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PHARMACOGNOSY, PHYTOCHEMISTRY AND PHARMACOLOGICAL STUDIES ON Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble Thesis submitted to the Pondicherry University In partial fulfillment of the requirements for the award of the Degree of Doctor of Philosophy in Botany By G. ANANDHI Under the guidance of Dr. A. PRAGASAM DEPARTMENT OF BOTANY KANCHI MAMUNIVAR CENTRE FOR POST-GRADUATE STUDIES PUDUCHERRY-605 008 August - 2014

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Page 1: PHARMACOGNOSY, PHYTOCHEMISTRY AND ...14.139.183.117/jspui/bitstream/1/2058/1/T5786.pdfPHARMACOGNOSY, PHYTOCHEMISTRY AND PHARMACOLOGICAL STUDIES ON Tricalysia sphaerocarpa (Dalzell

PHARMACOGNOSY, PHYTOCHEMISTRY AND

PHARMACOLOGICAL STUDIES ON

Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble

Thesis submitted to the

Pondicherry University

In partial fulfillment of the requirements

for the award of the Degree of

Doctor of Philosophy in Botany

By

G. ANANDHI

Under the guidance of

Dr. A. PRAGASAM

DEPARTMENT OF BOTANY

KANCHI MAMUNIVAR CENTRE FOR POST-GRADUATE STUDIES

PUDUCHERRY-605 008

August - 2014

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PHARMACOGNOSY, PHYTOCHEMISTRY AND

PHARMACOLOGICAL STUDIES ON

Tricalysia sphaerocarpa (Dalzell ex Hook. F,) Gamble

Thesis submitted to the

Pondicherry University

In partial fulfillment of the requirements

for the award of the Degree of

Doctor of Philosophy in Botany

Submitted by

G. ANANDHI

Under the guidance of

Dr. A. PRAGASAM

DEPARTMENT OF BOTANY

KANCHI MAMUNIVAR CENTRE FOR POST-GRADUATE STUDIES

PUDUCHERRY-605 008

August - 2014

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Dr. A. PRAGASAM

Research Supervisor

Department of Botany

Kanchi Mamunivar Centre for PG Studies

Puducherry-605 008

CERTIFICATE

This is to certify that the PhD research work entitled “Pharmacognosy,

Phytochemistry and Pharmacological studies on Tricalysia sphaerocarpa (Dalzell ex

Hook. f,) Gamble” is based on the original work done by Mrs. G. ANANDHI,

Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Puducherry

and this has not previously formed the basis for the award of any degree, diploma,

associateship, fellowship or any other similar title and it represents entirely an

independent work on the part of the candidate.

I further state that the entire thesis represents the independent work of G.

Anandhi and all the experimental techniques employed in this work were actually

undertaken by the candidate herself under my guidance.

Place: Puducherry (A. PRAGASAM)

Date:

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G. ANANDHI

Ph.D. Research Scholar

Department of Botany

Kanchi Mamunivar Centre for Post Graduate Studies

Puducherry- 605 008

DECLARATION

I, Mrs. G. ANANDHI hereby declare that the research work entitled

“Pharmacognosy, Phytochemistry and Pharmacological studies on Tricalysia

sphaerocarpa (Dalzell ex Hook. f,) Gamble” submitted for the award of the Degree of

Doctor of Philosophy in Botany is my original work and has not previously formed the

basis for the award of any degree, diploma, associateship, fellowship or any other similar

title.

(G. ANANDHI)

Place: Puducherry

Date:

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ACKNOWLEDGEMENT

I deeply express my sincere gratitude to my guide and research supervisor,

Dr. A. Pragasam, Department of Botany, Kanchi Mamunivar Centre for Post

Graduate Studies, Puducherry for his excellent guidance, inspiration, continued

support and critical perusal of thesis.

I would like to thank Dr.V.Ananthan the present Director,

Dr.R.Swaminathan, Dr.V.Ramassamy, Dr.E.M.Rajan and Dr.O.P.Shyma the

former Directors, Kanchi Mamunivar Centre for PG Studies, Puducherry for

providing necessary facilities to carry out my project successfully.

I extend my thanks to my doctoral committee members, Dr. D.

Ramamoorthy, Associate Professor, Department of Ecology & Environmental

Sciences, Pondicherry University, and Dr. B. K. Nayak, Associate Professor,

Department of Botany, Kanchi Mamunivar Centre for PG Studies, Puducherry, for

providing valuable suggestions during the doctoral committee meetings.

I am grateful to Dr.V. Jayachandran the present Head of the Department,

Dr.D.Kadamban and Dr.S.Nadanakunjidam former Heads of the Department of

Botany, Kanchi Mamunivar Centre for PG Studies, Puducherry, for their

encouragement.

I express my sincere thanks to the faculty member of Botany, Dr. K.

Rajendiran for giving constant support in completing my research programme.

My heartfelt thanks to my husband R. Sachithanantha Kumar and my

beloved parents for their constant encouragement and continued support to complete

the research work successfully.

Mrs. G. ANANDHI

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CONTENT TITLE Page No.

1. Introduction 1-11

1.1. Pharmacognosy

1.2. Phytochemistry

1.2.1. Phytochemical Revolution

1.3. Pharmacology

1.3.1. Antioxidant Activity

1.3.2. Anti - Depressant Activity

1.3.3. Anti - Diabetic Activity

2. Review of Literature 11-18

2.1. Family Rubiaceae

2.2. Objectives of the Present Study

3. Materials and Methods 19-50

3.1. Collection of Plant material

3.2. Taxonomy of the Species

3.3. Morphological Features

3.4. Ecology

3.5. Medicinal Uses

3.6. Chemical constituents isolated from the different species of Tricalysia

3.7. Pharmacognostical Studies

3.7.1. Anatomical studies

3.7.2. Histochemical Colour Reactions

3.7.3. Fluorescence Analysis

3.8. Phytochemistry

3.8.1. Physio - Chemical Constants

3.8.2. Preparation of the Extracts

3.8.3. Extractive Values

3.8.4. PH Determination of Powdered Drug

3.8.5. Preliminary Phytochemical Screening

3.8.6. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

3.9. Pharmacology

3.9.1. In vitro Antioxidant activity

3.9.1.1. Inhibitiory effects on DPPH Radical Assay

3.9.1.2. Hydrogen peroxide Assay

3.9.1.3. Superoxide dismutase (L-methionine and NBT) assay

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3.9.1.4. Iron Chelating Activity (FRAP)

3.9.2. In vivo Pharmacological Studies

3.9.2.1. Acute Toxicity Studies

3.9.2.2. Anti-Depressant Activity

3.9.2.2.1. Forced Swimming Test (FST)

3.9.2.2.2. Tail Suspension Test (TST)

3.9.2.2.3. Hole Board Test (HBT)

3.9.2.3. Anti Diabetic Activity

3.9.2.3.1. Screening of Hypoglycemic Activity in Normal Rats

3.9.2.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats

3.9.2.3.2.1. Single-Dose Short Term Study

3.9.2.3.2.2. Multi- Dose Long Term Study

3.9.2.3.3. Effect of Formulation Test Extract on Body Weight in Normal and

Alloxan Induced Diabetic Rats

3.9.2.3.4. Biochemical Parameters Determinations

3.9.2.3.5. Histopathological Studies

4. Results 51-76

4.1. Pharmacognosy

4.1.1. Anatomy

4.1.1.1. Leaf peeling

4.1.1.2. Venation Pattern

4.1.1.3. Quantitative Values of Foliar Epidermis

4.1.1.4. Stem Peeling

4.1.1.5. Maceration

4.1.1.6. Transverse Section of Leaf

4.1.1.7. Transverse Section of Stem

4.1.1.8. Transverse Section of Root

4.1.2. Histochemical Colour Reactions

4.1.3. Fluorescence Analysis

4.2. Phytochemistry

4.2.1. Physico-Chemical Parameters of Various Parts

4.2.2. Extractive Values

4.2.2.1. Batch Process

4.2.2.2. Successive Process

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 Methanolic Extract of Leaf

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4.2.5.2. GC-MS Analysis of Methanolic Extract of Stem

4.2.5.3. GC-MS Analysis of Methanolic Extract of Root

4.2.5.4. GC-MS Analysis of Methanolic Extract of Fruit

4.2.5.5. Comparative Analysis of Compounds Identified by GC-MS Analysis

4.3. Pharmacology

4.3.1. Invitro Antioxidant Activity

4.3.1.1. DPPH Scavenging Activity

4.3.1.2. Iron Chelating Activity (FRAP)

4.3.1.3. Hydrogen peroxide Assay

4.3.1.4. Superoxide dismutase (L-methionine and NBT) assay

4.3.2. In vivo Pharmacological Studies

4.3.2.1. Acute Toxicity Studies

4.3.2.2. Anti-Depressant Activity

4.3.2.2.1. Forced Swimming Test (FST)

4.3.2.2.2. Tail Suspension Test (TST)

4.3.2.2.3. Hole Board Test (HBT)

4.3.2.3. Anti Diabetic Activity

4.3.3.1. Screening of Hypoglycemic Activity in Normal Rats

4.3.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats

4.3.3.2.1. Single-Dose Short Term Study

4.3.3.2.2. Multi- Dose Long Term Study

4.3.3.3. Effect of Formulation Test Extract on Body Weight in Normal and

Alloxan Induced Diabetic Rats

4.3.3.4. Biochemical Parameters Determinations

4.3.3.5. Histopathological Studies

5. Discussion 77-93

5.1. Pharmacognosy

5.2. Phytochemistry

5.2.1. Phytochemical Screening

5.2.2. GC-MS Analysis

5.3. Pharmacognosy

5.3.1. Antioxidant Activity

5.3.2. Anti - Depressant Activity

5.3.3. Anti - Diabetic Activity

6. Summary and Conclusions 94-98

7. Bibliography 99-112

8. Publications

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List of Tables

Table 1: Quantitative values of foliar epidermis of Tricalysia sphaerocarpa

Table 2: Histochemical colour reactions of various parts of Tricalysia sphaerocarpa

Table 3 : Fluorescence analysis of Leaf powder of Tricalysia sphaerocarpa

Table 4 : Fluorescence analysis of Stem powder of Tricalysia sphaerocarpa

Table 5: Fluorescence analysis of Root powder of Tricalysia sphaerocarpa

Table 6: Fluorescence analysis of Fruit powder of Tricalysia sphaerocarpa

Table 7: Proximate analysis of various parts of Tricalysia sphaerocarpa

Table 8: Extractive values of various parts of Tricalysia sphaerocarpa by batch process Table 9: Extractive values of various parts of Tricalysia sphaerocarpa by successive

process Table 10: PH Determination of water extract of Tricalysia sphaerocarpa

Table 11: Phytochemical colour reactions of various extracts of Leaf of Tricalysia sphaerocarpa

Table 12: Phytochemical colour reactions of various extracts of Stem of Tricalysia

sphaerocarpa Table 13: Phytochemical colour reactions of various extracts of Root of Tricalysia

sphaerocarpa Table 14: Phytochemical colour reactions of various extracts of Fruit of Tricalysia

sphaerocarpa Table 15: GC-MS Analysis of Methanol extract of Leaf of Tricalysia sphaerocarpa

Table 16: GC-MS Analysis of Methanol extract of Stem of Tricalysia sphaerocarpa

Table 17: GC-MS Analysis of Methanol extract of Root of Tricalysia sphaerocarpa

Table 18: GC-MS Analysis of Methanol extract of Fruit of T. sphaerocarpa

Table 19: Combined table for GC-MS Analysis of Methanol extract of Tricalysia sphaerocarpa

Table 20: Chemical groups obtained from GC-MS Analysis of Methanol extract of

Tricalysia sphaerocarpa Table 21: Antioxidant activity of various extracts using DPPH assay

Table 22: Antioxidant activity of various extracts using Iron chelating activity

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Table 23: Antioxidant activity of various extracts using Hydrogen peroxide assay

Table 24: Antioxidant activity of various extracts using Superoxide dismutase assay

Table 25 : Effect of Methanolic extract of Tricalysia sphaerocarpa on Immobility time in FST

Table 26: Effect of Methanolic extract of Tricalysia sphaerocarpa in Immobility time in

TST Table 27: Effect of Methanolic extract of Tricalysia sphaerocarpa. in Hole Board Test

(HBT) Table 28: Effect of Test extract of Tricalysia sphaerocarpa on blood glucose level in

normal fasted rats Table 29: Effect of Test extract of Tricalysia sphaerocarpa on blood glucose level in

Alloxan-induced diabetic rats (Single-dose short term study) Table 30: Effect of multidose administration of Test extract of Tricalysia sphaerocarpa

on blood glucose level in Alloxan-induced diabetic rats (long term study of 15 days daily once)

Table 31: Effect of formulation Test extract of Tricalysia sphaerocarpa on body weight

in Normal and Alloxan induced diabetic rats Table 32: Effect of formulation Test extract of Tricalysia sphaerocarpa on Biochemical

parameters in Alloxan induced diabetic rats.

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List of Figures

Figure 1: Extractive values of various parts of Tricalysia sphaerocarpa by Batch process Figure 2: Extractive values of various parts of Tricalysia sphaerocarpa by Successive process Figure 3: GC-MS chromatogram of Methanolic Leaf extract of Tricalysia sphaerocarpa

Figure 4: GC-MS chromatogram of Methanolic Stem extract of Tricalysia sphaerocarpa

Figure 5: GC-MS chromatogram of Methanolic Root extract of Tricalysia sphaerocarpa

Figure 6: GC-MS chromatogram of Methanolic Fruit extract of Tricalysia sphaerocarpa

Figure 7: (a-f) Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

Figure 8: Anti oxidant activity - DPPH Scavenging assay

Figure 9: Anti oxidant activity - Iron chelating activity (FRAP)

Figure 10: Anti oxidant activity - Hydrogen peroxide assay

Figure 11: Anti oxidant activity - Superoxide dismutase (L-methionine and NBT assay)

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List of Plates

Plate 1: Morphology of Tricalysia sphaerocarpa

Plate 2: Leaves of Tricalysia sphaerocarpa

Plate 3: Size and orientation of stomata in Tricalysia sphaerocarpa

Plate 4: Vein islet and Veinlet termination of Tricalysia sphaerocarpa

Plate 5: T. S of Stem of Tricalysia sphaerocarpa

Plate 6: T. S of Root of Tricalysia sphaerocarpa

Plate 7: Macerated elements of stem of Tricalysia sphaerocarpa

Plate 8: Effect of Methanolic stem extract of Tricalysia sphaerocarpa –Anti-Depressant Activity

Plate 9: Effect of Methanolic stem extract of Tricalysia sphaerocarpa - Anti-Diabetic Activity -

Histopathological Studies

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1

CHAPTER 1

INTRODUCTION

Human beings came on this earth as guests of plants is a monumental

ancient aphorism. Nature is the supreme creation and man has completely been

dependent on plants. As population increased, he has learnt to implicit natural

resources and to make use of every bit of it. Man since creation has depended on

plants for food, drinks, shelter, clothing, equipment, dental care and medicine

(Gbile, 1986). In fact from the start of life to the last breath, almost every aspect

of human life is deeply associated with plants. Primitive man tried to cure

diseases from plants growing abundantly around him. His experience through trial

taught him a lot about the medicinal properties of different plants. India is

endowed with vast resources of medicinal and aromatic plants. These plants have

been used in Indian health systems. The great interest in the use and importance

of Indian medicinal plants by world health organization in many developing

countries has let to intensify efforts on the documentation of ethno medicinal data

of medicinal plants (Perumalsamy and Ignacimuthu, 2000).

Our forefathers were depending on plants for treatment of various diseases

before the introduction of orthodox medicine. Ancient literatures of world on

medicines suggest that the primitive people of antiquity and those of earlier

centuries have been using several kinds of medicinal plants for combating

diseases. China used drug plants as early as 5000 to 4000 BC. India has over

3000 year-old medicinal heritage based on herbs. The sacred Vedas and other

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ancient Indian treatises give many references of these medicinal plants. The

ancient Indian treatise ‘Rig veda’ deals with medicinal plants. Indians classified

plants into three groups on the basis of their usage as ‘Ubhdida’ (botanical),

‘Annapanandi’ (dietic) and ‘Virechandi’ (medicinal). Parashara

wrote’Virkshayerveda’ describing medicinal plants much before the beginning of

Christian era (Saxena, 1989). There are references of miracle herbs and wonder

drugs in the ancient Indian literatures which had magical properties and were used

to cure some of the incurable diseases from tip to the toe, to increase longevity

and even to bring the dead back to life. The charak and sushrut samhitras were

written between 700-200 BC, and include accounts of the discovery of medicinal

plants (Pandey and Verma, 2005). The Assyrians, Babylonians and Ancient

Hebrews were all familiar with the usage of plants. The Greeks were familiar with

many of the present day drugs, as evidenced by the works of Aristotle,

Hippocrates (Father of medicine), Pythagoras, Theophrastus, Pleny and Galen. In

77 BC Dioscorides wrote his great treastise, “De Materia Medica” which dealt

with the nature and properties of all the medicinal substances known at that time.

The foremost classical work in botany of medicinal flora in the world ‘Hortus

Malabaricus’ was written by Heinrich Van Rheede in Kerala, India. India is now

beginning to search her roots in the past and revive her lost glory of the traditional

system of medicine which flourished here for several centuries and contributed

much to the development of medicinal science to the world. From this crude

beginning the study of drugs and drug plants has progressed until now as

pharmacognosy and pharmacology which are the essential branches of medicine.

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The most valuable of the drug and drug plant has been standardized as a result of

the Pure Food and Drug act of 1906 (Hill, 1972).

Herbal medicine is known in every village and communes of India and

every village has elders both men and women who have acquired knowledge

about the medicinal properties of plants through long tradition and experience. In

the past, sickness was viewed as a punishment of the God and hence was treated

with prayers and rituals that included what may have been considered “magic

portion” prepared from local herbs (Sandhya et al., 2010). Plants produce a wide

variety of compounds that can act on different systems of the body and have high

therapeutic activity. More than 2,40,000 plants are considered to be growing in

different parts of the world. Only about 5-10 percent of them have been screened

for chemical or biological activity. Herbal medicine cures the root cause of a

disease and not merely providing symptomatic relief, as does the modern

synthetic medicine. Thus, traditional medicine not only cures but also rejuvenates

the body’s defense system. The medicine and aromatic plants sector has

traditionally occupied an important position in the socio-cultural, spiritual and

medicinal arena of rural and tribal lives of India (Battacharrya et al., 2005).

Nature keeps ready within its ‘green bag’ substances which would

promptly act to neutralize the effect of any such substance proving unsuitable and

non-compatible to the human body. Chemical investigations of wild medicinal

plants used by the indigenous people of world shows unknown compounds with

promising biological activity. Indigenous culture has provided several ‘miracle

plants’ of immense food and medicinal value to the modern civilization. Seventy

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four percent of 119 plant derived drugs were discovered as a result of chemical

studies to isolate the active substances responsible for their traditional use

(Farnsworth and Soejarto, 1991). So, plants, especially the higher plants contain a

variety of substances, which are useful as food additives, perfumes and in

treatment of various diseases as medicines due to their versatile therapeutic

potential (Mukherjee and Wahile, 2006). The active secondary metabolites

possess various medicinal applications as drugs or as model compounds for drug

synthesis. Large scale evaluation of the local flora exploited in traditional

medicine for various biological activities is a necessary first step in the isolation

and characterization of the active principle and further leading to drug

development. The identification of drug yielding plants, crude drugs obtained

from them, identification of crude drugs, extraction of the principle drugs, study

of their antimicrobial activities and their potential use as antioxidants are essential

to evolve new natural curatives instead of antibiotics. The worldwide experiments

in these fields are related to pharmacognosy, phytochemistry, and

pharmacological investigations.

1.1. Pharmacognosy:

Pharmacognosy is defined as the scientific and systematic study of

structural, physical, chemical and sensory characters of crude drugs along with

their history, method of cultivation, collection and preparation for the market

(Evans, 1996). Identification of drugs can be done by morphological, histological

and chemical testing. There are five methods of evaluation crude drugs namely

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Morphlolgical or Organoleptic, Microscopical or Histological, Physical, Chemical

and Biological.

Organoleptic evaluation means the study of morphological characteristics

by which the drugs are identified. It also includes those of colour, odour, taste,

consistency of powdered drug, size, shape etc. Micorscopical evaluation is useful

for organized drugs. If the drug is in entire form which we can take transverse or

longitudinal sections and study the cellular structures. Surface preparations can be

studied for stomata or trichomes. If the drug is in powder form, microscopic

identification is done to identify the parts of the crude drug. The measurement of

length, diameter of structures also helps in identification. Physical standards are

studied as under refractive index, moisture content, viscosity, melting point,

optical rotation and solubility of crude drugs. The evaluation of drug can be done

by chemical method such as assays, extractive values, volatile oil content, ash

content and drugs standardized by chemical tests. In the biological method of

estimation of potency of a crude drug is done by means of its effect on living

organisms such as other plants, animals, microbes etc.

1.2. Phytochemistry:

Phytochemistry includes drug development from natural origin,

establishment of botanical identity of herbs, phytochemical isolation and

identification, screening of herbal formulations and isolated compounds.

Phytochemicals (or) secondary metabolites are a wide range of low molecular

weight chemical compounds that are produced and accumulated by the plants.

These include alkaloids, phenolic acids, flavanoids, steroids, terpenoids and

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saponins. Phytochemical analysis of plants, used in folklore has yielded a number

of compounds with various pharmacological activities. Hence medicinal plants

are important substances for the study of their traditional uses through the

verification of pharmacological effects and can be natural composite sources that

act as a disease curing agents. About 3000 materials from 2764 plant species have

been screened for their pharmacological and chemotherapeutic properties (Anon,

1988). Traditionally used medicinal plants produce a variety of compounds of

known therapeutic properties (Iyengar, 1976; Harbone, 1989; Chopra et al.,

1992).

1.2.1. Phytochemical Revolution:

Even modern medicines and some very valuable drugs such as morphine,

digitoxin, reserpine, vinblastine, quinine etc. are obtained from the plants.

Cocaine derived from Erythroxylum cacao lead to the synthesis of procaine and

other related anesthetics. Salicin obtained from Salix purpurea, lead to the

synthesis of acetyl salicylic acid (aspirin). Morphine and codeine from Papaver

somniferum and P. bracteatum lead to the synthesis of pain killer. Anti cancerous

drug taxol is obtainted from Taxus wallichiana and T. buccata. Synthetic anti

cholinergic drugs like atropine and scopolamine are obtained from Atropa

belladonna and A. acuminata. Tinospora cordifolia has been reported to stimulate

indigenous insulin secretion by the pancreas (Gupta, 1967).

1.3. Pharmacology

Parmacology is the study of the relevant forms of knowledge, practice and

cultures implementing them the role of natural products, herbal medicines, tribal

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and traditional medicines is being increasingly appreciated during the recent years

for the prevention and cure of human ailments (Janardhanan et al., 2006)

1.3.1. Antioxidant Activity

Antioxidation compounds in food play an important role as a health

protecting factor. Scientific evidence suggests that antioxidants reduce the risk for

chronic diseases including cancer and heart disease. Primary sources of naturally

occurring antioxidants are whole grains, fruits and vegetables. Plant sourced food

antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and

phytoestrogens have been recognized as having the potential to reduce disease

risk. Most of the antioxidants in a typical diet are derived from plant sources and

belong to various classes of compounds with a wide variety of physical and

chemical properties. Some compounds, such as gallates, have strong antioxidant

activity, while others, such as the mono-phenols are weak antioxidants. The main

characteristic of an antioxidant is its ability to trap free radicals.

In recent years, there has been great interest in screening various plant

extracts for natural antioxidants because of their great free radical sacvenging

properties (Jia et al., 2007). Antioxidants neutralize reactive oxygen which cause

stress, diseases of our cells and inflict damage to biomolecules, resulting in aging

and genetic changes that lead to cancer. Common sources of antioxidants are

fruits, vegetables and medicinal plants. Therefore, a great number of different

spices and aromatic herbs have been investigated for antioxidant activity

(Erdemoglu et al., 2006). Antioxidants are widely used as food additives to

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provide protection against oxidative degradation of foods by free radicals (Gulcin

et al., 2002).

1.3.2. Anti - Depressant Activity

According to the World Health report (WHO, 2001), approximately 450

million people suffer from mental or behavioral disorder, yet only a small

minority of them receive even the most basic treatment. This amounts to 12.3% of

the global burden of disease, and will rise to 15% by 2020 (Reynolds, 2003).

Major depression, a debilitating psychiatric disorder, is predicted to be the second

most prevalent human illness by the year 2020. Various antidepressants, ranging

from monoaminoxidase inhibitors to recently developed dual reuptake inhibitors,

are prescribed for alleviating the symptoms of depression. The common

symptoms of major depression include depressed or irritable mood, decreased

interest in pleasurable activities, significant weight loss or gain, insomnia or

hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy,

feeling of worthlessness or excessive guilt, decreased concentrating power, and

increase in suicidal tendencies. Earlier, major depression was considered to be an

old-age disease. However, current trends reveal an increased percentage of

younger populations being affected from its consequences. Major depression is

relatively common among patients with a diagnosis of dementia (Ballard et al.,

1996, Stepaniuk et al., 2008) and also may pose a risk factor for development of

dementia (Kokmen et al., 1996). Despite the availability of these blockbuster

molecules, approximately 30% of depressed patients do not respond to the

existing drug therapies and the remaining 70% fail to achieve complete remission

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(Kulkarni et al., 2009). Herbal drug used in depression are Centella asiatica ,

Hypericum perforatum, Rhodiola rosea, Pfaffia paniculata, Rauwolfia serpentina,

Rhododendron molle, Schizandra chinesis, Thea sinensis, Uncaria tomentosa,

Valeriana officinalis and Withania somnifera (Mamedov, 2005). Moreover,

antidepressants are associated with a plethora of side effects and drug-drug/drug-

food interactions. In this context, novel approaches are being tried to find more

efficacious and safer drugs for the treatment of major depression.

1.3.3. Anti - Diabetic Activity

In India, the prevalence of diabetes mellitus is on increase and needs to be

addressed appropriately. A study of ancient literature indicates that diabetes

(madhumeha) was fairly well known and well conceived as an entity in India. The

knowledge of the system of diabetes mellitus, as the history reveals, existed with

the Indians since prehistoric age. 'Madhumeha' is a disease in which a patient

passes sweet urine and exhibits sweetness all over the body, i.e. in sweat, mucus,

breathe, blood, etc. Diabetes mellitus is a serious complex chronic condition that

is a major source of ill health worldwide. This metabolic disorder is characterized

by hyperglycemia and disturbances of carbohydrate, protein, and fat metabolisms,

secondary to an absolute or relative lack of the hormone insulin. Besides

hyperglycemia, several other factors including dislipidemia or hyperlipidemia are

involved in the development of micro and macrovascular complications of

diabetes that are the major causes of morbidity and death (Kameswararao, 2003).

According to WHO projections, the prevalence of diabetes is likely to increase by

35%. Currently, there are over 150 million diabetic patients worldwide and this is

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likely to increase to 300 million or more by the year 2025. Statistical projection

about India suggests that the number of diabetics will rise from 15 million in 1995

to 57 million in the year 2025, the highest number of diabetics in the world

(Satyanarayana, 2006). Reasons for this rise include increase in sedentary

lifestyle, consumption of energy-rich diet, obesity, higher life span, etc. Other

regions with greatest number of diabetics are Asia and Africa, where diabetes

mellitus rates could rise to twofold to threefold than the present rates (Eidi, 2006).

