INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,

JHANSI

CERTIFICATE

This is to certify that present dissertation work entitled

“ Phytochemical Screening and Pharmacological

investigation on the leaves of Clitoria ternatea” submitted

in fulfillment of the requirements for the award of the degree of

Master of Pharmacy in “Pharmacognosy” of Institute of

Pharmacy, Bundelkhand University, Jhansi, is a bonafide

work carried out by Ram kumar (En. No. B.U./03/B-2039), under

the guidance and supervision of

Dr. Raghuveer Irchhaiya, during the academic session 2008-

2009.

Date: Dr. S.K. Prajapati

Place: Jhansi Head & Reader,Institute of Pharmacy

Bundelkhand University

Jhansi (U.P.)

INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,

JHANSI

CERTIFICATE

This is to certify that present dissertation work entitled

“Phytochemical Screening and Pharmacological

investigation on the the leaves of Clitoria

ternatea”Submitted in fulfillment of the requirements for the

award of the degree of Master of Pharmacy in

“Pharmacognosy” of Institute of Pharmacy, Bundelkhand

University, Jhansi, is a bonafide work carried out by Ram

kumar (En. No. B.U./03/B-2039), the guidance and supervision of

Dr. Raghuveer Irchhaiya, during the academic session 2008-

2009.

Date:

Place: Jhansi Guide

Dr. Raghuveer IrchhaiyaReaderInstitute of PharmacyBundelkhand University, Jhansi, (U.P.)

INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,

JHANSI

DECLARATION

I hereby declare that this dissertation entitled

“Phytochemical Screening and Pharmacological

investigation on the leaves of Clitoria ternatea” is prepared

under the genial guidance and supervision of Dr. Raghuveer

Irchhaiya,Institute of pharmacy, Bundelkhand University, Jhansi,

(U.P.).

The same is submitted to Bundelkhand University, Jhansi in

partial fulfillment of the requirement for the degree of Master of

Pharmacy in Pharmacognosy.

I further declare that I have not submitted this dissertation

previously for award of any degree or Diploma to me.

Date: Ram kumar Place: Jhansi

Acknowledgement

I wish to express my sincere thanks to all those who assisted

with the completion of my dissertation work.

It is a matter pleasure to acknowledge my respected guide

Dr. Raghuveer Irchhaiya. It is because of his priceless

intellectual guidance, constructive ideas and for having given me

complete independence, affectionate encouragement to put my

desire and thought which paved the way for the successful

completion of this work. It is indeed privilege to work under him.

I express my special regards to Dr. S. K. Prajapati,( H.O.D)

Institute of Pharmacy, Bundelkhand University, Jhansi,for furnishing

me all the necessary facilities to carryout this work and for their

ever helping attitude during the course of this work.

I also express my special regards to Reader, Dr. S. K.

Jain,Institute of Pharmacy, Bundelkhand University, Jhansi, for

furnishing me all the necessary facilities to carry out this work.

I want to thank my mother Smt. Kishori Devi, my father

Mr. Tularam Niranjan and my elder sister Smt.Tara Devi and

her husband Mr Kamlesh Patel. my sister Smt.Sarita Devi and

her husband Mr .Keshavdas Patel , my younger sister Smt.

Brajesh Devi and her husband Mr. Manoj Patel and my elder

brother Mr.Vinay Nirajan and his wife Pratima Niranjan and my

fiancée Archana Patel for their inspiration and motivation

throughout my project works. I am also thankful to my nieces and

nephews for their love and cheering me during the project work.

I am also thankful to my village’s friend Dilip, Birju, Chhotu,

Jitendra, Pramod, Mahendra, Rinku, Shivraj, Lakshmikant, Aanand

and Mansingh for collecting the leaves of Clitoria ternatea.

I am thankful to Mr. Sunil Kumar Niranjan (Lecturer), Mr.Ramji

Swarnkar (Lecturer), Mr. Man Singh (Lecturer), Mr.Shashi Alok

(Lecturer), Mr. Prasant Mishra (Lecturer), Mr. Nandlal Singh

(Lecturer), Mr Sailendra Singh (Lecturer) & Mr. V. K. Dexit (Lecturer)

Institute of Pharmacy, B.U., Jhansi (U.P.), for their cooperation and

help during the course of this work.

I am also thankful to Mr. Ratan Yadav (Lab Assistant),

Mr.Brijkishore, Mr.Santosh Shivhare Mr. Sailes Soni, Mr. Dipak

Shukla for providing me all the chemicals, glassware, instruments

and laboratory facilities to carry out my complete dissertation work.

I take this opportunity to thank my seniors Mr. Chandrahass

yadav ,Mr.T.K. Vashishtha, Mr. Dharmendra Singh, Mr. Atul Vikram

Patel, Mr. Ramshankar and my classmates Mr.O.P.Goutam,

Mr.Himanshu Gurjar, Mr. Lokesh Meena, Mr.Sushil Saini, Mr. Om Pal

Singh, Mr. Brajesh Singh, Mr.Pawan Dhakar, Mr.Vinod Sahu,

Mr.Manish Pathodiya,Mr. Sanjay Dutt, Mr.K.L.Rathor, Mr. Pinkesh

Tiwari, Mr.Yogesh Maddesiya, Rohit and Anoop Gurjar Mrs.Savita

Varma, Mrs. Smita Khare, Mrs.Pratibha Mishra, Mrs. Tanuja,

Ms.Shweta Sachan, Mr. Raghvendra Mishra, & my junior Mr.

Santosh Kumar, Mr. Hemant, for their help and moral support.

I kindly thank to C.D.R.I..., Lucknow for providing FTIR, NMR,

and Mass Spectroscopy.

I am also thankful to Director, NISCAIR New Delhi for providing

library facilities.

I am also thankful to my heartiest friends Mr.Rajneesh Kumar,

Mr. Naveen Patel, Mr. Nitin Paliwal Mr. Ajay Seth, and Mr.Surendra

Patel for giving moral support and valuable advice during my project

work.

Finally I am indebted to those poor animals that took all the

suffering and gave me an opportunity to carrying out this duty and

all who were involved directly or indirectly to work tenure of mine.

I will be grateful to all these people for ever and always want

to live up to all their expectations.

(RAM KUMAR)

Certificate Head

Certificate Guide

Declaration

Acknowledgement

Abbreviations

List of Tables

List of Figures

S.No.PAGE

NO.

1.

INTRODUCTION

1.1 General

1.2 Plant Profile

1.3 Disease Profile

1-32

2. REVIEW OF LITERATURE 33-38

3. PLAN OF WORK 39

4. COLLECTION AND EXTRACTION 40-42

5. PHYTOCHEMICAL SCREENING

5.1 Qualitative Chemical Analysis

5.2 Thin Layer Chromatography

5.3 High Performance Thin Layer

43-65

CONTENTS

Chromatography

5.4 Column Chromatography

5.5 Characterization Of Isolated Compound

6.

PHARMACOLOGICAL INVESTIGATION

6.1 Evaluation of Antidiabetic Activity

6.2 Evaluation of Anti-Inflammatory Activity

66-77

7.

RESULT AND DISCUSSION

7.1 General

7.2 Phytochemical Screening

7.3 Evaluation of Antidiabetic Activity

7.4 Evaluation of Anti-Inflammatory Activity

78-80

8. SUMMARY AND CONCLUSION 81-83

BIBLIOGRAPHY 83-89

ENCLOSURE

ERRATA

LIST OF TABLES

TABLE NUMBER AND TABLE TITLE PAGE NO.

1.1 Diagnostic Criteria for IGT and IFG 18

1.2 List of Some Medicinal plants used in the

treatment of Diabetes

19

1.3 Cell derived mediators 28

1.4 Plasma derived factors 29

4.1 The characterization of methanolic extract 42

5.1 Qualitative Phytochemical analysis 48

5.2 TLC of Methanolic extract 53

5.3 Rf value of the spots of Methanolic extract 53

5.4 Column Chromatography of Isolated compound 59

5.5 Interpretation of IR Spectroscopy 61

5.6 Mass Spectra of Isolated compound 63

5.7 Interpretation of NMR Spectroscopy 64

6.1 The Antihyperglycemic effect of Methanolic

Extract on Alloxan induced Diabetic rats

70

6.2 The Antihyperglycemic effect of Methanolic

Extract

On Glucose Loaded rats

71

6.3 The Anti-inflammatory effect of Methanolic 76

Extract On Carrageenin-induced rat

LIST OF FIGURES

FIGURE NUMBER AND FIGURE TITLE PAGE NO.

1.1 Progress of inflammation 25

4.1 The Extraction procedure in schematic

manner

41

5.1 HPTLC peaks and HPTLC Chromatogram

with Rf values

57

5.2 IR spectra of compound (R9) 61

5.3 Mass Spectra of Isolated compound (R9) 62

5.4 NMR Spectra of Isolated compound (R9) 63

6.1 The Antihyperglycemic Effect of Methanolic

Extract on Alloxan induced Diabetic rats

70

6.2 The Antihyperglycemic Effect of Methanolic

extract on oral glucose tolerance test

72

6.3 Anti-inflammatory effect of Methanolic

Extract on Carrageenan-induced rat

77

LIST OF PHOTOGRAPHS

PHOTO NUMBER AND PHOTO TITLE PAGE NO.

1.Leaves and flower of Clitoria ternatea 8

2. TLC of Clitoria ternatea extract 54

Abbreviation

Abbreviatio

n usedMeaning of Abbreviation

ANOVA

WHO

Rf

TLC

FA

E A

CDRI

N C

D C

D M

OGTT

BGL

PG

HPTLC

FPG

h

CDA

ADA

CTLE

Analysis of variance

World Health Organization

Resolution Factor

Thin Layer Chromatography

Formic Acid

Ethyl acetate

Central Drug Research Institute

Normal Contral

Diabetic Contral

Diabetic Mellitus

Oral Glucose Tolerance Test

Blood Glucose Level

Plasma Glucose

High Performance Thin Layer

Chromatography

Fasting Plasma Glucose

Hour

Canadian Diabetes Association

American Diabetes Association

Conc

Dil

S.E.M

I P

I G T

NIDDM

ADM

IDDM

NMR

M S

I R

C C

T M M M

I H P

Q C

I D M A

N I S C A I R

Clitoria ternatea Leaves Extract

Concentrate

Dilute

Standard Error Mean

International Pharmacopoeia

Impaired glucose tolerance

Non-Insulin dependent Diabetes Mellitus

Administration

Insulin dependent Diabetes Mellitus

Nuclear Magnetic Resonance

Mass Spectroscopy

Infrared Resonance

Column Chromatography

Traditional Medicine Material medica

Indian herbal Pharmacopoeia

Quality Control

Indian Drug medical association

National Institute of Science Communication

and Information Resources

1. INTRODUCTION

1.1 HISTORY OF AYURVEDA:

1.1.1 History of Herbal Medicine: The history of herbal

medicines is as old as human civilization. The documents many of

which are of great antiquity, revealed that plants were used

medicinally in China, India, Egypt and Greece long before the

beginning of the christian era. One of the most famous surviving

remains it Papyrus Ebers, a scroll some go feet long and a foot wide,

dating back to the sixteenth century before christ, The text of

document is dominated by more than 800 formula and 700 different

drugs. The drugs such as acacia, castor oil and fenel are mentioned

along with apparent references to such compounds as iron oxide,

sodium chloride, sodium carbonate and sulphur.

Most of 6 medicinally active substances identified in the

nineteenth and the twentieth century’s were used in the form of

crude extract. In China, many medicinal plants had been in use

since 500 B.C. the oldest known herbal is pent saw written by

Emperor Shen Nung around 3000 B.C. It contains 365 drugs, one for

each day of the year. Indians also, worked meticulously to examine

and classify the herbals, which they come across, into groups called

Gunas Charaka made fifty groups of ten herbs each which according

to him would suffice an ordinary physician’s need similarly, Sushruta

arranged 760 herbs in 7 distinct sets based on some of their

common properties A large portion of the Indian populations even

today depends on the Indian system of medicine Ayurveda, an

ancient science of life. The well known treatises in Ayurveda are

Charak Samita and Sushuta Samita. (Purohit et. al., 2002)

India has an ancient heritage of traditional medicine; Materia

Medica of India provides lot of information on the folklore practices

and traditional aspects of therapeutically important natural

products. Indian traditional medicine is based on various system

including Ayurveda, Siddha and Unani.

The evaluation of these drugs in mostly based on

phytochemical, pharmacological and allied approaches including

various instrumental techniques like chromatography, microscopy

and others. These traditional system of Indian medicine have their

uniqueness, no doubt but there is a common thread running

through these system in their fundemental principles and

practices with the emerging interest in the world to adopt and

study the traditional system and to exploit their potentials based on

different health care system. The evaluation of the rich heritage of

the traditional medicine is essential. (Mukherjee P.K., 2002)

1.1.2 Importance of Herbal therapies:

Herbal medicines are prepared from a variety of plant

materials, leaves, stem, and root, bark and so on they usually

contain many biological active ingredients and are used primary for

treating mild or chronic ailments. Herbs can be prepared at home in

ways using either fresh or dried ingredients.

