PHARMACOGNOSTICAL, PHYTOCHEMICAL AND …repository-tnmgrmu.ac.in/2692/1/14050272012senthilkumar.pdf · CERTIFICATE This is to certify that the thesis entitled “PHARMACOGNOSTICAL,

PHARMACOGNOSTICAL, PHYTOCHEMICAL AND

PHARMACOLOGICAL SCREENING FOR

BAMBUSA VULGARIS (GRAMINEAE) AND

PANDANUS ODORATISSIMUS (PANDANACEAE)

Thesis submitted to

The Tamilnadu Dr. M.G.R. Medical University, Chennai

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

By

Mr. M.K.Senthil Kumar M.Pharm.,

Under the guidance of

Dr.L.Suseela, M.Pharm.,Ph.D

JANUARY 2012

C.L. BAID METHA COLLEGE OF PHARMACY,

THORAPAKKAM,

CHENNAI : 600 097.

CERTIFICATE

This is to certify that the thesis entitled “PHARMACOGNOSTICAL,

PHYTOCHEMICAL AND PHARMACOLOGICAL SCREENING FOR

BAMBUSA VULGARIS (GRAMINEAE) AND PANDANUS

ODORATISSIMUS (PANDANACEAE)” is a record of research work done by

Mr. M.K.Senthil Kumar M.Pharm, , under my guidance, and supervision at

C.L. Baid metha college of pharmacy, Thorapakkam, Chennai ., during the years

2008-2012 and this thesis has not previously formed the basis for the award of any

Degree, Diploma, Associateship, Fellowship or other similar title. I also certify

that the thesis represents independent work done by the candidate and this has not

formed in part or fully the basis for the award of any other previous research

degree.

Dr.L.Suseela, M.Pharm.,Ph.D

Principal (retd)

Madurai Medical College

College of Pharmacy

Madurai.

Date :

DECLARATION

I hereby declare that the thesis entitled PHARMACOGNOSTICAL,

PHYTOCHEMICAL AND PHARMACOLOGICAL SCREENING FOR

BAMBUSA VULGARIS (GRAMINEAE) AND PANDANUS

ODORATISSIMUS (PANDANACEAE) submitted by me for the award of

degree of Doctor of Philosophy in Pharmacy under the Tamilnadu Dr.MGR

Medical University, Chennai is the result of my original and independent work at

C.L. Baid metha college of pharmacy,Thorapakkam, Chennai ., during the years

2008 – 2012, under the supervision of Dr. L. Suseela, M.Pharm., Ph.D and has

not formed the basis for the award of any Degree, Diploma, Associateship,

Fellowship, or any other similar title, previously.

Date : M.K.. Senthil Kumar

ACKNOWLEDGEMENT

Every event, small or big in nature, is itself a creation. At the heart of every event,

lies, a reason and a motivating force or an inspiration. To a student, in whatever

walk of life he may be, this inspiration is always there through a guide, a mentor.

It gives me a deep-seated pleasure to express my sence of gratitude to my guide

Dr. L. Suseela, M.Pharm., Ph.D who suggested this interesting and challenging

field of investigation. I wish to express my gratitude to her for her effective

guidance and constant encouragement. I am very thankful for her excellent care,

continuous support and optimistic approach, which influenced me to accomplish

this work successfully.

I wish to express my heartfelt gratitude to Dr.V.Vaidhyalingam, M. Pharm.,

Ph.D.,Joint Director of Medical Education,Pharmacy, (retd)chennai , whose belief

and confidence in me gave me the courage to follow my dreams in the area of

research. I also express my sincere thanks to Mrs.Arivuselvi vaidhyalingam.

My heartful thanks to Dr.Shantha Arcot ,, M.Pharm., Ph.D., Principal, C.L.

Baid metha college of pharmacy, Thorapakkam, Chennai, for her untiring support

and encouragement during my study period.

My affectionate thanks to my loving friend Prof.V.Rajesh, Head, Dept of

Pharmacology,J.K.K.Nataraja college of pharmacy for his timely assistance in

Pharmacological work and also giving valuable suggestion in entire research

work. I also express my sincere thanks to

Dr.S.Suresh kumar, Head, Dept of Pharmacognosy, J.K.K.Nataraja college of

pharmacy and all my friends for their friendly help during my work.

I sincerely express my deep sence of gratitude and immence respect to secretary

and correspondent Smt. N. Sendamaraai, J.K.K.Rangammal charitable trust

and, , for her kind cooperation. I am immensely thankful to Dr. P. Perumal,

M.Pharm., Ph.D., Principal, J.K.K.nataraja College of Pharmacy,

komarapalayam, for his valuable suggestions in all aspect of my research work.

I specially thank my beloved father in law Mr.K.Rangasamy, my mother in law

Mrs.R.Dhanalakshmi, my uncle Mr. N.Rangasamy, my aunty Mrs. T.N.

Anusuya my father Mr.N.Krishnamurthy, my mother Mrs.M.Malligeswari for

their love and affection.

Words are inadequate to express gratitude to my wife Mrs.

R.suganthi.B.com.M.B.A and my loving daughters S.Mohanapreetha and

S.Rithika for their patience, and valuable support which made this commitment

feasible.

No words are adequate to express my thanks for the help done by Dr. Ashok

Godavarthi, and Mr. Pavan Kumar, Radiant Research Services Pvt. Ltd.

Bangalore.

(M.K. Senthil kumar)

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““““HIS HOLINESSHIS HOLINESSHIS HOLINESSHIS HOLINESS,,,,

AVATHARA VARISHTARAVATHARA VARISHTARAVATHARA VARISHTARAVATHARA VARISHTAR,,,,

SRISRISRISRI RAMAKRISHNA RAMAKRISHNA RAMAKRISHNA RAMAKRISHNA

PARAMAHAMSARPARAMAHAMSARPARAMAHAMSARPARAMAHAMSAR””””

CONTENTS

Chapter

No TITLE

PAGE

NO

1 INTRODUCTION 1

2 AIM AND OBJECTIVE 10

3 REVIEW OF LITERATURE 13

4 MATERIALS AND METHODS 26

5 RESULTS AND ANALYSIS 54

6 DISCUSSION 120

7 SUMMARY AND CONCLUSION 124

8 BIBLIOGRAPHY 129

LIST OF TABLES

TABLE

NO TITLE

PAGE

NO

1 Ash values 70

2 Extractive value 70

3 Moisture content 71

4 Phytochemical analysis of Bambusa vulgaris leaf extracts 72

5 Phytochemical analysis of Pandanus odoratissimus root

extracts

74

6 Body weight analysis of test drug treated rats 90

7 Macroscopic findings of animals from test drug treated

groups

91

8 Body weight analysis of test drug treated mice 93

9 Macroscopic findings of animals from test drug treated

groups 94

10 Anti pyretic effect of Methanol extract of Bambusa vulgaris on

Brewer’s yeast-induced pyrexia in rats 96

11 Effect of the methanol extract of Pandanus odoratissimusi

on the lethality of snake venom 99

12 Cytotoxic study of Bambusa vulgaris leaf extracts against

L6 cell line by MTT assay 103

13 In vitro glucose uptake studies in L-6 cell line 104

14 In situ glucose uptake studies in rat hemi diaphragm 106

15

In vivo antidiabetic activity of methanolic extracts of

Bambusa vulgaris leaf and Pandanus odoratissimus root

108

16

Effect of Bambusa vulgaris leaf and Pandanus

odoratissimus root on STZ induced changes on the serum

biochemical parameters

112

17

Effect of Bambusa vulgaris leaf and Pandanus

odoratissimus root on STZ induced changes on the serum

biochemical parameters 117

LIST OF FIGURES

FIGURE

NO

TITLE PAGE

NO

1.1 T S of lamina of Bambusa vulgaris leaf 59

1.2 T S of lamina through midrib of Bambusa vulgaris leaf 59

1.3 T S of lamina through midrib of Bambusa vulgaris leaf 59

2.1 T S of lamina through smaller lateral veins of Bambusa

vulgaris leaf 60

2.2 T S of lamina through smaller lateral veins of Bambusa

vulgaris leaf 60

2.3 T S of lamina through marginal part of Bambusa vulgaris

leaf 60

3.1 Lower epidermis of the leaf, showing the stomata of

Bambusa vulgaris leaf 61

3.2 Upper epidermis cells showing wavy cell wall of Bambusa

vulgaris leaf 61

3.3 Venation of the lamina of Bambusa vulgaris leaf 61

4.1 Surface view of the cleased leaves showing parallel veins of

Bambusa vulgaris leaf 62

4.2 Surface view of the cleased leaves showing parallel veins

of Bambusa vulgaris leaf

62

4.3 Crystals in the leaf mesophyll tissue of Bambusa vulgaris

leaf 62

5.1 TS of thin root-entire view of Pandanus odoratissimus root 63

5.2 TS of thick root showing the crystals with inner cortical

cells Pandanus odoratissimus root 63

6.1 TS of thin root –Cortical portion of Pandanus

odoratissimus root 64

6.2 TS of thin root-Stele-enlarged of Pandanus odoratissimus

root 64

7.1 TS of thick root –entire view of Pandanus odoratissimus

root 65

7.2 TS of thick root –a sector enlarged of Pandanus


8.1 TS of old root –Periderm and cortex of Pandanus


8.2 TS of old root-Stele of Pandanus odoratissimus root 66

9.1 TS of thick root – secondary xylem of Pandanus


10.1 Fibres of Pandanus odoratissimus root powder 68

10.2 Fibres and vessels of Pandanus odoratissimus root powder 68

10.3 A single vessel element of Pandanus odoratissimus root

powder 68

11 Anti pyretic effect of Methanol extract of Bambusa

vulgarison Brewer’s yeast induced pyrexia in rats 97

12 Effect of the methanol extract of Pandanus odoratissimusi

on the lethality of snake venom 100

13 In vitro glucose uptake effect of extracts of Bambusa

vulgaris in L-6 cell line 105

14 In vitro glucose uptake effect of extracts of Pandanus

odoratissimusi in L-6 cell line 105

15 Glucose uptake effect of extracts of Bambusa vulgaris in rat

hemi diaphragm 107

16 Glucose uptake effect of extracts of Pandanus

doratissimusi in rat hemidiaphragm 107

17 Glucose uptake effect of extracts of Pandanus

odoratissimusi in rat hemidiaphragm 109

18 Effect of methanolic extracts on blood glucose levels in

diabetic rats 109

19 Effect of methanolic extracts on serum HbA1c levels in

diabetic rats 110

20 Effect of methanolic extracts on serum CK levels in

diabetic rats 110

21 Effect of methanolic extracts on serum LDH levels in

diabetic rats 111

22 Effect of extracts on serum cholesterol levels in diabetic

rats 113

23 Effect of extracts on serum triglycerides levels in diabetic

rats 113

24 Effect of extracts on serum HDL levels in diabetic rats 114

25 Effect of extracts on serum LDL levels in diabetic rats 114

26 Effect of extracts on serum creatinine levels in diabetic rats 115

27 Effect of extracts on urea levels in diabetic rats 115

28 Effect of extracts on serum Alkaline phosphotase levels in

diabetic rats 116

29

Histopathology 117

ABBREVIATIONS

% : Percent

α : Alpha

β : Beta

µ : Micron

ºC : Degree Celsius

µg : Microgram

µg/ml : Microgram per milliliter

ALAT : Alanine aminotransferase

ANOVA : Analysis of variance

ASAT : Aspartate aminotransferase

ALP : Alkaline phosphatase

b.w. : Body weight

CCl4 : Carbon tetra chloride

DMSO : Dimethyl sulphoxide

et al., : and coworkers

Fig. : Figure

g : Gram

g/l : Grams per Litre

H2O : Water

H2O2 : Hydrogen peroxide

hr : Hour

kg. : Kilogram

l : Litre

IU/mg : International Unit per milligram

LD50 : Lethal dose (50)

LDH : Lactate dehydrogenase

Ltd. : Limited

mg/dl : Milligram per deciliter

mg/kg : Milligram per kilogram

SEM : Standard Error Mean

min : Minute

No. : Number

Pvt. : Private

Rf : Retention factor

SGOT : Serum Glutamate Oxaloacetate

Transaminase

SGPT : Serum Glutamate Pyruvate

Transaminase

Tc : Total cholesterol

TGL : Triglycerides

Tp : Total protein

U : Unit

U / l : Units per litre

UV / VIS : Ultra-Violet – Visible

WHO : World Health Organization

% : Percent

w / w : Weight / weight

Introduction

1

CHAPTER-1

INTRODUCTION

Fossil records date human use of plants as medicines at least to the Middle Paleolithic age some

60,000 years ago1. From that point the development of traditional medical systems incorporating

plants as a means of therapy can be traced back only as far as recorded documents of their

likeness. However, the value of these systems is much more than a significant anthropologic or

archeologic fact. Their value is as a methodology of medicinal agents, which, according to the

World Health Organization (WHO), almost 65% of the world’s population has incorporated into

their primary modality of health care2. The goals of using plants as sources of therapeutic agents

are a) to isolate bioactive compounds for direct use as drugs, e.g., digoxin, digitoxin, morphine,

reserpine, taxol, vinblastine, vincristine; b) to produce bioactive compounds of novel or known

structures as lead compounds for semisynthesis to produce patentable entities of higher activity

and/or lower toxicity, e.g., metformin, nabilone, oxycodon (and other narcotic analgesics),

taxotere, teniposide, verapamil, and amiodarone, which are based, respectively, on galegine, Δ9-

tetrahydrocannabinol, morphine, taxol, podophyllotoxin, khellin, and khellin; c) to use agents as

pharmacologic tools, e.g., lysergic acid diethylamide, mescaline, yohimbine; and d ) to use the

whole plant or part of it as a herbal remedy, e.g., cranberry, echinacea, feverfew, garlic, ginkgo

biloba, St. John’s wort, saw palmetto.

The number of higher plant species (angiosperms and gymnosperms) on this planet is estimated

at 250,000 , with a lower level at 215,0003 and an upper level as high as 500,000

4-5 (Jones et al,

2006; Drahl et al, 2005). Of these, only about 6% have been screened for biologic activity, and a

Introduction

2

reported 15% have been evaluated phytochemically 6. With high throughput screening methods

becoming more advanced and available, these numbers will change, but the primary

discriminator in evaluating one plant species versus another is the matter of approach to finding

leads. There are some broad starting points to selecting and obtaining plant material of potential

therapeutic interest. However, the goals of such an endeavor are straightforward. Plants have an

advantage in this area based on their long-term use by humans (often hundreds or thousands of

years). One might expect any bioactive compounds obtained from such plants to have low

human toxicity. Obviously, some of these plants may be toxic within a given endemic culture

that has no reporting system to document these effects. It is unlikely, however, that acute toxic

effects following the use of a plant in these cultures would not be noticed, and the plant would

then be used cautiously or not at all. Chronic toxic effects would be less likely to signal that the

plant should not be used. In addition, chemical diversity of secondary plant metabolites that

result from plant evolution may be equal or superior to that found in synthetic combinatorial

chemical libraries.

It was estimated that in 1991 in the United States, for every 10,000 pure compounds that are

biologically evaluated (primarily in vitro), 20 would be tested in animal models, and 10 of these

would be clinically evaluated, and only one would reach U.S. Food and Drug Administration

approval for marketing. The time required for this process was estimated as 10 years at a cost of

$231 million 7. Most large pharmaceutical manufacturers and some small biotechnology firms

have the ability to screen 1,000 or more substances per week using high throughput in vitro

assays. In addition to synthetic compounds from their own programs, some of these companies

screen plant, microbial, and marine organisms. Thus, the challenges facing these companies in

Introduction

3

acquiring organisms and extracts (vide infra) usually result in a failure to consider collection of

plants, especially if the acquisitions are based on ethnomedical use. It is time-consuming to

collect specific plants having an ethnomedical history. Despite these problems, one cannot

discount the past importance of plants as sources of structurally novel drugs.

For thousands of years, natural products have played an important role throughout the world in

treating and preventing human diseases. Natural product medicines have come from various

source materials including terrestrial plants, terrestrial microorganisms, marine organisms, and

terrestrial vertebrates and invertebrates 8. The value of natural products in this regard can be

assessed using 3 criteria: (1) the rate of introduction of new chemical entities of wide structural

diversity, including serving as templates for semisynthetic and total synthetic modification, (2)

the number of diseases treated or prevented by these substances, and (3) their frequency of use in

the treatment of disease.

An analysis of the origin of the drugs developed between 1981 and 2002 showed that natural

products or natural product-derived drugs comprised 28% of all new chemical entities (NCEs)

launched onto the market. In addition, 24% of these NCEs were synthetic or natural mimic

compounds, based on the study of pharmacophores related to natural products 9. This combined

percentage (52% of all NCEs) suggests that natural products are important sources for new drugs

and are also good lead compounds suitable for further modification during drug development.

The large proportion of natural products in drug discovery has stemmed from the diverse

structures and the intricate carbon skeletons of natural products. Since secondary metabolites

from natural sources have been elaborated within living systems, they are often perceived as

Introduction

4

showing more “drug-likeness and biological friendliness than totally synthetic molecules” 10

making them good candidates for further drug development 11-12

.