Evaluation of plant products to treat diabetes mellitus is of growing interest as

they contain many bioactive substances with therapeutic potential. In recent years,

several authors evaluated and identified the antidiabetic potential of traditionally

used Indian medicinal plants using experimental animals. Previous studies

confirmed the efficacy of several medicinal plants in diabetes mellitus. Although

a large number of medicinal plants have been already tested for their antidiabetic

effects, these effects remain to be investigated in several other Indian medicinal

plants(Sharma, 1994). Herbal remedies are considered convenient for

management of type 2 diabetes with postprandial hyperglycemia due to their

traditional acceptability and availability, low costs and lesser side effects.

The application of systems biology technologies and approaches, that is,

genomics, proteomics and metabolomics, to phytomedicine research may greatly

assist evidence-based phytotherapeutics, and such research may also lead to a

change of paradigm in the development and application of complex plant/

phytochemical compound mixtures in modern medicine (Ulrich et al., 2007). So

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the present work has been taken up to evaluate the medicinal potential of

Tricalysia spherocarpa (Dalzell ex Hook. F.) Gamble

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CHAPTER 2

REVIEW OF LITERATURE

Consumption of fruits and vegetables is shown to lower the risk for

chronic diseases such as cancer, cardiovascular diseases and stroke. The positive

health effects may be due to high contents of certain phenolic compounds in

plant-derived foods. Recently, phytochemicals and their effects on human health

have been intensively studied. In particular, a search for antioxidants,

hypoglycemic, and anticancer agents in vegetables, fruits, teas, spices and

medicinal herbs has attracted great attention.

A rich literature is available on the studies of Indian medicinal plants. The

studies of Chaudhuri (1965) on Calophyllum inophylum, Kannabiran and

Krishnamurthy (1972) on Anisomeles malabarica, Nayar et al., (1976) on

Aristalochia tagala, Krishnamurthy and Kannabiran (1982) on Caesalpinia crista,

Santha et al., (1988) on Nilgirianthus heyneanus, Ahmad (1994) on Jatropha

curcas, Nambiar et al., (1996) on Hemidesmus indicus, Seetharam et al., (1999)

on Eclipta alba, Annamalai et al., (2000) on Phyllanthus amarus, Srivastava

(2001) on Curcuma amada, Amerjothy (2003) on Spilanthes calva, Suseela and

Prema (2007) on Lagascea mollis, Devika and Sajitha (2007) on Phyllanthus

niruri, Jushi Singh (2011) on many endangered medicinal plants are some of the

examples to be mentioned.

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This chapter encompasses the published literature on medicinal uses and

various studies on the genus Tricalysia a member of the family Rubiaceae which

is chosen for the present study.

2.1. Family Rubiaceae

Rubiaceae are a family of flowering plants, variously called the coffee

family, madder family or bedstraw family. Members of the coffee family tend to

be concentrated in warmer and tropical climates around the world. Currently,

about 611 genera and more than 13,000 species are placed in Rubiaceae. This

makes it the fourth-largest family of flowering plants by number of species, and

fifth largest by number of genera. The group contains many commonly known

plants, including the economically important coffee (Coffea), quinine (Cinchona),

and gambier (Uncaria), the medicinal ipecacuanha (Carapichea ipecacuanha),

and the horticulturally valuable madder (Rubia), west indian jasmine (Ixora),

partridgeberry (Mitchella), Morinda, Gardenia, and Pentas.

During the survey of Rubiaceous taxa, it was investigated that most of the

plants of this family are of great medicinal value. Several ailments like ulcers,

dysentery, athlete’s foot, diabetes, whooping cough, bronchitis, asthma, migraine

etc. are successfully cured by the use of the plants. Some plants of family

Rubiaceae are of miraculous importance which are used in the treatment of snake

bite, scorpion sting, regulation of menses and securing the birth of male child. A

very poor attention has still been paid on family Rubiaceae regarding its

medicinal properties.

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The genus Tricalysia comprises of about 50 species in subtropical and

tropical regions of Asia and Africa (Xiao et al., 1987, He et al., 2002), 2 species

were reported from Western Ghats and Courtallum in Tinnevelly District of Tamil

Nadu state (Gamble, 1986). The International Plant Names Index (IPNI) includes

187 species of Tricalysia (http://www. Plant systematics. org). Tricalysia A. Rich.

comprises of about 10 spp. in Tropical Africa, Madagascar and few in Indomalaya

(George Usher, 1984). It is also found in central and south Maharashtra Sahyadris

(Almeida, 2001). Tricalysia sphaerocarpa (Dalzell ex Hook. F.) Gamble is not

recorded from the tropical dry ever green forest. This species is known only from

Western Ghats and its occurrence is uncommon for the entire east coast and could

be considered to be the relict of the past wetter regimes of the Cuddalore district

(Israel Oliver King, 2004).

Parthasarathy et al., (2008) reported that Tricalysia sphaerocarpa and

Lepisanthes tetraphylla are the dominant evergreen trees in Thirumanikuzhi

sacred grove (Cuddalore, Tamil Nadu). Tricalysia sphaerocarpa is the most

abundant species in Kuzhanthaikuppam (Cuddalore, Tamil Nadu), probably due

to past disturbance (Mani and Parthasarathy, 2006). The wild coffee Tricalysia

sphaerocarpa contributed 50 % of multistemmed individuals in Arasadikuppam

and the site was dominated by 33% of the stand (Venkateswaran and

Parthasarathy, 2003). Anbarashan and Parthasarathy (2013) have reported that

Tricalysia sphaerocarpa formed 72% of the forest stand density at S.Pudhoor.

The studies of Mike O. Soladoye et al., (2010) on ethnobotanical survey

of anti-cancer plants in Ogun state, Nigeria, revealed that the bark of Tricalysia

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macrophylla along with some other plants and the fruit juice of Citrus medica is

used to cure cancer. Chris Long (2005) studied the ethnobotanical uses of

Tricalysia capensis and Tricalysia lanceolata. Moshi et al., (2009) studied the

ethnobotanical uses of Tricalysia coriacea and Tricalysia coriacea sbsp.

Nyassae. George usher (1984) studied the ethnobotanical uses of Tricalysia

sphaerocarpa. Prajapat and Kumar (2005) studied the ethnobotanical uses of

Tricalysia sphaerocarpa and Tricalysia singularis.

The bioassay-guided fractionation scheme identified the triterpenoids

ursolic and oleanolic acids from Tricalysia niamniamensis Hiern, demonstrated

DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid.

Protolichesterinic acid, swertifrancheside and fulvoplumierin represent three

additional natural-product structural classes that inhibit hLI (human Ligase I).

Fagaronine chloride and certain flavonoids are also among the pure natural

products that were found to disrupt the activity of the enzyme, consistent with

their nucleic acid intercalative properties. Further analysis revealed the step of the

ligation reaction, indicating a direct interaction with the enzyme protein(Tan et

al., 1996).

He et al., (2002) isolated seven rearranged ent-Kaurane glycosides, named

tricalysiosides A-G(1-7) from the leaves of Tricalysia dubia collected from

Okinawa island. Their C-18 and 19 methyls were found to have rearranged to

form and alpha, beta- unsaturated gamma-lactone ring, with other functional

groups remotely located only on C-15,-16, and -17 of the five membered ring.

Using X-ray crystallographic analysis, the structure of tricalysioside A(1) was

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determined. On the basis of the crystal structure of 1, the structures of the other

tricalysiosides (2-7) were also established.

He et al., (2005) isolated eight ent-kaurane glucoside from the leaves of

Tricalysia dubia. The structure of tricalysioside H (1) was established by X-ray

crystallography and those of tricalysiosides I-O (2-8) were elucidated by analysis

of spectroscopic evidence.

Four rearranged ent-kaurane diterpenoid alkaloids, tricalysiamides A-D

(1-4) having a cafestol-type carbon framework were isolated from the wood of

Tricalysia dubia. Their absolute structures were determined on the basis of 2D

NMR spectroscopy, X-ray crystallographic analysis and chemical methods

(Nishimura et al., 2007).

Tamaki et al., (2008) isolated 2 new rearranged ent-kaurane derivatives

namely tricalysiolides H and I from the EtOAc-soluble fraction of an MeOH

extract of the stem of Tricalysia dubia, together with 5 known rearranged ent-

kauranes, i.e. tricalysiolides A-E, stigmast-4-en-6beta-ol-3-one, (+)-pinoresinol,

scopoletin and syringaldehyde. Their structure were elucidated from the

spectroscopic evidence, and their cytotoxicity toward KB cells and P-gp

inhibitory activity were assayed.

Shitomoto et al., (2010) isolated one new megastigmane gentiobioside

namely tricalysionoside A (1) and 3 sulfates, named sulfatricalysines A-C (2-4)

from the water-soluble fraction of a MeOH extract. Sulfatricalysines D-F(5-7)

and 3 new ent-kaurane glucosides namely tricalysiosides X-Z(8-10) from the 1-

BuOH –soluble fraction of a MeOH extract of leaves of Tricalysia dubia.

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Xu et al., (2010) isolated two new ent-kaurane glycosides namely

tricalysiosides V and W, with an acylated diasaccharide moiety at the C-3

position from the roots of Tricalysia okelensis and their structures established by

spectroscopic and chemical methods.

The studies of Anandhi and Pragasam (2013a) on pharmacognostical and

preliminary phytochemical studies on leaf extracts of Tricalysia sphaerocarpa

revealed the marked presence of carbohydrate, glycosides, alkaloids, tannin,

flavanoids, moderate presence of protein, phenol, terpenoids and saponin and

absence of triterpenoids, anthraquiones, catachins, coumarins. Anandhi and

Pragasam (2013b) identified 17 phytochemicals from the stem of Tricalysia

sphaerocarpa through GC-MS analysis. They have concluded that the plant is

highly valuable in medicinal usage for the treatment of various human ailments

along with the chemical constituents present in it. The compounds need further

research on toxicological aspects to develop safe drug. Anandhi et al., (2014)

identified 30 phytochemicals from the methanolic extract of leaves of Tricalysia

sphaerocarpa through GC-MS analysis among which fatty acid was the major

group consists of 9 compounds. Eicosanoic acid was found to be present as the

major compound with peak area 35.77% and retention time 21.865 min, followed

by octadecanoic acid (18.81%).

2.2. Objectives of the Present Study

Keeping the view of significances of traditional medicine in the field of

plant-based drug discovery, the important Indian medicinal plant, Tricalysia

sphaerocarpa was selected to carry out the following tasks.

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1. Pharmacognosy

• Anatomical studies

• Histochemical localization

• Fluorescence analysis

2. Phytochemistry

• Physico-chemical parameters of various parts

• Successive extractive and Batch extractive values

• Ph Determination of aqueous extract

• Preliminary Phytochemical Screening

• GC-MS analysis of methanolic extract of leaf, stem, root and fruit

3. Pharmacology

• In-vitro Antioxidant activity

• In-vivo studies such as Acute toxicity, Anti-depressant activity and Anti-diabetic

activities to develop new plant-based drug that may lead to therapeutic

significance.

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CHAPTER 3

MATERIALS AND METHODS

3.1. Collection of Plant material:

The plant of Tricalysia sphaerocarpa was collected from the sacred grove

of Thirumanikuzhi, of Cuddalore district, Tamil Nadu. The collected plant

material was botanically identified. The species identity conformation was

engaged at French Institute Herbarium (HIFP), Puducherry. The herbarium

specimen was prepared and deposited at the Department of Botany, Kanchi

Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, for future

reference.

3.2. Taxonomy of the Species

Tricalysia sphaerocarpa ( Dalzell ex Hook. F,) Gamble

Basianym :- Discospermum sphaerocarpum Dalzell ex Hook. F.

Common English name : Wild Coffee

Kingdom :- Plantae

Phylum Trachiophyta

Class Magnoliopsida

Order Gentianales

Family Rubiaceae

Genus Tricalysia

Species Tricalysia sphaerocarpa

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Synonyms:

Diplospora dalzellii (Thwaites) Hook. F.

Diplospora sphaerocarpa (Dalzell ex Hook. F.) Hook. F.

Discospermum dalzellii Thwaites

Tricalysia dalzellii (Thwaites) Alston

Vernacular names:

Tamil : irrukulimaram

Sri Lankans : vella

Kannadam : kaadukafibija.

3.3. Morphological Features (Plate 1)

Habit : Trees up to 15m tall

Trunk /bark : trunk flutted, bark whitish, smooth, fissured when matured; balze

yellowish. The tree outer bark is often attacked by termites giving creamish

appearance.

Branchlets : young branchlets angular to compressed, glabrous, apical bud usually

exudes yellow resin.

Leaves : Leaves dark green, simple, opposite decussate, stipules interpetiolar,

narrow triangular to 0.7 cm long, glabrous, petiole 0.6-1.5 cm long, slightly

canaliculated above, glabrous.

Lamina : 10-13 X 4-7 cm, elliptic to elliptic-ovate, apex acuminate with blunt tip

or obtuse, base attenuate, margin entire, coriaceous, glabrous, midrib raised above

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secondary nerves 5-8 pairs, hairy domatia present at axils, tertiary nerves broadly

reticulate.

Flowers : inflorescence axillary fascicles, flowers polygamodioecious, white,

scented, minute, calyx lobes oblong – Orbicular, Coralla lobes orbicular, stamens

sessile.

Fruits and Seeds : greenish yellow, berry globose upto 6 in. in diameter; the seeds

flat, smooth, much compressed, with membranous partitions, dispersed by

mammals and birds (Gamble 1921, Gamble 1993, Sasidharan 2004, Almeida

2001, Cook 1903).

3.4. Ecology

Trees in the evergreen forests up to 1000 m.

3.5. Medicinal Uses

The root decoction of Tricalysis pallens Hiern is drunk against malaria.

The root decoction of Tricalysia sp. Aff. mixed with leaf juice of Tricalysia

coricea sbsp. Nyassae is drank, and the body bathed with a root decoction for

malaria. The leaves/roots of Tricalysia coriacea (Benth.) Hiern are boiled and the

decoction drank for skin diseases and malaria/yellow fever (jaundice)(Moshi et

al., 2009). The bark of Tricalysia macrophylla K. Schum is used for the

management of cancer (Mike O. Soladoye et al., 2010). The roots of Tricalysia

capensis (rock jackal coffee) and T. lanceolata (jackal coffee) is used as an emetic

(Chris Long, 2005). The roasted seeds of T. coffeoides Good. Congo. are used

locally as a coffee substitute(George Usher, 1984). The roasted seeds of

Tricalysia sphaerocarpa taste and smell like coffee and the infusion of leaves of

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T.singularis (Korth.) K.Schum. is used as a beverage in Andala (Sumatra)

(Prajapat and Kumar, 2005). T.singularis (Korth.) K. Schum (alleopathically

enriched) + wild KaliMusli Curculigo orchioides is used as a traditional medicine

for sleep. Tricalysia Sphaerocarpa (Hook. F.) Gamble Null (alleopathically

enriched) + Tinospora giloy type 31 + white flowered Argemone satyanashi is

used traditionally used for sleep(Pankaj Oudhia’s Research documents).

3.6. Chemical constituents isolated from the different species of Tricalysia:

Tricalysia dubia

Leaf : tricalysiosides A-G(1-7), 8 ent-kaurane glucoside, tricalysionoside A (1)

and sulfatricalysines A-C (2-4)

Stem : 4 rearranged ent-kaurane diterpenoid alkaloids, tricalysiamides A-D (1-4),

tricalysiolides H – I, tricalysiolides A-E

Tricalysia okelensis

Root : tricalysiosides V and W

3.7. Pharmacognostical Studies:

The plant is collected from the wild growing in the natural environment of

Cuddalore district and identified using Flora of the Presidency of Madras

(Gamble, 1921). The fresh plant materials were collected and the morphological

features of the specimen were studied directly in the field and were photographed.

Leaf, stem and root were cut into small pieces and fixed in FAA (Formalin,

Acetic acid, and 70% ethyl alcohol in the ratio of 5ml:5ml:90ml, Johansen, 1940)

immediately after collection. Fresh parts of the plant mainly leaves, stem, root and

fruits were collected and kept in polythene bags. The materials collected were

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dried under shade in the laboratory for 3 to 4 days and the dried materials were

stored in dry polythene bags for carrying out pharmacognostical, phytochemical

and pharmacological investigations.

3.7.1. Anatomical Studies:

Free-hand sections of leaf, stem and root were also employed in the

present study. They were stained in 1% safranin, mounted in 50% glycerine and

sealed with DPX mountant.

To study the foliar epidermal morphology, peels were obtained form the

fresh leaf as well as fixed materials with the help of forceps or a razor. In addition

fixed as well as fresh young and mature leaves were cleared in 5% NaOH solution

for a period of 24-48 hours. They were washed in distilled water thoroughly and

allowed to remain in saturated chloral hydrate solution again for 24 to 48 hours.

They were further washed thoroughly with distilled water, stained with safranin

and mounted in 50% glycerine and sealed with DPX mountant.

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 µm 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 separating in about 24 hours. A thick glass rod with

rounded end was used to crush the material very gently. 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 safranin (1%). The macerated

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materials were kept in 1% safranin 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.

The following parameters were studied:

Epidermal cell number:

Epidermal cell number is the average number of epidermal cells/sq. mm.

For calculation the number of epidermal cells were counted in both the surfaces.

Stomatal number:

Stomatal number is the average number of stomata/sq. mm of epidermis of

the leaf (Evans, 1996). For calculation the number of stomata at different region

of the lower surface of leaf (hypostomatic) were counted.

Stomatal index :

Stomatal index is the percentage, with the number of stomata to the total

number of epidermal cell, each stoma being counted as one cell. Stomatal index is

calculated by using the following equation,

Where,

S.I=S/E+SX100

S.I= Stomatal index

S=Number of stomata per unit area

E=Number of epidermal cells in the same unit area.

Stomatal types:

The distribution of various stomatal types were studied at different regions

of abaxial and adaxial surfaces of leaves.

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Palisade Ratio:

Palisade ratio is defined as the average number of palisade calls beneath

each upper epidermal cell (Evans, 1996). The semi permanent mounts of cleared

leaves were employed for this study.

Vein-islet number:

Vein islet number is the number of vein-islets/ sq. mm of the leaf surface

midway between the midrib and the margin. This is constant for a given species

of the plant and used as a characteristic 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 the plant (Wallis, 1985, Evans, 1996).

Veinlet- termination number:

Veinlet-termination number is defined as the number of veinlet

termination/ sq. mm of the leaf surface midway between midrib and margin. To

study the veinlet termination number the method of Khandelwal (2008) was

adopted. The cleared leaves were used for calculating vein-islet number and

veinlet-termination number.

Photomicrography:

Photomicrography of peels and cleared leaves, free-hand section of leaf,

stem and root were taken using Olympic Nikon (Japan) Automatic Camera

attached to the microscope.

3.7.2. Histochemical Colour Reactions:

The histochemical colour reactions of leaves, stem and root of Tricalysia

sphaerocarpa were performed for the identification of major cell components

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(Johansen, 1940). For testing as far as possible, clear transparent solution were

used. Free-hand sections of plant materials were taken and treated with various

chemicals/ reagents to identify alkaloid, lignin, tannins, mucilage, starch and

proteins. The colour and results are recorded . The tests were as follows:

Lignin:

Fresh free-hand sections were mounted in 1 % neutral aqueous potassium

permanganate and allowed to stand for 15 min, washed thoroughly with water and

placed in 2 % HCl for 2 min, removed and washed with distilled water. Dilute

ammonia solution was added and covered with coverslip. Change to deep red

colour indicates the presence of lignin.

Tannin:

Fresh free-hand sections were placed in 1 % solution of Ferric chloride.

Change of blue to black colour indicates the presence of tannins.

Mucilage:

Fresh free-hand sections were treated with methylene blue reagent.

Change to blue colour indicates the presence of mucilage.

Starch:

Fresh free-hand sections were mounted in 1 % Iodine solution. Change to

blue colour, indicates the presence of starch.

Alkaloid:

Fresh free-hand section were mounted in 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).

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Proteins:

Fresh free-hand sections were stained in aqueous solution of picric acid,

covered and allowed to stand for 24 hours. Change to yellow colour indicates the

presence of proteins.

3.7.3. 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 leaves, fruits, and stem) was

observed in daylight and UV light (365 nm) using drug powder and various

solvent extracts of the drug (Pratt and Chase, 1949) as follows.

The drug powders were treated with the solvents like Benzene,

Chloroform, Acetone, Alcohol, and acid like 1N HCl, H2SO4, NaOH. Then they

were subjected to fluorescence analysis in day light and UV light. The results

were tabulated.

3.8. Phytochemistry:

3.8.1. Physio-Chemical Constants:

The authenticity of a crude drug is established with reference to the

descriptions of the pharmacopoeia or other official publications (BPC; USP) of

the country concerned. The quality and purity required is achieved by standards

(numerical values) also given in the official work of reference. The powdered

plant materials were morphologically and organoleptically screened and subjected

to physio-chemical analysis. The various parameters considered were:

Ash values (Anonymous, 1996; Khandelwal, 2008):

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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 form leaf and

stem. 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 weighted. 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 tabulated.

Determination of water soluble ash:

The ash obtained as described in the 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 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 recorded.

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Moisture content

The moisture content was determined by using the method of Anonymous

(1996) and Khandelwal (2008).

3.8.2. Preparation of the Extracts:

The collected materials were chopped into small pieces separately, shade-

dried, and coarsely powdered using a pulverizor. The coarse powder were

subjected to successive extraction with chloroform, diethylether, ethylacetate and

methanol by Soxhlet method. 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 4ºC. The resulted extracts were subjected to preliminary

phytochemical screening and GC-MS analysis.

3.8.3. Extractive Values :

Extractive values by (i) Batch process (Kokate, 1986) and (ii) Successive

process (Harborne, 1998) were calculated

3.8.4. 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.

3.8.5. Preliminary Phytochemical Screening

All the extracts were subjected to preliminary phytochemical tests

following the method of Harborne (1998) and Trease and Evans (1983).

Test for Alkaloid (Evans, 1996):

The substance 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

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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.

Test for Cardiac glycosides (Keller-Killiani test):

To 2 ml extract glacial 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.

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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.

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.

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Test for Phlobotannins:

Deposition of a red precipitate when an 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 redusing sugars.

Test for Non - Reducing Polysaccharides (Starch)(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.

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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 was 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.

3.8.6. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:

GC-MS analysis was performed with GC Clarus 500 Perkin Elmer

equipment. Compounds were separated on Elite-1 capillary column (100%

Dimethylpolysiloxane). Oven temperature was programmed as follows:

isothermal temperature at 50ºC for 2 minutes, then increased to 200ºC at the rate

of 10ºC/minutes, then increased up to 280ºC at the rate of 5ºC/minutes held for 9

minutes. Ionization of the sample components was performed in the El mode (70

eV). The carrier gas was helium (1ml/minutes) and the sample injected was 2μl.

The detector was Mass detector turbo mass gold-Perkin Elmer. The total running

time for GC was 36 minutes and software used was Turbomass 5.2. Using

computer searches on a NIST Ver.2.1 MS data library and comparing the

spectrum obtained through GC – MS compounds present in the plant samples

were identified.

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Identification of Compounds:

The individual compounds were identified from methanol extracts based

on direct comparison of the retention times and their mass spectra with the spectra

of known compounds stored in the spectral database, NIST (version year 2005).

3.9. Pharmacology

3.9.1. In Vitro Antioxidant Activity

3.9.1.1. Inhibitiory Effects on DPPH Radical Assay:

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was

selected due to its straight forwardness, quickness, sensitivity and reproducibility

(Sanja et al., 2008).DPPH is a nitrogen centered free radical scavenger that shows

strong absorbance at 517 nm. Deep violet coloured methanolic DPPH solution

changes to yellow colour in the presence of DPPH radical scavengers. DPPH

radical accepts an electron or hydrogen radical to become a stable diamagnetic

molecule. Extent of DPPH radical scavenged was determined by the decrease in

absorbance at 517 nm induced by antioxidants, due to the reaction between

antioxidant molecules and free radicals, which results in the scavenging of free

radicals by hydrogen donation (Chang et al., 2002). DPPH radical scavenging

activity of extract was determined according to the method reported by Blois

(1958). An aliquot of 0.5 ml of sample solution in methanol was mixed with 2.5

ml of 0.5 mM methanolic solution of DPPH. The mixture was shaken vigorously

and incubated for 37 minutes in the dark at room temperature. The absorbance

was measured at 517 nm using UV spectrophotometer. Butyl Hydroxyl Toluene

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(BHT) was used as a positive control. DPPH free radical scavenging ability (%)

was calculated by using the formula.

absorbance of control – absorbance of sample % of inhibition = --------------------------------------------------------

absorbance of control ×100.

3.9.1.2. Hydrogen peroxide Assay

Hydrogen peroxide content was determined by measuring the absorbance

of titanium-hydroperoxide complex. The extract was reacted with titanium

reagent and ammonium to form hydroperoxide-titanium complex. The complex

was dissolved in 1 M sulfuric acid and absorbance of the supernatant was

measured at 415 nm against blank. Concentration of hydrogen peroxide was

determined using the standard curve plotted with known concentration of

hydrogen peroxide.

3.9.1.3. Superoxide dismutase (L-methionine and NBT) Assay (SOD)

SOD activity was assayed by determining the inhibition rate of nitro blue

tetrazolium reduction with xanthine oxidase as a hydrogen peroxide generating

agent. The rate of NBT reduction is directly proportional to SOD levels. The

absorbance at 360 nm was noted and expressed the value as percentage of SOD

levels.

3.9.1.4. Iron Chelating Activity (FRAP)

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 1

ml of 0.05% O-Phenanthroline in methanol, 2 ml ferric chloride (200μM) and 2

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ml of various concentrations ranging from 50 to 500μg were incubated at room

temperature for 10 minutes and the absorbance of the same was measured at 510

nm. Ethylene diamine tetra acetic acid (EDTA 10mM) was used as a classical

metal chelator. The experiment was performed in triplicates.

3.10. In vivo Pharmacological Studies

Selection of extracts

The extractive values is high in methanol solvent, hence the methanol

extract of the stem of T. sphaerocarpa was selected for in vivo pharmacological

studies.

Animals

Young adult male Wister rats, 8 weeks old were used as experimental

model. The weight of each of the animals on the first day of experiment was 120-

180 grams. They were randomly housed 6 per gauge and maintained in 10:14

light: dark cycle and given access to food and water ad libitum. All injections in

this study were performed once daily between 8.00 AM and 9.00 AM. The

experimental protocals were carried out at K.M.C.H. College of Pharmacy,

Coimbatore, Tamil Nadu, India, approved by the Institutional Animals Ethics

Committee (IAEC. NO: 793/ 03/ C/ CP CSEA/112-2013).

Drugs and chemicals

Imipramine hydrochloride (Sigma-Aldrich, St Louis, USA) was used as

reference standard for antidepressant activity.

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Administration of the extracts

Suspensions of methanolic extract was prepared in distilled water using

Tween-80 (0.2% v/v) as the suspending agent. The extract was administered in

different doses of 100 and 200 mg/kg of body weight to rats by oral route, 45

minutes before the test procedures for pre-pharmacological screening as per

Organization for the Economic Cooperation Development (OECD) 423

guidelines. Control groups were given only the vehicle (0.2% v/v Tween-80

solution) in volume equivalent to that of the plant extracts.

3.10.1. Acute Toxicity Studies

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 mice

(18-25g) and observed for signs of behavioral, Neurological toxicity and mortality

after 14 days. All the parameters were thoroughly checked and dose for the

further studies was calculated as per the Organization for the Economic

Cooperation Development 423 guidelines (OECD). After the conduct of acute

toxicological studies the dose of methanolic extract was decided i.e. 100mg/kg,

200mg/ kg. Oral route was selected for the administration of drugs. The procedure

was followed as per OECD 423 guidelines.