In the united states today, herbal remedies are not regulated

and come in unpredictable strengths the amount of active

ingredients varies greatly, depending on whether more than are

species of the herb is used and how and when the herb is gathered

and prepared. Because some herbs can be toxic or carcinogenic, all

herbs should be used under the guidance of a health care

practitioner familiar with herbal medicine.

Across the spectrum of alternative medicine, the use of herbs

is varied: Naturopathic medicine, traditional Chinese medicine, and

Ayurvedic medicine, all differ in how diseases are diagnosed and

which herbal remedies are prescribed.

Plants are considered to be medicinal if they possess

pharmacological activities of possible therapeutic use. These

activities are after known as a result of millennia of trial and error

but they have to be carefully investigated if we wish to develop new

drugs that meet the criteria of modern treatment.

The identification of the active principles of medicinal plant

investigation of the extract in order to ensure that they are safe

effective and of constant activity.

The isolation of these active principles and the determination of

their structure in order that they may be synthesized structurally

modified or simple extracted more efficiently.

The methodology of research into medicinal plants must be

rigorous. Often simple technical errors undermine the value of

research on natural products. There are many who believe that a

little rapid research is sufficient to confirm the reputation of a plant

and who then attempt to proceed from there towards lucrative

industrial production. (Mukherjee P.K., 2002)

1.1.3 Efficacy of Herbal Medicinal Products:

Phytomedicines consist of many chemical constituents with

complex pharmacological effects on the body. They are used

continuously for many decades or centuries, often in ways that

differ from these conventional medical prescribing researches.

Development in phytotherapy has suffered through lack of patent

protection, and the diversity and relatively small scale of the indus-

tries involved compared to the rest of the pharmaceutical industry,

established guidelines for assessing the efficacy and safety of

phytomedicines. The differing regional uses of traditional herbal

remedies present extra difficulties for the harmonization of quality

procedure around the world. Therapeutic efficacy and clinical trials

are two most important criteria for the development of herbal drugs.

Although preliminary assessments of effect can be obtain

through the results of in vitro costing and experiments on animals,

authorities licensing new medicine for public use require evidence of

their effects on human beings. Only carefully planned clinical trials

that minimize experimental bias are able to satisfy these

requirements. Most herbal remedies can call on a tradition of

popular use, which has in practice, allowed manufacturers to submit

relevant bibliographic evidence in reviewing their earlier licenses of

right. (Mukherjee P.K., 2002)

1.1.4 Safety in Herbal Drugs:

Major differences in the assessment of quality, safety and

efficacy would hinder free circulation of herbal medicinal products

may represent risk for consumers.

The complexity of herbal drug preparations and the

interpretation of bibliographic data on safety and efficacy reflecting

the experience gathered during long term use are best addressed

by involving specific expertise and experience. Safety and efficacy

of complex biological products such as herbal medicinal products

are directly linked to Pharmaceutical details such as the way of

production and the specification of extracts.

1.1.5 World Situation on Herbal Medicine:

Traditional medicine is a very important part of health care.

Most population in the developing countries still relies mainly on

indigenous traditional medicine for satisfying their primary health

care needs. Traditional medicine has not however been

incorporated in most national health systems and the potential of

services provided by traditional practitioners is far from being fully

utilized herbal medicines are of great important to the health

individuals and communities but their quality assurance need to be

developed. During last decade, in many developed countries, there

has also been a growing interest in herbal medicine, acupuncture

and alternative systems of medicine. Consequently, an increase in

international trade in herbal medicines and other types of traditional

medicines has occurred proper use of these different types of

medicine has therefore become concern. (Mukherjee P.K., 2002)

1.1.6 The utility of Plants in Current Therapy:

Despite the enormous availability of medicines and, above all

of pharmaceutical specialties, plants have a place in current therapy

as can be justified at least by the four following reasons. There is

renewed interest in using plants in therapy. Such is the case of

Artemisia (Klayman, 1985), a source of quinine. Behind the

therapeutic success of chloroquine and its synthetic derivatives in

the treatment of malaria, the use of quinine passed into a chapter in

the history of medicine. Though a biological phenomenon, that is

now well studied, even at the molecular level, bacteria and

parasites can develop resistance to chemotherapeutics that is, they

undergo selection that results in resistance to a particular chemical

compound. This process has occurred in part, with species of

plasmodium, the causative agent of malaria, to a point that

synthetic antimalarial drugs have lost such a significant part of their

efficiency in the last quarter of the twentieth century that it has

often been necessary to return to the use of quinine.

Currently, there is such a great demand for the plant alkaloids

that extraction laboratories cannot satisfy the growing demand,

maximized now that malaria has again become a great health risk in

topical area. The demand has been accentuated further by the

assistance of the insect vector, to identify their main active

chemical compounds. That sample, even through or partial one

reveals the enormous empirical traditional knowledge about

medicinal plants. Most of this knowledge is verbal and only

incompletely incorporated in historical and folklore work. The

aboriginal knowledge is the fruit of centuries and in capacity of

chemists to modify a molecular structure is almost unlimited the

capacity to invent or create new structures.

Phytochemical investigation carried out during the 1970s

and 1980s have discovered a number of alkaloids and other

pharmacologically active substances that are currently being

studies and that can possible serve as models for new synthetic

compounds. (Barz and Ellis, 1980)

Traditional medicine depends on a number of plants that are

currently used in scientific medicine although they have not yet

been improved upon. Such is the case of digitals purpurea L. and D.

lanata Ehrh. Many other drugs exist to which therapeutic effect

have been attributed As is well known, synthetic chemistry has until

now had little success in obtaining drugs effective in the treatment

of various viral disease, even though immunotherapy has achieved

great successful. We still do not have vaccines for all viral diseases.

It is possible that plants may be useful to treat this disease. An

example from Ecuator is laniqua (Marggricarpis seto-sus Ruiz and

Pavon), the roots of which, in infusion are used in the symptomatic

treatment of measles. Furthermore, numerous plants are known for

certain antineoplastics effects. (Cassady and Douros, 1980)

1.1.7 Differences between Herbs and Other Drugs:

Herbs are different in several respects from the type of

purified therapeutics agents we have become accustomed to call

drugs in the last half of the twentieth the century. In the first place,

they are more dilute than the concentrated chemicals that are

familiar use in the form of aspirin tablets or tetracycline capsules. A

simple example will illustrate the difference. One can take caffeine

for its stimulatory effects on the central nervous system. The usual

dose is 200mg contained in one or two small tablets, depending on

their strength or it is possible to get the some effect by drinking a

caffeine containing beverage such as coffee or tea. Dilution is not

the only difference that just be considered in utilizing medicinal

herbs. In addition to physiologically inert substances such as

cellulose and starch, herbs often contain addition active principles

that may be closely related both chemically and therapeutically to

the active constituents primary responsible for its effects. (James E.

et. al., 2002)

1.1.8 Indian Trade in Medicinal Plants:

The exports of medicinal plants and herbs from India has

been quite substantial in the last few years., India has been the

major supplier of medicinal plants in the world market till 1976

when it was relegated to the second position by South Korea. With

exports worth only Rs. 15 crores during 1978-79. The quantum of

export has dropped to almost half of what it was in 1976-77, when

India exported medicinal plants worth of Rs. 29 crores. During 1988-

89, India exported crude drugs alone to the tune of about 62 crores.

The items of export value are opium, psyllium husks and seeds,

Vinca rosea, Kuth roots, Nux-vomica, Galanga and Senna leaves and

pods. India is the second largest producer of castor seed in the

world. Producing about 1,25,000 tonnes per annum.

With development of phytochemical industry in India,

domestic requirement for various medicinal plants of grew

considerably. Consequently, the Government of India has adopted

restrictive export policy in respect of those crude drugs which were

indiscriminately exploited in the forest.

In accordance with the policy the exports of rauwalfia,

podophyllum, Indian rhubarb, dioscorea, saussurea etc. from India

were restricted. The export of these drugs is, however, permitted by

firms obtaining certificates from the chief conservator of forests or

officer autherized by him that the material is of plantation as

nursery origin.

Apart from requirements of medicinal plants for internal

consumption, India exports crude drugs mainly to developed

countries viz, USA, Germany, France, Switzerland, U.K. and Japan.

Who share between them 75 to 80 percent of the total export of

crude drugs? From India, the principal herbal drugs that have been

finding a good market in foreign countries are Aconite, Aloe,

Bellodena Acorus, Cinchona, Cassia tora, Dioscorea, Digitals,

Ephedra, Plantago (Isabgol), Cassia (Senna) etc.

The total value of export of crude drugs, Ayurvedic put up for

retail have increase from Rs. 394 crores in 1996-97 to Rs. 446

crores in 1998-99. (Purohit et. al., 2002)

1.2 PLANT PROFILE:

Photograph 1: leaves and flower of Clitoria ternatea:

1.2.1 Taxonomical Hierarchy:

Botanical Name : Clitoria ternatea L.

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Fabales

Family : Fabaceae

Subfamily : Faboideae

Tribe : Cicereae

Genus : Clitoria

Species : C. ternetea.

1.2.2 Vernacular Name:

Sanskrit : Aparajita, Girikarnika

English : Clitoria, Butterfly pea.

Hindi : Aparajita

Tamil : Kannikkoti

Telgu : Gilagarnika

Kan : Karnike

Malyalam : Samkhupuspam, Aral, Malaya Mukki

1.2.3 Description:

A rambling, pretty, indigenous climber up to 2-3m in height,

extensively grown in gardens for its flowers and also found

commonly as an escape in hedges and thickets throughout India,

up to an altitude of 1500m and in the Andaman Islands. Stem

scandent; leaves pinnately 5-foliolate, 6-13 cm long; leaflets ovate

or oblong, 2-5 cm long flowers papilionaceous, white or bright blue

with yellow or orange centre pods flat, beaked, seeds yellowish

brown, subglobose.

Though a hardly perennial, the climber is grown in gardens as

an annual and trained on bowers or trellises. The white and blue-

flowered types cross naturally resulting in a variety of colors and

single and double forms. (Wealth of India, 2001)

1.2.4 Habitat: Throughout India are hedges and thickets, also

cultivated in gardens.

1.2.5 Propagation: By Seeds

1.2.6 Part Used: Roots, leaves seeds.

Leaves: A good-looking perennial twining herb with terete stems

and branches, leaves compound, imparipinnate, leaflets 5-7, sub-

coriaceous, elliptic- oblong, obtuse.The shoots, leaves and tender

pods are eaten as vegetable in Kerala, and in the Philippines.

Flower Variety: The flower has an almond taste, cooling, acrid,

laxative, alexiteric, anthelminitic tonic to the brain, good for eyes

dise-ases, ulcers of the cornea, tuberculosis glands, elephantiasis,

head ache, cures, tridosha leucoderma burning sensation, pains,

bilious-ness, inflammation, ulcers, “Kapha” snake bites.

Blue Flowered Variety: The root is bitter and has all the

properties of that of the white flowered variety; in addition, it is

aphrodisiac; was density severe bronchitis asthma consumption

useful in as cites and abdominal enlargement (Ayurveda) the roots

purgative and diuretic useful in as cites (Unani).

1.2.7 Cultivation:

The climber yields green fodder throughout the year,

particularly during dry period. It can be grown as a forage legume

either alone or with perennial fodder grasses in Punjab, Rajasthan,

Uttar Pradesh, Gujarat, Maharashtra, Madhya Pradesh, Andhra

Pradesh, Tamil Nadu and Karnataka, and has been recommended

as a forage legume in the Andamans., It is being introduced a

drought resistant pasture in arid and semi-arid regions. The plant is

also suitable as a green manure and cover-crop. Besides

suppressing many perennial weeds, it enriches the soil by fixing

nitrogen.

The seeds are sown in August in rows 30 cm. apart. The crop

starts yielding green fodder (dry matter 21.8%) in 60 days, the first

cutting yielding 20-24 tonnes/ha. Protection from frost and drought

is necessary to ensure fodder supply throughout the year. The green

fodder can also be made into hay, each cutting yielding C. 5.4

tonnes/ha. The hay is used as good quality maintenance feed for

both growing and adult stock and is relished by sheep and goats.

1.2.8 Chemical Constituents:

The high calcium concentration in the plant showed that it can

be exploited as a significant source of calcium brewed as herbal

drink. The presence of stigmast-4-ene-3, 6, diene is reported from

the plant. The roots contain taraxerol and teraxerone. The leaves

contain 3- monoglucoside, 3-rutinoside, 3-neohesperidoside, 3-o-

rhamnosyl- glucoside, 3-o-rhamnosyl galactoside of Kaempferol,

besides kaempferol 3-o- rhamnosyl-o-rhamnosyl- glucoside.It also

contain aparajitin and β- sitosterol. The blue flowers contain

delphinidin-3,5-diglucoside delphinidin-3-β glucoside and its 3-

methyl derivative, malvidin-3β- glucoside, Kaempferol and cyanin

chloride ; the white flowers yield only kaempferol, other substances

present in the seeds are: p-hydroxycinnamic acid, flavonol-3-

glycoside, ethyl-α-D galactopyranoside, adenosine, 3,5,7,4- tetra

hydroxyflavone-3- rhamnoglucoside, a polypeptide, hexacosanol, β-

sitosterol and an anthoxanthin glucoside the seeds also contain

oligosaccharides or flatulene.