Scrutiny of medical indications by source of compounds has demonstrated that natural products

and related drugs are used to treat 87% of all categorized human diseases (48/55), including as

antibacterial, anticancer, anticoagulant, antiparasitic, and immunosuppressant agents, among

others 13

. There was no introduction of any natural products or related drugs for 7 drug categories

(anesthetic, antianginal, antihistamine, anxiolytic, chelator and antidote, diuretic, and hypnotic)

during 1981 to 2002. In the case of antibacterial agents, natural products have made significant

contributions as either direct treatments or templates for synthetic modification. Of the 90 drugs

of that type that became commercially available in the United States or were approved

worldwide from 1982 to 2002, ~79% can be traced to a natural product origin 14

.

Frequency of use of natural products in the treatment and/or prevention of disease can be

measured by the number and/or economic value of prescriptions, from which the extent of

preference and/or effectiveness of drugs can be estimated indirectly. According to a study, 84 of

a representative 150 prescription drugs in the United States fell into the category of natural

products and related drugs 15

. They were prescribed predominantly as anti-

allergy/pulmonary/respiratory agents, analgesics, cardiovascular drugs, and for infectious

diseases. Another study found that natural products or related substances accounted for 40%,

24%, and 26%, respectively, of the top 35 worldwide ethical drug sales from 2000, 2001, and

2002 16

. Of these natural product-based drugs, paclitaxel, a plant-derived anticancer drug, had

sales of $1.6 billion in 2000. The sales of 2 categories of plant-derived cancer chemotherapeutic

agents were responsible for approximately one third of the total anticancer drug sales worldwide,

Introduction

5

or just under $3 billion dollars in 2002; namely, the taxanes, paclitaxel and docetaxel, and the

camptothecin derivatives, irinotecan and topotecan 17-18

.

This short review covers new drugs derived from natural sources launched in the 6-year period

from 2000 to 2005, and drug candidates from natural sources in clinical trials during the same

time period arranged according to their origin (terrestrial plants, terrestrial microorganisms,

marine organisms, and other natural sources). For drug candidates in clinical trials 19

, only

examples of new chemical templates of potential cancer chemotherapeutic drugs will be

mentioned.

Drug discovery from terrestrial plants

Terrestrial plants, especially higher plants, have a long history of use in the treatment of human

diseases. Several well-known species, including licorice (Glycyrrhiza glabra), myrrh

(Commiphora species), and poppy capsule latex (Papaver somniferum), were referred to by the

first known written record on clay tablets from Mesopotamia in 2600 BC, and these plants are

still in use today for the treatment of various diseases as ingredients of official drugs or herbal

preparations used in systems of traditional medicine 20

. Furthermore, morphine, codeine,

noscapine (narcotine), and papaverine isolated from P. somniferum were developed as single

chemical drugs and are still clinically used. Hemisuccinate carbenoxolone sodium, a semi-

synthetic derivative of glycyrrhetic acid found in licorice, is prescribed for the treatment of

gastric and duodenal ulcers in various countries 21

.

Historical experiences with plants as therapeutic tools have helped to introduce single chemical

entities in modern medicine. Plants, especially those with ethnopharmacological uses, have been

Introduction

6

the primary sources of medicines for early drug discovery. In fact, a recent analysis by Fabricant

and Farnsworth showed that the uses of 80% of 122 plant-derived drugs were related to their

original ethnopharmacological purposes 22

. Current drug discovery from terrestrial plants has

mainly relied on bioactivity-guided isolation methods, which, for example, have led to

discoveries of the important anticancer agents, paclitaxel from Taxus brevifolia and

camptothecin from Camptotheca acuminate 23

.

In fact, over 120 pharmaceutical products in use are obtained from the plants 24

. A large number

of therapeutic activities are mediated by these drugs, and a host of drugs in use are still obtained

from plants in which they are synthesized. Examples include, cardiotonic glycosides (Digitalis

glycosides), anticholinergics (belladonna type tropane alkaloids), analgesics and antitussives

(opium alkaloids), antihypertensives (reserpine), cholinergics (physostigmine, pilocarpine),

antimalarials (cinchona alkaloids), antigout (colchicines), anesthetic (cocaine), skeletal muscle

relaxant (tubocurarine), and anticancer agents (paclitaxel, vincristine, vinorelbine, teniposide,

and analogs of camptothecin) 25

.

Analysis of the number and sources of anticancer and anti-infective agents, reviewed mainly in

Annual Reports of Medicinal Chemistry from 1984 to 1995, indicates that over 60% of the

approved drugs and pre-NDA candidates (for the period 1989-1995), excluding biologics,

developed in these diseases areas are of natural origin. According to Newman et al 26

that during

the period of 1981-2002 vast majority of New Chemical Entities (NCEs) i.e. 10% are unmodified

natural products, 68% are derived from natural products source (semisynthetic) and 1 (1%) is by

total synthesis, but originally modeled on natural products parent and natural product mimic.

79% of the whole is from the natural products source in one or the other way. Thus natural

Introduction

7

products have been playing an invaluable role in the drug discovery process, particularly in the

areas of metabolic disorders, cancer and infectious diseases.

Examples of plant-derived compounds currently in clinical trials

From terrestrial plant-derived secondary metabolites, several new chemical entities are

undergoing clinical trials including four that are derivatives of known anticancer drugs like

camptothecin, paclitaxel, epipodophyllotoxin, and vinblastine 27

. In addition, combretastatin A4,

isolated from the South African medicinal tree, Combretum caffrum (Combretaceae), was

derivatized to combretastatin A4 phosphate and AVE-8062 28-29

. These analogs bind to tubulin

leading to morphological changes and then disrupt tumor vasculature, and are in phase II trials 30-

31. Homoharringtonine , a cephalotaxus alkaloid from the tree, Cephalotaxus harringtonia found

in mainland China 32

, is an inhibitor of protein synthesis and is reported to have activity against

hematologic malignancies 33

. Ingenol 3-O-angelate, an analog of the polyhydroxy diterpenoid,

ingenol, which was originally obtained from Euphorbia peplus (known as “petty spurge” in

England or “radium weed” in Australia), is a potential topical chemotherapeutic agent for skin

cancer and exhibits its action through activation of protein kinase C 34-35

. Phenoxodiol, a

synthetic analog of daidzein, a well known isoflavone from soyabean (Glycine max), is being

developed as a therapy for cervical, ovarian, prostate, renal, and vaginal cancers, and induces

apoptosis through inhibition of anti-apoptotic proteins including XIAP and FLIP 36

. Phenoxodiol

is currently undergoing clinical studies in the United States and Australia 37

. Protopanaxadiol, a

derivative of a triterpene aglycone of several saponins from ginseng (Panax ginseng), exhibits its

apoptotic effects on cancer cells through various signaling pathways, and is also reported to be

cytotoxic against multidrug resistant tumors 38-39

. Triptolide, a diterpene triepoxide, was isolated

Introduction

8

from Tripterygium wilfordii, and has been used for autoimmune and inflammatory diseases in the

People’s Republic of China 40

. PG490–88 (12, 14-succinyl triptolide sodium salt), a

semisynthetic analog of triptolide, exerts antiproliferative and proapoptotic activities on primary

human prostatic epithelial cells as well as tumor regression of colon and lung xenografts 41

.

Indian Scenario

India is a treasure chest of biodiversity which hosts a large variety of plants and has been

identified as one of the eight important “Vavilorian” centres of origin and crop diversity. Its

diversity is unmatched due to the presence of 16 different agroclimatic zones, 10 vegetative

zones and 15 biotic provinces. The country has 15000-18000 flowering plants, 23000 fungi,

2500 algae, 1600 lichnens, 1800 bryophytes and 30 million micro-organisms. India also has

equivalent to ¾ of its land exclusive economic zone in the ocean, harbouring a large variety of

flora and fauna, many of them with therapeutic properties. Although its total land area is only

2.4 percent of the total geographical area of the world, the country accounts for eight percent of

the total global biodiversity with an estimated 49000 species of plants of which 4900 are

endemic 42

. The ecosystems of the Himalayas, the Khasi and Mizo hills of northeastern India, the

Vindhya and Satpura ranges of northern peninsular India, and the Western Ghats contain nearly

90 percent of the country's higher plant species and are therefore of special importance to

traditional medicine. Although, a good proportion of species of Medicinal Plants (MP) do occur

throughout the country, peninsular Indian forests and the Western Ghats are highly significant

with respect to varietal richness 43

. About 1500 plants with medicinal uses are mentioned in

ancient texts and around 800 plants have been used in traditional medicine.

Introduction

9

Peninsular India extending downwards from Gujarat, Madhya Pradesh and Southern Bihar was

once dominated by a continuum of tropical forests, namely: thorn forests, dry deciduous forests,

moist deciduous forests, dry evergreen forests, wet evergreen forests and semi-evergreen forests.

The complexity with respect to soils, topography and climate has created an exceptional variety

of bio-mass and specialized habitats within this region. The ecosystems of southern peninsular

India including the southern Western Ghats contain more than 6000 species of higher plants

including an estimated 2000 endemic species. Of these, 2500 species representing over 1000

genera and 250 families have been used in Indian systems of medicine 44

.

The classical Indian texts include Rigveda, Atherveda, Charak Samhita and sushruta samhita.

The herbal medicines/Traditional medicaments have, therefore, been derived from rich traditions

of ancient civilizations and scientific heritage. Ancient literature also mentions herbal medicines

for age-related diseases namely memory loss, osteoporosis, diabetic wounds, immune and liver

disorders, etc. for which no modern medicine or only or palliative therapy is available. Herbal

medicines have stood the test of time for their safety, efficacy, cultural acceptability and lesser

side effects. The chemical constituents present in them are a part of the physiological functions

of living flora and hence they are believed to have better compatibility with the human body. It

has been observed that, the plant materials selected based on their traditional claims was found to

be biologically active. So, the appreciation of the significance of natural products as sources for

structurally novel and mechanistically unique drugs and enormous biodiversity of India

prompted our interest in evaluating the traditional medicinal plants for their potential biological

properties.

Aim and Objective

10

CHAPTER-2

AIM AND OBJECTIVE

The use of traditional medicines and medicinal plants in most developing countries as

therapeutic agents for the maintenance of good health has been widely observed. Interest

in medicinal plants as a re-emerging health aid has been fuelled by the rising costs of

prescription drugs in the maintenance of personal health and well being and the

bioprospecting of new plant-derived drugs. The ongoing growing recognition of

medicinal plants is due to several reasons, including escalating faith in herbal medicine

and also least risk of side effects when compared to modern drugs.

However among the estimated 2,50,000 to 4,00,000 plant species, only 6 % have been

studied for biological activity and about 15 % have been investigated phytochemically.

This shows a need for planned activity guided phyto-pharmacological evaluation of

herbal drugs, since most of the modern drugs has its natural product prototype. Correct

identification and quality assurance of the starting materials is an essential prerequisite to

ensure reproducible quality of herbal medicine which will contribute to its safety and

efficacy, so standardization of a plant material becomes at most important which can be

done by pharmacognostic studies.

Aim and Objective

11

In these views, the present work is undertaken based on its traditional claims to study the

Pharmacognostical, Phytochemical and Pharmacological screening for Bambusa vulgaris

Schrad (gramineae) and Pandanus odoratissimus Linn.f (Pandanaceae).

Plan of work

Based on the objectives, the research work was planned as described below.

Phase I: Literature survey and collection of plant material

1. To screen the geographical source and the availability of the Bambusa vulgaris

Schrad (Graminae) and Pandanus odoratissimus Linn.f (Pandanaceae) in India.

2. Identification, authentication and collection of plant material

Phase II: Pharmacognostical studies on plant parts and powders

1. To evaluate the pharmacognostical characters of Bambusa vulgaris leaves and

Pandanus odoratissimus root

Phase III: Phytochemical evaluation of extracts

1. Preparation of extracts by using standard techniques

2. To evaluate the phytochemical constituents present in the extracts

3. To estimate the total Phenolic content

Phase IV: Pharmacological screening based on traditional claims

1. Determination of Acute oral toxicity study of methanolic extract of Bambusa

vulgaris leaves and Pandanus odoratissimus root.

2. Determination of venom neutralizing potential of Pandanus odoratissimus root

extract.

Aim and Objective

12

3. Evaluation of antipyretic activity of methanolic extract of Bambusa vulgaris

leaves.

4. Determination of in vitro cytotoxicity of Bambusa vulgaris leaves, and

Pandanus odoratissimus root using hexane, benzene, chloroform,

ethylacetate,and methanol extracts in skeletal muscle cell line.

5. In vitro glucose uptake studies for Bambusa vulgaris leaves and Pandanus

odoratissimus root using hexane, benzene, chloroform, ethylacetate, and

methanol extracts in skeletal muscle cell line.

6. Glucose uptake studies in rat hemi diaphragm for Bambusa vulgaris leaves, and

Pandanus odoratissimus root using hexane, benzene, chloroform, ethylacetate

and methanol extracts.

7. In vivo anti-diabetic activity for methanolic extracts of Bambusa vulgaris leaves

and Pandanus odoratissimus root.

Phase V: Isolation and characterization of phytoconstituents

1. Isolation of phytoconstituents by column chromatography from the bioactive

extracts and fractions.

2. Identification and characterization of isolates by different spectral studies.

Review of Literature

13

CHAPTER-3

REVIEW OF LITERATURE

Since the beginning of human civilization, medicinal plants have been used by mankind

for its therapeutic value. Nature has been a source of medicinal agents for thousands of

years and an impressive number of modern drugs have been isolated from natural

sources. Many of these isolations were based on the uses of the agents in traditional

medicine. The plant-based, traditional medicine systems continues to play an essential

role in health care, with about 80% of the world’s inhabitants relying mainly on

traditional medicines for their primary health care 45

. India has several traditional medical

systems, such as Ayurveda and Unani, which has survived through more than 3000 years,

mainly using plant-based drugs. The materia medica of these systems contains a rich

heritage of indigenous herbal practices that have helped to sustain the health of most rural

people of India. The ancient texts like Rig Veda (4500-1600 BC) and Atharva Veda

mention the use of several plants as medicine. The books on ayurvedic medicine such as

Charaka Samhita and Susruta Samhita refer to the use of more than 700 herbs 46

.

The use of traditional medicines and medicinal plants in most developing countries as

therapeutic agents for the maintenance of good health has been widely observed 47

.

Modern pharmacopoeia still contains at least 25% drugs derived from plants and many

others, which are synthetic analogues, built on prototype compounds isolated from plants.

Interest in medicinal plants as a re-emerging health aid has been fuelled by the rising

costs of prescription drugs in the maintenance of personal health and well being and the


14

bioprospecting of new plant-derived drugs 48

. The ongoing growing recognition of

medicinal plants is due to several reasons, including escalating faith in herbal medicine

49. Furthermore, an increasing reliance on the use of medicinal plants in the industrialized

societies has been traced to the extraction and development of drugs and

chemotherapeutics from these plants as well as from traditionally used herbal remedies 50

.

The medicinal properties of plants could be based on the antioxidant, antimicrobial,

antipyretic effects of the phytochemicals in them 51-52

. According to World Health

Organization, medicinal plants would be the best source to obtain a variety of drugs.

Therefore, such plants should be investigated to better understand their properties, safety

and efficacy 53

. The traditional knowledge especially on the medicinal uses of plants has

provided many important drugs of modern day 54-56

.

In India, the Ayurvedic system has described a large number of such medicines based on

plants or plant product and the determination of their morphological and pharmacological

or pharmacognostical characters can provide a better understanding of their active

principles and mode of action. However a large number of plants have not been studied in

detail for their chemical constituents, pharmacological properties of the extracts, and their

pharmacognostical characterization.

Traditional medicinal claims of Bambusa vulgaris and Pandanus odoratissimus

Even though Bambusa vulgaris is known for its usage in fencing and construction, the

Bambusa vulgaris had been used as an important medicinal plant for a long period of

time. Since generations, it is an integral part of many important herbal formulations that


15

are used in traditional systems of medicine. Many Orientals think it has medicinal values.

Medieval alchemists in Europe extracted tabachir, a poison anti-dote from the species.

Javanese people use water preserved in Golden Bamboo tubes as cure for jaundice 57

. In

Nigeria, a drink of macerated leaves is taken against sexually transmitted diseases, and in

Congo, leaves are used as part of treatment against measles. A chloroform extract of

leaves is active against Mycobacterium tuberculosis. The leaves of Bambusa vulgaris

have been used in Indian folk medicine to treat various inflammatory conditions. Other

traditional uses are astringent, emmanogogue, vulnerary, and febrifuge to heal the

wounds and also to control diarrhea in cattle 58

. Manna is a crystalline substance found

inside the bamboo and leaves are used in ayurvedic medicine in ptosis and paralytic

complaints 59

.

Leaves have been claimed to be used as astringent, ophthalmic solution, sudorific and

febrifuge. In Nigerian folklore medicine, bamboo is claimed to be used as an

emmenagogue, abortifacient, appetizer and for managing respiratory diseases as well as

gonorrhea. Leaves are used in Ayurvedic medicine in ptosis and paralytic complaints 60-

62.

