000), Srivastava (2001), Amerjothy
(2003), Dubey et al., (2004), Shanthi, et al., (2006), Suseela and Prema (2007),
Devika and Sajitha (2007) are some of the examples to be mentioned.
This chapter encompasses the published literature on medicinal uses and
various studies on the genus Adenia, a member of the family Passifloraceaewhich is
chosen for the present study.
26
2.1. Family Passifloraceae
The International Plant Names Index (IPNI) includes 27 plant genera and 694
accepted species (http://www.plantsystematics.org). The family Passifloraceae is also
known as passion flower family because of the popular species Passiflora edulis. The
species of this genus are distributed in the warm temperate and tropical regions of the
New World; they are much rarer in Asia, Australia, and tropical Africa. Several
species are grown in the tropics for their edible fruits, the most widely grown being
Passiflora edulis (Passion fruit or purple granadilla) (McGuire, 1999). Many other are
grown outdoors in the warmer parts of the world or in the glasshouses for their exotic
flowers (Dhawan et al., 2004).
2.1.1. Passiflora
The plants of genus Passiflora are shrubs and herbs, mostly climbers with
auxillary tendrils. Stem herbaceous or woody, generally climbing, very rarely
arborescent. The British Herbal Pharmacopoeia, United States Homoeopathic
Pharmacopoeia, Homoeopathic Pharmacopoeia of India, Pharmacopoeia Helvetica,
Deutsches Arzneibuch, Homeopathie, Pharmacopee Francaise and many of the herbal
compendiums and Materia Medica contain data on species of Passiflora.
2.1.2. Ethnopharmacology of Passiflora
In Argentine folk medicine, the aerial parts of Passiflora caerulea are used as
mild anti-microbial agents in diseases like catarrh and pneumonia (Anesini and Perez,
1993). Passiflora capsularis is a reputed emmenagogue (Felter and Lloyd, 1983).
Passiflora contrayerva is a reputed counter-poison, deobstruent and cordial (Felter
and Lloyd, 1983). Passiflora edulis has been used as a sedative, diuretic, anthelmintic,
anti-diarrheal, stimulant, tonic and also in the treatment of hypertension, menopausal
27
symptoms, colic of infants in South America (Chopra et al., 1956). In Madeire, the
fruit of Passiflora edulis is regarded as a digestive stimulant and is used as a remedy
for gastric carcinoma (Watt and Breyer-Brandwijk, 1962). In Nagaland (India), fresh
leaves of Passiflora edulis are boiled in little amount of water and the extract is drunk
for the treatment of dysentry and hypertension (Jamir et al., 1999). Fruits are eaten to
get relief from constipation. Passiflora foetida leaf infusion has been used to treat
hysteria and insomnia in Nigeria (Nwosu, 1999). Passiflora incarnata is a popular
traditional European remedy (Handler, 1962) as well as a homoeopathic medicine
(Rawat, 1987) for insomnia, anxiety, and has been used as a sedative tea in North
America (Bergner, 1995). The juice of Passiflora maliformis is used for intermittent
fevers in Brazil. Passiflora quadrangularis is used throughout the Caribbean as a
sedative and for headaches. Leaf tea is taken for high blood pressure and diabetes
(Seaforth et al., 1983). In Mauritius and Rodrigues, bath of leaf-docoction of
Passiflora suberosa is used to treat skin diseases. Root decoction is used as an
emmenagogne and is useful in hysteria (Fakim et al., 1993). In Central America,
stems of Passiflora pedunculata the aerial parts of Passiflora sexflora and Passiflora
vitifolia have been used against snakebites (Morton, 1981).
2.1.3. Phytochemical constituent of Passiflora
Alkaloids, phenols, glycosyl flavonoids and cyanogenic compounds have been
reported from this genus. Literature survey has revealed that a number of
phytochemical reports are available on the following species of Passiflora. Passiflora
incarnata (Gavasheli et al., 1974), Passiflora adenopoda (Spencer et al., 1986),
Passiflora alata (Ulubelen et al., 1982), Passiflora apetala (Olafsdottir et al., 1997),
Passiflora biflora (McCormicket al., 1983), Passiflora bryonioides (Poethke et al.,
1970), Passiflora caerulea (Speroni et al., 1996), Passiflora capsularis (Fischer et al.,
28
1982), Passiflora coactilis (Escobar et al., 1983), Passiflora cochinchinensis (Ma et
al., 1982), Passifloraco linvauxii (Adsersen et al., 1993), Passiflora foetida
(Echeverriet al., 1985), Passiflora hybrida (Jaroszewski,et al., 1989), Passiflora
lechenaultii (Bombardelli et al., 1975), Passiflora molliseria (Uzcategui, 1985),
Passiflora mollissima (Froehlich et al., 1989), Passiflora platyloba (Ayanoglu et al.,
1982).
Adenia wightiana
Foreign
Seven female hypertensive subjects treated with aqueous extract of Adenia
cissempeloidesreportsshowed low systolic blood pressure, serum alpha-
hydroxybutyrate dehydrogenase and creatine kinase and the levels of total and
conjugated bilirubin were also significantly lower. The plant preparation appears not
to have hepatotoxic or nephrotoxic effects. This study indicates that Adenia
cissempeloides reduced the systolic blood pressure with little or no effect on the
diastolic and potent source of plant drug to be used for cardiac muscle function.
(Nyarkoet al., 1990). Anti-viral activity of whole plant of Adenia cardiophylla among
one of the selected chinese medicinal plants against infections was reported by Lynn
yip (1993). Adenia gummifera was reported to be used in psychiatry to treat
symptoms like insanity, mental disease, hysteria, delirium, convulsions, epilepsy, fits,
divining, trance, dream induction, hallucinatory, stimulants, anti-depressants in the
South Africa population (Sobiecki, 2002).
High level of total phenolic compounds of Adenia gummifera and the
antioxidant properties and could serve as free radical inhibitors or scavengers, acting
possibly as primary antioxidants and the folklore use of this was justified (Adedapoet
al., 2008). Ethnomedicinal use of Adenia lobata used for cough and colic was
29
experimentally confirmed by in-vitro antimicrobial activity of the plant extract against
beta lactam resistant bacteria (Gangoue-Pieboji et al., 2009). Christian Agyarea et al.,
(2009) reported the ethno-medicinal use of the decoction of whole plant Adenia
cissampeloides and in-vitro confirmation of ethno-pharmacological use of the same
for Stings/bites healing in Bosomtwi-Atwima-wanwoma area of Ghana.
Ethnobotanical use of whole plant of Adenia lobata was reported for the treatment of
infertility in male sexual impotence and it is used as an aphrodisiac. Stem of Adenia
cissampeloides was reported to cure infertility in females (Aku Diame et al., 2010)
Panyaphuet al., (2011) had listed Adenia penangiana as one of the plants used
in Northern Thailand as postpartum herbal bath recipes by the Mien (Yao) community
and their phytochemical tests revealed the presence of phenolic compounds,
flavonoids, triperpene and lactone glycosides. In the study of conservation of some
commercialized succulent species of Madagascar A.firingalavensis, A. olaboensis
have been reported as vulnerable and Adenia subsessifolia Perrier have been reported
to be endangered. (Ravaomanalinaet al., 2011). The study suggests that A. lobata is
apotential source of flavonoids which possess antioxidant potential that can counteract
the oxidative damage induced by the illnesses for which the populations use it to take
care of themselves (Marcelet al., 2011).De Wetet al., (2012) have reported the
method of preparation and administration of the medicinal use of root of Adenia
gummifera to cure HIV and gonorrhea in Northern Maputaland, Kwazulu-Natal
Province of South Africa.
Different classes of chemical compounds in the extract of Adenia
cissampeloides such as alkaloids, quassinoids, sesquiterpene, lactones, and
flavonoids have been found to exhibit in-vitro anti-plasmodial activity through several
mechanisms (Annanet al., 2012). The study on anti-hyperglycaemic activity of
30
ethanolic extract of the stem has revealed the isolate palmitic acid exhibited promising
anti-hyperglycaemic activity in streptozotocin-induced diabetic rats (Sarkodie et al.,
2013). Adebiyiet al., (2013) have reported the aqueous extract of Adenia
cissampeloides as a pain modulator in mice. The Ethno-botanical studies of Kaingu,et
al., (2014) reported Adenia gummifera roots and stem bark boiled in water and
consumption of half-a-glass of the decoction daily for three days to treat fibroids.
Decoctions, infusions, concoctions and vapour of roots, leaves, stem, bark, flowers,
and oil of the plants Adenia venenata has been reported to use for the treatment of
headache and migraine in Nigeria. (Saganuwan, 2014). Srichaiwong et al., (2014)
have reported that the leaf top and fruits of Adenia viridiflora is being consumed as
natural food in the Chi Basin of Thailand.
Nateshan Anilet al., (2015) reported the phyto-constituent like Tetraphyllin B
(barterin), Epitetraphyllin B (volkenin), Gummiferol and Proanthocyanidins from
Adenia gummifera which are active against lymphatic filariasis. Aboh et. al.,(2015)
have studied the leaf of Adenia cissapeloides in the ethno medical practices when
administered within the appropriate toxicity levels for humans and animals. The
antimicrobial results showed that the extracts were highly bacterial and fungicidal and
of broad spectrum activities.An ethno-medicinal study shows that the whole plant of
Adenia cissampeloides and leaf of Adenia rumicifolia have been reported to be
effective against plasmodium and can be used to treat malarial diseases (Lawalet al.,
2015).
The phytochemical screening of leaf and stem of Adenia cissampeloides
investigated by Okunyeet al., (2015) showed the presence of saponins, alkaloids,
tannins, flavonoids and steroids. The leaf has the highest compound yield when
compared stem. The noted antibacterial and antifungal activity of Adenia
31
cissampeloides against Staphylococcus aureus, Esherichia coli, Pseudomonas
aeruginosa, Candida albicans and Aspergillus nigersuggest potential use of the plant
extracts for treatment of gastroenteritis and other associated infections.The leaves of
Adenia rumicifolia boiled and the extract of the boiled leaf was kept in the mouth for
3-5 minutes to cure tooth-ache and the same formulation is used as a drink to cure
swollen neck in the folklore medicine of Wonegizi, Ziama Clan-Lofa County, Liberia
(Kpadehyeaet al., 2015).
India
Vijayan et al., (2007) have recorded the traditional remedies being followed
by the Kani Tribes of Kottoor Reserve Forest, Agasthyavanam, Thiruvananthapuram,
Kerala. They have reported that forty grams of tuber paste/powder of Adenia hondala
was reported to be taken with cow/coconut milk to promote lactation. Ramachandran,
(2007) reported that the vernacular name of the wild edible climber Adenia wightiana
as Kanvalikizhangu and it is one of the 74 plants being utilized by the tribal
community of Anamalai Hills of Coimbatore District of Western Ghats of Tamilnadu.
Parthasarathyet al., (2008) have reported Adenia wightiana as one of the lianas
species out of a total of 47 lianas investigated in 75 patches of Tropical Dry
Evergreen Forests of Peninsular India. An ethanobotanical survey conducted by
Kottaimuthuet al., 2008 in Southern Eastern Ghats of Tamilnadu inhabitated by
valaiyan tribals consumes Adenia wightiana as one of the wild plant out of 63 plants
enumerated by them in that area. They have reported the vernacular name of the plant
is tamil as “Perum Kurattai” and 10 ml of filtered juice of tuber is administered to
cure peptic ulcers.Anandhanet al., (2011) had listed Adenia wightiana as one of the
species in the list of plants of Floral Diversity of Pudukkottai Narthamalai Hillock,
Tamilnadu, India. Khajjaet al., (2011) showed that the fruits of Adenia palmata to
32
contain Toxalbumin (Cyanogenic glycoside) and Emulsin (enzyme) which can be
used as an indicator to identify the plants in forensic study.
The roots of Adenia digitata (modeccin), Adenia volkensii (volkensin) and
caudex of Adenia goetzii (lectin like protein), Adenia lanceolata (lanceolin), Adenia
stenodactyla (stenodactylin) were reported as a source of toxic and non-toxic type 2
Ribosomal Inactivating Protein (RIP). They have also reported that juice of leaves and
root of Adenia hondala were used in skin troubles and the root tuber for urinary
disorders (Sobiya Raj D and Jannet Vennila, 2013). The study states that out of 67
belonging to 44 families used by the tribes of Vythiri Taluk of Wayanad District of
Kerala, Adenia hondala a member of Passifloraceae is one among the important wild
species. Eating the boiled tuber of Adenia hondalahave been reported to cure hernia
(Devi Prasadet al., 2013). A detail diagnostic characters and instrumental validation
for authentication of a traditionally important herbal drug Adenia hondala as one of
the constitutents of “Vidari” in local Indian market. Qualitative screening of
phytochemicals indicated the presence of Carbohydrates, Glycosides, Saponins,
Phytosterols, Flavanoids, Proteins, Gums & mucilages and absence of Phenolic acids
and Tannins in all the plant used in Vidari (Shilpashreeet al., 2015). Out of 52 liana
species reported Adenia wightiana was one among them in the tropical dry evergreen
forest on the Coromandel Coast of India (Vivek et al., 2015). Out of 194 wild
medicinal plants Adenia hondala has been reported to be one among 30 climbers
investigated by Prashanth Kumar et al., (2016). As reported by them extract of leaves
and root of this plant are used to treat skin troubles, whereas, root tuber is used to treat
the urinary disorders.
33
2.2. Objectives of the Present Study
Keeping the view of significances of traditional medicine in the field of plant-
based drug discovery that leads to therapeutic significances, an important Indian
medicinal plant Adeniawightiana(Passifloraceae)was selected to carry out the
following tasks.
Pharmacognosy
Anatomical studies of leaf, petiole, stem, root and root tubers for proper
identification
Microscopic analysis for authentic identification of crude drugs
Physico-chemical constants of the drug
Histochemical localization of phytochemicals
Extractive values of leaves, stem, root in various solvents
Phytochemistry
Preliminary phytochemical screening of various crude extracts of leaves, stem
and root
GC-MS analysis of methanol extracts of methanolic leaves, stem and root to
identify the bioactive phytochemicals
Pharmacology
In vitro antioxidant activity of the drug
In vitro antimicrobial activity of the drug
In vitro cytotoxicity study to reveal the anti-cancerous activity of the drug
In vitro anti-inflammatory activity
34
In-vivoAcute toxicity study to know the toxicity level of the drug
In-vivo anti-ulcer to find out the efficacy of the drug to treat gasteroentric
problem
In-vivo anti-inflammatory activities to unveil the potentiality of the drug to
treat inflammation
The in-vitro studies will help to develop new plant based drug
Regeneration of the plant in an eco-restored site
35
CHAPTER 3
MATERIALS AND METHODS
3.1. Collection of plant materials
The plant specimens of Adenia wightiana were collected from the Tropical
Dry Evergreen Forests (TDEF) of Puthupet about 13 km north of Pondicheny on the
way to Marakkanam on the East Coast Road to Chennai. The whole patch of Puthupet
green vegetation in 20 ha is protected on the basis of religious faith. It is a relic of a
TDEF forest, housing 104 plant species belonging to 44 families (Sethi, 1993).
The plant was identified with the help of the Flora of the Presidency of Madras
(Gamble, 1935) and the Flora of Tamil Nadu Carnatic (Mathew, 1983). Care was
taken to select healthy plants and normal organs. It was photographed and herbarium
specimens were prepared. Species identity confirmation was conducted in Auro
Herbarium, Auroville, Tamilnadu and the herbarium specimen was deposited there.
3.2. Taxonomy of the plant species Adenia wightiana
Kingdom : Plantae
Division : Angiospermae
Class : Dicotyledonae
Subclass : Polypetalae
Series : Calyciflorae
Order : Passiflorales
Family : Passifloraceae
Genus : Adenia
Species : Adenia wightiana(Wall. ex Wight & Arn.) Engl.
Basionym
36
Modecca wightiana Wall.ex Wight & Arn.
Synonym
Modecca wightiana Wall.ex Wight & Arn.
Vernacular name
Tamil: Perum kurattai
Sri Lanka: Pothu hondala
3.3. Morphological features (Plate 1)
Habit: Vine; branchlets glabrous.
Leaves
Broadly ovate, sometimes lanceolate, lobed or not, 3.5-8 x 3-7.5 cm,
chartaceous, 3-5 nerved at base, glabrescent, base cuneate-cordate or truncate, margin
entire, apex subacute; petiole 1.5-4.5 cm, with a gland at the leaf-base; stipules
triangular.
Flowers
Polygamo-monoecious or dioecious.Calyx-tube campanulate or tubular; lobes
4 or 5. Petals 4 or 5, inserted at the throat or base of calyx-tube. Corona a fringe of
short hairs at the base of petals. Male: Stamens 4 or 5, basally connate, inserted at the
base of calyx-tube; pistillode obscure or 0. Female: ovary stipitate or sub-sessile;
ovules numerous on 3 parietal placentae; staminodes 4 or 5. Capsule globose, 3 x 2.5
cm; seeds numerous, ovoid, to 5 mm (Matthew, 1981).
Root: Tuberous
Distribution: Peninsula, Sri Lanka.
37
3.4. Pharmacological Studies
The fresh plant materials were collected and the morphological features of the
specimen were studied directly in the field and were photographed. The required
samples of different organs were cut from the plant and fixed in FAA (5ml Formalin,
5ml Glacial Acetic acid, 90 ml of 70% Ethyl alcohol (Johansen, 1940) immediately
after collection. Fresh parts of the plant mainly leaves, stem and root were collected
and kept in polythene bags for further laboratory studies. The materials collected were
dried under shade in the laboratory for 3 to 4 days and the dried materials were stored
in dry polythene bags to carry out pharmacognostical, phytochemical and
pharmacological investigations.
3.4.1. Anatomical studies
Different parts of the plants were cut and fixed in FAA. After 24 hours of
fixing, the specimens were dehydrated with graded series of tertiary-butyl alcohol
(TBA) as per the schedule (Sass, 1940). Infiltration of the specimens was done by
gradual addition of paraffin wax (melting point 58-60˚C) until TBA solution attained
super saturation. The specimens were cast into paraffin blocks. The paraffin
embedded specimens were sectioned with the help of Rotary Microtome. The
thickness of the sections was 10-12 µm. Dewaxing of the sections was done by
customary procedure (Johansen, 1940). The sections were stained with Toluidine blue
(O’Brien et. al., 1964). Since Toluidine blue is polychromatic stain, it rendered pink
colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to
mucilage, blue to the protein bodies etc. Wherever necessary sections were stained
with safranin, fast-green and iodine, potassium iodide for starch.
To study the epidermal cells, stomatal morphology and venation pattern
paradermal sections (sections taken parallel to the surface of leaf) as well as clearing
38
of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration
employing Jeffrey’s maceration fluid (Sass, 1940) were done. Glycerine mounted
temporary preparations were made for macerated/cleared materials. Powdered
material of different parts were cleared with sodium hydroxide and mounted in
glycerine medium after staining. Different cell components were studied and the
dimensions were measured.
Maceration technique
Maceration was carried out with the stem and root materials, following
Jeffrey’s method (Johansen, 1940). This method involves, cutting the material (either
fresh or dry) into slices of about 300 micrometer in thick and boiling repeatedly until
free from air, then macerated in a solution of equal parts of 10% aqueous Nitric acid
and 10% aqueous chromic acid. The time varies according to the material, and cells
begin to separate in about 24 hours. A thick glass rod with rounded end was used to
crush the material very gently. It was washed very thoroughly with water to remove
the acids. The use of a centrifuge is advisable in order to speed up the process. The
material was stained with saffranin (1%). The macerated materials were kept in 1%
saffranin for about 6 hours and rinsed thoroughly in water. From this macerated
material, a few drops of the stained macerate were taken, mounted in glycerine and
sealed with DPX mountant.
Stomatal Number
Stomatal number is the average number of stomata per square mm of the
epidermis of the leaf (Evans, 1996). A piece of leaf (middle part) was cleared by
boiling with alkaline solution. The upper and lower epidermal layers were peeled
separately. The peeled epidermis was placed on slide and mounted with glycerine.
39
With the help of stage micrometer one square millimeter was drawn. The prepared
slide was placed on the stage. Epidermal cells and stomata are traced. The number of
stomata lying in the area of one square millimeter are counted including the cell if at
least half of its area lying with in the square. Average number of stomata per
millimeter square is calculated by tracing four different fields.
Stomatal Index (SI)
It is the percentage which the numbers of stomata form to the total number of
epidermal cells, each stomata being counted as one cell. Stomatal index can be
calculated by using following equation.
Stomatal Index = ( )
푥100
Where,
S = the number of stomata per unit area
E = the number of epidermal cells in the same unit area (including trichomes)
Stomatal type
The distribution of various stomatal types was studied at different regions of
abaxial and adaxial surfaces of leaves.
Palisade ratio
Palisade ratio is defined as the average number of palisade cells beneath each
upper epidermal cell (Trease &Evans, 1996). The semi-permanent mounts of cleared
leaves were employed for this study.
40
Vein-Islet Number
A vein-islet is the small area of green tissue surrounded by the veinlets. The
vein-islet number is the average number of vein-islets/mm2 of a leaf surface midway
between the midrib and the margin of the leaf. This is constant for a given species of
the plant and used as a characteristic feature for the identification of the allied species.
This number is independent of the size of the leaf and does not alter with the age of
plant. A piece of leaf was cleared by boiling in chloral hydrate solution. Camera
Lucida and drawing board was arranged for the drawings. With the help of stage
Micrometer one square millimeter was drawn. The cleared leaf was mounted on the
slide and a drop of glycerin water was added then covered with cover slip. The above
prepared slide was placed on the stage of the microscope. Veins are traced which are
included within the square. The outlines of those islets which overlap two adjacent
sides of the square are also traced. The number of vein-islets in the square millimeter
is counted. The islets which are intersected by the sides of square are included on two
adjacent sides and excluded on other two sides (Wallis, 1985, Evans, 1996).
Veinlet Termination Number
It is defined as the veinlet termination per square millimeter of the leaf
surface, midway between midrib of the leaf and its margin. A vein termination is the
ultimate free termination of veinlet. A piece of leaf was cleared by boiling in chloral
hydrate solution. With the help of stage micrometer one square millimeter was
drawn. The cleared leaf was mounted on the slide and a drop of glycerin water was
added then covered with cover slip. The above prepared slide was placed on the stage
of the microscope. Veins are traced which are included within the square. The
outlines of those islets which overlap two adjacent sides of the square are also traced.
41
The number of vein-islets in the square millimeter is counted. The islets which are
intersected by the sides of square are included on two adjacent sides and excluded on
other two sides (Wallis, 1985, Evans, 1996). To study the veinlet termination number
the method of Khandelwal (2002) was adopted.
Photomicrographs
Microscopic description of tissues was supplemented with micrographs
wherever necessary. Photographs of different magnifications were taken with Nikon
labphoto 2 microscopic unit. For normal observations bright field was used. For the
study of crystals, starch grains and lignified cells, polarized light was employed. Since
these structures have birefringent property, under polarized light they appeared bright
against dark background. Magnifications of the figures are indicated by the scale-bars.
3.4.2. Histo-chemical colour reaction
Histo-chemical methods are employed in the identification, density of
accumulation and distribution of chemical compounds within biological cells and
tissues in different organs under microscopes using the color-stainreaction technique
and photographic documentation (Hassan and El-Awadi, 2013). Histo-chemical
colour reaction of leaf, stem and root of Adenia wightiana were performed according
to Johansen (1940). Free-hand sections of plant materials were taken and treated with
various chemical reagents to identify lignin, tannin, mucilage, starch, alkaloid and
protein. The colour and results are recorded. The methodology of the test performed
to detect the presence of histochemical substances is as follows.
42
Lignin
Fresh free-hand sections of leaf, stem and root of Adenia wightiana were
mounted in 1% neutral aqueous potassium permanganate and allowed to stand for 15
minutes, washed thoroughly with water and placed in 2% HCl for 2 minutes, removed
and washed with distilled water. Dilute ammonia solution was added and covered
with coverslip. Change to colour to deep red indicates the presence of lignin.
Tannin
Fresh free hand sections of leaf, stem and root of Adenia wightiana were
placed in 1% solution of Ferric chloride. Change of blue to black colour indicates the
presence of tannins.
Mucilage
Fresh free hand sections of leaf, stem and root of Adenia wightiana were
treated with methylene blue reagent. Change of colour to blue, indicates the presence
of mucilage.
Starch
Fresh free hand sections of leaf, stem and root of Adenia wightiana were
mounted in 1% Iodine solution. Change of colour to blue, indicates the presence of
starch.
Alkaloid
Fresh free hand sections of leaf, stem and root of Adenia wightiana were
mounted in Meyer’s reagent (36 g of Mercuric chloride was dissolved in 60 ml of
43
water and added to a solution of 5g of Potassium iodide in 20 ml of water and made
up to 100 ml). Change of colour to reddish brown indicates the presence of alkaloid.
Proteins
Fresh free hand sections of leaf, stem and root of Adenia wightiana were
stained in aqueous solution of picric acid, covered and allowed to stand for 24 hours.
Change of colour yellow indicates the presence of proteins.
3.4.4. Fluorescence analysis
Quantitative fluorescence analysis utilizes the fluorescence produced by a
compound in day light and ultraviolet light for quantitative evaluation (Evans, 1996).
Fluorescence analysis of the drug dried leaf, stem and root) was observed in daylight
and UV light (254 & 365 nm) using drug powder and various solvent extracts of the
drug (Pratt and Chase, 1949) as follows.
Dried powder of leaf, stem and root of Adenia wightiana was treated with
different solvents namely 1N aqueous Sodium hyrdroxide, 1 N alcoholic Sodium
hydroxide, and acids namely 50% 1 N Hydrochloric acid, 50% Sulphuric acid and
Nitric acid separately and then these extracts were subjected to fluorescence analysis
in visible day light and UV light. The results were tabulated.
3.5. Phytochemistry
3.5.1. Physico-chemicalconstants
The authenticity of a crude drug is established with reference to the
descriptions of the pharmacopoeia or other publications of the country concerned. The
quality and purity required is achieved by standards (numerical values) also given in
44
the official work of reference. The powdered plant materials were morphologically
and organoleptically screened and subjected to physico-chemical analysis. The
various parameters considered were:
Ash values
Ash values are helpful in determining the quality and purity of a crude drug,
especially in the powdered form. The total ash, acid insoluble ash, water soluble ash
and sulphated ash of the leaf, stem and root of the plant was performed (Khandelwal,
2002).