1.2.9 Uses:

The leaves are useful in ophthalmopathy, tubercular glands,

amentia, hemicrania, burning sensation, strangury, helminthiasis,

leprosy, leucoderma, elephantiasis, inflammation, vitiated

conditions of pitta, bronchitis, asthma, pulmonary tuberculosis,

ascites, ulcers, visceromegaly and fevers. The roots are bitter,

refrigerant, ophthalmic, laxative, intellect promoting, alexeteric,

diuretic, anthelmintic, depurative, aphrodisiac and tonic. The leaves

are also useful in otalgia hepatopathy and eruptions. The seeds are

cathartic and are useful in visceralgia.

1.3 DISEASE PROFILE:

1.3.1 INTRODUCTION OF DIABETES:

The knowledge regarding diabetes existed since Vedic period

and treatment of diabetes has been mentioned in “Sushruta

Samhita” in 200 B.C. Diabetes mellitus was known to ancient Indian

physicians as ‘Madhumeha’. This term Diabetes mellitus hails its

origin from a Greek word “diabainess” which means “to pass

through” and a Latin word “mellitus” meaning “sweetened” with

honey. Many herbal products including several metals and minerals

have been described for the care of Diabetes mellitus in ancient

literature. A medicinal plant, Galega officinalis, led to discovery and

synthesis of metformin. The modern medicine extensively used to

control blood glucose level. The earliest mention of the medicinal

use of plants is available in the ‘Rig Veda, which was written

between 4500 and 1600 B.C. In the ‘Atharva Veda’ (1500 B.C.). We

find more varied use of drugs. It is Ayurveda which is considered as

an ‘Upa Veda’ in which definite properties of drugs and their uses

have been given in great detail. (Kumar et .al., 2005)

Many oral hypoglycemic agents, such as biguanides and

sulfonylurea are available along with insulin for the treatment of

diabetes mellitus, but these synthetic agents can produce serious

side effects, and in addition, they are not suitable for use during

pregnancy. Since ancient times, diabetes has been treated orally

with several medicinal plants or their extracts based on folklore

medicine. These herbal remedies are apparently effective, produce

minimal or no side effects in clinical experience and are of relatively

low costs as compared to oral synthetic hypoglycemic agents. There

is a growing tendency all over the world to shift from synthetic to

natural products including medicinal plants. Today more than 25%

prescriptions issued in most of the developed countries of Europe

and the United States contains one or more plant drugs. Several

species of plants have been described as having ant diabetic

property. In Indian Materia Medica, 42 medicinal plants have been

recorded for the treatment of diabetes. (Indian Materia Medica,

1954)

According to the National Bureau of plant Genetic Resources,

the world Health Organization has listed more than 21,000 plant

species used around the world for medicinal purposes of which 8000

species of medicinal plants exists in this country. In Africa up to 80%

of the population uses traditional medicine for primary health care.

In Germany, 90% of the population has used a natural remedy at

some point of their life.

Diabetes mellitus (DM) is a widespread disorder, which has long

been recognized in the history of medicine, before the advent of

insulin and oral hypoglycemic drugs, the major form of treatment

involved the use of paints. More than 400 plants are known to have

been recommended ad recent investigations have affirmed the

potential value of some of these treatments. (Baily and Day, 1989)

Diabetes is not new to the medical world. It has been known

since antiquity, almost from 1500 BC. According to Charaka (2nd

century B.C.,) It is called PRAMEHA, i.e. the causative significant of

heredity, obesity and lack of physical activity; clinical features such

as thirst and dryness of mouth, peculiar odour, burning sensation or

lack of sensation in the hands and feet and onset of boils. (Reddy,

2005)

Diabetes mellitus is a group of endocrine syndromes

characterized by hyperglycemia; altered metabolism of lipids, carb-

ohydrates, and proteins, and an increased risk of complications from

vascular disease. Most patients can be classified clinically as having

either type I diabetes mellitus (type I DM formerly known as insulin-

dependent diabetes of IDDM) or type II diabetes mellitus (type II DM

formerly known as non-insulin dependent diabetes of NIDDM).

(Goodman and Gilman, 2001)

1.3.1.1 Type of Diabetes, causes and their treatment:

A. Type I Diabetes:

(Insulin dependent diabetes mellitus IDDM) Insulin dependent

diabetes most commonly afflicts juveniles, but it can also occur in

adults. The disease is characterized by an absolute deficiency of

insulin caused by massive β-cell lesions or necrosis. Loss of β-cell

function may be due to invasion by viruses, the action of chemical

toxin or usually through the actions of autoimmune antibodies

directed against the β-cells. As a result of the destruction of β-cells,

the pancreas fails to responsed to ingestion of glucose and the type

1 diabetes shows classic symptoms of insulin deficiency (Polydipsia,

Polyphagia and Polyuria). Type I diabetes requires exogenous insulin

to avoid hyperglycemia and life threatening ketoacidosis.

a) Cause of Type I Diabetes:

A burst of insulin secretion normally occurs after ingestion of a

meal in response to transient increase in the levels of circulating

glucose and amino acids. In the absorptive period. Low basal levels

of circulating insulin are maintained through β-cells secretion.

However, the type I diabetes has virtually to functional β-cells and

can neither respond o variation in circulating fuels nor maintain

even a basal nephropathy and retinopathy are directly related to the

extent of glycemic control.

b) Treatment of Type I Diabetes:

Type I diabetes requires exogenous (injected) insulin in order

to control hyperglycemia, maintain acceptable levels of glycosylated

haemoglobin and avoid ketoacidosis. The goal of administering

insulin to type I diabetes is to maintain blood glucose concentration

as close to possible and to avoid wide swings in blood glucose levels

that may contribute to long term complications. The use of portable

blood glucose analyzers facilitates close self-monitoring and

treatment.

B. Type II Diabetes:

(Non-Insulin dependent diabetes, mellitus, NIDDM) Most

diabetic are in this category, genetic factors, rather than viruses or

autoimmune antibodies are apparently casua1. The metabolic

alterations observed are milder than those described for IDDM (for

example, NIDDM patients typically are not ketosis) but the long term

clinical consequences can be just as devasting (for example,

vascular complications and subsequent infection can lead to

amputation of the lower limbs.)

a) Cause of Type II diabetes:

In NIDDM the pancreas retains some β-cells function, resulting

in variable insulin levels that are insufficient to maintain

homeostasis patients with type II diabetes are often obese. Type II

diabetes is frequently accompanied by target organ insulin

resistance that limits responsiveness to both endogenous and

exogenous insulin. In some cases, insulin resistance is due to a

decreased number or mutations of insulin receptors.

b) Treatment of Type II diabetes:

The goal in treating type II diabetes is to maintain blood gluc-

ose concentration within normal limits and to prevent the

development of long term complication of disease weight reduction.

Exercise and dietary modification decrease insulin resistance and

correct the hyperglycemia of Type II diabetes in some patients

however most are dependent on pharmacological intervention with

oral hypoglycemic agents. Insulin therapy may be required to

achieve satisfactory serum glucose levels sulphonylureas and

bigunide are two most commonly prescribed oral treatment option.

(Mary et. al., 2000)

C) Type III Gestational Diabetdes:

Gestational diabetes mellitus is an operational classification

(rather than a pathophysiologic condition) identifying women who

develop diabetes mellitus during gestation (Women with diabetes

mellitus before pregnancy are said to have “pregestational

diabetes” and are not included in this group). Women who develop

type I diabetes mellitus during pregnancy and women with

undiagnosed asymptomatic type II diabetes mellitus that is

discovered during pregnancy are classified with gestational diabetes

mellitus. However most women classified with gestational diabetes

mellitus have normal glucose homeostasis during the first half of the

pregnancy and develop a relative insulin deficiency during the last

half of the pregnancy leading to hyperglycemia. The hyperglycemia

resolves in most women after delivery but places them at increase

risk of developing type II diabetes mellitus later in life. (National

diabetes data group, 1995)

1.3.1.2 Characterization of Diabetes Mellitus:

It is metabolic disorder characterized by hyperglycaemia,

glycosuria, hyperlipemia, negative nitrogen balance and sometimes

ketonemia, A wide spread pathologic change is thickening of

capillary basement membrane, increase in vessel wall matrix and

cellular proliferation early atherosclerosis, sclerosis of glomerular

capillaries, retinopathy, neuropathy and peripheral vascular

insufficiency. (Tripathi K.D., 1985)

A. Atherosclerosis:

The diabetic has a two to three fold higher risk of dying

prematurely of atherosclerosis that a non diabetic individual

Foremost is the reduction of LDL-cholesterol levels and triglyceride

while increasing HDL- cholesterol levels. [Mohan Harsh, 2004]

B. Diabetic Neuropathy:

Diabetic neuropathies are among the most frequent

complications of long term diabetes. Loss of peripheral nerve

function. Tingling sensations, number loss of pain, and muscle

weakness may occur as a result of diabetic retinopathies. In rats

increasing the sorbitol concentration in the sciatic nerve is directly

related to decreasing nerve conduction velocity, possible as a result

of decreeased myoinostiol concentration.

C. Diabetic Retinopathy:

World Diabetes Day is celebrated every year on 14 November,

which is incidentally the birthday of Frederick Banting, who together

with Charles Best discovered insulin in the year 1921. This year’s

theme of world diabetes Day was to address retinopathy one of the

complications of DM. Your Eyes and Diabetes: Don’t Lost sight of the

Risks. Diabetic retinopathy is a serious eye disease that can result in

blindness. The retinopathic lesions are divided into background or

“simple” retinopathy (consisting of microaneurysms, haemorrhages,

exudates and retinal edema) and proliferative or malignant (with

newly formed vessels, scaring retinitis proliferens vitreous

haemorrhage and retinal detachement).

D. Diabetic Nephropathy:

This is a common complication and a leading cause of death in

DM. Four types of possible overlapping lesions develop: glomerulo

sclerosis arteriosclerosis of the efferent and afferent arteriosclerosis

of the renal artery and its intrarenal branches; and peritubular

deposits of glycogen fat and mucopolysaccharides. Periodic

monitoring of diabetic patients kidney function (uric acid, creatinine

and creatininme clearance) is important.

E. Diabetic Foot Ulcers:

Ischemia and peripheral neuropathy are the key factors in the

development of diabetic foot ulcers. However foot ulcers are largely

preventable through proper foot care, the avoidance of injury and

tobacco, in any form and employing methods to improve local

circulation. Tobacco constricts the peripheral blood vessels and can

cause Burger’s disease. (Yue et. al., 1984)

1.3.1.3 Diagnosis:

A number of tests for hyperglycemia have been assessed as

diagno-stic tools for DM, namely fasting plasma glucose (FPG),

casual plasma glucose (PG), 2- hour plasma glucose in a 75 gram

oral glucose tolerance test (OGTT) and haemoglobin Ale (Ale). At the

present time, well established diagnostic criteria for DM exist

utilizing all of the above, except Ale. These criteria, which are

supported by the Canadian Diabetes Association (CDA), America

Diabetes Association (ADA) and the World Health Organization, in

the absence of unequivocal hyperglycemia, the diagnosis needs to

be confirmed by repeat testing on a separate day. The diagnostic

criteria for DM are useful to identify patients at risk for developing

the micro vascular complications of diabetes. However there has

been increasing recognition that even lesser degrees of

abnormalities of glucose metabolism are associated with an

increased risk of developing cardio vascular complications and of

course, diabetes. Thus the concept of impaired glucose (IFG) was

introduced. The diagnostic criteria for IFG and IGT have evolved

over time with the emergence of data suggesting a further

lowering of the threshold fasting glucose value from 6.1 to 5.6

mmol/L by the ADA in 2002, However, discrepancies remain

between organization and the CDA’s current definition of IFG is

fasting plasma glucose of 6.1-6.9 mmol/L and IGT is defined as a

2-hour value based on the 75 g OGTT of 7.8-11.0 mmol/L.

Diagnostic criteria for diabetes mellitus according to the Canadian

Diabetes Association, American Diabetes Association and World

Health Organization.

FPG = 7.0 mmol/L

Fasting = no. caloric intake for at least 8 hours.

OR

Casual PG= 11.1 mmol/L + symptoms of diabetes

OR

2hPG in a 75g OGTT = 11.1 mmol/L.

A confirmatory laboratory glucose test must be done in all cases

on another day in the absence of unequivocal hyperglycemia

accomp-anied by acute metabolic decomposition symptoms of

diabetes as polyuria, polydipsia and unexplained weight loss.