Pandanus odoratissimus is said to be a restorative, deodorant, indolent and phylactic,

promoting a feeling of wellbeing and acting as a counter to tropical lassitude. It may be

chewed as a breath sweetener or used as a preservative on foods. It is also said to have

healthful properties. In Ayurveda, the therapeutic activities of Pandanus

odoratissimus have been mentioned to treat for various ailments 63

. Pandanus


16

odoratissimus root have been claimed to be used as bitter, sweet, acrid, thermogenic,

emollient, dupurative, procreant, antiseptic, cephalic, aphrodisiac, carminative,

stomachic, suppurative, anodyne, deodorant, urinary astringent, vulnerary, sudorific,

febrifuge and tonic. They are useful in vitiated conditions of kapha and pitta,skin

diseases, leprosy, cephalalgia, coxalgia, otalgia, wounds, ulcers, dyspepsia, flatulence,

colic, fever, diabetes, strerility spontaneous abortion and general debility. The roots

considered as antidote to snake bite; Tribal/Folklore practitioners use this drug to treat

many ailments. The root and flowers of the drug Pandanus odoratissimus acts as an

abortifacient and it is indicated for the treatment of skin diseases, leprosy, scabies and

syphilis 64

. A mixture of the dried root powder along with one spoonful turmeric juice and

supernatant from a clean lime water extract, taken early in the morning orally for one

week will cure urinary disorders 65

.While the root decoction has diuretic activity 66

. In

Villupuram, India, the traditional medicinal system is very efficient for successfully

treating jaundice, female sterility and rheumatism. The leaves of Pandanus spp. are a

natural antioxidant and Pandanus extracts are capable of retarding oxidation.

In the Marshall Islands this plant is used for a number of conditions related to the female

reproductive organs. In infants with jaundice, restlessness and colic, the juice squeezed

from the aerial roots together with Centella asiatica is given to the infant in a dose of one

teaspoon and then the rest is rub over the whole body of the infant. For oral thrush the

juice of the soft part of the aerial root is squeezed into the child’s mouth 67

. In Palau

Island a drink prepared from the root alleviates stomachache while the leaves can help

relieve vomiting. In Kiribati decoction of the root is a remedy for haemorrhoids, In the


17

Marshall Islands the male flower is believed to have aphrodisiac properties 68

. Water

distilled from the flowering tops is considered an antispasmodic while at the same time

helps in relieve of faintness and giddiness. The oil extracted from the flowering tops

of Pandanus odoratissimus is used to treat earaches and otorrhoea. The leaves are remedy

for cold/flu, asthma, boils and cancer in Kiribati.

Pharmacognosy

"Pharmacognosy" derives from two Greek words, "pharmakon" or drug, and "gnosis" or

knowledge. Like many contemporary fields of science, Pharmacognosy has undergone

significant changes in recent years and today represents a highly interdisciplinary science,

which is one of five major areas of pharmaceutical education. Its scope includes the study

of the physical, chemical, biochemical and biological properties of drugs, drug

substances, or potential drugs or drug substances of natural origin as well as the search

for new drugs from natural sources.

After decades of serious obsession with the modern medicinal system, people have

started looking at the ancient healing systems; however a key obstacle, which has

hindered the acceptance of the alternative medicines in the developed countries, is the

lack of documentation and stringent quality control. There is a need for documentation of

research work carried out on traditional medicines 69

. With this backdrop, it becomes

extremely important to make an effort towards standardization of the plant material to be

used as medicine. The process of standardization can be achieved by stepwise

pharmacognostic studies 70

.These studies help in identification and authentication of the


18

plant material. Correct identification and quality assurance of the starting materials is an

essential prerequisite to ensure reproducible quality of herbal medicine which will

contribute to its safety and efficacy. Simple pharmacognostic techniques used in

standardization of plant material include its morphological, anatomical and biochemical

characteristics 71

.

To differentiate the two plants, Shakoor et al 72

carried out the pharmacognostic study and

identifies the two plants as different on the basis of anatomical characters, chemical

composition and compared the pharmaceutical and medical applications. In the same

context, Ashraf and Riaz 73

described the, botanical chemical, medicinal and

pharmacological background of various Nymphaea species to resolve the controversy

regarding application of common indigenous name Nilofar. Reviewing the reported

literature on various Nymphaea species, botanical name N. alba was assigned to Nilofar.

Based on the two main forms of indigenous drugs (parts) viz. double stained transverse

section and dry powder of Conyza ambigua, Eclipta alba and Sonchus asper were studied

by 74

.

Similar work was carried out for Bamboo species, where anatomical and histological

features were studied from all the species known from Taiwan to suggest a new

classification of Bambuseae 75

.In another study, cultivated Bambusa vulgaris of two and

four year old was studied for their anatomy and physical properties to know degrees of

variation correlation with age. The leaf of Pandanus odoratissimus was also studied for

histological, physical and powdered characteristics 76

.


19

Phytochemistry

“Phyto” is the Greek word for plant. Phytochemistry, evolved from natural products

chemistry is confined to the study of products elaborated by plants and it has developed

as a distinct discipline between natural product organic chemistry and plant biochemistry

in recent years. It deals with the study of chemical structure of plant constituents, their

biosynthesis, metabolism, natural distribution and biological functions. The fact that only

less than 10% of about 7.5 lakhs species of plants on earth has been investigated indicates

the opportunity provided and challenges thrown open to phytochemists. The task of the

phytochemist is compounded in accomplishing the characterization of very small quantity

of the compounds isolable from plants. Phytochemistry also enjoys the application of

modern research for the scientific investigation of ancestral empirical knowledge. It has

found wide and varying application in about all fields of life and civilization. Its

involvement in the field of food and nutrition, agriculture medicine and cosmetics, is well

known for years. Its contribution even in calmingly remote areas such as plant

physiology, plant pathology, plant ecology, palaeobotany, plant genetics, plant

systematics and plant evolution has been increasingly felt 77-82

.

There are many “families” of phytochemicals, the most important of these bioactive

constituents of plants are alkaloids, tannins, flavonoids, and Phenolic compounds 83

and

they help the human body in a variety of ways. Phytochemicals may protect human from

a host of diseases. Phytochemicals are non-nutritive plant chemicals that have protective

or disease preventive properties. Plant produces these chemicals to protect itself but


20

recent research demonstrates that many phytochemicals can protect humans against

diseases. There are many phytochemicals in fruits and herbs and each works differently.

Recovery of bioactive compounds from plant materials is typically accomplished through

different extraction techniques taking into account their chemistry and uneven

distribution in the plant matrix. For example, standard methods of extraction, separation

and chemical characterization of flavonoid compounds are described by Tracey as well as

Harborne 84

. Systematic procedure for the flavonoid identification employing

chromatographic methods of analysis and chemical and spectral methods of identification

have been explained by Geissman 85

, Harborne 86

, Mabry 87

, Markham 88

and Linskens

and Jackson 89

.

The conventional chromatographic methods like column, paper and thin layer are still in

use for separation and purification of the flavonoid compounds. Increase in speed and

efficiency in the separation of mixtures had been achieved by high pressure liquid

chromatography (HPLC). Among the separation techniques applied to flavonoids HPLC

has the advantage over to other techniques in regard to sensitivity, rapidity and easy

quantification.

Various phytoconstituents have been isolated from past few years from both the species

of Bambusa vulgaris and Pandanus odoratissimus. Preliminary phytochemical screening

of the aqueous extract of Bambusa vulgaris leaves revealed the presence of bioactive

agents such as alkaloids, tannins, phenolics, glycosides, saponins, flavonoids and


21

anthraquinones, Quantitative analysis of the phytochemicals indicated that the aqueous

extract of the leaves of Bambusa vulgaris consisted largely of alkaloids, while the

flavonoids were the least, where as pet ether extract showed the presence of phytosterols

and tannins 90

.

The roots of Pandanus odoratissimus have showed the presence of various

phytochemicals, two phenolic compounds, four lignan type compounds plus a new

benzofuran derivative was isolated from methanolic root extract. Among them,

pinoresinol and 3, 4-bis (4-hydroxy-3-methoxybenzyl) tetrahydrofuran showed strong

antioxidative activities. The new compounds were identified as 4-hydroxy-3-(2′, 3′-

dihydroxy-3′-methylbutyl)-benzoic acid methyl ester and 3-hydroxy-2-isopropenyl-

dihydrobenzofuran-5-carboxylic acid methyl ester, by spectroscopic analysis 91

, other

phytoconstituents such as 2-phenyl ethyl alcohol, 2-phenyl ethyl methyl ether, terpinen-4-

ol, 3-hydroxy-2-isopropenyl-dihydrobenzofuran-5-carboxylic acid methyl ester, 3-

methyl-3-buten-1-yl acetate, 3-methyl-3-buten-1-yl cinnamate, 3-methyl-2-buten-1-yl

acetate, 3-methyl-2-buten-1-yl cinnamate, 25-diene-3-one, alpha terpienol, beta carotene,

beta sitosterol, benzyl benzoate, pinoresinol, germacrene B, stigmasterol, viridine,

vitamin C 92

.

Venom Neutralizing study

Snake bite is a major socio-medical problem of tropical countries, especially in India.

20,000 deaths per year have been reported in India 93

. Snake bite treatment is as variable

as the bite & its symptoms. The one & only medical treatment available is the usage of


22

antisera, but the usage of snake venom antisera has its own limitations. Due to its high

cost & lack of availability, it is difficult for the rural patients to access antisera. Further,

due to its storage difficulty & short expiry, its use is restricted. Snake venom antiserum or

AVS has administration problem, the exact dosage of AVS is also not clear. AVS

administration is often associated with hypersensitive reactions (early & late) which need

further medical attention 94

.

Various plants have been worked out as an antidote for snake envenomation, some of

which possesses strong neutralizing activity whereas others possess moderate activity

against snake venom. Plants like Pluchea indica, Hemidesmus indicus95-96

, Strychnous

nux vomica 97

, Emblica officinalis, Vitex negundu 98

& Curcuma aromatica possess

strong neutralizing capacities, whereas Aristolochia indica, Andrographis paniculata,

Dolichondron sp., Crotolaria juncea, Croton tiglium, Moringa oliefera possess moderate

snake venom neutralizing capacity. So far no open scientific literature that addresses the

anti venom potential activity is available for Pandanus odoratissimus.

Antipyretic study

Pyrexia or fever is caused as a secondary impact of infection, malignancy or other

diseased states. It is the body’s natural defense to create an environment where infection

agent or damaged tissue cannot survive. Most of the antipyretic drugs inhibit Cox-2

expression to reduce the elevated body temperature by inhibiting prostaglandin E2

(PGE2) biosynthesis. Moreover these synthetic agents irreversibly inhibit Cox-2 with

high selectivity but are toxic to the hepatic cells, glomeruli, cortex of brain and heart


23

muscles, whereas the natural Cox-2 inhibitors have lower selectivity with fewer side

effects. A natural antipyretic agent with reduced or no toxicity is therefore essential 99-100

.

Medicinal plants contain so many chemical compounds which are the major source of

therapeutic agents to cure human diseases. Recent discovery and advancement in

medicinal and aromatic plants have lead to the enhancement of health care of mankind.

Various medicinal plants like Neem, Arjuna, Aswagandha, Tulsi, etc. traditionally used

for treating fever. The extract prepared from the heartwood of Acacia catechu, stem bark

and leaves of Bauhinia racemosus, Cleome viscosa etc. reported to have antipyretic

activity in rats 101

. So far no open scientific literature that addresses the anti pyretic

potential activity is available for Bambusa vulgaris.

Antidiabetic study

Diabetes is a syndrome characterized by deranged carbohydrate metabolism resulting in

abnormally high blood sugar level (hyperglycemia). It is caused by hereditary, increasing

age, poor diet, imperfect digestion, obesity, sedentary lifestyle, stress, drug-induced,

infection in pancreas, hypertension, high serum lipid and lipoproteins, less glucose

utilization and other factors.

There are three main types of diabetes:

• Type 1 diabetes: results from the body's failure to produce insulin, and presently

requires the person to inject insulin. (Also referred to as insulin-dependent

diabetes mellitus, IDDM for short, and juvenile diabetes.)


24

• Type 2 diabetes: results from insulin resistance, a condition in which cells fail to

use insulin properly, sometimes combined with an absolute insulin deficiency.

(Formerly referred to as non-insulin-dependent diabetes mellitus, NIDDM for

short, and adult-onset diabetes.)

• Gestational diabetes: is when pregnant women, who have never had diabetes

before, have a high blood glucose level during pregnancy. It may precede

development of type 2 DM.

The treatment of diabetes with synthetic drugs is costly and chances of side effects are

high. For example, long-term use of Exenetide (Byetta) has lead to side effects such as

nausea, vomiting, diarrhea, dizziness, headache, jittery feeling and acidity. Sulfonylureas

cause abdominal upset, headache and hypersensitivity, while Metformin 102

causes

diarrhea, nausea, gas, weakness, indigestion, abdominal discomfort and headache.

Thiazolidinediones has side effects like, upper respiratory infections and sinusitis,

headache, mild anemia, retention of fluid in the body which may lead to heart failure and

muscle pain.

Ayurveda and other traditional medicinal system for the treatment of diabetes describe a

number of plants used as herbal drugs. Hence, they play an important role as alternative

medicine due to less side effects and low cost. The active principles present in medicinal

plants have been reported to possess pancreatic beta cells regenerating, insulin releasing

and fighting the problem of insulin resistance 103

. Aloe vera juice stimulates the release of

insulin from the beta-cells in human, Acacia catechu wood extract enhances the

regeneration of pancreatic beta cells in rabbits, Momordica charantia fruit extract


25

enhances insulin secretion by the islets of Langerhans etc. A significant proportion of

these plants have been observed to possess potent antioxidant activity, which may

contribute to anti-diabetic property in strepotozotocin /alloxan, induced animal model 104

.

Not only in Ayurveda, but also in several other traditional systems of medicine, it is

described that plants useful in diabetes also possess strong antioxidant/free-radical

scavenging properties.

Previous reports on Bambusa vulgaris has showed its antidiabetic potential by various

extracts against different models, the aqueous extract was tested against for its

hypoglycemic activity, where the effect of the extract was superior to tolbutamide 105

. So

far no open scientific literature that addresses the antidiabetic potential of is available for

Pandanus odoratissimus.

Materials and Methods

26

CHAPTER-4

MATERIALS AND METHODS

PHARMACOGNOSY

Collection of specimens and identification

All the plant materials were collected from the ABS botanical research centre, Karipatti,

Salem. They were identified and authenticated by Dr. P. Jayaraman, Director, Medicinal

Plant Research unit and plant anatomy Research centre, Chennai India. Voucher

specimens of the plants have been deposited in the herbarium.

Pharmacognostical studies on Bambusa vulgaris leaf and Pandanus odoratissimus

root

The collected healthy plant specimens for the proposed study were carefully selected.

The samples of different parts were cut and removed from the plant and fixed in FAA

(Farmalin-5ml+ Acetic acid-5ml + 70% Ethyl alcohol-90ml).After 24 hrs of fixing, the

specimens were dehydrated with graded series of tertiary –Butyl alcohol as per the

schedule given by Sass, 1940. Infiltration of the specimens was carried by gradual

addition of paraffin wax (melting point 58-600C) until TBA solution attained super

saturation. The specimens were cast into paraffin blocks.

Sectioning

The paraffin embedded specimens were sectioned with the help of Rotary Microtome.

The thickness of the sections was 10-12 μm. Dewaxing of the sections was by customary


27

procedure 106

. The sections were stained with Toluidine blue as per the method published

by 107

. Since Toluidine blue is a polychromatic stain, the staining results were remarkably

good; and some cytochemical reactions were also obtained. The dye rendered pink colour

to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the

mucilage, blue to the protein bodies etc. wherever necessary sections were also stained

with safranin and Fast-green and IKI(for Starch).

For studying the stomatal morphology, venation pattern and trichome distribution,

paradermal sections (sections taken parallel to the surface of leaf) as well as clearing of

leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing

Jeffrey’s maceration fluid 108

were prepared. Glycerine mounted temporary preparations

were made for macerated/cleared materials. Powdered materials of different parts were

cleared with NaoH and mounted in glycerin medium after staining. Different cell

component were studied and measured.

Photomicrographs

Microscopic descriptions of tissues are supplemented with micrographs wherever

necessary. Photographs of different magnifications were taken with Nikon labphoto 2

microscopic Unit. For normal observations bright field was used. For the study of

crystals, starch grains and lignified cells, polarized light was employed. Since these

structures have birefringent property, under polarized light they appear bright against

dark background. Magnifications of the figures are indicated by the scale-bars.


28

Descriptive terms of the anatomical features are as given in the standard Anatomy books

109.

Pharmacognostical evaluation of selected plants was carried out in order to establish the

identity and standardization of the plants. Microscopical characters were studied as per

standard protocol.

Physiochemical evaluation

Bambusa vulgaris leaf and Pandanus odoratissimus root were used for analysis for

physiochemical parameters such as ash values, extractive values and moisture content.