Determination of Total Ash
About 2 g of the crude drug powder is accurately weighed in a silica crucible
which is previously ignited and weighed. The powdered drug is spread in a fine layer
at the bottom of the crucible. The crucible is incinerated at a temperature not
exceeding 450° C until free from carbon. The crucible is cooled and weighed. The
procedure is repeated to a constant weight. The percentage of the total ash is
calculated with reference to the air-dried drug.
Determination of Acid Insoluble ash
The total Ash values were determined by the ash obtained from leaf , stem and
root. When it is boiled separately with 25 ml of Hydrochloric acid for a few minutes
the insoluble ash is collected on an ashless filter paper and washed with hot water.
The insoluble ash is transferred to the pre-weighed silica crucible, ignited, cooled and
weighed. The procedure is repeated to the constant weight. The percentage of acid
insoluble ash was calculated with reference to the air-dried drug. The results were
recorded.
45
Acid insoluble ash value (%) =
푥100
Determination of Water soluble ash
The ash obtained by the method of determination of total ash is boiled for five
minutes with 25 ml of water. The insoluble matter was collected on an ashless filter
paper ignited, cooled and weighed. The weight of the insoluble matter is subtracted
from the weight of the total ash. The difference in weight was considered as the water
soluble ash. The percentage of water soluble ash is calculated with reference to air-
dried drug. The results were tabulated.
Water soluble ash (%) =
푥100
Determination of Sulphated ash
One gram of crude drug in a crucible, ignited gently until the substance is
thoroughly charred. Cooled and the residue was moistened with 1 ml of concentrated
sulphuric acid, heated gently until white fumes are no longer evolved and ignited
them at 800°C until black particles disappear. Allow the crucible to cool, few drops of
sulphuric acid were added and then heated. This operation was repeated until two
successive weights do not differ by more than 0.5 mg.
Moisture content
An accurately weighed quantity of about 2 g of powdered drug was taken in a
petriplate and distributed evenly. The petriplate kept open in the oven and the sample
at a temperature between 100° to 105°C. Then it was cooled to room temperature,
weighed and recorded the percentage loss on drying was calculated using the
following formula (Khandelwal, 2002).
46
Loss on drying (%) =
푥100
3.5.2. Prepartation of the Extract
The leaf, stem and root parts of freshly collected plant materials were chopped
into small pieces separately, shade-dried and coarsely powdered using a pulverizor.
The coarse powder were subjected to successive extraction with Methanol, Ethyl
acetate, Chloroform and Petroleum ether by using Soxhlet apparatus. The extracts
were collected and distilled off on a water bath at atmospheric pressure and the last
trace of the solvents was removed in vacuo and stored at 4°C. The resultant extracts
were subjected to preliminary phytochemical screening and GC-MS anlaysis.
Extractive Values
Crude drug extraction of the leaf, stem and root of Adenia wightiana using
four different solvents Methanol, Ethyl acetate, Chloroform and Diethyl ether was
performed by Batch process (Kokate, 1986).
3.5.3. pH Determination of Powdered Drug
One gram of the accurately weighed powdered drug was dissolved in water
and filtered. pH of the filtrate was determined by using digital pH meter.
3.5.4. Preliminary Phytochemical Screening
All the extracts were subjected to preliminary phytochemical tests following
the method of Harborne (1973) and Trease and Evans (1983).
47
Test for Alkaloid (Evans, 1996)
The extract was mixed with little amount of dilute hydrochloric acid and
Meyer’s reagent (36 g of mercuric chloride was dissolved in 60 ml of water and added
to a solution of 5 g potassium iodide in 20 ml of water and made up to 100 ml).
Formation of white precipitate is the indication for the presence of alkaloid.
Test for Anthraquinone (Modified Berntrager’s test)
To 5 ml extract, 5 ml of 5 % FeCl3 and 5 ml dilute HCl were added and heated
for 5 minutes in boiling water bath. Then it was cooled and benzene or any organic
solvent was added and shook well. Organic layers were separated and equal volume
of dilute ammonia was added. Ammonical layer shows pinkish red colour and
indicates the presence of anthraquinone.
Test for Aminoacids (Ninhydrin test)
Three ml test solution was heated and 3 drops of 5% Ninhydrin solution was
added in boiling water bath for 10 minutes. Purple or bluish colour indicates the
presence of amino acids.
Test for Catechins
To the substance, a drop of Ehrlich’ reagent (para- dimethyl amino
benzaldehyde) was added which turns into pink colour indicating the presence of
catechins.
48
Test for Cardiac glycosides (Keller-Killiani test)
To 2 ml extract acetic acid, one drop of 5% ferric chloride and concentrated
Sulphuric acid were added. Reddish brown colour appears at junction of the two
liquid layers and upper layer appears bluish green.
Test for Coumarins
To the substance, a drop of sodium sulphate was added which turns into
yellow colour indicating the presence of Coumarins.
Test for flavonoids
To a little of the substance or powder in alcohol, 10 % sodium hydroxide
solution or ammonia was added. Dark yellow colouration is the indication of
presence of flavonoids.
Test for Glycosides
A small amount of the drug is mixed with a little anthrone on a watch glass.
One drop of concentrated sulphuric acid was added to that and a paste is prepared
when warmed gently over water bath. The appearance of dark green colouration
indicates the presence of glycosides.
Test for Gums, Oils, and Resins
The test solution was applied on filter paper which develops a transparent
appearance on the filter paper indicating the presence of oils, gums and resins.
49
Test for Phenol
To the powder substance, a few drops of alcohol and ferric chloride solution
were added. Bluish green or red colour is the indication of the presence of phenol.
Test for Proteins (Biuret test)
To 3 ml of test solution, 4 % NaOH and few drops of 1 % CuSO4 solution
were added. Violet or pink colour indicates the presence of proteins.
Test for Phlobotannins
Deposition of a red precipitate when aqueous extract of plant sample boiled
with 1 % aqueous HCl is the evidence for the presence of phlobotannins.
Test for Saponins
A little of the substance is shaken with water and copious lather formation is
the indication for the presence of saponins.
Test for Steroids (Liebermann’s reaction)
Three ml extract was mixed with 3 ml of acetic anhydride then heated and
cooled. A few drops of concentrated sulphuric acid was added. The appearance of
blue colour indicates the presence of steroids.
Test for Reducing Sugars (Fehling’s test)
The substance is mixed with Fehling’s solution A and B. Formation of a red
colouration is the indication for the presence of reducing sugars.
50
Test for Non-Reducing (Iodine test)
To 3ml test solution a few drops of dilute iodine solution was added. The
appearance of blue colour and disappearance on boiling and reappears on cooling
indicates the presence of non-reducing sugars.
Test for Tannins (Mace, 1963)
The substance is mixed with basic lead acetate solution. Formation of a white
precipitate is the indication for the presence of tannins.
Test for Terpenoids (Salkowski test)
To 5 ml test solution, 2 ml of chloroform and 3 ml of concentrated sulphuric
acid were carefully added to form a layer. A reddish brown colour formation
indicates the presence of terpenoids.
Test for Triterpenoids
Two or three granules of tin metal were dissolved in 2 ml of thionyl chloride
solution. Then one ml of extract was added to it. The formation of pink colour
indicates the presence of triterpenoids.
3.5.5. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Around 25 g powdered leaf, stem and root of the selected plant was soaked in
30 ml of methanol overnight and then filtered through filter paper. The filtrate is then
concentrated by flushing nitrogen gas into the solution and was concentrated to 1 ml.
The concentrate was again filtered in the Whatmann No. 41 filter paper along with 2 g
Sodium sulfate to remove the sediments and traces of water in the filtrate. The
chemical composition of methanolic extract of leaf, stem and root of Adenia
51
wightiana was investigated through Gas Chromatography-Mass Spectrometry/Mass
Spectrometry Electron Ionization (GC-MS/EI) mode.
The GC-MS/MS is a Scion 436-GC Bruker model coupled with a Triple
quadruple mass spectrophotometer with fused silica capillary column BR-5MS (5%
Diphenyl/95% Dimethyl polysiloxane) and Length: 30m; Internal diameter: 0.25 mm;
Thickness: 0.25 μm. Helium gas (99.999%) was used as the carrier gas at a constant
flow rate of 1 ml/min and an injection volume of 2 μl was employed (split ratio of
10:1). The column oven temperature program was as follows: 110° C hold for 3.50
min, Up to 200° C at the rate of 10° C/min-No hold, Up to 280° C at the rate of 5
°C/min hold for 12 min. Injector temperature 280° C and total GC running time was
40.50 min. This last increase was to clean the column from any residues. The mass
spectrometer was operated in the positive electron ionization (EI) mode with
ionization energy of 70eV. The solvent delay was 0-3.5 min. A scan interval of 0.5
seconds and fragments from m/z 50 to 500 Da was programmed. The inlet
temperature was set at 290° C, source temperature 250° C. The relative percentage
amount of each component was calculated by comparing its average peak area to the
total areas. Software adopted to handle mass spectra and chromatograms was MS
Work station 8. The NIST Version 2.0 library database of National Institute Standard
and Technology (NIST) having more than 62,000 patterns was used for identifying
the chemical components. The spectrum of the unknown component was compared
with the spectrum of the known components stored in the NIST library. The name,
molecular weight and structure of the components of the test materials were
ascertained. The GC-MS/MS was performed by Food Safety and Quality Testing
Laboratory, Institute of crop processing technology, Thanjavur (Srinivasan et al.,
2016).
52
3.6. Pharmacology
3.6.1. In-vitro antimicrobial study
Agar Well Diffusion Method
Pure cultures of all experimental bacteria and fungi were obtained from the
Microbial Type Culture Collection (MTCC), Institute of Microbial Technology
(IMTECH), Chandigarh. The pure bacterial cultures were maintained on Mueller
Hinton Agar medium and fungal culture on Sabouraud Dextrose Agar medium. Each
bacterial and fungal culture was further maintained by sub-culturing regularly on the
same medium and stored at 4°C before use in experiments.
Agar well diffusion method is widely used to evaluate the antimicrobial
activity of plants or microbial extracts (Magaldi et al., 2004, Valgas et al., 2007). The
methanolic extract of leaf, stem and root of Adenia wightiana were tested by Agar
Well Diffusion Method. A cork borer was sterilized by autoclaving or disinfecting it
by rising in alcohol followed by sterile water (6-mm) holes were punched aseptically
in nutrient agar plate, SDA plate by using cork borer. The cotton swabs were dipped
into the broth culture of the test organisms and were gently squeezed against the
inside of the tube to remove excess fluid. Bacillus subtilis, Staphylococcus aureus,
Escherichia coli, Klebsiella pneumonia, were swabbed on Mueller Hinton Agar
plates, Candida albicans and Aspergillus niger was swabbed on Sabouraud Dextrose
Agar plates. Swabbing was done in outside diameter of the plates. The plates were
allowed to dry for about 5 minutes. Then the methanolic extracts of leaf, stem and
root of Adenia wightiana (30 µl) were added in 4 wells of different petri plates
containing different microbial organism. The plates were incubated at 37°C for 18-24
hour for bacterial pathogens and 28°C for 48 hours fungal pathogens. The zone of
53
inhibition was measured in millimetre, using a ruler on the underside of the plate. The
zone size was recorded and all the cultures were discarded and autoclaved (Chamanet
al., 2013).
3.6.2. In-vitro Antioxidant Activity
3.6.2.1. DPPH
Radical scavenging activity of plant extracts against stable 1,1-diphenil-2-
picrylhydrazyl (DPPH) was determined by the slightly modified method of Brand-
Williams et al 1995 (Brand-Williams, et al., 1995). DPPH reacts with an antioxidant
compound, which can donate hydrogen, and reduce DPPH. The change in colour
(from deep violet to light yellow) was measured at 517 nm on a UV visible light
spectrophotometer. The solution of DPPH in methanol 6 × 10-5 M was prepared fresh
before UV measurements. Three ml of this solution was mixed with different
concentration of plant extracts. The samples were kept in dark for 15 minutes at room
temperature and the decrease in absorbance was measured. The experiment was
carried out in triplicate. Radical scavenging activity was calculated by the following
formula (Senevirathneet al., 2006).
% Inhibition = [(AB –AA)/A B] × 100
Where
AB = absorption of blank sample (Ascorbic acid) (time = 0 min)
AA = absorption of test extract solution (time =15 mins)
3.6.2.2. Ferric Reducing Antioxidant Power (FRAP) Assay
The method of Benzie and Strain (1996) was adopted for the assay. The
principle is based on the formation of O-phenanthroline-Fe2+ complex and its
disruption in the presence of chelating agents. The reaction mixture containing one ml
54
of 0.05% O-phenanthroline in methanol, 2 ml ferric chloride (200 µM) and 2 ml of
various concentrations ranging from 50 to 500 µg were incubated at room temperature
for 10 minutes and the absorbance of the same was measured at 510 nm. Ascorbic
acid was used as standard. The experiment was performed in triplicates.
3.6.2.3. Phosphomolybdenum assay
10mg of plant extract was dissolved in 1ml of DMSO. 100µl from the
prepared sample was taken and 1ml of reagent solution was added to it and incubated
in a water bath at 95°C for 90 min. After 90 min, the absorbance of the solution was
read at 695 nm. Ascorbic acid (10mg/ml DMSO) was used as standard. The
Phosphomolybdenum reduction potential (PRP) of the studied extracts were reported
in percentage.
3.6.3. In-vitro cytotoxicity activity (MTT assay)
Selection of extracts
The extractive value was high in methanol solvent, hence the methanol extract
of the leaf, stem and root were selected for in-vitro and in-vivo pharmacological
studies.
Cell lines and culture conditions
MCF-7 were procured from National Centre for Cell Science at Pune and
maintained in 10% FBS, antibiotic 2% (penicillin or streptomycin) in a humidified
atmosphere of 5% CO2 at 37°C until confluent. The stock cultures were grown in
culture flask and the experiments were carried out in 96 well plate.
MTT assay
55
The MTT assay is based on the cleavage of the soluble yellow tetrazolium salt
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into a blue
colored formazan by the mitochondrial enzyme succinate dehydrogenase. This assay
is extensively used for measuring cell survival and proliferation. There is a direct
proportionality between the formazan produced and the number of viable cells.
However, it depends on the cell type, cellular metabolism, and incubation time with
MTT. This method is based on the capacity of mitochondrial enzymes of viable cells
to reduce the yellow soluble salt MTT to purple blue insoluble formazan precipitate
which is quantified spectrophotometrically at 570 nm after dissolving in DMSO. Cells
are plated on to 96 well plates and allowed to grow in CO2 incubator for 24 hrs (37°C,
5% CO2). The medium is then removed and replaced by fresh medium containing
different concentrations of leaf extract for 48 hrs. The cells are incubated for 24-48
hrs (37°C, 5% CO2). Then, 20 μL MTT stock solutions (5 mg/mL in PBS) added to
each well and incubated for 4 hour. The medium is removed and 200 μL DMSO is
added to each well to dissolve the MTT metabolic product. Then the plate is shaken at
150 rpm for 5 minutes, and the optical density is measured at 570 nm (Thakkar et. al.,
2014).
% Cell viability=([O.D of control−O.D of test compound]/[O.D. of control]) × 100
3.6.4. Invitro anti-inflammatory activity
The anti-inflammatory activity of methanolic extract of Adenia wightiana was
assessed by in-vitro HRBC membrane stabilization method. Blood was collected from
healthy volunteers. The collected blood was mixed with equal volume of Alsever’s
solution (Dextrose 2%, Sodium citrate 0.8%, Citric acid 0.05%, Sodium chloride
0.42% and Distilled water 100 ml) and centrifuged with isosaline. To 1 ml of HRBC
56
(Human Red Blood Cell) suspension equal volume of test drug in three different
concentrations was added. All the assay mixtures were incubated at 37°C for 30
minutes and centrifuged. The haemoglobin content in the supernatant solution was
estimated by using spectrophotometer at 560nm. The percentage of haemolysis was
calculated by using the following formula (James et. al., 2009).
Percentage of haemolysis =
푥100
The percentage of HRBC membrane stabilization or protection was calculated by
using the following formula,
Percentage protection = 100 -
푥100
In-vivo pharmacological studies
Animals
Adult Wistar Albino rats of either sex (150-200g) were used for the present
investigation. Animals were housed under standard environmental conditions at
temperature (25±2°C) and light and dark (12:12 h). Rats were fed with standard pellet
diet (Goldmohur brand, MS Hindustan lever Ltd., Mumbai, India) and water ad
libitum. The experimental protocols were carried out at Padmavathi College of
Pharmacy and Research Institute, Dharmapuri, Tamilnadu approved by the
Institutional Animals Ethics Committee.
3.6.5. Acute Toxicity Study
Methanolic extract in the doses of 500, 1000 and 2000 mg/kg were given
orally for the assessment of acute toxicological studies to different groups of albino
rats (150-200g) and observed for signs of behavioral, neurological toxicity and
57
mortality. All the parameters were thoroughly checked and dose for the further studies
was calculated as per the Organisation for the Economic Cooperation Development
423 guidelines (OECD). After the conduct of acute toxicological studies a single dose
of methanolic extract was decided i.e. 200 mg/kg.
3.6.5.1. Invivo anti-inflammatory activity
Albino rats of either sex weighing 150-200 grams were divided into six groups
of three animals each. The dosage of the drugs administered to the different groups
were as follows. Group 1 - Control (normal saline 0.5 ml/kg), Group 2 - 0.1 ml of 1%
Carrageenan, Group 3 - 0.1 ml of 1% Carrageenan and Indomethacin (20 mg/kg/i.p),
Group 4 - Methanolic extract of leaf (200 mg/kg/i.p), Group 5 - Methanolic extract of
stem (200 mg/kg/i.p), Group 6 - Methanolic extract of root (200 mg/kg/i.p). All the
drugs were administered orally. Indomethacin served as the reference standard for
anti-inflammatory drug. After one hour of the administration of the drugs, 0.1 ml of
1% w/v carrageenan solution in normal saline was injected into the sub plantar tissue
of the left hind paw of the rat and the right hind paw was served as the control. The
paw volume of the rats were measured in the digital plethysmograph (Ugo basile,
Italy), at the end of 0 min., 60min., 120min., 180min. The percentage increase in paw
edema of the treated groups was compared with that of the control and the inhibitory
effect of the drugs was studied. The relative potency of the drugs under investigation
was calculated based upon the percentage change of the inflammation. Percentage
change in activity was calculated using the formula,
% change in activity = [(Vc-Vt)/Vc] × 100
58
Where,
Vt the percentage represents the percentage difference in increased paw volume after
the administration of test drugs to the rats and
Vc represents difference of increased volume in the control groups.
3.6.5.2. Invivo Anti-Ulcerous Study
The Wistar albino rats weighing between 150-200g were divided into five
groups each of 3 animals.After the fasting period the rats were anaesthetized with di
ethyl ether. The abdomen was opened and the pyloric end was ligated with a thread.
All the samples were given 60 minutes prior to pyloric ligation. Group 1 received 1%
Carboxy Methyl Cellulose (1ml/kg,p.o.) act as a control. Group 2 received
omeprazole (20 mg/kg, p.o.) act as a standard. Group 3 received methanolic leaf
extract of Adenia wightiana (200 mg/kg, p.o). Group 4 received methanolic stem
extract of Adenia wightiana (200 mg/kg, p.o). Group 5 received methanolic root
extract of Adenia wightiana (200mg/kg, p.o.). After 4 hours by pyloric ligation all the
animals were sacrificed to observe gastric lesions.The gastric juice was collected and
centrifuged at 1000 rpm for 10 minutes. The volume of gastric juice (ml) as well as
pH of gastric juice was noted.The gastric ulcer score was recorded.Gastric content
were assayed for total acidity by titration against 0.01N NaOH using phenophthalein
as indicator. The volume of gastric content was measured and the total acidity and
free acidity were estimated (Kulkarni, 1985).
Regeneration of the plant
Regeneration of the plant Adenia wightianawas done in Project EcoLake,an
ecorestoration project near Oussudu lake of Sri Aurobindo Ashram, Pondicherry.
Young stem cuttings of Adenia wightianawere used for vegetative propagation. The
59
stem cuttings were planted in the earthern pots with garden soil. After development of
new shoots and leaves they were removed from the pot and planted in the soil under
shade. They established themselves in the habitat.
60
CHAPTER 4
RESULTS
4.1. Pharmacognosy
4.1.1. Anatomy
The anatomical study of Adenia wightiana includes the leaf epidermal study,
venation pattern of leaf, powder microscopic study and transverse section of leaf,
stem and root (Plate 2-4).
4.1.1.1. Leaf epidermis in surface view
Surface view of the epidermal cells was studied in paradermal sections. The
adaxial epidermis consists of polyhedral thick walled cells with straight anticlinal
walls. Tannin cells and large, irregularly lobed, deeply staining sclereids are found
distributed here and there. The epidermal cells have several successive annular
cuticular striations and form small conical mounds. The abaxial epidermis is
stomatiferous, the epidermal cells are larger in size and have fairly thick wavy
anticlinal walls. The annular cuticular striations are more prominent on the cells of the
abaxial epidermis. The stomata are of paracytic type and 15x10 µm in size. Of the two
subsidiary cells one is much smaller and the other is larger (Plate 2).
4.1.1.2. Quantitative values of foliar epidermis
The mean number of epidermal cells per square millimeter in the abaxial
surface of Adenia wightiana is 542.0±8.84 and adaxial surface is 963±6.76. The mean
number of stomata per square millimeter on the abaxial surface is 312.0±7.16 and
61
stomata completely absent on the adaxial surface. The stomatal index found to be
022.9±4.37. the palisade to spongy ratio is 05.4±0.24 (Table 1).
4.1.1.3. Venation
The venation is densely reticulate, veins are thin and wavy, vein islets are
wide and the vein boundaries are not well defined. The vein terminations occur in all
vein-islets. The termination is much branched repeatedly forming large, dendroid
outline. The vein islet number is 40.8±3.85 and the veinlet termination number is
88.4±5.78 (Table 1).
4.1.1.4. Powder microscopy
The powder preparation of the plant material exhibited the following
inclusions when examined under the microscope. Xylem fibres are of libriform type,
long, uniformly thick with lignified walls and narrow lumen. They are about 500 µm
long and less than 5 µm wide. Fibre-tracheids are 550 µm long and 20 µm wide,
similar to fibres but with wide lumen and dense slit like lateral wall pittings. The pits
occur in two or three vertical rows. The tracheids are about 450 µm long and 30 µm
wide with thick lateral walls and wide cell lumen and blunt ends. They have bordered
pits densely distributed on the lateral walls. Vessel elements are 220-270 µm long and
common in the powder. They are short, cylindrical and wide with multiseriate,
elliptical pits on the lateral walls. The end wall may be horizontal or slightly oblique
with wide circular simple perforation plate (Plate 4).
62
4.1.1.5. Transverese section of Leaf
The lamina is 110 µm thick, dorsiventral and hypostomatic. The adaxial
epidermis consists of thick and wide cylindrical cells. The abaxial epidermis is thin
and made of rectangular thin walled cells with thick conical cuticular projections. The
mesophyll consists of single row of vertically elongated conical and compact palisade
cells on the adaxial side and 3 or 4 layers of small, lobed spongy parenchyma with
large intercellular spaces on the abaxial side. The spherical idioblasts containing
prismatic calcium oxalate crystals are sparsely distributed in the palisade tissue of the
lamina each measures 70 to 100 µm in diameter and may have 1-3 druses (Plate 2).
4.1.1.6. Transverse section of Leaf- midrib
The leaf in cross section is thin and bifacial with plano-convex midrib. The
midrib is 230 µm thick, flat on the adaxial side and convex and semi-circular on the
abaxial side. The adaxial epidermis of the midrib is thin with short cylindrical thick
walled cells. The abaxial epidermis is thick with squarish thick walled cells and
prominent conical, cuticular projections. The ground tissue of the midrib is
parenchymatous, the cells are wide, angular, compact and thick walled. The vascular
strand is single and collateral. The xylem strand includes about four short vertical
lines of xylem elements which are angular and thick walled. Phloem occurs in thin arc
at the lower end of the xylem strand (Plate 2).
4.1.1.7. Transverse section of Petiole
The petiole is roughly circular in cross section. It is 1.5 mm in diameter and
exhibits dorsiventral polarity. The epidermis has barrel shaped, fairly thick epidermal
cells with prominent cuticle. Internal to the epidermis there are 3 or 4 layers of small
63
collenchyma cells. The remaining ground tissue is parenchymatous and made of thin
walled angular and compact cells. The vascular system consists of a pair of adaxial
small accessory vascular bundles. The main vascular strand includes wide and deep
bowl shaped vascular bundle and fairly thick wedge shaped medullary vascular
bundle which is situated above the concavity of the main bundle. All the bundles are
collateral with well-developed vertical line of xylem elements which are circular or
elliptical, wide and thick walled. Phloem occurs in the form of thin band on the outer
end of each vascular bundle (Plate 3).
4.1.1.8. Transverse section of Stem
The stem is circular in transverse section and measures 6.5 mm in thickness.
The epidermal layer is thin with cylindrical cells and thick cuticle. The cortical zone
is thick and parenchymatous with discontinuous, discrete masses of cortical fibres.
The vascular tissues occur in wide six segments with narrow parenchymatous gaps in
between the segments. The vascular segments are collateral with outer phloem and
inner mass of xylem. Phloem consists of fairly wide thick walled, angular compact
cells which are diffuse in distribution. Xylem strands include secondary xylem as well
as primary xylem. The secondary xylem consists of fairly large number of wide
circular, solitary vessels and xylem fibres. The secondary xylem vessels are 90-150
µm wide. The primary xylem vessels are comparatively narrow, fairly thick walled
and occur in tangential row in the inner border. Pith is wide, parenchymatous and
homogenous (Plate 3).
64
4.1.1.9. Transverse section of Root
The root is 8.5 mm thick. It consists of outer thick, highly fissured periderm
followed by cortex. The vascular cylinder includes secondary phloem and secondary
xylem enclosing narrow pith. The periderm is irregularly fissured at several places,
thin at certain places and thick in other places. It includes thin walled, tubular,
suberized phellem cells. The secondary phloem is very thick measuring one mm in
radial plane. It consists of outer portion of collapsed phloem and inner intact phloem.
The collapsed phloem includes crushed and obliterated sieve elements which are seen
as dark thin tangential or irregular markings. In the intact phloem, the sieve elements
are intact and they occur in wide circular masses. The secondary phloem is followed
by thick wavy cylinder of secondary xylem which is cleaved into many thick radial
segments by the wide, dilated xylem rays. In the segmented xylem the segments
include numerous solitary or paired vessels. The vessels are wide, fairly thick walled,
circular or angular and are 100-170 µm in diameter. The ground tissue of the
secondary xylem includes xylem fibres and thin tangential segments of parenchyma.