3.10.2. Anti-Depressant Activity

3.10.2.1. Forced Swimming Test (FST)

Rats of either sex (120-150g) were individually forced to swim in an open

cylindrical container (diameter 10 cm, height 25 cm), containing 19 cm of water

at 25±1 °C. All the rats of either sex were divided in to four different groups of 6

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in each group (n=6). The first group assigned as control received only vehicle

(Distilled water, 5ml/kg). The other two groups received acute oral dose of

methanolic extract (100 and 200mg/kg). The fourth group received standard drug

Imipramine (30 mg/kg). The total duration of immobility was recorded during the

last 6 minutes of the 10-minutes period. Each rat was judged to be immobile when

it ceased struggling and remained floating motionless in the water, making only

those movements necessary to keep its head above water. A decrease in the

duration of immobility is indicative of an antidepressant like effect.

3.10.2.2. Tail Suspension Test (TST)

All the rats of either sex (120-150g) were divided into five different

groups. The first group assigned as control received only vehicle (Distilled water,

5ml/kg). The other two groups received acute doses of methanolic extract (100

and 200 mg/kg). The fourth group received standard drug Imipramine (30 mg/kg).

The total duration of immobility induced by tail suspension was measured

according to the methods described by Steru et al., (1985). Briefly, rat both

acoustically and visually isolated were suspended 50 cm above the floor by

adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time

was recorded during a 6-minutes period. Rats were considered immobile only

when they hung passively and were motionless.

3.10.2.3. Hole Board Test (HBT)

Experiment was conducted, 30minutes after injection of control vehicle,

methanolic extract 100 and 200mg/kg and diazepam (3mg/kg) by placing mouse

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on a wooden board. The number of head dips and time spent in each dip was

recorded during 3minutes trial.

Statistical analysis

The immobility time in tail suspension test and forced swimming test was

analyzed with ANOVA (Analysis of Variance), further comparisons between

vehicle and drug-treatment groups were performed using the Dunnett's t-test.

Results are expressed as the mean ± SEM. Analyses were performed using the

software SPSS (Statistical program for Social Sciences) version 13 for windows.

The level of statistical significance adopted was **P<0.01, when compared with

the control group.

3.10.3. Anti Diabetic Activity

3.10.3.1. Screening of Hypoglycemic Activity in Normal Rats

Normal fasted rats: Normal albino rats (150-180 g) were first used for the

screening of the herbal drug for hypoglycemic activity. Overnight fasted normal

rats were randomly divided into 5 groups of 6 rats each. The group I served as

control, which received vehicle i.e. 1% Gum acacia solution (1ml/kg, orally).

Group II, III and IV were treated orally with Test extract 125, 250 and 500 mg/kg,

respectively. Group V received Glibenclamide 5 mg/kg orally.

Experimental Design

Group 1 - Treated orally with 1% Gum acacia solution, 1ml/kg

Group 2 - Treated orally with Test extract, 125mg/kg

Group 3 - Treated orally with Test extract, 250mg/kg

Group 4 - Treated orally with Test extract, 500mg/kg

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Group 5 - Treated orally with Glibenclamide 5mg/kg

Blood samples were collected from tail vein prior and 1, 2, 4 and 6 hours

after treatment. Fasting blood glucose (FBG) was determined by the glucose

oxidase method using CONTOURTMTS Blood Glucose Meter with same test

strips. The percentage (%) fall in blood glucose level was also calculated at peak

hour of effect.

3.10.3.2. Anti-Diabetic Activity in Experimentally Induced Diabetic Rats:

Induction of experimental diabetes

Overnight fasted albino rats (150-180 g) were made diabetic by injecting

Alloxan monohydrate (in the ice cold normal saline) intra peritonially (i.p) at a

dose of 150 mg/kg body weight. Diabetes was confirmed in Alloxan injected rats

by measuring the fasting blood glucose concentration, 72 hours after the

Alloxanization. Rats with blood glucose level above 250 mg/dl were considered

to be diabetic and were used in this study.

Experimental design

The diabetic rats were divided into 5 groups of 6 rats each.

Group 1 - Normal control and received vehicle i.e. 1% Gum acacia Solution,

1ml/kg/BW

Group 2 - Diabetic control and received 1% Gum acacia solution, 1ml/kg/BW

Group 3 - Treated orally with Test extract, 125mg/kg/BW

Group 4 - Treated orally with Test extract, 250mg/kg/BW

Group 5 - Treated orally with Test extract, 500mg/kg/BW

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Group 6 - Received Glibenclamide 5mg/kg/BW, orally on 3rd day after

alloxanation (i.e. 1st day of treatment)

Group 1 and 2 served as normal and diabetic control respectively and received

vehicle (1ml/kg, po). Group 3, 4 and 5 were treated orally with Test extract, 125,

250 and 500 mg/kg BW respectively. Group 6 received Glibenclamide 5 mg/kg,

orally on 3rd day after alloxanization (i.e. 1st day of treatment).

3.10.3.2.1. In single-dose, short term study :

Fasting Blood Glucose was estimated from the tail vein prior and 1, 3 and

6 hr after administration of test drugs and vehicle.

3.10.3.2.2. In multi dose long term study:

The same animals were continued with the same dose of vehicle, test

extract and Glibenclamide once daily for 15 days. Fasting Blood Glucose in the

blood was collected and measured at 24 hours after the previous dose on 3, 6, 9,

12 and 16th day.

3.10.3.3. Effect of extract in Body Weight in Normal and Alloxan Induced

Diabetic Rats

3.10.3.4. Biochemical Parameters Determinations

After 15 days of treatment, overnight fasted rats were sacrificed and blood

was collected. The serum was separated and analyzed for lysosomal enzymes

such as transaminases (Serum Glutamate Oxaloacetate Transaminase, SGOT and

Serum Glutamate Pyruvate Transaminase, SGPT), and Alkaline Phosphatase

(ALP), by colorimetric method.

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The pancreas were dissected out and washed with ice-cold saline

immediately. A portion of pancreatic tissue was homogenized and the extract was

used for the estimation of enzymatic antioxidants (Catalase, CAT and Glutathione

Peroxidase, GPX) activities including Lipid Peroxidation (LPO) process to see the

effect of 15 days treatment with test extract.

Determination of Blood Glucose:

The test provides a quantitative measurement of glucose in blood

from 10 to 600 mg/dl as described in the manual of manufacturer Bayer polychem

(India) Limited (CONTOURTMTS Blood Glucose Meter with same Test Strips )

as follows:

Principle:

The CONTOUR TS blood glucose test is based on measurement of

electrical current caused by the reaction of glucose with the reagents on the

electrode of the strip. The blood sample was drawn into the tip of the test strip

through capillary action. Glucose in the sample reacts with FAD glucose

dehydrogenase (FAD-GDH) and potassium ferricyanide. Electrons were

generated, producing a current that is proportional to the glucose in the sample.

After the reaction time, the glucose concentration in the sample is displayed. No

calculation is required.

Chemical Composition: FAD glucose dehydrogenase (Aspergillus sp., 2.0 U/test

strip), 6%; potassium ferricyanide 56%; Non-reactive ingredients 38%.

Determination of Serum glutamate oxaloacetate transaminase (SGOT)

Method using SGOT kit.

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Principle

SGOT catalyses the following reaction

SGOT α -Keto glutarate + L-asparate L -glutamate + Oxalacetate pH 7.4 Oxaloacetate Alkaline 2,4 dinitrophenyl + hydrazone 2,4 DNPH Medium (Brown coloured)

Oxaloacetate formed in the reaction is spontaneously converted into

pyruvic acid. Rate of reaction is then determined by the estimation of pyruvic acid

using dinitrophenyl hydrazine. Dinitrophenyl hydrazine (DNPH) formed was

estimated at 505 nm. The unreacted α-keto glutarate also gives coloured product

with color reagent but the intensity was much less than that of pyruvate and hence

it was negligible.

Reagents

Reagent 1: Buffered alanine α-KG substrate, pH 7.4

Reagent 2: DNPH colour reagent

Reagent 3: Sodium hydroxide 4 N

Reagent 4: Working pyruvate standard, 2mM

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Procedure

Tube No. 1 2 3 4 5

Enzyme activity (units/ml) 0 24 61 114 190

Reagent 1 0.5 0.45 0.4 0.35 0.3

Reagent 4 - 0.05 0.1 0.15 0.2

Purified water (ml) 0.1 0.1 0.1 0.1 0.1

Reagent 2 0.5 0.5 0.5 0.5 0.5

Mix well and allow to stand at room temperature for 20 minutes.

Solution I (ml) 5.0 5.0 5.0 5.0 5.0

Mixed well by inversion. Allowed to stand at room temperature for 20 minutes.

and measured the absorbance of all the five tubes against purified water on a

colorimeter using a green filter.

Test procedure

Reagents Blank Test

Reagent 1: Buffered alanine, pH 7.4 0.5ml 0.5ml

Incubate at 37˚C for 5 minutes.

Serum 0.1 ml

Mix well and incubate at 37˚C for 60 minutes.

Reagent 2: DNPH colour reagent 0.5ml 0.5ml

Mix well and allow to stand at room temp. for 20 minutes.

Distilled water 0.1 ml 0.1 ml

Working sodium hydroxide 5.0 ml 5.0 ml

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Mixed well and allowed to stand at room temperature for 10 minutes. Estimated

with the help of spectrophotometer at 505nm and expressed as IU/l.

Determination of Serum glutamic pyruvic transaminase (SGPT)

Method using SGPT kit.

Principle

SGPT (ALT) catalyses the following reaction

SGPT α -Keto glutarate + L-alanine L -glutamate + pyruvate pH 7.4

Pyurate Alkaline 2,4 dinitrophenyl + hydrazone 2,4 DNPH Medium (Brown coloured)

Pyruvate was coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to give the

corresponding hydrazone, which gives the brown color in alkaline medium and

this can be measured colorimetrically.

Reagents

Reagent 1: Buffered alanine α-KG substrate, pH 7.4

Reagent 2: DNPH colour reagent

Reagent 3: Sodium hydroxide 4 N

Reagent 4: Working pyruvate standard, 2mM

Preparation of working solutions

Solution I: Dilute 1 ml of reagent 3 to 10 ml with purified water.

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Procedure

Tube No. 1 2 3 4 5

Enzyme activity (units/ml) 0 28 57 97 100

Reagent 1 0.5 0.45 0.4 0.35 0.3

Reagent 4 - 0.05 0.1 0.15 0.2

Purified water (ml) 0.1 0.1 0.1 0.1 0.1

Reagent 2 0.5 0.5 0.5 0.5 0.5

Mix well and allow to stand at room temperature for 20 minutes

Solution I (ml) 5.0 5.0 5.0 5.0 5.0

Mixed well by inversion. Allowed to stand at room temperature for 20 minutes

and measured the absorbance of all the five tubes against purified water on a

colorimeter using a green filter.

Test procedure

Reagents Blank Test

Reagent 1: Buffered alanine, pH 7.4 0.5ml 0.5ml

Incubate at 37˚C for 5 minutes.

Serum 0.1 ml

Mix well and incubate at 37˚C for 60 minutes.

Reagent 2: DNPH colour reagent 0.5ml 0.5ml

Mix well and allow to stand at room temp. for 20 minutes.

Distilled water 0.1 ml 0.1 ml

Working sodium hydroxide 5.0 ml 5.0 ml

Mix well and allow to stand at room temperature for 10 minutes. Estimated with

the help of spectrophotometer at 505nm and expressed as IU/L

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Determination of Serum alkaline phosphatase (SALP)

The alkaline phosphates level was estimated by p-Nitrophenyl

phosphate (PNPP) method.

Principle

The determination of the activity of alkaline phosphatase in serum based

on the hydrolysis of p- nitrophenyl phosphate (PNPP) by the enzyme with the

formation of free p- nitrophenol.This compound was yellow in alkaline solution.

The formation of yellow colour can be spectrophotometrically readapt 405 nm,

which was directly proportional to the enzymatic activity of alkaline phosphatase

in serum / plasma.

Alkaline Phosphatase PNPP + H2O P- nitrophenol + Phosphate

The method has been recommended by the German Society of Clinical Chemistry

and by the committee on enzyme of the Scandinavian Society of Clinical

Chemistry and Clinical Physiology.

Reagents

Reagents 1: Substrate

Reagents 2: Buffer

Preparation of working solution

Dissolve each vial content (Reagent 1) of dry substance with 3.0 ml of

buffer (Reagent 2). Mixed to dissolve by slow stirring to ensure uniform mixing.

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Procedure

Test(T) Blank(B)

Working reagent 1.0 ml Distilled water

Sample 20 µl Distilled water

Mix well and read the absorbance at 60, 90, 120 and 150 seconds at 405 nm.

Determine the A /minutes from the linear part of the assay.

Calculation

IU /L of Alkaline phosphatase = A /minutes × 2713

Where F=2713was calculated on the basis of molar extinction coefficient for p-

nitrophenol and total assay volume to sample volume.

We can also measure the change of optical density directly from Bio Chemical

analyzer at 405 nm.

Measurement of Lipid Peroxidation (LPO)

The concentration of thiobarbituric acid reactive substances (TBARS) was

measured (lipid peroxidation product maondialdehyde (MDA) was estimated) in

liver using the method of Okhawa et al., (1979). One ml of the sample was mixed

with 0.2 ml 4 % (w/v) sodium dodecyl sulfate, 1.5 ml 20% acetic acid in 0.27 M

hydrochloric acid (pH 3.5) and 15 ml of 0.8% thiobarbituric acid (TBA, pH 7.4).

The mixture was heated in a hot water bath at 85˚C for 1 hour. The intensity of

the pink colour developed was read against a reagent blank at 532 nm following

centrifugation at 1200 g for 10 minutes. The concentration was expressed as n

moles of MDA per mg of protein using 1,1,3,3,-tetra-ethoxypropane as the

standard.

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Determination of Glutathione- Peroxidase activity:

The reaction mixture contained 0.1 M reduced glutathione, 10 U/ml of

glutathione reductase, 2 mM nicotinamide adenine dinucleotide phosphate

reduced (NADPH), 0.05 M phosphate buffer (pH 7.0) and 7 Mm t-butyl

hydroperoxide. Decrease in absorbance of NADPH was measured as GPx activity

at 340 nm. One unit of GPx is equal to the number of nano moles of NADPH

oxidized/utilized per minutes at 25˚C.

Measurement of Catalase (CAT)

In animals, catalase was present in all major body organs, especially being

concentrated in liver and erythrocyte. During β-oxidation of fatty acids by

flavoprotien dehydrogenase, hydrogen peroxidewas generated, which was

accepted upon by catalase present in peroxisomes. (Nichollas and Schonbaum,

1963).

The catalase activity was assayed by the method of catalase catalyses the

rapid decomposition of hydrogen peroxide to water.

2H2O2 2H2O + O2

The decomposition of hydrogen peroxide by catalase proceeds at one of the

highest rates known for enzymatic reactions.

Reagents

Dichromate-acetic acid reagent: Five percent of potassium dichromate was

prepared with acetic acid (1:3 v/v in distilled water).

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Phosphate buffer - 0.01M, pH 7.0: 173 mg of disodium hydrogen phosphate and

122 mg of sodium dihydrogen phosphate were dissolved in 61 ml and 39 ml of

distilled water respectively and made up to 200 ml with distilled water.

Hydrogen peroxide – 0.2M: 2.27 ml h hydrogen peroxide was made upto 100 ml

with distilled water.

Procedure

To 0.1 ml of liver homogenate 1.0 ml of each phosphate buffer and

hydrogen peroxide were added and a timer started. The reaction was arrested by

the addition of 0.2 ml dichromate acetic acid reagent. Standard hydrogen peroxide

in the range of 4 to 20 µm were taken and treated similarly. The tubes were heated

in a boiling water bath for 10 minutes. The green color developed was read at 570

nm in a Double beam UV-VIS spectrometer (Perkin Elmer), Germany. Catalase

activity was expressed as IU/L.

3.10.3.5. Histopathological Studies:

Pancreas were isolated and preserved in 10% formalin. Section of

the pancreas tissues were made, stained with Haematoxylin and Eosin reagent and

observed under low and high power objective for histopathological changes. The

alteration and changes in the histology of pancreas were shown in vide plate and

the results with photomicrograph were given in the result section.

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CHAPTER 4

RESULTS

4.1. Pharmacognosy

4.1.1. Anatomy

The anatomical studies of Tricalysia sphaerocarpa includes the epidermal

peels of the leaf and stem, clearing of leaf, maceration of stem and transverse

section of leaf, stem and root.

4.1.1.1. Leaf peeling:

Epidermal cell number, stomatal number, stomatal index and palisade

ratio were calculated and presented in the table 1.

Adaxial epidermis: Cells larger than those on the abaxial epidermis, irregularly

shaped, walls thick, sinous, stomata absent, trichomes absent (Plate 2).

Abaxial epidermis: Cells smaller and irregularly shaped in the intercostal region,

elongated in the costal region, walls thick, deeply sinous, stomata more frequent,

irregularly distributed, variously oriented, occur in various sizes in the intercostal

region, less frequent and large in size in the costal region, slightly elongated,

rubiaceous type, edges very thick (Plate 2). Giant stomata, blind stomata, half

stomata, medium size stomata, small size stomata and degenerated stomata are

also encountered. Rare occurrence of unicellular, conical, straight trichomes are

observed (Plate 3).

4.1.1.2. Venation Pattern:

In cleared lamina, the venation system was studied. The veins are thin and

straight. The islets are variable in shape and size. Veins reticulate, showing lateral

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branches, vein-islet faily large, each islet containing 3-4 termination points. The

vein islet number and veinlet-termination number were 93.8 ± 6.85 and 57.4 ±

3.78 respectively (Table 1) (Plate 4).

4.1.1.3. Quantitative Values of Foliar Epidermis

The mean number of epidermal cells/mm2 is 1122.8 ± 2.84 in adaxial

surface whereas it is 1414 ± 3.76 in abaxial surface. The stomata were

encountered only in the abxial surface. The number of stomata /mm2 was 336 ±

8.43. The stomatal index was 26.9 ± 4.37. The palisade to spongy ratio was 6.2 ±

2.82.

4.1.1.4. Stem Peeling:

Cells larger, axially enlongated, arranged in longitudinal rows, septate,

walls very thick, septa thin, stomata rare, small, rubiaceous, trichomes absent

(Plate 4).

4.1.1.5. Maceration:

Fibres

The fibres are libriform type, with thick lignified walls and fairly wide

lumen. The fibre is uniform in thickness and they become tapering at the ends

(Plate 7).

Vessel Elements

The vessel elements are narrow, long and cylindrical. The end wall

perforation is simple, circular, mostly oblique or horizontal. Vessel elements are

tailed at one end or at both ends.(Plate 7).

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Table 1: Quantitative values of foliar epidermis of Tricalysia sphaerocarpa

Quantitative values Abaxial Adaxial Epidermal cell/mm²² 1122.8±2.84 1414±3.76 Stomata/mm²² 336±8.43 - Stomatal index 26.9±4.37 - Palisade ratio 6.2±2.82 Vein islet number 93.8±3.85 Veinlet termination number 57.4±3.78

Note : All the values are expressed as mean ± SEM(n=6)

Table 2: Histochemical colour reactions of various parts of Tricalysia sphaerocarpa

Test for Chemicals/reagents used Status of the substance leaf stem root

Starch Iodine solution + + + alkaloid Meyer’s reagent + + + Proteins Aqueous picric acid solution + + + Tannin Dilute ferric chloride + + - Lignin 1% potassiumpermanganate,

2% HCl, dil. Ammonia - - +

Mucilage Methylene blue reagent - - - Note : + = present; - = absent.

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4.1.1.6. Transverse Section of Leaf:

Leaf in T. S. is dorsiventral, upper epidermis single layered, cells tabular

in the leaf blade region, hemispherical in the midrib region, cuticle thick

continous in the blade region and arched in the midrib region. Cuticle covers the

radial walls also to some extent. Stomata absent. Lower epidermis single layered,

epidermal cells tabular in the blade region, outer tangential walls deeply arched in

the midrib region. Midrib region raised on the abaxial side into a simple hump.

Stomata sunken. Mesophyll differentiated into upper single layered palisade and

lower spongy parenchymatous ground tissue, group of scleroids and fibres are

observed in the midrib region. Xylem occurs in the form of an arch in the midrib

region surrounded by phloem on the abaxial side. The ground cells are rich in

starch grains, groups of tannineferous idioblasts are evident (Plate 2).

4.1.1.7. Transverse Section of Stem:

The young stem in cross section is mostly dumble shaped. Epidermis is

unilayered, epidermal cells hemispherical surrounded by a thick cuticle. Cuticle

covers the radial walls also to some extent. Hypodermis collenchymatous in

discontinous patches. Cortex parenchymatous, followed by a layer of

sclerenchyma cells (scleroids). Secondary phloem continuous with patches of

sclerenchyma fibres. Secondary xylem continuous with elliptical or oval shaped

vessels. Vessels discrete arranged in radial rows. Vessel members narrow, pitted,

simple perforation plate with long tail. Pith parenchymatous with abundant starch

grains. Tannineferous idioblasts are distributed in cortex, phloem and pith. The

idioblast in the pith are larger than the other (Plate 5).

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4.1.1.8. Transverse Section of Root:

The root in cross section is circular. The rhizodermis is peeled off. The 8-

10 layers of parenchymatous cells are seen beneath the rhizodermis. Secondary

xylem and phloem are present. Rays are clearly seen. Xylem seen in the centre

and the phloem towards the periphery. The secondary xylem occupies wide major

portion of the root. It includes densely crowded, diffusely distributed wide,

circular, thick walled, narrow xylem fibres. The vessels include both wide and

narrow elements (Plate 6).

4.1.2. Histochemical Colour Reactions:

The histochemical localization tests revealed the presence of starch,

alkaloid and protein in all the plant parts studied. Tannin is present in leaf and

stem, lignin is present only in root and the mucilage is absent in all the plant parts

studied (Table 2).

4.1.3. Fluorescence Analysis

The fluorescence analysis of various parts of plant in different solvents

and chemical reagents observed under ordinary day light and UV light are given

in Table 3-6.

Leaf

Powdered leaf material was green in day light and yellow under UV light.

Similarly, it was dark green in acetone, benzene and chloroform, green in ethanol

and water, yellowish green in n-butyl alcohol. Under UV light, orange colour in

acetone, benzene, chloroform, ethanol and n-butyl alcohol whereas dark brown in

water.

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Under day light, reddish brown was developed in 10% ferric chloride,

50% sulphuric acid, 50% nitric acid, dark green in 10% aqueous NaOH, green in

1N HCl, 5% ammonia and 1% thionyl chloride. Under UV light, black colour was

observed in 10% ferric chloride and 50% nitric acid, dark brown in 50% sulphuric

acid, brown in 10% aqueous NaOH, green in 1N HCl and 5% ammonia, dark

green in 1% thionyl chloride.

Stem

Powdered stem material was creamy white in day light and pale yellow

under UV light. Similarly, it was yellow in acetone, n-butyl alcohol and ethanol,

pale yellow in benzene, dark yellow in chloroform, creamy white in water. Under

UV light, stem powder was orange colour in acetone, creamy white in benzene

and n-butyl alcohol, pale yellow in chloroform, dark yellow in ethanol, whereas

pale yellow in water.

Under day light, orange colour was developed in 10% ferric chloride,

reddish brown 50% sulphuric acid and 50% nitric acid, yellow in 10% aqueous

NaOH, creamy white in 1N HCl, pale yellow in 5% ammonia and light yellow in

1% thionyl chloride. Under UV light, black colour was observed in 10% ferric

chloride, 50% nitric acid and 50% sulphuric acid, creamy white in 10% aqueous

NaOH, yellow in 1N HCl and 5% ammonia, brown in 1% thionyl chloride.

Root

Powdered root material was creamy white in day light, and pale yellow

under UV light. Similarly, in day light, it was yellow in acetone, benzene,

chloroform, ethanol and n-butyl alcohol, creamy white in water. Under UV light,

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Table 3 : Fluorescence analysis of Leaf powder of Tricalysia sphaerocarpa

Chemicals Leaf Day light UVlight

Powder as such Green Yellow Solvent

Acetone Dark green Orange Benzene Dark green Orange Chloroform Dark green Orange Ethanol Green Orange n-butyl alcohol Yellowish green Orange Water Green Dark brown green

Reagents 10% FerricChloride Reddish brown Black 50% Sulphuric Acid Reddish brown Dark brown 50%Nitric acid Reddish brown Black 10%aq. NaOH Dark green Brown 1 NHCl Green Green 5% Ammonia Green Green 1% Thionyl Chloride Green Dark green

Table 4 : Fluorescence analysis of Stem powder of Tricalysia sphaerocarpa

Chemicals Stem Day light UVlight

Powder as such Creamy white Pale yellow Solvent Acetone Yellow Orange Benzene Pale yellow Creamy white Chloroform Dark yellow Pale yellow Ethanol Yellow Dark yellow n-butyl alcohol Yellow Creamy white Water Creamy white Pale yellow

Reagents 10% FerricChloride Orange Black 50% Sulphuric Acid Reddish brown Black 50%Nitric acid Reddish brown Black 10%aq. NaOH Yellow Creamy white 1 NHCl Creamy white Yellow 5% Ammonia Pale yellow Yellow 1% Thionyl Chloride Light yellow Brown

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stem powder was orange colour in acetone, yellow in benzene and water, pale

yellow in chloroform, dark yellow in ethanol and n-butyl alcohol.

Under day light, reddish brown colour was developed in 10% ferric

chloride and 50% sulphuric acid, orange in 50% nitric acid, yellow in 10%

aqueous NaOH and 5% ammonia and greenish yellow in 1N HCl, and light

yellow in 1% thionyl chloride. Under UV light, black colour was observed in

10% ferric chloride and 50% sulphuric acid, brown in 50% nitric acid and 1%

thionyl chloride, dark yellow in 10% aqueous NaOH, pale yellow in 1N HCl and

green in 5% ammonia.

Fruit

Fruit powder was light brown in day light, and creamy white under UV

light. Similarly, in day light, it was yellow in acetone, benzene and chloroform,

pale yellow in ethanol, light brown in n-butyl alcohol and water. Under UV light,

stem powder was green colour in acetone, creamy white in benzene, chloroform,

ethanol and n-butyl alcohol and yellow in water.

Under day light, orange colour was developed in 10% ferric chloride and

50% nitric acid, reddish brown in 50% sulphuric acid, light brown in 10%

aqueous NaOH, pale yellow in 5% ammonia and 1N HCl and yellow in 1%

thionyl chloride. Under UV light, brown colour was observed in 10% ferric

chloride and 50% sulphuric acid, dark brown in 50% nitric acid, creamy white in

10% aqueous NaOH, yellow in 1N HCl and 5% ammonia and black in 1% thionyl

chloride.

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Table 5: Fluorescence analysis of Root powder of Tricalysia sphaerocarpa

Chemicals Root Day light UVlight

Powder as such Creamy white Pale yellow Solvent

Acetone Yellow Orange Benzene Yellow Yellow Chloroform Yellow Pale yellow Ethanol Yellow Dark yellow n-butyl alcohol Yellow Dark yellow Water Creamy white Yellow

Reagents 10% FerricChloride Reddish brown Black 50% Sulphuric Acid Reddish brown Black 50%Nitric acid Orange Brown 10%aq. NaOH Yellow Dark yellow 1 NHCl Greenish yellow Pale yellow 5% Ammonia Yellow Green 1% Thionyl Chloride Light yellow Brown

Table 6: Fluorescence analysis of Fruit powder of Tricalysia sphaerocarpa Chemicals Fruit

Day light UVlight Powder as such Light Brown Creamy white

Solvent Acetone Yellow Green Benzene Yellow Creamy white Chloroform Yellow Creamy white Ethanol Pale yellow Creamy white n-butyl alcohol Light Brown Creamy white Water Light Brown Yellow

Reagents 10%FerricChloride Orange Brown 50% Sulphuric Acid Reddish brown Brown 50%Nitric acid Orange Dark brown 10%aq. NaOH Light Brown Creamy white 1 NHCl Pale yellow Yellow 5% Ammonia Pale yellow Yellow 1% Thionyl Chloride Yellow Black

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4.2. Phytochemistry

4.2.1. Physico-Chemical Parameters of Various Parts

The physico-chemical parameters such as moisture content, total ash, acid

insoluble ash and water soluble ash of leaf, stem, root and fruit of Tricalysia

sphaerocarpa were analysed and presented in the Table 7.