Determination of Ash Values 110

Ash values such as total ash, acid insoluble ash, water soluble ash, acid soluble and

sulfated ash were determined. The total ash determination method is designed to measure

the total amount of material remaining after ignition. This includes both “physiological

ash”, which is derived from the plant tissue itself, and “non physiological ash”, which is

the residue of extraneous matter adhering to the plant surface.

For determination of ash values, powder was prepared of roots (Pandanus odoratissimus)

and leaf (Bambusa vulgaris) and shifted through sieve no. 20 and following tests were

performed.


29

Determination of Total Ash

About 3 g each of powdered parts were accurately weighed and taken separately in silica

crucible, which was previously ignited and weighed. The powder was spread as a fine

layer on the bottom of crucible. The powder was incinerated gradually by increasing

temperature to make it dull red hot until free from carbon. The crucible was cooled and

weighed. The procedure was repeated to get constant weight. The percentage of total ash

was calculated with reference to the air dried powder.

Acid Insoluble Ash

The ash obtained as described above was boiled with 25 ml of 2N HCl for 5 minutes. The

insoluble ash was collected on an ash less filter paper and washed with hot water. The

insoluble ash was transferred into a crucible, ignited and weighed. The procedure was

repeated to get a constant weight. The percentage of acid insoluble ash was calculated

with reference to the air dried drug.

Water Soluble Ash

The ash obtained as described for the total ash, was boiled for 5 minutes with 25 ml of

water. The insoluble matter was collected on ash less filter paper and washed with hot

water. The insoluble ash was transferred into silica crucible, ignited for 15 min. and

weighed. The procedure was repeated to get a constant weight. The weight of insoluble

matter was subtracted form the weight of total ash. The difference of weight was

considered as water soluble ash. The percentage of water soluble ash was calculated with

reference to air dried parts respectively.


30

Acid soluble Ash

Total ash treated with dilute hydrochloric acid reacts with minerals to form soluble salts

and the insoluble residue consists mainly of silica, as acid insoluble ash.

To the crucible/silica dish containing the total ash obtained by the previous test, 25ml of

HCl (N70g/l) was added, covered with a watch glass and boiled gently for 5 min on a hot

plate or burner. Watch glass was rinsed with 5ml of hot water and washings were added

to the crucible. Insoluble matter was collected on an ash less filter paper by filtration and

rinsed repeatedly with hot water until the filtrate was found to be neutral/free from acid.

Filter paper containing the insoluble matter was transferred to the original crucible, dried

on a hot plate and ignited to a constant weight in the muffle furnace at 450°-500°C. Silica

dish was allowed to cool in desiccator for 30 min and weighed without delay. Content of

acid insoluble ash as % was calculated as followed.

Where, A = sample weight in g

B = wt. of dish + contents after drying (g)

C = wt. in g. of empty dish.

Sulphated ash

A silica crucible was heated to red for 10 min. and was allowed to cool in a desiccator

and weighed. A gram of substance was accurately weighed and transferred to the

crucible. It was ignited gently at first, until the substance was thoroughly charred. Then

the residue was cooled and moistened with 1 ml of concentrated sulfuric acid, heated

Acid insoluble ash % = (B-C)

A × 100


31

gently until white fumes are no longer evolved and ignited at 800 oC ± 25

oC until all

black particles have disappeared. The ignition was conducted in a place protected from

air currents. The crucible was allowed to cool. A few drops of concentrated sulfuric acid

were added and heated. Ignited as before and was allowed to cool and weighed. The

operation was repeated until two successive weighing do not differ by more than 0.5 mg.

Determination of Extractive values 111-112

Water Soluble Extractive

Five grams of the each raw material was added to 50mL of water 80oc in a stoppered

flask. It was shaken well and allowed to stand for 10min. It was cooled to 15oc and 2 g of

Kiesulghur was added and filtered, 5 mL of the filterate was transferred to a tarred

evaporating basin. The solvent was evaporated on a water bath, for ½ h and then dried in

steam for 2 h and weighed. The percentage of water soluble extractive was calculated

with reference to the air dried powdered plant material.

Alcohol Soluble Extractive

Five grams of each raw material was macerated with 100mL of 70% alcohol in a closed

flask for 24h, shaken frequently during 6h and allowed to stand for 18h. It was filtered

rapidly taking precatutions against loss of alcohol and 25 mL of the filterate was

evaporated to dryness in tarred flat bottomed shallow dish and dried at 105o and

weighed. The percentage of alcohol soluble extractive was calculated with reference to

air- dried powdered formulation.


32

Determination of Moisture Content or Loss on Drying

About 5 g of each raw material were accurately weighed. The air dried material was

taken in a previously dried and tarred flat weighting bottle in IR moisture balance and the

temperature was adjusted to 105oC and heating was done for 5 minutes. The procedure

was repeated for three times for different samples and the loss in weight of the

formulation was calculated with respect to the original weight.

The formula used for calculating LOD is = W1/W2 x100

W1-weight of raw material after heating

W2- Original weight of the raw material


33

PHYTOCHEMISTRY

Preparation of Extracts

The dry leaf powder of B. vulgaris and root powder of P. odoratissimus 500 gms each

were subjected to soxhlet extraction. Using hexane, benzene, chloroform, ethylacetate

and methanol for 48 hours. The extracts were concentrated to dryness under reduced

pressure and controlled temperature (40-50 °C) using Buchi R-153 Rotavapour and

preserved in a desiccator until further use.

Qualitative Phytochemical Screening

The different qualitative tests were performed for establishing profile of the given extract

for its chemical composition. The following tests were performed on extracts to detect

various phyto constituents present in them.

Detection for carbohydrates 113

500 mg of extract was dissolved in 5 ml of distilled water and filtered. The filtrate was

used to test the presence of carbohydrates.

Molisch’s test

Molish reagent: 10 gm of alpha napthol was dissolved in 100 ml of 95% methanol to

prepare Molish reagent

To the extract, two drops of Molish reagent and few drops of concentrated H2SO4 is

added, formation of purple-violet ring indicates the presence of carbohydrates.


34

Detection of Glycosides 114

0.5 gm of the extract was hydrolyzed with 20 ml of HCl (0.1 N) and filtered. The filterate

was used to test the presence of Glycosides.

a. Modified Borntrager’s test

To 1 ml of filterate, 2 ml of 1% ferric chloride solution was added in a test tube and

heated for 10 minutes in boiling water bath. The mixture was cooled and shaken with

equal volumes of Benzene. The Benzene layer was separated and treated with half of its

volume of ammonia solution. Formation of rose pink or cherry red colour in the

ammonical layer indicates the presence of anthranol glycoside.

b. Keller-Killiani test: To the extract, few drops of glacial acetic acid and one drop of

5% FeCl3 and concentrated H2SO4 was added, formation of reddish brown colour at the

junction of two liquid layers and upper layer turned bluish green indicates the presence of

glycosides.

Detection of Saponins 115

a. Foam test: 1 ml of extract was diluted to make up to 20 ml with distilled water and

slowly shaked in a graduated cyclinder for 15 minutes. 1 one cm layer of foam indicates

the presence of saponins.


35

Detection of Alkaloids 113,116

0.5 gm of the extract was dissolved in 10 ml of dilute HCL (0.1N) and filtered. The

filterate was used to test the presence of alkaloids.

a. Mayer’s test

Mayer’s reagent: readily available from Sd fine chemicals, Mumbai.

Filtrate was treated with Meyer’s reagent; formation of yellow cream colored precipitate

indicates the presence of alkaloids.

b. Dragendrodroff’s test

Dragendroff’s reagent:

i) Dissolve 8 gm of bismuth subnitrate in 20 ml of nitric acid.

ii) Dissolve 27.2 gm of Potassium iodide in 50 ml of distilled water, mix (a) and

(b) and adjust the volume to 100 ml with distilled water.

Filtrate was treated with Dragendroff’s reagent; formation of red colored precipitate

indicates the presenc of alkaloids.

Detection of Flavonoids 117

a. Alkaline reagent test:

To 100 mg 0f extract, few drops of NaOH solution was added in a test tube. Formation of

intense yellow color that becomes colorless on addition of few drops of of dilute HCl

indicates the presence of Flavonoids.


36

Detection of Phenolics and Tannins 115

100 mg of extract was boiled with 1 ml of distilled water and filtered. The filterate was

used for the following test,

a. Ferric chloride test: To 2 ml of filtrate, 2 ml of 1% ferric chloride solution was added

in a test tube. Formation of bluish black color indicates the presence of phenolic nucleus.

b. Test for Tannins: To the extract 0.5 ml NaOH was added, formation of precipitate

indicates the presence of tannins.

Detection of Phytosterols and Triterpenoids 116-118

0.5 gm of extract was treated with 10 ml chloroform and filtered. The filterate was used

to test the presence of Phytosterols and Triterpenoids.

a. Leibermann’s test: To 2 ml of filtrate in hot alcohol, few drops of acetic anhydride

was added. Formation of brown precipitate indicate the presence of sterols.

b. Leiberman-Bucharat test: To the extract, few drops of acetic acid and concentrated

H2SO4 were added, deep red ring at the junction of two layers indicates the presence of

triterpenes.

c. Salkowaski test: To the extract solution few drops of Conc Sulphuric acid was added

and shaken and allowed to stand, lower layer turns red indicating the presence of sterols.


37

Detection of fixed oils and fats 113

a. Oily spot test: One drop of extract was placed on filter paper and solvent was allowed

to evaporate. An oily stain on filter paper indicates the presence of fixed oil.

Estimation of Total phenol content

Total phenol content of the extracts was determined by using the Folin-Ciocalteu Method

119. This test is based on the oxidation of Phenolic groups with phosphomolybdic and

phosphotungstic acids. After oxidation the green– blue complex formed was measured at

750 nm.

Chemicals and Reagents

1. Folin-Ciocalteu Reagent

Commercially available Folin-Ciocalteu reagent was diluted (1:10) with distilled

water and used.

2. Sodium carbonate

20.25 g of sodium carbonate was dissolved in 100 ml of distilled water and used (0.7

M).

Preparation of test and standard solutions

The plant extracts (50 mg each) were dissolved separately in 50 ml of methanol. These

solutions were serially diluted with methanol to obtain lower dilutions. Gallic acid

monohydrate (50 mg) was dissolved in 50 ml of distilled water. It was serially diluted

with water to obtain lower dilutions.


38

Procedure

In a test tube, 200 μl of the extract (1 mg/ml) was mixed with 1 ml of Folin-Ciocalteu

reagent and 800 μl of sodium carbonate. After shaking, it was kept for 2 h for reaction.

The absorbance was measured at 750 nm. Using gallic acid monohydrate, standard curve

was prepared and linearity was obtained in the range of 10-50 μg/ml. Using the standard

curve the total phenol content of the extract was determined and expressed as gallic acid

equivalent in mg/g of the extract.

Isolation and characterization of phytoconstituent from methanolic extract of

Bambusa vulgaris leaves 120

Definition

“Technique employed for the separation of mixture by continuous distribution of the

components between two phases, when the chromatographic operations are carried using

column, it is called column chromatography”.

Choosing the solvent system (Standardization of mobile solvent)

To standardize the mobile solvent, initially TLC study was performed for separation of

compounds. For this different combination of mobile phases were tried with two solvent

systems at variable proportions viz., Hexane, Chloroform, Ethyl acetate, Methanol. It was

concluded that Chloroform and methanol solvent system showed good separation, so this

combination was chosen as mobile phase for column chromatography.


39

Loading of column (wet packing)

Column was packed with slurry of silica gel of mesh size 60-120 (SD fine chemicals,

Mumbai) with chloroform. Column length is 100 cm and diameter is 3 cm. on top of

silica bed, sample 15 Gms was loaded. On top of the sample cotton was placed to avoid

any disturbance to the sample bed.

Elution of the column

Based on TLC results column elution was started with Chloroform but initially

chloroform solvent was eluted for small quantity for correct distribution of activated

sample in the column and later Chloroform and Chloroform: Methanol solvent

combination was followed with increasing polarity. Fractions were collected in 50 ml

portions and monitored on TLC and the fraction showing similar spots were pooled.

Pattern of column elution

Solvent ratio Fraction

no TLC result

Eluted

sample

color

Solvent ratio

For TLC

Chloroform 1 to 3 No spots Colorless Chloroform: Methanol:9:1

Chloroform 4 Multiple spots

(2-3 spots)

Light

straw

colour

Chloroform: Methanol:9:1

Chloroform 5-6 Multiple spots

(2-3 spots)

Dark

Straw Chloroform: Methanol:9:1

Chloroform 7-9 Multiple spots

(2-3 spots)

Light

straw

colour

Chloroform: Methanol:9:1


40

Chloroform:

Methanol:

9.5:0.5

10-21 No spots colorless Chloroform: Methanol:

9.5:0.5

Chloroform:

Methanol:

9.5:0.5

22 3 bands with

tailing pattern

Straw

colour

Chloroform: Methanol:

9.5:0.5

Chloroform:

Methanol:

9.5:0.5

23

Single band

Flourescent

orange

Green

colour


9.5:0.5

Chloroform:

Methanol:

9.5:0.5

24

3 bands,

Flourescent

Orange, Light

blue and Reddish

orange.

Green

Colour


9.5:0.5

Chloroform:

Methanol:

9.5:0.5

25-26

3 bands,

Flourescent light

Orange, Light

blue and Reddish

orange.

Dark

Green

Colour

Chloroform: Methanol :

9.5:0.5

Chloroform:

Methanol:

9.5:0.5

27-55 Multiple spots

(2-3 spots)

straw

colour


9:1

Evaporation of fractions

During the above column elution process, the fraction 23 has a single banding pattern

which was confirmed by TLC study with various mobile phases. So it was kept for

evaporation to dryness in room temperature. After drying the dried residue was scrapped

off once again checked for its purity and named as S2. The remaining fractions were not


41

worked out because of lower yield as well impurity. The compounds were sent for

spectral analysis i.e., IR, MASS, C13

NMR & 1H NMR for structural elucidation.

Pandanus odoratissimus root material bioactivity guided identification for

antidiabetic compound or an active fraction 120

Column chromatography purification of methanolic extract

Adsorption of sample on silica gel

The methanolic extract was dissolved in methanol and mixed with dry activated silica

gel, after air drying the sample; it was used for purification process. Adsorbed sample

kept for complete drying and later used for column elution.

Choosing the solvent system (Standardization of mobile solvent)

To standardize the mobile solvent, initially TLC study was performed for separation of

compounds. For this different combination of mobile phases were tried with two solvent

systems at variable proportions viz., Hexane, Chloroform, Ethyl acetate, Methanol. It was

concluded that Hexane, Chloroform and methanol solvent system showed good

separation, so this combination was chosen as mobile phase for column chromatography.

Loading of column (wet packing)

Column was packed with slurry of silica gel of mesh size 60-120 (Sd fine chemicals,

Mumbai) with Hexane. Column length was 100 cm and diameter is 3 cm. on top of silica


42

bed, activated sample was loaded and cotton was placed on top of it to avoid any

disturbance to the sample bed.

Elution of the column

Initially Hexane solvent was eluted for small quantity for correct distribution of activated

sample in the column and later with two solvent combinations with increasing order of

polarity was used.

Based on preliminary TLC observations, column elution was started with Hexane,

Chloroform and Methanol combinations Fractions were collected in 50 ml portions and

all the fractions were pooled wit respect to their mobile phase.

Pattern of column elution

Solvent ratio Fraction no TLC result

Eluted

sample color

Hexane 1 Multiple spots Green

Chloroform 2 Multiple spots Straw

Chloroform: Methanol:70:30 3 Multiple spots Brown

Chloroform: Methanol:50:50 4 Multiple spots Brown

Chloroform: Methanol:30: 70 5 Multiple spots Brown

Methanol 6 Multiple spots Brown


43

From above elution process, the fractions were pooled as per their mobile phase

concentrations, all the pooled fractions were evaporated under reduced pressure. Later

each fraction was tested for its toxicity in L6 cell line by MTT assay (Table no 1).

After obtaining the CTC50 value, the above fractions were tested for its antidiabetic study

by Glucose uptake study by L 6 cell lines (Table no 2). From the Glucose uptake study it

was found out that the Methanol fraction showed good antidiabetic activity followed by

Chloroform: Methanol: 30: 70 fractions with % glucose uptake of 15.54 and 12.42 over

the control. So the Methanol fraction was further preceded for purification by preparative

TLC.

Purification of phytoconstituents from methanolic fraction by preparative TLC

The mobile phase was standardized for the Methanol fraction fractions, Hexane:

Chloroform: Methanol: 2:4:1 was used for separating phytoconstituents. After

developing/separating the TLC plate was visualized under UV light, the bands at Rf value

13.5 (FBR) was scrapped off and the scrapped band was dissolved in methanol for two to

three times, later it was sent for spectral analysis.


44

BIOLOGICAL ACTIVITIES

Acute toxicity studies

Animals

Adult healthy female Sprague–Dawley rats with body mass of approximately 200–225 g

were used. Adult healthy female mice weighing 20±2g were used. The animals were

conditioned at room temperature and at natural photoperiods for 1 week before study. A

commercial balanced diet and tap water ad- libitum were provided. Room temperature

was maintained at 22±2°C with light and dark cycle of 12/12 h. The experiments were

conducted as per the guidelines of CPCSEA, Chennai, India (approval no

APTUS/IAEC/252/11)

.