Table: 1.1 represents the Diagnostic criteria for impaired

glucose tolerance (IGT) and impaired fasting glucose (IFG) according

to recommendation from the Canadian Diabetes Association (CDA),

American Diabetes Association (ADA) and The World Health

Organization (WHO).

CDA ADA WHO

IGT2hPH OGTT7.8-11.0

2hPG OGTT 7.8-11.0

FPG < 7.0 AND 2hPG OGTT 7.8-11.0

IFG FPG 6.1-6.9 FPG 5.6-6.9FPG 6. 1-6.9 AND 2hPG < 7.8

IFG and IGTBoth of the above criteria

Both of the above criteria

N/A

FPG = fasting plasma glucose

2hPG OGTT = 2 hour plasma glucose in a 75 g oral glucose

tolerance test – all values are in mmol/L.

PG = plasma glucose

OGTT = oral glucose tolerance test

1.3.1.4 Traditional approach of diabetes therapy using

plants:

History of medicine dates back practically to the existence of

human civilization. Natural products, including plants, animals, and

minerals have been the basis of treatment of human disease.

According to the World Health Organization more than70% of the

world population must use traditional medicine to satisfy their

principal health needs. The current accepted modern medicine or

allopathy has gradually developed over the years by scientific and

observational efforts of scientists. However, the basis of its

development remains rooted in traditional medicine and therapies.

Numerous drugs have entered in “International Pharmacopoeia”

structures, which may be used as templates for the development of

new drugs. Natural products are prescribed widely because of their

effectiveness, less side effects and relatively low cost. A great

numbers of medicinal plants used in the control of diabetes mellitus

have been reported.

Table.1.2: List of some Medicinal plants used in the

treatment of Diabetes: (Khan et. al., 2005)

S.No

Plant Name FamilyUseful Part

1. Abroma augusta L.f. Sterculiaceae Bark, Flower

2. Annona squamosa L. Annonaceae Leaves

3. Barleria cristata L. Acanthaceae Roots

4. Beta vulgaris L. Betulaceae Bark

5. Calamug rotang L. Arecaceae Bark

6. Cannabis sativa L. CannabinacaeResin & Leaves

7. Desmodium gyrans L. Papilionaceae Roots

8. Dioscorea alata L. Dioscoreaceae Rhizome

9. Eryngium foelidum L. ApiaceaeWhole

plant

10. Ficus fistulosa L. Moraceae Fruit

11. Gymnema sylvestrisAsclepiadacea

eLeaves

12. Hordeum Vulgare L. Poaceae Seed

13. Ipomaea balatus L. ConvolvulaceaeTuberus Roots

14. Juslicia adhatoda L. Acanthaceae Leaves

15. Kyllianga bulbosa CyperaceaeWhole

plant

16. Lysium barbala L. Solanaceae Fruits

17. Momordica charanlia Cucurbitaceae Fruit

18. Nepeta cataria L. LamiaceaeLeaves & Flowering

19. Oplopanax horridum Umbelliferae Root

20. Picrorhiza kurrooa Scrophulariaceae Herb

21. Quercus lineala Blume Fagaceae Stem bark

22. Rotula aquatica Lour. Boraginaceae Root

23. Swertia chirata Gentianaceae Whole Plant

24.Trigonellafoenum

graecum L.Papilionaceae Seed

Some steroidal plants used for the purpose are barks of various

species of ficus, the roots of ginseng, fenugreek, and the fruit and

seed of various Cucurbitaceae families. It also includes famous

Momordica charantia or Kerala fruit. (Ansari, 2005)

Other plants which are most effective and most commonly

studied in relation to diabetes and their complication are Allium

cepa, Allium sativum, Aloe Vera, Cajannus Cajan, Gymnema Sylve-

stris, Ocimum Sanctum and Tinospora Cordifolia. (Grover et. al.,

2002)

1.3.1.5 Statistics of Diabetes:

It is alarming that India is fast assuming the mantle of being

the diabetic capital of the world. We have the largest number of

diabetics in the world and the number of new cases has increased

from 1% before 1970 to 8% in the 80’s and is still growing as per

ICMR studies. (Reddy, 2005). India is expected to have 40 million

people with diabetes by the year 2010 and 57.2 million by 2025.

There are more than 125 million persons with diabetes in the world

today, and by 2010 this number is expected to approach 220

million. (Amos et. al., 1997) Some investigators expect the

incidence to double by 2035. Types I and II are both increasing

frequently. Diabetics are 25 times more likely to develop heart

attacks and twice as likely to get strokes as compared to non-

diabetics. About one or two million patients have type I the

remaining 80 to 90% of diabetic patients have type II diabetes.

(Goodman and Gillman, 2001)

1.3.2 INTRODUCTION OF INFLAMMATION:

Inflammation is a complex pathophysiological process

mediated by a variety of singling molecules produced by leukocytes,

macrophages and mast cells as well as by the activation of

complement factors, which bring about edema formation as a result

of extravasations of fluid, proteins etc and pain at the site of

inflammation (White, 1999).

The Roman writer Celsus named the famous four Cardinal Signs

of inflammation as:

Rubor (redness)

Tumor (swelling/ edema)

Color (heat)

Dolor (pain)

The fifth sign function lasea (loss of function) was later added

by Virchow.

1.3.2.1 Types of inflammation:

Depending upon the defense capacity of host and duration of

response inflammation can be classified in to following types;

A. Acute inflammation:

In acute inflammation short duration in which PMN are the

main cells for early body reaction leads to accumulation of fluid and

migration of leucocytes and platelets to the affected site followed by

repair.

Pathophysiology of acute inflammation

The earliest response to tissue injury leads to alterations

including hemodynamic changes and changes in vascular

permeability.

1. Transient vasoconstriction

Irrespective of type of injury of arterioles vasoconstriction may

last longer from 3-5 second with mild form of injury to 5 minutes for

severe form of injury.

2. Persistent progressive vasodilatation

Vasodilatation results in increased blood volume in

microvascular bed in arterioles within half an hour of injury due to

Histamine, 5-HT which are responsible for redness and warmth at

site of acute inflammation.

3. Elevation of local hydrostatic pressure

Progressive vasodilatation may lead local hydrostatic pressure

resulting in transudation of fluid in extracellular space which is

responsible for swelling.

4. Stasis of microcirculation

Slowing is attributed to increased permeability of

microvascular that results in increased in the concentration of red

cells, and thus, raised blood viscosity.

5. Leukocyte migration

Leukocyte sticks to the vascular endothelium due to factors

such as selectin, intigrin and ICAM-1. After attaching to the

endothelial wall these secretes collagenase which cause breakdown

of endothelium and basement membrane and escape out towards

the inflammation site this known as emigration. Chemokinenes are

substances which attract leucocytes toward inflammation site are IL-

2, LT-B4, PF-4 MCP-1.

6. Phagocytosis

Phagocytosis is defined as the process of engulfing of the solid

particulate material by the cells. There are two types of phagocytic

cells, these are PMNs and macrophages and tissue macrophages.

B. Chronic inflammation:

Chronic inflammation is a prolonged reaction arising when the

acute response is insufficient to eliminate proinflammatory agents.

It includes a proliferation of fibroblast & infiltration of the

neutrophiles and exudation. Chronic inflammation has two types

such as specific and non specific can be caused by following 3 ways:

Chronic inflammation following acute inflammation

Recurrent attacks of acute inflammation

Chronic inflammation starting de novo

General features of chronic inflammation

Though there may be differences in chronic inflammatory

response depending upon the tissue involved and causative

organisms, these are the general characteristic of chronic

inflammation;

1. Mononuclear cell infiltration:

Chronic inflammatory lesions are infiltrated by mononuclear

inflammatory cells like phagocytes and lymphoid cells. Phagocytes

are represented by circulating monocytes, tissue macrophages,

epithelioid cells and multinucleated giant cells. The macrophages

compromise the most important cells in chronic inflammation. On

activation they release several biological active substances such as;

acid and neutral proteases, oxygen-derived reactive metabolites

and cytokines. These products bring tissue destruction,

neovascularisation and fibrosis.

2. Tissue destruction or necrosis

Tissue destruction brought about by activated macrophages

which release of a variety of factors like protease, elastase,

collagenase, lipase, reactive oxygen radicals, cytokines (IL-1, IL-8

and TNF), nitric oxide, angiogenesis growth factors etc.

3. Proliferative changes

As results of necrosis, proliferation of small blood vessels and

fibroblasts is simulated resulting in the formation of inflammatory

granulation tissue. Eventually, healing by fibrosis and collagen lying

takes place.

Fig-1.1: Progress of inflammation

Neutrophils migrate from blood vessels to the inflamed tissue via

chemotaxis, where they remove pathogens through phagocytosis

and degranulation.

1.3.2.2 Morphological changes in inflammation:

Specific patterns of acute and chronic inflammation are seen

during particular situations that arise in the body as shown below.

1. Granulomatous inflammation

It is characterized by the formation of granulomas, they are

the result of a limited but diverse number of diseases, among which

are tuberculosis, leprosy, and syphilis.

2. Fibrinous inflammation

Inflammation resulting in a large increase in vascular

permeability allows the blood vessels to pass through fibrin. If an

appropriate procoagulative stimulus is present, such as cancer cells,

fibrinous exudate is deposited. This is commonly seen in serous

cavities, where the conversion of fibrinous exudates into a scar can

occur between serous membranes, limiting their function.

3. Purulent inflammation

Infection of pyrogenic bacteria such as staphylococci causes

inflammation resulting in large amount of pus which consists of

neutrophils, dead cells, and fluid. Large, localized collections of pus

enclosed by surrounding tissues are called abscesses.

4. Serous inflammation

It is characterized by the copious effusion of non-viscous

serous fluid, commonly produced by mesothelial cells of serous

membranes, but may which also be derived from blood plasma. Skin

blisters exemplify this pattern of inflammation.

5. Ulcerative inflammation:

Inflammation occurring near an epithelium can result in the

necrotic loss of tissue from the surface, exposing lower layers.

Excavation in the epithelium is known ulcer.

1.3.2.3 Chemical mediators of inflammation:

These are a large number of endogenous compounds which

can enhance vascular permeability. These are broadly classified

into 2 groups:

A. Cell-derived mediators (Table 1.3)

Vasoactive amines (Histamine, 5-HT)

Arachidonic acid metabolites (Eichosanoids)

Lysosomal components

Platelet activating factors

Cytokines (IL-1, TNF-α, TNF-β, IF-γ and chemokines)

Nitric oxide and oxygen metabolites

B. Plasma-derived mediators (plasma protease) (Table

1.4)

The kinin system

The clotting system

The fibrinolytic system

The complement system

5-Hydroxytryptamine

It is present in tissue like chrommaffin cells of GIT, spleen,

nervous tissue, mast cells and platelets. The actions of 5-HT are

similar to histamine but less potent mediators than histamine in

increasing vascular permeability and vasodilatation.

Platelet activating factor (PAF)

It is released from IgE-sensitized basophiles or mast cells,

other leucocytes, endothelium and platelets. Apart from its action

on platelets aggregation and release reaction, the actions of PAF as

mediators of inflammation are increased vascular permeability,

adhesion of leucocytes to endothelium, Chemotaxis and cell-

mediated immunity to the irritant, implying thereby the role of

hypersensitivity in granulomotous inflammation.

Prostaglandins

PGs play a significant role in different phase of inflammatory

reactions. PGs elicit pain by direct stimulation of sensory nerve

ending and also sensitize sensory nerve endings to other pain

provoking stimuli (Campbell et al., 1991). Especially PGE was

reported to act on cell membrane during inflammatory condition

leading to destabilization in lipoprotein structure of cell membrane

(Bhaskar et al., 1987). PGI2 plays an important role in vascular

function because, like nitric oxide, it inhibits platelet adhesion to the

vascular endothelium and is a strong vasodilator. Thromboxanes

and leukotrienes produce vasoconstriction and are important

modulators.

Table: 1.3 Cell derived mediator

Name Type Source Description

Lysosome granules

Enzymes Granulocytes

These cells contain a large variety of enzymes which perform a number of functions. Granules can be classified as either specific or azurophilic depending upon the contents, and are able to break down a number of substances, some of which may be plasma-derived proteins which allow these enzymes to act as inflammatory mediators.

HistamineVasoactive

amine

Mast cells, basophils, platelets

Stored in preformed granules, histamine is released in response to a number of stimuli. It causes arteriole dilation and increased venous permeability.

IFN-γ CytokineT-cells, NK

cells

Antiviral, immunoregulatory, and anti-tumour properties. This interferon was originally called macrophage-activating factor, and is especially important in the maintenance of chronic inflammation.

IL-8 Chemokine Primarily macrophag

e

Activationand chemoattraction of neutrophils, with a weak

effect on monocytes and eosinophils.

Leukotriene B4

Eicosanoid Leukocytes

Able to mediate leukocyte adhesion and activation, allowing them to bind to the endothelium and migrate across it. In neutrophils, it is also a potent chemoattractant, and is able to induce the formation of reactive oxygen species and the release of lysosome enzymes by these cells.