Druse idioblasts are distributed in the parenchyma cells (Plate 4).
4.1.1.10. Transverse section of Root tuber
Because of the storage function of the root tuber, the structure is unusual in
general. The tuber contains more quarters of storage parenchyma than vascular
tissues. The tuber consists of a thick dark superficial layer and less distinct periderm
layers. The cortical zone is wide and parenchymatous. The cortical tissue includes
cells of various shape and size and highly distorted in orientation. The central part of
the tuber consists of large number of small clusters of vessels and fibres. The vessel
clusters are distorted and occur in different orientation. The cluster includes two to
65
four vessels which are ensheathed by thick walled lignified fibres. The vessels are
wide, circular and fairly thick walled. They are 120-170 µm wide. Towards the
periphery of the tuber, the vessel clusters become reduced in number and are radially
stretched. Each vessel cluster is associated with a narrow, radially oriented phloem
strand. The phloem strand consists of small, angular thick walled sieve elements. The
ground tissue in between the vascular strands is composed of proliferated cells of the
cambial derivatives. The cells possess dense accumulation of the starch grains and
large druse idioblasts (Plate 4).
4.1.1.11. Distribution of crystals and starch grains
The druse idioblasts of spherical calcium oxalate crystals are fairly abundant
in the mesophyll of leaf and different tissues of the root and root tuber. The druse
idioblasts in the leaf are large and spherical with 1-3 druses. In root they are mainly
distributed in the parenchyma of phloem and ray parenchyma of xylem. In the root
tuber they are distributed in the ground parenchyma. The cells bearing the druses in
root and root tuber are similar to the ordinary cells in size and shape. They are
irregularly distributed and range from 20-25 µm in diameter. The parenchyma cells of
the root tuber contain dense accumulation of the starch grains. They are cylindrical
and longitudinally four lobed with dark cross marks on the polar ends.
4.1.2. Histochemical colour reaction
The histochemical localization tests revealed the presence of starch, alkaloid,
protein, tannin in leaf, stem and root of Adenia wightiana. Lignin was found to be
present in stem and leaf only. Mucilage was not reported from any of the parts (Table
2).
66
4.1.3. Fluorescence analysis
The fluorescence analysis of leaf, stem and root parts of Adenia wightiana in different
in different chemical reagents observed under ordinary and UV light are given in
Table 3-5.
Leaf
The dried leaf powder of Adenia wightiana produced green colour in visible
light and light green colour on exposing to UV light. When treated with 1N aq.
NaOH, 1N HCl, 50% H2SO4 , 50%HNO3 produced bright green and light green in
visible light and light green respectively (Table-3).
Stem
The dried stem powder of Adenia wightiana developed light brown colour on
exposing to both visible and UV light. Drug on treating with 1N aq. NaOH produced
light brown in both light. On treating with chemicals like 1N HCl, 50% H2SO4,
50%HNO3 it showed brown colouration (Table 4).
Root
The dried leaf powder of Adenia wightiana was observed to produce light
brown and dark brown in visible light and UV light respectively. The drug on treating
with 1N aq. NaOH develops light brown colouration in both light. When treated with
1N HCl, 50% H2SO4, 50%HNO3 shows dark brown colour in both light (Table-5).
67
4.2. Phytochemistry
4.2.1. Physico-chemicalanalysis
The leaf, stem and root of Adenia wightiana was subjected to Physico-
chemicalanalysis which includes parameters like loss on drying, total ash, acid
insoluble ash, water soluble ash and sulfated ash. The results were given in table 6.
Leaf
In leaf moisture content, total ash, acid insoluble ash, water soluble ash and
sulfated ash were found to be 20.0±0.8, 16.0±0.63, 09.0±0.31, 09.4±0.54 and
10.0±0.46 in percentage respectively.
Stem
In stem moisture content, total ash, acid insoluble ash, water soluble ash and
sulfated ash were found to be 10.1±0.50, 04.5±0.42, 1.01±0.20, 04.0±0.29 and
02.5±0.40 in percentage respectively.
Root
In root moisture content, total ash, acid insoluble ash, water soluble ash and
sulfated ash were found to be 10.4±0.70, 09.4±0.30, 04.0±0.40, 06.0±0.08 and
02.0±0.82 in percentage respectively.
4.2.2. Extractive values
The extractive values of dried leaf, stem and root powder of Adenia wightiana
on extraction with Methanol, Ethyl acetate, Chloroform, Diethyl ether are given in
table 7.
68
Leaf
In leaf, the highest extractive value was recorded in Methanol (23.0±0.84)
followed by Ethyl acetate (21.6±0.95), chloroform (14.0±0.81), and the lowest
extractive value was found in Diethyl ether (11.2±0.57).
Stem
In stem, the highest extractive value was obtained in Methanol (19.8±0.75)
followed by Ethyl acetate (18.1±0.64), Chloroform (12.3±0.88) and the lowest
extractive value was recorded in Diethyl ether (10.5±0.49).
Root
In root, the highest extractive value was obtained in Methanol (21.0±0.23)
followed by Ethyl acetate (20.1±0.98), Diethyl ether (13.0±0.22) and the lowest
extractive value was recorded in Chloroform (10.6±0.42).
4.2.3. pH determination of powdered drug
The pH value of aqueous extract of powdered drug of leaf, stem and root of
Adenia wightiana are given in the table 8. The pH value of leaf, stem and root are
6.8±0.04, 6.4±0.11 and 6.7±0.05 respectively.
4.2.4. Preliminary phytochemical screening
Preliminary phytochemical screening of leaf, stem and root extract of Adenia
wightiana in four different solvents namely Methanol, Ethyl acetae, Chloroform and
Diethyl ether were subjected for the various tests to confirm the presence or absence
of alkaloid, anthraquinone, aminoacid, cardiac glycoside, catechin, coumarin,
flavonoid, gums, oils & resins, glycoside, non-reducing sugar, protein, phlobotannin,
69
phenol, quinone, reducing sugar, saponin, steroid, tannin, terpenoid and triterpenoid
(Tables 9-11).
Leaf
The preliminary phytochemical studies of methanolic extract of leaf showed
the presence of alkaloid, anthroquinone, aminoacid, cardiac glycoside, coumarin,
flavonoid, glycoside, non-reducing sugar, protein, phenol, and tannin. In Ethyl acetate
extract, alkaloids, anthraquinone, aminoacids, cardiac glycosides, coumarins,
flavonoids, glycosides, non-reducing sugar, protein, quinone, reducing sugar and
tannin. In Chloroform extract, glycoside, phenol, saponin, steroid and terpenoid were
present. In Diethyl ether extract, coumarin, glycoside, steroid and triterpenoid.
Catechin, gum, oils & resin and phlobotannin were absent in all the leaf extracts
(Table 9).
Stem
The preliminary phytochemical studies of methanolic extract of stem showed
the presence of alkaloid, aminoacid, protein, phlobotannin, reducing sugar, tannin and
terpenoid. In Ethyl acetate extract, glycosides, protein, phenol and reducing sugar. In
Chloroform glycoside and steroid were present and in Diethyl ether extract, glycoside
was the only phytochemical present. Anthraquinone, cardiac glycoside, catechin,
coumarin, flavonoid, gums, oils & resins, non-reducing sugar, quinone, saponin,
steroid and triterpenoid were found to be absent in all the extracts of stem (Table 10).
Root
The preliminary phytochemical studies of methanolic extract of leaf showed
the presence of alkaloid, aminoacid, catechin, flavonoid, protein, phenol, quinone,
reducing sugar and tannin. In Ethyl acetate extract, glycosides, protein, quinone,
70
reducing sugar and terpenoid were present. In Chloroform extract, anthraquinone,
aminoacid, gums, oils & resins, glycoside, protein, quinone, steroid and triterpenoid
were present. In Diethyl ether extract, gums, oils & resins, glycoside and quinone
were present. Cardiac glycoside, coumarin, non-reducing sugar, phlobotannin,
saponin, steroid, terpenoid and triterpenoid were absent in all the root extracts (Table
11).
4.2.5. GC-MS analysis
Phytochemicals were best extracted in methanol because of its high polarity.
Hence, the methanol extract of leaf, stem and root of Adenia wightiana were
subjected to GC-MS analysis to detect the possible compounds present in the active
fraction (Table 12-14) (Fig. 1-3).
4.2.5.1. GC-MS analysis of leaf
A total of 25 chemical compounds were found to be present in the methanolic
extract of leaf. 2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-
trimethyltridecyl)-,[2R-[2R*(4R*,8R*)]] was found to be present as major constituent
with the peak area 16.52% and retention time 29.52 minutes followed by dl-α-
Tocopherol with the peak area 16.38% and retention time 32.75 followed by β-
Sitosterol with the peak are 15.39% and retention time 37.22 retention time. 4H-
Pyran-4-one,2, 3-dihydro-3, 5-dihydroxy-6-methyl- was found to be with least peak
area of 0.26% with a retention time of 4.70 (Table 12, Fig.1,4).
4.2.5.2. GC-MS analysis of stem
A total of 32 chemical compounds were found to be present in the methanolic
extract of leaf. Stigmasterol was found to be present as major constituent with the peak
area 12.56% and retention time 35.56 minutes followed by 5-
71
Hydroxymethylfurfuralwith the peak area 11.46% and retention time 6.06 followed by
β-Sitosterol with the peak are 15.39% and retention time 37.22 retention time. Trans-
Geranylgeraniol was found to be with least peak area of 0.03% with a retention time of
27.70 (Table 13, Fig. 2,5).
4.2.5.3. GC-MS analysis of root
A total of 32 chemical compounds were found to be present in the methanolic
extract of leaf. Thiophene,2-propyl- was found to be present as major constituent with
the peak area 22.63% and retention time 6.09 minutes followed by 9-
Octadecenoicacid(Z)-,methylester with the peak area 11.37% and retention time 17.45
followed by S)-(+)-2',3'-Dideoxyribonolactone with the peak are 9.30% and retention
time 5.60 retention time. Cholestan-3-ol,2-methylene-,(3β, 5α) was found to be with
least peak area of 0.03% with a retention time of 21.10 (Table 14, Fig. 3,6).
4.3. Pharmacology
4.3.1. In-vitro antioxidant activity
4.3.1.1. DPPH scavenging activity
The anti-oxidant activity of Methanol, Ethyl acetate, Chloroform and Diethyl
ether leaf extract of Adenia wightiana were subjected to DPPH assay in three different
concentrations (10 µg/ml, 50 µg/ml, 100 µg/ml). All the extracts showed dose
concentration dependent activity in all the tested concentration and the results are
given in the tables 21-23 (Fig.7).
Leaf
The anti-oxidant acitivity of leaf extract of Adenia wightiana by DPPH assay
showed maximum percentage of radical scavenging activity in the Ethyl acetate
extract (49.56±2.15) and minimum in Diethyl ether extract (26.94±2.11) in 10 µg/ml
72
concentration. Highest activity was found in Ethyl acetate extract (60.66±2.33) and
the least in Diethyl ether extract (45.85±1.89) in 50 µg/ml concentration. In 100
µg/ml concentration, highest activity was showed by Ethyl acetate extract
(81.19±4.32) and lowest activity in Ethyl acetate (66.31±3.29). All the extracts
showed dose concentration dependent activity in all the tested concentration and the
results are given in the table 21.
Stem
The anti-oxidant acitivity of leaf extract of Adenia wightiana by DPPH assay
showed maximum percentage of radical scavenging activity in the Methanol extract
(40.15±2.66) and minimum in Ethyl acetate extract (23.58±1.85) in 10 µg/ml
concentration. Highest activity was found in methanol extract (59.68±2.10) and the
least in Ethyl acetate extract (44.21±1.55) in 50 µg/ml concentration. In 100 µg/ml
concentration, highest activity was showed by methanol extract (70.17±2.54) and
lowest activity in Ethyl acetate (67.78±3.21). All the extracts showed dose
concentration dependent activity in all the tested concentration and the results are
given in the table 22.
Root
The anti-oxidant acitivity of leaf extract of Adenia wightiana by DPPH assay
showed maximum percentage of radical scavenging activity in the Methanol extract
(48.58±2.14) and minimum in Diethyl ether extract (25.36±1.25) in 10 µg/ml
concentration. Highest activity was found in methanol extract (67.32±3.85) and the
least in Diethyl ether extract (34.55±1.99) in 50 µg/ml concentration. In 100 µg/ml
concentration, highest activity was showed by methanol extract (88.52±3.58) and
lowest activity in Diethyl ether (59.63±4.25). All the extracts showed dose
73
concentration dependent activity in all the tested concentration and the results are
given in the table 23.
4.3.1.2. FRAP method
The anti-oxidant activity of Methanol, Ethyl acetate, Chloroform and Diethyl
ether leaf extract of Adenia wightiana were subjected to FRAP method in three
different concentrations (10 µg/ml, 50 µg/ml, 100 µg/ml). All the extracts showed
dose concentration dependent activity in all the tested concentration and the results
are given in the tables 24-26 (Fig. 8).
Leaf
The anti-oxidant acitivity of leaf extract of Adenia wightiana by FRAP
method showed maximum percentage of total antioxidant capacity in the Methanol
extract (17.64±0.17) and minimum in Diethyl ether extract (09.44±0.01) in 10 µg/ml
concentration. Highest activity was found in Methanol extract (33.2±0.81) and the
least in Diethyl ether extract (21.97±0.13) in 50 µg/ml concentration. In 100 µg/ml
concentration, highest activity was showed by Methanol extract (72.5±1.65) and
lowest activity in Chloroform extract (66.31±3.29). All the extracts showed dose
concentration dependent activity in all the tested concentration and the results are
given in the table 24.
Stem
The anti-oxidant acitivity of leaf extract of Adenia wightiana by FRAP
method showed maximum percentage of total antioxidant capacity in the Methanol
extract (18.46±1.38) and minimum in Ethyl acetate extract (13.58±1.09) in 10 µg/ml
concentration. Highest activity was found in methanol extract (42.8±1.54) and the
least in Ethyl acetate extract (22.24±1.84) in 50 µg/ml concentration. In 100 µg/ml
74
concentration, highest activity was showed by methanol extract (88.41±2.94) and
lowest activity in Ethyl acetate extract (43.17±1.27). All the extracts showed dose
concentration dependent activity in all the tested concentration and the results are
given in the table 25.
Root
The anti-oxidant acitivity of leaf extract of Adenia wightiana by FRAP
method showed maximum percentage of total antioxidant capacity in the Methanol
extract (24.06±0.19) and minimum in Diethyl ether extract (12.56±1.21) in 10 µg/ml
concentration. Highest activity was found in methanol extract (32.58±3.14) and the
least in Diethyl ether extract (22.67±4.12) in 50 µg/ml concentration. In 100 µg/ml
concentration, highest activity was showed by methanol extract (66.73±2.45) and
lowest activity in Diethyl ether extract (43.11±2.17). All the extracts showed dose
concentration dependent activity in all the tested concentration and the results are
given in the table 26.
4.3.1.3. Phosphomolybdenum assay
The anti-oxidant activity of Methanol, Ethyl acetate, Chloroform and Diethyl
ether leaf extract of Adenia wightiana were subjected to Phospho molybdenum assay
in three different concentrations (10 µg/ml, 50 µg/ml, 100 µg/ml). All the extracts
showed dose concentration dependent activity in all the tested concentration and the
results are given in the tables 27-29 (Fig. 9).
Leaf
The anti-oxidant acitivity of leaf extract of Adenia wightiana by
Phosphomolybdenum assay showed maximum percentage of total antioxidant
capacity in the Ethyl acetate extract (87.26±2.16) and minimum in chloroform extract
75
(5.33±1.09) in 10 µg/ml concentration. Highest activity was found in Ethyl acetate
extract (42.63±1.59) and the least in Chloroform extract (21.97±0.13) in 50 µg/ml
concentration. In 100 µg/ml concentration, highest activity was showed by Methanol
extract (87.26±2.16) and lowest activity in Chloroform extract (43.56±1.58). All the
extracts showed dose concentration dependent activity in all the tested concentration
and the results are given in the table 27.
Stem
The anti-oxidant acitivity of leaf extract of Adenia wightiana by
Phosphomolybdenum assay showed maximum percentage of total antioxidant
capacity in the Ethyl acetate extract (9.87±1.31) and minimum in Chloroform extract
(4.78±1.58) in 10 µg/ml concentration. Highest activity was found in methanol extract
(40.09±1.92) and the least in Diethyl ether extract (22.20±1.76) in 50 µg/ml
concentration. In 100 µg/ml concentration, highest activity was showed by
Chloroform extract (81.10±2.99) and lowest activity in Diethyl ether extract
(44.72±2.57). All the extracts showed dose concentration dependent activity in all the
tested concentration and the results are given in the table 28.
Root
The anti-oxidant acitivity of leaf extract of Adenia wightiana by
Phosphomolybdenum assay showed maximum percentage of total antioxidant
capacity in the Chloroform extract (10.21±1.84) and minimum in Diethyl ether extract
(3.49±1.20) in 10 µg/ml concentration. Highest activity was found in Ethyl acetate
extract (41.25±1.28) and the least in Diethyl ether extract (15.28±2.95) in 50 µg/ml
concentration. In 100 µg/ml concentration, highest activity was showed by methanol
extract (83.57±2.16) and lowest activity in Diethyl ether extract (32.08±1.20). All the
76
extracts showed dose concentration dependent activity in all the tested concentration
and the results are given in the table 29.
4.3.1.4. Antimicrobial activity of Adenia wightiana
The zone of inhibition obtained in the antimicrobial acitivity of methanolic
leaf, stem and root extract of Adenia wightiana against two Gram positive bacteria
(Bacillus subtilis MTCC 441 andStaphylococcus aureus MTCC 6908), two Gram
negative bacteria (Escherichia coli MTCC 406 andKlebsiella pneumonia MTCC 530),
two fungus (Aspergillus niger MTCC 1344 and Candida albicans MTCC 227) in
four different concentration (0.5 mg, 1 mg, 2 mg and 3 mg) were recorded. All the
extracts showed dose concentration dependent activity in all the tested concentration
(Table 30-32, Plate 5-7).
Leaf
In leaf extract, Bacillus subtilis showed maximum zone of size 13±1.28 mm at
4 mg concentration and minimum of 11±1.02 mm at 2 mg concentration in
comparison to positive control (Ciproflaxacin) 28±1.29 mm. Staphylococcus aureus
showed highest zone value of 17±1.95 mm at 4 mg and lowest value of 10±1.09 mm
at 1 mg in comparison to positive control 24±1.84 mm. In Escherichia coli, the leaf
extract showed the highest zone of 16±1.87 mm and the lowest zone of 12±2.51 mm
for the 4 mg and 2 mg respectively whereas the positive control was 22±1.33 mm. In
Klebsiella pneumoniae, the leaf extract showed inhibition for all the three
concentration 1mg, 2 mg and 4 mg and the values are 12±1.20mm, 15±1.58 mm,
18±1.02 mm in comparison to the positive control 22±1.54 mm. All the four
concentration of leaf extract revealed activity against Aspergillus nigeras 11±1.04
mm, 12±1.50 mm, 13±0.98 mm and 15±1.06 mm whereas the positive control was
77
26±2.16 mm. Zones were obtained only in one concentration of 4 mg with a value of
14±1.11 mm for which the positive control (Nystatin)value is 24±1.35 mm. All the
extracts showed dose concentration dependent activity in all the tested
concentration(Table 30, Plate 5).
Stem
In stem extract, Bacillus subtilis showed maximum zone of size 10±1.70 mm
only at 4 mg concentration in comparison to positive control (Ciproflaxacin) 25±1.81
mm. Staphylococcus aureus showed highest zone value of 11±1.51 mm only at 4 mg
in comparison to positive control 29±1.98 mm. In Escherichia coli, the leaf extract
showed the highest zone of size 17±1.62 mm and the lowest zone of size 10±0.15 mm
for the 4 mg and 0.5 mg respectively whereas the positive control was 24±2.00 mm.
In Klebsiella pneumoniae, the leaf extract showed inhibition for only at 4 mg and the
value was 12±1.80 mm in comparison to the positive control 33±3.10 mm. Two
concentration of leaf extract revealed activity against Aspergillus nigeras 9±0.89 mm,
14±0.65 mm, whereas the positive control was 34±2.83 mm. Two zones were
obtained for two concentrations at 2 mg and 4 mg with a value of 11±1.21 mm and
13±1.33 mm respectively for which the positive control (Nystatin)value is 25±2.06
mm.All the extracts showed dose concentration dependent activity in all the tested
concentration (Table 31, Plate 6).
Root
In root extract, Bacillus subtilis showed maximum zone of size 16±1.07 mm at
4 mg concentration and minimum of 13±1.10 mm at 2 mg concentration in
comparison to positive control (Ciproflaxacin) 27±1.49 mm. Staphylococcus aureus
showed only one zone of 14±1.09 mm at 4 mg in comparison to positive control
78
24±1.02 mm. In Escherichia coli, the leaf extract showed the highest zone of 17±1.01
mm and the lowest zone of 13±0.86 mm for the 4 mg and 2 mg respectively whereas
the positive control was 25±1.08 mm. In Klebsiella pneumoniae, the leaf extract
showed inhibition only in two concentration 2 mg and 4 mg and the values are
11±0.19 mm, 18±1.05 mm, respectively in comparison to the positive control 27±1.69
mm. Only one concentration of leaf extract revealed activity against Aspergillus
nigeras 13±1.21 mm whereas the positive control for the same was 24±1.80 mm.
Zones were obtained in two concentration of 2 mg and 4 mg with a value of 9±0.88
mm and 14±1.08 mm respectively for which the positive control(Nystatin) value is
27±1.11 mm.All the extracts showed dose concentration dependent activity in all the
tested concentration(Table 32, Plate 7).
Minimum Inhibitory Concentration
A graph plotted between the values of zone of inhibition and the concentration
of the extract used for the antimicrobial study has revealed the minimum inhibitory
concentration of the methanolic extract of leaf, stem and root of Adenia wightiana to
act against two Gram positive bacteria (Bacillus subtilis MTCC 441 and
Staphylococcus aureus MTCC 6908), two Gram negative bacteria (Escherichia coli
MTCC 406 and Klebsiella pneumonia MTCC 530), two fungus (Aspergillus niger
MTCC 1344 and Candida albicans MTCC 227) were recorded. The highest value of
minimum inhibitory concentration of methanolic leaf extract was 3.84±0.18 mg for K.
pneumonia followed by S. aureus (3.55±0.87 mg) and the lowest for C. albicans
(1.36±0.55 mg). The highest value of minimum inhibitory concentration of
methanolic stem extract was 3.70±0.73 mg for E. coli followed by K. pneumonia
(2.10±0.27 mg) and the lowest for B. subtilis(1.22±0.11 mg). The highest value of
minimum inhibitory concentration of methanolic root extract was 2.67±0.47 mg for E.
79
coli followed by K. pneumoniae(2.46±0.15 mg) and the lowest for A. niger(1.31±0.26
mg) (Table 33).
4.3.1.5. In-vitro anti-inflammatory study
In-vitro anti-inflammatory study of methanolic leaf, stem and root extract of
Adenia wightiana by HRBC method in five different concentrations (200 µg/ml, 400
µg/ml, 600 µg/ml, 800 µg/ml, 1000 µg/ml) were recorded. In leaf, stem and root
highest percentage of membrane stabilization was found in maximum concentration
(1000 µg/ml) and lowest percentage of protection in the minimum concentration (200
µg/ml). Lowest percentage of protection was found in 200 µg/ml for leaf (45.2±1.78),
stem (53.3±1.43) and root (55.7±1.27). Highest percentage of protection was found
in 1000 µg/ml for leaf (79.4±2.47), stem (81.4±2.51) and root (85.7±1.62). All the
extracts showed dose concentrations dependent activity in all the tested concentration
(Table 34).
4.3.1.6. In-vitro cytotoxicity study
In-vitro cytotoxicity study for the methanolic leaf, stem and root extract of
Adenia wightiana against breast cancer cell lines (MCF-7) in five different
concentrations (10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml and 250 µg/ml) are given in
the table 35. The percentage of cell viability at 10 µg/ml for leaf is 95.4±1.10, stem is
98.0±2.40 and root is 92.4±1.30 was maximum and at 250 µg/ml for leaf is
43.6±0.85, stem is 69.3±1.02 and root is 62.2±3.13 was minimum. All the extracts
showed dose concentration dependent activity in all the tested concentration
80
4.3.2. Invivo Pharmacological Study
4.3.2.1. Acute toxicity study
There was no mortality and sign of toxicity observed at 2gm/kg given to the
group of animals. From the toxicity studies it was found that the methanolic leaf, stem
and root extract of Adenia wightiana proved to be non toxic at tested dose level and
well tolerated by the experimental animals as their LD50 cut off values greater than
2000mg/kg. Hence 1/10th (200mg/kg) of this dose was selected for further study. The
gross behavioral studies of the formulations were made and the results were shown in
table 39. It was noted that the methanolic leaf, stem and root of Adenia wightiana
does not affect the spontaneous activity when compared to control. The normal
behavior shown by the organism for the treated dose of 200 mg/kg of the body weight
shows that the drug is non-steroidal, does not cross the blood brain barrier and it acts
peripherally (Table 36).
4.3.2.2. In-vivo anti-inflammatory study
Carrageenan induced paw edema test was conducted to study the in-vivo anti-
inflammatory activity of the methanolic leaf, stem and root extract (200 mg/kg/i.p) of
Adenia wightiana in comparison with the standard drug indomethacin (200 mg/kg/i.p)
for a duration of 3 hours and the results were given in the table 37.
Group 1 showed no change in the size of the paw volume and it remained the
same (0.45±0.03 mm) till the end of the study. Group 2 treated with 0.1 ml of 1%
Carrageenan showed gradual increase in size of the paw volume. It showed 0.40±0.04
mm at 0 hour, 0.73±0.01 mm at 1 hour after the treatment, 0.86±0.02 mm at 2 hour
after the treatment, 0.87±0.01 mm at 3 hour after the treatment. Group 3 treated with
0.1 ml of 1% Carrageenan and Indomethacin (20 mg/kg/i.p) showed 0.44±0.06 mm at
81
0 hour, 0.61±0.07 mm at 1 hour after the treatment, 0.72±0.01 mm at 2 hour after the
treatment, 0.47±0.08 mm at 3 hour after the treatment. Group 4 treated with 0.1 ml of
1% Carrageenan and Methanolic extract of leaf (200 mg/kg/i.p) showed 0.43±0.01
mm at 0 hour, 0.70±0.09 mm at 1 hour after treatment, 0.87±0.08 mm at 2 hour after
treatment, 0.56±0.03 mm at 3 hour after treatment. Group 5 treated with 0.1 ml of 1%
Carrageenan and Methanolic extract of stem (200 mg/kg/i.p) showed 0.48±0.03 mm
at 0 hour, 0.63±0.02 mm at 1 hour after treatment, 0.81±0.06 mm at 2 hour after
treatment, 0.59±0.05 mm at 3 hour after treatment. Group 6 treated with 0.1 ml of 1%
Carrageenan and Methanolic extract of root (200 mg/kg/i.p) showed that 0.42±0.01
mm at 0 hour, 0.67±0.05 mm at 1 hour after treatment, 0.83±0.02 mm at 2 hour after
treatment, 0.54±0.03 mm at 3 hour after treatment.