Treatment

The animals received a single dose of the test item by oral administration at 2000 mg/kg

body weight, after being fasted for approximately 18.0 hours but with free access to

water. Food was provided again at approximately 3.0 hours after dosing for both the

Steps. The administration volume was 10 mL/kg body weight. The animals were dosed

using 18 G oral Stainless steel feeding tubes.

The animals were observed daily during the acclimatization period and mortality/viability

and clinical signs were recorded. All animals were observed for clinical signs during first

30 minutes and at approximately 1, 2, 3 and 4 hours after administration on test day 0 and

once daily during test days 1-14. Mortality/viability was recorded twice daily during days

1-14 (at least once on day of sacrifice). Body weights were recorded on test day 0 (prior


45

to administration), test days 7 and 14. All animals were necropsied and examined

macroscopically.

Necroscopy

All animals were sacrificed at the end of the observation period by carbon dioxide in

euthanasia chamber and discarded after the gross/macroscopic pathological changes were

observed and recorded. No organs or tissues were retained.

Evaluation of venom neutralization activity 121

Selection and Maintenance of Animals

Female Swiss albino mice (20±2 g, 8-10 weeks old) were obtained from the animal house

of Aptus Biosciences Private Limited, Hyderabad. Mice were housed in open top cages

and maintained on food and water ad labium. Room temperature was maintained at

22±2°C with light and dark cycle of 12/12 h. The experiments were conducted as per the

guidelines of CPCSEA, Chennai, India (approval no APTUS/IAEC/253/11)

Venom

The snake venom was obtained from Irula Snake Catcher’s Industrial Co-operative

Society Ltd, Chennai and was preserved at 4°C. Before use, the venom was dissolved in

saline, centrifuged at 2000rpm for 10 min and supernatant was used for anti-venom

studies. Venom concentration was expressed in terms of dry weight.


46

Snake venom antiserum

Lyophilized polyvalent snake venom antiserum (as standard reference serum) was

obtained from vins Bioproducts Ltd, Eradur, Medak dist, Andhra Pradesh, India. Before

use the antiserum was dissolved in 10ml of water for injection.

Determination of median lethal dose (LD50) of venom

Animals, total of 48 were divided into 6 groups (n=8) i.e. Group I to Group VI. Group I

was kept as control received 0.2 ml of saline intra peritonially (i.p.). Group II to VI

received different concentrations of venom ranging from 100 to 20 µg in saline solution

(0.2 ml i.p.). LD50 was determined by the standard method with the confidence limit at

50% probability by the analysis of deaths occurring within 24 h of venom injection 122

.

Experimental Design

The animals, total of 56 were divided into seven groups (n=8) i.e. Group I to Group VII.

Except the Group I, rest of the groups received LD50 dose of venom prepared in saline

(0.2ml) at zero hour. This was followed by i.p. administration of antiserum and plant

extract as described below,

Group I: Normal control and received saline (0.2ml).

Group II: Venom control and received saline (0.2ml).

Group III: Positive control was treated with dil. snake venom antiserum (0.2ml).

Group IV: Treated with Methanolic extract (250 mg/kg b. wt)

Group V: Treated with Methanolic extract (500 mg/kg b. wt)


47

Group VI: Treated with Methanolic extract (750 mg/kg b. wt)

Group VII: Treated with Methanolic extract (1000 mg/kg b. wt)

After 24 h of treatment, the number of mice survived in each group was counted. The

efficacy of the plant extracts was evaluated against the venom induced lethality and

expressed in percentage of survival and increase in survival rate by extract treatment.

Antipyretic studies 123

Animals

Adult healthy male wistar rats with body mass of approximately 150-200 g were used.

The animals were conditioned at room temperature and at natural photoperiods for 1

week before study. A commercial balanced diet and tap water ad- libitum were provided.

The experiments were conducted as per the guidelines of CPCSEA, Chennai, India

(approval no APTUS/IAEC/254/11)

Treatment

The antipyretic activity of formulations was evaluated using Brewer’s yeast induced

pyrexia in male albino rats of wistar strain weighing between 150-200 gms. Fever was

induced by subcutaneous injecting 10 ml/kg of 20 percent aqueous suspension of

Brewer’s yeast in normal saline after measuring the rectal temperature using the digital

thermometer. Eighteen hours (0 h) after the yeast injection, the animals were again placed

in individual cages for recording the rectal temperature. The different groups of rats were

treated orally with polyherbal formulations at doses of 500 and 250 mg/kg body weight.


48

The animals of control group were administered orally the suspension of 2% aqueous

solution of Tween 80 a volume of 2 ml/kg body weight. The animals of Positive control

group were received the standard prototype antipyretic agent, paracetamol (150 mg/kg

body weight) orally. The rats were restrained for their rectal temperature to be recorded at

the 0 h immediately before formulation or vehicle or paracetamol administration and

again at regular time intervals for next six hours.

Statistical analysis

The results of the experiment were expressed as mean ± SE in each test. The data were

evaluated by one-way Analysis of Variance (ANOVA) followed by Tukey’s multiple

pair-wise comparison tests to assess the statistical significance, using software ANOVA

ver. 0.98.

Anti diabetic studies

In vitro anti-diabetic studies 124

Chemicals

3-(4, 5–dimethyl thiazol–2–yl)–5–diphenyl tetrazolium bromide (MTT), Fetal Bovine

serum (FBS), Phosphate Buffered Saline (PBS), Bovine Serum Albumin (BSA), D-

glucose, Dulbecco’s Modified Eagle’s Medium (DMEM), Metformin and Trypsin were

obtained from Sigma Aldrich Co, St Louis, USA. EDTA, Antibiotics from Hi-Media

Laboratories Ltd., Mumbai. Insulin (Torrent Pharmaceuticals, 40IU/ml) was purchased

from a drug store. Dimethyl Sulfoxide (DMSO) and Propanol from E.Merck Ltd.,

Mumbai, India.


49

Cell lines and Culture medium

L-6 (Rat, Skeletal muscle) cell culture was procured from National Centre for Cell

Sciences (NCCS), Pune, India. Stock cells of L-6 were cultured in DMEM supplemented

with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin

(100 µg/ml) and amphotericin B (5 µg/ml) in an humidified atmosphere of 5% CO2 at

37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin,

0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture

flasks and all experiments were carried out in 96 microtitre plates (Tarsons India Pvt.

Ltd., Kolkata, India).

Preparation of Test Solutions

For in vitro studies, each weighed test drugs were separately dissolved in distilled DMSO

and volume was made up with DMEM supplemented with 2% inactivated FBS to obtain

a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two fold

dilutions were prepared from this for carrying out cytotoxic studies.

Cytotoxicity studies

The 24 hr cell cultures with 70-80% confluency in 96 well plates were used for the study.

100 µl of each dilution of the test drugs were added in quadruplicate in 96 well plate and

cell controls maintained in same number. The culture were incubated at 370 C with 5%

CO2 for 24 hrs and the cultures were observed microscopically for any visible change in

morphology of cells and observations were recorded. The cell viability assay was

determined by MTT assay (francis and Rita). The percentage cytotoxicity caused by each


50

dilution of the drug was determined and Cytotoxic Concentration 50 (CTC50) values

determined by interpolation method. The non-toxic concentrations of test drugs, i.e.

concentrations below CTC50 value were taken for glucose uptake studies.

In vitro glucose uptake assay

Glucose uptake activity of test drugs were determined in differentiated L6 cells 125-126

. In

brief, the 24 hr cell cultures with 70-80% confluency in 40mm petri plates were allowed

to differentiate by maintaining in DMEM with 2% FBS for 4-6 days. The extent of

differentiation was established by observing multinucleation of cells. The differentiated

cells were serum starved over night and at the time of experiment cells were washed with

HEPES buffered Krebs Ringer Phosphate solution (KRP buffer) once and incubated with

KRP buffer with 0.1% BSA for 30min at 370C. Cells were treated with different non-

toxic concentrations of test and standard drugs for 30 min along with negative controls at

370C. 20µl of D-glucose solution was added simultaneously to each well and incubated at

370C for 30 min. After incubation, the uptake of the glucose was terminated by aspiration

of solutions from wells and washing thrice with ice-cold KRP buffer solution. Cells were

lysed with 0.1M NaOH solution and an aliquot of cell lysates were used to measure the

cell-associated glucose. The glucose levels in cell lysates were measured using glucose

assay kit (Biovision Inc, USA). Three independent experimental values in duplicates

were taken to determine the percentage enhancement of glucose uptake over controls 127-

128.


51

In situ glucose uptake studies in rat hemi diaphragm 129

Glucose uptake by rat hemi-diaphragm was estimated by the methods described earlier

130-131 with some modification. Albino rats of either sex weighing between 160-180 gm

were selected. The animals were maintained on a standard pellet diet (water ad libitum),

and fasted overnight. The animals were sacrificed by decapitation and diaphragms were

dissected out quickly with minimal trauma and divided into two halves. The hemi

diaphragms were then rinsed in cold Tyrode solution (without glucose) to remove any

blood clots and were placed in small culture tubes containing 2ml Tyrode solution with

2% glucose and incubated for 30 minutes at 370C. Twelve sets containing five numbers

of graduated test tubes (n=5) each and treated with negative, positive controls (Insulin)

and with different extracts at 200 µg/ml. Two diaphragms from the same animal were not

used for the same set of experiment. Following incubation, the hemi-diaphragms were

taken out and weighed. The glucose content of the incubated medium was measured by

GOD-POD method. The uptake of glucose was calculated in mg/g of moist tissue/30 min.

Glucose uptake per gram of tissue was calculated as the difference between the initial and

final glucose content in the incubated medium.

In vivo anti-diabetic studies 132

Animals

Adult healthy male Sprague–Dawley rats with body mass of approximately 200–225 g

were used. The animals were conditioned at room temperature and at natural

photoperiods for 1 week before study. A commercial balanced diet and tap water ad-


52

libitum were provided. The experiments were conducted as per the guidelines of

CPCSEA, Chennai, India (approval no APTUS/IAEC/254/11)

Induction of diabetes

The animals were initially divided into two groups, the first group (6) received saline

solution intraperitonially (i.p.) and it was kept as control. The second group (36 rats) was

injected with a single intravenous dose of streptozotocin (STZ) at 40 mg/kg of body

weight, dissolved in 0.01 M citrate buffer, pH 4.5, immediately before use. Three days

later blood glucose levels were determined in this group in whole blood samples

collected from the tip of the tail.

Treatment

The animals which received saline were named as Group I, normal controls were fed with

normal diet. The diabetic rats were devided into Group II-VII, in which Group II served

as positive control without any treatment. Group III and IV were treated with different

doses of methanolic extract (500 and 1000 mg/kg) of Bambusa vulgaris Schrad leaves.

Group V and VI were treated with different doses of methanolic extract (500 and 1000

mg/kg) of Pandanus odoratissimus Linn.f. root. Group VII were treated with the standard

drug Gliclazide at 25mg/kg b.w. Treatment was carried out for 4 weeks.

Blood and tissue collection

At the end of the experiment, rats were fasted overnight and anesthetized with sodium

pentothal (intraperitoneally) and 2.5 ml of blood was withdrawn through the retro-orbital


53

plexus using a glass capillary and collected in tubes and subjected to blood serum

analysis. The animals were sacrificed and pancreas was collected to study

histopathological parameters.

Preparation of hemolysate

Collected blood was centrifuged for 10 minutes at 3000 rpm. Plasma and serum was

separated for the analsysis. The plasma thus obtained was used for glucose and

glycosylated hemoglobin (HbA1C) using commercial kits. The serum samples were used

for the estimation of creatine kinase (CK) and lactate dehydrogenase (LDH) using

commercial kits.

Histopathology

The pancreas were fixed in 10% neutral buffered formalin, and 4 ftm paraffin sections

were cut and stained with haematoxylin and eosin (H&E). Degeneration and necrotic

damage produced by STZ was observed microscopically.

Results and Analysis

54

CHAPTER-5

RESULTS AND ANALYSIS

PHARMACOGNOSTICAL STUDIES ON BAMBUSA VULGARIS LEAF AND

PANDANUS ODORATISSIMUS ROOT

Bambusa vulgaris leaf microscopy study

The leaf consists of a median, less prominent midrib and uniformly thick lamina (fig 1.1). The

vascular bundle of the midrib is slightly larger than the vascular bundles of the lateral veins (fig

1.1). the vascular bundle of the midrib consists of two wide metaxylem elements and short row

of proto xylem elements; a wide circular mass of phloem elements is situated in between the

metaxylem elements; a thick mass of fibres occurs beneath the phloem and small are of fibres is

situated on the adaxial end of the vascular strand.(fig 1.2). The vascular bundles of the lateral

veins vary in size, some being larger others being smaller. The larger lateral vein-bundles are

circular with two large metaxylem elements and wide mass of phloem elements. The vascular

strand has parenchymatous bundle sheath with wide, circular hyline cells (fig 1.3). A flat pade of

two layers of fibres is situated at the lower end of the bundle; a thin vertical pillar of

sclerenchyma cells is also seen on the upper of the vascular strand. The vascular strand is 90µm

wide. Smaller vascular bundlesof the lateral veins use also circular with less prominent xylem

elements, lightly dilated parenchymatous bundle sheath and a thickpad of sclerenchyma located

on the lower end of the vascular strand. The palisade cells are horizontally transcurrent along

adaxial end of the vascular strands (fig2.1, 2).


55

Epidermal layers

The adaxial epidermal cells are barrel shaped with thick smooth cuticle (fig 2.1, 2). The cells are

barrel shaped. The abaxial epidermal cells are circular with with heavy cuticle. The cuticle

develops thick, peg like outgrowth on each epidermal cells. (fig.1.3) the cells are 13µm thick.

The ground tissue is differentiated into adaxial and abaxial zones of mesophyll tissue, and a

median row of air-chambers (fig 1.1; 2.1, 2).the adaxial mesophyll tissue consists of three layers

of short, compact cylindrical cells; the abaxial zone has two or three layers of spherical compact

cells. Both abaxial and axial cells have dense chloroplasts. The median air chambers are wide

and rectangular, separated laterally by thick septa and vascular bundles of the lateral veins.

Bulli form cells

Some of the adaxial epidermal cells are highly dilated into vertically elongated triangular, thin

walled cells. A group of 3-5 cells form such dilated cluster which are called bulliform cells or

motor cells(fig.1.3; 2.1,2) of the cluster of cells,the central cell is the highest and the lateral cells

are short. In sectional view, the bulliform cells appear pyramidal in shape. The bulliform

apparatus is 60µm in heightand 40µm in width.

Leaf margin

The marginal portion of the lamina is thin and conical; the mesophyll tissue consists of four

layers of short compact cells; the air chambers are totally absent. At the extreme end of the

margin is a wide circular canal, which does not contain any inclusion (fig2.3).


56

Stomata and epidermal cells

The stomata are monocot-type; the stoma consists of two dumb – bell shaped guardcells with

one semicircular subsidiary cells on either side of the guard cells.the stomata occur in

longitudinal lines (fig3.1).the epidermal cells are rectangular , arranged in longitudinal files.

Their walls are closely undulate (fig 3.2).

Venation

Uniformly thick veins run parallel to each other along straight lines (fig.3.3). The veins include

xylem elements of spiral, annular and scalar form lateral thickenings. Bundle sheath cells are

seen all along the lateral sides of the veins –bundles. The cells are rectangular, thin walled and

hyaline. (fig.3.3). the midrib is thicker than the lateral veins (fig 4.1, 2).the veins are linned by

thin sclereids at certain places.

Crystals

Calcium oxalate crystals are abundant in the mesophyll tissue. The crystals are druses. They are

scattered and random in distribution (fig.4.3).

Pandanus odoratissimus root microscopy study

Both thin root and thick root were studied. The thin root measuring 600 micro meter in diameter

is undulate in cross sectional view (fig.5.1) .The epidermal layer is not well defined. The cortex

consists of outer zone of compact cells and inner zone of aerenchyma. The outer cortical zone

consists of 6 or 7 layers of thin walled, compact shrunken cells .The outer layer of the cortex

consists mucilaginous coating and assumes the function of the epidermis (fig.5.1, 6.1) .Thinner


57

cortex consists of two or three layers of small air-chambers separated by thin uniseriate

filaments. In the intersecting regions of the separation filaments occur small circular clusters of

thick walled fibres (fig.6).

The stele is circular measuring 210 micro meter thick.It consists of well defined endodermoid

layer of spindle shaped cells, lacking the characteristic annular thickenings. Inner to the

endodermoid layer is a thin hyaline layer of pericycle. The stele consists of about 9 exarch xylem

strands with 3 elements in each strand and alternating 9 small groups of phloem elements. The

ground tissue of the stele consists of sclerenchyma cells. The xylem elements are angular and

wide. The sclerenchyma elements are thick walled and lignified.