Nitric oxideSoluble

gas

Macrophage endothelial cells, some

neurons

Potent vasodilator, relaxes smooth muscle, reduces platelet aggregation, aids in leukocyte recruitment, direct antimicrobial activity in high concentrations.

Prostaglandins

Eicosanoid Mast cellsA group of lipids which can cause vasodilation, fever, and pain.

Table: 1.4 Plasma derived factors

M Name Type d by Description

Bradykinin Kinin system

A vasoactive protein which is able

to induce vasodilation, increase

vascular permeability, cause

smooth muscle contraction, and

induce pain.

C3 Complement

system

Cleaves to produce C3a and C3b.

C3a stimulates hishistamine

release by mast cells, thereby

producing vasvasodilation. C3b is

able to bind to bacterial cell walls

an act as an opsonin, which marks

the invader as a target for

phagocytosis.

C5aComplement

system

Stimulates histamine release by

mast cells, thereby producing

vasodilation. It is also able to act

as a chemoattractant to direct

cells via chemotaxis to the site of

inflammation.

Factor XII

(Hageman

Factor)

Liver

A protein which circulates

inactively, until activated by

collagen, platelets, or exposed

basement membranes via

conformational change. When

activated, it in turn is able to

activate three plasma systems

involved in inflammation: the kinin

system, fibrinolysis system, and

coagulation system.

Membrane

attack complex

Complement

system

A complex of the complement

proteins C5b, C6, C7, C8, and

multiple units of C9. The

combination and activation of this

range of complement proteins

forms the membrane attack

complex, which is able to insert

into bacterial cell walls and causes

cell lysis with ensuing death.

Plasmin Fibrinolysis Able to break down fibrin clots,

systemcleave complement protein C3,

and activate Factor XII.

ThrombinCoagulation

system

Cleaves the soluble plasma protein

fibrinogen to produce insoluble

fibrin, which aggregates to form a

blood clot. Thrombin can also bind

to cells via the PAR1 receptor to

trigger several other inflammatory

responses, such as production of

chemokines and nitric oxide.

1.3.2.4 Inflammatory associated disorders:

Asthma

Autoimmune diseases

Chronic prostatitis

Glomerulonephritis

Hypersensitivities

Inflammatory bowel diseases

Pelvic inflammatory disease

Rheumatoid arthritis

Transplant rejection

1.3.2.5 Drugs used as analgesic and anti-inflammatory

agents:

1. Opioid used as analgesic: Morphine, Pholcodine, Pethidine,

Fentayl

2. Corticosteroids: Betamethasone, Betanosolone

3. Drugs that act on COX

a. Nonselective COX inhibitors

Salicylates: Aspirin, Diflunisal

Pyrozolone derivatives: Phenylbutazone, Oxyphenbutazone

Indole derivatives: Indomethacin, sulindac

Propionic acid derivatives: Ibuprofen, Naproxen,

Ketoprofen

Anthranilic acid derivative: Mephenamic acid

Aryl-acetic derivatives: Diclofenac

Oxicam derivatives: Piroxicam, Tenoxicam

Pyrollo-pyrrole derivatives: Ketorol

b. COX-2 Inhibitors

Preferential inhibitors: Nimesulide, Meloxicam, Nabumetone

Selective COX-2 inhibotors: Celocoxib, Rofecoxib, Valdecoxib

2. LITERATURE REIVEW

Naeem A., et al., (2007) reported an alteration high yielding

purification method for clitoria ternatea lectin, In our previous

publication we had reported the purification and characterization

of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose

affinity column, designated CTA [A. Neem, S. Haque, R.H. Khan.

Protein J., 2007] Since CTA binds B-D- galactosides, this lectin can

be used as valuable tool for glycobiology studies in biomedical and

cancer research. So an attempt was made for a high yielding

alternative purification method employing the use of asialoftuin CL

agarose column for the above mentioned lectin, designated CTL.

Kogawa K., et al., (2007) reported Biosynthesis of malonylated

flavonoid glycosides on the basis of malonyltransferase activity in

the petals of clitoria ternatea. The crude malonyltransferase from

the petals of Clitoria ternatea was characterized enzymatically to

investigate its role on the biosynthetic pathways of anthocyanins

and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB)

and mauve flower variety (WM) accumulate polyacylated

anthocyanins (ternatins) and delphinidin3-O- (6”-O-malonyl)-β-

glucoside which is one of the precursors of ternatins, respectively

Moreover WM, accumulates minor delphinidin glycosides-3-O-B

glucoside, 3.-O (2” O-a-rhamnosyl)-B- glucoside .

Juma H.K., et al., (2006) reported evaluation of clitoria, Gliricida

and Mucuna as nitrogen supplements to Napier grass basal diet in

relation to the performance of lactating Jersey cows. A study was

carried out at the Kenya Agricultural Research Institute Mywapa in

Coastal lowland Kenya to evaluate the effects of supplementing

Napier Grass variety Bana (Pennisetum Purpureum ) with clitoria

ternatea (Clitoria), Gliricidia sepium (Gilricidia) and Mucuna

Pruriuens (Mucuna) on feed intake, diet, digestibility and milk yield

of lactating Jersey cows.

Parimaladeve B. et al., (2004) reported evaluation of antipyretic

potential of clitoria ternatea L. extract in rats. The methanol extract

of Clitoria ternatea L. root (MECTR) blue flowered variety (family:

faba-ceae), was evaluated for its anti-pyretic potential on normal

body temperature and yeast induced pyrexia in albino rats. Yeast

suspension (10ml/kg body wt.) increased rectal temperature after

19 hours of subcutaneous injection. The extract at doses of 200,300

and 400 mg/kg. Body wt., p.o., produced significant reduction in

normal body temperature and yeast provoked elevated temperature

in a dose dependent manner. The effect extended up to 5 hours

after the drug administration. The anti- pyretic effect of the extract

was comparable to that of paracetamol (150 mg/Kg. body wt., p.o.,)

a standard anti-pyretic agent.

Nataraja K., et al., (2005) reported screening of antibacterial

activity in the extract of clitoria ternatea. Hexane methanol and

water extracts of leaf, stem and roots of white flowered variety of

clitoria ternatea (Linn.) (Febaceae) used by Indian traditional healers

for treating ulcer, eye infections, bronchitis, tuberculosis and/or anti-

inflammatory properties were screened for in vitro antibacterial

activities.

Kelemu S., et al., (2005) reported evaluation of antipyretic

potential of clitoria ternatea L. extract in rats. The tropical forage

legume clitoria ternatea (L.) has important agronomic traits such as

adaptation to a wide range of soil conditions and resistance to

drought. It is resistant to a number of pathogens and pests. These

import-ant traits gave reasons to look more closely at the plant. A

highly basic small protein was purified from seeds of C. ternatea to

homogeneity by using ultrafiltration with Centricon-3 membrane

tubes and preparative granulated- bed isoelectric focusing (IEF). A

single protein band was obtained on both sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) and IEF gels.

Shroff S.K., et al., (2003) reported clitoria ternatea and the CNS.

The present investigation was aimed at determining the spectrum of

activity of the methanolic extact of Clitoria ternatia (CT) on the CNS.

The CT was studied for its effect on cognitive behavior, anxiety,

depression, stress and convulsions induced by pentylenetetrazol

(PTZ) and maximum electroshock (MES) to explain these effects the

effect of CT was also studied on behavior mediated by dopamine

(DA), noradrenaline, serotonin and acetylcholine. The extract

decreased time requ-ired to occupy the central platform (transfer

latency TL) in the elevated plus maze (EPM) and increased

discrimination index in the object recognition test, indicating no

tropic activity.

Nagappa A.N. et al., (2003) studied antidiabetic activity of

Terminalia catappa Linn. Fruits. In view of alleged antidiabetic

potential, effect of the petroleum ether, methanol, and aqueous

extracts of Terminalia catappa Linn (combretaceae) fruit, on fasting

blood sugar levels and serum biochemical analysis in alloxan

induced diabetic rats were investigated. All the three extracts of

Terminalia catappa produced a significant antidiabetic activity at

dose levels 1/5 of their lethal doses. Concurrent histological studies

of the pancreas of these animals showed comparable regeneration

by methanolic and aqueous extracts which were earlier, necrosed

by alloxan.

Babu V. et al., (2003) reported antidiabetic activity of ethanol

extract of Cassia kleinii leaf in streptozotocin induced of diabetic

rats and isolation of an active fraction and toxicity evaluation of the

extract.

Kazuma K. et al., (2003) reported malonylated flavonal

glycosides from the petals of clitoria ternatea. Three flavonol

glycosides kaempf-erol 3-O-(2”-O-a-rhamnosyl-6-O-malonyl)-β-

glucoside, querce-tin 3-O-(2---O-a rhamnosyl-6”-O-malonyl-β-

glucoside and myricetin 3-O- (2”-6-di-O-a-rhamnosyl)-β-glucoside

were isolated from the petals of Clitoria ternatea cv., Double Blue,

Together with eleven known flavonol glycosides. Their structures

were identified using UV, MS and NMR spectroscopy.

Boominathan R. et al., (2003) reported Anti- Inflammatory,

analgesic and antipyretic properties of Clitoria ternatea root. Clitoria

ternatea roots methanol extract when given by oral route to rats

was found to inhibit both the rat paw oedema caused by carrageen

in and vascular permeability induced by acetic acid in rats.

Moreover the extract exhibited a significant inhibition in yeast

induced pyrexia in rats. In the acetic acid- induced writhing

response, the extract markedly reduced the number of writhings at

doses of 200 and 400 mg/kg. (p.o.,) in mice.

Kazuma K. et al., (2003) reported flavonoid composition related

to petal color in different lines of Clitoria ternatea. Flavonoids in the

petals of several Clitoria ternatea lines with different petal colors

were investigated with LC/MS/Ms. Delphinidin 3-O-(2”O-a-rhamnosyl-

6-O-malonyl)-B- glucoside was newly isolated from the petals of a

mauve line (wm) together with three known anthocyanins. They

were identified structurally using UV, Ms, and NMR spectroscopy.

Although ternatins a group of 15 (poly) acylated delphinidin

glucosides were identified in all the blue petal lines Double Blue and

Albiflora WM, accumulated delphinidin glucosides were identified in

all the blue petals lines.

Rai K.S. et al., (2002) reported clitoria ternatea root extract

enhances a cetylcholine content in rat hippocampus. Treatment with

100 mg/Kg. of clitoria ternatea aqueous root extract (CTR,) for 30

days in neonatal and young adult age groups of rat, significantly

increased acetylcholine (ACh) content in their hippocampi as

compared to age matched controls. Increase in ACh content in their

hippocampus may be the neurochemical basis for their improved

learning and memory.

Chaudhari U.S. and Hutke V., (2002) reported Ethano-medico-

botanical information on some plants used by melghat tribes of

Amravati district, Maharashtra. The paper deals with ethnobotanical

uses of 14 plant species among the Korian and Gond tribes living in

Melghat forests of Amravati district. The study comprises

information on traditional formulations, modes of administration and

the ailments for which they are effective. Use of root of clitoria

ternatea mixed with hen blood and honey in chronic cough is found

to be a unique method of cure. Chlorophytum borivilianum and

Plumbago are preferred as medicines, moreover leaves are

generally uses food.

Terahara N. et al., (1996) Reported Five new anthocyanins,

Ternatins A3, B4, B3B2, and D2 from clitoria ternatea flowers.: Five

new ternatins 1-5 have been isolated from Clitoria ternatea flowers,

and the structures have been determined by chemical spectroscopic

methods as delphinidin 3-GCG-5-GCG-3-GCG-5CG3’- and 3’-GCGC-

side chains respectively in which G is D-glucose and C is a coumaric

acid. Pigment 1 had symmetric 3’5- side chains. Compounds 3 and 4

are structural isomers. These trernatins were shown to form an intra

molecular stacking between the aglycon ring and the 3’5-‘side

chains in solution.

Bavaliya N.K., (1993) reported poisonous lagurnes of Rajasthan 5

The paper deals with 33 poisonous leguminous species which are

toxic to men, animals, fishes and livestock or other living things

while enumerating the species are arranged alphabetically, with

their habit common/local name (s) toxic part of the plant toxic to

which living organism and their distribution in state of Rajasthan in

a tabular form.

Terahara N. et al., (1990) Reported acylated anthocyanims of

clitoria ternatia flowers and their acyl moieties. Two acyl moieties

prepared by alkaline deacylation or H2O2 oxidation of ternatin

mixture from Clitoria ternatea flowers, were determined as E-4-O-B-

D glucopyranoisyl p-coumaric acid and 6-O-malonylD-

glucopyranose respectively through FABMS and NMR. Furthermore

six ternatins A1, A2, B1, B2, D1 and D2 in C. ternmatea flowers

were isolated by reversed phase HPLC and their structures were

partly characterized as highly acylated delphinidin derivatives.

Venkatesh S. et. al., (1969) reported antidiabetic activity of

helicteres isora root. The different extracts of the roots of helicteres

isora (Family Sterculiaceae) were tested for antidiabetic activity by

glucose tolerance test in normal rats and alloxan induced diabetic

rats. alloxan diabetic rats the maximum reduction in blood glucose

was observed after 3h at a dose level 250 mg/kg of body weight.