Leaf

In leaf the moisture content, total ash, acid insoluble ash and water soluble

ash were 15 %, 4 %, 0.84 % and 3.24 % respectively.

Stem

In stem the moisture content, total ash, acid insoluble ash and water

soluble ash were 20 %, 5.16 %, 1.06 %, and 3.90 % respectively .

Root

In root the values for similar parameters were 18 %, 3.26 %, 0.38 %, and

2.28 % respectively .

Fruit

In fruit the moisture content, total ash, acid insoluble ash and water

soluble ash were 17.8 %, 1.5 %, 0.5 %, and 1.1 % respectively.

4.2.2. Extractive Values

The results of extractive values are given in Table 8 and 9 and figure 1 and 2.

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Table 7: Proximate analysis of various parts of Tricalysia sphaerocarpa: Parameter Results %

Leaf Stem Root Fruit Loss of drying 15 20 18 17.8 Total ash 4 5.16 3.26 1.5

Acid insoluble ash 0.84 1.06 0.38 0.5 Water soluble ash 3.24 3.90 2.28 1.1 Table 8: Extractive values of various parts of Tricalysia sphaerocarpa by batch

process. Solvent Values in %

Leaf Stem Root Fruit Acetone 15 18 17 13.8 Benzene 11.5 11 8 5 Chloroform 11 9 8 6 Diethyl ether 9 7 5 3 Ethanol 26 20 15 10 n-butyl alcohol 20 10 11.4 9 Methanol 32 30 20 26 Water 30 20 19 20

Table 9: Extractive values of various parts of Tricalysia sphaerocarpa by successive

process. Solvent Values in %

Leaf Stem Root Fruit Chloroform 11.3 14 8.7 5 Diethyl ether 11 9 7.8 4.6 Ethyl acetate 18.2 20 15.5 12 Methanol 20 22.9 17.5 17.9

Table 10: PH Determination of water extract of Tricalysia sphaerocarpa

Various parts PH Leaf 6.4 Stem 6.7 Root 6.8 Fruit 6.8

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4.2.2.1. Batch Process

Leaf

In leaf, the highest extractive value was recorded in methanol (32%)

followed by aqueous extract (30%), ethanol (26%), n-butanol(20%),

acetone(15%), benzene(11.5%), chloroform(11%) and the lowest extractive value

was found in diethylether (9%).

Stem

In stem, the highest value was seen in methanol (30%), followed by

aqueous extract (20%) ethanol (20%) acetone (18%), benzene (11%), n-butanol

(10%), chloroform (9%) and the lowest in diethylether (7%).

Root

In root, the highest extractive value was recorded in methonal (20%),

followed by aqueous extract (19%), when compared to other solvents like

acetone(17%), ethanol(15%), n-butanol(11.4%), benzene(8%), chloroform(8%)

and diethylether(5%).

Fruit

In fruit, the highest extractive value was recorded in methonal (26%),

followed by aqueous extract (20%), acetone(13.8%), ethanol (10%), n-butanol

(9%), chloroform (6%), benzene(5%) and the lowest value was found in

diethylether(3%).

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4.2.2.2. Successive Process

Leaf

In leaf, the highest extractive value was recorded in methonal (20%)

followed by ethyl acetate (18.2%), chloroform (11.3%), and diethylether (11%).

Stem

In stem, the highest value was seen in methanol extract (22.9%) when

compared to other solvents like ethyl acetate (20%), chloroform (14%), and

diethylether (9%).

Root

In root, the highest value was recorded in methanol (17.5%), when

compared to other solvents like ethyl acetate (15.5%), chloroform (8.7%), and

diethylether (7.8%).

Fruit

In fruit, the highest value was seen in methanol (17.9%), when compared

to other solvents like ethyl acetate (12%), chloroform (5%), and diethylether

(4.6%).

4.2.3. PH Determination of Powdered Drug

The water extract of the powdered drug of various parts like leaf, stem,

root and fruit were slightly acidic in nature. It showed that the aqueous extract

contains more number of acidic compounds (Table 10).

4.2.4. Preliminary Phytochemical Screening

Preliminary phytochemical screenings of various extracts are given in

Table 11-14.

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Leaf

The preliminary phytochemical studies in methanol, aqueous and powder

drug showed similar results. It revealed the presence of cardiac glycosides,

glycosides, alkaloids, protein, phenolic group, steroid, saponins, reducing sugar,

non-reducing polysaccharide, flavanoids and terpenoids in methanol, aqueous and

the powder drug. Aminoacids, quinones, phlobatannins, triterpenoids,

anthraquiones, catachins, coumarins and tannins were absent. In diethylether

extract, only the alkaloids were present and others were absent. In chloroform

extract, alkaloids, cardiac glycosides, flavonoids, reducing sugar, non-reducing

polysaccharide (starch), glycosides, protein, phenolic group, saponins, terpenoids

were present and the other phytocompounds were absent. In ethylacetate extract,

only cardiac glycosides, glycosides, reducing sugar, non-reducing polysaccharide

(starch) and steroids were present and the others absent. Gums, oils and resins are

absent in all the extracts.

Stem

The preliminary phytochemical studies in methanol, aqueous and powder

drug showed similar results. It revealed the presence of cardiac glycosides,

glycosides, alkaloids, protein, phenolic group, steroid, saponins, flavanoids,

reducing sugar, non-reducing polysaccharide (starch) and terpenoids in methanol,

aqueous and the powder drug and absence of aminoacids, quinones,

phlobatannins, triterpenoids, anthraquiones, catachins, coumarins and tannins. In

diethylether extract, only the alkaloids were present and other phytocompounds

were absent. In chloroform extract, alkaloids, cardiac glycosides, flavonoids,

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Table 11: Phytochemical colour reactions of various extracts of stem of Tricalysia sphaerocarpa

Phytochemicals Diethylether extract

Chloroform extract

Ethylacetate extract

Methanol extract

Aqueous extract

Powder as such

Alkaloids ++ ++ - ++ ++ ++ Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides

- ++ ++ ++ ++ ++

Catechins - - - - - - Coumarins - - - - - - Flavonoids - ++ - ++ ++ ++ Gums, oils and resins

- - - - - -

Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides

- ++ ++ ++ ++ ++

Proteins - + - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - ++ ++ + + Tannins - - - - - - Terpenoids - + - + + + Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.

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Table 12: Phytochemical colour reactions of various extracts of leaf of Tricalysia sphaerocarpa

Phytochemicals Diethylether extract

Chloroform extract

Ethylacetate extract

Methanol extract

Aqueous extract

Powder as such

Alkaloids ++ ++ - ++ ++ ++ Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides

- ++ ++ ++ ++ ++

Catechins - - - - - - Coumarins - - - - - - Flavonoids - ++ - ++ ++ ++ Gums, oils and resins

- - - - - -

Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides

- ++ ++ ++ ++ ++

Proteins - + - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - ++ ++ + + Tannins - - - - - - Terpenoids - + - + + + Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.

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glycosides, protein, phenolic group, reducing sugar, non-reducing polysaccharide

(starch), saponins, terpenoids are present and the others chemical compounds

were absent. In ethylacetate extract, only cardiac glycosides, glycosides, reducing

sugar, non-reducing polysaccharide (starch) and steroids were present and the

others were absent. Gums, oils and resins are absent in all the extracts.

Root

The preliminary phytochemical studies in methanol, aqueous and powder

drug showed similar results. It revealed the presence of cardiac glycosides,

glycosides, reducing sugar, non-reducing polysaccharide (starch), alkaloids,

protein, phenolic group and saponins in methanol, aqueous and the powder drug

and absence of terpenoids, steroids, triterpenoids, anthraquiones, catachins,

coumarins, aminoacids, quinones, phlobatannins and tannins. In diethylether

extract, the phytoconstituents were absent. In chloroform extract, alkaloids,

cardiac glycosides, flavonoids, reducing sugar, non-reducing polysaccharide

(starch), glycosides, protein, phenolic group, saponins were present and the others

were absent. In ethylacetate extract, only cardiac glycosides, reducing sugar, non-

reducing polysaccharide (starch) and glycosides were present and the others were

absent. Gums, oils and resins are absent in all the extracts.

Fruit

The preliminary phytochemical studies in methanol, aqueous showed

similar results. It revealed the presence of cardiac glycosides, glycosides,

reducing sugar, non-reducing polysaccharide (starch), alkaloids, protein, phenolic

group, saponins, steroids and flavanoids in methanol and aqueous extract and the

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Table 13: Phytochemical colour reactions of various extracts of root of Tricalysia sphaerocarpa

Phytochemicals Diethylether extract

Chloroform extract

Ethylacetate extract

Methanol extract

Aqueous extract

Powder as such

Alkaloids - + - + + + Anthraquinones - - - - - - Amino acids - - - - - - Cardiac glycosides - ++ + ++ ++ + Catechins - - - - - - Coumarins - - - - - - Flavonoids - - - - - - Gums, oils and resins

- - - - - -

Glycosides - ++ + ++ ++ ++ Non reducing polysaccharides

- ++ ++ ++ ++ ++

Proteins - + - + + + Pholobatannins - - - - - - Phenolic group - + - + + + Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - - - - - Tannins - - - - - - Terpenoids - - - - - - Triterpenoids - - - - - - Note : ++ = marked present; + = moderate present; - = absent.

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Table 14: Phytochemical colour reactions of various extracts of fruit of Tricalysia sphaerocarpa

Phytochemicals Diethylether extract

Chloroform extract

Ethylacetate extract

Methanol extract

Aqueous extract

Powder as such

Alkaloids - + - + ++ + Anthraquinones - - - - - - Amino acids - - - + - - Cardiac glycosides

- ++ + + ++ ++

Catechins - - - - - - Coumarins - - - - - - Flavonoids - + - + ++ + Gums, oils and resins

- - - - - -

Glycosides - ++ ++ ++ ++ ++ Non reducing polysaccharides

- ++ ++ ++ ++ ++

Proteins - ++ - ++ ++ + Pholobatannins - - - - - - Phenolic group - + - + + - Quinones - - - - - - Reducing sugars ++ ++ ++ ++ ++ Saponins - + - + ++ ++ Steroids - - - + + - Tannins - - - - - - Terpenoids - - - - - - Triterpenoids - - - - - - Note : ++ = marked presence; + = moderate presence; - = absent.

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other phytocompounds were absent. The powder drug showed the presence of

cardiac glycosides, glycosides, alkaloids, reducing sugar, non-reducing

polysaccharide (starch), protein, phenolic group, saponins, and flavanoids and

absence of aminoacids, quinones, phlobatannins, steroids, triterpenoids,

anthraquiones, catachins, coumarins and tannins. In diethylether extract, all the

phytocompounds were absent. In chloroform extract, alkaloids, reducing sugar,

non-reducing polysaccharide (starch), cardiac glycosides, flavonoids, glycosides,

protein, phenolic group, saponins were present and the others were absent. In

ethylacetate extract, only cardiac glycosides, reducing sugar, non-reducing

polysaccharide (starch) and glycosides were present and the others were absent.

Gums, oils and resins are absent in all the extracts.

4.2.5. GC-MS Analysis

Phytochemicals were best extracted in methanol because of its high

polarity. Hence, the methanol extract of leaves, stem, root and fruit were

subjected to GC-MS analysis to detect the possible compounds present in the

active fraction.

4.2.5.1. GC-MS Analysis of Methanolic Extract of Leaf

Totally 30 chemical compounds were identified from the methanolic

extract of leaf. Of which 9 belong to fatty acids (Oleic acid, Octadecanoic acid,

Nonadecanoic acid, n-Hexadecanoic acid, Tetradecanoic acid, 9,12,15-

Octadecatrienic acid, (Z,Z,Z)-, 9,12-Octadecadienoic acid (Z,Z)-, Eicosanoic acid,

Docosanoic acid), four to aliphatic and aromatic bicyclics

(Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-,[1R-1α,2β,5α]-,2,2-Dimethyl indene, 2,3-

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dihydro-, Bicyclo[3.1.1]heptane,2,6,6-trimethyl-, 2AH-Cyclobut[a]indene-2a-

carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester), two each to aromatic

hydrocarbons groups(2-(2-Hydroxyphenyl)buta-1,3-diene, Phenanthro [3,2-

b]furan-7,11-dione, 1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-,(+)-, (2-

Methoxyphenyl) carbamic acid, naphthalene-2-yl ester), aromatic nitrile groups

(-Ethylbenzonitrile, 2,5-Dimethylbenzonitrile), aromatic dicarboxylic esters

groups (Bis(2-ethylhexyl) phthalate, Phthalic acid, di(2-propylpentyl ester)). Of

which one compound belonged to each of the class terpenoids (Squalene),

barbiturates (cyclobarbital), aromatic alcohols group(Dibenzo[a,c]phenazin-10-

ol), aliphatic aldehydes group (9,17-Octadecadienal, (Z)-, 1H-Benzimidazole, 5,6-

dimethyl-), aromatic ketones group (Chrysene-1,7(2H,8H)-dione, 3,4,9,10-

tetrahydro-2,8-dimethyl-, tert-Butyl (5-isoproply-2-methylphenoxy)

dimethylsilane), aromatic ethers (2,3,5,6-Tetrafluoroanisole), phenolic group(4,6-

Bis(1,1-dimethylethyl)-4´-methyl-1-1´-biphenyl-2-ol) and to pyrimidinedione

group (2,4(1H,3H)-Pyrimidinedione, 5(trifluoromethyl)-). Among this, eicosanoic

acid was found to be present as major constituent with the peak area 35.77% and

retention time 21.86 minutes, followed by octadecanoic acid with the peak area

18.81 % and retention time 20.09 minutes, and followed by 9,12-octadecatrienoic

acid,(z,z)- and 9,17-octadecadienal, (z)- with the peak area 11.54 % and retention

time 19.97 minutes. 1H-Benzimidazole, 5,6-dimethyl-, 2,2-Dimethylindene,2,3,-

dihydro- and 2-(2-Hydroxyphenyl) buta-1,3-diene was found to be as least

quantity with the peak area 0.51 % and retention time 22.8 minutes (Table 15 and

Figure 3).

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Table 15: GC-MS Analysis of Methanol Extract of Leaf of Tricalysia sphaerocarpa

No. Name of the compound Molecular formula

Molecular weight

RT Peak area %

Aliphatic & Aromatic bicyclics 1 Bicyclo[3.1.1]heptane, 2,6,6-

trimethyl-, [1R-1α,2β,5α]- C10H18 138 16.942 0.91

2 2,2-Dimethylindene,2,3-dihydro-

C11H14 146 22.882 0.51

3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-

C10H18 138 16.942 0.91

4 2AH-Cyclobut[a]indene-2a-carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester

C13H14O2 202 13.456 2.89

Aromatic nitriles 5 -Ethylbenzonitrile C9H9N 131 13.456 2.89 6 2,5-Dimethylbenzonitrile C9H9N 131 13.456 2.89 Aromatic ethers 7 2,3,5,6-Tetrafluoroanisole C7H4F4O 180 13.863 1.05 Pyrimidinedione 8 2,4(1H,3H)-

Pyrimidinedione, 5(trifluoromethyl)-

C5H3F3N2O2 180 13.863 1.05

Fatty acids 9 Oleic acid C18H34O2 282 20.979 1.10 10 Octadecanoic acid C18H32O2 284 20.093 18.81 11 Nonadecanoic acid C19H38O2 298 20.979 1.10 12 n-Hexadecanoic acid C16H32O2 256 18.176 10.41 13 Tetradecanoic acid C14H28O2 228 18.176 10.41 14 9,12,15-Octadecatrienic acid,

(Z,Z,Z)- C18H30O2 278 15.01 12.32

15 9,12-Octadecadienoic acid (Z,Z)-

19.977 11.54

16 Eicosanoic acid C20H40O2 312 21.865 35.77 17 Docosanoic acid C22H44O2 340 23.477 0.77 Aliphatic aldehydes 18 9,17-Octadecadienal, (Z)- C18H32O 19.977 11.54 19 1H-Benzimidazole, 5,6-

dimethyl- C9H10N2 146 22.882 0.51

Aromatic hydrocarbons 20 2-(2-Hydroxyphenyl)buta-

1,3-diene C10H10 130 22.882 0.51

21 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-hexahydro-

C19H20O3 296 23.274 1.62

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4,4,8-trimethyl-, (+)- 22 (2-Methoxyphenyl)carbamic

acid, naphthalene-2-yl ester C17H15O3N 281 23.129 1.38

Aromatic ketones 23 Chrysene-1,7(2H,8H)-dione,

3,4,9,10-tetrahydro-2,8-dimethyl-

C20H20O2 292 23.129 1.38

24 tert-Butyl(5-isoproply-2-methylphenoxy)dimethylsilane

C16H28OSi 264 23.129 1.38

Aromatic dicarboxylic esters 25 Bis(2-ethylhexyl) phthalate C24H38O4 390 23.216 1.42 26 Phthalic acid, di(2-

propylpentyl ester) C24H38O4 390 23.216 1.42

Barbiturates 27 cyclobarbital C12H16N2O3 236 23.477 0.77 Terpenoids 28 Squalene C30H50 410 25.322 10.87 Phenolics 29 4,6-Bis(1,1-dimethylethyl)-

4´-methyl-1-1´-biphenyl-2-ol C19H28O 272 23.274 1.62

Aromatic alcohols 30 Dibenzo[a,c]phenazin-10-ol C20H12N2O 296 23.274 1.62

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4.2.5.2. GC-MS Analysis of Methanolic Extract of Stem

Totally 17 chemical compounds were identified from the methanolic

extract of stem. Of which one belongs to aliphatic hydrocarbons groups

(2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-), four

to steroid groups (Androstane-3,16-diol,(3β,5α,16α)-, Ergost-5-en-3-ol,(3β)-,

Stigmasterol, Τ-Sitosterol), five to fatty acid esters (Hexadecanoic acid, methyl

ester, 9,12-Octadecadienoic acid, methyl ester, (E,E)-, 6,9,12-Octadecatrienoic

acid, methyl ester, Octadecanoic acid, methyl ester, Eicosanoic acid, methyl ester)

three to fatty acid group (9,12,15-Octadecatrienic acid, (Z,Z,Z)-, Octadecanoic

acid, n-Hexadecanoic acid). Of which one compound belongs to the class sugars

(3-O-Methyl-d-glucose), one to the class tocopherols (t-Tocopherol), one to

aromatic nitrile (4-Hydroxy-3-methyl-beta-phenylcinnamonitrile). Among this,

octadecanoic acid was found to be present as major constituent with the peak area

29.88% and retention time 15.33 minutes, followed by n-hexadecanoic acid with

the peak area 15.10 % and retention time 12.80 minutes, and followed by 9,12,15-

octadecatrienoic acid,(z,z,z)- with the peak area 12.32 % and retention time 15.01

minutes. Hexadecanoic acid, methylester was found to be as least quantity with

the peak area 0.90 % and retention time 12.21 minutes (Table 16, Figure 4).

4.2.5.3. GC-MS Analysis of Methanolic Extract of Root

Totally 8 compounds were identified from the methanolic extract of root.

Of which five belongs to heterocyclics groups (3-chloro-2,4-dimethyl -12- thia-

1,5,6a,11, tetraaza-indeno[2,1-a]fluorine, oxitriptan, dl-5-hydroxytryptophan, 2-

methyl-5-p-dimethylaminophenyl oxadiazol, Benzo(b)thiophene,4-methyl), one

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Table 16: GC-MS Analysis of Methanol Extract of Stem of Tricalysia sphaerocarpa

No. Name of the compound Molecular formula

Molecular weight

RT Peak area %

Sugars 1 3-O-Methyl-d-glucose C7H14O6 194 11.06 6.18 Aromatic acids & esters 2 1,2-Benzenedicarboxylic

acid, bis(2-methylproplyl) ester

C16H22O4 278 11.59 1.66

Steroids 3 Androstane-3,16-

diol,(3β,5α,16α)- C19H32O2 292 21.65 1.01

4 Ergost-5-en-3-ol,(3β)- C28H48O 400 29.71 4.40 5 Stigmasterol C29H48O 412 30.17 1.45 6 Τ-Sitosterol C29H50O 414 31.31 9.81 Fatty acid esters 7 Hexadecanoic acid, methyl

ester C17H34O2 270 12.21 0.90

8 9,12-Octadecadienoic acid, methyl ester, (E,E)-

C19H34O2 294 14.23 1.89

9 6,9,12-Octadecatrienoic acid, methyl ester

C19H32O2 292 14.31 1.03

10 Octadecanoic acid, methyl ester

C19H38O2 298 14.64 1.57

11 Eicosanoic acid, methyl ester

C21H42O2 326 17.34 1.42

Fatty acid 12 9,12,15-Octadecatrienic

acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32

13 Octadecanoic acid C18H36O2 284 15.33 29.88 14 n-Hexadecanoic acid C16H32O2 256 12.80 15.10 Aromatic nitrile 15 4-Hydroxy-3-methyl-beta-

phenylcinnamonitrile C16H13NO 235 22.07 1.94

Aliphatic hydrocarbons 16 2,6,10,14,18,22-

Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-

C30H50 410 24.02 5.99

Tocopherols 17 Τ-Tocopherol C28H48O2 416 28.26 3.45

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to aromatic carboxylic ester groups (Benzoic acid, 4-(3-hydroxy-3-methyl-1-

butynyl)-methyl ester), one to fatty esters (Hexadecanoic acid,

1a,2,5,5,5a,6,9,10,10a, octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-

dioxo-1H-2,8a-methano cyclopenta (a) cyclopropa (e) cyclodecen-5-yl ester) and

one to triterpenoid group (9,19-cyclolanostane-6,7-dione,3-acetoxy-). Among

this, benzo(b)thiophene, 4-methyl was found to be present as major constituent

with the peak area 100% and retention time 18.58 minutes, followed by 2-methyl-

5-p-dimethylaminophenyl oxadiazol with the same peak area and retention time

17.8 minutes, and followed by oxitriptan with peak area 100% and retention time

17.13 minutes. 3-chloro-2,4-dimethyl-12-thia-1,5,6a,11-tetraaza-indeno(2,1-a)

fluorine was found to be as in least quantity with the peak area 28 % and

retention time 7.94 minutes respectively (Table 17, Figure 5).

4.2.5.4. GC-MS Analysis of Methanolic Extract of Fruit

Totally 10 compounds were identified from the methanolic extract of fruit.

Of which three belongs to heterocyclics group (N-[4-(4-chlorophenyl)isothiazol-

5yl]-1-methylpiperidin-2-imine, N-[2-(1-piperazyl)ethyl]-N-[2-

thiophosphatoethyl]-1,3-propanamine, 5,8,15,18,23-pentaoxa-1,12-diazabicyclo

(10,8,5)-pentacosane), one to aliphatic aldehyde groups (4-octadecenal), one to

thiosulphate group (S,S1-3,8-diazaundecamethylene bis[hydrogenthiosulfate]),

one to thiophosphates group (2-[3-cyclohexylaminopropylamino]ethyl

thiophosphate), one to antibiotic (Deoxyspergualin) and three to others i.e.

unclassified (4,13,20-tri-O-methylphorbol 12-acetate, EPPS, 2-myristynoyl

pantetheine). Among this, S,S1-3,8-Diazaundecamethylene bis[hydrogen

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Table 17: GC-MS Analysis of Methanol Extract of Root of Tricalysia sphaerocarpa

No. Name of the compound Molecular

formula Molecular weight

RT Peak area %

Heterocyclics 1 3-chloro-2,4-dimethyl-

12-thia-1,5,6a,11,tetraaza-indeno[2,1-a]fluorine

C16H10N4SCl 325.5 7.94 28

2 dl-5-hydroxytryptophan C11H12N2O3 220.2 16.6 100 3 oxitriptan C11H12N2O3 220.2 17.13 100 4 2-methyl-5-p-

dimethylaminophenyl oxadiazol

C11H13N3O 203.2 17.8 100

5 Benzo(b)thiophene,4-methyl

C9H8S 148.2 18.58 100

Aromatic carboxylic ester 6 Benzoic acid,4-(3-

hydroxy-3-methyl-1-butynyl)-methyl ester

C13H14O3 218.3 12.53 61.7

Fatty esters 7 Hexadecanoic

acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester

C36H52O 580 22.98 10.4

Triterpenoids 8 9,19-cyclolanostane-6,7-

dione,3-acetoxy- C32H50O4 498.7 11.86 14.9

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Table 18: GC-MS Analysis of Methanol Extract of Fruit of T. sphaerocarpa

No. Name of the compound Molecular formula

Molecular weight

RT Peak area %

Antibiotics 1 Deoxyspergualin C17H37N7O3 512.96 9.27 25.7 Aliphatic aldehydes 2 4-octadecenal C18H34O 266.5 14.43 18.8 Heterocyclics 3 N-[4-(4-chlorophenyl)

isothiazol-5yl]-1-methylpiperidin-2-imine

C15H16ClN3S 305.8 15.02 16

4 N-[2-(1-piperazyl)ethyl]-N-[2-thiophosphatoethyl] -1,3-propanamine

C11H27N4O3PS 326.4 12.08 12.4

5 5,8,15,18,23-pentaoxa-1,12-diazabicyclo (10,8,5)-pentacosane

C18H36N2O5 370 19.15 61.2

Thiophosphates 6 2-[3-cyclohexylamino

propylamino]ethyl thiophosphate

C11H25N2SO4 296.4 18.9 47.9

Thiosulphates 7 S,S1-3,8-

diazaundecamethylene bis[hydrogenthiosulfate]

C9H22N2O6S4 382.5 17.2 83.7

Others 8 4,13,20-tri-O-

methylphorbol 12-acetate C24H35O7 435 25.97 12.5

9 EPPS C9H20N2SO4 252 20.63 15.9 10 2-myristynoyl

pantetheine C25H44N2O5S 484.7 10.88 15.4

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thiosulfate] was found to be present as major constituent with the peak area 83.7%

and retention time 17.2 minutes, followed by 5,8,15,18,23-pentaoxa-1,12-

diazabicyclo(10,8,5)-pentacosane with the peak area 61.2% and retention time

19.15 minutes. N-[2-[1-piperazyl]ethyl]-N-[2-thiophosphatoethyl]-1,3-

propanamine was found to be as in least quantity with the peak area 12.4 % and

retention time 12.08 minutes respectively. Deoxyspergualin is found to be an

antibiotic with the peak area 9.27 % and retention time 25.7 minutes (Table 18,

Figure 6).