The thick root is 2.5 mm in diameter. It is circular with wavy outer surface. The epidermis

remains intact at certain places; it consists of spindle shaped cells with thick cuticle. Inner to the

epidermis is a wide zone of storied periderm (or storied cork).It includes about 8 layers of tabular

suberised cells which do not form regular radial seviation. The cortex wide comprises outer

compact cells and minor several radial rows of air chambers. Small groups of sclerenchyma cells

are located both in the outer ground tissue and inner aerenchyma cells. The sclerenchyma

clusters include thick walled, lignified fibres (fig.7.1, 2; 8.1).

The stele consists of an endodermoid layer of barrel shaped cells; inner to the endodermoid layer

is a thin layer of parenchyma cells which is the pericycle (fig.8.2). The ground tissue of the stele

is sclerenchymatous mixed with wide, thin walled parenchyma cells. Small clusters of highly

thick walled fibres are also present with ground tissue. There are several wide circular vessels


58

diffusely distributed in the pith. These central wide elements are metaxylem cells. Along the

periphery of the stele is the protoxylem. Alternating with the protoxylem strands are several

phloem islands (fig.9). The metaxylem cells are wide and elliptical or circular in outline. The

cells are up to 150 micro meter in diameter.

Root Powder microscopic results

The root powder includes fibres, and vessel elements. Fibres are either wide or narrow. The wide

fibres are thin walled with wide lumen. They are about 700 micrometer long and 30 micrometer

wide. The narrow fibres have thick walls and narrow lumen. They are up to 850 micrometer long

and 10 micrometer thick (fig.10.1, 2).

Vessel elements are long, narrow and coiled (fig.6). They have either annular or spiral

thickenings. The metaxylem elements are vessels with wide shorter cells and wide elliptical

simple perforations at the end walls. The lateral walls have circular bordered pits (fig.7). These

elements are up to 250 micrometer long.

Crystals

Calcium oxalate crystals are abundant with inner cortical cells (fig.5.2) .The crystals are either

raphide bundles or sphaero crystals .They occur in the cells outer to the endodermoid layer. They

are random in distribution and do not occur in specialized cells.


59

Fig 1.1 (10X): T S of lamina of Bambusa vulgaris leaf

Fig 1.2 (40X): T S of lamina through midrib of Bambusa vulgaris leaf

Fig 1.3 (40X): T S of lamina through lateral veins and bulliform apparatus of Bambusa

vulgaris

(AdE: Adaxial epidermis ; Ads: Adaxial side ;B C, Bulliform cells ; E C : Echinate cuticle ;L V : Lateral

veins; M R : Midrib; M X : Metaxylem; Ph; Phloem; Sc; Scleren chyma;V B: Vascular bundle; X : Xylem)


60

Fig 2.1 (40X): T S of lamina through smaller lateral veins of Bambusa vulgaris leaf.

Fig 2.2 (40X): T S of lamina through smaller lateral veins of Bambusa vulgaris leaf.

Fig 2.3 (40X): T S of lamina through marginal part of Bambusa vulgaris leaf

(AbE: Abaxial Epidermis; Abs: Abaxial side; A C: Air-chamber; AdE :Adaxial epidermis ; Ads : Adaxial

side; B C : Bulli form cells; B S: Bundle sheath; Cu: Cuticle; L M : Leaf- margin ; L V: Lateral vein ; M T

: Mesophyll tissue; Ph: phloem; SC: Sclerenchyma, X: Xylem)


61

Fig 3.1 (10X): Lower epidermis of the leaf, showing the stomata of Bambusa vulgaris leaf

Fig 3.2 (40X): Upper epidermis cells showing wavy cell wall of Bambusa vulgaris leaf

Fig 3.3 (40X): Venation of the lamina of Bambusa vulgaris leaf

(E C: Epidermal cells; B C: Bundle sheath cells; Scl: sclereid ; St : Stomata; Ve: Veins)


62

Fig 4.1 (40X): Surface view of the cleased

leaves showing parallel veins of Bambusa

vulgaris leaf

Fig 4.3 (10X): Crystals in the leaf mesophyll tissue of Bambusa vulgaris leaf

(Cr: Crystals; L V, Lateral vein; M R : Midrib; Ve :veins)

Fig 4.2 (10X): Surface view of the

cleased leaves showing parallel veins

of Bambusa vulgaris leaf


63

Fig 5.1 (10X): TS of thin root-entire view of Pandanus odoratissimus root.

Fig 5.2 (10X): TS of thick root showing the crystals with inner cortical cells Pandanus

odoratissimus root

(AC- Air Chamber; Co-Cortex; Cr-Crystals; St-Stele)


64

Fig 6.1 (40X): TS of thin root –Cortical portion of Pandanus odoratissimus root

Fig 6.2 (40X): TS of thin root-Stele-enlarged of Pandanus odoratissimus root

(AC-air chamber; En-Endodermoid layer ;MX- Metaxylem; Pe- Periderm; Ph-Phloem; PX- Protoxylem; SC-

Sclerenchyma)


65

Fig 7.1 (40X): TS of thick root –entire view of Pandanus odoratissimus root

Fig 7.2 (40X): TS of thick root –a sector enlarged of Pandanus odoratissimus root

(AC-Air chamber; ACo- Aerenchymatous Cortex ;En – Endodermis ; MX- Metaxylem; Pe- Periderm; PF –

Partition filament; Ph-Phloem; PX- Protoxylem; Sc-Sclerenchyma strands; St-Stele)


66

Fig 8.1 (10X): TS of old root –Periderm and cortex of Pandanus odoratissimus root

Fig 8.2 (40X): TS of old root-Stele of Pandanus odoratissimus root

(AC-Air Chamber; ICo-inner Cortex; En – Endodermis ; MX- Metaxylem; Pe- Periderm; ; PX- Protoxylem;

OCo-Outer Cortex ;SC-Sclerenchyma)


67

Fig 9.1 (40X): TS of thick root – secondary xylem of Pandanus odoratissimus root

(MX- Metaxylem; Ph-Phloem; PX- Protoxylem; XF-Xylem Fibres)


68

Fig 10.1 (10X): Fibres of Pandanus odoratissimus root powder

Fig 10.2 (10X): Fibres and vessels of Pandanus odoratissimus root powder

Fig 10.3 (40X): A single vessel element of Pandanus odoratissimus root powder

(NFi- Narrow fibre; VE- Vessel element; WFi- Wide Fibre; XE- Xylem Element)


69

Physiochemical evaluation

For physiochemical evaluation, Bambusa vulgaris leaf and Pandanus odoratissimus root were

used for analysis for physiochemical parameters such as ash values, extractive values and

moisture content.

The total ash determination method is designed to measure the total amount of material

remaining after ignition. This includes both “physiological ash”, which is derived from the plant

tissue itself, and “non physiological ash”, which is the residue of extraneous matter adhering to

the plant surface.

The physiochemical parameters of plant materials viz., ash values (total ash, water soluble ash,

acid insoluble ash, and sulphated ash) were performed as per WHO bulletin, 2002 and recorded

in table.

Determination of Ash values

The ash values was determined as per standard procedure for Bambusa vulgaris leaf and

Pandanus odoratissimus root material and expressed interms of percentage found to be 4.14

(total ash), 0.76 (water soluble ash), 0.84 (acid insoluble ash), 3.30( acid soluble ash), 1.38

(sulphated ash) for Bambusa vulgaris leaf. Similarly ash values were also determined for

Pandanus odoratissimus root material and found to be higher than Bambusa vulgaris leaf viz.,

9.51(total ash), 1.23 (water soluble ash), 6.19 (acid insoluble ash), 3.30(acid soluble ash), 2.57

(sulphated ash).


70

Table 1: Ash values

Sample

Ash value %

Total

Ash

Water

soluble ash

Acid

insoluble ash

Acid

soluble ash

Sulphated

ash

Bambusa vulgaris

leaves

4.14 0.76 0.84 3.30 1.38

Pandanus

odoratissimus root 9.51 1.23 6.19 3.30 2.57

Determination of Extractive values

Both Bambusa vulgaris leaf and Pandanus odoratissimus root material was studied for extractive

value and found to be nearly equal. The alcohol soluble extractives for Bambusa vulgaris leaf

and Pandanus odoratissimus root found to be 8 and 10 % respectively. Similarly the water

soluble extractives found to be 11.1 and 12 % respectively (Table 2).

Table 2: Extractive value

Plant material Extractive value in %

Alcohol Water

Bambusa vulgaris 8% 11.1

Pandanus odoratissimus 10% 12


71

Determination of Moisture Content or Loss on Drying

The moisture content was found to be 9 and 7 % for Bambusa vulgaris leaf and Pandanus

odoratissimus root material respectively (Table 3).

Table 3: Moisture content

Plant material Moisture content in %

Bambusa vulgaris 9

Pandanus odoratissimus 7

PHYTOCHEMISTRY

Preparation of Extract

For phytochemical study the dry leaf powder of Bambusa vulgaris and root powder of Pandanus

odoratissimus extracted with different solvents was dried under reduced pressure and the average

extractive value was found to be 1.4% (hexane), 0.6% (benzene), 3% (chloroform), 1.4% (ethyl

acetate) and 6 % (methanol) for Bambusa vulgaris and 2 % (hexane), 1.8% (benzene), 3.5% (

chloroform), 2 % (ethyl acetate) and 8 % (methanol) for Pandanus odoratissimus.

Qualitative Phytochemical Screening

The qualitative chemical tests were performed on the both plant extracts to detect the various

phytoconstituents present in them as per the standard procedures and findings were recorded.

The qualitative chemical tests on the methanolic extract of Bambusa vulgaris leaves revealed the

presence of carbohydrates, glycosides, saponins, alkaloids, flavonoids, phenolics and tannins,

phytosterols and triterpenoids, fixed oils and fats, where as remaining extracts showed the


72

presence for Phytosterols and Triterpenoids, methanol and chloroform extracts showed the

presence for carbohydrates (Table 4).

Similar results were observed for the methanolic extract of Pandanus odoratissimus showed the

presence of carbohydrates, saponins, alkaloids, phenolics and tannins, phytosterols and

triterpenoids in methanolic extract. Whereas remaining extracts showed the presence for

phytosterols and triterpenoids, only methanol and chloroform extracts showed the presence for

carbohydrates (Table 5).

Table 4: Phytochemical analysis of Bambusa vulgaris leaf extracts

Sl/No Test M C E B H

1 Test for carbohydrates

a. Molisch’s test

+ + + - -

2 Test for Glycosides

b. Modified Borntrager’s test

c. Keller-Killiani test

+

+

-

-

-

-

-

-

-

-

3 Test for Saponins

a. Foam test

+ - - - -

4 Test for Alkaloids

a. Mayer’s test

b. Dragendrodroff’s test

+

+

-

-

-

-

-

-

-

-

5 Test for Flavonoids

a. Alkaline reagent test

+ + - - -


73

6 Test for Phenolics and Tannins

a. Ferric chloride test

b. Test for Tannins

-

+

-

-

-

-

-

-

-

-

7 Test for Phytosterols and Triterpenoids

a. Leiberman-Bucharat test

b. Salkowaski test

+

-

+

+

+

+

+

+

+

+

8 Test for fixed oils and fats

a. Oily spot test

+ - - - +

(+) Present, (-) Absent

M= Methanol extract, C= Chloroform, E=Ethyl acetate, B=Benzene, H=Hexane


74

Table 5: Phytochemical analysis of Pandanus odoratissimus root extracts

Sl.No Test M C E B H

1

Test for carbohydrates

a. Molisch’s test

+ + - - -

2

Test for Glycosides

a. Modified Borntrager’s test

b. Keller-Killiani test

-

-

-

-

-

-

-

-

-

-

3

Test for Saponins

a. Foam test

+

-

-

-

-

4

Test for Alkaloids

b. Mayer’s test

c. Dragendrodroff’s test

+

+

-

-

-

-

-

-

-

-

5

Test for Flavonoids

a. Alkaline reagent test

-

+

-

-

-

6

Test for Phenolics and Tannins

a. Ferric chloride test

b. Test for Tannins

-

+

-

-

-

-

-

-

-

-

7

Test for Phytosterols and

Triterpenoids

a. Leiberman-Bucharat test

+

+

+

+

+


75

b. Salkowaski test - + + + +

8

Test for fixed oils and fats

a. Oily spot test

-

-

-

-

-

(+) Present, (-) Absent

M= Methanol extract, C= Chloroform, E=Ethyl acetate, B=Benzene, H=Hexane

Estimation of Total phenol content

Both methanolic extracts were estimated for the total phenol content by Folin – ciocalteu

method. The total Phenolic content was found to be in Methanolic extract of Bambusa vulgaris

leaves 4.4±0.81 and Pandasmus odorantismus root 4.8±0.17 gallic acid equivalent in mg/g of the

dried extract.

Isolation and characterization of phytoconstituent from methanolic extracts of Bambusa

vulgaris leaves

The leaf of Bambusa vulgaris was extracted with methanol, concentrated under reduced pressure.

The methanol extract was then subjected for column chromatography with chloroform and

methanol solvent combinations with silica gel of 60-120 mesh size to afford 55 fractions. From

the fraction no 23, a single compound S2 was obtained, later by spectral analysis it was found to

be Palmitic acid.


76

Interpretation and observation of S 1 sample:

The compound in its IR spectrum exhibits absorption bands at 3400 cm-1

(broad band,

characteristic of –COOH group), 1712 cm-1

for a carbonyl group, 1455, 1374 and 722 cm-1

suggesting the presence of methylene groups.

In its 1H-NMR spectrum shows a triplet at δ 0.84 for three protons due to a terminal methyl

group adjacent to a methylene group. The multiplet signal at δ 1.86 shows the presence of a

methylene group adjacent to a carbonyl group. A strong singlets at δ 1.29 is due to the presence

of long chain methylene groups in the compound.

13C-NMR spectrum exhibits a signal at δ 12.08 for a methyl group and the signal at δ 29.00 due

to long chain methylene groups which confirms the assignment made in the 1H-NMR spectrum.

The negative mode APCI-MS indicates the molecular weight to be 256 by exhibiting a signal at

m/z 255 for [M-H]-1

ion. Based on the above data the structure of the compound may be of a

saturated fatty acid i.e. Palmitic acid

Physical properties and other details of isolated compound

1. S2:

Solubility: Chloroform

Nature: Dark green powder

Rf value: 0.75 (Chloroform: methanol: 9.5:0.5)

Detection: UV Long wavelength: Fluorescent Yellowish orange.


77

Spectral analysis:

1) S2:

1H NMR of S2:


78


79

C13

NMR of S 2:


80

Mass of S2:


81

IR of S2

E:\

STA

FF

\OU

TS

IDE

SA

MP

LE

\S-2

.0

S-2

In

str

um

ent

type a

nd /

or

accessory

22/0

2/2

011

3956.67

3903.70

3811.48

3767.18

3382.57

3332.56

3100.15

2920.24

2854.06

2344.00

2112.38

2052.99

1712.07

1456.72

1374.01

1240.07

1162.30

1088.24

1047.67

908.42

781.04

722.98

676.38

622.87

1000

1500

2000

2500

3000

3500

Wavenum

ber

cm

-1

889092949698100

Transmittance [%]

P

ag

e 1

/1


82

Pandanus odoratissimus root material bioactivity guided identification for antidiabetic

compound or an active fraction

The dried roots of Pandanus odoratissimus was extracted extracted with methnol to obtain

brown waxy material upon evaporation was subjected for purification by column

chromatography with 60-120 mesh size silica gel to afford 6 different fraction eluted with

Hexane, chloroform and methanol solvent combinations. All six fractions were studied for

Glucose uptake study by L 6 cell lines. From the Glucose uptake study it was found out that the

Methanol fraction showed good antidiabetic activity followed by Chloroform: Methanol: 30: 70

fractions with % glucose uptake of 15.54 and 12.42 over the control. So the Methanol fraction

was further preceded for purification by preparative TLC. After separation by TLC a brown band

with mobile phase Hexane: Chloroform: Methanol: 2:4:1 was scrapped off and dissolved with

methanol to obtain FBR which later upon spectral analysis it was found out to be Heptadecanoic

acid ethyl ester.

Interpretation and observation of FBR sample

The Compound in its ESI-MS (positive mode) spectrum exhibits a peak at m/z 322 for an ion

[M+Na] +

suggesting a molecular weight of 299.

In its 1H-NMR spectrum it showed peaks at δ 0.85 showing the presence of methyl groups in the

compound. The large singlet at δ 1.2 and the signals at δ 1.80 were due to the long chain

methylene groups. The signal at δ 1.99 is due to a methylene adjacent to a carbonyl group. The

signal at δ 4.00 may be due to the protons attached to oxygen function.


83

In the 13

C-NMR the signals at δ 20.00 is due to methyl group, at δ 28.00 to 31.00 are due to the

methylene carbons. The signal at δ 70.00 is due to the carbon attached to the oxygen function.

The signal at δ 170.00 confirms the presence of a carbonyl group.