There was no increase in paw volume in Group 1. The increase in paw volume
at the end of the study in the study Group were 0.47±0.02 mm in Group 2 treated
with 0.1 ml of 1% Carrageenan, 0.03±0.01 mm in Group 3 treated with 0.1 ml of 1%
Carrageenan and Indomethacin (20 mg/kg/i.p), 0.13±0.01 mm in Group 4 treated
with 0.1 ml of 1% Carrageenan and Methanolic extract of leaf (200 mg/kg/i.p),
0.11±0.01 in Group 5 treated with 0.1 ml of 1% Carrageenan and Methanolic extract
of stem (200 mg/kg/i.p) and 0.12±0.01 in Group 6 treated with 0.1 ml of 1%
Carrageenan and Methanolic extract of root (200 mg/kg/i.p). The percentage change
in activity for the in-vivo anti-inflammatory study for the leaf extract was 42.22%,
stem extract was 39.99% and root extract was 36.66% in comparison to the standard
24.44%.
82
4.3.2.3. In-vivo anti-ulcerous study
In-vivo anti-ulcerous study of methanolic leaf, stem and root extract of Adenia
wightiana reveals the result of various parameters like enteric pH, ulcer index,
percentage protection of the drug in comparison to standard. The enteric volume,
enteric pH, total acidity, free acidity and ulcer index value of Group 1 were 5.53±0.25
ml, 2.50±0.10, 68.00±2.64, 55.66±1.52 and 10 respectively. The values of Group 2
treated with Omeprazole (20 mg/kg/p.o) were 3.00±0.10 ml, 3.66±0.20, 57.33±1.52,
42.33±1.32 and 5.8 and for Group 3 treated with Methanolic extract of leaf (200
mg/kg/p.o) were 3.39±0.47 ml, 3.50±0.51, 61.87±3.17, 45.02±3.68 and 6.5
respectively. The results for Group 4 treated with Methanolic extract of stem (200
mg/kg/p.o) were 3.45±0.34 ml, 3.40±0.33, 63.66±2.61, 44.50±2.54 and 6.3 and for
Group 5 treated with Methanolic extract of root (200 mg/kg/p.o) were 3.50±0.20 ml,
3.50±0.26, 65.33±2.08, 44.66±1.50 and 6.2. The percentage of protection showed by
the leaf extract is 35, stem is 37 and the root is 38 in comparison to standard drug
omeprazole (42%) (Table 38).
Regeneration of the plant
Adenia wightiana established itself in the new habitat by vegetative
propagation through stem cuttings (Plate 8).
83
CHAPTER 5
DISCUSSION
Plants are the self-sufficient industries making product using the raw materials
from their surrounding trapping the energy from the sun. The products produced by
them are food and drugs being consumed by all the heterotrophs for their existence. A
single plant possesses many chemical compounds which has various biological
activities. There are thousands of plant species possessing myriads of chemical
compounds for various ailments of the humankind yet to be unfolded. The main duty
to acclaim the plant potentiality with the compounds possessed by them for their
biological activity is to establish the pharmacognosy, phytochemistry and
pharmacology of the drug. In this ray of discovery, the present work has been carried
out to bring out the medicinal potential of Adenia wightiana.
5.1. Pharmacognosy
Pharmacognosy is a simple and reliable tool, by which complete information
of the crude drug can be obtained (Gokhale, 1979, Trease and Evans, 2002).
Standardization is an essential measure of quality, purity and authenticity. The
standardization of crude drug is an integral part for establishing its correct identity.
Before any crude can be include in an herbal pharmacopoeia, pharmacognostic
parameters and standards must be established. In order to standardize a drug, various
botanical, pharmacognostical and phytochemical parameters like macroscopical,
microscopical, powder characteristics, organoleptic characters, ash values, extractive
values, vitamin studies, fatty acids, heavy metal analysis and preliminary qualitative
and quantitative phytochemical analysis of different solvent extracts must be analysed
(Bhattacharya and Zaman, 2009). As there is no pharmacognostic, phytochemical and
pharmacological work recorded so far on Adenia wightiana, the present work was
84
undertaken to lay down the standards which could be useful for establishing its
authenticity.
Anatomy is of primary importance for all aspects of research in plant sciences
such as morphogenesis, physiology, ecology, taxonomy, evolution and genetics
(Tamilselvi et al., 2011). Though the anatomy of 58 species of Adenia was described
(Hearn, 2009), the complete anatomy of vegetative parts of the species Adenia
wightiana not reported so far. The present study brings out the microscopic
observation of anatomy of leaf, petiole, stem, root, root tuber and crude powder for
proper identification of the plant which will be useful in systematic botany and
pharmacognosy. The salient features which will be useful for its specific identification
viz. dorsiventral, hypostomatic leaf with paracytic stomata measuring 15x10 µm with
distinctly unequal subsidiary cells; The clear vein islets and dendroid vein
terminations; Irregularly lobed, deeply staining scelereids and tannin cells in the upper
epidermis of the lamina; Large spherical crystalliferous idioblasts with 1-3 druses in
the mesophyll of leaf; The idioblasts in the root and root tuber are similar to the
cortical cells in size and shape; Successive concentric cuticular striations in the
epidermal cells of leaf; A pair of adaxial small accessory vascular bundles in the
petiole; Vascular cylinder of the stem cleaved into about six wide fan shaped
segments separated by wide vascular rays; Root tubers exhibiting anomalous
structures with division of the vascular tissue into discrete clusters and highly
proliferated ground parenchyma; Wide, circular vessel elements with transverse or
slightly inclined end walls with simple perforations and alternate lateral wall pitting.
Fibre-tracheids with elliptical lateral wall pitting; Abundant axial parenchyma;
Diffuse porous wood frequent with solitary vessels, sometimes in pairs. Large number
85
of cylindrical and longitudinally four lobed starch grains with dark cross shaped
polarimarks in the root tubers.
According to Selvam (2013) the type and distribution of starch grains are
characteristic and species specific. Many workers such as Edeoga et al., (1996),
Mbagwu et al., (2006)stressed that epidermal and cuticular traits of plants epidermal
cells, type and arrangement of stomata, size and shape of trichomes and number of
vascular bundles could serve as vital tools in solving taxonomic problems in
Angiosperms. The location of the crystals within a taxon is also often very specific
and may be represented as a taxonomic character (Lersten and Horner, 2000). The
quantitative analysis of epidermis revealed unique characteristic features for
identification of plants such as the number of epidermal cells per square millimeter,
number of stomata per square millimeter, stomatal index and palisade ratio. The
studies of Chukwumaet al., 2014 on Taxonomic value of the leaf micro-morphology
and quantitative phytochemistry of Clitoria ternatea and Centrosema pubescens
(Papilionoideae, Fabaceae) suggested the importance of the microscopic characters
for identification of the plant and the crude drug.
Histochemistry or cytochemistry deals with localization of chemical
compounds within thecells by means of specific colors of the compounds. Staining
the cells with different stains ordyes, which render the compounds visible under the
microscope, makes the specific colorreaction compounds. The importance of
histochemistry in solving critical biosystematics problems is as popular as the use of
other markers. According to botanical literatures, the use of histochemical characters
in taxonomic conclusions is now a common practice (Baker, 1966, Bowes, 1996). In
the present study the histochemical localization tests revealed the presence of starch,
alkaloid, protein and tannin in leaf, stem and root of Adenia wightiana. Lignin was
86
found to be present only in stem and root. The leaf, stem and root were devoid of
mucilaginous substance. The studies of Parthaet al., (2015) in Adenanthera pavonina
and that of Linga Rao et al.,Svensonia hyderobadensis revealed similar results.The
histochemical analysis is highly essential that will help the pharmacognosists to locate
chemical substances and their properties in terms of cells, tissues and parts of plants
(Johansen, 1940).
Fluorescence analysis is one of the Pharmacognostic procedures useful in the
identification of authentic samples and recognizing adulterants (Tyler et al., 1976).
The merits of simplicity and rapidity of the process makes it a valuable analytical tool
in the identification of plant samples and crude drugs (Denston, 1946). In
fluorescence analysisof Adenia wightiana, the leaf powder developed green and light
green in visible light and UV light respectively. The stem powder showed light brown
in both visible and UV light. The root powder developed light brown and dark brown
in visible and UV light. Similar results were reported by Partha et al., (2015) in
Adenanthera pavonina.
Physico-chemical evaluation of crude drug involves the determination of the
identity, purity and quality. Purity depends upon the absence of foreign matter,
whether organic or inorganic. While quality refers essentially to the concentration of
the active constituents in the drug that makes it valuable to medicine (Chase and Pratt,
1949). The pharmacognostical studies of the plant drugs focused on bringing out the
diagnostic characters will be immense help in the proper identification and
standardization of different botanical species of the plant origin. The
pharmacognostical parameters are major and reliable criteria for confirmation of the
identity and determination of quality and purity of the crude drugs that plays a major
role to establish (Rajan et al., 2013).
87
Of the physico-chemicalparameters studied the moisture content of the leaf
was high (20.0±0.81). The earlier reportsuggests that moisture content upto 12%,
which is not very high, would discourage the growth of bacteria, fungi or yeast
(Bhattacharya and Zaman et al., 2009). The total ash is particularly important in the
evaluation of purity of drugs, i.e. the presence or absence of foreign inorganic matter
such as metallic salts and/or silica (Musa et al., 2006). The total ash (16.0±0.63), acid
insoluble ash (09.0±0.31), water soluble ash (09.4±0.54) and sulfated ash (10.0±0.46)
were high in leaf when compared to stem and root of Adenia wightiana. Physico-
chemical properties such as moisture, ash content, acid soluble, acid insoluble, acid
value of ethanol extracts of Citrullus vulgaris (seeds), Cucumis melo (seeds), Moringa
oleifera (Fruit), Raphanus sativus (seeds) and Zea mays (Corn silk) were studied
(Mirza, et al., 2003).
The extractive values of methanol extract were high when compared to other
solvents namely Ethyl acetate, Chloroform and Diethyl ether. High alcohol soluble
and water soluble extractive values revealed the presence of polar substance like
phenols, tannins and glycosides. Okunye, et al., (2015) reported the extraction yield
from Adenia cissampeloides leaves as the highest compared to the yield obtained from
the stem, in which n-hexane leaf extract was 6.76%, and ethanolic leaf extract was
5.99%, while the aqueous leaf extract was 3.9%. The extraction yield obtained from
the stem were recorded as follows, n-hexane stem extract was 3.26% and ethanolic
stem extract which gave 3.64% while aqueous stem extract was 3.36%.
88
5.2. Phytochemistry
5.2.1. Phytochemical analysis
The concentration of phytochemicals is different in different parts of the same
plant and in different plants. The therapeutic efficacy of plants is because of these
compounds which include alkaloids, flavonoids, saponins, terpenoids, steroids,
phlobatannins, glycosides, tannins, etc. All these secondary metabolites are known for
curing one or other diseases. Alkaloids are known for antispasmodic, antimalarial,
analgesic, diuretic activity. Terpenoids are reported to have antiviral, anthelmintic,
antibacterial, anticancer, antimalarial, anti-inflammatory properties. They are also
known for inhibition of cholesterol synthesis and possess insecticidal properties hence
useful for storing agricultural products. Saponins are known for anti-inflammatory,
antiviral, plant defence and for cholesterol reducing property. Phlobatanins possess
astringent properties. Glycosides are reported for antifungal and antibacterial
properties. Phenols and flavonoids are known for their antioxidant, anti-allergic,
antibacterial, etc. (Padalia and Chanda 2015, Moteriya et al., 2015).
The preliminary phytochemical screening of leaf, stem and root of Adenia
wightiana in the Methanol, Ethyl acetae, Chloroform and Diethyl ether revealed the
presence of alkaloid, anthraquinone, aminoacid, cardiac glycoside, catechin,
coumarin, flavonoid, gums, oils & resins, glycoside, non-reducing sugar, protein,
phlobotannin, phenol, quinone, reducing sugar, saponin, steroid, tannin, terpenoid and
triterpenoid. In Adenia wightiana the preliminary phytochemical study of Ethyl
acetate extract of leaf reveals the presence of more phyto-constituents than other
solvents. Methanolic extract of stem extract shows positive results for the presence of
more phyto-chemical substance. In root both methanol and chloroform extract has
shown more positivity when compared to others. The studies of Prajapati (2003) in
89
Adhatoda vasica, Ilavarasan et al., 2006 in Ricinus communis, Vasudevan et al., 2007
in Thespesia populnea, Dandeet al, 2010 in Sesbania sesban, Santoshverma et. al.,
2010 in Aconitum heterophyllum, Okunye et al., 2015 in Adenia cissampeloides,
Partha et al., 2015 in Adenanthera pavonina revealed the presence of similar
compounds. The phytochemical analysis of Adenia wightiana reveals that it contains
vital chemicals to cure various diseases.
5.2.2. GC-MS analysis
The GC-MS analysis of methanolic extract of leaf, stem and root of Adenia
wightiana revealed the presence of 89 chemical compounds. Of them 25 from leaf, 32
from stem and 32 from root were reported to be present. (Tables 12-14).These 89
chemical compounds belong to 18 different chemical compounds groups (Aldehyde,
Alkaloid, Aromatic compound, Carbohydrate, Carboxylic acid, Nitrogenous base,
Ester, Fatty acid, Flavonoid, Furan, Glycoside, Hydrocarbon, Ketone, Phenol, Steroid,
Terpenes, Triterpene, Vitamin. These chemical compounds have been reported to
possess 208 biological activities by similar research studies all over the world in other
plants.
5.2.2.1. Comparative analysis of compounds present in leaf, stem and root
A total of 89 chemical compounds were identified altogether from the leaf,
stem and root of Adenia wightiana through GC-MS analysis. Out of 89 compounds 25
compounds were identified from leaf and 32 compounds from stem and root each.
Twenty five chemical compounds out of 89 were present commonly in leaf, stem and
root or stem and root. Six chemical compounds were commonly present in leaf, stem
and root and 13 chemical compounds were common in both stem and root but were
absent in leaf. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl; Hexadecanoic
90
acid, methyl ester; Trans-Geranylgeraniol; Campesterol; Stigmasterol; β-Sitosterol are
the chemical compounds found to be commonly present in leaf, stem and root.
Thymine; S)-(+)-2',3'-Dideoxyribonolactone;Phenol,2,6-dimethoxy;Phenol,3,4,5-
trimethoxy; n-Hexadecanoicacid; 9,12-Octadecadienoicacid,methylester;9-
Octadecenoicacid(Z)-,methylester;Cholestan-3-ol,2-methylene-,(3β,5α);1,2-
Benzenedicarboxylicacid,mono(2-ethylhexyl)ester; 17.alfa.,21β-28,30-Bisnorhopane;
Vitamin E;β-Amyrin; 4,22-Stigmastadiene-3-one were present both in stem and root
(Table 15).
5.2.2.2. Chemical compound groups present in GC-MS analysis
A total of 89 chemical compounds belonging to 18 different chemical groups
were identified from the GC-MS analysis of the entire plant. A maximum of 17
chemical compounds belongs to ester group followed by 14 steroid groups and 8
terpene groups. At least one each chemical compound belongs to alkaloid, aromatic
compound, glycoside and hydrocarbon. Out of 18 different chemical groups 25
chemical compounds of leaf comes under 13 groups, 32 chemical compounds of stem
comes under 12 groups and 32 chemical compounds of root comes under 14 groups
(Tables 19).
Leaf
A total of 25 chemical compounds were found to be present in the methanolic
extract of leaf. Twenty five chemical compound present in the leaf belonged to 13
different chemical compound groups. Out of 25 chemical compounds present, 7
chemical compoundone each belongs to alkaloid, aromatic compound, carbohydrate,
flavonoid, furan, hydrocarbon and triterpenoid groups. Two belongs to carbohydrate
91
group, 2 belongs to ketone group, 2 belongs to terpenoid group, 4 belongs to Ester of
fatty acid group, 4 belongs to steroid group and other 4 belongs to vitamin group.
Stem
A total of 32 chemical compounds were found to be present in the methanolic
extract of stem. Out of 32 chemical compounds present, 6 chemical compound of one
each belongs to aldehyde, carboxylic acid, Nitrogenous base, flavonoid, furan and
vitamin group. Two belongs to carbohydrate group. Four chemical compounds
belong to terpene group. Five chemical compounds belong to Ester group, 5 belong to
fatty acid group, 5 belong to phenol group, and the other 5 belongs to steroid group.
Root
A total of 32 chemical compounds were found to be present in the methanolic
extract of root. Out of 32 chemical compounds present, 6 chemical compound of one
each belongs to Nitrogenous base, furan, glycoside, ketone, triterpene and vitamin
group. Two belongs to aldehyde group, 2 belong to fatty acid group, 2 belong to
flavonoid group, 2 belong to phenol and 2 belong to terpene group. Three chemical
compounds belong to carbohydrate group. Five chemical compounds belong to
steroid group. Eight chemical compounds belong to ester group.
5.2.2.3. Chemical compounds and their biological activities in GC-MS analysis
The 89 chemical compounds present in the methanolic extract of leaf, stem
and root of Adenia wightiana have been reported to possess 208 biological activities
by various similar GC-MS research analysis done in different plants all over the
world. Out of 208 biological activities shown chemical compounds present in the leaf
shows 61, stem shows 78 and root 69. A maximum of 29 chemical compounds show
92
anti-oxidant activity followed by 23 to anti-inflammatory, 20 to antimicrobial and 20
to hypocholesterolemia (Tables 20).
Leaf
Out of 208 biological activities shown by 89 chemical compounds present in
the entire plant, 25 chemical compounds of leaf have been reported to possess 61
biological activities. A maximum of 11 chemical compounds shown to possess anti-
oxidant activity followed by 6 chemical compounds to anti-inflammatory activity and
6 to anti-tumour activity. At least one chemical compound were found to possess anti-
arthritic, anti-bronchitis, anti-dermatitic, anti-diabetic, anti-HIV reverse transcriptase,
anti-leukemic, anti-malarial, anti-parasitic, anti-parkinsonism, anti-proliferative, anti-
spasmodic, anti-ulcerogenic, anti-viral, cardioprotective, cytotoxic, hepatoprotective
activity.
Stem
Out of 208 biological activities shown by 89 chemical compounds present in
the entire plant, 32 chemical compounds of stem have been reported to possess 78
biological activities. A maximum of 10 chemical compounds were shown to possess
anti-oxidant activity followed by 9 chemical compounds to anti-inflammatory
activity, 9 to anti-microbial activity and 9 to hypocholesterolemic. At least one
chemical compound was found to possess anti-acne, anti-diabetic, anti-fungal, anti-
helminthic, anti-leukemic, anti-malarial, antinoceptive, anti-proliferative, anti-septic,
anti-spasmodic.
93
Root
Out of 208 biological activities shown by 89 chemical compounds present in
the entire plant, 32 chemical compounds of root have been reported to possess 69
biological activities. A maximum of 8 chemical compounds shown to possess anti-
cancer, 8 to anti-inflammatory and 8 to anit-oxidant activity followed by 6 to possess
hypocholesterolemic. At least one chemical compound were found to possess anti-
ageing, anti-carcinogenic, anti-dote, anti-hypersensitive, anti-leukemic, anti-lipimic,
antinoceptive, anti-proliferative, anti-septic, anti-viral and vasodilating activity.
Alkaloids have been reported to have analgesic, anti-hypertensive and anti-
microbial activities (Mallikharjuna et al., 2007, Zongo et al., 2009). Cardiac and
steroid glycosides are used in the treatment of congestive heart failure and arrhythmia
(Dewick, 2002). Saponins have anti-inflammatory, anti-melanogenic and anti-
spasmodic activities that could protect against skin cancer and inhibit human colon
cancer (Corea et al., 2005, Kawabata et al., 2011). Flavonoids have strong antioxidant
effect and show anti-allergic, anti-inflammatory, anti – microbial and anti – cancer
activities (Kim et al., 2004, Cushnieet al., 2005, Aregullin et al., 2006). Tannins have
also been reported to exhibit anti-viral, anti-bacterial and anti-tumor activities
(Akiyama et al., 2001, Luet al., 2004).GC-MS analysis of the whole plant of
Sarcostemma secamone whole plant revealed the presence of 9, 12, Octadecadienoic
acid (Z, Z)-, phenyl methyl ester and 9-Octadecanoic acid (Z)-, phenyl methyl ester.
These compounds may have the role in anti-inflammatory effects
(Thangakrishnakumari et al., 2012).
In the present study similar compounds were identified in Adenia wightiana.
The phytosterols namely Campesterol, Stigmasterol, Lanosterol, β-Sitosterol and
94
Cholestan-3-ol, 2-methylene-, (3β, 5α)- have been reported for the treatment of
diabetic mellitus by lowering fasting blood glucose levels by cortisol inhibition
(Devaraj and Jialal, 2006). The methanolic extract of Adenia wightiana contains
Campesterol, Stigmasterol, β-Sitosterol and Cholestan-3-ol, 2-methylene-, (3β, 5α).
Palmitic acid, linoleic acid, linolenic acid are essential fatty acids found in animals
and plants which are primarily used to produce hormone like substances that regulate
a wide range of functions, including blood pressure, blood clotting, blood lipid levels,
the immune response, and the inflammation response to injury orinfection (Altieri et
al., 2009). Adenia wightiana contains 23 chemical compounds for anti-inflammation,
20 for hypocholesteremia and one for anti-hypertension (Friedelan-3-one). McAnuff
et al., (2005) reported that β-sitosterol positively influences a diabetic state by directly
lowering fasting blood glucose levels by cortisol inhibition. The present investigation
reveals the presence of such a chemical which can be harnessed for diabetes.
5.3. Pharmacology
5.3.1. Antioxidant activity
The phytochemicals have been found to act as anti-oxidants by scavenging
free-radicals and many have therapeutic potential for free radical association disorders
such as coronary heart disease, inflammation, stroke, diabetes milletus and cancer
(Lee et al., 2000, Scalbert et al., 2005). Therefore it is important to assess the anti-
oxidant activity of the medicinal plants.
The anti-oxidant activity of Methanol, Ethyl acetate, Chloroform, Diethyl
ether extracts of leaf, stem and root of Adenia wightianawas assayed by DPPH, FRAP
and Phosphomolybdenum methods. Antioxidant activity was highest in methanol
extract of leaf, stem and root in comparison to all other solvent extracts for the
percentage radical scavenging activity by DPPH and FRAP assay. The highest
95
percentage of total antioxidant capacity by phosphomolybdenum assay was recorded
for the Ethyl acetate extract of leaf and root and in stem it was recorded in methanolic
extract. Predominance of anti-oxidant activity in leaf, stem and root of Adenia
wightiana may be due to abundant presence of phenol, flavonoid, tannin and steroid.
Antioxidant activity increased proportionally to the polyphenol content.
According to recent reports, a highly positive relationship between total
phenols and antioxidant activity appears to be the trend in many plant species
(Oktayet al., 2003). The effect of antioxidants on DPPH is thought to be due to their
hydrogen donating ability. Although the DPPH radical scavenging abilities of the
extracts were significantly lower than those of ascorbic acid and BHT in the methanol
extracts of the stems of Acokanthera oppositifolia and Adenia gummifera, it was
evident that the extracts did show the proton-donating ability and could serve as free
radical inhibitors or scavengers, acting possibly as primary antioxidants (Adedapoet
al., 2008). The antioxidant activity of these solvent fractions that were obtained from
the two varieties of Adenia lobata is due mainly to the free radical – scavenging
activity of their phenolic compounds such as flavonoids and tannins (Rahmanet al.,
2007, Hasan, et al., 2009). Oxidative damage by free radicals has been reported to be
the fundamental mechanism underlying various diseases (Aruoma, 2003). For
instance, in carcinogenesis, reactive oxygen species (ROS) are responsible for
initiating this process by causing DNA damage (Tsaoet al., 2004). The presence of
antioxidant activity in the various solvent fractions of Adenia lobata varieties reveals
why these plants are used for the treatment of cancer in some places in eastern Nigeria
(Agoreyo et al., 2012).
96
5.3.2. Anti-microbial activity
In-vitro anti-microbial activity of methanolic extract of leaf, stem and root of
Adenia wightiana was performed against Bacillus subtilis,Staphylococcus aureus,
Escherichia coli,Klebsiella pneumonia, Aspergillusniger, Candida albicans. In-vitro
anti-microbial activity of methanolic leaf extract of Adenia wightiana was higher
against the fungus Aspergillus niger showing zone of inhibition for all the four
concentration. The methanolic extract of stem showed maximum zone of inhibition
for the Gram negative bacteria E. coli in all the four concentration. In root, none of
the studied microorganisms developed zone of inhibition both in 0.5 mg and 1 mg
concentration but all showed activity in higher concentration. The antimicrobial
activity may be due to the presence of 20 different phytochemical compounds
reported through GC-MS analysis.
In most medicinal plants, the therapeutic value is due to the presence of one
or more secondary metabolites like tannins, phenols, phenolic acids, quinones,
flavonoids, coumarins, alkaloids which are synthesized by plants in response to
microbial infection and activity produced is due to concentration in the plant part used
(Gibbon, 2003). Similar results were obtained by the antimicrobial work on Adenia
cissampeloides on stock cultures of selected bacteria species both Gram-positive and
Gram- negative such as Staphylococcus aureus, Bacillus sp, Salmonella sp, Klebsiella
aerogenes, Pseudomonas pyocyania, Enterobacter aerogenes, Proteus vulgaris,
Streptococcus sp including fungal cultures such as Aspergillus niger, Aspergillus
flavusand Candida albicans (Abohet al., 2015).