3. PLAN OF WORK

1- Literature survey of selected medicinal plant.

2 - Collection and Authentication of Clitoria ternatea leaves.

3 - Phytochemical Investigation:

A. Extraction of drug powder

B. Phytochemical test.

C. Thin layer chromatography, HPTLC and Column

Chromatography.

D. Identification and characterization of constituent by H-

NMR, IR and Mass spectroscopy.

4 - Assesment of antidiabetic activity.

5 - Assesment of anti-inflammatory activity.

6 - Statistical Analysis.

4. COLLECTION & EXTRACTION

4.1 COLLECTION AND AUTHENTICATION OF CRUDE DRUG

The fresh leaves of Clitoria ternatea was collected during the

month of September 2008, from my village Kailiya and gandoli

(Distt-Jalaun), the Kush Nursury, Gwalior Road, Jhansi and from the

Institute of Pharmacy, Bundelkhand University, Jhansi The plant

materials was taxonomically identified and authenticated by Dr.

Gaurav Nigam, Botany Department, Bundelkhand University, Jhansi.

Herbarium and Museum Division with ref. no. BU/BOT /376/24-01-

2009.

4.2 EXTRACTION:

The leaves of Clitoria ternatea were shaded dried until

cracking sound was observed during breakage, and then these are

made into coarsely powdered from using dry grinder. The powdered

leaves of the plant (600 gm.) was packed in soxhlet apparatus and

continuously extracted with petroleum ether (40-600C) till complete

extraction, after completion of extraction the solvent was

removed by distillation and then concentrated extract obtained was

dried under reduced pressure using rotatory evaporator at

temperature not exceeding 400C and then give moderate heating

on water bath. A pale green extract approximate 18 gm. was

obtained. From the drug, petroleum ether was removed and the

defatted drug was extracted with methanol till complete extraction,

after completion of extraction the solvent was removed by

distillation and then concentrated extract obtained dried under

reduced pressure at temperature not exceeding 400C and then give

moderate heating on water bath. The methanolic extract obtained

was greenish black in colour, weighed about 40 gm. The both

petroleum ether and methanolic extract was kept in petridish and it

was stored in desiccator at cool place (Mukherjee, 2002).

Powdered Crude Drug

Extracted with Petroleum Ether

(40-600C)

Defatted Powdered Drug Petroleum Ether Extract

Dried in Hot Air Oven Below 500 C

Extracted with Methanol Solvent removed by Distillation

Extracted Drug (Discarded) Methanolic extract

Traces of solvent removed under

Reduced pressure

Transferred remaining to a tarred dish

and dried to constant weight

Extract of Drug was collected and stored

in a Dessicator at room temperature

Fig.4.1 The Extraction procedure in schematic manner.

Table:4.1 The characteristics of methanolic extract.

S.

No.Characteristics

Methanolic

extract

Pet. Ether

extract

1. Extractive Value (%) 6.66 % 3%

2. Physical appearance Semisolid mass Semisolid mass

3. Colour Greenish black Yellowish green

4. Odour Odourless Odourless

5. Taste bitter bitter

5. PHYTOCHEMICAL SCREENING

The systematic phytochemical investigations not only help in

revealing the active components but also help in the synthesis of

better and newer analogues and congeners of higher therapeutics

activities or the various active principals isolated from plants. The

products of the investigations sometimes prove to be significant

than the ordinary plant constituent.

It is desirable not only for the discovery of new therapeutic

agents but also because such information may lead to the new

source of economically useful material and intermediates for the

synthesis of complex chemical substances. Again isolation of the

compound which is not necessarily of any intrinsic value in itself

but has a novel chemical structure may be stimulate the chemist

to modify the molecule to obtain semi synthetic substances having

medicinal and other useful properties.

Modern pharmacognosy has been developed rapidly to the

improvement made in the technology of isolation process which

includes the development techniques such as column, paper, thin

layer, gas, liquid, high performance liquid and droplet counter-

current chromatographic procedure. These methods have allowed

the rapid isolation of compounds, which are previously difficult to

obtain by classical procedures. The most important factor has been

the development of new spectroscopic techniques which are used to

identify structures of the isolated compounds, by which it become

easy to develop the new molecules and it is beneficial for the

research point of view, and now-a-days every research laboratory

having the latest techniques which help in the development of new

compounds.

PLANT PHYTOCHEMICAL SCREENING:

1. Qualitative chemical analysis.

2. Thin Layer Chromatography

3. High Performance Thin Layer Chromatography

4. Isolation of active constituent.

a) Column Chromatography

5. Characterization by

a) IR Spectroscopy

b) Mass Spectroscopy

c) Proton NMR

5.1 QUALITATIVE CHEMICAL ANALYSIS:

The plant extracts were subjected to preliminary

phytochemical screening for the detection of various plant

constituents present in the leaves of Clitoria ternatea.

a)Test for alkaloids:

Stirrer a small portion of the methanolic extract with a

few drops of dilute hydrochloric acid and filter. The filtrate were

tested with various alkaloid reagents such as Mayer’s reagent

(cream precipitate) Dragendroff’s reagent (orange brown

precipitate) and Wagner reagent (reddish brown precipitate).

Mayer’s reagent: Few drops of Mayer’s reagent were added

in each of the extract and observed formation of the white or cream

colored precipitates.

Dragendorff’s reagent: Few drops of dragendorff’s reagent

were added in each of the extract and observed formation of the

orange yellow or brown colored precipitates.

Wagner’s reagent: Few drops of Wagner reagent were

added in each of the extract and observed formation of the reddish

brown precipitates.

b) Test for Carbohydrates: Dissolve small quantities of

methanolic extract in 4 ml of distilled water and filter. The filtrate

may be subjected to Molisch’s test to detect the presence of

carbohydrates.

Molisch’s test: To small quantity of extract few drops of α-

napthol (20% in ethyl alcohol) were added. Then about 1 ml of

concentrated sulphuric acid was added along the side of the

tube. Reddish violet ring appeared at the junction of two

layers. It indicates the presence of carbohydrates.

Fehling’s Test: The 1 ml of Fehling’s reagent (copper

sulphate in alkaline conditions) was added to the filtrate of the

extract in distilled water and heated in a steam bath. Brick red

precipitates appeared which confirm the presence of

carbohydrates.

C) Test for Glycosides:

Hydrolyse small portion of the extract with dilute hydrochloric

acid for a few hours in water bath and subjected to hydrolysate to

Liebermann Burchard’s, Keller Killiani, Sodium nitrosoprusside and

Borntrager’s tests to detect the presence of different glycoside.

Small quantity of the extract was taken separately and subjected to

the following tests.

Keller Killiani Test: The 1 ml of glacial acetic acid containing

traces of FeCl3 and 1 ml of concentrated H2SO4 was added to

the extract carefully.

Bluish green colour appeared which confirm the presence of

glycosides in the extract.

Sodium nitrosoprusside test: The extract was made

alkaline with few drops of 10% sodium hydroxide and then

freshly prepared sodium nitrosoprusside solution was added

to it. Blue colour confirms the presence of glycosides in the

extract.

Borntrager’s test: The 1ml of benzene and 0.5 ml of dilute

amonia solution were added to the extract. A reddish pink

colour was obtained which show the presence of glycosides in

the extract.

D) Test for phenolic compounds and tannins:

Take small quantities of alcoholic extract in water and test for

the presence of phenolic compounds and tannins with dilute

ferric chloride solution (5%) and lead acetate test.

Ferric chloride test: On addition of ferric chloride solution

(5%) green or blue colour was observed, due to the presence

of phenolic compounds and tannins. No colour appeared which

shows the absence of phenolic compounds.

Lead Acetate test: Few drops of lead acetate solution (5%)

were added to the alcoholic extract. white precipitate was

appeared which confirm the presence of phenolic compounds.

E) Test for flavonoids:

Ammonia test: Filter paper strip were dipped in the alcoholic

solutions of the extract and ammoniated. The filter paper

changed its colour to yellow which indicates the presence of

flavonoids.

Pew test for flavonoids: To the small portion of the extract,

a piece of metallic magnesium/zinc was added followed by

addition of 2 drops of concentrated hydrochloric acids. A

brownish colour confirmed the presence of flavonoids in all

the extract.

F) Test for proteins and free amino acids:

Add small portion of alcoholic extract in a few ml of distilled

water and subjected the solution to million’s, Biuret and

Ninhydrin tests.

Million’s test: To the small portion of extract 5-6 drops of

million’s reagent (solution of mercury nitrate and nitrous acid)

were added. A red colour precipitate appeared which confirms

the presence of proteins and free amino acids.

Ninhydrin test: To the extract, lead acetate solution was

added to precipitate tannins and filtered. The filtrate was

spotted on a paper chromatogram, sprayed with ninhydrin

reagent and dried at 1100C for 5 minutes. Violet spots were

seen which confirm the presence of proteins and free amino

acids.

Biuret test: 1 ml of 40% sodium hydroxide solution and

2dropsof 1% copper sulphate solution was added to the

extract (1 ml.). The formation of violet color indicates the

presence of proteins.

Xanthoprotein test: 1ml of concentrated nitric acid was

added to the extract. Boil the white precipitate, if any, and

cool. Added 20% of sodium hydroxide or ammonia solution.

Orange color indicates the presence of aromatic amino acids.

G) Test for saponin:

Dilute small portion of alcoholic extract with distilled water to 20

ml and shake in a graduated cylinder for 15 minutes. A one -

centimeter layer of foam indicates the presence of saponin.

H) Test for Steroids:

Lieberman Burchard’s test: Dissolved the extract in 2 ml of

chloroform in a dry test tube. Added 10 drops of acetic

anhydride and 2 drops of concentrated sulphuric acid. The

solution becomes red, then blue and finally bluish green,

indicating the presence of steroids.

Salkowski test: Dissolved the extract in chloroform and

added equal volumes of concentrated sulphuric acid. The

formation of bluish red to cherry red color in chloroform layer

and green fluorescence in the acid layer represents the

steroid components in the tested extract.

Table: 5.1 Qualitative Phytochemical Analysis:

S.No

.Tests

Methanolic

extract

Pet. Ether

extract

1.

Alkaloids

Dragendorff’s test

Wagner’s test

Mayer’s test

Hager’s test

--

--

--

--

--

--

--

--

2.Carbohydrates

Molisch’s test

Fehling’s test

+

--

+

--

3.Proteins

Biuret test

Xanthoprotein test

+

--

+

--

4. Amino acid

Ninhydrin test+ --

5. Flavonoids

Shinoda test + --

6.Phenolic

compounds-- --

7.Glycoside

Keller Killiani test

Borntrager’s test

+

+

--

--

5.2 THIN LAYER CHROMATOGRAPHY:

5.2.1 General:

The technique thin layer chromatography was first introduced by

Izmailov and Shraiber in 1938. They used this technique for

separating plant extract on 2 mm thick and firm adhesive layer of

alumina set on glass plate. Condon, Gorden and Martin (1944)

started using filter papers. Williams carried out chromatograph on

adsorbent layer sandwiched between two glass plates; one of them

has a small hole through which solutions and developing solvents

were applied to the layer.

In 1958, Stahl demonstrated applications of TLC, a method

based on adsorption chromatography which is at important

analytical tool for qualitative and quantitative analysis of a number

of phytochemical substances. TLC is the first important step for

phytochemical screening. It also serves as a pilot technique for

column chromatography. (Chatwal, 2004)

TLC is the method mainly uses to investigate the presence of

chemical constituent qualitatively and quantitatively in the plant

extract. It is used to investigate alkaloids, glycosides, isoprenoids,

lipid component, sugar and their derivates etc. This component can

also run with standards for the investigation. It is an easy, versatile

and reliable method to establish authenticity, identity and purity.

(Kokate et. al., 2000)

5.2.2 Basic Principles of TLC:

Separation by TLC is effected by the application of the

mixture or extract as a spot or thin line on to a sorbent that has

applied to a backing plate. Analytical TLC plates (thickness 0.1-0.2

mm) are commercially available; e.g., the commonest analytical

silica gel plate is the 20x20 cm. Plastic or aluminum backed

Kieselgel 60 F254 plate, which has a 0.2 mm thickness of silica

sorbent. The plate is then placed into a tank with sufficient suitable

solvent to just wet the lower edge of the plate-sorbent but not

enough to wet the part of the plate where the spots were applied.

The solvent from then migrates up the plates through the sorbent

by the capillary action. A process known as development

The information provided a finished chromatography includes

the migrating behavior of the separated substances. It is given in

the form of the Rf value (relative to front)

Distance travelled by spot

Rf=

Distance travelled by solvent front

(Mukherjee P. K., 2002)

5.2.3 Methods & equipments of Thin Layer

Chromatography Apparatus:

The apparatus employed consisted of the following components:

1. Rectangular glass chambers (30 x 15 x 8 cm) with ground

glass rim on which a glass lid was placed. Grease was

applied on the rim of the chamber to make the glass jar

airtight.