Based on the above data the structure of the compound may be Heptadecanoic acid ethyl ester

Physical properties and other details of isolated compound

1. FBR

Solubility: Methanol

Nature: Yellowishbrown powder

Rf value: 0.75 (Chloroform: methanol: 9.5:0.5)

Detection: UV Long wavelength: Brown


84

Spectral analysis:

1) FBR:

1H NMR of FBR:


85


86

C13

NMR of FBR:


87

Mass of FBR:


88

IR of S2


89

BIOLOGICAL STUDY

ACUTE TOXICITY STUDIES

All animals were survived in step I and step II until the end of the experimental period. All the

animals dosed at 2000 mg/kg body weight did not show evident toxicity throughout the

experimental period (Table 7 and 9). The animals which were survived throughout the

experiment increased their body weight by day 14 as compared to day 0 (Table 7 and 9). No

abnormalities were detected for all the animals at necropsy. Based on the results, the median

lethal doses (LD50) of Bambusa vulgaris and Pandanus odoratissimus were greater than

2000mg/kg body weight and are classified as category 4.


90

Table 6: Body weight analysis of test drug treated rats

Sl.

No

Test drug Group

Dose

(mg/kg

bw)

Animal

Numbers

Sex

Test day 0

(treatment)

(g)

Test day

7

(g)

Test day

14

(g)

1

Bambusa

vulgaris

I 2000

R1 Female 220.23 241.22 252.38

R2 Female 221.24 241.89 252.13

R3 Female 202.28 240.92 251.68

II 2000

R4 Female 222.41 243.11 263.61

R5 Female 223.12 243.68 253.84

R6 Female 222.51 243.00 253.38

2 Pandanus

odoratissimus

I 2000

R7 Female 211.83 222.95 254.06

R8 Female 206.12 231.41 261.16

R9 Female 205.04 231.78 260.85

II 2000

R10 Female 212.12 240.54 255.72

R11 Female 211.35 231.35 253.24

R12 Female 201.54 231.54 252.63

mg/kg = miligram/kilogram, g = gram


91

Sl.

No Test drug Group

Dose

(mg/kg

bw)

Animal

Number

Sex

Mode of

death

Macroscopic

findings

1

Bambusa

vulgaris

I 2000

R1 Female

Terminal

Sacrifice

No abnormalities

Detected

R2 Female

Terminal

Sacrifice

No abnormalities

Detected

R3 Female

Terminal

Sacrifice

No abnormalities

Detected

II 2000

R4 Female

Terminal

Sacrifice

No abnormalities

Detected

R5 Female

Terminal

Sacrifice

No abnormalities

Detected

R6 Female

Terminal

Sacrifice

No abnormalities

Detected

2

Pandanus

odoratissimu

s

I 2000

R7 Female

Terminal

Sacrifice

No abnormalities

Detected

R8 Female

Terminal

Sacrifice

No abnormalities

Detected

R9 Female

Terminal

Sacrifice

No abnormalities

Detected

Table 7: Macroscopic findings of animals from test drug treated groups.


92

mg/kg = miligram/kilogram bw = body weight

II

2000

R10 Female

Terminal

Sacrifice

No abnormalities

Detected

R11 Female

Terminal

Sacrifice

No abnormalities

Detected

R12 Female

Terminal

Sacrifice

No abnormalities

Detected


93

Table 8: Body weight analysis of test drug treated mice

Sl.

No

Test drug Group

Dose

(mg/kg

bw)

Animal

Numbers

Sex

Test day 0

(treatment)

(g)

Test day

7

(g)

Test day

14

(g)

1

Bambusa

vulgaris

I 2000

M1 Female 220.23 241.22 252.38

M2 Female 221.24 241.89 252.13

M3 Female 202.28 240.92 251.68

II 2000

M4 Female 222.41 243.11 263.61

M5 Female 223.12 243.68 253.84

M6 Female 222.51 243.00 253.38

2 Pandanus

odoratissimus

I 2000

M7 Female 211.83 222.95 254.06

M8 Female 206.12 231.41 261.16

M9 Female 205.04 231.78 260.85

II 2000

M10 Female 212.12 240.54 255.72

M11 Female 211.35 231.35 253.24

M12 Female 201.54 231.54 252.63

mg/kg = miligram/kilogram, g = gram


94

Table 9: Macroscopic findings of animals from test drug treated groups.

Sl.

No Test drug Group

Dose

(mg/kg

bw)

Animal

Number Sex Mode of death

Macroscopic

findings

1 Bambusa

vulgaris

I 2000

M1 Female

Terminal

Sacrifice No abnormalities

Detected

M2 Female Terminal

Sacrifice

No abnormalities

Detected

M3 Female Terminal

Sacrifice

No abnormalities

Detected

II 2000

M4 Female Terminal

Sacrifice

No abnormalities

Detected

M5 Female Terminal

Sacrifice

No abnormalities

Detected

M6 Female Terminal

Sacrifice

No abnormalities

Detected

2 Pandanus

odoratissimus

I 2000

M7 Female

Terminal

Sacrifice No abnormalities

Detected

M8 Female Terminal

Sacrifice

No abnormalities

Detected

M9 Female Terminal

Sacrifice

No abnormalities

Detected

II 2000

M10 Female Terminal

Sacrifice

No abnormalities

Detected

M11 Female Terminal

Sacrifice

No abnormalities

Detected

M12 Female Terminal

Sacrifice

No abnormalities

Detected

mg/kg = miligram/kilogram bw = body weight


95

ANTIPYRETIC STUDIES

The experimental rats showed a marked increase in rectal temperature 18 h after the Brewer’s

yeast injection. In the study it is observed that methonolic of Bambusa vulgaris exerted their

antipyretic effect with varied efficacy. Bambusa vulgaris at 1000 mg/ kg b wt. exhibited

significant antipyretic activity (Table no 10 and Fig 11). Both the test doses of Bambusa vulgaris

caused reduction in temperature from 2 h onwards and by the end of 5th

hour temperatures of

both the groups brought down to normal. In case of Paracetamol treated group, significant

decrease towards normal in body temperature was observed from the 1 h onwards and normal

body temperature was maintained then on. Where as in pyrexia control group, the elevated body

temperature maintained throughout the study with marginal reduction at the end of the study.


96

Table 10: Anti pyretic effect of Methanol extract of Bambusa vulgaris on Brewer’s yeast-induced pyrexia in rats

Values are mean ± S.E.M. n= 6 animals in each group. values are significantly different from paracetamol intoxicated group.

ns; p*<0.05; p

**<0.01; p

***<0.001. (ANOVA, followed by Dunnett’s test). a: temperature just before yeast injection; b:

temperature just before drug administration; MEBV: Methanol extract of Bambusa vulgaris

Groups Treatment Dose

Rectal temperature 0c at time (hr)

-18a 0

b 1 2 3 4 5 6

Group I

2% w/v

acacia

5

ml/kg

37.07 ±

0.05

38.27 ±

0.12

38.22 ±

0.07

38.08 ±

0.06

38.02 ±

0.04

38.05 ±

0.02

38.03 ±

0.03

38.02 ±

0.07

Group II

Methanol

extract

500

mg/kg

37.07 ±

0.04

38.20 ±

0.08ns

38.35 ±

0.07ns

38.0 ±

0.04ns

37.80 ±

0.04*

37.82 ±

0.03***

37.38 ±

0.04***

37.18 ±

0.04***

Group III

Methanol

extract

1000

mg/kg

37.15 ±

0.05

38.40 ±

0.09ns

38.35 ±

0.06ns

37.82 ±

0.03**

37.47 ±

0.03***

37.30 ±

0.03***

37.13 ±

0.04***

37.12 ±

0.03***

Group IV Paracetamol 150

mg/kg

37.15 ±

0.06

38.32 ±

0.04ns

37.92 ±

0.01*

37.87 ±

0.02**

37.45 ±

0.03***

37.30 ±

0.02***

37.22 ±

0.03***

37.05 ±

0.02***


97

Fig 11: Anti pyretic effect of Methanol extract of Bambusa vulgaris on Brewer’s yeast induced pyrexia in rats


98

VENOM NEUTRALIZING ACTIVITY

From the studies, LD50 of snake venom was established at 60 µg/mouse (20 g body weight) i.p.

For the determination of venom neutralizing effect of extracts LD50 was employed as lethal test

dose. Whereas based on the observations from acute toxicity studies, test doses 1000, 750, 500

and 250 mg/ kg b. wt of methanolic extract of Pandanus odoratissimus were selected and studied

for their venom neutralizing potency. The methanolic extract exhibited venom neutralizing effect

in dose-dependant manner (Table 11). The methanolic extract exhibited significant venom

antagonistic effect at the higher test dose i.e at 1000 mg/kg b. wt. by exhibiting the percent

increase in survival rate by 75 % (Figure 12). Whereas, at 750 mg/ kg b. wt dose it showed

moderate activity with 25 % increase in survival of animals. At the lower doses, methanolic

extract failed to offer protection to the animals. Treatment with standard snake venom antiserum

has protected all the animals from the lethal effects of venom


99

Table 11: Effect of the methanol extract of Pandanus odoratissimusi on the lethality of

snake venom.

Sl.

No

Groups

(n=8)

Treatment % Survival

% Increase

in survival

rate

1 I Normal control 100.00 -

2 II Venom control 50.00 -

3 III

Postive control

(Snake venom antiserum)

100.00 100.00

4 IV

Methanolic extract

(250 mg/kg b.wt)

50.00 0.00

5 V

Methanolic extract

(500 mg/kg b. wt)

50.00 0.00

6 VI

Methanolic extract

(750 mg/kg b. wt)

62.50 25.00

7 VII

Methanolic extract

(1000 mg/kg b. wt)

87.50 75.00


100

Fig 12: Effect of the methanol extract of Pandanus odoratissimusi on the lethality of snake

venom.


101

ANTI-DIABETIC STUDIES

Different extracts of Bambusa vulgaris and Pandanus odoratissimus were investigated for their

glucose uptake enhancing properties in vitro in L-6 cell line. Methanolic extracts of both the plants

enhanced the glucose uptake in L-6 cells over control. Among the extracts, methanolic extracts of

Bambusa vulgaris and Pandanus odoratissimus exhibited better glucose uptake enhancement

properties with 13.50 ± 3.10 and 28.99 ± 3.56 percent over control (Table 13, fig.13 and 14).

Standard drugs, Insulin and Metformin enhanced the glucose uptake significantly. Glucose

uptake studies in isolated rat hemi diaphragm also revealed the similar results. Methanolic

extracts of Bambusa vulgaris and Pandanus odoratissimus enhanced the glucose uptake by 11.25

± 1.35 and 19.86 ± 1.86 percent over control (Table 14, fig. 15 and 16). Whereas, other extracts

enhanced glucose uptake moderately with values ranging between 5.45 to 9.80 percent over

control.

In vivo studies with methanolic extracts of Bambusa vulgaris and Pandanus odoratissimus against

Streptozotocin (STZ) induced diabetic rat model. It was observed that, in STZ-induced diabetic rats

a significant decrease in body weight gain when compared to controls. However, diabetic rats

treated with Bambusa vulgaris and Pandanus odoratissimus showed augmented body weight

when compared with STZ alone treated rats (Table 15). The blood glucose increased in STZ-

diabetic rats as compared to normal rats (fig.17). However, treatment of STZ-diabetic rats with

Pandanus odoratissimus significantly reduced the hyperglycemia when compared with STZ alone

treated rats (Table 15, fig.18). Whereas, Bambusa vulgaris was found to be moderately active at

its higher test dose i.e 1000 mg/ kg. HbAIC levels were higher in the STZ-induced diabetic rats

compared to the control rats (fig.19). The supplementation of 1000 mg/kg of Pandanus


102

odoratissimus decreased the HbAIC level of the STZ induced diabetic rats. In STZ-diabetic rats

the activities of serum CK and LDH were significantly increased (p < 0.05). The administration

of 1000mg/kg dose of Pandanus odoratissimus to STZ-diabetics rats decreased the activity of

LDH significantly, when compared other test groups (fig.21). However, the serum CK did not

return to the basal level compared to STZ controls (Table 15).

STZ treatment elevated the levels of serum cholesterol, triglycerides, LDL, creatinine, urea, and

ALP. It was observed that the treatment with Pandanus odoratissimus had brought down the test

parameters towards normal levels in dose dependant manner. Whereas, Bambusa vulgaris failed

to bring down these parameters towards normal (Table 16, fig.22-28). HDL levels were poorly

elevated by Pandanus odoratissimus and Bambusa vulgaris treatment, when compared to STZ

treated control group.

Based on these in vivo results, methanolic extract of Pandanus odoratissimus was processed

further to identify the phytoconstituents present in it. Fractions from the methanolic extract of

Pandanus odoratissimus were further studied in vitro to determine their glucose uptake enhancement

properties. It was observed that, methanol (100%) fraction exhibited significant enhancement in

glucose uptake in L-6 cells with 38.55 ± 4.32 percent over control (Table 17 and Fig 30). Other

fractions, Chloroform:Methanol (50:50) and Chloroform:Methanol (30:70) also enhanced the glucose

uptake by 23.44 ± 2.49 and 24.84 ± 2.95 percent over control.


103

Table no 12: Cytotoxic study of Bambusa vulgaris leaf extracts against L6 cell line by

MTT assay

Sl. No Plant

Name of

Extract

CTC50

( µg/ml)

1

Bambusa vulgaris

Hexane >1000

2 Benzene >1000

3 Ethyl acetate 493.16 ± 18.46

4 Chloroform 513.00 ± 16.85

5 Methanol 456.00 ± 10.56

6

Pandanus

odoratissimus

Hexane >1000

7 Benzene 856.75 ± 24.50

8 Ethyl acetate 786.00 ± 19.00

9 Chloroform 650.50 ± 23.50

10 Methanol 510.75 ± 17.00


104

Table 13: In vitro glucose uptake studies in L-6 cell line

Sl.No Name of the extract

Test concentration

(µg/ml)

% glucose uptake

over control

Bambusa vulgaris extracts

1. Hexane 200 3.97 ± 1.03

2. Benzene 200 8.45 ± 1.35

3. Ethyl acetate 200 5.50 ± 0.75

4. Chloroform 200 7.56 ± 2.35

5. Methanol 200 13.50 ± 3.10

Pandanus odoratissimusi extracts

6. Hexane 200 8.57 ± 2.65

7. Benzene 200 7.30 ± 1.85

8. Ethyl acetate 200 8.45 ± 2.54

9. Chloroform 200 11.00 ± 3.76

10. Methanol 200 28.99 ± 3.56

Standard drugs

11. Insulin 1 IU/ml 131.50 ± 17.62

12. Metformin 100 68.35 ± 11.45


105

Fig 13: In vitro glucose uptake effect of extracts of Bambusa vulgaris in L-6 cell line

Fig 14: In vitro glucose uptake effect of extracts of Pandanus odoratissimusi in L-6 cell line


106

Table 14: In situ glucose uptake studies in rat hemi diaphragm

Sl.No Name of the extract

Test concentration

(µg/ml)

% glucose uptake

over control ± SE

Bambusa vulgaris extracts

1. Hexane 200 6.59 ± 0.35

2. Benzene 200 7.95 ± 1.10

3. Ethyl acetate 200 5.75 ± 0.64

4. Chloroform 200 9.10 ± 1.56

5. Methanol 200 11.25 ± 1.35

Pandanus odoratissimusi extracts

6. Hexane 200 5.45 ± 0.95

7. Benzene 200 7.95 ± 2.10

8. Ethyl acetate 200 7.56 ± 1.85

9. Chloroform 200 9.80 ± 2.36

10. Methanol 200 19.86 ± 1.86

Standard drugs

11. Insulin 1 IU/ml 23.45 ± 4.13


107

Fig 15: Glucose uptake effect of extracts of Bambusa vulgaris in rat hemi diaphragm

Fig 16: Glucose uptake effect of extracts of Pandanus odoratissimusi in rat hemi

diaphragm


108

Table 15: In vivo antidiabetic activity of methanolic extracts of Bambusa vulgaris leaf and Pandanus odoratissimus root.