Theworks of Shanthi et al., (2006) on the antimicrobial activity of
Andrographis lineata and A. echioides with Staphylococcus aureus, Salmonella typhi,
Vibriocholorae and Shigella revealed similar results. Their studies further revealed
97
that the ethanolic extract of the plants produced better results than the
aqueousextracts. Essawi and Sroun (2000) have also reported similar results. The
studies of Sivakumar and Alagesaboopathi (2006) on the antimicrobial activity of two
different forms of Abrus precatorius against Staphylococcus aureus, Escherichia coli
and Candida albicans showed better inhibitory effects of the methanol extract of the
seeds against the microbes studied. According to them the phytochemicals terpenoids,
saponins, flavonoids and alkaloids might be responsible for the antimicrobial activity.
But the works of Neelam Jain and Bohra (2006) on the antimicrobial activity of
Delphinium ajacis against human and plant pathogenic bacteria revealed that the
aqueous extract was more effective.
5.3.3. In-vitro cytotoxicity study
A great number of in-vitro and in-vivo methods have been developed to
measure the efficiency of natural anti-cancer compounds either as pure compounds or
as plant extracts. In vitro methods like, Tryphan blue dye exclusion assay, LDH
(Lactic dehydrogenase) assay, MTT assay, XTT assay and Sulforhodamine B assay
are most commonly used for estimating anticancer properties of natural products from
medicinal plants. Among all in vitro methods MTT assay is most popular for
estimating anticancer activity.
MTT assay of methanolic extract of leaf, stem and root of Adenia wightiana
was performed to bring out the anti-cancerous activity. The present study shows the
stem to possess highest percentage of cell viability followed by root and leaf. This
activity may due to the presence of 17 different chemical compounds present in
Adenia wightiana (Tables 19-21). In vitro exposures of MCF-7 cells with various
concentrations of Syzygium cumini extract significantly suppressed MCF-7 cancer cell
growth in a dose-dependent manner (Tripathy et al., 2015).
98
5.3.4. In-vivo acute toxicity study
The methanolic extracts of leaf, stem and root of Adenia wightiana were
administered orally to the test animals for acute toxicity study. Acute oral toxicity was
performed by following OECD-423 guidelines (acute toxic class method), albino rats
(n=3) of either sex selected by random sampling were used for acute toxicity study
(OECD, 2002). In acute toxicity study there were no mortality and sign of toxicity
observed at 2gm/kg given to the group of animals. From the toxicity studies it was
found that the methanolic leaf, stem and root extract of Adenia wightiana proved to
be non-toxic at tested dose level and well tolerated by the experimental animals as
their LD50 cut off values greater than 2000 mg/kg. Non-toxicity of the drug was
confirmed with normal activities shown by the organism in the gross behavioural
study.
5.3.5. In-vivo, anti-inflammatory study
Carrageenan induced hind paw edema is the standard experimental model of
acute inflammation. Carrageenan is the phlogistic agent of choice for testing anti-
inflammatory drugs as it is not known to be antigenic and is devoid of apparent
systemic effects. Moreover, the experimental model exhibits a high degree of
reproducibility (Winter et al.,1962).
In-vivo, anti-inflammatory study by carrageenan induced paw edema test
shows that highest percentage change in activity by the leaf extract, followed by stem
and root extract. The results of the study were comparable to that of the standard drug.
The anti-inflammatory activity of the drug may be due to the presence of different
chemical compounds such as 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl;
2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,[2R-
[2R*(4R*,8R*)]]; β-Tocopherol; 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol;
99
9,12-Octadecadienoicacid(Z,Z); Stigmasterol; Phenol,3,4,5-trimethoxy; 9-
Octadecenoicacid(Z)-,methylester; Friedelan-3-one reported from Adenia wightiana.
The studies of Thangakrishnakumariet al., 2012 in Sarcostemma secamone
revealed the presence of phytol, 9, 12, Octadecadienoic acid (Z, Z)-, phenyl methyl
ester and 9-Octadecanoic acid (Z)-, phenyl methyl ester. Similar results of presence
of phytochemicals compounds were found in the GC-MS study of Adenia wightiana.
In the present study, the anti-inflammatory activity of Adenia wightiana can be
attributed to the above chemical constituents.
Regeneration of Adenia wightiana
Since the plant is wild and rare in occurrence, its regeneration by vegetative
propagation using stem cuttings was tried. They established successfully in the new
environment. Such efforts will help to maintain the wealth of the medicinal plants in
the natural habitat even after their collection for therapeutic purposes.
100
CHAPTER 6
SUMMARY AND CONCLUSION
Plants are the self-sufficient industries making product using the raw materials
from their surrounding trapping the energy from the sun. The products produced by
them are food and drugs being consumed by all the heterotrophs for their existence. A
single plant possesses many chemical compounds which have various biological
activities. There are thousands of plant species possessing myriads of chemical
compounds for various ailments of the humankind yet to be unfolded. The main duty
to acclaim the plant potentiality with the compounds possessed by them for their
biological activity is to establish the pharmacognosy, phytochemistry and
pharmacology of the drug. In this ray of discovery, the present work has been carried
out to bring out the medicinal potential of Adenia wightiana.
The outcome of the study is summarized under three heads namely
pharmacognostical studies for easy identification of plant species, phytochemical
analysis to identify the natural chemical compounds responsible for the medicinal
potentiality and pharmacology to understand the mode of action of those natural
chemical compounds with biological system by both in-vitro and in-vivo methods.
Adenia wightiana is a tendril climber belongs to passion fruit family
(Passifloraceae), which is called perumkurattai in Tamil.
The pharmacognostical parameters like leaf constants, microscopy, physico-
chemicalanalaysis, fluorescence analysis are a few of the basic protocol for the
standardization of crude drugs. Hence, in the present study the pharmacognostical
standardization has been performed.
Microscopical analysis of cleared leaf showed characteristic vein islets and
dendroid vein terminations. The examination of the macerated material showed the
101
characteristic fibres and vessel elements. The frequency of epidermal cells per unit
area on the abaxial and adaxial surfaces, the frequency of stomata, stomatal index,
occurrence of stomata on the lower epidermis, paracytic type of stomata with unequal
guard cells, palisade ratio are characteristics to Adenia wightiana.
The anatomical studies revealed the presence of irregularly lobed, deeply
staining scelereids and tannin cells in the upper epidermis of the lamina. Large
spherical crystalliferous idioblasts with 1-3 druses in the mesophyll of leaf. The
idioblasts in the root and root tuber are similar to the cortical cells in size and shape.
Successive concentric cuticular striations in the epidermal cells of leaf.A pair of
adaxial small accessory vascular bundles in the petiole. Vascular cylinder of the stem
cleaved into about six wide fan shaped segments separated by wide vascular rays.
Root tubers exhibiting anomalous structures with division of the vascular tissue into
discrete clusters and highly proliferated ground parenchyma. Wide, circular vessel
elements with transverse or slightly inclined end walls with simple perforations and
alternate lateral wall pitting.Fibre-tracheids with elliptical lateral wall
pitting.Abundant axial parenchyma. Diffuse porous wood frequent with solitary
vessels, sometimes in pairs. Large number of cylindrical and longitudinally four lobed
starch grains with dark cross shaped polarimarks in the root tubers. All the characters
are typical to this plant which would be very useful in correct botanical identity of
crude samples.
The histochemical localization tests revealed the presence of starch, alkaloid,
protein and tannin in leaf, stem and root of Adenia wightiana. Lignin was found to be
present only in stem and root. The leaf, stem and root were devoid of mucilaginous
substance.
102
In the fluorescence analysis, leaf powder developed green and light green in
visible light and light green. The stem powder showed light brown in both visible and
UV light. The root powder developed light brown and dark brown in visible and UV
light.
Of the physico-chemicalparameters studied the moisture content, total ash,
acid insoluble ash, water soluble ash and sulfated ash were high in leaf when
compared to stem and root of Adenia wightiana.The extractive values of methanol
extract were high when compared to other solvents namely Ethyl acetate, Chloroform
and Diethyl ether.
The preliminary phytochemical study of Ethyl acetate extract of leaf reveals
the presence of more phyto-constituents than other solvents. Methanol extract of stem
extract shows positive results for the presence of more phyto-chemical substance. In
root both methanol and chloroform extract has shown more positivity when compared
to others.
A total of 89 chemical compounds were present altogether in leaf, stem and
root of Adeniawightiana. Out of 89, 25 from leaf, 32 from stem and 32 from root were
observed. These 89 chemical compounds belong to 18 different groups of chemical
compounds. Eighty nine chemical compounds of Adenia wightiana have been
reported to possess 208 biological activities by similar research studies all over the
world in other plants.
Antioxidant activity was highest in methanol extract of leaf, stem and root in
comparison to all other solvent extracts for the percentage radical scavenging activity
by DPPH assay. In FRAP method, methanol extract of leaf, stem and root showed
highest percentage of total antioxidant capacity when compared to all other solvent
extracts. In phosphomolybdenum assay the highest percentage of total antioxidant
103
capacity was found in the Ethyl acetate extract of leaf and root and in stem it was in
methanolic extract. The predominance of anti-oxidant activity of Adenia wightiana
may be due to the presence of phenol, flavonoid, tannin and steroid in abundance.
In-vitro anti-microbial activity shows that methanolic leaf extract of Adenia
wightiana was very effective against the fungus Aspergillus niger. The methanolic
extract of stem showed maximum anti-microbial activity against the Gram negative
bacterium E. coli. In root, none of the studied micro-organisms developed zone of
inhibition both in lower concentration but all showed activity in higher
concentrations. The antibacterial activity may be due to the presence of 20 different
phytochemical compounds in the plant.
In-vitro anti-cancerous studies show that stem to possess highest percentage of
cell viability followed by root and leaf showing anti-cancerous activity. This may be
due to the presence of 17 different chemical compounds.
In acute toxicity study there were no mortality and sign of toxicity observed at
2gm/kg given to the groups of animals. From the toxicity studies it was found that the
methanolic leaf, stem and root extracts of Adenia wightiana proved to be non-toxic at
tested dose level and well tolerated by the experimental animals as their LD50 cut off
values were greater than 2000mg/kg. Non-toxicity of the drug was confirmed with
normal activity shown by the organism in the Gross Behavioural study.
In-vivo, anti-inflammatory study by carrageenan induced paw edema test
shows that highest percentage change in activity by the leaf extract, followed by stem
and root. The results of the study were comparable to that of the standard drug. The
anti-inflammatory activity of the drug may be due to the presence of 23 different
chemical compounds present in Adeniawightiana.
104
In-vivo, anti-ulcerous study by pylorus ligation method shows that highest
percentage of protection against ulcer by the methanolic extract of root followed by
stem and leaf. The results of the study were comparable to that of the standard drug.
The anti-ulcerous activity of the drug may be due to the presence of chemical
compound in Adenia wightiana.
Regeneration of Adenia wightiana was done in an Eco-Restored site called
Lake Estate near Puducherry by vegetative propogation.
Thus the study concludes that the plant may be used as a potential drug for
inflammation, ulcer, cancer, antioxidation and anti-microbial activity. The
pharmaconostical, phytochemical and pharmacological studies which are reported for
the first time from Adenia wightiana, may be useful for new drug discovery.
105
CHAPTER 7
REFERENCES
Abubakar N and Majinda RT. 2016. GC-MS Analysis and Preliminary Antimicrobial
Activity of Albizia adianthifolia (Schumach) and Pterocarpus angolensis (DC)
Mustapha.Medicines.3(3).
Abu-Dahab R and Afifi F. 2007. Antiproliferative activity of selected medicinal plants of
Jordan against a breast adenocarcinoma cell line (MCF7). Sci. Pharm. 75:121-136.
Acute oral toxicity-acute toxic class method, OECD guideline for testing of chemicals,
OECD/OECD 423, 17th December 2001.
Adebiyi OO, Adebiyi OA, Oyeyipo IP, Obembe O and Oladokun O. 2013. Aqueous extract
of Adenia cissampeloides modulates Pain in Mice. International Journal of
Pharmaceutical & Biological Archives.4(5):918–922.
Adedapo AA, Jimoh FO, Afolayan AJ and Masika PJ. 2008. Antioxidant activities and
phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia
and Adenia gummifera. BMC Complementary and Alternative Medicine.8:54.
Adsersen A, Brimer L, Olsen CE and Jaroszewski JW. 1993. Cyanogenesis of Passiflora
colinvauxii, a species endemic to the Galapagos Islands. Phytochemistry33:365–367.
Agoreyo BO, Okoro NC and Choudhary MI. 2012.Preliminary phytochemical analyses of
two varieties of Adenia lobata (Jacq) and the anti-oxidant activity of their various
solvent fractions. Journal of Pure and Applied Sciences. 5(1):182–186.
106
Agyarea C, Asase A, Lechtenbergc M, Niehues M, Deters A and Hensel A. 2009. An
ethnopharmacological survey and in vitro confirmation of ethnopharmacological use
of medicinal plants used for wound healing in Bosomtwi-Atwima-Kwanwoma area,
Ghana. Journal of Ethnopharmacology.125: 393–403.
Ahmad J. 1994. Pharmacognostical and ethno-botanical studies on the stem bark of Jatropha
curcus Linn. (Unani name: Bagherenda): Its comparison with J. gossypifolia on the
basis of phloem fibre distribution in the bark of both species, Int. Cong. Curr. Prog.
Med. Aromat. Pl. Res. Calcutta.p. 57.
Akerele O. 1992. WHO guidelines for the assessment of herbal medicine.Fitoterapia.62:99-
110.
Akiyama H, Fuji K, Yamasaki O, Oono T and Iwatsuki K. 2001.Antibacterial action of
several tannins against Staphylococcus aureus.Journal of Antimicrobial
Chemotherapy.48:487–491.
Aku Diame GL. 2010. Ethnobotany and Ecological Studies of Plants used for Reproductive
Health: A Case Study at Bia Biosphere Reserve in the Western Region of Ghana.
Altameme HJ, Hameed IH and Abu-Serag NA. 2015. Analysis of Bioactive phytochemical
compounds of two medicinal plants, Equisetum arvense and Alchemila valgaris seeds
using Gas Chromatography-Mass Spectrometry and Fourier-Transform Infrared
Spectroscopy. Malays. Appl. Biol. 44(4): 47–58.
Altieri C, Bevilacqua A, Cardillo D and Sinigagha M. 2009.Effectiveness of fatty acids and
their monoglycerides against gram-negative pathogens. Int J Food Sci Tech. 44:359–
366.
107
Amerjothy S. 2003. An anotomists contribution towards Botanical standardization of the drug
Akkara Kara (Spilanthes calva DC) Asteraceae, Proceedings of National Conference
on Siddha Medicine for all Ages, Courtallum.p.83.
Anandhan V and Komalavalli N. 2011. Floral Diversity of Pudukkottai Narthamalai Hillock,
TamilNadu, India: A Comparative study. Indian Journal of Natural
Sciences.11(7):413-433.
Anesini C and Perez C. 1993.Screening of plants used in argentine folk medicine for
antimicrobial activity.Journal of Ethnopharmacology.39:19–128.
Anil N and Talluri VR. 2015. Lymphatic Filariasis: Drug targets and Nematicidal Plants. J.
Pharm. Sci. & Res. 7(11):928-933.
Annamalai A, Murugesan K, Jayaraman P and Lalithakumari D. 2000. Pharmacognostic
studies on Bhumyamalaki (Phylianthus amarus Schum and Thonn.). Proceedings of
International Congress on “Ayurveda – 2000”: Tamil Nadu, India. p.166.
Annan K, Sarpong K, Asare C, Dickson R, Amponsah K and Gyan B. 2012. In vitro anti-
plasmodial activity of three herbal remedies for malaria in Ghana: Adenia
cissampeloides (Planch.) Harms.Termina liaivorensis A. Chev and Elaeis guineensis
Jacq.Pharmacognosy Res. 4:225-9.
Apu AS, Bhuyan SH, Prova SS and Muhit MA. 2012. Anti-inflammatory activity of
medicinal plants native to Bangladesh: A review Journal of Applied Pharmaceutical
Science. 2(02):07-10.
108
Aregullin M, Berry JP and Cadena L. 2006.Anti-oxidant activity and inhibition of human
cancer cells by the herbal product, ARCOMIG. Journal of Medical Sciences.6:229 –
234.
Aruoma OI. 2003. Methodological considerations for characterizing potential antioxidant
actions of bioactive components in food plants. Mutation Research.9:523–524.
Ayanoglu E, Ulubelen A, Mabry TJ, Dellamonica G and Chopin J. 1982.O-Glycosylated C-
glycosyl flavones from Passiflora platyloba. Phytochemistry.21:799–801.
Baker JR (1966). Cytological technique 5th Eds, Methven, London.pp.125.
Benzie IF and Strain JJ. 1996. The ferric reducing ability of plasma (FRAP) as a measure of
‘‘antioxidant power’’: the FRAP assay. Analytical Biochemistry.239: 70–76.
Bergner P 1995. Passionflower. Medical Herbalism 7: 13-14.
Bhattacharya S and Zaman MK. 2009.Pharmacognostical evaluation of Zanthoxylum
nitidum bark. Int J Pharm Tech Res. 1:292–298.
Bombardelli E, Bonati A, Gabetta B, Martinelli E and Mustich G. 1975.
Bowes BG (1996). A colour atlas of plant structure.Manson, London, UK.
Brand-Williams W, Cuvelier ME, Berset C. 1995 Use of free radical method to evaluate
antioxidant activity. Lebensm Wiss Technology.28:25-30.
Brudenell AJP, Griffiths H, Rossiter JT and Baker DA. 1999. The Phloem Mobility of
Glucosinolates. Journal of Experimental Botany.50(335):745-756.
109
Burke A, Smyth E and Fitz Gerald GA. 2005.Analgesic antipyretic agents; pharmacotherapy
of gout. In L.B. Brunton, J.S. Lazo & K.L. Parker (Ed.) Goodman & Gilman's the
Pharmacological Basis of Therapeutics. New York: McGraw-Hill. pp. 671-715.
Chaman S, Sharma G, Shalini and Reshi AK. 2013. Study of Antimicrobial Properties of
Catharanthus roseus by Agar Well Diffusion Method. Int. Res J Pharm. App Sci.
3(5):65-68.
Chanda S. 2014. Importance of pharmacognostic study of medicinal plants: An overview.
Journal of Pharmacognosy and Phytochemistry. 2(5):69-73.
Chase CR and Pratt RJ. 1949. Florescence of powdered vegetable drugs. Indian Journal of
Experimental Biology.33(6):428-432.
Chopra RN, Nayar SL and Chopra IC. 1956. Glossary of Indian Medicinal Plants. CSIR,
New Delhi, India, pp. 186–187.
Chukwuma EC, Soladoye MO and Abdus Salaam KRP. 2014. Taxonomic value of the leaf
micro-morphology and quantitative phytochemistry of Clitoria ternatea and
Centrosema pubescens (Papilionoideae, Fabaceae). Phytologia Balcanica. 20(1):3–8.
Corea G, Fattorusso E, Lanzotti V, Capasso R and Izzo AA. 2005. Anti-spasmodic saponins
from bulbs of red onion, Allium cepa L. var. tropea. Journal of Agricultural and Food
Chemistry.53:935-940.
Cushnie TPT and Lamb AJ. 2005. Antimicrobial activity of flavonoids. International Journal
of Antimicrobial Agents. 26:343-356.
110
Dande PR, Talekar VS and Chakraborthy GS. 2010. Evaluation of crude saponins extract
from leaves of sesbania sesban (L.) Merr.for topical anti-inflammatory activity.Int J
Res Pharm Sci. 1:296-299.
Dandekar R, Fegade B and Bhaskar VH. 2015. GC-MS analysis of phytoconstituents in
alcohol extract of Epiphyllum oxypetalum leaves. Journal of Pharmacognosy and
Phytochemistry.4(1):149-154.
De Wet H, Nzama VN and Van Vuuren SF. 2012. Medicinal plants used for the treatment of
sexually transmitted infections by lay people in northern Maputaland, KwaZulu–Natal
Province, South Africa. South African Journal of Botany.78:12–20.
Denston TC. 1946. A textbook of Pharmacognosy. Sir Isaac Pitman & Sons, Ltd, London. pp.
46-51.
Devaraj S and Jialal I. 2006. The role of dietary supplementation with plant sterols and
stanols in the prevention of cardiovascular disease. Nutr Rev. 64:348–354.
Devi Prasad AG and Shyma TB. 2013. Medicinal plants used by the tribes of Vythiri taluk,
Wayanad district (Kerala state) for the treatment of human and domestic animal
ailments. J. Med. Plants Res. 7(20): 1439-1451.
Devika R and Sajitha V. 2007.Phytochemical and Pharmacological investigations of
Phyllanthus niruri - A preliminary screening. J. Phytol. Res. 20(1): 133-135.
Dewick PM. 2002. Medicinal Natural Products: A biosynthetic approach (2nded). Wiley,
Chichester, UK. pp. 241-243.
111
Dhawan K, Dhawan S, Sharma A. 2004. Passiflora: a review update. Journal of
Ethnopharmacology.94:1–23.
Doss A. 2009. Preliminary phytochemical screening of some Indian medicinal plants.Anc Sci
Life.29:12-16.
Dubey NK, Kumar R and Tripathi P. 2004. Global promotion of herbal medicines: India’s
opportunity. Current Science.86: 37-41.
Duke, James A. 1992. Handbook of phytochemical constituents of GRAS herbs and other
economic plants. Boca Raton, FL. CRC Press.
Easwaran L and Alex Ramani V. 2014.Phytochemical Examination and GC-MS studies of
the medicinal plant - Naravelia zeylanica International Journal of Research and
Development in Pharmacy and Life Sciences.3(5):1180-1188.
Echeverri F and Suarez GE. 1985. Flavonoids from the surface of Passiflora foetida L.
(Passifloraceae). Actual Biology.14:58–60.
Edeoga HO, Ogbebor NO, Amayo AO. 1996. Pollen morphology of some Nigerian species
of Aneilema R. Br. and Ludwigia L. New Bot. 23: 223–31.
Escobar LK, Liut YL and Mabry TJ. 1983. C-glycosylflavonoids from Passiflora coactilis.
Phytochemistry.22:796–797.
Essawi T, Srour M. 2000. Screening of some Palestinian medicinal plants for antibacterial
activity.Journal of Ethnopharmacology 70, 343-349.
Evans WC. 1996. Trease and Evans Pharmacognosy. Fifth Edition. Elsevier, New Delhi.
112
Ezhilan BP, Neelamegam R. 2012. GC-MS analysis of phytocomponents in the ethanol
extract of Polygonum chinense L. Pharmacognosy Research. 4(1).
Fakim AG, Sewraj M, Gueho J and Dulloo E. 1993.Medical ethnobotany of some weeds of
Mauritius and Rodrigues.Journal of Ethnopharmacology. 39, 175–185.
Farnsworth NR, Akerele O, Bingel AS, Soejarto DD and Guo, Z., 1985. Medicinal plants in
therapy. Bulletin of the World Health Organization.63:965–981.
Felter HW and Lloyd JU. 1983. King’s American Dispensatory 1898. Reprint by Eclectic
Medical Publications, Portland.
Fernie AR, Trethewey RN, Krotzky AJ and Willmitzer L. 2004. Metabolite profiling: From
diagnostics to systems biology. Nat Rev Mol Cell Biol. 5:763–9.
Fischer FC, Fung SY and Lankhorst PP. 1982.Cyanogenesis in Passifloraceae. II. Cyanogenic
compounds from Passiflora capsularis, P. warmingii and P. perfoliata. Planta
Medica. 45:42–45.
Froehlich O, Duque C, Schreier P. 1989. Volatile constituents of Curuba (Passiflora
mollissima) fruit.Journal of Agriculture and Food Chemistry.37:421–425.
113
Gamble JS. 1935. Flora of the Presidency of Madras.Vol.I, II & III.Botanical Survey of India,
Calcutta, India.
Gangoue-Pieboji J, Eze N, Ngongang Djintchui A, Ngameni B, Tsabang N, Pegnyemb D,
Biyiti L, Ngassam P, Koulla-Shiro S and Galleni M. 2009. The in-vitro antimicrobial
activity of some medicinal plants against β-lactam-resistant bacteria. J Infect Dev
Ctries. 3(9): 671-680.
Gavasheli NM, Moniava II and Eristavi LI. 1974. Aminoacids from P. incarnata cultivated in
the Georgian SSR. Khimiya Prirodnykh Soedinenii. 10:266.
Gibbons S. 2003. An overview of plant extracts as potential therapeutics. Expert Opin.Ther.
Pat. 13(4):489-497.
Gnanavel V and Mary Saral A. 2013. GC-MS analysis of petroleum ether and ethanol leaf
extracts from Abrus precatorius Linn. Int J Pharm Bio Sci. 4(3):37-44.
Gokhale SB. 1979. Text book of Pharmacognosy. Nirali Prakashan, Pune. pp. 246-268.
Handler N. 1962. Psychomimetic Medicine, The First Hahnemann Symposium. Lea and
Febiger, Philadelphia.
Harborne JB. 1973. Pytochemical methods. Chapman and Hall, London.
Hasan SMR, Hossain MM, Akter R, Jamila M, Mazumder MEH and Rahman S. 2009.DPPH
free radical scavenging activity of some Bangladeshi medicinal plants.Journal of
Medicinal Plants Research.3:875-879.
Hassan EA and El-Awadi ME. 2013. Brief Review on the Application of Histochemical
Methods in Different Aspects of Plant Research. Nat Sci. 11(12):54-67.
114
Hausladen A and Stamler JS. 1999. Nitrosative stress. Method in Enzymology.300:389- 395.
Hearn D J. 2009.Descriptive anatomy and evolutionary patterns of anatomical diversification
in Adenia.A Journal of systematic and Evolutionary Botany.27(10):5-16.
Hema R, Kumaravel S and Alagusundaram K. 2011. GC/MS Determination of Bioactive
Components of Murraya koenigii. Journal of American Science.7(1).
Ilavarasan R, Mallika M and Venkataraman S. 2006. Anti-inflammatory and free radical
scavenging activity of Ricinus communis root extract. J Ethnopharmacol.103:478-
480.
Jain, Neelam and Bohra, A. (2006). Antibacterial activity of plant part extracts of
Delphinium ajacis Linn. against a human and a plant pathogenic bacterium. Ad. Plant
Sci., 19 (1) : 171 – 176.
James O, Nnacheta OP, Wara HS and Aliyu UR. 2009. In-vitro and in-vivo studies on the
anti-oxidative activities, membrane stabilization and cytotoxicity of water spinach
(Ipomoea aquatic Forsk) from Ibaji ponds, Nigeria. Int J of Pharmtech
Research.1(3):474-482.
Jamir TT, Sharma HK and Dolui AK. 1999. Folklore medicinal plants of Nagaland, India.
Fitoterapia.70:395–401.