4.2.5.5. Comparative Analysis of Compounds Identified by GC-MS Analysis

Totally 65 compounds were identified from various parts of Tricalysia

sphaerocarpa through GC-MS analysis. Among this, 30 compounds were isolated

from leaf, 17 from stem, 10 from fruit and 8 from root. Octadecanoic acid, n-

hexadecanoic acid and 9,12,15- octadecatrienoic acid (Z,Z,Z-) are the common

compounds seen both in stem and leaf (Table 19). Totally 26 different groups of

compounds are seen. Among this 12 compounds belongs to fatty acid group, 8

compounds belongs to heterocyclics, 6 compounds belongs to fatty acid esters, 4

belongs to steroids, 4 belongs to aliphatic & aromatic bicyclics, 3 belongs to

aromatic hydrocarbons, 3 belongs to aromatic nitriles, 3 belongs to unclassified, 2

belongs to aliphatic aldehydes, 2 belongs to aromatic ketones, 2 belongs to

aromatic dicarboxylic esters, and one compound each belongs to triterpenoid,

antibiotic thiosulphates, thiophosphates, alphatic hydrocarbons, aromatic esters,

tocopherol, aromatic acid & esters, sugars, aromatic alcohols, phenolics,

terpenoids, barbiturates, pyrimidinedione and aromatic ethers. Fatty acid group

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Table 19: Combined Table for GC-MS Analysis of Methanol Extract of Tricalysia sphaerocarpa

No. Name of the compound Leaf Stem root fruit activity 1 Bicyclo[3.1.1]heptane,

2,6,6-trimethyl-, [1R-1α,2β,5α]-

+ - - - No activity found

2 2,2-Dimethylindene,2,3-dihydro-

+ - - - No activity found

3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-

+ - - - No activity found

4 2AH-Cyclobut[a]indene-2a-carboxylic acid, 1,2,7,7a-tetrahydro, methyl ester

+ - - - No activity found

5 -Ethylbenzonitrile + - - - No activity found 6 4-Hydroxy-3-methyl-

beta-phenylcinnamonitrile

- + - - No activity found

7 2,5-Dimethylbenzonitrile + - - - No activity found 8 2,3,5,6-

Tetrafluoroanisole + - - - Raw material for

bioenergy, biomedicines 9 2,4(1H,3H)-

Pyrimidinedione, 5(trifluoromethyl)-

+ - - - Antiviral

10 Oleic acid + - - - Antibacterial, Antifungal 11 Octadecanoic acid + + - - Antibacterial, Antifungal 12 9,12,15-Octadecatrienic

acid, (Z,Z,Z)- + + - - No activity found

13 9,12-Octadecadienoic acid (Z,Z)-

+ - - - Anti-inflammatory, hypocholesterolemic, cancer preventive, insectifuge, antiarthritic, hepatoprotective, antiandrogenic, nematicide, antihistaminic, antieczemic

14 9,17-Octadecadienal, (Z)-

+ - - - antimicrobial

15 9,12-Octadecadienoic acid, methyl ester, (E,E)-

- + - - No activity found

16 6,9,12-Octadecatrienoic acid, methyl ester

- + - - No activity found

17 4-octadecenal - - - + No activity found 18 Octadecanoic acid,

methyl ester - + - - No activity found

19 Nonadecanoic acid + - - - Cytotoxic activities

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20 Eicosanoic acid + - - - No activity found 21 Eicosanoic acid, methyl

ester - + - - No activity found

22 n-Hexadecanoic acid + + - - Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, Lubricant, Antiandrogenic, anti inflammatory, Flavor, Hemolytic, 5-Alpha reductase inhibitor

23 Hexadecanoic acid, methyl ester

- + - - No activity found

24 Hexadecanoic acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester

- - + - No activity found

25 Tetradecanoic acid + - - - Antioxidant, Cancer preventive, Cosmetic, Nematicide, Lubricant, Hypercholesterolemic

26 Docosanoic acid + - - - Antimicrobial, lubricant, hair conditioners

27 1H-Benzimidazole, 5,6-dimethyl-

+ - - - No activity found

28 2-(2-Hydroxyphenyl)buta-1,3-diene

+ - - - No activity found

29 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-, (+)-

+ - - - No activity found

30 (2-Methoxyphenyl)carbamic acid, naphthalene-2-yl ester

+ - - - No activity found

31 Chrysene-1,7(2H,8H)-dione, 3,4,9,10-tetrahydro-2,8-dimethyl-

+ - - - No activity found

32 tert-Butyl(5-isoproply-2-methylphenoxy)dimethylsilane

+ - - - No activity found

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33 Bis(2-ethylhexyl) phthalate

+ - - - No activity found

34 Phthalic acid, di(2-propylpentyl ester)

+ - - - Antibacterial, Oral toxicity during pregnancy and sucking in the Long-Evans Rat

35 cyclobarbital + - - - Anesthetic, anticonvulsant, sedative, hypnotic, veterinary euthanasia agent

36 Squalene + - - - Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immunostimulant, Chemo preventive, Lipoxygenaseinhibitor, Pesticide

37 4,6-Bis(1,1-dimethylethyl)-4´-methyl-1-1´-biphenyl-2-ol

+ - - - No activity found

38 Dibenzo[a,c]phenazin-10-ol

+ - - - No activity found

39 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-

- + - - No activity found

40 3-O-Methyl-d-glucose - + - - No activity found 41 1,2-Benzenedicarboxylic

acid, bis(2-methylproplyl) ester

- + - - Used in preparation of perfumes and cosmetics, plasticized vinyl seats on furniture, cars, and clothing including jackets, raincoats, and boots and used in textiles, as dyestuffs, cosmetics, and glass making

42 Androstane-3,16-diol,(3β,5α,16α)-

- + - - No activity found

43 Ergost-5-en-3-ol,(3β)- - + - - No activity found 44 Stigmasterol - + - - Antimicrobial, Diuretic,

Antiinflammatory, Antiasthma, Antiarthritic, Anticancer

45 τ-Sitosterol - + - - Antibacterical activity

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46 τ-Tocopherol - + - - Vitamin E, cosmetics 47 Deoxyspergualin - - - + Antitumour,

immunosuppressive activity

48 2-myristynoyl pantetheine

- - - + No activity found

49 N-[2-(1-piperazyl)ethyl]-N-[2-thiophosphatoethyl]-1,3-propanamine

- - - + No activity found

50 N-[4-(4-chlorophenyl)isothiazol-5yl]-1-methylpiperidin-2-imine

- - - + antipsychotic activity

51 5,8,15,18,23-pentaoxa-1,12-diazabicyclo(10,8,5)-pentacosane

- - - + No activity found

52 2-[3-cyclohexylaminopropylamino]ethyl thiophosphate

- - - + No activity found

53 EPPS - - - + No activity found 54 S,S1-3,8-

diazaundecamethylene bis[hydrogenthiosulfate]

- - - + No activity found

55 4,13,20-tri-O-methylphorbol 12-acetate

- - - + No activity found

56 3-chloro-2,4-dimethyl-12-thia-1,5,6a,11,tetraaza-indeno[2,1-a]fluorine

- - + - No activity found

57 dl-5-hydroxytryptophan - - + - insomnia, depression, anxiety, lower blood pressure in Hypertension patients, anti inflammatory, stimulate the production of antibodies

58 oxitriptan - - + - No activity found 59 2-methyl-5-p-

dimethylaminophenyl oxadiazol

- - + - antifungal

60 Benzo(b)thiophene,4-methyl

- - + - No activity found

61 Benzoic acid,4-(3-hydroxy-3-methyl-1-butynyl)-methyl ester

- - + - No activity found

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62 9,19-cyclolanostane-6,7-dione,3-acetoxy-

- - + - immunosuppressive effect

*SOURCE: earlier reported Note : + = present; - = absent.

Table 20: Chemicals groups obtained from GC-MS Analysis of Methanol Extract of Tricalysia sphaerocarpa

Sl.No

Group No. of compounds Total of compounds leaf stem root fruit

1 Aliphatic & aromatic bicyclics

4 - - - 4

2 Aromatic nitriles 2 1 - - 3 3 Aromatic ethers 1 - - - 1 4 Pyrimidinedione 1 - - - 1 5 Fatty acids 9 3 - - 12 6 Aliphatic aldehydes 2 - - 1 3 7 Aromatic hydrocarbons 3 - - 3 8 Aromatic ketones 2 - - 2 9 Aromatic dicarboxylic

esters 2 - - - 2

10 barbiturates 1 - - - 1 11 terpenoids 1 - - - 1 12 Phenolics 1 - - - 1 13 Aromatic alcohols 1 - - - 1 14 sugars - 1 - - 1 15 Aromatic acids & esters - 1 - 1 16 steroids - 4 - - 4 17 Fatty acid esters - 5 1 - 6 18 Aliphatic hydrocarbons - 1 - - 1 19 tocopherols - 1 - - 1 20 heterocyclics - - 5 3 8 21 Aromatic esters - - 1 - 1 22 Thiophosphates - - - 1 1 23 Thiosulphates - - - 1 1 24 antibiotic - - - 1 1 25 triterpenoids - - 1 - 1 26 others 3 3 Total 30 17 8 10 65

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plays a major role with 12 different chemical constituents with therapeutic

significance (Table 20).

4.3. Pharmacology

4.3.1. In-vitro Antioxidant Activity

4.3.1.1. DPPH Scavenging Activity

In stem, maximum activity was observed in chloroform extract. The

percentage of scavenging activities at 100µg/ml, 50µg/ml and at 10µg/ml were

observed to be 87.73 ± 8.37, 67.22 ± 7.23 and 58.68 ± 6.47 respectively. In

methanol extract, the percentage of scavenging activity at 100µg/ml was 71.62 ±

7.76, 62.43 ± 6.74 at 50µg/ml and 51.59 ± 5.95 at 10µg/ml. In petroleum ether

extract, the percentage of scavenging activity at 100µg/ml was 66.63 ± 7.23,

42.65 ± 5.82 at 50µg/ml and 28.67 ± 5.76 at 10µg/ml. In water extract, the

percentage of scavenging activity at 100µg/ml, 50µg/ml and 10µg/ml were 51.49

± 5.85, 47.48 ± 4.92 and 35.53 ± 4.81 respectively. All the extracts showed dose

concentration dependent activity in all the tested concentration. The significantly

higher activity was observed in the chloroform extract and the results are

presented in the table 21 and figure 7.

4.3.1.2. Iron Chelating activity (FRAP Assay)

In stem, maximum value of Iron Chelating activity was observed in

chloroform extract as 66.46 ± 6.21 at 100µg/ml, 41.24 ± 3.98 at 50µg/ml and

25.52 ± 1.95 at 10µg/ml. In methanol extract, the percentage of scavenging

activity at 100µg/ml was 61.77 ± 5.98, 49.87 ± 4.76 at 50µg/ml and 32.02 ± 2.84

at 10µg/ml. In water extract, the percentage of scavenging activity at 100µg/ml

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Table 21: Antioxidant activity of various extracts using DPPH assay: Solvent % of radicle scavenging

10µg/ml 50µg/ml 100µg/ml Petroleum ether 28.674 ± 5.76 42.650 ± 5.82 66.638± 7.23 Chloroform 58.688±6.47 67.224± 7.23 87.728± 8.37 Methanol 51.595±5.95 62.439± 6.74 71.624± 7.76 Water 35.553±4.61 47.485± 4.92 51.496± 5.85 BHT 47.393± 5.89 69.784± 7.76 89.720± 8.80 All the values are expressed as mean ± SEM(n=6) Table 22: Antioxidant activity of various extracts using Iron chelating activity Solvent % of radicle scavenging

10µg/ml 50µg/ml 100µg/ml Petroleum ether 10.789± 1.79 29.456± 2.46 38.493± 2.79 Chloroform 25.523± 1.95 41.240± 3.98 66.458±6.21 Methanol 32.029± 2.84 49.879± 4.76 61.776± 5.98 Water 29.555± 3.65 40.788± 3.62 55.466± 5.72 EDTA 59.437± 5.77 70.125± 6.75 95.459± 8.39 All the values are expressed as mean ± SEM(n=6) Table 23: Antioxidant activity of various extracts using Hydrogen peroxide assay Solvent % of radicle scavenging

10µg/ml 50µg/ml 100µg/ml Petroleum ether 0.240±1.65 0.578±3.54 0.724±7.58 Chloroform 0.442±2.79 0.712±5.71 0.993±8.32 Methanol 0.378±2.43 0.562±3.45 0.728±7.61 Water 0.153±1.30 0.310±2.73 0.478±3.26 Ascorbic acid 0.994±1.18 1.120±8.41 1.440±8.72 All the values are expressed as mean ± SEM(n=6) Table 24: Antioxidant activity of various extracts using Superoxide dismutase assay Solvent % of radicle scavenging

10µg/ml 50µg/ml 100µg/ml Petroleum ether 1.988±3.75 2.590±4.21 4.778±6.79 Chloroform 2.322±3.98 5.330±4.65 6.901±6.37 Methanol 4.784±4.06 6.120±5.39 7.953±6.59 Water 2.850±3.22 4.875±6.32 5.445±5.25 Ascorbic acid 5.510±4.81 8.010±7.51 9.847±8.73 All the values are expressed as mean ± SEM(n=6)

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was 55.46 ± 5.72, 40.78 ± 3.62 at 50µg/ml and 29.55 ± 3.65 at 10µg/ml. In

petroleum ether extract, the percentage of scavenging activity at 100µg/ml was

38.49 ± 2.97, 29.45 ± 2.46 at 50µg/ml and 10.78 ± 1.79 at 10µg/ml. All the

extracts showed dose concentration dependent activity in all the tested

concentration. The significantly higher activity was observed in the chloroform

extract and the results are presented in the table 22 and figure 8.

4.3.1.3. Hydrogen peroxide Assay

The results of hydrogen peroxide assay were presented in the table 23 and

figure 9. In stem, all the extracts showed dose concentration dependent activity in

all the tested concentrations. Chloroform extract showed the maximum value as

0.99 ± 8.32 at 100µg/ml, 0.71 ± 5.91 at 50µg/ml and 0.44 ± 2.79 at 10µg/ml,

moderate value was observed in methanol extract as 0.73 ± 7.61 at 100µg/ml,

0.56 ± 3.45 at 50µg/ml and 0.37 ± 2.43 at 10µg/ml followed by petroleum ether

extract as 0.72 ± 7.58 at 100µg/ml, 0.57 ± 3.54 at 50µg/ml and 0.24 ± 1.65 at

10µg/ml and the minimum value was observed in water extract as 0.47 ± 3.26 at

100µg/ml, 0.31 ± 2.73 at 50µg/ml and 0.15 ± 1.30 at 10µg/ml respectively. The

chloroform extract showed the high performance activity.

4.3.1.4. Superoxide dismutase (L- methionine and NBT Assay)

In stem, the maximum Superoxide dismutase activity was observed in

methanol extract as 7.95 ± 6.59 at 100µg/ml, 6.12 ± 5.39 at 50µg/ml and 4.78 ±

4.06 at 10µg/ml followed by the chloroform extract as 6.90 ± 6.37 at 100µg/ml,

5.33 ± 4.65 at 50µg/ml and 2.32 ± 3.98 at 10µg/ml, water extract showed 5.45 ±

5.95 at 100µg/ml, 4.87 ± 5.01 at 50µg/ml and 2.85 ± 3.22 at 10µg/ml and the

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minimum value was observed in petroleum ether extract as 4.78 ± 6.79 at

100µg/ml, 2.59 ± 4.21 at 50µg/ml and 1.98 ± 3.96 at 10µg/ml respectively. It

shows that the methanolic extract to be the most potential scavenger. The results

of Superoxide dismutase assay were presented in the table 24 and figure 10.

4.3.2. In-vivo Pharmacological studies

4.3.2.1. Acute toxicity

No mortality was observed in the animals treated with 2000 mg/kg

methanol extract of stem. There were no signs of any toxicity.

4.3.3. Anti-depressant activity

The behavioral despair model was performed in order to investigate the

ability of this herbal drug in the elevation of suppressed mood, which is quite

common in today’s scenario. The results obtained from FST (Forced swimming

test), TST (Tail suspension test) and HBT (Hole board test) clearly reveled the

fact that this drug is potentially quite useful in cases of depression (Table 25-27).

The present findings suggested that methanolic extract when administered at an

acute oral dose of 200 mg/kg of body weight (P<0.01) reduced the immobility

time by 135 seconds as compared to the immobility time of control i.e. 190

seconds the time shown by animals treated with extract was found to be 170

seconds when it was compared with control and standard. The decrease in the

immobility time was quite close to that of standard. The time of mobility was

increased by methanolic extract at a dose of 200 mg/ml, shown the immobility

time **140 seconds (P<0.01) to that of standard **135 seconds (P<0.01). These

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Table 25 : Effect of methanolic extract of Tricalysia sphaerocarpa on Immobility time in FST

Group no. Drug treatment Dose mg/kg Immobility period

I Contol (Distilled water) 5ml/kg 190 sec

II Methanolic extract 100 mg/kg 170 sec.

III Methanolic extract 200 mg/kg 135 sec.

IV Standard drug 30 mg/kg 140 sec.

Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01.

Table 26 : Effect of methanolic extract of T. sphaerocarpa in Immobility time in TST

Group no. Drug treatment Dose Immobility period I Control (Distilled water) 5ml/kg 195 sec.

II Methanolic extract 100 mg/kg 170 sec.

III Methanolic extract 200 mg/kg 165 sec.

IV Standard drug 30 mg/kg 135 sec.

Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01. Table 27: Effect of methanolic extract of T. sphaerocarpa. in Hole Board Test (HBT) Group Dose(i.p;mg/kg) No. of head dippings

per 3 min No. of line crossings per 3min

Control(distilled water)

5ml/kg 22 ± 1.43 18 ± 0.9

Standard(Diazepam) 4mg/kg 8 ± 0.71 10 ± 1.24

Test group-1 100mg/kg 18 ± 1.04 15 ± 0.55

Test group-2 200mg/kg 13 ± 1.01 14 ± 0.55

Results are expressed as mean ±S.E.M [n=6]. Test group compared with control group p<0.01. test group compared with standard group p<0.01.

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results show that after standard i.e. Imipramine HCl (30 mg/kg), the methanolic

extract is most potent amongst all the treated groups.

Findings on tail suspension test were quite comparable to the previous FS

test. It is quite evident that none of the drug treated animals showed excellent

results compared to the standard. The immobility of Imipramine HCL (P<0.01) 30

mg/ kg was found to be 135 seconds. In this test the time of animals treated with

methanolic extract 100 mg/kg was found to be 170 seconds (P<0.01) when it was

compared to the control group of animals which was 195 seconds. The

immobility time of methanolic extract when given an acute dose of 200 mg/kg

each of body weight significantly reduced the time of immobility by 135 seconds

(P<0.01). The present findings on HBT suggested that methanolic extract when

administered at an acute oral dose of 200 mg/kg of body weight (P<0.01) reduced

the number of head dippings by 13 per 3 min and the number of line crossing per

3 minutes is 14. Adminstration of an acute oral dose of 100 mg/kg of body weight

(P<0.01) reduced the number of head dipping by 18 per 3 min and the number of

line crossing per 3 min is 15 as compared to control number of head dipping by

23 per 3 minutes and the number of line crossing per 3 min is 18 and as standard,

number of head dipping by 8 per 3 minutes and the number of line crossing per 3

minutes is 10. The results clearly revel the fact that standard treated animals

showed better response as compared to the plant extract treated groups but even

though methanolic extract 200 mg/kg treated group showed better response as

compared to standard drug treated group of animals (Plate 8).

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4.3.2.3. Anti - diabetic Activity

4.3.2.3.1. Effect of Methanolic Stem Extract on Blood Glucose Level in

Normal Fasted Rats

All doses of methanolic stem extract of Tricalysia sphaerocarpa in normal

fasted rats, significantly (P<0.05) reduced the blood glucose levels up to 4 hour

except the lowest dose. The impairment of blood glucose levels of Tricalysia

sphaerocarpa was marked and dose dependent at each time point. The normal

control group showed 60.3 ± 1.70 mg/dl at 0 hour, 62.5 ± 2.12 mg/dl one hour

after the treatment, 61.0 ± 1.24 mg/dl 2 hours after the treatment, 60.2 ± 1.14

mg/dl after 4 hour of treatment, 58.2 ± 2.62 mg/dl after 6 hour of treatment. The

group treated with 125 mg/kg showed 62.0 ± 4.80 mg/dl after the treatment of 0

hour, 62.0 ± 1.90 mg/dl after the treatment of 1 hour, 61.0 ± 5.17 mg/dl after 2

hour of treatment, 59.0 ± 1.80 mg/dl after 4 hour of treatment, 60.3 ± 2.70 mg/dl

after 6 hour of treatment. The group treated with 250 mg/kg showed 64.1 ± 2.72

mg/dl after the treatment of 0 hour, 65.0 ± 1.22 mg/dl after the treatment of 1

hour, 63.5 ± 2.62 mg/dl after 2 hour of treatment, 60.3 ± 2.14 mg/dl after 4 hour

of treatment, 62.0 ± 1.40 mg/dl after 6 hour of treatment. The group treated with

500 mg/kg showed 70.0 ± 2.12 mg/dl after the treatment of 0 hour, 68.0 ± 2.01

mg/dl after the treatment of 1 hour, 64.1 ± 1.70 mg/dl after 2 hour of treatment,

59.0 ± 6.20 mg/dl after 4 hour of treatment, 63.0 ± 2.12 mg/dl after 6 hour of

treatment. The group treated with glibenclamide 5 mg/kg showed 61.1 ± 3.18

mg/dl at 0 hour the treatment, 57.0 ± 1.20 mg/dl after the treatment of 1 hour,

51.3 ± 2.24 mg/dl after 2 hour of treatment, 46.3 ± 2.34 mg/dl after 4 hour of

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Table 28 : Effect of Test extract on blood glucose level in normal fasted rats Group Treatment (dose mg/kg, po)

Blood Glucose level (mg/dl) 0 hr 1 hr 2 hr 4 hr 6 hr

Normal control 60.3 ± 1.70 62.5 ± 2.12 61.0 ±1.24 60.2 ±1.14 58.2 ±2.62 Test extract (125) 62.0 ± 4.80 62.0 ±1.90

(1.5) 61.0 ±5.17 (4.5)

59.0 ±1.80 (10.6)

60.3 ±2.70a

(8.6)

Test extract (250) 64.1 ± 2.72 65.0 ±1.22 (3.1)

63.5 ±2.62 a

(5.3) 60.3 ±2.14 b

(10.3) 62.0 ±1.40b

(5.1)

Test extract (500) 70.0 ± 2.12 68.0 ±2.01a

(5.5) 64.1 ±1.70 b

(10.9) 59.0 ±6.20 b

(18.0) 63.0 ±2.12b

(12.5)

Glibenclamide (5) 61.1 ± 3.18 57.0 ± 1.20 (6.7)

51.3 ±2.24 b

(16.0) 46.3 ±2.34 b

(24.2) 50.3 ±2.00 b

(17.6) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to 0 hr. Table 29 : Effect of Test extract on blood glucose level in Alloxan-induced diabetic rats (Single-dose short term study)

Group & Treatment

(dose mg/kg, po)

Blood Glucose Level (mg/dl)

0 hr 1 hr 3 hr 6 hr

Normal control 60.8±1.60 61.6±2.02 62.5±1.64 60.1±1.20

Diabetic control 295.8±1.96 a 306.1±2.80a

(-3.7) 299.6±1.26a

(-1.5) 300.3±1.82a

(-1.7) Test extract (125) 309.5±2.07 270.0±1.80 b

(11.9) 212.0±1.60 b

(31.4) 202.0±2.21 b

(32.1) Test extract (250) 302.3±1.00 241.0±1.12 b

(21.1) 200.3±1.06 b

(32.8) 199.0±2.10 b

(34.2) Test extract (500) 300.0±1.46 210.1±2.00 b

(29.7) 132.5±2.20 b

(54.8) 150.1±2.31 b

(49.3) Glibenclamide (5) 280.0±2.20 198.0±1.20 b

(29.4) 100.5±1.87 b

(60.1) 120.8±2.60 b

(54.1) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to 0 hr. P value: <0.01; compared to a normal group, b diabetic group

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treatment, 50.3 ± 2.00 mg/dl after 6 hour of treatment. The maximum

hypoglycemic activity was induced by 500 mg/kg dose at 4 hour by 18%.

However, the effects of other tested doses of Tricalysia sphaerocarpa were less

than the reference drug (Table 28).

4.3.2.3.2. Effect of Test Extract on Blood Glucose Level in Alloxan-induced

Diabetic Rats

4.3.2.3.2.1. Effect of single dose administration of Test extract on blood

glucose level in Alloxan-induced diabetic rats (Short Term Study):

All doses of methanolic stem extract of T. sphaerocarpa in alloxan-

induced diabetic rats, significantly (P<0.01) reduced the blood glucose levels up

to 6 hour except the lowest dose. The normal control group showed 60.8 ± 1.60

mg/dl at 0 hour, 61.6 ± 2.02 mg/dl after the treatment of 1 hour, 62.5 ± 1.64 mg/dl

after 3 hour of treatment, 60.1 ± 1.20 mg/dl after 6 hour of treatment. The group

treated with 125 mg/kg showed 270.0 ± 1.80 mg/dl after the treatment of 1 hour,

212.0 ± 1.60 mg/dl after 3 hour of treatment, 202.0 ± 1.60 mg/dl after 6 hour of

treatment. The group treated with 250 mg/kg showed 241.0 ± 1.12 mg/dl after the

treatment of 1 hour, 200.3 ± 1.06 mg/dl after 3 hour of treatment, 199.0 ± 2.10

mg/dl after 6 hour of treatment. The group treated with 500 mg/kg showed 210.1

± 2.00 mg/dl after the treatment of 1 hour, 132.5 ± 2.20 mg/dl after 3 hour of

treatment, 150.1 ± 2.31 mg/dl after 6 hour of treatment. The group treated with

glibenclamide 5 mg/kg showed 198.0 ± 1.20 mg/dl after the treatment of 1 hour,

100.5 ± 1.87 mg/dl after 3 hour of treatment, 120.8 ± 2.60 mg/dl after 6 hour of

treatment. The diabetic control group showed 295.8 ± 1.96 mg/dl at 0 hour, 306.0

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± 2.80 mg/dl after the treatment of 1 hour, 299.6 ± 1.26 mg/dl after 3 hour of

treatment, 300.3 ± 1.82 mg/dl after 6 hour of treatment (Table 29).

4.3.2.3.2.2. Effect of multidose administration of Test extract on blood

glucose level in Alloxan-induced diabetic rats (long term study of 15 days

daily once)

The methanol extract of T. sphaerocarpa showed significant (P<0.01)

plasma glucose lowering effect. The present study indicates that alloxan induced

tissue injury is reversed by continuous administration of T. sphaerocarpa extract

with subsequent decrease in blood sugar. Alloxan treated diabetic rats showed

significant increase in the level of fasting plasma glucose levels when compared

to normal rats. Alloxan generate free radicals in the body, leads to tissue damage

including pancreas and would be responsible for increased blood sugar seen in the

animals. Oral administration of T. sphaerocarpa methanolic extract of 500 mg/kg

showed significant (P<0.01) plasma glucose lowering effect in 12 and 16 days of

treatment. The normal control group showed 56.1 ± 1.02 mg/dl on 3rd day, 61.0 ±

1.20 mg/dl on 6th day, 61.0 ± 0.82 mg/dl on day 9, 62.8 ± 2.10 mg/dl on day 12

and 58.9 ± 4.40 mg/dl on 16th day of treatment. The group treated with 125mg/kg

showed 291.2 ± 2.26 mg/dl on 3rd day, 280.5 ± 1.40 mg/dl on 6th day, 260.2 ±

2.30 mg/dl on day 9, 230.0 ± 4.02 mg/dl on day 12 and 192.1 ± 0.62 mg/dl on

16th day of treatment. The group treated with 250mg/kg showed 290.2 ± 0.01

mg/dl on 3rd day, 270.2 ± 4.20 mg/dl on 6th day, 228.0 ± 0.48 mg/dl on day 9,

184.0 ± 2.70 mg/dl on day 12 and 172.2 ± 1.50 mg/dl on 16th day of treatment.