2. Glass Sheets (20 x 5 cm) used for the preparation of thin

layer plates.

3. Sprayer for detection.

5.2.4 Preparation of Plates:

Silica gel G was used as the adsorbent. Slurry of it was prepared

with distilled water in a glass pestle mortar. The slurry was poured

on the clean and dry glass plates and spread on the plate as a

uniform coating using a glass rod. These plates were then placed on

a leveled surface in the horizontal position and allowed to air dry for

20-25 minutes.

5.2.5 Activation of Plates:

When the plates were dried they were placed in an oven.

Maintained at 1100 C for 30 minutes. The prepared plates were

stored in a closed desiccated cabinet and removed only when

required for use.

5.2.6 Preparation of Samples:

About 100 mg of test material was dissolved in 10 ml of the

respective solvent and was used for the TLC studies.

5.2.7 Application of Spots:

The spots were applied on the activated plate at a distance of 2

cm from one end of the plate and 3cm from each other with the help

of a fine capillary tube or diameter less than 1mm. The solvent was

removed from spot by air drying. The position of the origin was

marked.

5.2.8 Saturation of TLC chamber:

The inner wall of the chamber was lined with filter paper from all

side except the front face to maintain a solvent saturated

atmosphere. The solvent system was poured into the chamber up to

a height of 1 cm from the base. The mouth of the chamber was then

closed with a rectangular glass plate and made airtight with grease.

The chamber was then allowed to stand till the filter paper became

completely wetted with solvent vapors.

5.2.9 Development of Chromatograms:

Chromatograms were developed by one way ascending TLC. The

plate carrying spots was placed squarely in the developing chamber

and the lid was replaced as quickly as possible to minimize

disturbance of the solvent saturated atmosphere. The developing

solvent was allowed to travel up the plate until it reached the

desired level (10 to 15 cm). The plate was then removed from the

chamber; solvent front was marked and dried in air at room

temperature.

5.2.10 Detection of Spots:

The number and position of the various constituents present in

the mixtures was determined by spraying the plate with the 1%

vanalin in sulphuric acid and the plate was heated at 1100C for 10

minutes and the spots were marked. Rf value was calculated for well

defined spots.

5.2.11 Advantages of TLC:

a) TLC is an elegantly simple procedure for chromatography in

all kinds of solid-liquid and liquid- liquid systems.

b) TLC can be performed on an analytical as well as on a

preparative scale.

c) It may be applied to almost the entire spectrum of chemical

compounds.

d) TLC can be used for uncovering adulterations of food as well

as decomposition of foods and drugs caused by improper

storage or incorrect use, because of its great resolving power.

e) TLC is of great help in chemical taxonomy.

f) In chemical laboratory, morphological appearance of tissues

can be associated with their chemical composition as detected

by chromatographic patterns of tissue extracts.

g) The great advantages of TLC are often most profitably

exploited when TLC is employed in conjugation with other

methods of analysis. (Sethi, 2005)

5.2.12 Thin Layer Chromatography of Methanolic Extract:

100 mg of methanolic extract was weighted and dissolved in 10

ml of methanol and filtered. Filterate was taken as sample for TLC.

Table 5.2: TLC of Methanolic extract:

S. No Solvent systemNumber of spots

Resolution

1Chloroform: Ethylacetate

( 6:4 )4

spotGood

2Benzene: Diethylether

(3:7)4

spotGood

3Ethylacetate: : Methanol: Water

(7:2:1)4

spotGood

4Benzene: Ethylacetate

(8.5:1.5)4 spot Good

5Benzene: Methanol: Formic Acid

(8.5:1:0.5)5

spotExcellent

Adsorbent – Activated Silicagel-G

Detecting agent – Iodine

According to combination tried above it was found that Benzene:

Methanol: Formic Acid (8.5:1:0.5) may be the best solvent.

Table 5.3: Rf value of the spots of methanolic extract:

S. No. Rf value Developed in iodine

1. 0.12 Brownish

2. 0.24 Brownish

3. 0.57 Brownish

4. 0.68 Brownish

5. 0.92 Brownish

Photo: 2 TLC of Clitoria ternatea extract

Detecting agent – Iodine

5.3 High Performance Thin Layer Chromatography:

5.3.1 General:

Standardized manufacturing procedures and suitable analytical

tools are required to establish the necessary framework for quality

control in herbals. Among those tools separation techniques

including high performance liquid chromatography (HPLC), high

performance thin layer chromatography (HPTLC) and capillary

electrophoresis are the most widely used to establish reference

fingerprints of herbs, against which raw materials can be evaluated

and finished products can be assayed.

High performance thin layer chromatography also known under

the synonym planar chromatography which is a modern, powerful

analytical technique with separation power and reproducibility

superior to TLC.

5.3.2 Advantages of HPTLC:

Unsurpassed flexibility by design and being an off line

technique, HPTLC is extremely flexible with following advantages.

Choice of Detection.

Cost and time efficiency

User friendliness and Result Presentation

One time use of the TLC plate.

5.3.3 Requirements for HPTLC standardization:

For the analysis of herbals, HPTLC offers a number of

advantages. This technique is especially suitable for comparison of

samples on scanning densitometry or video technology. It has

become a cost and time effective alternative to HPLC.

Fingerprint analysis by HPTLC or HPLC is one of the most

powerful tools to link the botanical identify to the chemical

constituent profile of the plant, in combination with microscopic

investigations the fingerprint provides for a convenient identity

check. It can also be used to detect adulterations in raw materials.

This technique further describes the quality of the herb and the

herbal preparation. High performance thin layer chromatography

can also be employed for quantitative determination of such marker

compounds.

The production of most herbals preparations includes some

extraction process. It is essential for quality assurance that this

extraction is standardized, the quantity of marker compounds of

their relative abundance assayed by HPTLC or HPLC which are the

principle methods of monitoring. When choosing marker compounds

for a particular herb or herbal preparation. It is of critical

importance that chemically well characterized standards are

available for their qualification. It is often impossible to separate all

components of a plant extract completely.

A useful method for this purpose could be adopting the following

parameters as standard.

Place HPTLC plate 10 x 10 cm or 20 x 10 cm.

Sample application: application of bands (5-10 mm in length)

using the spray on technique.

Chamber saturated twin-trough or flat bottom chamber.

Developing distance 50-60 mm

Derivatization by immersion

Fig: 5.1 HPTLC of Clitoria ternatea extract

5.3.4 Result:

HPTLC of extract show the ten peaks confirming that the ten

compound may be present in the methanolic extract of the leaves of

Clitoria ternatea.

5.4 COLUMN CHROMATOGRAPHY:

It is used for separation and isolation of different constituents of

the methanolic extract of Clitoria ternatea.

5.4.1 Apparatus:

A glass column of 60 cm. length and 2.9 m diameter was taken

it was thoroughly cleaned, dried and checked for any type of

leakage. At the lower end of the column about one inch bed of glass

wool was placed for collection of elutes. Clean and dry beaker (100

ml) was uses.

5.4.2 Adsorbent:

Silica gel (60-120 Mesh, Merck) was used for column

Chromatography. The powder was activated at 1100C for an hour in

hot air oven prior to use in the column.

5.4.3 Packing of the Column:

The wet packing method was adopted. Initially the lower end of

the glass column was plugged with glass wool. Methanol was poured

on to the glass wool to release any air bubbles, which might be

trapped with the flat end of a packing rod. A portion of the slurry of

activated silica gel in hexane was poured in to the column.It should

be added continuousily. The side of the column was tapped gently

with a glass rod to even the compaction of the particles as the silica

gel settled. The outlet of the column was then adjusted so that the

eluent was continuously released but a small solvent head was

maintained on the top of the column.

5.4.4 Eluent:

Hexane, Benzene Methanol and Ethanol are generally used for

elution of column with increasing polarity.

5.4.5 Application of Sample:

The sample was prepared in respective solvent and added

slowly by the sides of the glass column without disturbing the

column packing. Then outlet of column was opened until the sample

got absorbed in silica gel in column.

5.4.6 Column Chromatography of Methanolic extract:

5.4.6.1 Preparation of Sample:

Methanolic extract was dried in reduced pressure and dissolve in

minimum quantity of methanol, mixed with silica gel, then dried,

and applied in the column and eluted with Benzene: Methanol

(90:10)

5.4.6.2 Collection of Samples in Volumetric:

First approximately 500ml, solvent Hexane: Benzene was

prepared for eluting column, which is collected in 25 ml. volumetric

flask and TLC was performed for each volumetric flask. The samples

showing the same TLC pattern were mixed as shown in table 5.4

Table 5.4: Column Chromatography of isolated compound

S.No.

EluteVolume

collected (ml)

No of spot

Code

1. Hexane: Benzene (95:5) 1-4 No spot R-1

2. Hexane: Benzene (90:10) 5-10 No spot R-2

3. Hexane: Benzene (85:15) 11-20 No spot R-3

4. Hexane: Benzene (75:25) 21-30 One spot R-4

5. Hexane: Benzene (50:50) 31-40 No spot R-5

6. Benzene (100) 41-50 No spot R-6

7. Benzene: Methanol (95:5) 51-60 No spot R-7

8. Benzene: Methanol (90:10) 61-70 Two spot R-8

9. Benzene: Methanol (85:15) 71-80 One spot R-9

10 Benzene: Methanol (75:25) 81-90 Three spot R-10

11. Benzene: Methanol (50:50) 91-100 No spot R-11

12 Methanol (100) 101-110 No spot R-12

The entire fraction (1to 40) were subjected to TLC using solvent

system Hexane: Benzene and entire fraction (41to 100) were

subjected to TLC using solvent system Benzene: Methanol. Fraction

R-9 showed single spot and was in sufficient quantity for analysis,

hence selected for further characterization. Other fraction R-1, R-2,

R-5 showed no spot and fraction R-4 was not studied due to very

minimum quantity of the isolates so the fractions were not applied

for further isolation.

5.5 CHARACTERIZATION OF ISOLATED COMPOUND:

The compound which is isolated in column chromatography is

characterized by the analytical techniques such as Infrared

spectroscopy, NMR spectroscopy and Mass spectroscopy.

5.5.1 Infrared Spectroscopy

Infrared spectroscopy is generally sensitive to the presence of

functional groups in the samples. The most powerful aspects of

Infrared spectroscopy is that it allows identification of unknown

compound. IR spectroscopy of compound (R-9) was performed in

CDRI, Lucknow. Spectra of compound have shown in figure 5.3. The

interpretation that can be made from spectra has shown in table 5.5

Fig: 5.2 IR spectra of compound (R-9)

Table 5.5: Interpretation of IR Spectroscopy

Wave number (cm-1) Functional Group

3357.3 O-H Stretching alcohol and phenols

2945.2 C-H Stretching Alkane

2833.3 C-H Stretching in aldehyde

1453.4 C-H Bending in Alkane

1114.3 C-N Vibration in Aliphatic

1030.3 C-OH

5.5.2 Mass Spectroscopy:

The mass spectroscopy of compound (R-9) was performed at

C.D.R.I. Lucknow, the mass spectra is used to determine the

possible fragmentation in the compound. The mass spectra of

compound have shown in Fig. 5.4 the spectra exhibited various

peeks suggesting fragmentation pattern.

Fragmentation data of isolated compound (R-9): 81, 95, 109,

154.

Fig: 5.3 Mass Spectra of Isolated compound (R-9)

Table 5.6: Interpretation of Mass Spectroscopy

m/z Relative intencity

95 100

109 85

154 70

81 65

5.5.3 NMR Spectroscopy:

The NMR spectroscopy of compound (R-9) was performed at

C.D.R.I. Lucknow, the mass spectra is used to determine the

possible Proton in the compound. The NMR spectra of compound

have shown in Fig. 5.5

Fig: 5.4 NMR Spectra of Isolated compound (R9)

Table 5.7: Interpretation of NMR Spectroscopy

δ(ppm) of Std.

Compouund

δ(ppm) of Isolated

Compouund

Inference No.of Protons

8.91 8.30 s 1H

6.66 6.68 d 1H

6.86 6.89 d 1H

7.74 7.54 s 2H

3.96 3.94 dd 1H

3.71 3.71 t 1H

3.47 3.48 t 1H

3.50 3.48 ddd 1H

3.83 3.83 dd 1H

3.66 3.66 dd 1H

3.30 3.26 t 1H

3.63 3.66 dd 1H

0.91 0.92 d 3H

Analytical Result of Isolated Compound R-9:

In 3-neohesperidoside, functional group OH, CH, C-OH, hydroxy,

alcohal, Vinylic present and elemental analysis of isolated

compound show C=37.19%, H=12.88%.

According to above study, the isolated compound may be 3-

neohesperidoside, colour has yellowish green and Rf values of

isolated compound 0.49 and elemental analysis of isolated

compound show C=37.19%, H=12.88%.