Janakiraman N, Johnson M, Sahaya Sathish S. 2012. GC-MS analysis of bioactive
constituents of Peristrophe bicalyculata (Retz.)Nees.(Acanthaceae).Asian Pacific
Journal of Tropical Biomedicine.46-49.
115
Jaroszewski JW and Fog E. 1989. Cyclopentenoid cyanohydrin glycosides.Part 10. Sulfate
esters of cyclopentenoid cyanohydrin glycosides. Phytochemistry.28:1527–1528.
Johansen D A. 1940. Plant Microtechnique. McGraw Hill Book Co. New York. pp. 523.
Jun L, Stefan W and Chunlin X. 2015. A review of bioactive plant polysaccharides:
Biological activities, functionalization, and biomedical application. Bioactive
Carbohydr.Diet Fibre.5(1): 31–61.
Kadhim MJ, Sosa AA and Hameed IHJ. Evaluation of anti-bacterial activity and bioactive
chemical analysis of Ocimum basilicum using Fourier transform infrared (FT-IR) and
gas chromatography mass spectrometry (GC-MS) techniques. Pharmacognosy
Phytother.8(6):127-146.
Kaingu CK, Mbaria J, Oduma JA and Kiama SG. 2014. Ethnobotanical study of medicinal
plants traditionally used in Tana River County for management of illnesses. Asian
Journal of Complementary and Alternative Medicine.2(2):1-5.
Kalaiarasan A and Ahmed John S. 2011.GC-MS analysis of Bulbophyllum Kaitense
Rechib.Pseudobulbs Estern Ghats of India.International Journal of Chemistry and
Applications.3(3):215-220.
Karthikeyan V, Baskaran A and Sebastian Rajasekaran C. 2016.Gas Chromatography-Mass
Spectrometry (GC-MS) Analysis of Ethanolic Extracts of Barleria acuminata
Nees.International Journal of Pharmaceutical Research.6(2).
Kawabata T, Cui MY, Hasegawa T, Takano F and Ohta T. 2011.Anti–inflammatory and anti–
melanogenic steroidal saponin glycosides from Fenugreek (Trigonella foenum –
graecum L.) seeds.Planta Medica.77: 705–710.
116
Khajja BS, Sharma M, Singh R and Mathur GK. 2011. Forensic Study of Indian
Toxicological Plants as Botanical Weapon (BW): A Review. J Environment Analytic
Toxicol.1:112.
Khandelwal KR. 2002. Practical Pharmacognosy Techniques & Experiments.9th Edition,
Nirali Prakashan. pp.156-161.
Kim HP, Son KH, Chang HW and Kang SS. 2004.Anti–inflammatory plant flavonoids and
cellular action mechanisms.Journal of Pharmacological Science.96: 229–245.
Kokate CK. 1986. Practical pharmacognosy. Vallabh Prakashan. New Delhi. 112-113.
Kottaimuthu R. 2008. Ethnobotany of the Valaiyans of Karandamalai, Dindigul District,
Tamil Nadu, India.Ethnobotanical Leaflets.12:195-203.
Kpadehyea JT, Fernando ES, Tinio CE and Buot IE. 2015. Ethnobotany Survey of the
Wonegizi, Ziama Clan-Lofa County, Liberia. Electronic Journal of Biology.11(4):
165-175.
Kulkarni SK. 1985. Handbook of Experimental Pharmacology.3rdEdn., Vallabh Prakashan,
NewDelhi.
Lawal B, Shittu OK, Kabiru AY, Jigam AA, Umar MB, Berinyuy EB and Alozieuwa BU.
2015. Potential antimalarials from African natural products: A review. J Intercult
Ethnopharmacol.4(4): 318-343.
Lee YM, Kim H, Hong EK, Kang BH and Kim SJ. 2000. Water extract of 1:1 mixture of
Phellodendron cortexandAralia cortex has inhibitory effects on oxidative stress in
kidney of diabetic rats. Journal of Ethnopharmacology.73:429-436.
117
Lersten NR and Horner HT. 2000. Calcium oxalate crystals types and trends in their
distribution patterns in leaves of Prunus (Rosaceae: Prunoideae). Plant Syst. Evol.
224:83-96.
Linga Rao M and Savithramma N. Histochemical studies of Svensonia hyderobadensis
(Walp.) mold – A rare medicinal plant taxon. World Journal of pharmacy and
pharmaceutical sciences.2(5):3631-3640.
Lu L, Liu SW, Jiang SB and Wu SG. 2004. Tannin inhibits HIV – 1 entry by targeting gp 41.
Acta Pharmacological Sinica.25:213 – 218.
Lynn yip. 1993. Adenia cardiophylla (Mast.) Engl. – infectious hepatitis – whole plant –
Antiviral activities of selected Chinese medicinal plants – Ph.D. thesis.
Ma Y, Liang B, Hu B and Wu X. 1982. Isolation and identification of components of the
water-soluble fraction of Guangdong [China] snake bite drug. Zhongcaoyao.13: 1–4.
Magaldi S, Mata-Essayag S and Hartung de Capriles C. 2004.Well diffusion for antifungal
susceptibility testing. Int. J. Infect. Dis. 8:39–45.
Mallikharjuna PB, Rajanna LN, Seetharam YN and Sharanabasappa GK. 2007.
Phytochemical studies of Strychnos potatorum L. f., a medicinal plant. E- Journal of
chemistry. 4:510–518.
Manju Madhavan. 2015. Phytochemical Constituents of Leaves of Spatholobus parviflorus a
Rare Threatened Climber of South India. Int. J. Pharmacognosy and
Phytochem.Res.7(5).
118
Marcel KK, Janat Akhanovna MB, Yves-Alain B, MarcGabin DB and Tra Jérôme ZB. 2011.
In vitro antioxidant activities of total flavonoids extracts from leaves and stems of
Adenia lobata (Jacq.) Engl. (Passifloraceae). J. Pharmacognosy Phytother. 3(1): 8-12.
Mathew KM. 1983. The Flora of Tamilnadu Karnatic. Vol. I. Polypetalae. Rapinat
Herbarium, St. John’s College, Tiruchirappalli. India. pp. 688.
Matthew KM. 1981. The Flora of Tamil Nadu Carnatic, Vol. 1 - 3. The Rapinat Herbarium,
St, Joseph’s College, Thiruchirapally, Tamilnadu.
Mbagwu FN, Edeoga HO. 2006. Palynological studies on some Nigerian species of Vigna
savi. Journal of Biological Sciences.6(6):112–5.
McAnuff MA, Harding WW, Omoruyi FO, Jacobs H, Morrison EY and Asemota HN.
2005. Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter
yam, Dioscorea polygonoides. Food ChemToxicol.43:1667–1672.
McCormick S and Mabry TJ. 1983. O- & C-Glycosylflavones from Passiflora biflora.
Phytochemistry.22:798–799.
McGuire CM, 1999. P. incarnata (Passifloraceae): a new fruit crop. Economic Botany
53:161–176.
Medicinal Plants.International Journal of Microbiology.Article ID 519590, 14 pages.
Meyer JJM, Afolayan AJ, Taylor MB and Engelbrecht L. 1996. Inhibition of herpes simplex
virus type 1 by aqueous extracts from shoots of Helichrysum qureonites. Journal of
Ethnopharmacology.52: 41-43.
119
Mirza M, Kalhoro MA, Yaqeen Z, Sarfaraz TB and Qadri RB. 2003. Physico-chemical
studies of indigenous diuretic medicinal plants Citrullus vulgaris Schrad, Cucumis
melo Linn, Cymbopogon citratus (DC) Stapf, Moringa oleifera Lam, Raphanus
sativus Linn and Zea mays Linn. Pakistan Journal of Pharmacology.20(1):9-16.
Mohammed GJ, Omran AM and Hussein HM. 2016. Anti-bacterial and Phytochemical
Analysis of Piper nigrum using Gas Chromatography – Mass Spectrum and Fourier-
Transform Infrared Spectroscopy. International Journal of Pharmacognosy and
Phytochemical Research.8(6):977-996.
Morton JF. 1981. Atlas of Medicinal Plants of Middle America. Springfield, IL, p. 1281.
Moteriya P, Satasiya R and Chanda S. 2015.Screening of phytochemical constituents in some
ornamental flowers of Saurashtra region.J Phcog Phytochem.3(5):112- 120.
Musa KY, Katsayal AU, Ahmed A, Mohammed Z and Danmalam UH. 2006.
Pharmacognostic investigation of the leaves of Gisekia pharnacioides. African
Journal of Biotechnology.5(1):959-957.
Nambiar VPK, Sabie VP, Jayanthi A and Rajendrakumar K. (1996). Pharmacognostical
studies on Indian Sarasaparilla–Hemidesmus indicus (Linn.) R. Br. Aryavidhyan. 10:
77-93.
Nascimento GGF, Locatelli J, Freitas PC and Silva GL. 2000.Antibacterial activity of plant
extracts and phytochemicals on antibiotic-resistant bacteria.Braz J
Microbiol.31(1):247-56.
120
Neelima N, Sudhakar M, Patil MB and Lakshmi BVS. 2012. Anti-ulcer activity and HPTLC
analysis of Mangifera indica leaves. International Journal of Pharmaceutical and
Phytopharmacological Research. 1(4):146–155.
Nikhila GS, Sangeetha G, Preetha TS and Swapna TS. 2016. GC-MS analysis of
phytochemical compounds present in the rhizome of Gloriosa superba L. Journal of
Pharmacognosy and Phytochemistry. 5(5):17-20.
Nwosu MO. 1999. Herbs for mental disorders.Fitoterapia.70:58–63.
Nyarko AA and Marian E. Addyt. 1990. Effect of aqueous extract of Adenia cissampeloides
on blood pressure and serum analytes of hypertensive patients. Phytotherapy
Research. 4(1): 25-28.
O’Brien T P, Feder N and Mc Cull ME. 1964. Polychromatic staining of plant cell walls by
toluidine blue-O.Protoplasma. 59:364-373.
Aboh AA, Egbulefu AVI and Chudi OPA. 2015. Antimicrobial analysis and structural
elucidation of chloroform leaf extract of Adenia cissanpeloides. International Journal
of Pharmaceutical Chemistry.5:03.
Oktay M, Gulcin I and Kufrevioglu ÖI. 2003. Determination of in vitro anti-oxidant activity
of fennel (Foeniculum vulgare) seed extracts. Lebens-Wiss Technologie. 36: 263-271.
Okunye OL, Odeleye FO and Jayeola BM. 2015.A Study of the Antimicrobial Potency of
Adenia Cissampeloides extracts on Bacteria and Fungi of Clinical Importance.
Journal of Natural Sciences Research.5(12):90-94.
121
Olafsdottir ES, Thorgeirsdottir E and Jaroszewski, JW. 1997. Isolation and identification of
cyclopentene cyanohydrin bis-glycosides from three Passiflora species. European
Journal of Pharmaceutical Science.5:46.
Padalia H and Chanda S. 2015. Compsrative phytochemical analysis of aerial parts of A.
procumbeans, F. dichotoma, S. sponteneum, S. nigra and T. angustifolia. J Phcog
Phytochem.4(2).
Pandey P, Mehta R and Upadhyay R. 2013. Physico-chemical and preliminary phytochemical
screening of Psoralea corylifolia.Arch Appl Sci Res. 5:261-265.
Panyaphu K, Sirisa-ard P, Na Ubol P, Nathakarnkitkul S, Chansakaow S and Van On T.
2011. Phytochemical, antioxidant and antibacterial activities of medicinal plants used
in Northern Thailand as postpartum herbal bath recipes by the Mien (Yao)
community. Phytopharmacology.2(1):92-105.
Partha G and Rahaman CH. 2015.Pharmacognostic, phytochemical and anti-oxidant Studies
of Adenanthera pavonina L. International Journal of Pharmacognosy and
Phytochemical Research.7(1):30-37.
Parthasarathy N, Arthur Selwyn M and Udayakumar M. 2008. Tropical dry evergreen forests
of peninsular India: ecology and conservation significance. Tropical Conservation
Science.1(2):89-110.
Poethke VW, Schwarz C and Gerlach H. 1970.Substances of Passiflora incarnata
1.(Constituents of Passiflora bryonioides).Alkaloids Planta Medica.18:303–314.
122
Prabhadevi V, Sahaya Sathish S, Johnson M, Venkatramani B and Janakiraman N. 2012.
Phytochemical studies on Allamanda cathartica L. using GC-MS. Asian Pacific
Journal of Tropical Biomedicine. 550-554.
Prajapati ND. 2003. A Handbook of Medicinal Plants, Agrobois Publication, India.
Prashanth Kumar GM and Shiddamallayya N. 2016. Survey of wild medicinal plants of
Hassan district, Karnataka. Journal of Medicinal Plants Studies.4(1): 91-102.
prevention of diseases. Critical Reviews in Food Science and Nutrition.45:287-306.
Priya K, Krishankumari S and Vijaykumar M. 2013. Cyathulaprostrata: A potent source of
anti-cancer agent against Dalton’s ascites in Swiss albino mice. Asian Pacific J Trop
Med. 6:776-779.
Rahman MAA and Moon SS. 2007. Anti-oxidant polyphenol glycosides from the plant
Drabane morosa. Bulletin of the Korean Chemical Society.28:827- 831.
Rajarajeswari N, RamaLakshmi S and Muthuchelian K. 2011. GC-MS Analysis of bioactive
components from the ethanolic leaf extract of Canthium dicoccum (Gaertn.) Teijsm &
Binn.J. Chem. Pharm. Res. 3(3):792-798.
Rajan M, Senthil Kumar N and Jeyabalan G. 2013. Evaluation of pharmacognostical,
preliminary phytochemical studies on Blepharis repens (Vahl) Roth. Inter. J. of
Phytotherapy.3(2):82-90.
Rajeswari J and Rani S. 2014. GC-MS Analysis of Phytochemical Compounds in the
Ethanolic Extract of Root of Lawsonia inermis Linn. Int. J. ChemTech Res.
15(07):389-399.
123
Rajeswari J and Rani S. 2014. GC-MS analysis of whole plant of Leptadenia reticulate. Int.
J. PharmTech Res. 6(7):2043-2050.
Ramachandran VS. 2007. Wild edible plant of the Anamalais Coimbatore district Western
Ghats, Tamilnadu. Indian Journal of Traditional Knowledge.6(1):173-176.
Ravaomanalina BH, Rakotonavalona A and Rakouth B. 2011. Conservation status of some
commercialized succulent species of Madagascar. Malagasy Nature.5:59-67.
Rawat PS. 1987. Select Your Dose and Potency. B. Jain Publishers (P) Ltd., New Delhi, pp.
481–482.
Rukangira. 2004. Overview on Medicinal Plants and Traditional Medicine in Africa.
Saganuwan SA. 2014. Nigerian plants that are used for treatment of headache and migraine.
The Journal of Headache and Pain.15(1):33.
Sakagami Y and Kajimura K. 2002.Bactericidal activities of disinfectants against
vancomycin-resistant enterococci.J Hosp Infec.50(2):140-4.
Santha TK, Pattanshetty JK and Gopakumar K. 1988. Pharmacognostical studies on the root
of Sahachara – Nilgrianthus heyneanus (Nees) Bremel. (Acanthaceae).Ancient
Science of Life. 7: 139-144.
Sarkodie JA, Fleischer TC, Edoh DA, Dickson RA, Mensah MLK, Annan K, Woode E,
Koffour GA, Appiah AA and Brew-Daniels H. 2013. Anti-hyperglycaemic activity of
Ethanolic extract of the stem of Adenia lobata Engl (Passifloraceae).Int J Pharm Sci
Res. 4(4): 1370-1377.
124
Sass J E. 1940. Elements of Botanical Microtechnique. McGraw Hill Book Co. New York.
Pp. 222.
Scalbert A, Manach C, Remesy C and Morand C. 2005. Dietary polyphenols and the
Seaforth CE, Adams CD and Sylvester Y. 1983.A Guide for the Medicinal Plants of Trinidad
& Tobago. Commonwealth Secreteriate, Marlborough House, Pall Mall, London.
Seetharam YN, Venugopal RB and Vijay.1999. Pharmacognostic studies on Eclipta alba
Hassk. Aryavaidhyan.13: 97-102.
Selvam ABD. 2013. Standardization of descriptive terminology of starch granules with
reference to identification of raw drugs. International Journal of Pharmaceutical
Research and Development.5(7):1-6.
Senevirathne M, Kim S, Siriwardhana N, Hwan J, Lee K and Jeon Y. 2006. Antioxidant
potential of Ecklonia cava on reactive oxygen species, metal chelating, reducing
power and lipid peroxidation inhibition.Food Science and Technology.12:27-38.
Sermakkani M and Thangapandian V. 2012.GC-MS analysis of Cassia italica leaf methanol
extract. Asian J Pharm Clin Res. 5(2):90-94.
Sethi, P. 1993. Phytosociology of a tropical dry evergreen forest patch in the Puthupet
sacred grove, Coromandel coast, Tamil Nadu. MS Thesis. Pondicherry: Pondicherry
Univ.
Shaikh BT and Hatcher J. 2005. Complementary and alternative medicine in Pakistan:
prospects and limitations. Evidence-based Complementary and Alternative
Medicine.2(2):139-142.
125
Shanthi R, Alagesaboopathi C and Rajasekara pandian M. 2006.Antimicrobial activity of
Andrograohis lineata and Andrographis echioides of the Shevaroy Hills of Salem
District, Tamil Nadu.Ad. Plant Sci. 19(20): 371-375.
Shilpashree VK, Raman Dang and Kuntal Das. 2015. Pharmacognostic authentication and
constituents’ validation by HPLC for four different plant species of Vidari marketed
in India. South Pacific Journal of Pharma and Bioscience.3(1):217-229.
Sivakumar R, Alagesaboopathi C. 2006. Antimicrobial activity of two different forms of
Abrus precatorius L. Ad. Plant Sci., 19: 409-413.
Sobiecki JF. 2002. A preliminary inventory of plants used for psychoactive purposes in
southern African healing traditions. Trans. Roy. Soc. S. Afr. 57(1&2):1–24.
Sobiya Raj D and Jannet Vennila J. 2013. Progress in Ribosomal Inactivating Protein (Rip)
Studies: Recent Review of Potential Applications. Internation Journal of Pharma and
Biosciences.3(3):88-100.
Spencer KC and Seigler DS. 1985. Passibiflorin, eoipassibiflorin and passitrifasciatin:
cyclopentenoid cyanogenic glycosides from Passiflora. Phytochemistry.24:981–986.
Speroni E, Billi R, Perellino NC and Minghetti A. 1996.Role of chrysin in the sedative
effects of P. incarnata L. Phytotherapy Research.10:98–100.
Srichaiwong P, Kwewjai L and Kroeksakul P. 2014. A Study on the Biodiversity of Natural
Food Production to Support Community Upstream of Chi Basin, Thailand.Asian
Social Science.10(2):145-156.
126
Srinivasan K and Kumaravel S. 2016.Unraveling the Potential Phytochemical Compounds of
Gymnema sylvestre Through GC-MS Study.International Journal of Pharmacy and
Pharmaceutical Sciences.8(1):1-4.
Srivastava AK. 2001. Pharmacognostic studies on Curcuma amada Roxb.,Sachitra
Ayurveda.54 :144-147.
Sulochana S, Meyyappan RM and Singaravadivel K. 2016.Phytochemical Screening and GC-
MS Analysis of Garudan Samba Traditional Rice Variety.International Journal of
Environmental & Agriculture Research.2(4).
Suseela A and Prema S. 2007. Pharmacognostic studies on Lagascea mollis. J. Phytol.
Res.20(1): 95-102.
Tamilselvi N, Dhamotharan R, Krishnamoorthy P and Shivakumar. 2011. Anatomical studies
of Indigofera aspalathoides Vahl. (Fabaceae).Journal of Chemical and
Pharmaceutical Research.3(2):738-746.
Tayade AB, Dhar P, Kumar J, Sharma M, Rajinder S, Chauhan, Chaurasia OP and
Srivastava RB. 2013. Chemometric Profile of Root Extracts of Rhodiola
imbricata Edgew with Hyphenated Gas Chromatography Mass Spectrometric
Technique.PLoS One.8(1):527-97.
Thangakrishnakumari S, Muthukumarasamy S and Mohan VR. 2012. GC-MS determination
of bioactive compounds of Sarcostemma secamone (L) Bennet (Asclepiadaceae).
Science research reporter.3:187-191.
127
Thakkar KN, Prasad AK, Nayak J, Iyer SV and Kumar S. 2014. Antioxidant and in-vitro
cytotoxic activity of extracts of aerial parts of Cocculus hirsutus (L) using cell line
cultures (breast cell line). The Journal of Phytopharmacology.3(6): 395-399.
Tiwari P, Kumar B, Kaur M, Kaur G and Kaur H. 2011.Int. pharm. Sciencia. 1:98-106.
Trease GE and Evans WC. 1983. Pharmacognosy. London: Bailliere Tindal.
Trease GE and Evans WC. 1996. Pharmacognosy. London: 14th ed. Bailliere Tindall and Co.
pp. 545.
Trease GE and Evans WC. 2002. Pharmacognosy. 15th Edn. Saunders. pp. 214-393.
Tripathy G and Pradhan D. 2015.In-vitro anti-breast cancer activity of Syzygium cumini
against MCF-7 Cell Line.Journal of Innovations in Pharmaceuticals and Biological
Sciences. 2(2):119-124.
Tsao AS, Kim ES and Hong WK. 2004.Chemoprevention of cancer CA.A Cancer Journal for
Clinicians. 54:150–180.
Tyler VE, Brady LR and Robbers JE. 1976. Pharmacognosy. Lea & Febiger,
Philadelphia.pp.24.
Ulubelen A, Oksuz S and Mabry TJ. 1982. C-glucosylflavonoids from Passiflora pittieri, P.
alata, P. ambigua and Adenia mannii. Journal of Natural Products.45:783.
Uzcategui AE. 1985. Determination of ascorbic acid in some exotic fruits and vegetables
from Eucador by HPLC. Poletecnica.10:221–234.
128
Valgas C, De Souza SM and Smânia EFA.2007.Screening methods to determine antibacterial
activity of natural products.Braz. J. Microbiol. 38: 369–380.
Vasudevan M, Gunnam KK and Parle M. 2007. Antinociceptive and anti-inflammatory
effects of Thespesia populnea bark extract. J Ethnopharmacol.109:264-270.
Verma S, Ojha S and Raish M. 2010.Anti-inflammatory
activity of Aconituim heterophyllum on cotton pellet-induced granuloma in rats.J
Medicinal Plants Research.4:1566-1569.
Vijayan A, Liju VB, Reena John JV, Parthipan B and Renuka. 2007. Traditional remedies of
Kani tribes of Kottoor reserve forest, Agasthyavanam, Thiruvananthapuram, Kerala.
Indian Journal of Trdaitional Knowledge.6(4): 589-594.
Vimala G and Gricilda Shoba F. 2014.A Review on Antiulcer Activity of Few Indian
Medicinal Plants.International Journal of Microbiology.Article ID 519590, 14 pages.
Vivek P and Parthasarathy N. 2015. Diversity and carbon stock assessment of trees and
lianas in tropical dry evergreen forest on the Coromandel Coast of India. Tropical
plant Research.2(3): 230–239.
Wagay NA and Rothe SP. 2016.Investigations on secondary metabolites of Alhagi
pseudalhagi (M. Bieb.)Desv.ex B. Keller & Shap.Leaves using GC-MS. Journal of
Pharmacognosy and Phytochemistry.5(5):114-118.
Wallis TE. 1985. Text book of Pharmacognosy. New Delhi: 5th ed. CBS Publishers. pp. 566
Watt JM and Breyer-Brandwijk MG. 1962.The Medicinal and Poisonous Plants of Southern
and Eastern Africa. Edinburg, Livingston, pp. 826–830.
129
Winter CA, Risley EA and Nuss GW. 1962. Carrageenan-induced edema in the hind paw of
rat as an assay for anti-inflammatory activity. Proc Soc. Exp. Biol. Ther. 111:544-547.
World Health Organization. 1999. WHO Monographs on selected medicinal plants. Vol.
1&2. World Health Organization, Geneva.
World Health Organization. 2002. Policy perspective on medicines traditional medicines
growing needs and potential. WHO Geneva. 2:1-6.
World Health Organization. 2011. Cancer. Retrieved from http:// www.who. int/ mediacentre
/fact sheets /fs297/en/infectious diseases in human
Zongo C, Akomo EO, Savadogo A, Obame LC, Koudou J and Traore AS. 2009. In vitro
anti-bacterial properties of total alkaloids extract from Mitragyna inermis (Willd) O.
Kuntze, a West African traditional medicinal plant. Asian Journal of Plant Sciences.
8: 172-177.
Figure 4: Structure of chemical compounds present in GC-MS analysis of methanolic extract of leaf of Adeniawightiana
4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
4-Ethylbenzoicacid
6-Methylenebicyclo[3.2.0]hept-3-en-2-one Thiophene,3-nitro-2-(2-thienylsulfonyl)-
Quinoline,2-ethyl- α-Santonin
γ-Tocopherol 9-Eicosyne
Ethaneperoxoicacid,1-cyano-1-[2-(2-phenyl-1,3-dioxolan-2-yl)ethyl]pentylester
17-Octadecynoicacid
Hexadecanoicacid,methylester 9,12-Octadecadienoylchloride,(Z,Z)-
Trans-Geranylgeraniol 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol, (3β,5Z,7E)-
2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,[2R-
[2R*(4R*,8R*)]]-
Lup-20(29)-en-3-one
dl-α-Tocopherol Campesterol
Stigmasterol β-Sitosterol
Figure 5Structure of chemical compounds present in GC-MS analysis of methanolic extract of stem of Adeniawightiana
Thymine 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-
6-methyl-
S)-(+)-2',3'-Dideoxyribonolactone 5-Hydroxymethylfurfural
2-Methoxy-4-vinylphenol Phenol,2,6-dimethoxy-
Propanedioicacid,3-thienyl- β-Sitosterol
Ascaridole d-Mannose
OleicAcid Phenol,3,4,5-trimethoxy-
Phenol,2,6-dimethoxy-4-(2-propenyl)- 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol
Tetradecanoicacid Thiophene,2-isobutyl-5-isopentyl-
Hexadecanoicacid,methylester n-Hexadecanoicacid
9,12-Octadecadienoicacid,methylester 9-Octadecenoicacid(Z)-,methylester
9,12-Octadecadienoicacid(Z,Z)- [1,1'-Bicyclopropyl]-2-octanoicacid,2'-hexyl-
,methylester
β-Amyrin 1,2-Benzenedicarboxylicacid,mono(2-
ethylhexyl)ester
Trans-Geranylgeraniol 4,22-Stigmastadiene-3-one
Vitamin E Campesterol
Stigmasterol
Figure 6 Structure of chemical compounds present in GC-MS analysis of methanolic extract of root of Adeniawightiana
Thymine Stigmasterol
4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
S)-(+)-2',3'-Dideoxyribonolactone
Thiophene,2-propyl- Butanedioicacid,2-hydroxy-3-methyl-,dimethylester
Cyclohexanecarboxylicacid,2-hydroxy-,ethylester
Phenol,2,6-dimethoxy-
2-Methyl-oct-2-enedial Lactose
(E,Z,Z)-2,4,7-Tridecatrienal Phenol,3,4,5-trimethoxy-
Hexanoicacid,2-phenylethylester β-Amyrin
n-Hexadecanoicacid Hexadecanoicacid,methylester
4,22-Stigmastadiene-3-one 9-Octadecenoicacid(Z)-,methylester
Heptadecanoicacid,16-methyl-,methylester β-Sitosterol
1,2-Benzenedicarboxylicacid,mono(2-
ethylhexyl)ester Friedelan-3-one
Trans-Geranylgeraniol 9,12-Octadecadienoicacid,methylester
Vitamin E Campesterol
Reference: NIST, Chemspider.