The group treated with 500mg/kg showed 280.0 ± 2.21 mg/dl on 3rd day, 252.2 ±

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Table 30 : Effect of multidose administration of Test extract on blood glucose level in Alloxan-induced diabetic rats (long term study of 15 days daily once)

Group & treatment

(dose mg/kg; p.o)

Blood glucose level (mg/dl)

Day 3

Day 6

Day 9

Day 12

Day 16

Normal control

56.1±1.02 61.0±1.20 61.0±0.82 62.8±2.10 58.9±4.40

Diabetic control

312.5 ±1.20 a

306.1±2.12a

(1.7) 290.8 ± 0.42a

(6.8) 279.2 ± 2.12a

(10.3) 278.2 ± 2.20a

(11.96) Test extract (125)

291.2 ± 2.26 b

280.5± 1.40c

(4.4) 260.2 ± 2.30c

(11.3) 230.0 ± 4.02c

(27.1) 192.1 ± 0.62c

(33.5)

Test extract (250)

290.2 ± 0.01 c

270.2± 4.20c

(6.4) 228.0 ± 0.48c

(20.7) 184.0 ± 2.70c

(35.1) 172.2 ± 1.50c

(39.9) Test extract (500)

280.0 ± 2.21 c

252.2± 2.12c

(10.6) 220.0 ± 2.12c

(22.6) 182.4 ± 2.10c

(36.4) 162.2 ± 2.10c

(35.7) Glibenclamide(5)

278.5 ± 2.30 c

240.2± 2.12c

(1.5) 210.6 ± 1.40c

(22.5) 182.5 ± 0.86c

(35.8) 164.5 ± 2.12c

(42.9) Values are mean ± SEM from 6 animals in each group. Figure in parenthesis indicates % fall in BGL as compared to Day 3. p values: <0.01, as compared to a normal group; c

diabetic control group b<0.05 compared to diabetic group.

Table 31 : Effect of formulation Test extract on body weight in Normal and Alloxan induced diabetic rats

Group & treatment (dose,

mg/kg; p.o)

Initial Body weight (g)

Final Body weight (g)

% increased/ decreased of body weight

Normal control 80.22±2.70 102.00±8.22 + 18.30

Diabetic control 99.60±10.20 60.12±2.90 - 41.92

Test extract (125) 82.60±12.34 50.82±8.92 - 34.80

Test extract (250) 84.22±10.22 63.44±12.12 - 25.44

Test extract (500) 88.12±8.44 74.10±4.22 - 19.00

Glibenclamide (5) 90.00±11.02 76.22±8.52 - 15.98

values are mean ± SEM from 6 animals in each group Where + indicates % increase of body weight.

- Indicates % decrease of body weight.

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2.12 mg/dl on 6th day, 220.0 ± 2.12 mg/dl on day 9, 182.4 ± 2.10 mg/dl on day 12

and 162.2 ± 2.10 mg/dl on 16th day of treatment. The group treated with

glibenclamide 5mg/kg showed 278.5 ± 2.30 mg/dl on 3rd day, 240.2 ± 2.12 mg/dl

on 6th day, 210.6 ± 1.40 mg/dl on day 9, 182.5 ± 0.86 mg/dl on day 12 and 164.5

± 2.12 mg/dl on 16th day of treatment. The diabetic control group showed 312.5 ±

1.20 mg/dl on 3rd day, 306.1 ± 2.12 mg/dl on 6th day, 290.8 ± 2.12 mg/dl on day

9, 279.2 ± 2.12 mg/dl on day 12 and 278.2 ± 2.20 mg/dl on 16th day of treatment

(Table 30).

4.3.3.3. Effect of Formulation Test Extract on Body Weight in Normal and

Alloxan Induced Diabetic Rats

In the anti-diabetic activity, the effects methanolic extract of T.

sphaerocarpa on body weight was measured and were compared with normal and

diabetic control groups. Oral administration of the extract at the dose of 500

mg/kg showed a significant (P<0.01) decrease in body weight. In normal control

groups, the initial body weight was 80.22 ± 2.70 g, the final body weight was

102.00 ± 8.22 g and the percentage of body weight was increased by 18.30 In

diabetic control groups, the initial body weight was 99.60 ± 10.20 g, the final

body weight was 60.12 ± 2.90 g and the percentage of body weight was decreased

by -41.92. The groups treated with 125 mg/kg shows, the initial body weight was

82.60 ± 12.34 g, the final body weight was 50.82 ± 8.92 g and the percentage of

body weight was decreased by -34.80. In the groups treated with 250 mg/kg

shows, the initial body weight was 84.22 ± 10.22 g, the final body weight was

63.44 ± 12.12 g and the percentage of body weight was decreased by -25.44. The

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groups treated with 500 mg/kg shows, the initial body weight was 88.12 ± 8.44 g,

the final body weight was 74.10 ± 4.22 g and the percentage of body weight was

decreased by -19.00. The groups treated with glibenclamide 5 mg/kg shows, the

initial body weight was 90.00 ± 11.02 g, the final body weight was 76.22 ± 8.52 g

and the percentage of body weight was decreased by -15.98 (Table 31).

4.3.3.4. Effect of Formulation Test Extract on Bio-Chemical Parameters in

Alloxan Induced Diabetic Rats.

In normal control groups, the lysosome enzyme levels, SGOT (72.12 ±

0.12 IU/L), SGPT (41.08 ± 0.36 IU/L), Alkaline phosphatise (132.90 ± 0.25

IU/L), and also in % of lipid peroxidation (60.22 ± 1.02). It also reduced the

enzymatic antioxidants like CAT (3.16 ± 0.14 U/mg), GPx (2.56 ± 0.10 U/mg). In

diabetic control groups, the lysosome enzyme levels, SGOT (138.20 ± 0.18 IU/L),

SGPT (98.16 ± 0.44 IU/L), Alkaline phosphatise (250.23 ± 0.22 IU/L), and also

in % of lipid peroxidation (102.0 ± 1.59). It also reduced the enzymatic

antioxidants like CAT (1.88 ± 0.169 U/mg), GPx (1.83 ± 0.175 U/mg). The group

treated with 125 mg/kg showed the lysosome enzyme levels, SGOT (115.8 ± 0.16

IU/L), SGPT (75.16 ± 0.58 IU/L), Alkaline phosphatise (216.30 ± 0.42 IU/L), and

also in % of lipid peroxidation (80.56 ± 0.04). It also reduced the enzymatic

antioxidants like CAT (2.20 ± 0.817 U/mg), GPx (2.10 ± 0.09 U/mg). The group

treated with 250 mg/kg showed the lysosome enzyme levels, SGOT (105.23 ±

0.06 IU/L), SGPT (64.66 ± 0.60 IU/L), Alkaline phosphatise (194.20 ± 0.14

IU/L), and also in % of lipid peroxidation (74.36 ± 0.15). It also reduced the

enzymatic antioxidants like CAT (2.46 ± 0.069 U/mg), GPx (2.26 ± 0.04 U/mg).

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Table 32 : Effect of formulation Test extract on biochemical parameters in Alloxan induced diabetic rats.

Group & treatment(dose

mg/kg, po)

SGOT (IU/L)

SGPT (IU/L)

Alkaline phosphatise (IU/L)

% Lipid Peroxi-dation

CAT (U/mg)

GPX (U/mg)

Normal control 72.12 ± 0.12

41.08 ± 0.36

132.90 ± 0.25

60.22 ± 1.02

3.16 ± 0.14

2.56 ± 0.10

Diabetic control

138.20 ± 0.18 a

98.1 6± 0.44 a

250.23 ± 0.22 a

102.0 ± 1.59

1.88 ± 0.169 a

1.83 ± 0.175 a

Test extract 125

115.8 ± 0.16b

75.16 ± 0.58 b

216.30 ± 0.42 b

80.56 ± 0.04 b

2.20 ± 0.817 b

2.10 ± 0.09 b

Test extract 250

105.23 ± 0.06 b

64.66 ± 0.60 b

194.20 ± 0.14 b

74.36 ± 0.15 b

2.46 ± 0.069 b

2.26 ± 0.04 b

Test extract 500

90.36 ± 0.06 b

55.34 ± 0.45 b

169.20 ± 0.17 b

65.32 ± 0.58 b

2.76 ± 0.31 b

2.68 ± 0.17 b

Glibenclamide (5)

83.33 ± 0.18 b

48.30 ± 0.24 b

155.30 ± 0.40 b

60.93 ± 0.36 b

2.80 ± 0.23 b

2.50 ± 0.15 b

Values were expressed as Mean ± SEM of 6 rats in each group. p value: <0.01; compared to a normal group b diabetic

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The group treated with 500mg/kg showed the decrease the lysosome enzyme

levels, SGOT (90.36 ± 0.06 IU/L), SGPT (55.34 ± 0.45 IU/L), Alkaline

phosphatise (169.20 ± 0.17 IU/L), and also in % of lipid peroxidation (65.32 ±

0.58). It also reduced the enzymatic antioxidants like CAT (2.76 ± 0.31 U/mg),

GPx (2.68 ± 0.17 U/mg). All the concentrations tested have dose dependent

activity in the experimental animals (Table 32).

4.3.3.5. Histopathological Studies

Microscopically examined pancreas section of normal rat group and

diabetic control group showed that normal architecture of pancreas with acini of

serous epithelial cells along with nest of endocrine cells separated by

fibrocollaoenous, stroma into lobules. No fibrosis or inflammation was found.

Pancreas section of rat treated with Test extract (125 mg/kg and 250 mg/kg)

showed that normal architecture of pancreas with acini of serous epithelial cells

along with nest of endocrine cells separated by fibrocollagenous, stroma into

lobules. No fibrosis or inflammation was found. The test extract (500 mg/kg)

showed stroma into Mules like the standard Glibenclamide (Plate 9).

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CHAPTER 5

DISCUSSION

Natural products have been the most successful source of drugs for ever.

Researches on drug discovery especially in phytodrug investigation have become

one of the frontier areas in phytochemistry. Plants are probably the best cell

factories that can be exploited to produce a wide range of chemical compounds,

especially the low molecular weight secondary metabolites (Hadacek, 2002).

5.1. Pharmacognosy :

Pharmacognosy is defined as the scientific and systematic study of

structural, physical, chemical and sensory characters of crude drugs along with

their history, method of cultivation, collection and preparation for the market

(Evans, 1996). Identification of drugs can be done by morphological, histological

and chemical testing. There are five methods of evaluation of crude drugs namely

Morphlolgical or Organoleptic, Microscopical or Histological, Physical, Chemical

and Biological.

Micromorphology of vegetative and reproductive plant organs is the

object of research to resolve the taxonomic problems of critical species and

genera. Of the several traits on leaf surface, the stomata are perhaps the most

significant from the point of view of systematic and phylogeny. Stomata that are

highly characteristic of the epidermis occur in widely divergent parts of the plants

including common foliage leaves. Stomatal size is an ecologically important

attribute. The type, size, distribution and frequency of stomata have been

recognized to be specific to the taxa below the family and these characters were

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used as significant parameters in the angiosperm taxonomy as well as phylogeny.

The importance of epidermal cell characters is now well established in taxonomic

considerations of angiosperms (Parveen et al., 2000). Microscopical evaluation of

the plant drugs helps to identify the organized drugs by their known histological

characters and used to confirm the structural details of the drugs from plant origin.

The anatomical features of T. sphaerocarpa such as the astomatic nature

of the adaxial epidermis of leaf, the epidermal cell number, stomatiferous abaxial

epidermis, rubiaceous type of stomata in various sizes, orientation and frequency,

palisade spongy ratious, vein islet number, veinlet termination number and

unicellular conical trichomes of the leaf traits are the characteristic to the plant.

The macerated elements showed characteristic vessel elements with tails on one

side or on both sides. Anatomy of the leaf, stem and root reveled unique features

such as hemispherical epidermal cells on the stem and midrib region of leaf with

arches of thick cuticle, discrete vessels in the secondary xylem, distribution of

tannineferous idioblasts. All these characters are typical to this plant which

would be very useful in correct botanical identification of crude samples.

Histochemical localization methods provides the authentic data on the

availability of chemical compounds by simple and quick methods. Histochemical

analysis is highly essential that will aid the pharmacognosist to locate chemical

substances and its properties in terms of cells, tissues and parts (Johansen, 1940).

Histochemical localization is performed for starch, alkaloid, protein,

tannin and lignin. The present study reports the presence of starch, alkaloid and

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protein in leaf, stem and root; tannin in leaf and stem. Lignin only in root.

Mucilage is totally absent in all the plant part studied.

Fluorescence analysis showed that, the leaf powder is mostly green to

dark green in daylight whereas it is orange in UV light. In stem, it is mostly

yellow in both day light and UV light. In root, it is mostly yellow in day light and

pale yellow to dark yellow in UV light. In fruit, it is mostly light brown in day

light and creamy white to brown in UV light. When physical and chemical

methods are insufficient, as often happens with the powdered drugs, there are

methods such as fluorescence studies and microchemical tests to identify the

powdered drugs and their adulterants. In addition to this preliminary

phytochemical, histochemical tests using free hand sections of fresh parts,

microtome sectioning are used in identifying the adultered ones. Maceration

methods also included, in which the type of vessels, tracheids, fibres etc. are

considered in determining the genuiness of the drug (Kulkarni and Surekha,

1981). In Tricalysia sphaerocarpa, the libriform type of fibres, with thick

lignified walls and fairly very narrow lumen are seen.

A glimpse at the literature reveals that there is very little information on

the anatomical features of Rubiaceae members. Virtually Tricalysia sphaerocarpa

the present test plant has remained unexplored. Therefore, the present study marks

the first comprehensive report on the anatomical features of leaves, stem and root

of the test plant. Along with the physico-chemical studies, it would pave the way

for its botanical identification and to distinguish from adulterants and

substitutions during herbal drug quality control.

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5.2. Phytochemistry :

Physicochemical properties are important parameters in detecting

adulteration on improper handling of the drug. In the evaluation of crude drug,

ash value and extractive values are important parameters. The estimation of ash

value is useful for detecting low-grade products, exhausted drugs and the drug

with excess of sandy matter. The determination of extractive values with array of

solvent gives information about extractable polar and non polar as well as total

extractable plant constituents (Rajan et al., 2013). Physicochemical 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. The present study reveals that the moisture

content, total ash, acid insoluble ash and water soluble ash are high in stem when

compared to leaf, root and fruit.

5.2.1. Phytochemical Screening

The preliminary phytochemical colour reactions reveals the marked

presence of alkaloids, cardiac glycosides, flavonoids, glycosides, reducing sugar

and non-reducing polysaccharide(starch) in methanolic extract, water extract and

powder as such in leaf, stem, root and fruit. Proteins, phenolic group, steroids, and

saponins are very low or sparingly observed in leaf, stem, root and fruit.

Terpenoids are present in leaf, stem and root, but it is absent in fruit. Flavonoids

encompass a huge array of biologically active compounds that are omnipresent in

plants, many of which have been used in established eastern medicine for

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thousands of years (Gurudev Singh Raina, 2013). Flavonoides are also shown to

inhibit microbes which are resistant to antibiotics (Linuma et al., 1994).

Phenols the aromatic compounds with hydroxyl group are widespread in

plant kingdom. They occur in all parts of the plants. Phenols are said to offer

resistance to diseases and pests in plants. Phenolic compounds could be a major

determinant of antioxidant potentials of food plants and could therefore be a

natural source of antioxidants and therefore phenolic compounds have been

associated with the health benefits derived from consuming high levels of fruits

and vegetables (Ka¨ hko¨ nen et al., 1999). Hence presence of phenolic

compounds in Tricalysia sphaerocarpa plays an important role in antioxidation.

Saponins are a special class of glycosides which have soapy characteristics

(Fluck, 1973), and also an active antifungal agents (Sadipo et al., 1991). Tannins

are water – soluble polyphenols that are present in many plant foods and

precipitate proteins. Tannins have been reported to prevent the development of

microorganisms (antimicrobial agents) by precipitating microbial protein and

making nutritional proteins unavailable for them (Sadipo et al., 1991). The growth

of many fungi, yeasts, bacteria and viruses is inhibited by tannins (Chung et al.,

1998). Tannins are reported to have various physiological effects like anti–irritant,

antisecretolytic, antiphlogistic, antimicrobial and antiparasitic effects. Tannins are

known to be useful in the treatment of inflamed or ulcerated tissues and they have

remarkable activity in cancer prevention (Ruch et al., 1989; Motar et al., 1985).

The phytochemical analysis conducted on Helichrysum longifolium extract

revealed the presence of tannins, flavonoids, steroids and saponins (Aiyegoro and

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Okoh, 2010). Phytochemical screening of the plants (Carica papaya, Magnifera

indica, Psidium guajava, Vernonia amygdalina) revealed the presence of

flavonoids, terpenoids, saponins, tannins and reducing sugars (Ayoola et al.,

2008). The studies of Shakeri et al., (2012) on phytochemical screening revealed

the presence of alkaloid, flavonoid, saponin, terpenoid, steroid and sterols in the

extracts of aerial parts of Anabasis aphylla.

5.2.2. GC-MS Analysis

Methanolic extract of leaves, stem, root and fruit were subjected to GC-

MS analysis. This analysis were carried out to detect the possible compounds

present in the active fraction. Phytochemicals were extracted best in methanol

(Bhaigyabati et al., 2011).

Leaf

In the present study, from the methanol extract of leaf, totally 30 chemical

compounds were identified of which 9 belong to fatty acids, four to aliphatic and

aromatic bicyclics, two each to aromatic hydrocarbons groups, aromatic nitrile

groups, aromatic dicarboxylic esters groups. Of which one compound belonged to

each of the class terpenoids, barbiturates, aromatic alcohols group, aliphatic

aldehydes group, aromatic ketones group, aromatic ethers, phenolic group and to

pyrimidinedione group. Among this, eicosanoic acid is found to be present as

major constituent, followed by octadecanoic acid. Oleic acid is an unsaturated

fatty acid present in several plants and being unsaturated is considered as a

healthy source of fat in the diet. Many fatty acids are known to have antibacterial

and antifungal properties (Russel, 1991). Dodecanoic acid, tetradecanoic acid,

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hexadecanoic acid, octadecanoic acid and oleic acids are among the fatty acids

known to have potential antibacterial and antifungal activity (McGraw et al.,

2002; Seidel and Taylor, 2004). Oleic acid has been found to be fungistatic

against a wide spectrum of moulds and yeasts. For example, it was observed to

cause a delay of 6-8 hour in the germination of fungal spores, and was also found

to be effective at concentrations as low as 0.7% v/v (Sheba et al., 1999). It has

also been disclosed that these fatty acids have potential antibacterial and

antifungal principle for clinical application (Altieri et al., 2008). Docosanoic acid

also called as Behenic acid is a normal carboxylic acid, which is a saturated fatty

acid. Commercially, behenic acid is often used to give hair conditioners and

moisturizers due to their smoothing properties. It is also used in lubricating oils,

as solvent evaporation retarder in paint removers. As Amide an anti-foam is used

in the manufacturing of detergents, floor polishes and dripless candles. Reduction

of behenic acid yields behenyl alcohol. Pracaxi oil from the seeds of Pentaclethre

macroloba is a natural product with one of the highest concentrations of behenic

acid, and is used in hair conditioners. Nonadecanoic acid found in ox fats and

vegetables oils is used by certain insects as a phermone. Nonadecanoic acid has

also been reported from the genus Streptomyces, along with its biological

functions as anti-tumor agent and inhibition of IL-12 production (Yoo et al.,

2002). Nonadecanoic acid has already been isolated from several sources,

including a fungus (Juzlova et al., 1996), marine sponge (Mishra et al., 1996), and

plant (Hogg and Gillan, 1984; Fukunaga et al., 1989), and exhibits inhibitory

effects on fibrinolysis and plasmin activity (Kawashiri et al., 1986).

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Squalene, an isoprenoid compound structurally similar to beta-carotene, is

an intermediate metabolite in the synthesis of cholesterol. In humans, about 60

percent of dietary squalene is absorbed. It is transported in serum generally in

association with very low density lipoproteins and is distributed ubiquitously in

human tissues, with the greatest concentration in the skin, where it is one of the

major components of skin surface lipids. Squalene is not very susceptible to

peroxidation and appears to function in the skin as a quencher of singlet oxygen,

protecting human skin surface from lipid peroxidation due to exposure to UV and

other sources of ionizing radiation. Supplementation of squalene to mice has

resulted in marked increases in cellular and non-specific immune functions in a

dose-dependent manner. Squalene may also act as a "sink" for highly lipophilic

xenobiotics. Since it is a nonpolar substance, it has a higher affinity for un-ionized

drugs. In animals, supplementation of the diet with squalene can reduce

cholesterol and triglyceride levels. In humans, squalene might be a useful addition

to potentiate the effects of some cholesterol-lowering drugs. The primary

therapeutic use of squalene currently is as an adjunctive therapy in a variety of

cancers. Although epidemiological, experimental and animal evidence suggests

anti-cancer properties, to date no human trials have been conducted to verify the

role this nutrient might have in cancer therapy regimens. Phthalic acid, di(2-

propyl pentyl)ester and squalene was found in the wood extractives of Melaleuca

leucadendrda (Xu et al., 2013). Squalene has already been reported from the

acetone extract of the glandular hair of fruit of Mallotus philippensis (Velanganni,

2012), and also from the root of Bulbophyllum kaitense (Kalairasan et al., 2012).

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n-hexadecanoic acid has anti inflammatory property. The rigorous use of

medicated oils rich in n-hexadecanoic acid for the treatment of rheumatic

symptoms in the traditional medical system of India, Ayurveda (Aparna et al.,

2012). n-hexadecanoic acid, oleic acid are the two major compounds present in

the oil of Chasmanthera dependens (Modupe Ogunlesi et al., 2010). 9,12,15-

octadecatrenoic acid, methyl ester, (Z,Z,Z-), oleic acid, 9,12-octadecadienoic

acid(Z,Z)- has been reported from the ethanolic extract of stem and root of the

plant Mallotus philippensis (Velanganni et al., 2011). 2,3,5,6,tetrafluoroanisole

was already reported from Dinochloa puberula by Py-GC/MS and it used as raw

material for bioenergy and rare biomedicines (Qiang et al., 2008). 2,4,(1H,3H)-

Pyrimidinedione is used as an antiviral agent, and it reduces cellular cytotoxicity

and inhibits HIV type 1 and HIV type 2 (Buckheit et al., 2007). Phthalic acid,

di(2-propyl pentyl)ester and oleic acid was identified from the chloroform extract

of marine Kocuria sp. SRS88 by GC/MS and the chloroform extract showed

antibacterial activity (Ranganathan Sahadevan et al., 2014). Ethyl benzonitrile

was found to be the major constituents of the methanolic extract of leaf of

Gaultheria fragrantissima (Padmavathy et al., 2014). The oil yielded from the

ethanolic extract of the seeds of Brachystegia eurycoma showed n-hexadecanoic

acid, octadecanoic acid, docosanoic acid, beter-sitosterol, eicosanoic acid and the

oil showed antibacterial activity (Okenwa Uchenna Igwe et al., 2013).

Cyclobarbital is used as an anesthetic, anticonvulsant, sedative, hypnotic,

veterinary euthanasia agent.

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Stem

From the methanol extract of stem, totally 17 chemical compounds were

identified of which one belongs to aliphatic hydrocarbons groups, four to steroid

groups, five to fatty acid esters. Of which one compound belongs to the class

sugars, one to the class tocopherols, one to aromatic nitrile. Among this,

octadecanoic acid was found to be present as major constituent, followed by n-

hexadecanoic acid. n-hexadecanoic acid has also reported from the stem of

Bulbophyllum kaitense (Kalairasan and Ahmed John, 2011). n-hexadecanoic

acid, octadecanoic acid, 9,12,15- octadecatrenoic acid, methyl ester, (Z,Z,Z-),

oleic acid, 9,12-octadecadienoic acid(Z,Z)- has been reported the ethanolic extract

of stem and root of the plant Mallotus philippensis (Velanganni and Kadamban,

2011). Steroid are abundant in nature, many derivatives of steroid have

physiological activity (Vollhardt et al., 1994). Steroids are used in medicine in the

treatment of cancer, arthritis or allergies and in birth control (Okwu et al., 2010).

Stigmasterol isolated from plants were reported to be involved in the synthesis of

many hormones like progesterone, androgens, estrogens and corticoids with

several pharmacological prospects such as antiosteoarthritic,

antihypercholestrolemic, antitumor, hypoglycaemic, antimutagenic, antioxidant,

anti-inflammatory and CNS effects. Stigmasterol does seem to be play a role in

reducing inflammation, which may because it is a precursor to chemical

compounds which can limit inflammatory processes. Sterols like stigmasterol

have also been recommended for their cholesterol lowering abilities, although

more study is needed to determine which compounds perform this function, and

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how they work in the body. It has been already reported form the pseudobulb of

Bulbophyllum kaitense (Kalairasan and Ahmed John, 2011). Sitosterol is already

recorded from the ethanolic extract of leaf of Mallotus philippensis (Velanganni

et al., 2011). τ-Tocopherol is the analog of tocopherol (vitamin E). In cosmetics

and personal care products, tocopherol and other ingredients made from

tocopherol, including tocopherol esters are used in the formulation of lipstick, eye

shadow, blushers, face powders and foundations, moisturizers, skin care products,

bath soaps and detergents, hair conditioners, and many other products. Similar

results were reported from the pseudobulb of Bulbophyllum kaitense (Kalairasan

and Ahmed John, 2011).

Root

Totally 8 compounds were identified from the methanol extract. Of which

five belongs to heterocyclics groups, one to aromatic ester groups, one to fatty

esters and one to triterpenoid group. Among this, benzo(b)thiophene, 4-methyl

was found to be present as major constituent, followed by 2-methyl-5-p-

dimethylaminophenyl oxadiazol. Oxadiazol is a compound showing strong

antifungal activity (Zhang et al., 2013). 9,19-Cyclolanostane derivates was also

isolated from the roots of Actaea pachypoda. Cyclolanostane Triterpene

diglycosides isolated from the aerial parts of Cimicifuga foetida shows

immunosuppressive effect (Pan et al., 2009). Cyclolanostane Triterpene was also

isolated from the ethanolic extract of the stems of Kadsura heteroclite (Wang et

al., 2006). Tryptophan is a compound which is useful for insomnia, depression

and anxiety. Its also lower blood pressure in Hypertension patients, stimulate the

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production of antibodies, reduce inflammation. Tryptophan supplementation may

inhibit the development of full-blown AIDS in persons infected with HIV virus

(www.biogenesis-antiaging.com).

Fruit

Totally 10 compounds were identified of which three belong to

heterocyclic group, one to aliphatic aldehyde groups, one to thiosulphate group,

one to thiophosphates group, one to antibiotic and three to the other unclassified

groups. Among this, S,S-3,8-Diazaundecamethylene bis[hydrogen thiosulfate]

was found to be present as major constituent, followed by 5,8,15,18,23-pentaoxa-

1,12-diazabicyclo(10,8,5)-pentacosane. Deoxyspergualin (DOS), a substance

composed of a guanidinic and a spergmidine moiety, was originally described as

an antitumor agent(Takeuchiii et al., 1981). Deoxyspergualin (DSG) has been

found to have an antitumour and immunosuppressive activity. It also acts as an

antimalarial agent (Ramya et al., 2007). Methyldeoxyspergualin (MeDSG) in in

vitro culture studies of DSG shows good stability in aqueous solution and retains

strong immunosuppressive activity (Odaka et al., 1998). Methylpiperidin isolated

from plants shows antipsychotic therapeutic potential (Fuchigami et al., 2012).