Group Treatment Body weight

(g)

Blood glucose

(mg/dl ) HbA1C (%) CK (IU/L) LDH (IU/L)

Group 1 Control 284.67 ± 12.45 115.00 ± 4.78 1.43 ± 0.16 204.48 ± 18.15 91.17 ± 5.47

Group 2 STZ treated

control 155.50 ± 16.00

a 351.17 ± 5.71

a 4.29 ± 0.84

a 284.57 ± 74.23

a 161.67 ± 16.42

a

Group 3 STZ + BV

(500 mg/kg)

157.33 ± 6.68 304.83 ± 21.41 b 4.27 ± 0.54 272.28 ± 50.27 129.42 ± 13.27

b

Group 4 STZ + BV

(1000 mg/kg)

189.17 ± 8.93b

179.00 ± 8.03 b

3.68 ± 0.58 271.90 ± 28.07 127.28 ± 13.72 b

Group 5 STZ + PO

(500 mg/kg)

220.00 ± 10.51b 191.00 ± 6.13

b 3.03 ± 0.29

b 275.11 ± 30.62 115.1 ± 27.04

b

Group 6 STZ + PO

(1000 mg/kg)

244.50 ± 11.79 b 146.5 ± 10.08

b 2.59 ± 0.67

b 239.17 ± 23.86 101.84 ± 21.3

b

Group 7 STZ + GLI

(25 mg/kg)

240.5 ± 14.52 b

146.50 ± 12.08 b 2.91 ± 0.69

b 262.35 ± 41.58 131.10 ± 25.90

BV: Bambusa vulgaris, PO: Pandanus odoratissimus

p values <0.05, a - when compared with a normal control; b – when compared with STZ treated


109

Fig 17: Effect of methanolic extracts on body weight in diabetic rats

Fig 18: Effect of methanolic extracts on blood glucose levels in diabetic rats


110

Fig 19: Effect of methanolic extracts on serum HbA1c levels in diabetic rats

Fig 20: Effect of methanolic extracts on serum CK levels in diabetic rats


111

Fig 21: Effect of methanolic extracts on serum LDH levels in diabetic rats


112

Table 16: Effect of Bambusa vulgaris leaf and Pandanus odoratissimus root on STZ induced changes on the serum biochemical

parameters

Treatment Cholestrol TG HDL LDL Creatineine Urea ALP

Group 1 Control 152.5 ± 3.0 82 ± 3.4 37 ± 1.7 94.3 ± 3.0 0.49 ± 0.02 23.0 ± 2.1 115.6 ± 3.3

Group 2 STZ treated

control 269.5 ± 3.7

a 191.8 ± 4.9

a 30 ± 2.6

a 187 ± 7.0

1.40 ± 0.04

a 59.3 ± 4.8

a 317.1 ± 6.1

a

Group 3 STZ + BV

(500 mg/kg) 214.6 ± 17

b 184.1 ± 8.8 30 ± 1.5 161.8 ± 5 1.14 ± 0.11

b 46.8 ± 3.1

b 270.5 ± 14

b

Group 4 STZ + BV

(1000 mg/kg) 203.0 ± 7.8

b 177 ± 11 27.3 ± 2.1 154.8 ± 5 1.12 ± 0.11

b 39 ± 4.2

b 224.8 ± 8

b

Group 5 STZ + PO

(500 mg/kg) 169 ± 16.2 138.50 ± 1

b 30.50 ± 1.8 131.6 ± 8

b 0.74 ± 0.11

b 31.0 ± 1

b 202.8 ± 21

Group 6 STZ + PO

(1000 mg/kg) 171.3 ± 10 135.17 ± 9

b 29.50 ± 1.7 114 ± 8

b 0.69 ± 0.07

b 33.6 ± 3

b 170.5 ± 15

Group 7 STZ + Gli (25

mg/kg) 158.0 ± 7.1

b 172.1 ± 11

b 50.3 ± 2.3

b 78.1 ± 6

0.60 ± 0.05

b 34.2 ± 2.4

b 126.8 ± 4.3

b

BV: Bambusa vulgaris, PO: Pandanus odoratissimus

p values <0.05, a - when compared with a normal control; b – when compared with STZ treated


113

Fig 22: Effect of extracts on serum cholesterol levels in diabetic rats.

Fig 23: Effect of extracts on serum triglycerides levels in diabetic rats


114

Fig 24: Effect of extracts on serum HDL levels in diabetic rats

Fig 25: Effect of extracts on serum LDL levels in diabetic rats


115

Fig 26: Effect of extracts on serum creatinine levels in diabetic rats

Fig 27: Effect of extracts on urea levels in diabetic rats


116

Fig 28: Effect of extracts on serum Alkaline phosphotase levels in diabetic rats


117

Table no 17: In vitro glucose uptake activities of fractions from methanolic extract of

Pandanus odoratissimus root against L6 cell line

Sl.No Fraction name CTC50 in µg/ml

Test

concentration

(µg/ml)

% glucose

uptake over

control

1 Chloroform (100%) 712.65 ± 23.50 200 10.76 ± 1.45

2 Chloroform: Methanol:

(70:30)

678.15 ± 12.56 200 9.04 ± 0.75


(50:50)

524.50 ± 8.50 200 23.44 ± 2.49


(30:70)

576.00 ± 14.50 200 24.84 ± 2.95

5 Methanol (100%) 459.00 ± 11.50

200 38.85 ± 4.32

6 Insulin - 1 IU/ml 98.76 ± 11.30

Fig.29: Histopathology


118

Fig 30: In vitro glucose uptake effect of fractions from methanolic extract of

Pandanus odoratissimus in L-6 cell line

Discussion

119

CHAPTER-6

DISCUSSION

After decades of serious obsession with the modern medicinal system, people have started

looking at the ancient healing systems; however a key obstacle, which has hindered the

acceptance of the alternative medicines in the developed countries, is the lack of documentation

and stringent quality control. There is a need for documentation of research work carried out on

traditional medicines 69

. With this backdrop, it becomes extremely important to make an effort

towards standardization of the plant material to be used as medicine. The process of

standardization can be achieved by stepwise pharmacognostic studies 70

.These studies help in

identification and authentication of the plant material. Correct identification and quality

assurance of the starting materials is an essential prerequisite to ensure reproducible quality of

herbal medicine which will contribute to its safety and efficacy. Simple pharmacognostic

techniques used in standardization of plant material include its morphological, anatomical and

biochemical characteristics 71

.

Two traditional medicinal plants Bambusa vulgaris and Pandanus odoratissimus were selected

based on their traditional use in treating different ailments. Bambusa vulgaris has been

mentioned as feberifuge and its effectiveness against jaundice, measules etc. Particullarly in

India, it has been used in treating wounds, inflammations etc. Pandanus odoratissimus was

mentioned for its traditional uses in treating snakebite, skin disorders, lepracy, urinary disorders

jaundice etc. Pandanus odoratissimus was reported to have potent antioxidant properties.

With an objective to evaluate and confirm the biological efficacy, leaves of Bambusa vulgaris

and root of Pandanus odoratissimus were selected and different extracts were prepared, studied

Discussion

120

for their biological activities. Based on the traditional claims, initially Bambusa vulgaris was

tested for its antipyretic activity against brewer yeast induced pyrexia. Methanolic extract of

Bambusa vulgaris exhibited significant antipyretic activity. It has reduced the body temperature

from 2 h onwards and by 5 h the body temperature was normal.

Methanolic extract of Pandanus odoratissimus was tested for venom neutralizing potential

against snake venom. It was observed that at 750 mg/kg dose, the extract offered 75 percent

protection against lethal effect of snake venom. These observations confirmed the earlier

traditional claims on Pandanus odoratissimus as an anti-dote. The methanolic extract of

Pandanus odoratissimus exhibited dose dependant effect in protecting the animal from the lethal

effects of venom.

In anti-diabetic studies, different extracts of Bambusa vulgaris and Pandanus odoratissimus were

initially screened for their glucose uptake activity in L-6 cells. Methanolic extracts from both the

plants exhibited higher glucose uptake with 13.50 ± 3.10 and 28.99 ± 3.56 percent over control.

Other extracts from both the plants exhibited moderate to poor efficacy. Even the phytochemical

analysis revealed that the methanloic extracts are rich with active phytoconstituents and the

results confirms the same. Based on these in vitro results the methanolic extracts of Bambusa

vulgaris and Pandanus odoratissimus were studied for their antidiabetic activity in STZ induced

diabetic rat models. Both the extracts exhibited dose dependant activity and Pandanus

odoratissimus was found to be potent among the two plants. Pandanus odoratissimus at

1000mg/kg dose has regulated the diabetic parameters like, body weight of diabetic rats, blood

glucose, HbA1c and LDH. Even the biochemical analysis on lipid profile of diabetic rats

Discussion

121

revealed that Pandanus odoratissimus at 1000mg/kg dose restored all the parameters towards

normal.

From the pharmacological studies it was understood that the methanolic extracts of Bambusa

vulgaris and Pandanus odoratissimus were active. Both of these extracts were further processed

to identify the active principles responsible for their activity. Suitable techniques like

fractionization, column chromatography and analytical techniques were employed to isolate and

identify the phytoconstituents responsible for the activities.

From Bambusa vulgaris we were able to isolate the pure compound Palmitic acid. Simulatiously,

methanolic extract of Pandanus odoratissimus was further fractionated and glucose uptake

potential of those fractions were evaluated. Among the fractions, methanolic fraction (100%)

was found to have better glucose uptake potential with 38.85 ± 4.32 percent over control.

Further, methanolic fraction (100%) was processed through column chromatography to isolate

pure compound Heptadecanoic acid ethyl ester.

Present study revealed the pharmacological properties of both the plants. The method for

selection of plants based on traditional usage is once again proved to be beneficial in selection of

medicinal plants for pharmacological evaluation. From the present studies it has been understood

that Bambusa vulgaris has got potent antipyretic properties and phytochemical analaysis

followed by isolation led to the isolation of a compound Palmitic acid. Methanolic extract of

Pandanus odoratissimus exhibited potent venom neutralizing and anti-diabetic properties. As

discussed earlier, Pandanus odoratissimus is mentioned as anti-dote and present study confirms

Discussion

122

the same and it is a good finding from the present study. These results will help in developing a

product for treating the lethal effects of venom. Same time, Pandanus odoratissimus exhibited

potent anti-diabetic properties. So, further studies and phytochemical investigations are required

to understand any other phytochemical behind these biological activities.

Summary and Conclusion

123

CHAPTER-7

SUMMARY AND CONCLUSION

Despite the recent interest in molecular modeling, combinatorial chemistry, and other synthetic

chemistry techniques by pharmaceutical companies and funding organizations, natural products,

particularly medicinal plants, remains an important source of new drugs, new drug leads, and

new chemical entities. It is evident that, natural products have played a vital role in drug

discovery, by contributing a wide variety of phytochemicals for the treatment of cancer,

cardiovascular diseases, infections related with viral and microbial origin and other health

disorders.

After collection and authentication of the plant material, the plant materials were analysed for

pharmacognostic and physiochemical parameters. Both Bambusa vulgaris leaf and Pandanus

odoratissimus root material were studied for extractive value and found to be nearly equal. The

alcohol soluble extractives for Bambusa vulgaris leaf and Pandanus odoratissimus root found to

be 8 and 10 % respectively. Similarly the water soluble extractives found to be 11.1 and 12 %

respectively.

For phytochemical study the dry leaf powder of Bambusa vulgaris and root powder of Pandanus

odoratissimus extracted with different solvents was dried under reduced pressure and the average

extractive value was found to be 1.4% (hexane), 0.6% (benzene), 3% (chloroform), 1.4% (ethyl

acetate) and 6 % (methanol) for Bambusa vulgaris and 2 % (hexane), 1.8% (benzene), 3.5% (

chloroform), 2 % (ethyl acetate) and 8 % (methanol) for Pandanus odoratissimus.


124

The qualitative chemical tests were performed on the both plant extracts to detect the various

phytoconstituents present in them as per the standard procedures and findings were recorded.

The qualitative chemical tests on the methanolic extract of Bambusa vulgaris Schrad leaves

revealed the presence of carbohydrates, glycosides, saponins, alkaloids, flavonoids, phenolics

and tannins, phytosterols and triterpenoids, fixed oils and fats, where as remaining extracts

showed the presence for Phytosterols and Triterpenoids, methanol and chloroform extracts

showed the presence for carbohydrates.

Similar results were observed for the methanolic extract of Pandanus odoratissimus showed the

presence of carbohydrates, saponins, alkaloids, phenolics and tannins, phytosterols and

triterpenoids in methanolic extract. Whereas remaining extracts showed the presence for

phytosterols and triterpenoids, only methanol and chloroform extracts showed the presence for

carbohydrates.

The methanolic extract from the both the plants were selected for in vivo studies for antipyretic,

anti-venom and antidiabetic activities. Prior to in vivo studies, acute toxicity studies were

performed in rats and mice as per standard protocol. In acute toxicity studies, all animals were

survived in step I and step II until the end of the experimental period. All the animals dosed at

2000 mg/kg body weight did not show evident toxicity throughout the experimental period

(Table 7 and 9). The animals which were survived throughout the experiment increased their

body weight by day 14 as compared to day 0 (Table 7 and 9). No abnormalities were detected for

all the animals at necropsy. Based on the results, the median lethal doses (LD50) of Bambusa


125

vulgaris and Pandanus odoratissimus were greater than 2000mg/kg body weight and are

classified as category 4.

The methanolic extract of Bambusa vulgaris was studied for its antipyretic properties. The

experimental rats showed a marked increase in rectal temperature 18 h after the Brewer’s yeast

injection. In the study it is observed that methonolic of Bambusa vulgaris exerted their

antipyretic effect with varied efficacy. Bambusa vulgaris at 1000 mg/ kg b wt. exhibited

significant antipyretic activity (Table no 10 and Fig.11). Both the test doses of Bambusa vulgaris

caused reduction in temperature from 2 h onwards and by the end of 5th

hour temperatures of

both the groups brought down to normal.

The methanolic extract of Pandanus odoratissimus was studied for its venom neutralizing

potential. Test doses 1000, 750, 500 and 250 mg/ kg b. wt of methanolic extract of Pandanus

odoratissimus were selected and studied for their venom neutralizing potency. The methanolic

extract exhibited venom neutralizing effect in dose-dependant manner (Table 11). The

methanolic extract exhibited significant venom antagonistic effect at the higher test dose i.e at

1000 mg/kg b. wt. by exhibiting the percent increase in survival rate by 75 %. Whereas, at 750

mg/ kg b. wt dose it showed moderate activity with 25 % increase in survival of animals. At the

lower doses, methanolic extract failed to offer protection to the animals. Treatment with standard

snake venom antiserum has protected all the animals from the lethal effects of venom.

In anti-diabetic studies, different extracts of Bambusa vulgaris and Pandanus odoratissimus were

investigated for their glucose uptake enhancing properties in vitro in L-6 cell line. Methanolic


126

extracts of both the plants enhanced the glucose uptake in L-6 cells over control. Among the extracts,

methanolic extracts of Bambusa vulgaris and Pandanus odoratissimus exhibited better glucose

uptake enhancement properties with 13.50 ± 3.10 and 28.99 ± 3.56 percent over control. Glucose

uptake studies in isolated rat hemi diaphragm also revealed the similar results. Methanolic

extracts of Bambusa vulgaris and Pandanus odoratissimus enhanced the glucose uptake by 11.25

± 1.35 and 19.86 ± 1.86 percent over control.

In vivo studies with methanolic extracts of Bambusa vulgaris and Pandanus odoratissimus against

Streptozotocin (STZ) induced diabetic rat model. Diabetic rats treated with Bambusa vulgaris and

Pandanus odoratissimus showed augmented body weight when compared with STZ alone treated

rats. The blood glucose increased in STZ-diabetic rats as compared to normal rats. However,

treatment of STZ-diabetic rats with Pandanus odoratissimus significantly reduced the

hyperglycemia when compared with STZ alone treated rats. Whereas, Bambusa vulgaris was

found to be moderately active at its higher test dose i.e 1000 mg/ kg. HbAIC levels were higher

in the STZ-induced diabetic rats compared to the control rats. The supplementation of 1000

mg/kg of Pandanus odoratissimus decreased the HbAIC level of the STZ induced diabetic rats. In

STZ-diabetic rats the activities of serum CK and LDH were significantly increased (p < 0.05).

The administration of 1000mg/kg dose of Pandanus odoratissimus to STZ-diabetics rats

decreased the activity of LDH significantly, when compared other test groups. However, the

serum CK did not return to the basal level compared to STZ controls.

STZ treatment elevated the levels of serum cholesterol, triglycerides, LDL, creatinine, urea, and

ALP. It was observed that the treatment with Pandanus odoratissimus had brought down the test


127

parameters towards normal levels in dose dependant manner. Whereas, Bambusa vulgaris failed

to bring down these parameters towards normal. HDL levels were poorly elevated by Pandanus

odoratissimus and Bambusa vulgaris treatment, when compared to STZ treated control group.

Based on these in vivo results, methanolic extract of Pandanus odoratissimus was processed

further to identify the phytoconstituents present in it. Fractions from the methanolic extract of

Pandanus odoratissimus were further studied in vitro to determine their glucose uptake enhancement

properties. It was observed that, methanol (100%) fraction exhibited significant enhancement in

glucose uptake in L-6 cells with 38.55 ± 4.32 percent over control (Table 17 and Fig 30). Other

fractions, Chloroform:Methanol (50:50) and Chloroform:Methanol (30:70) also enhanced the glucose

uptake by 23.44 ± 2.49 and 24.84 ± 2.95 percent over control.

Based on the pharmacological reports, methanolic extracts of Pandanus odoratissimus and

Bambusa vulgaris were further processed for the isolation of active phytoconstituents. We were

successful in isolating Palmitic acid and Heptadecanoic acid ethyl ester from Bambusa vulgaris

and Pandanus odoratissimus, respectively.

In conclusion, methanolic extracts of Pandanus odoratissimus and Bambusa vulgaris were

evaluated for different biological activities. Methanolic extract of Bambusa vulgaris was found

to have significant antipyretic properties. Whereas, Pandanus odoratissimus exhibited potent

venom neutralizing and antidiabetic properties. Its venom neutralizing activity confirms its

traditional claim as an anti-dote. Whereas, its anti-diabetic activity was observed for the first time

and good finding of present study.

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