Figure 7 Anti-oxidant activity – DPPH Scavening assay
0
20
40
60
80
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
LEAF EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
0
20
40
60
80
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
STEM EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
0
20
40
60
80
100
Methanol Ethyl acetate
Chloroform Diethyl ether
Ascorbic acid
% O
F AC
TIVI
TY
SOLVENTS
ROOT EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
Figure 8 Anti-oxidant activity – FRAP assay
0
20
40
60
80
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
LEAF EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
020406080
100120
Methanol Ethyl acetate
Chloroform Diethyl ether
Ascorbic acid
% O
F AC
TIVI
TY
SOLVENTS
STEM EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
0
20
40
60
80
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
ROOT EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
Figure 9 Anti-oxidant activity - Phosphomolybdenum assay
0102030405060708090
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
LEAF EXTRACT
Solvent 10 µg/ml
Solvent 50 µg/ml
Solvent 100 µg/ml
0
20
40
60
80
100
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
STEM EXTRACT
Solvent 10 µl
Solvent 50 µl
Solvent 100 µl
020406080
100120
Methanol Ethyl acetate Chloroform Diethyl etherAscorbic acid
% O
F AC
TIVI
TY
SOLVENTS
ROOT EXTRACT
Solvent 10 µl
Solvent 50 µl
Solvent 100 µl
Table 1: Quantitative values of foliar epidermis of Adenia wightiana
Values are expressed as mean±SEM (n=6)
Table 2: Histo-chemical colour reactions of leaf, stem and root of Adenia wightiana Sl. No. Test for Chemicals/reagents Status of the substance
Leaf Stem Root 1. Starch Iodine solution + + + 2. Alkaloid Meyer’s reagent + + + 3. Protein Aqueous Picric acid solution + + + 4. Tannin Dil. Ferric chloride + + + 5. Lignin 1% KMnO4 , 2 % HCl, dil. NH3 - + + 6. Mucilage Methylene blue - - -
Sl. No. Quantitative values Abaxial Adaxial
1 Epidermal cell/mm² 542.0±8.84 963±6.76
2 Stomata/mm² 312.0±7.16 -
3 Stomatal index 022.9±4.37 -
4 Palisade ratio 05.4±0.24
5 Vein islet number 40.8±3.85
6 Veinlet termination number 88.4±5.78
Table 3. Fluorescence analysis of leaf powder of Adenia wightiana
Sl. No. Chemical composition Visible Light UV Light 1 Drug Green Light Green 2 Drug + 1N aq. NaOH Bright Green Light Green 3 Drug + 1NHCl Bright Green Light Green 4 Drug+50% H2SO4 Bright Green Light Green 5 Drug+50%HNO3 Bright Green Light Green
Table 4. Fluorescence Analysis of stem powder of Adenia wightiana
Sl.No. Chemical composition Visible Light UV Light 1 Drug Light Brown Light Brown 2 Drug + 1N aq. NaOH Light Brown Light Brown 3 Drug + 1NHCl Brown Brown 4 Drug+50% H2SO4 Brown Brown 5 Drug+50%HNO3 Brown Brown
Table 5. Fluorescence Analysis of root powder of Adenia wightiana
Sl.No. Chemical composition Visible Light UV Light 1 Drug Light Brown Dark Brown 2 Drug + 1N aq. NaOH Light Brown Light Brown 3 Drug + 1NHCl Dark Brown Dark Brown 4 Drug+50% H2SO4 Dark Brown Dark Brown 5 Drug+50%HNO3 Dark Brown Dark Brown
Table 6. Physico-chemical analysis of leaf, stem and root of Adenia wightiana
Sl. No.
Parameters Results in % Leaf Stem Root
1. Loss on drying 20.0±0.81 10.1±0.50 10.4±0.70 2. Total Ash 16.0±0.63 04.5±0.42 09.4±0.30 3. Acid Insoluble Ash 09.0±0.31 1.01±0.20 04.0±0.40 4. Water soluble ash 09.4±0.54 04.0±0.29 06.0±0.08 5. Sulfated ash 10.0±0.46 02.5±0.40 02.0±0.82
Values are expressed as mean±SEM (n=3)
Table 7. Extractive values of leaf, stem and root of Adenia wightiana by batch process
Sl. No.
Solvents Results in % Leaf Stem Root
1. Methanol 23.0±0.84 19.8±0.75 21.0±0.23 2. Ethyl acetate 21.6±0.95 18.1±0.64 20.1±0.98 3. Chloroform 14.0±0.81 12.3±0.88 10.6±0.42 4. Diethyl ether 11.2±0.57 10.5±0.49 13.0±0.22
Values are expressed as mean±SEM (n=3)
Table 8. pH Determination of leaf, stem and root of Adenia wightiana
Sl. No.
Parts of the plant pH Value
1. Leaf 6.8±0.04 2. Stem 6.4±0.11 3. Root 6.7±0.05
Values are expressed as mean±SEM (n=3)
Table 9. Phytochemical screening of various solvent extracts of leaf of Adenia wightiana S. No. Phytochemicals Leaf
Methanol Ethyl Acetate
Chloroform Diethyl Ether
1. Alkaloids + + - - 2. Anthraquinones + + - - 3. Amino acids + + - - 4. Cardiac glycosides + + - - 5. Catechins - - - - 6. Coumarins + + - + 7. Flavonoids + + - - 8. Gums, oils and resins - - - - 9. Glycosides + + + + 10. Non reducing sugar + + - - 11. Proteins + + - - 12. Pholobatannins - - - - 13. Phenolic group + - + - 14. Quinones - + - - 15. Reducing sugars - + - - 16. Saponins - - + - 17. Steroids - - + + 18. Tannins + + - - 19. Terpenoids - - + - 20. Triterpenoids - - - +
Note: (+) Indicates Presence (-) Indicates Absence
Table 10. Phytochemical screening of various solvent extracts of stem of Adenia wightiana
S. No.
Phytochemicals Stem Methanol Ethyl
Acetate Chloroform Diethyl
Ether 1. Alkaloids + - - - 2. Anthraquinones - - - - 3. Amino acids + - - - 4. Cardiac glycosides - - - - 5. Catechins - - - - 6. Coumarins - - - - 7. Flavonoids - - - - 8. Gums, oils and resins - - - - 9. Glycosides - + + + 10. Non reducing sugar - - - - 11. Proteins + + - - 12. Pholobatannins + - - - 13. Phenolic group - + - - 14. Quinones - - - - 15. Reducing sugars + + - - 16. Saponins - - - - 17. Steroids - - + - 18. Tannins + - - - 19. Terpenoids + - - - 20. Triterpenoids - - - -
Note: (+) Indicates Presence (-) Indicates Absence
Table 11. Phytochemical screening of various solvent extracts of root of Adenia wightiana
S. No.
Phytochemicals Root Methanol Ethyl
Acetate Chloroform Diethyl
Ether 1. Alkaloids + - - - 2. Anthraquinones - - + - 3. Amino acids + - + - 4. Cardiac glycosides - - - - 5. Catechins + - - - 6. Coumarins - - - - 7. Flavonoids + - - - 8. Gums, oils and resins - - + + 9. Glycosides - + + + 10. Non reducing sugar - - - - 11. Proteins + + + - 12. Pholobatannins - - - - 13. Phenolic group + - - - 14. Quinones + + + + 15. Reducing sugars + + - - 16. Saponins - - - - 17. Steroids - - + - 18. Tannins + - - - 19. Terpenoids - + - - 20. Triterpenoids - - + -
Note: (+) Indicates Presence (-) Indicates Absence
Table 12: GC-MS analysis of methanolic extract of leaf of Adenia wightiana
No.
RT
Name of the compound Molecular
formula Molecular
Weight
Peak Area
% 1. 4.70 4H-Pyran-4-one,2,3-
dihydro-3,5-dihydroxy-6-methyl-
C6H8O4 144 0.26
2. 5.30 5-Hydroxymethyl dihydrofuran-2-one
C5H8O3 116 0.60
3. 5.79 6-Methylenebicyclo[3.2.0] hept-3-en-2-one
C8H8O 120 0.98
4. 7.23 4-Ethylbenzoicacid C9H10O2 150 2.10 5. 8.31 l-Gala-l-ido-octose C8H16O8 240 0.98 6. 9.34 Thiophene,3-nitro-2-(2-
thienylsulfonyl)- C8H5NO4S3 275 0.46
7. 10.96 Quinoline,2-ethyl- C11H11N 157 0.82 8. 12.36 α-Santonin C15H18O3 246 0.81 9. 13.30 Ppropiolicacid,3-
(1-hydroxy-2-isopropyl-5-methylcyclohexyl)-
C13H20O3 224 1.91
10. 13.94 9-Eicosyne C20H38 278 1.48 11. 14.25 Ethaneperoxoicacid,1-
cyano-1-[2-(2-phenyl-1,3-dioxolan-2-yl)ethyl]pentylester
C19H25NO5 347 0.78
12. 14.47 17-Octadecynoicacid C18H32O2 280 0.64 13. 15.05 Hexadecanoicacid,
methylester C17H34O2 270 1.27
14. 17.60 9,12-Octadecadienoylchloride, (Z,Z)-
C18H31ClO 298 4.65
15. 27.70 Trans-Geranylgeraniol C20H34O 290 3.95 16. 28.57 Benzene,1-[1,1-
dimethylethyl]-4-[2-propenyloxy]-
C13H18O 190 1.40
17. 29.52 2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,[2R-[2R*(4R*,8R*)]]-
C27H46O2 402 16.52
18. 30.94 β-Tocopherol C28H48O2 416 5.46 19. 31.23 γ-Tocopherol C28H48O2 416 6.21 20. 31.52 9,10-Secocholesta-5,7,10
(19)-triene-3,24,25-triol, (3β,5Z,7E)-
C27H44O3 416 1.74
21. 32.75 dl-α-Tocopherol C29H50O2 430 16.38 22. 34.85 Campesterol C28H48O 400 1.88 23. 35.56 Stigmasterol C29H48O 412 8.03 24. 37.22 β-Sitosterol C29H50O 414 15.39 25. 38.77 Lup-20(29)-en-3-one C30H48O 424 5.30
Table 13: GC-MS analysis of methanolic extract of stem of Adenia wightiana
No.
RT
Nameofthecompound Molecular
formula Molecular
Weight
PeakArea
% 1. 3.71 Thymine C5H6N2O2 126 1.64 2. 4.70 4H-Pyran-4-
one,2,3-dihydro-3,5-dihydroxy-6-methyl-
C6H8O4 144 3.94
3. 5.62 S)-(+)-2',3'-Dideoxyribonolactone
C5H8O3 116 9.27
4. 6.06 5-Hydroxymethylfurfural C6H6O3 126 11.46 5. 7.25 2-Methoxy-4-vinylphenol C9H10O2 150 0.57 6. 7.77 Phenol,2,6-dimethoxy- C8H10O3 154 3.63 7. 8.76 Propanedioicacid,3-
thienyl- C7H6O4S 186 0.49
8. 8.94 Pyrrolizin-1,7-dione-6-carboxylicacid,methyl(ester)
C9H11NO4 197 0.09
9. 9.16 Ascaridole C10H16O2 168 0.52 10. 10.31 d-Mannose C6H12O6 180 0.62 11. 10.71 OleicAcid C18H34O2 282 1.41 12. 11.29 Phenol,3,4,5-trimethoxy- C9H12O4 184 1.61 13. 12.37 Phenol,2,6-dimethoxy-4-
(2-propenyl)- C11H14O3 194 1.89
14. 12.90 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol
C10H12O3 180 2.85
15. 13.07 Tetradecanoicacid C14H28O2 228 1.13 16. 13.13 Thiophene,2-isobutyl-5-
isopentyl- C13H22S 210 1.02
17. 15.05 Hexadecanoicacid,methylester
C17H34O2 270 2.84
18. 15.70 n-Hexadecanoicacid C16H32O2 256 8.46 19. 17.36 9,12-
Octadecadienoicacid,methylester
C19H34O2 294 2.76
20. 17.45 9-Octadecenoicacid(Z)-,methylester
C19H36O2 296 2.14
21. 18.25 9,12-Octadecadienoicacid(Z,Z)-
C18H32O2 280 3.81
22. 20.23 [1,1'-Bicyclopropyl]-2-octanoicacid,2'-hexyl-,methylester
C21H38O2 322 0.52
23. 21.10 Cholestan-3-ol,2-methylene-,(3β,5α)-
C28H48O 400 0.52
24. 23.76 1,2-Benzenedicarboxylicacid,mono(2-ethylhexyl)ester
C16H22O4 278 0.25
25. 27.70 Trans-Geranylgeraniol C20H34O 290 0.03 26. 29.50 17.alfa.,21β-28,30-
Bisnorhopane C28H48 384 0.10
27. 32.74 Vitamin E C29H50O2 430 2.57 28. 34.85 Campesterol C28H48O 400 7.47 29. 35.56 Stigmasterol C29H48O 412 12.56 30. 37.22 β-Sitosterol C29H50O 414 10.33 31. 38.22 β-Amyrin C30H50O 426 1.71 32. 39.13 4,22-Stigmastadiene-3-one C29H46O 410 1.79
Table 14: GC-MS analysis of methanolic extract of root of Adenia wightiana
No.
RT
Nameofthecompound Molecular
Formulae Molecular
Weight
PeakArea
% 1. 3.71 Thymine C5H6N2O2 126 3.18 2. 4.20 6-Acetyl-β-d-mannose C8H14O7 222 0.29 3. 4.70 4H-Pyran-4-
one,2,3-dihydro-3,5-dihydroxy-6- m e t h y l -
C6H8O4 144 6.03
4. 5.60 S)-(+)-2',3'-Dideoxyribonolactone
C5H8O3 116 9.30
5. 6.09 Thiophene,2-propyl- C7H10S 126 22.63 6. 6.27 Butanedioicacid,2-
hydroxy-3-methyl-,dimethylester
C7H12O5 176 3.80
7. 7.39 Cyclohexanecarboxylicacid,2-hydroxy-,ethylester
C9H16O3 172 2.19
8. 7.77 Phenol,2,6-dimethoxy- C8H10O3 154 1.41 9. 9.14 2-Methyl-oct-2-enedial C9H14O2 154 0.80 10. 10.29 Lactose C12H22O11 342 2.73 11. 10.87 (E,Z,Z)-2,4,7-Tridecatrienal C13H20O 192 0.99 12. 11.29 Phenol,3,4,5-trimethoxy- C9H12O4 184 0.85 13. 11.53 Desulphosinigrin C10H17NO6S 279 0.94 14. 12.08 α-D-
Glucopyranoside,O-α-D- lucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl
C18H32O16 504 1.09
15. 14.57 Hexanoicacid,2-phenylethylester
C14H20O2 220 0.37
16. 15.05 Hexadecanoicacid,methylester
C17H34O2 270 3.08
17. 15.70 n-Hexadecanoicacid C16H32O2 256 0.14 18. 16.19 5,8,11-
Heptadecatriynoicacid,methylester
C18H24O2 272 1.14
19. 17.36 9,12-Octadecadienoicacid,methylester
C19H34O2 294 5.77
20. 17.45 9-Octadecenoicacid(Z)-,methylester
C19H36O2 296 11.37
21. 17.82 Heptadecanoicacid,16-methyl-,methylester
C19H38O2 298 0.46
22. 21.10 Cholestan-3-ol,2-methylene-,(3β,5α)-
C28H48O 400 0.03
23. 23.76 1,2-Benzenedicarboxylicacid,mono (2-ethylhexyl)ester
C16H22O4 278 0.58
24. 26.94 Friedelan-3-one C30H50O 426 7.25 25. 27.70 Trans-Geranylgeraniol C20H34O 290 0.12 26. 29.50 17.alfa.,21β-28,30-
Bisnorhopane C28H48 384 0.56
27. 32.74 Vitamin E C29H50O2 430 2.28 28. 34.85 Campesterol C28H48O 400 1.17 29. 35.56 Stigmasterol C29H48O 412 3.71 30. 37.22 β-Sitosterol C29H50O 414 2.58 31. 38.22 β-Amyrin C30H50O 426 0.91 32. 39.13 4,22-Stigmastadiene-3-one C29H46O 410 2.25
Table 15: A comparative analysis of compounds in GC-MS analysis of methanolic extract of leaf, stem and root of Adenia wightiana
Sl. No.
Name of the compound Leaf Stem Root
1. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
+ + +
2. 5-Hydroxymethyldihydrofuran-2-one + - - 3. 6-Methylenebicyclo[3.2.0]hept-3-en-2-one + - - 4. 4-Ethylbenzoicacid + - - 5. l-Gala-l-ido-octose + - - 6. Thiophene,3-nitro-2-(2-thienylsulfonyl)- + - - 7. Quinoline,2-ethyl- + - - 8. α-Santonin + - - 9. Ppropiolicacid,3-(1-hydroxy-2-
isopropyl-5- methylcyclohexyl)-
+ - -
10. 9-Eicosyne + - - 11. Ethaneperoxoicacid,1-cyano-1-[2-(2-
phenyl-1,3-dioxolan-2-yl)ethyl]pentylester
+ - -
12. 17-Octadecynoicacid + - - 13. Hexadecanoicacid,methylester + + + 14. 9,12-Octadecadienoylchloride,(Z,Z)- + - - 15. Trans-Geranylgeraniol + + + 16. Benzene,1-[1,1-dimethylethyl]-4-[2-
propenyloxy]- + - -
17. 2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,[2R-[2R*(4R*,8R*)]]-
+ - -
18. β-Tocopherol + - - 19. γ-Tocopherol + - - 20. 9,10-Secocholesta-5,7,10(19)-triene-3,24,25-
triol, (3β,5Z,7E)-
+ - -
21. dl-α-Tocopherol + - - 22. Campesterol + + + 23. Stigmasterol + + + 24. β-Sitosterol + + + 25. Lup-20(29)-en-3-one + - - 26. Thymine - + + 27. S)-(+)-2',3'-Dideoxyribonolactone - + + 28. 5-Hydroxymethylfurfural - - - 29. 2-Methoxy-4-vinylphenol - - - 30. Phenol,2,6-dimethoxy- - + + 31. Propanedioicacid,3-thienyl- - - - 32. Pyrrolizin-1,7-dione-6-
carboxylicacid,methyl(ester) - - -
33. Ascaridole - - - 34. d-Mannose - - - 35. OleicAcid - - - 36. Phenol,3,4,5-trimethoxy- - + + 37. Phenol,2,6-dimethoxy-4-(2-propenyl)- - - - 38. 4-((1E)-3-Hydroxy-1-propenyl)-2-
methoxyphenol - - -
39. Tetradecanoicacid - - - 40. Thiophene,2-isobutyl-5-isopentyl- - - - 41. n-Hexadecanoicacid - + + 42. 9,12-Octadecadienoicacid,methylester - + + 43. 9-Octadecenoicacid(Z)-,methylester - + + 44. 9,12-Octadecadienoicacid(Z,Z)- - - - 45. [1,1'-Bicyclopropyl]-2-
octanoicacid,2'-hexyl-,methylester - - -
46. Cholestan-3-ol,2-methylene-,(3β,5α)- - + + 47. 1,2-Benzenedicarboxylicacid,mono(2-
ethylhexyl)ester - + +
48. 17.alfa.,21β-28,30-Bisnorhopane - + + 49. Vitamin E - + + 50. β-Amyrin - + + 51. 4,22-Stigmastadiene-3-one - + + 52. 6-Acetyl-β-d-mannose - - + 53. Thiophene,2-propyl- - - + 54. Butanedioicacid,2-hydroxy-3-methyl-
,dimethylester - - +
55. Cyclohexanecarboxylicacid,2-hydroxy-,ethylester
- - +
56. 2-Methyl-oct-2-enedial - - + 57. Lactose - - + 58. (E,Z,Z)-2,4,7-Tridecatrienal - - + 59. Desulphosinigrin - - + 60. α-D-Glucopyranoside,O-α-D-
lucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl
- - +
61. Hexanoicacid,2-phenylethylester - - + 62. 5,8,11-Heptadecatriynoicacid,methylester - - + 63. Heptadecanoicacid,16-methyl-,methylester - - + 64. Friedelan-3-one - - +
Note: (+) Indicates Presence (-) Indicates Absence
Table 16: Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of leaf of Adenia wightiana
Sl. No.
Nameofthecompound Compound
group Biological activity Reference
1. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
Flavonoid Antimicrobial Anti-inflammatory Antiproliferative
Antioxidant
Rajeswari et al., 2014
2. 5-Hydroxymethyl dihydrofuran-2-one
Ketone Not reported -
3. 6-Methylenebicyclo [3.2.0]hept-3-en-2-one
Ketone Bacteriostatic Fungistatic
Antiparasitic
Mohammed et al., 2016
4. 4-Ethylbenzoicacid Carboxylic acid Not reported - 5. l-Gala-l-ido-octose Carbohydrate Anti-dementia Jun et al.,
2015 6. Thiophene,3-nitro-2-
(2-thienylsulfonyl)- Furans Bacteriostatic
Antimalarial Fungicidal Herbicidal
Jun et al., 2015
7. Quinoline,2-ethyl- Alkaloid Not reported - 8. α-Santonin Terpenoid Anti-fungal,
Anti-helminthic -
9. Ppropiolicacid,3-(1-hydroxy-2-isopropyl-5-methylcyclohexyl)-
carboxylic acid Anti-angiogenic Antitumor
Antimicrobial
Mohammed et al., 2016
10. 9-Eicosyne Hydrocarbon Antimicrobial Antioxidant Antitumor
Cancer-preventive Pesticide
Fathima, et. al., 2016
11. Ethaneperoxoicacid,1-cyano-1-[2-(2-phenyl-1,3-dioxolan-2-yl)ethyl]pentylester
Ester of fatty acid Anti-microbial Janakiraman, et. al., 2012
12. 17-Octadecynoicacid Ester of fatty acid Antioxidant Nematicide
Fathima, et. al., 2016
13. Hexadecanoicacid,methylester
Ester of fatty acid Antioxidant Hypocholesterolemic
Sermakanni, et. al., 2012
14. 9,12-Octadecadienoylchloride,(Z,Z)-
Ester of fatty acid Not reported -
15. Trans-Geranylgeraniol Terpenoid Antifungal Vijyalakshmi,
Antinoceptive Antilipimic Antitumor
potent inhibitor of Mycobacterium
tuberculosis
et. al., 2014
16. Benzene,1-[1,1-dimethylethyl]-4-[2-propenyloxy]-
Aromatic compound
Not reported -
17. 2H-1-Benzopyran-6-ol,3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-,[2R-[2R*(4R*,8R*)]]-
Vitamin E Antiinflammatory Antioxidant Antitumor Anticancer
Kalaiarasan, et. al., 2011
18. β-Tocopherol Vitamin E Antioxidant Antiinflammatory
Rajani et. al., 2015
19. γ-Tocopherol Vitamin E Antioxidant Antiinflammatory Cardio-protective
Anti-cancer
Rajani et. al., 2015
20. 9,10-Secocholesta- 5,7,10 (19)-triene-3,24,25- triol, (3β,5Z,7E)-
Steriod Antiviral Anti-Parkinsonism
Altameme et al., 2015
21. dl-α-Tocopherol Vitamin E
Antiageing Analgesic
Antidiabatic Antiinflammatory
Antioxidant Antidermatitic Antileukemic
Hepatoprotective Hypocholesterolemic
Antiulcerogenic Vasodilator
Antispasmodic Antibronchitic
Karthikeyan et al., 2016
22. Campesterol Steroid Antioxidant, Hypocholesterolemic
Tayade, et. al., 2013
23. Stigmasterol Steroid Antioxidant Hypocholesterolemic
Anti-osteoarthitic Cyto-toxicity Anti tumour
Anti-HIV reverse transcriptase
Wagay, et. al., 2016
24. β-Sitosterol Steroid Anti-oxidant Analgesic
Anti-inflammatory Hypocholesterolemic
Sulochana, et. al., 2016
25. Lup-20(29)-en-3-one Triterpenoid Not reported -
Table 17: Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of stem of Adenia wightiana
Sl. No.