Octadecanoic acid, n- hexadecanoic acid and 9,12,15- octadecatrienoic

acid (Z,Z,Z-) are the common compounds seen both in stem and leaf. Most of the

compounds obtained through GC-MS analysis from the methanolic extract of

leaf, stem, root and fruit of Tricalysia sphaerocarpa show antibacterial, antifungal

and antiviral properties. Some of them have antitumour, anti-inflammatory,

antiasthma, antiarthritic, diuretic, antipsychotic, anesthetic, anticonvulsant,

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sedative, antiarthritic, antioxidant and anticancer properties and hence the plant

Tricalysia sphaerocarpa have high medicinal value.

5.3. Pharmacology

5.3.1. Antioxidant Activity

The antioxidant activity of various extacts (petroleum ether, chloroform,

methanol, water) of Tricalysia sphaerocarpa was determined by DPPH, FRAP,

Hydrogen peroxide and SOD Scavenging assay. The present study reveals, the

maximum activity in chloroform extract, followed by the methanolic extract using

DPPH, FRAP and Hydrogen peroxide assay, but in SOD assay, the maximum

activity was observed in methanolic extract. Using DPPH radical scavenging

method, methanolic extract of some medicinal plants like Camellia sinensis,

Eugenia caryophyllus, Piper cubeba, Zingiber officinale, Trigonella foenum-

graecum and Elettaria cardamonum was found to have significant antioxidant

activity (Khalae et al., 2008). Al-fartosy (2011) reported strong antioxidant

activity as well as strong reducing power (increase in the extract concentration

increases the activity) and ferrous ion chelating abilities from the methanolic

extract of Inula graveolensa. Higher antioxidant potential of the Samanea saman

extracts (petroleum ether, ethyl acetate, chloroform, aqueous and HCl extracts)

was observed in both DPPH scavenging assay and reducing power assay

(Arulpriya et al., 2010). Crataegus. monogyna flowers, leaves and fruits had H2O2

radical scavenging, total antioxidant activity (Keser et al., 2012). Antioxidant

potential of various extracts of Cassia fistula was determined by the DPPH,

FRAP, Fe3+ reducing power, and hydrogen peroxide scavenging assay.

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Methanolic extracts of Cassia fistula showed the highest amount of reducing

capacity (Irshad et al., 2012). Riaz et al., (2012) reported highest total antioxidant

activity from the chloroform fraction of Dodonaea viscose. The methanolic

extract of Leucas plukenetti Whole plant plays an important role in the

modulation of oxidative stress (Subhangkar Nandy et al., 2012). The methanolic

leaf extract of Moringa peregrine exhibited the scavenging acativity on DPPH

assay (Dehshahri et al., 2012). The ethyl acetate fraction of Tagetes erecta

ethanol extract was found to be the most effective in DPPH assay (Miglena

Valyova et al., 2012).

5.3.2. Anti - Depressant Activity

The present findings obtained from FST, TST and HBT clearly reveal the

methanolic extract of Tricalysia sphaerocarpa elevate the suppressed mood in

animal models. The decrease in the immobility time was quite close to that of the

standard i.e. Imipramine. It clearly reveals that the animals treated with methanol

extract 200 mg/kg showed better response than those treated with standard drug.

Saroj Kothari et al., (2010) reported the methanolic extract of Aegle marmelos

leaf showed significant antidepressant and anxiolytic activities. All doses of the

aqueous extract of Melissa officinalis, produced a significant reduction in

immobility along with an increase in climbing behavior which is similar to those

which have been observed with imipramine (Emamghoreishi and Talebianpour,

2009). The methanol extract at the dose of 100mg/kg of the leaves of Citrus

paradise var. foster markedly increased the average time spent in the open arms in

EPM and methanol extract at the dose of 400mg/kg showed a significant decrease

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in the time spent immobile by mice in FST (Vikas Gupta et al., 2009). The

methanolic extract of Foeniculum vulgare possesses significant antidepressant

activity due to its reduction in the immobility period (Jamwal Neetu Singh et al.,

2013). The ethanolic extract of Caryophyllus aromaticus proposed antidepressant-

like effect of higher dose concentration (200mg/kg) and significantly increased

swimming time and decreased immobility time (Sangavai et al., 2013). Caffeine,

as a psychomotor stimulant, suggest a possible positive effect on dopaminergic

activity of caffeine augmentation (10 mg/kg or lower dose) with antidepressant

agents for depression treatment (Pravin Popatrao Kale et al., 2010). Celastrus

paniculatus seed oil showed significant antidepressant-like activity (Valeca et al.,

2014).

5.3.3. Anti - Diabetic Activity

The present study revealed that all dosess of Tricalysia sphaerocarpa

methanolic stem extract in normal fasted rats, significantly(P<0.05) reduced the

blood glucose levels up to 6 hr. except the lowest dose. The maximum

hypoglycemic activity was induced by 500 mg/kg dose at 4 hr. by 18%. In

alloxan-induced diabetic rats, it significantly(P<0.01) reduced the blood glucose

levels up to 3 hour except the lowest dose. The maximum hypoglycemic activity

was induced by 500 mg/kg dose at 3 hour. The present study indicates that

alloxan induced tissue injury is reversed by continuous administration of T.

sphaerocarpa extract with subsequent decrease in blood sugar. Oral

administration of T. sphaerocarpa methanolic extract of 500 mg/kg showed

significant (P<0.01) plasma glucose lowering effect in 12 and 16 days of

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treatment. Oral administration of the extract showed a significant (P<0.01)

decrease in body weight in all the tested concentrations, compared to control

groups. The group treated with 500 mg/kg showed the decrease the lysosome

enzyme levels, SGOT, SGPT, Alkaline phosphatise, and also in % of lipid

peroxidation. It also reduced the enzymatic antioxidants like CAT, GPx,

compared to diabetic controls. Microscopically examined pancreas section of

normal rat group, diabetic control group, Test extract (125, 250 and 500 mg/kg)

and the standard showed that normal architecture of pancreas with acini of serous

epithelial cells along with nest of endocrine cells separated by fibrocollaoenous,

stroma into lobules. No fibrosis or inflammation was found. Similar results

reported by previous workers are in conformitry into the present findings. The

antidiabetic potential of the methanolic extract of Operculina turpethum stem and

root was evaluated in the Streptozotocin- induced type 2 diabetic models

(Pulipaka et al., 2012). The antidiabetic effects of the methanol and acetone

extract of Acalypha indica Linn. was evaluated in normal and Alloxan induced

diabetic rats. Decreased blood glucose level of the test animals shows that the

extract exhibit significant antidiabetic activity when compared to diabetic control

group (Masih et al., 2011). The aqueous and methanolic extract of Gongronema

latifolium leaves showed antidiabetic activity in alloxan induced diabetic rats

(Akah et al., 2011). The methanolic and ethanolic extracts 200 mg/kg b.wt. of

seeds of Annona squamosa as significant hypoglycemic activity in both normal

and Alloxan induced diabetic rats (Ravinder Sangala et al., 2011). Aqueous and

cold extracts of Terminalia catappa exhibited significant anti-hyperglycemic

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activities in alloxan-induced hyperglycemic rats without significant change in

body weight. They also improved conditions of DM as indicated by parameters

like bodyweight, and lipid profiles along with serum (Ahmed et al., 2005).

Manikandan et al., (2013) reported the methanolic extract of Psidium guajava

leaves, showed its in vitro anti-diabetic activity. The aqueous and methanolic

extracts of aerial parts, viz. leaves, stem and seeds of the plant, Cassia

occidentalis possessed anti-hyperglycemic/ anti-diabetic activity against alloxan

induced animal model (Arya et al., 2013). The methanol extract of Costus

pictus(120mg/kg.p.o.) showed significant (p<0.001) reductions of blood glucose

and serum enzymes (SGOT, SGPT, ALP) in alloxan induced diabetic rats

(Nandhakumar Jothivel et al., 2007).

Thus the present study will be useful in the following ways.

In proper botanical identification of the crude drug of the plant studied

through pharmacognostical investigation

To identify the chemicals responsible for the medicinal properties of the

plant through various phytochemical studies

Pharmacological investigations on antioxidant activity, antidepressant

activity and antidiabetic activity of Tricalysia sphaerocarpa will provide

the first scientific report in medicinal science

It will also provide clues for new drug discovery.

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Figure 1: Extractive values of various parts of Tricalysia sphaerocarpa by Batch process.

Figure 2: Extractive values of various parts of Tricalysia sphaerocarpa by Successive process

0

5

10

15

20

25

Leaf Stem Root Fruit

Values in %

Chloroform Diethyl ether Ethyl acetate Methanol

0

5

10

15

20

25

30

35

Leaf Stem Root Fruit

Values in %Acetone Benzene Chloroform Diethyl etherEthanol n-butyl alcohol Methanol Water

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Figure 3: GC-MS chromatogram of Methanolic Leaf Extract of Tricalysia sphaerocarpa

Figure 4: GC-MS chromatogram of Methanolic Stem Extract of Tricalysia sphaerocarpa

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Figure 5: GC-MS chromatogram of Methanolic Root Extract of Tricalysia sphaerocarpa

Figure 6: GC-MS chromatogram of Methanolic Fruit Extract of Tricalysia sphaerocarpa

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Figure7(a): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

2,5-Dimethylbenzonitrile 2-Ethylbenzonitrile 2,3,5,6-Tetrafluoro-4-Methylanisole

n-Hexadecanoic acid Tetradecanoic acid

Eicosanoic acid Oleic acid 9,12-Octadecadienoic acid(Z,Z-)

Docosanoic acid Bis(2-Ethylhexyl)Phthalate 3-O-Methyl-D-Glucose

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Figure7(b): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

Eicosanoic acid,methyl ester Octadecanoic acid

Ergost-5-en-3-ol Stigmasterol Cyclobarbital

1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester

9,12,15-octadecatrienoic acid, (zzz)- 9,12-Octadecadienoic acid, methyl ester,(E,E)-

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Figure7(c): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

Hexadecanoic acid, methyl ester octadecanoic acid, methyl ester

9Z-octadeca-9,17-dienal 1H-Benzimidazole, 5,6-dimethyl-

1H-Indene-2-ethanol,2,3-dihydro- Oxitriptan

Benzoic acid,4-(3-hydroxy-3-methyl-1-butynyl)- methyl ester

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Figure7(d): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

2-[3-cyclohexylaminopropylamino]ethyl thiophosphate EPPS

S,S1-3,8-diazaundecamethylene bis[hydrogenthiosulfate]

2-methyl-5-p-dimethylaminophenyl oxadiazol Benzo(b)thiophene,4-methyl

Hexadecanoic acid,1a,2,5,5,5a,6,9,10,10a,octahydro-4-(hydroxymethyl)-1,1,7,9-tetramethyl-6,11-dioxo-1H-2,8a-methanocyclopenta(a)cyclopropa(e)cyclodecen-5-yl ester

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Figure 7(e): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

4,13,20-tri-O-methylphorbol 12-acetate 2-myristynoyl pantetheine

5,8,15,18,23-pentaoxa-1,12-diazabicyclo dl-5-hydroxytryptophan (10,8,5)-pentacosane

3-chloro-2,4-dimethyl-12-thia-1,5,6a,11,tetraaza- indeno[2,1-a]fluorine

4-octadecenal

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Figure7(f): Compounds identified from GC-MS analysis of Tricalysia sphaerocarpa

N-[4-(4-chlorophenyl)isothiazol-5yl] Deoxyspergualin -1-methylpiperidin-2-imine

Squalene

Phthalic acid, di(2-propylpentyl) ester 9,17-Octadecadienal, (Z)-

Nonadecanoic acid

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Figure 8: Anti oxidant activity -DPPH Scavenging assay:

0102030405060708090

100

10µg/ml 50µg/ml 100µg/ml

% of radicle scavenging

Petroleum ether Chloroform Methanol Water BHT

Figure 9: Anti oxidant activity - Iron chelating activity (FRAP)

0

20

40

60

80

100

120

10µg/ml 50µg/ml 100µg/ml

% of radicle scavenging

Petroleum ether Chloroform Methanol Water EDTA

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Figure 10: Anti oxidant activity - Hydrogen peroxide assay

00.20.40.60.8

11.21.41.6

10µg/ml 50µg/ml 100µg/ml

% of radicle scavenging

Petroleum ether Chloroform Methanol Water Ascorbic acid

Figure 11: Anti oxidant activity - Superoxide dismutase (L-methionine and NBT assay)

02468

1012

10µg/ml 50µg/ml 100µg/ml

% of radicle scavenging

Petroleum ether Chloroform Methanol Water Ascorbic acid

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CHAPTER 6

SUMMARY AND CONCLUSIONS

Chemical investigations of wild medicinal plants used by the indigenous

people of world show unknown compounds with promising biological activities.

Phytochemical analysis of plants, used in folklore has yielded a number of

compounds with various pharmacological activities. Hence medicinal plants are

important substances for the study of their traditional uses through the

verification of pharmacological effects and can be natural composite sources that

can act as disease curing agents. The integrated research in drug discovery have

attracted and provided multidisciplinary research platforms. The present work has

been concentrated to bring out the medicinal potential of Tricalysia

sphaerocarpa.

The important conclusion derived from this study is summarized in three

aspects viz, 1. Pharmacognostical studies for easy identification of plant species,

2. Phytochemical analysis to identify the chemicals responsible for the medicinal

properties of the plant, 3. To understand the Pharmacological properties of the

chemicals through in vivo and in vitro studies i.e., the antioxidant, antidepressant

activity and antidiabetic activity.

Tricalysia sphaerocarpa belongs to the coffee family (Rubiaceae), which

is commonly called as wild coffee, locally called as irrukulimaram in tamil, vella

by Srilankans and kadukafibija in kanada. The roasted seeds of Tricalysia

sphaerocarpa are used as a coffee substitute. Along with Tinospora giloy,

Argemone satyanashi, Tricalysia sphaerocarpa is traditionally used for sleep.

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Pharmacognostical parameters like leaf constants, microscopy, physico-

chemical analysis, fluorescence analysis are a few of the basic protocol for

standardization of crude drugs. Hence, in the present work the pharmacognostical

standardization has been performed.

Microscopical analysis of cleared leaf showed the characteristic

polyhedral vein islets and vein terminations. The examination of the macerated

materials showed the characterstic fibres, vessel elements and broken parenchyma

cells. The epidermal studies revealed that the number of epidermal cells/mm2

,type of trichome, occurrence of stomata on the lower surface only, the number of

stomata /mm2 , type of stomata, the stomatal index are characteristic to this plant.

The anatomical studies revealed the presence of hemispherical shaped

epidermal cells with very thick arches of cuticle in leaf and stem that extends to

the radial walls also. Presence of tannineferous idioblast, discrete vessel elements

with simple perforation and tails at one or both ends are the salient features

present in Tricalysia sphaerocarpa. 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 and protein in all the plant parts studied, tannin in leaf and stem, lignin

only in root and the absence of mucilage in all the parts.

In fluorescence analysis, leaf powder is mostly green to dark green in

daylight whereas it is orange in UV light. In stem, it is mostly yellow in both day

light and UV light. In root, it is mostly yellow in day light and pale yellow to dark

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yellow in UV light . In fruit, it is mostly light brown in day light and creamy

white to brown in UV light.

Of the physico-chemical parameters studied the moisture content (20%),

total ash (5.16%), acid insoluble ash (1.06%) and water soluble ash (3.90%) were

high in stem when compared to all other parts of the plant. In both the batch

process and successive process the highest extractive values were recorded in

methanol extract of leaf, stem, root and fruit, when compared to other solvents.

The preliminary phytochemical screening of leaves, stem, root and fruit

revealed the presence of phytoconstituents like alkaloids, carbohydrates,

flavonoids, glycosides, proteins, phenolic group and saponins in methanolic

extract, water extract and powder as such.

By GC-MS analysis, totally 65 chemical compounds (17 chemical

compounds from stem, 30 from leaf, 8 from root and 10 compounds from fruit)

were identified from the methanolic extract. Octadecanoic acid, n- hexadecanoic

acid and 9,12,15- octadecatrienoic acid (Z,Z,Z-) are the three common

compounds seen both in stem and leaf.

Antioxidant activity was significantly higher in the chloroform extract,

followed by the methanolic extract of stem. By DPPH scavenging method, the

percentage of scavenging activity was found to be more in chloroform extract,

followed by methanol, petroleum ether and water extracts. All the extracts

showed dose concentration dependent activity in all the tested concentration. By

FRAP assay, the maximum value was observed in chloroform extract, followed

by methanol, water and petroleum ether extracts. By Hydrogen peroxide assay,

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Chloroform extract showed the maximum value, followed by methanol,

petroleum ether and water extracts. By superoxide dismutase assay, the maximum

value was observed in methanol extract, followed by chloroform, water and the

minimum value was observed in petroleum ether extract. This activity may be due

to the presence of secondary metabolities such as flavonoids, tannins, and

steroids, all of which are known antioxidants.

In acute toxicity, no mortality was observed in the animals treated with the

dose of 2000 mg/kg methanol extract of stem. There were no signs of any

toxicity. The studies on anti-depressant activity clearly revealed that the animals

treated with methanol extract at 200 mg/kg had better response than those treated

with standard drug (Imipramine). In anti-diabetic activity, the methanolic extract

of stem, significantly reduced the blood glucose level in both normal fasted rats

and alloxan induced diabetic rats. It also reduces the body weight, and decrease

the enzymatic levels like SGOT, SGPT, Alkaline phosphatise, CAT and GPX. It

also reduced the lipid peroxidation level in all the tested concentrations.

Microscopically examined pancreas section of normal rat group, diabetic control

group, test extract (125, 250 and 500 mg/kg) and the standard showed the normal

architecture of pancreas with acini of serous epithelial cells along with nest of

endocrine cells separated by fibrocollaoenous, stroma into lobules. No fibrosis or

inflammation was found. The various extracts (petroleum ether, chloroform and

methanol) of stem possess significant antioxidant activity and the methanolic stem

extract possesses significant anti-depressant and anti-diabetic activities.

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Thus the study concludes that the plant may be used as a potential drug for

antioxidation, depression and diabetes. The results of pharmaconostical,

phytochemical and pharmacological studies which are reported for the first time

from Tricalysia sphaerocarpa, may pave way for new drugs discovery.

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Plate 1

Morphology of Tricalysia sphaerocarpa

Habit Basal portion

Trunk A twig with fruits

T.S and L.S of fruit showing flat seeds

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Plate 2

Leaves of Tricalysia sphaerocarpa

Dorsal surface Ventral surface

T.S. of Lamina

Midrib Leaf blade

Cu: Cuticle; Ep: Epidermis; X:Xylem; Ph:Phloem; GT:Ground tissue; Pc: Palisade cells; Sc:Spongy cells

Leaf epidermal peel of Tricalysia sphaerocarpa

Adaxial Epidermis without Stomata Abaxial Epidermis with Stomata

ICR: Inter Costal Region; CR: Costal Region

--------EEpp

----------GGTT

----XX ----------PPhh

--------CCuu --------CCuu --------PPcc

--------CCRR

--------SScc

----IICCRR

----------XX

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Plate 3

Size and Orientation of Stomata in Tricalysia sphaerocarpa

Various sizes of Stomata

Stomatal opening Stomata enlarged

Degenerated Stomata Unicellular Trichome GS: Giant Stomata; MS: Medium sized Stomata; SS: Small Stomata; BS: Blind Stomata; DS: Degenerated Stomata; HS: Half Stomata.

----MMSS

------BBSS

--------GGSS

----SSSS

------GGSS

------MMSS

------SSSS ------HHSS

--------DDSS

------DDSS

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Plate 4

Vein islet and Veinlet termination of Tricalysia sphaerocarpa

Stem epidermal peel of Tricalysia sphaerocarpa

VI: Vein islet; VLT: Veinlet termination; S:Stomata.

--------VVLLTT

--------SS

--------VVII

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Plate 5

T.S. of Stem of Tricalysia sphaerocarpa

Portion enlarged Outer portion

Epidermis enlarged Xylem showing vessels

Pith portion enlarged Sc: Sclerenchyma cells; Ve: Vessel element; TI: Tannineferous Idioblast.

--------SScc

--------VVee

--------VVee

--------TTII

--------CCuu

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Plate 6

T.S. of Root of Tricalysia sphaerocarpa

Entire view portion enlarged

Xylem showing vessels SG: Starch grains; Ve:Vessel.

--------SSGG

--------VVee

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Plate 7

Macerated elements of stem of Tricalysia sphaerocarpa

Vessel elements Single tailed vessel member Single fibre

Vessel member with pits Double tailed vessel member

Parenchyma cells Trachea P: Pits; PP: Perforation plate; Ta: Tail.

--------PP

----PPPP

--------TTaa

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Plate 8

Effect of Methanolic Stem extract of Tricalysia sphaerocarpa

Anti - Depressant Activity

Forced swimming test (FST)

Tail suspension test (TST)

Hole Board Test (HBT)

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Plate 9

Effect of Methanolic Stem extract of Tricalysia sphaerocarpa

Anti –Diabetic Activity

Histopathological Studies

Normal Control Diabetic Control Test extract (125)

Test extract (250) Test extract (500) Glibenclamide (5mg)

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CHAPTER 7

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Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.

~ 47 ~

e - ISSN - 2249-7722

Print ISSN - 2249-7730

International Journal of Phytotherapy

www.phytotherapyjournal.com

PHARMACOGNOSTICAL AND PRELIMINARY

PHYTOCHEMICAL STUDIES ON TRICALYSIA SPHAEROCARPA

(DALZELL) GAMBLE

G. Anandhi* and A. Pragasam

Department of Plant Science, Kanchi Mamunivar centre for post graduate studies, Lawspet, Puducherry-605008.

INTRODUCTION

Human beings come on this earth as guests of

plants is a monumental ancient aphorism. Since time

immemorial, nature is own supreme creation, man has

completely been dependent on plants and as citizen

developed, he has learnt to emplit natural resources and to

make use of every bit of it. Infact from the start of life to

the last breath, almost every aspect of human life is

deeply associated with plants. Primitive man tried to cure

diseases from plants growing abundantly around him. His

experience through trial and taught him a lot about the

medicinal properties of different plants.

Survey on medicinal plants used by ethnic

people of north western Tanzania revealed the use of

Tricalysia sphaerocarpa (Dalzell)Gamble for various

ailments. Tricalysia pallensa root decoction is drunk

against malaria. Tricalysia coricea sbsp. Nyassaea root

decoction mixed with leaf juice is drunk, and the body

bathed with a root decoction. Tricalysia coriacea is also

used for skin diseases, epixstasis and malaria/ yellow

fever (jaundice). To the best of our knowledge, nobody

has investigated form the angle of anatomy,

histochemistry, phytochemistry, the present study was

taken up.

MATERIALS AND METHODS

Anatomical works were carried out in leaf, stem

and root by preparing the peelings and transverse

sections. Mature leaves were cleared by using 5% NaOH

and chloral hydrate solution, washed in water, stained and

mounted in 50% glycerine. Maceration was carried out

with stem and root materials following Jeffrey’s method

[1]. Histochemical color reactions were done by treating

free hand sections with different reagents. Phytochemical

tests were done with dried powdered drugs as well as

different solvent extracts [2].

RESULTS

Morphological features-A small tree with smooth leaves

and very small flowers. Leaves dark green,

Corresponding Author:-G. Anandhi Email: [email protected]

ABSTRACT

The present work has been taken up to study the crude drug of Tricalysia sphaerocarpa (Dalzell)Gamble of

the family Rubiaceae. The morphological characters of the plant; the anatomical characters of the leaf, stem, and root,

microscopic observations of the crude drug; qualitative analysis of primary and secondary metabolites such as

carbohydrates, alkaloids, tannins etc., of the powder as well as different solvent extracts of leaf and ash values were

studied. Qualitative phytochemical observations revealed the presence of many primary and secondary metabolites.

The values calculates/ data collected could be used for the identification and standardization of the powdered drug of

this taxon.

Key words: Tricalysia sphaerocarpa, Pharmacognosy, Phytochemistry.

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Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.

~ 48 ~

Flowers minute, white, scented, Fruits greenish yellow,

berry globose, the seeds flat, smooth, leaves elliptic or

lancaolate, obtusely acute, smooth, the main nerves about

6-8 pairs, not prominent, nor the reticulation.

Anatomical features:-

Leaf peelings:

Adaxial : Cells small in size, cell wall undulate, thin, no

stomata.

Abaxial: Intercostal region: cells irregular, wall undulate,

cell medium sized, stomata rubiaceous type, different in

size.

Costal region: Cells small when compared to the

intercostal region, cell wall undulate, giant size stomata

are seen, unicellular hairs present. Epidermal cell number,

stomatal number, stomatal index, palisade ratio, vein islet

number and veinlet termination number have been

calculated and presented in the Table 1.

Stem peeling: Cells fairly large, cell wall thick, straight,

basal cells very broad, tip cells tapering, stomata frequent,

rubiaceous type.

Venation pattern: Veins reticulate, showing lateral

branches; cells elongated with thin walls; vein-islet fairly

large, each islet containing 3-4 termination points. Vein-

islet number and veinlet termination number are presented

in Table 1.

Transverse section: Leaf: The midrib portion of the leaf

is not much differentiated form the lamina. It is slightly

thicker than the lamina. The xylem is omega shaped with

10-15 rays of xylem cells. Phloem is seen on the abaxial

surface. Both the epidermis is uniseriate, with thick

cuticle. 2 layers of palisade parenchyma are seen below

the upper epidermis. Stomata are seen only on the abaxial

surface.

Stem: The stem in its outline is mostly dumble shaped.

Epidermis is unilayered with high cuticle. Cortex is

parenchymatous with 4-6 layered. Even the very younsg

stem undergoes secondary thickening. The secondary

xylem and phloem are continous. A single layer of

scleroids are seen as an outer ring. The pith is very broad

and formed of circular parenchymatous cells.

Root: The root in its outline is circular and formed of 8-

10 layers of parenchyma cells. The rhizodermis peeled

off. Secondary xylem and phloem are present. There is no

cortex. Rays are clearly seen. Xylem seen in the centre

and the phloem towards the periphery.

Phytochemical studies - The preliminary phytochemical

studies in methanol, aqueous and powder drug revealed

the marked presence of carbohydrate, glycosides,

alkaloids, tannin, flavanoids, moderate presence of

protein, phenol, terpenoids and saponin and absence of

triterpenoids, anthraquiones, catachins, coumarins (Table

4).

Histochemical colour reactions - Presence of starch,

protein and tannin and absence of lignin and mucilage

(Table 3). The total ash, acid insoluble ash and water

soluble ash were 4 percent, 0.84 percent and 3.24 percent

respectively (Table 2). Fluorescence analysis shows

mostly dark green in day light whereas orange in UV light

(Table 5).

Table 1. Quantitative values of foliar epidermis

Quantitative Values Abaxial Epidermis Adaxial Epidermis

Epidermal cell/mm² 1122.8/sq.mm 1414/sq.mm

Stomata/mm² 336/sq.mm -

Stomatal index 26.9 -

Palisade ratio 6.2 -

Vein islet number 93.8 -

Veinlet termination number 57.4 -

Table 2. Analytical ash values of leaf of T. sphaerocarpa

Parameter Results (%)

Total ash 4

Acid insoluble ash 0.84

Water soluble ash 3.24

Table 3. Histochemical colour reactions of leaf of T. sphaerocarpa

Test for Status of the substance

Starch +

Proteins +

Tannin +

Lignin -

Mucilage -

(+) Indicates presence; (-) Indicates absence.

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Inter. J. of Phytotherapy / Vol 3 / Issue 2 / 2013 / 47-49.

~ 49 ~

Table 4. Phytochemical colour reactions of leaf of T. sphaerocarpa.

