An investigation on the Pharmacognosy, Phytochemistry and ...

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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LIST OF PLATES

Plate No. TITLE

1 : Adenia wightiana– Morphology

2 : Adenia wightiana– Anatomy



5 : Antimicrobial activity of methanolic leaf extract



8 : Regeneration of Adeniawightiana

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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

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i.p. - intra peritoneal

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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

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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

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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

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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

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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

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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

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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).

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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.

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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.

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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

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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


treated with methylene blue reagent. Change of colour to blue, indicates the presence

of mucilage.

Starch


mounted in 1% Iodine solution. Change of colour to blue, indicates the presence of

starch.

Alkaloid


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


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



Root

In root moisture content, total ash, acid insoluble ash, water soluble ash and



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


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


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


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


given in the table 22.

Root


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


least in Diethyl ether extract (34.55±1.99) in 50 µg/ml concentration. In 100 µg/ml


lowest activity in Diethyl ether (59.63±4.25). All the extracts showed dose

73



4.3.1.2. FRAP method


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


concentration. Highest activity was found in Methanol extract (33.2±0.81) and the


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



Stem



extract (18.46±1.38) and minimum in Ethyl acetate extract (13.58±1.09) in 10 µg/ml


least in Ethyl acetate extract (22.24±1.84) in 50 µg/ml concentration. In 100 µg/ml

74


lowest activity in Ethyl acetate extract (43.17±1.27). All the extracts showed dose



Root







lowest activity in Diethyl ether extract (43.11±2.17). All the extracts showed dose



4.3.1.3. Phosphomolybdenum assay


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

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(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



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



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


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


(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%.

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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

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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

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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


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

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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


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


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

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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


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


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

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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

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Figure 1 GC-MS chromatogram of methanolic leaf extract of Adeniawightiana

Figure 2 GC-MS chromatogram of methanolic stem extract of Adeniawightiana

Figure 3GC-MS chromatogram of methanolic root extract of Adeniawightiana

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


% 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


% 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


% 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


% 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


% O

F AC

TIVI

TY

SOLVENTS

STEM EXTRACT

Solvent 10 µl

Solvent 50 µl

Solvent 100 µl

020406080

100120


% 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


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


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


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 - - - -


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 - - + -


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 - - +


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


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


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


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


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


13. Phenol,2,6-dimethoxy-4-(2-propenyl)-

Phenol Not reported

14. 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol

Phenol

Antimicrobial antioxidant

antiinflammatory


15. Tetradecanoicacid Fatty acid Antioxidant Cancer preventive

Nematicide Hypocholesterolemic


16. Thiophene,2-isobutyl-5-isopentyl-

Furans Not reported

17. Hexadecanoicacid,methylester

Ester Antioxidant Hypocholesterolemic


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


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


29. Stigmasterol Steroid Antioxidant Hypoglycemic Antimicrobial

Anticancer Antiarthritic Antiasthama

Antiinflammatory Diuretic

Dandekar, et. al., 2015




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


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


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


16. Hexadecanoicacid, methylester

Fatty acid ester

Antioxidant Hypocholesterolemic


17. n-Hexadecanoicacid Fatty acid (Palmitic acid)

Antioxidant Hypocholesterolemic

Hema, et. al., 2011

18. 5,8,11-Heptadecatriynoicacid,methylester


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


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


29. Stigmasterol Steroid Antioxidant Hypoglycemic Antimicrobial

Dandekar, et. al., 2015

Anticancer Antiarthritic Antiasthama

Anti-inflammatory Diuretic




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


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



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



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



Table 25: Antioxidant activity of stem extract of Adenia wightiana - FRAP assay

Sl. No.




Table 26: Antioxidant activity of root extract of Adenia wightiana - FRAP assay

Sl. No.




Table 27: Antioxidant activity of leaf extract of Adenia wightiana –

Phosphomolybdenum assay Sl. No.




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



Table 29: Antioxidant activity of root extract of Adenia wightiana – Phosphomolybdenum assay


Sl. No.

Solvent % of Total Antioxidant Capacity 10 µl 50 µl 100 µl


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


MTCC 441 - - - 10±1.70 25±1.81


- - - 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


- - - 12±1.80 33±3.10


- - 9±0.89 14±0.65 34±2.83


- - 11±1.21 13±1.33 25±2.06


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


MTCC 441 - - 13±1.10 16±1.07 27±1.49


- - - 14±1.09 24±1.02

3. E. coli MTCC 406

- - 13±0.86 17±1.01 25±1.08


- - 11±0.19 18±1.05 27±1.69


- - - 13±1.21 24±1.80


- - 9±0.88 14±1.08 27±1.11


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


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


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


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.


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


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


Escherichia coli Klebsiella pneumonia

Aspergillus niger Candida albicans

PLATE 7 ANTIMICROBIAL ACTIVITY OFMETHANOLIC ROOT EXTRACT


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

LIST OF PLATES

An investigation on the Pharmacognosy, Phytochemistry and ...

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