Nameofthecompound Compound group
Biological
activity
Reference
1. Thymine Nitrogenous base
Not reported Prabhadevi, et. al., 2012
2. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-
Flavonoid Antimicrobial Antiinflammatory Antiproliferative
Antioxidant
Rajeswari, et. al., 2014
3. S)-(+)-2',3'-Dideoxyribonolactone
carbohydrate Antimicrobial Easwaran & Ramani 2014
4. 5-Hydroxymethylfurfural Aldehyde Antimicrobial antiinflammatory
Karthikeyan et al.,2016
5. 2-Methoxy-4-vinylphenol Phenol Flavor, Perfumery
Rajeswari, et. al., 2015
6. Phenol,2,6-dimethoxy- Phenol Antimicrobial Nikhila, et. al., 2016
7. Propanedioicacid,3-thienyl-
carboxylic acid Not reported
8. Pyrrolizin-1,7-dione-6-carboxylicacid,methyl(ester)
Ester Anti-Viral Anti-Tumor
Mohammed, et. al., 2016
9. Ascaridole Terpenes Anthelmintic Antimalarial
Tayade, et. al., 2013
10. d-Mannose carbohydrate Preservative Prabhadevi, et. al., 2012
11. OleicAcid Fatty acid Anticancer Sulochana, et. al., 2016
12. Phenol,3,4,5-trimethoxy- Phenol Antioxidant Analgesic
Antibacterial Anti-inflammatory
Antiseptic Vasodilator Antiviral
Cancer preventive
Karthikeyan et al.,2016
13. Phenol,2,6-dimethoxy-4-(2-propenyl)-
Phenol Not reported
14. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol
Phenol
Antimicrobial antioxidant
antiinflammatory
Karthikeyan et al.,2016
15. Tetradecanoicacid Fatty acid Antioxidant Cancer preventive
Nematicide Hypocholesterolemic
Karthikeyan et al.,2016
16. Thiophene,2-isobutyl-5-isopentyl-
Furans Not reported
17. Hexadecanoicacid,methylester
Ester Antioxidant Hypocholesterolemic
Sermakanni, et. al., 2012
18. n-Hexadecanoicacid Fatty acid (Palmitic acid)
Antioxidant Hypocholesterolemi
c Nematicide Anti-androgenic
Hema, et. al., 2011
19. 9,12-Octadecadienoicacid,methylester
Ester Hepatoprotective Antihistaminic
Hypocholesterolemic Antieczemic
Gnanavel, et. al., 2013
20. 9-Octadecenoicacid(Z)-,methylester
Fatty acid Antiinflammatory Antiandrogenic
Cancer preventive Hypocholesterolemic
Rajeswari, et. al., 2015
21. 9,12-Octadecadienoicacid(Z,Z)-
Fatty acid (Linoleic acid)
Antiinflammatory Nematicide
Hypocholesterolemic Cancer preventive Hepatoprotective Antihistaminic
Antiacne Antiarthritic Antieczemic
Manju Madhavan et.,
al., 2015
22. [1,1'-Bicyclopropyl]-2-octanoicacid,2'-hexyl-,methylester
Ester Hypocholesterolemic Gnanavel, et. al., 2013
23. Cholestan-3-ol,2-methylene-,(3β,5α)-
Steroid Antimicrobial Anticancer
Diuretic Antiasthma, Antiarthritic
Manju Madhavan, et.
al., 2015
24. 1,2-Benzenedicarboxylicacid,mono(2-ethylhexyl)ester
(Plasticizer compound)
Ester
Antimicrobial Ezhilan, et. al., 2011
25. Trans-Geranylgeraniol Terpenes Antifungal Antinoceptive
Antilipimic Antitumor
Vijyalakshmi, et. al., 2014
26. 17.alfa.,21β-28,30-Bisnorhopane
Terpenes Not reported
27. Vitamin E Vitamin E Analgesic Anti-diabetic
Antiinflammatory Antioxidant
Antidermatitic Antileukemic
Antitumor Anticancer
Hepatoprotective Antispasmodic
Santhosh Kumar, et. al.,
2014
28. Campesterol Steroid Antioxidant Hypocholesterolemic
Tayade, et. al., 2013
29. Stigmasterol Steroid Antioxidant Hypoglycemic Antimicrobial
Anticancer Antiarthritic Antiasthama
Antiinflammatory Diuretic
Dandekar, et. al., 2015
30. β-Sitosterol Steroid Anti-oxidant Analgesic
Anti-inflammatory Hypocholesterolemic
Sulochana, et. al., 2016
31. β-Amyrin Terpenes Anti-tumor Srinivasan, et. al., 2015
32. 4,22-Stigmastadiene-3-one Steriod Antimicrobial Dandekar, et. al., 2015
Table 18: Group name and biological activity of the compounds present in the GC-MS analysis of methanolic extract of root of Adenia wightiana
Sl. No.
Nameofthecompound Compound
group Biological
Activity Reference
1. Thymine Nitrogenous base
Not reported Prabhadevi, et. al., 2012
2. 6-Acetyl-β-d-mannose Carbohydrate Anti-carcinogenic Kadhim, et. al., 2016
3. 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6- m e t h y l -
Flavonoid Antimicrobial Anti-inflammatory Antiproliferative
Antioxidant
Rajeswari, et. al., 2014
4. S)-(+)-2',3'-Dideoxyribonolactone
Carbohydrate Antimicrobial Easwaran & Ramani 2014
5. Thiophene,2-propyl- Furan Not reported - 6. Butanedioicacid,2-
hydroxy-3-methyl-,dimethylester
Ester Not reported -
7. Cyclohexanecarboxylicacid,2-hydroxy-, ethylester
Ester Antipyretic, Antiinflammatory
Kadhim, et. al., 2016
8. Phenol,2,6-dimethoxy- Phenol Antimicrobial
Nikhila, et. al., 2016
9. 2-Methyl-oct-2-enedial Aldehyde Not reported - 10. Lactose Carbohydrate Preservative
Nutritive Muthuchelia
n, et. al., 2011
11. (E,Z,Z)-2,4,7-Tridecatrienal
Aldehyde Anticancer Antitumour
Antidote
Duke, 1992
12. Phenol,3,4,5-trimethoxy- Phenol Antioxidant Analgesic
Antibacterial Antiinflammatory
Antiseptic Vasodilator
Antiviral Cancer preventive
Karthikeyan et al.,2016
13. Desulphosinigrin Glycoside/ Glucosinolate
Aid in phloem mobility of plants
Brundenell, et. al., 1999
14. α-D-Glucopyranoside,O-α-D- lucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl
Flavanoid Anti-diabetic
15. Hexanoicacid,2-phenylethylester
Ester Not reported -
16. Hexadecanoicacid, methylester
Fatty acid ester
Antioxidant Hypocholesterolemic
Sermakanni, et. al., 2012
17. n-Hexadecanoicacid Fatty acid (Palmitic acid)
Antioxidant Hypocholesterolemic
Hema, et. al., 2011
18. 5,8,11-Heptadecatriynoicacid,methylester
Ester Not reported -
19. 9,12-Octadecadienoicacid,methylester
Oleic acid methyl ester
Hepatoprotective Antihistaminic
Hypocholesterolemic Antieczemic
Gnanavel, et. al., 2013
20. 9-Octadecenoicacid(Z)-,methylester
Ester Anti-inflammatory Antiandrogenic
Cancer preventive Hypocholesterolemic
Rajeswari, et. al., 2014
21. Heptadecanoicacid,16-methyl-,methylester
Fatty acid Not reported -
22. Cholestan-3-ol,2-methylene-,(3β,5α)-
Steroid Antimicrobial anticancer Diuretic
Antiasthma, Antiarthritic
Manju Madhavan, et. al., 2015
23. 1,2-Benzenedicarboxylicacid,mono (2-ethylhexyl)ester
Ester (Plasticizer compound)
Antimicrobial Ezhilan, et. al., 2011
24. Friedelan-3-one Ketone Antibacterial Antifungal
Anti-inflammatory Analgesic
Antipyretic Antihypertensive
Abubakar, et. al., 2016
25. Trans-Geranylgeraniol Terpenes Antifungal Antinoceptive
Antilipimic Antitumor
Vijyalakshmi, et. al., 2014
26. 17.alfa.,21β-28,30-Bisnorhopane
Terpenes Not reported
27. Vitamin E Vitamin E Antiageing Analgesic
Antidiabetic Antiinflammatory
Antioxidant Antileukemic
Prabhadevi, et. al., 2012
28. Campesterol Steroid Antioxidant Hypocholesterolemic
Tayade, et. al., 2013
29. Stigmasterol Steroid Antioxidant Hypoglycemic Antimicrobial
Dandekar, et. al., 2015
Anticancer Antiarthritic Antiasthama
Anti-inflammatory Diuretic
30. β-Sitosterol Steroid Anti-oxidant Analgesic
Anti-inflammatory Hypocholesterolemic
Sulochana, et. al., 2016
31. β-Amyrin Triterpene Anti-tumor Srinivasan, et. al., 2015
32. 4,22-Stigmastadiene-3-one
Steroid Antimicrobial Dandekar, et. al., 2015
Table 19: A comparative analysis total number of chemical compounds groups present in the GC-MS analysis of Adenia wightiana
Sl. No. Compound group Leaf Stem Root Entire plant 1. Aldehyde - 1 2 3 2. Alkaloid 1 - - 1 3. Aromatic compound 1 - - 1 4. Carbohydrate 1 2 3 6 5. Carboxylic acid 2 1 - 3 6. Nitrogenous base - 1 1 2 7. Ester 4 5 8 17 8. Fatty acid - 5 2 7 9. Flavonoid 1 1 2 4 10. Furan 1 1 1 3 11. Glycoside - - 1 1 12. Hydrocarbon 1 - - 1 13. Ketone 2 - 1 3 14. Phenol - 5 2 7 15. Steroid 4 5 5 14 16. Terpenes 2 4 2 8 17. Triterpene 1 - 1 2 18. Vitamin 4 1 1 6
Total 25 32 32 89
Table 20: Biological activities possessed by the chemical compounds present in the GC-MS analysis of Adenia wightiana
Sl. No. Compound group Leaf Stem Root Entire plant 1. Analgesic 2 3 4 9 2. Anti-acne - 1 - 1 3. Anti-ageing - - 1 1 4. Anti-arthritic 1 3 2 6 5. Anti-asthama - 2 2 4 6. Anti-bacterial - - 2 2 7. Anti-bronchitis 1 - - 1 8. Anti-cancer 4 5 8 17 9. Anti-carcinogenic - - 1 1 10. Anti-dermatitic 1 2 - 3 11. Anti-diabetic 1 1 - 2 12. Anti-dote - - 1 1 13. Anti-eczemic - 2 - 2 14. Anti-fungal 4 1 2 7 15. Anti-helminthic - 1 - 1 16. Anti-histaminic - 2 - 2 17. Anti-HIV 1 - - 1 18. Anti-hypertensive - - 1 1 19. Anti-inflammatory 6 9 8 23 20. Anti-leukemic 1 1 1 3 21. Anti-lipimic - - 1 1 22. Anti-malarial 1 1 - 2 23. Anti-microbial 4 9 7 20 24. Anti-noceptive - 1 1 2 25. Anti-oxidant 11 10 8 29 26. Anti-parasitic 1 - - 1 27. Anti-parkinsonism 1 - - 1 28. Anti-proliferative 1 1 1 3 29. Anti-pyretic - - 2 2 30. Anti-septic - 1 1 2 31. Anti-spasmodic 1 1 2 32. Anti-tumor 6 4 3 13 33. Anti-ulcerogenic 1 - - 1 34. Anti-viral 1 2 1 4 35. Bacteriostatic 3 - - 3 36. Cancer preventive - 4 2 6 37. Cardioprotective 1 - - 1 38. Cytotoxicity activity 1 - - 1 39. Diuretic - 2 2 40. Hepatoprotective 1 2 3 41. Hypocholesterolemic 5 9 6 20 42. Vasodilator - - 1 1
Total 61 78 69 208
Table 21: Antioxidant activity of leaf extract of Adenia wightiana - DPPH Assay
Sl. No. Solvent % of radical scavenging 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 33.46±1.25 54.12±2.98 73.54±1.09 2. Ethyl acetate 49.56±2.15 60.66±2.33 81.19±4.32 3. Chloroform 41.36±2.65 58.54±3.87 69.24±2.44 4. Diethyl ether 26.94±2.11 45.85±1.89 66.31±3.29 5. Ascorbic acid 58.16±1.07 76.28±2.65 92.13±1.55
Values are expressed as mean±SEM (n=3)
Table 22: Antioxidant activity of stem extract of Adenia wightiana - DPPH Assay
Sl. No. Solvent % radical scavenging 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 40.15±2.66 59.68±2.10 70.17±2.54 2. Ethyl acetate 23.58±1.85 44.21±1.55 67.78±3.21 3. Chloroform 27.61±2.64 45.63±1.61 72.24±2.11 4. Diethyl ether 29.45±1.24 47.21±2.51 69.56±1.20 5. Ascorbic acid 51.87±2.33 65.94±1.09 86.32±1.32
Values are expressed as mean±SEM (n=3)
Table 23: Antioxidant activity of root extract of Adenia wightiana - DPPH Assay
Sl. No. Solvent % radical scavenging 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 48.58±2.14 67.32±3.85 88.52±3.58 2. Ethyl acetate 28.76±1.98 49.32±2.15 87.81±2.87 3. Chloroform 31.18±2.17 52.29±2.86 71.64±1.54 4. Diethyl ether 25.36±1.25 34.55±1.99 59.63±4.25 5. Ascorbic acid 54.74±2.59 74.25±3.17 91.36±2.99
Values are expressed as mean±SEM (n=3)
Table 24: Antioxidant activity of leaf extract of Adenia wightiana - FRAP assay
Sl. No.
Solvent % of Total Antioxidant Capacity 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 17.64±0.17 33.2±0.81 72.5±1.65 2. Ethyl acetate 14.39±0.11 27.69±0.65 66.77±1.98 3. Chloroform 10.68±0.01 24.53±0.15 50.21±1.21 4. Diethyl ether 09.44±0.01 21.97±0.13 64.00±1.25 5. Ascorbic acid 26.47±1.32 44.25±2.12 89.14±2.34
Values are expressed as mean±SEM (n=3)
Table 25: Antioxidant activity of stem extract of Adenia wightiana - FRAP assay
Sl. No.
Solvent % of Total Antioxidant Capacity 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 18.46±1.38 42.8±1.54 88.41±2.94 2. Ethyl acetate 13.58±1.09 22.24±1.84 43.17±1.27 3. Chloroform 14.33±1.20 26.22±1.08 50.02±2.88 4. Diethyl ether 17.76±1.19 31.56±1.64 66.84±2.84 5. Ascorbic acid 32.63±1.67 53.57±1.97 95.89±2.55
Values are expressed as mean±SEM (n=3)
Table 26: Antioxidant activity of root extract of Adenia wightiana - FRAP assay
Sl. No.
Solvent % of Total Antioxidant Capacity 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 24.06±0.19 32.58±3.14 66.73±2.45 2. Ethyl acetate 12.56±1.21 22.67±4.12 43.11±2.17 3. Chloroform 19.07±0.21 23.59±2.15 50.21±3.01 4. Diethyl ether 16.11±0.58 29.96±3.45 60.08±2.57 5. Ascorbic acid 28.21±1.25 43.74±2.19 87.65±4.10
Values are expressed as mean±SEM (n=3)
Table 27: Antioxidant activity of leaf extract of Adenia wightiana –
Phosphomolybdenum assay Sl. No.
Solvent % of Total Antioxidant Capacity 10 µg/ml 50 µg/ml 100 µg/ml
1. Methanol 7.88±1.80 34.54±2.17 67.58±2.23 2. Ethyl acetate 9.11±1.20 42.63±1.59 87.26±2.16 3. Chloroform 5.33±1.09 21.82±2.76 43.56±1.58 4. Diethyl ether 6.55±1.31 30.91±1.32 61.24±2.00 5. Ascorbic acid 15.52±1.21 56.28±1.19 92.08±2.87
Values are expressed as mean±SEM (n=3)
Table 28: Antioxidant activity of stem extract of Adenia wightiana – Phosphomolybdenum assay Sl. No.
Solvent % of Total Antioxidant Capacity 10 µl 50 µl 100 µl
1. Methanol 6.04±1.15 40.09±1.92 64.27±2.14 2. Ethyl acetate 9.87±1.31 31.67±2.51 48.54±3.19 3. Chloroform 4.78±1.58 24.32±1.25 81.10±2.99 4. Diethyl ether 5.91±1.12 22.20±1.76 44.72±2.57 5. Ascorbic acid 17.28±1.35 49.43±2.64 90.71±2.32
Values are expressed as mean±SEM (n=3)
Table 29: Antioxidant activity of root extract of Adenia wightiana – Phosphomolybdenum assay
Values are expressed as mean±SEM (n=3)
Sl. No.
Solvent % of Total Antioxidant Capacity 10 µl 50 µl 100 µl
1. Methanol 5.64±1.13 31.08±2.14 83.57±2.16 2. Ethyl acetate 6.84±1.07 41.25±1.28 63.69±3.21 3. Chloroform 10.21±1.84 22.91±2.71 46.27±2.64 4. Diethyl ether 3.49±1.20 15.28±2.95 32.08±1.20 5. Ascorbic acid 20.76±1.22 61.33±1.11 98.28±2.01
Table 30: Antimicrobial activity of methanolic leaf extract of Adenia wightiana
Sl. No.
Microbial strain
Zone of Inhibition (mm) 0.5 mg 1 mg 2 mg 4 mg Positive
control 1. B. subtilis
MTCC 441 - - 11±1.02 13±1.28 28±1.29
2. S. aureus MTCC 6908
- 10±1.09 14±1.24 17±1.95 24±1.84
3. E. coli MTCC 406
- - 12±2.51 16±1.87 22±1.33
4. K. pneumoniae MTCC 530
- 12±1.20 15±1.58 17±1.02 22±1.54
5. A. niger MTCC 1344
11±1.04 12±1.50 13±0.98 15±1.06 26±2.16
6. C. albicans MTCC 227
- - - 14±1.11 24±1.35
Values are expressed as mean±SEM (n=3) Positive control for bacteria – Ciproflaxacin Positive control for fungi – Nystatin
Table 31: Antimicrobial activity of methanolic stem extract of Adenia wightiana
Sl. No.
Microbial strain
Zone of Inhibition (mm) 0.5 mg 1 mg 2 mg 4 mg Positive
control 1. B. subtilis
MTCC 441 - - - 10±1.70 25±1.81
2. S. aureus MTCC 6908
- - - 11±1.51 29±1.98
3. E. coli MTCC 406
10±0.15 12±0.95 14±1.21 17±1.62 24±2.00
4. K. pneumoniae MTCC 530
- - - 12±1.80 33±3.10
5. A. niger MTCC 1344
- - 9±0.89 14±0.65 34±2.83
6. C. albicans MTCC 227
- - 11±1.21 13±1.33 25±2.06
Values are expressed as mean±SEM (n=3) Positive control for bacteria – Ciproflaxacin Positive control for fungi – Nystatin
Table 32: Antimicrobial activity of methanolic root extract of Adenia wightiana
Sl. No. Microbial strain Zone of Inhibition (mm) 0.5 mg 1 mg 2 mg 4 mg Positive
control 1. B. subtilis
MTCC 441 - - 13±1.10 16±1.07 27±1.49
2. S. aureus MTCC 6908
- - - 14±1.09 24±1.02
3. E. coli MTCC 406
- - 13±0.86 17±1.01 25±1.08
4. K. pneumoniae MTCC 530
- - 11±0.19 18±1.05 27±1.69
5. A. niger MTCC 1344
- - - 13±1.21 24±1.80
6. C. albicans MTCC 227
- - 9±0.88 14±1.08 27±1.11
Values are expressed as mean±SEM (n=3) Positive control for bacteria – Ciproflaxacin Positive control for fungi – Nystatin
Table 33: Minimum Inhibitory Concentration of antimicrobial activity of methanolic leaf, stem and root extract of Adenia wightiana Sl. No. Microbial strain Minimum Inhibitory Concentration (in mg)
Leaf Stem Root 1. B. subtilis
MTCC 441 2.03±0.20 1.22±0.11 2.37±0.38 2. S. aureus
MTCC 6908 3.55±0.87 1.24±0.66 1.46±0.65 3. E. coli
MTCC 406 2.37±0.61 3.70±0.73 2.67±0.47 4. K. pneumoniae
MTCC 530 3.84±0.18 2.10±0.27 2.46±0.15 5. A. niger
MTCC 1344 3.51±0.14 1.95±0.76 1.31±0.26 6. C. albicans
MTCC 227 1.36±0.55 2.03±0.58 1.95±0.22 Values are expressed as mean±SEM (n=3)
Table 34: In-vitro anti-inflammatory study of methanolic leaf, stem and root extract of Adenia wightiana
Sl. No.
Concentration (µg/ml) % Protection
Leaf Stem Root
Standard (Diclofenac sodium)
1 200 45.2±1.78 53.3±1.43 55.7±1.27 65.7±2.16 2 400 59.8±1.65 61.6±2.61 66.1±2.31 71.4±1.72 3 600 71.0±1.32 65.0±1.48 71.6±1.86 82.2±2.61 4 800 75.1±2.13 76.1±3.11 80.1±1.91 89.8±2.11 5 1000 79.4±2.47 81.4±2.51 85.7±1.62 98.9±1.32
Values are expressed as mean±SEM (n=3) Table 35: In-vitro cytotoxicity study of methanolic leaf, stem and root extract of Adenia wightiana
Sl. No. Concentration (µg/ml)
% of cell viability Leaf Stem Root Standard
(Doxorubicin) 1. 10 95.4±1.10 98.0±2.40 92.4±1.30 71.13±1.22 2. 25 85.0±3.65 87.2±4.81 82.7±1.94 63.51±1.62 3. 50 68.1±2.63 81.4±1.41 79.6±1.55 40.35±1.15 4. 100 47.2±2.43 76.9±3.84 70.1±2.90 22.81±1.24 5. 250 43.6±0.85 69.3±1.02 62.2±3.13 2.10±0.61
Values are expressed as mean±SEM (n=3)
Table 36: Animal gross behavioural study methanolic leaf, stem and root extract of Adenia wightiana
Sl. No.
Behaviour
Before drug
After drug (in minutes) 30 60 90 120 180
1. Alertness & awareness
N
N
N
N
N
N
2. Sound response N N N N N N 3. Touch response N N N N N N 4. Pain response N N N N N N 5. Gait N N N N N N 6. Grip strength N N N N N N 7. Pinna reflex N N N N N N 8. Righting reflex N N N N N N
N – Normal; Values are expressed as mean±SEM (n=3)
Table 37: In-vivo anti-inflammatory study of methanolic leaf, stem and root extract of Adenia wightiana
Sl. No.
Treatment TIME (hours)
0 1 2 3
Increase inpaw volume (mm)
% change in activity
1 Group 1 0.45±0.03 0.45±0.03 0.45±0.03 0.45±0.03 0.00 - 2 Group 2 0.40±0.04 0.73±0.01 0.86±0.02 0.87±0.01 0.47±0.02 58.88 3 Group 3 0.44±0.06 0.61±0.07 0.72±0.01 0.47±0.08 0.03±0.01 24.44 4 Group 4 0.43±0.01 0.70±0.09 0.87±0.08 0.56±0.03 0.13±0.01 42.22 5 Group 5 0.48±0.03 0.63±0.02 0.81±0.06 0.59±0.05 0.11±0.01 39.99 6 Group 6 0.42±0.01 0.67±0.05 0.83±0.02 0.54±0.03 0.12±0.01 36.66
Values are expressed as mean±SEM (n=3)
Table 38: In-vivo anti-ulcerous study of methanolic leaf, stem and root extract of Adenia wightiana
Sl. No.
Groups Enteric volume
(ml)
Enteric pH
Total acidity
Free acidity
Ulcer index
% protection
1. Group 1 5.53±0.25 2.50±0.10 68.00±2.64 55.66±1.52 10 - 2. Group 2 3.00±0.10 3.66±0.20 57.33±1.52 42.33±1.32 5.8 42 3. Group 3 3.39±0.47 3.50±0.51 61.87±3.17 45.02±3.68 6.5 35 4. Group 4 3.45±0.34 3.40±0.33 63.66±2.61 44.50±2.54 6.3 37 5. Group 5 3.50±0.20 3.50±0.26 65.33±2.08 44.66±1.50 6.2 38
Values are expressed as mean±SEM (n=3)
PLATE 1 ADENIA WIGHTIANA - MORPHOLOGY
NATURAL HABITAT A TWIG WITH FLOWERS
A TWIG WITH FRUIT A LEAF WITH GLAND AT THE BASE
STEM ROOT TUBER
PLATE 2 ADENIA WIGHTIANA – ANATOMY
T.S. OF LEAF CRYSTALS IN LAMINA
CUTICULAR STRIATION OF EPIDERMAL CELLS
EPIDERMIS SHOWING STOMATA
VEIN ISLET AND VEIN TERMINATION LOBED SCELERIDS OF THE LAMINA
AdE-Adaxial Epidermis, AdS-Adaxial side, , CM-Cuticular Mound, Cr-Crystal, GC-Guard Cell, La-Lamina, MR-Midrib, PM-Palisade Mesophyll, SC-Subsidiary Cell, Scl-Sclereid, VB-Vascular Bundle, VI-Vein Islet, VT –Vein Termination.
PLATE 3 ADENIA WIGHTIANA – ANATOMY
VEINS AND CRYSTALS IN LAMINA CRYSTAL IDIOBLASTS IN LAMINA
T.S. OF PETIOLE - ENTIRE T.S. OF PETIOLE – ENLARGED
T.S. OF STEM - ENTIRE T.S. OF STEM – ENLARGED
Cr-Crystal, , VB-Vascular Bundle, VT –Vein Termination. Ep-Epidermis, AdAB – Adaxial Accessory Bundle, CF-Cortical Fibre, Co-Cortex, DCr-Druce Crystal, ID-Idioblast, GT-Ground Tissue, MB-Median Bundle, Pi-Pith, PX-Primary Xylem, SPh-Secondary Phloem, , SX-Secondary Xylem, Ph-Phloem
PLATE 4 ADENIA WIGHTIANA – ANATOMY
T.S. OF ROOT - ENTIRE T.S. OF ROOT – ENLARGED
CRYSTALS IN ROOT TUBER STARCH GRAINS IN ROOT TUBER
VESSEL ELEMENTS TRACHEID
Cr-Crystal, Co-Cortex, Pe-Perforation, Pi-Pith, PX-Primary Xylem, SPh-Secondary Phloem, SG-Starch Grains, Tr-Tracheid, VE-Vessel Element, Ph-Phloem, XF-Xylem fibre,
PLATE 5
ANTIMICROBIAL ACTIVITY OFMETHANOLIC LEAF EXTRACT
Bacillus subtilis Staphylococcus aureus
Escherichia coli Klebsiellapneumonia
Aspergillus niger Candida albicans
PLATE 6
ANTIMICROBIAL ACTIVITY OF METHANOLIC STEM EXTRACT
Bacillus subtilis Staphylococcus aureus
Escherichia coli Klebsiella pneumonia
Aspergillus niger Candida albicans
PLATE 7 ANTIMICROBIAL ACTIVITY OFMETHANOLIC ROOT EXTRACT
Bacillus subtilis Staphylococcus aureus
Escherichia coli Klebsiella pneumonia
Aspergillusniger Candida albicans
PLATE 8 REGENERATION OF ADENIA WIGHTIANA
UNEARTHENED ROOT TUBER WITH SHOOT PLANTED STEM CUTTING
GROWTH FROM ROOT TUBER GROWTH FROM STEM CUTTING
WELL GROWN PLANT IN NATURAL HABITAT FLOWERING AND FRUITING STAGE
top related