Phytochemicals Chloroform

extract

Diethylether

extract

Ethylacetate

extract

Methanol

extract

Aqueous

extract

Powder as

such

Alkaloids ++ ++ - ++ ++ ++

Anthraquinones - - - - - -

Carbohydrates ++ - ++ ++ ++ ++

Catechins - - - - - -

Coumarins - - - - - -

Flavonoids ++ - - ++ ++ ++

Gums, oils and resins - - - - - -

Glycosides ++ - ++ ++ ++ ++

Proteins + - - ++ ++ +

Phenolic group + - - + + +

Saponins + - - + + +

Tannins + - - + + +

Terpenoids + - - + + +

Triterpenoids - - - - - -

(++) Marked presence; (+) Moderate presence, (-) Absence

Table 5. Fluorescence analysis of leaf of T. sphaerocarpa.

Chemicals Leaf

Day light UVlight

Powder as such Green Yellow

Solvent

Acetone Dark green Orange

Benzene Dark green Orange

Chloroform Dark green Orange

Ethanol Green Orange

n-butyl alcohol Yellowish green Orange

Water Green Dark brown green

Reagents

10% FerricChloride Reddish brown Black

50% Sulphuric Acid Reddish brown Dark brown

1 NHCl Green Green

5% Ammonia Green Green

1% Thionyl Chloride Green Dark green

DISCUSSION AND CONCLUSION

The qualitative microscopic characters are useful

in the identification of the crude drug sample [3]. This

features are believed to be constant for a given species

[4]. Hence, the need for evolving criteria for standard

samples of crude drugs has become very important in

pharmacognosy. Methanolic extract, aqueous extract and

crude drug powder shows the similar results in the

phytochemical analysis. The plant T. sphaerocarpa was

subjected to pharmacognosticalstudies to identify the

plant materials and to differentiate them from the spurious

crude drugs. In light of the above, a combination of

characters such as epidermal cell number, stomatal

number, stomatal index, palisade ratio, venation pattern,

vein-islet numbers, vein-let termination number are found

to be very significant micro-morphological characters in

the identification of crude drug of Tricalysia

sphaerocarpa could be successfully used for the

identification of the powdered drug of this taxon.

REFERENCES

1. Johensen DA. Plant Microtechnique. Mc Graw Hill Book Co. inc, New York, 1940.

2. Khandelwal KR, Pawar AP, Kokate CK, Gokhale SB. Practical pharmacognosy techniques and experiments, IIIed .

Nirali Prakashan. 1996, 140-141.

3. Trease GE and Evans WC. Pharmacognosy, XIII ed. WB Saunders Ltd UK. 1996, 516-547.

4. Suseela A and Pream S. Pharmacognostic studies on Lagascea mollis. J. Phytol. Res, 20(1), 2007, 95-102.

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Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.

65

PHYTOCHEMICAL SCREENING OF THE METHANOLIC

EXTRACT OF STEM OF TRICALYSIA SPHAEROCARPA (DALZELL

EX HOOK. F,) GAMBLE

G. Anandhi and Dr. A. Pragasam

Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Lawspet, Puducherry, India.

INTRODUCTION

Primitive man tried to cure diseases from plants

growing abundantly around him. His experience through

trial and taught him a lot about the medicinal properties of

different plants. The active secondary metabolites possess

various medicinal applications as drugs or as model

compounds for drug synthesis. Phytochemical analysis of

plants, used in folklore has yielded a number of

compounds with various pharmacological activities.

Hence medicinal plants are important substances for the

study of their traditional uses through the verification of

pharmacological effects and can be natural composite

sources that act a disease curing agents. Review of

literature revealed no work as been done form the angle of

histochemical analysis, preliminary phytochemical

screening and identification of phytocomponents by Gas

Chromatography-Mass Spectrometry (GC-MS) analysis

on this plant. Hence it was decided to do so.

MATERIALS AND METHODS

Collection of Plant material

The plant of Tricalysia sphaerocapa was

collected from the sacred grove of Thirumanikuzhi, of

Cuddalore district, Tamil Nadu. The collected plant

material was botanically identified. The species

conformation was engaged at French Institute Herbarium

(HIFP), Puducherry. The herbarium specimen was

prepared and deposited at the Department of Botany,

Kanchi Mamunivar Centre for Post Graduate Studies,

Lawspet, Puducherry, for future reference.

Preparation of the Extracts

The collected materials (stem) were chopped into

small pieces separately, shade-dried, and coarsely

powdered using a pulverizer. The coarse powder was

subjected to successive extraction with chloroform,

diethyl ether, ethyl acetate and methanol by Soxhlet

method. The extracts were collected and distilled off on a

water bath at atmospheric pressure and the last trace of

Corresponding Author:-G. Anandhi Email: [email protected]

International Journal of Pharmacotherapy

www.ijopjournal.com

ISSN 2249 - 7765

Print ISSN 2249 - 7773

ABSTRACT

Tricalysia is a genus of the plant family Rubiaceae. Approximately 50 species distributed in subtropical and

tropical regions in Asia and Africa. Some of these used as folk fore medicine as sedative, emetic, malaria/yellow fever,

skin diseases and also for urine disorders. Tricalysia sphaerocarpa is commonly known as wild coffee and its synonym

was Discospermum sphaerocarpum Dalzell ex Hook. F. Powdered materials were subjected to successive extraction with

chloroform, diethyl ether, ethyl acetate and methanol by soxhlet method for preliminary phytochemical screening and

methanol extract is used for GC-MS analysis to investigate the chemical components present in it. Totally 17 chemical

compounds were identified, among which 5 belongs to fatty acid esters groups, 4 belongs to steroid groups. Of which

octadecanoic acid (29.88%), n-hexadecanoic acid (15.10 %), 9, 12, 15-octadecatrienoic acid,(z,z,z)-(12.32 %) were the

major constituent identified.

Key words : GC-MS analysis, Tricalysia sphaerocarpa, Discospermum sphaerocarpum, Methanol extract.

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Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.

66

the solvents was removed in vacuum and stored at 4ºC.

The resulted extracts were subjected to preliminary

phytochemical screening and GC-MS analysis.

Histochemical color reactions were done by treating free

hand sections of stem with different reagents.

Phytochemical tests were done with dried powdered drugs

as well as different solvent extracts [1].

Gas Chromatography-Mass Spectrometry (GC-MS)

analysis

GC-MS analysis was performed with GC Clarus

500 Perkin Elmer equipment. Compounds were separated

on Elite-1 capillary column (100%

Dimethylpolysiloxane). Oven temperature was

programmed as follows: isothermal temperature at 50ºC

for 2min, then increased to 200ºC at the rate of 10ºC/min,

then increased up to 280ºC at the rate of 5ºC/min held for

9 min. Ionization of the sample components was

performed in the El mode (70 eV). The carrier gas was

helium (1ml/min) and the sample injected was 2μl. The

detector was Mass detector turbo mass gold-Perkin Elmer.

The total running time for GC was 36 min and software

used was Turbomass 5.2. Using computer searches on a

NIST Ver.2.1 MS data library and comparing the

spectrum obtained through GC –MS compounds present

in the plants sample were identified.

Identification of Compounds

The individual compounds were identified from

methanol extract based on direct comparison of the

retention times and their mass spectra with the spectra of

known compounds stored in the spectral database, NIST

(version year 2005).

RESULTS

Phytochemical screening

The preliminary phytochemical study in

methanol, aqueous and powder drug shows the similar

results. It revealed the presence of carbohydrate,

glycosides, alkaloids, protein, phenolic group, steroid,

saponins, flavanoids and terpenoids in methanol, aqueous

and the powder drug and absence of triterpenoids,

anthraquiones, catachins, coumarins and tannins. In

diethyl ether extract, only the carbohydrate is present and

others are absent. In chloroform extract, alkaloids,

carbohydrates, flavonoids, glycosides, protein, phenolic

group, saponins, terpenoids are present. In ethyl acetate

extract, only carbohydrates, glycosides and steroids are

present(Table 1).

Table 1. Phytochemical colour reactions of stem of T. sphaerocarpa.

Phytochemicals Chloroform

extract

Diethyl ether

extract

Ethyl acetate

extract

Methanol

extract

Aqueous

extract

Powder as

such

Alkaloids ++ ++ - ++ ++ ++

Anthraquinones - - - - - -

Carbohydrates ++ - ++ ++ ++ ++

Catechins - - - - - -

Coumarins - - - - - -

Flavonoids ++ - - ++ ++ ++

Gums, oils and resins - - - - - -

Glycosides ++ - ++ ++ ++ ++

Proteins + - - ++ ++ +

Phenolic group + - - + + +

Saponins + - - + ++ ++

Steroids - - ++ ++ + +

Tannins - - - - - -

Terpenoids + - - + + +

Triterpenoids - - - - - -

Table 2. Histochemical colour reactions of stem of T. sphaerocarpa.

Test for Chemicals/reagents used Status of the substance

Starch Iodine solution +

Proteins Aqueous picric acid solution +

Tannin Dilute ferric chloride -

Lignin 1% potassium permanganate, 2% HCl, dil. Ammonia -

Mucilage Methylene blue reagent -

(‘+’’+’) presence, ‘-‘ absence)

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Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.

67

Table 3. Analytical ash values of stem of T. sphaerocarpa.

Parameter Results %

Total ash 5.16

Acid insoluble ash 1.06

Water soluble ash 3.90

Table 4. Fluorescence analysis of stem of T. sphaerocarpa.

Chemicals Stem

Day light UVlight

Powder as such Creamy white Pale yellow

Solvent

Acetone Yellow orange

Benzene Pale yellow Creamy white

Chloroform Dark yellow Pale yellow

Ethanol Yellow Dark yellow

n-butyl alcohol yellow Creamy white

Water Creamy white Pale yellow

Reagents

10% Ferric Chloride Orange Black

50% Sulphuric Acid Reddish brown Black

1 NHCl Creamy white Yellow

5% Ammonia Pale yellow Yellow

1% Thionyl Chloride Light yellow Brown

Table 5. GC-MS Analysis of methanol extract of stem of T. sphaerocarpa

No. Name of the compound Molecular

formula

Molecular

weight

RT Peak

area %

Sugars

1 3-O-Methyl-d-glucose C7H14O6 194 11.06 6.18

Aromatic acids & esters

2 1,2-Benzenedicarboxylic acid, bis(2-methylproplyl) ester C16H22O4 278 11.59 1.66

Steroids

3 Androstane-3,16-diol,(3β,5α,16α)- C19H32O2 292 21.65 1.01

4 Ergost-5-en-3-ol,(3β)- C28H48O 400 29.71 4.40

5 Stigmasterol C29H48O 412 30.17 1.45

6 τ-Sitosterol C29H50O 414 31.31 9.81

Fatty acid esters

7 Hexadecanoic acid, methyl ester C17H34O2 270 12.21 0.90

8 9,12-Octadecadienoic acid, methyl ester, (E,E)- C19H34O2 294 14.23 1.89

9 6,9,12-Octadecatrienoic acid, methyl ester C19H32O2 292 14.31 1.03

10 Octadecanoic acid, methyl ester C19H38O2 298 14.64 1.57

11 Eicosanoic acid, methyl ester C21H42O2 326 17.34 1.42

Fatty acids

12 9,12,15-Octadecatrienic acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32

13 Octadecanoic acid C18H36O2 284 15.33 29.88

14 n-Hexadecanoic acid C16H32O2 256 12.80 15.10

Aromatic nitrile

15 4-Hydroxy-3-methyl-beta-phenylcinnamonitrile C16H13NO 235 22.07 1.94

Aliphatic hydrocarbons

16 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-

hexamethyl-, (all-E)-

C30H50 410 24.02 5.99

Tocopherols

17 τ-Tocopherol C28H48O2 416 28.26 3.45

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Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.

68

Fig 1. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the methanolic extract of stem of T.

sphaerocarpa

Fig 2. Structure of some phytocomponents

N-Hexadecanoic Acid

3-O-Methyl-D-Glucose

Eicosanoic Acid, Methyl Ester

Ergot-5-En-3-Ol

Stigmasterol

Sitosterol

Histochemical colour reactions

It reveals the presence of starch, protein and

absence of tannin, lignin and mucilage (Table 2). The

total ash, acid insoluble ash and water soluble ash were

5.16 percent, 1.06 percent and 3.90 percent respectively

(Table 3). Fluorescence analysis shows mostly yellow to

white in day light whereas black in UV light (Table 4).

Gas Chromatography-Mass Spectrometry (GC-MS)

analysis

Totally 17 chemical compounds were identified

of which 1 belongs to aliphatic hydrocarbons groups, 4

belongs to steroid groups, 5 belongs to fatty acid esters.Of

which1 compound belonged to the class sugars,

1compound belongs to the class tocopherol, 1 belongs to

aromatic nitrile.Among this, octadecanoic acidwas found

to be present as major constituent with the peak area

29.88% and retention time 15.33 minutes, followed by n-

hexadecanoic acid with the peak area 15.10 % and

retention time 12.80 minutes, and followed by 9,12,15-

octadecatrienoic acid,(z,z,z)- with the peak area 12.32 %

and retention time 15.01 minutes. Hexadecanoic acid,

methylester was found to be as least quantity with the

peakarea 0.90 % and retention time 12.21 minutes

respectively (Table 5; Fig.1). Some of the important

structures of phytocomponents were given below (Fig.2).

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Inter. J. of Pharmacotherapy / 3(2), 2013, 65-69.

69

DISCUSSION

Ash values, fluorescence analysis and

histochemical studies were used for the standardization of

drug. Stigmasterol isolated from plants were reported to

be involved in the synthesis of many hormones like

progesterone, androgens, estrogens and corticoids [2] with

several pharmacological prospects such as

antiosteoarthritic, antihypercholestrolemic, antitumor,

hypoglycaemic, antimutagenic, antioxidant, anti-

inflammatory and CNS effects [3-6]. τ-Tocopherol is the

analog of tocopherol (vitamin E). Dodecanoic acid,

tetradecanoic acid, hexadecanoic acid and octadecanoic

acid are among the fatty acids known to have potential

antibacterial and antifungal activity [7-8]. Moreover, the

presence of various bioactive compounds confirms the

application of T. sphaerocarpa for various ailments by

traditional practitioners. Similarly, the same studies were

previously reported by the plants like Alseodaphne

semecarpifolia, Stylosanthes fruticosa, Cassia auriculata,

Wrightia tinctoria, Vernonia cinerea, Hugonia mystax [9-

15]. However, isolation of individual phytochemical

constituents may proceed to find a novel drug. In addition

to this, the results of the GCMS profile can be used as

phytochemical tool for the identification of the bioactive

components.

CONCLUSION

From the present study, it was concluded that the

plant T. sphaerocarpa are highly valuable in medicinal

usage for the treatment of various human ailments along

with the chemical constituents present in it. The

compounds needs further research on toxicological

aspects to develop safe drug.

ACKNOWLEDGEMENT

Authors thanks Mr. S. Kumaravel, Manager,

Quality Control, Food Testing Laboratory, Indian Institute

of Crop Processing Technology (IICPT), Thanjavur for

providing facilities to carry out the work.

REFERENCES

1. Khandelwal KR, Pawar AP, Kokate CK and Gokhale SB. Practical Pharmacognosy- techniques and experiments, III edn.

Nirali Prakashan. 1996, 140-141.

2. Kaur S, Singh HP, Batish DR, Kohli RK. J. Med. Plants Res., 5(19), 2011, 4788-4793.

3. Marquis VO, Adanlawo TA, Olaniyi AA. Planta Med., 31(4), 1977, 367-74.

4. Prestwich GD, Eng WS, Roe RM, Hammock BD. Arch. Biochem. Biophys., 228, 1984, 639.

5. Svoboda JA, Rees HH, Thompson MJ, Hoggard N. Steroids, 53(3-5), 1989, 329-43.

6. Chowdhury R, Rashid RB, Sohrab MH, Hasan CM. Pharmazie, 58(4), 2003, 272-273.

7. McGraw LJ, Jager AK, Van Staden J, Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia 73,

2002, 431-433.

8. Seidel V, Taylor PW, In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria.

Int. J. Antimicrob. Agen., 23,2004,613-619.

9. Gook-Che J, Myoung-Soon P, Do-Young Y, Chul-Ho S, Hong-Sig S, Soo Jong U. Exp. Mol. Med., 37(2), 2005, 111-

120.

10. Charles A, Leo Stanly A, Joseph M, Alex Ramani V. Asian J. Plant Sci. Res., 1(4), 2011, 25-32.

11. Paul John Peter M, Yesu Raj J, Prabhu Sicis VP, Joy V, Saravanan J, Sakthivel S. Asian J. Plant Sci.Res., 2(3), 2012,

243-253.

12. Yesu Raj J, Paul John Peter M and Joy M. Asian J. Plant Sci. Res., 2(2), 2012, 187-192.

13. Jayamathi T, Komalavalli N, Pandiyarajan V. Asian J. Plant Sci. Res., 2(6), 2012 688-691.

14. Abirami P, Rajendran A. European J. Exp. Biol., 2(1), 2012, 9-12.

15. Vimalavady A, Kadavul K.Euro. J. Exp. Bio., 3(1), 2013, 73-80.

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Vol 5 | Issue 1 | 2014 | 53-56.

53

e - ISSN 2249-7544

Print ISSN 2229-7464

CHARACTERIZATION OF THE METHANOLIC EXTRACT OF

LEAVES OF TRICALYSIA SPHAEROCARPA (DALZELL EX HOOK.F.)

GAMBLE BY GC-MS

Anandhi G1*, Pragasam A

1, Prakash Yoganandam G

2

1Department of Botany, Kanchi Mamunivar Centre for Post Graduate Studies, Puducherry, India.

2College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry, India.

ABSTRACT

Tricalysia sphaerocarpa is commonly known as wild coffee and its basianym was Discospermum sphaerocarpum

Dalzell ex Hook. F. Gas Chromatography-Mass Spectrometry is an important technique used for metabolic profiling in plants

and also used for the qualitative and quantitative estimation of organic compounds. Totally 30 chemical compounds were

identified from the methanolic extract of the leaves of Tricalysia sphaerocarpa, among which fatty acid is the major group

consists of 9 compounds. Eicosanoic acid was found to be present as the major compound with peak area 35.77% and retention

time 21.865minutes, followed by octadecanoic acid (18.81%).

Keywords: GC-MS analysis, Tricalysia sphaerocarpa, Discospermum sphaerocarpum, Methanol extract.

INTRODUCTION

The genus Tricalysia, (Rubiaceae) comprises 50

species distributed in subtropical and tropical regions in

Asia and Africa, of which 2 species has been reported form

India [1]. Some of these used as folk fore medicine as

sedative, emetic, malaria, yellow fever, skin diseases and

also for urine disorders. Medicinal plants are important

substances for the study of their traditional uses through

the verification of pharmacological effects and can be

natural composite sources that act a disease curing agents.

So phytochemical investigation on the extract for their

main phytocompounds is very vital. Hence in the present

study, the methanolic extract of leaves of Tricalysia

sphaerocarpa were screened for Gas Chromatography-

Mass Spectrometry.

MATERIALS AND METHODS

Collection of Plant material

The plant of Tricalysia sphaerocapa was

collected from the sacred grove of Thirumanikuzhi, of

Cuddalore district, Tamil Nadu. The collected plant

materials were botanically identified. The species

confirmation was engaged at French Institute Herbarium

(HIFP), Puducherry. The herbarium specimen was

prepared and deposited at the Department of Botany,

Kanchi Mamunivar Centre for Post Graduate Studies,

Lawspet, Puducherry, for future reference.

Gas Chromatography-Mass Spectrometry (GC-MS)

analysis

GC-MS analysis was performed with GC Clarus

500 Perkin Elmer equipment. Compounds were separated

on Elite-1 capillary column (100% Dimethylpolysiloxane).

Oven temperature was programmed as follows: isothermal

temperature at 50ºC for 2min, then increased to 200ºC at

the rate of 10ºC/min, then increased up to 280ºC at the rate

of 5ºC/min held for 9 min. Ionization of the sample

components was performed in the El mode (70 eV). The

carrier gas was helium (1ml/min) and the sample injected

was 2μl. The detector was Mass detector turbo mass gold-

Perkin Elmer. The total running time for GC was 36 min

and software used was Turbomass 5.2. Using computer

searches on a NIST Ver.2.1 MS data library and

comparing the spectrum obtained through GC –MS

compounds present in the plants sample were identified [2-

3].

Identification of Compounds

The individual compounds were identified from

methanolic extracts based on direct comparison of the

retention times and their mass spectra with the spectra of

known compounds stored in the spectral database, NIST

(Version year 2005).

RESULTS

The compounds were identified by GC-MS

Corresponding Author: Anandhi G Email:- [email protected]

INTERNATIONAL JOURNAL

OF

PHYTOPHARMACY RESEARCH www.phytopharmacyresearch.com

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Vol 5 | Issue 1 | 2014 | 53-56.

54

analysis enumerated with molecular formula, retention

time, molecular weight and peak area% (Table 1; Fig.1).

GC-MS analysis of an methanolic extract of leaves of

Tricalysia sphaerocapa showed 30 compounds. Of which

9 compounds belongs to the group fatty acids, 4 belongs to

aliphatic & aromatic bicyclics, 3 belongs to aromatic

hydrocarbons, 2 belongs to aromatic nitriles, aliphatic

aldehydes, aromatic ketones and aromatic dicarboxylic

esters each and 1 compounds belongs to aromatic alcohols,

phenolics, terpenoids, barbiturates, pyrimidinedione,

aromatic ethers each. Among this, Eicosanoic acid was

found to be present as the major compound with peak area

35.77% and retention time 21.865minutes followed by

Octadecanoic acid with peak area 18.81% and retention

time 20.093 minutes, followed by 9,12,15- octadecatrienic

acid with peak area 12.32% and retention time

15.01minutes. 2,2-Dimethylindene,2,3-dihydro-, 2-(2-

Hydroxyphenyl)buta-1,3-diene, 1,2,3,4,8,9-hexahydro-

4,4,8-trimethyl-,(+)- was found to be as least quantity with

the peak area 0.51% and retention time 22.882 minutes.

Some of the important structure of phytocomponents was

given below (Fig. 2).

Table 1. GC-MS Analysis of methanolic extract of leaf of T. sphaerocarpa

No. Name of the compound Molecular formula Molecular

weight RT Peak area %

Aliphatic & Aromatic bicyclics

1 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl-, [1R-

1α,2β,5α]- C10H18 138 16.942 0.91

2 2,2-Dimethylindene,2,3-dihydro- C11H14 146 22.882 0.51

3 Bicyclo[3.1.1]heptane, 2,6,6-trimethyl- C10H18 138 16.942 0.91

4 2AH-Cyclobut[a]indene-2a-carboxylic acid,

1,2,7,7a-tetrahydro, methyl ester C13H14O2 202 13.456 2.89

Aromatic nitriles

5 -Ethylbenzonitrile C9H9N 131 13.456 2.89

6 2,5-Dimethylbenzonitrile C9H9N 131 13.456 2.89

Aromatic ethers

7 2,3,5,6-Tetrafluoroanisole C7H4F4O 180 13.863 1.05

Pyrimidinedione

8 2,4(1H,3H)-Pyrimidinedione, 5(trifluoromethyl)- C5H3F3N2O2 180 13.863 1.05

Fatty acids

9 Oleic acid C18H34O2 282 20.979 1.10

10 Octadecanoic acid C18H32O2 284 20.093 18.81

11 Nonadecanoic acid C19H38O2 298 20.979 1.10

12 n-Hexadecanoic acid C16H32O2 256 18.176 10.41

13 Tetradecanoic acid C14H28O2 228 18.176 10.41

14 9,12,15-Octadecatrienic acid, (Z,Z,Z)- C18H30O2 278 15.01 12.32

15 9,12-Octadecadienoic acid (Z,Z)- 19.977 11.54

16 Eicosanoic acid C20H40O2 312 21.865 35.77

17 Docosanoic acid C22H44O2 340 23.477 0.77

Aliphatic aldehydes

18 9,17-Octadecadienal, (Z)- C18H32O 19.977 11.54

19 1H-Benzimidazole, 5,6-dimethyl- C9H10N2 146 22.882 0.51

Aromatic hydrocarbons

20 2-(2-Hydroxyphenyl)buta-1,3-diene 22.882 0.51

21 Phenanthro[3,2-b]furan-7,11-dione,1,2,3,4,8,9-

hexahydro-4,4,8-trimethyl-, (+)- C19H20O3 296 23.274 1.62

22 (2-Methoxyphenyl)carbamic acid, naphthalene-2-

yl ester C17H15O3N 281 23.129 1.38

Aromatic ketones

23 Chrysene-1,7(2H,8H)-dione, 3,4,9,10-tetrahydro-

2,8-dimethyl- C20H20O2 292 23.129 1.38

24 tert-Butyl(5-isoproply-2-

methylphenoxy)dimethylsilane C16H28OSi 264 23.129 1.38

Aromatic dicarboxylic esters

25 Bis(2-ethylhexyl) phthalate C24H38O4 390 23.216 1.42

26 Phthalic acid, di(2-propylpentyl ester) C24H38O4 390 23.216 1.42

Barbiturates

27 cyclobarbital C12H16N2O3 236 23.477 0.77

Terpenoids

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28 Squalene C30H50 410 25.322 10.87

Phenolics

29 4,6-Bis(1,1-dimethylethyl)-4´-methyl-1-1´-

biphenyl-2-ol C19H28O 272 23.274 1.62

Aromatic alcohols

30 Dibenzo[a,c]phenazin-10-ol C20H12N2O 296 23.274 1.62

Fig 1. Gas Chromatography - Mass Spectrometry (GC-MS) Chromatogram of the methanolic extract of leaves of T.

sphaerocarpa

Fig 2. Structure of Some Important Phytocompounds

Squalene Docosanoic acid Eicosanoic acid

Oleic acid n-Hexadecanoic acid 2,5-Dimethylbenzonitrile

2-Ethylbenzonitrile Cyclobarbital 2,3,5,6-Tetrafluoro-4-

methylanisole

Octadecanoic acid 1H-Benzimidazole,5,6-dimethyl- (9Z)-octadeca-9,17-dienal

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DISCUSSION

n-Hexadecanoic acid is also known as Palmitic

acid. n-Hexadecanoic acid, Octadecanoic acid, 9,12-

octadecadienoic acid (Z,Z)-, 9,12,15-octadecatrienoic acid ,

methyl ester (Z,Z,Z)- was already reported in the leaf of

Mallotus philippensis [4]. 9,12,15-octadecatrienoic acid ,

n-Hexadecanoic acid, Octadecanoic acid was also reported

in the methanolic extract of stem of Tricalysia

sphaerocarpa [5]. Secondary metabolites in plant products

are responsible for several biological activities in man and

animals [6]. The active components usually interfere with

growth and metabolism of microorganisms in a negative

manner [7]. Phenolic compounds and steroidal compounds

which are more effective in higher concentrations

inhibiting the growth of all fungi[8]. The fatty acids being

effective in the treatment of asthma, rheumatoid arthritis,

inflammatory bowel diseases [9]. Esters are functionally

used in the design of “Prodrugs” [10], terpenes are anti-

allergic [11] and antimicrobial agents [12]. Squalene is the

Triterpene compound showed activity against

Antibacterial, Antioxidant, Antitumor, cancer preventive,

Immunostimulant, Chemo preventive, Lipoxygenase

inhibitor, pesticide [13].

CONCLUSION

From the present study, it was concluded that the

plant T. sphaerocarpa are highly valuable in medicinal

usage for the treatment of various human ailments along

with the chemical constituents present in it. The

compounds needs further research on toxicological aspects

to develop a safe drug.

ACKNOWLEDGEMENT

Authors are thankful to Mr. P. Gopal, Technical

Manager, Sargam Laboratory Pvt. Ltd. Guindy, Chennai

for providing facilities to carry out the work.

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