Chemical name – 3-neohesperidoside

Molecular formula- C30H33O19

6. PHARMACOLOGICAL INVESTIGATIONS

6.1 EVALUATION OF ANTIDIABETIC ACTIVITY:

6.1.1 EXPERIMENTAL METHODS OF DIABETES:

1. Alloxan Induced Diabetes:

A. Purpose and Rational:

It has been described mainly for dogs; rabbits, and rats,

guinea pigs have been found resistant to it. In most species,

triphasic time course is observed a rise of glucose found by a

decrease, probably due to depletion of islets from insulin, again

followed by sustained increase of blood glucose.

B. Procedure:

a) Rabbits: Weighing 2.0 to 3.5 Kg are infused via ear with 150

mg/Kg alloxan monohydrates (5.0 gm/100 ml. pH 4.5) for 10

minutes resulting in 70% of the animals become hyperglycemic and

uricosuric.

b) Rats of wistar or Sprange-Dawley strain: weighing 150-

200 gm are injected subcutaneous with 100-175 mg/Kg alloxan.

c) Male Beagle dogs: Weighing 15-20 Kg are injected

intravenously with 60 mg/Kg alloxan, subsequently animals receive

daily 1000 ml 5 Glucose solution with 10 I.U. regular insulin for one

week and canned food ad libitum.

2. Streptozotocin Induced Diabetes:

A. Purpose and Rational:

The antibiotic streptozotocin is an antidiabetic having diabe-

togenic activity. The compound turned out to be specifically

cytotoxic to β-cells of the pancreas.

B. Procedure:

Male wistar rats weighing 150-220 gm fed with standard diet

were injected with 60 mg/kg streptozotocin intravenously. Six to

eight hour after streptozotocin injection, the serum insulin values

are increased up to 4 times resulting in hypoglycemic phase. This is

followed by persistant hyperglycemia. Severity and onset of

diabetes symptoms depend on the dosage of streptozotocin.

Although 60 mg/kg dosage of streptozotocin cause hyperglycemia in

24-48 hours upto 800 mg % due to β-cells degranulation yet a

steady state is reached in 10 to 14 days allowing the use of animals

for pharmacological test. Other authors have also described the

modification of method in other animals.

3. Hormone Induced Diabetes:

A. Growth Hormone Induced Diabetes:

Pure anterior pituitary growth hormone shows diabetogenic

action in cats. Rats of any age subjected to a similar treatment do

not become diabetic but grow faster and shows striking hypertrophy

of the pancreatic islets.

B. Corticosteroid Induced Diabetes:

Forced fed rats treated with cortisone causes hyperglycemia

and glycosuria. In the guinea pig and rabbit, experimental corticoid

diabetes could be obtained without forced feeding. In the rats, the

adrenal cortex stimulated by corticotrophin has the capacity to

secrete steroids which induced steroids diabetes.

4. Other Diabetogenic Compounds:

Various chelators like dithiazone, gold thioglucose and mono-

sodium glutamate in a single i.v. dose of 40-100 mg/kg to cats,

rabbits, hampster, rats cause a triphasic diabetic state in rabbit.

Initial phase is hyperglycemic and normoglycemic and again

permanent hyperglycemic in 24-72 hours due to complete or partial

degranulation of β-cells.

Many other methods are also described by author. The

method which we have used here is alloxan induction method and

oral glucose tolerance test. (Vogel, 2004)

6.1.2 EXPERIMENTAL WORK:

1. Effect of methanolic extract on alloxan induced diabetic rats.

2. Effect of methanolic extract on glucose loaded rats.

A. Animals:

The adult male albino rats of weight 180-240 gm were

selected for the study. All animals were procured from disease free

animal house, Institute of Pharmacy, Bundelkhand University, Jhansi.

The Institute of Pharmacy is approved by Institutional Animal Ethical

Committee (716/02/a/CPCSEA). The animals were housed in

polypropylene cages, 5 per cage with free access to standard

laboratory diet and water ad libitum. The rats were maintained

under standard laboratory conditions at 25±20C relative humidity

50±15% and normal photo period (12 h dark/ 12h light) were used

for experiment.

B. Drugs:

Alloxan of CDH, New Delhi was used for the inducted of

diabetes and was obtained from Department of Pharmacy and the

standard drug i.e. glibenclamide was send by Sun Pharmaceutical

Industries, J & K.

C. Extraction of Plant:

The powder of leaves of Clitoria ternatea was subjected to

extraction in methanol. The extract was then concentrated at

reduced pressure and used for the experimentation.

D. Preparation of Dose:

The Dose of 200 mg/kg and 400 mg/kg of methanol extract

was selected for the test. All the doses was given orally after

making emulsion in vehicle i.e. 1% acacia gum and the standard

drug i.e. glibenclamide was given orally (10 mg/kg) in the vehicle.

1. Effect of Methanolic extract on alloxan induced

diabeticrats:

A) Induction of experimental diabetes:

Diabetes mellitus was induced by administering

intraparitoneal injection of alloxan monohydrate 120 mg/kg

(Nagappa A. N.,2003) to the overnight fasted rats. Five days after

administration of alloxan, fasting blood glucose of 300 to 450 mg/dl

were included in the study.

B) Sample collection:

Blood sample were collected from tail nipping and glucose

level was determined by an automatic electronic glucometer

(Accuchek comfort). (Vats et al., 2002)

C) Procedure:

After checking the fasting blood glucose in overnight fasted

diabetic rats. They were divided into five groups of five rats each

and one group of non-diabetic rats.

All the doses were given in the following manner

1st Group- normal control group received vehicle.

2nd Group-diabetic control received vehicle.

3rd Group-Received alcoholic extract at dose of 200 mg/Kg

orally.

4th Group- Received alcoholic extract at dose of 400 mg/Kg.

orally.

5th Group- Received standard drug i.e. Glibenclamide (10

mg /Kg. in Vehicle) orally. [Nagappa A.N., 2003]

The treatment was continued for 3 hours. During the period

water was supplied ad libitum. All the doses were administered

orally by the oral feeding needle. The effect of extract on Blood

glucose levels was estimated on overnight fasted rats on 0 hour, 1

hour, 2 hr and 3 hr by the method described before. The general

behaviors of the animals were recorded. The blood glucose level in

(Mean ± SEM) is shown in the Table 6.1.

Table 6.1: The Antihyperglycemic effect of Methanolic

Extract on Alloxan induced Diabetic rats.

GPDose Blood Glucose Level (mg/dl) at hr

0 hr 1 hr 2 hr 3 hr

I N.C75.75±3.93

75.56±2.20

76.63±1.59

76.06±1.48

II D.C343.37±8.04

342.19±6.37

340.52±5.48

333.69±4.57

IIICTLE

(200mg/kg)340.82± 4.51

289.95±3.01***

272.48±3.72***

260.01±4.98***

IVCTLE

(400mg/kg)347.52 ±4.92

293.11±2.76***

271.52±2.48***

256.19±2.50***

VGlibenclamid

e(10mg/kg)

346.35±4.28

287.90±2.51***

253.46 ±2.77***

238.67±2.36***

N.C. = Normal Control ;

D.C. = Diabetic Control

CTLE= Clitoria ternatea Leaves Extract

***P < 0.001 show significant when compare with group II

Fig: 6.1 The Antihyperglycemic effect of Methanolic

extract on alloxan induced diabetic rats.

2. Effect of Methanolic extract on oral glucose tolerance

test:

The hypoglycemic effect of methanolic extract of Clitoria

ternatea leaves was study on glucose loaded rats.

Protocol:

In this glucose tolerance test fasted normal rats were divided

into sifour groups of five animals each, Group I served as control

and received vehicle. Group IV received standard drug

glibenclamide at an oral dose of 10 mg/kg and Group II and III

received methanolic extract orally at a dose of 200 mg/kg and 400

mg/kg respectively. The rats of all the groups were given glucose

(4g/kg), 30min after the extract and drug administration Blood

samples were collected by tail nipping just prior to glucose loading

and blood glucose levels were measured by Accuchek Comfort

glucometer. Basal value is those after which glucose was

administered.

Table: 6.2 The Antihyperglycemic effect of Methanolic Extract On Glucose Loaded

rats

GP

Dose

Blood Glucose Level (mg/dl) at minutes

0minutes

30 minutes

60 minutes

120 minutes

IControl (4g/kg)

75.92±2.21

177.50±4.38

151.89±3.54

126.32±3.61

IICTLE

(200mg/kg)71.52±1.37

159.50±3.73**

135.68 ±2.10***

110.37±1.64**

IIICTLE

(400mg/kg)77.30±3.07

153.40±2.52***

130.73±2.38***

101.74±1.60***

IVGlibenclamide

(10mg/ kg)81.85±2.52

147.01±2.00***

119.81±2.86***

86.97±3.03***

CTLE= Clitoria ternatea Leaves Extract

***P < 0.001 show significant when compare with group I

Fig. 6.2: The Antihyperglycemic effect of Methanolic

extract on Glucose loaded rats.

6.1.3 STATISTICAL ANALYSIS:

The data were statistically evaluated using one way Anova.

expressed as Mean ± SEM followed by Tukey test using the Graph

pad instant Demo (Data set 1.IS) version P. values of 0.05 or less

were considered to be significant.

Result:

The methanolic extract of the drug showed marked effect for

decreasing the blood glucose level and rectifying the problem like

fatigue and irritation associated with the disease. Two concentration

of the extract were used for the investigation i.e. 400 mg/kg and

200 mg/kg against the standard glibenclamide 10 mg/kg dose

showed 23.12 % decrease in blood glucose level, 200mg /kg showed

21.92% decrease and standard drug showed 28.52% decrease

during the study of two week when compare with the standard drug.

400mg/kg dose of methanolic extract was near about as effective as

standard drug (glibenclamide).

When the activity of extract was done by the glucose

tolerance test in glucose loaded rats, the methanolic extract

400mg/kg showed significant effect on the blood glucose level but

extract of 200 mg/kg did not show the significant decrease in blood

glucose level. The value of p is less than 0.001 except in 200 mg/kg

in glucose tolerance test.

6.2 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY:

A. Screening of the acute inflammatory agents:

1. Ultraviolet erythema in guinea pig

After cleaning of back skin they are chemically depilated by a

suspension of barium sulfide. The guinea pigs are placed in a

leather cuff with a hole of 1.5 to 2.5 cm size punched in it, allowing

the ultraviolet radiation to reach only this area. The erythema is

scored 2 and 4 h after exposure (Yawalkar, 1991).

2. Paw edema

After injecting 0.1 ml of 1% solutions of carrageenan into the

plantar side of the left hind paw. The paw volume measured by

plethysmometer after injection, and for a specific time period after

challenge.

B.Screening of the chronic inflammatory agents:

1. Granuloma formation

After sacrificed of animal on 8th day cotton pellet are removed

that was placed on both sides in scapular region on first day. After

drying at 60°C for 24 h, net dry weight is determined (Ismail et al.,

1997).

2. Sponge implantation technique

Standard size and weight (10.0 ±0.02 mg) sponges are inserted

into dorsal cavities by insertion of blunt forceps. For estimation of

the fluid phase of sponge are exudates, e.g. protein content and

enzyme levels are noted.

3. Glass rod granulomas

The glass rods together with the surrounding connective tissue is

removed from sacrificed animal in which rods are placed in caudal

region by blunted forceps for 7 days before under goes

histopathological study (Vogel et al., 1990)

Animals:

Albino adult male rat weighing 220-280 gm were used for

assessment of anti-inflammatory activity. All animals supplied by

Central Drug Research Institute, Lucknow and kept at animal house

B.U. Jhansi. There were maintained standard environmental

condition (R.H. - 55-65%, room temperature 25±2°C and 12 hr light/

dark cycle) and were fed with standard pellet diet and water ad

libitum. Each experiment group constitute of six animal housed in

separate cages. All experiments were carried out with the consent

of Institutional Animal Ethical committee of the institute Approved

with reff, no. (716/02/a/CPCSEA)

Drugs and Chemicals:

Carrageenan (Himedia, Mumbai), Diclofenac (Alfa Remedies,

Ambala), The Methanol, Chloroform and Petroleum ether is provided

by Institute of Pharmacy, Bundelkhand University Jhansi.

Acute toxicity Study:

The limit test for acute toxicity was carried out at 2000 mg/kg

oral dose of CTLE in group of three rats (OECD 423 guidelines). The

rats undergoes for 2 hr behavioral, neurological and autonomic

profiles and morbid state. There also notice mortality rate in

duration 24 hours.

Doses and Treatments:

Rats were divided into different groups (n= 5). Diclofenac

(10mg/kg) was administered orally in mice and rats in acetic acid

induced writhing and carrageenan induced oedema. The control

groups received 0.9% saline. The dose of 1% carrageenan was

taken as 0.1 ml subplantar administered in rats is introduced in

animals as i.p (10ml/kg).

The rats were divided into four groups of five animals each in

a group to receive various treatments as mentioned bellow.

The characteristics of the groups are as follows:

Group 1: Control (Normal) rats given only saline.

Group 2: control standard group receive Indomethacin

Group 3: Test group receive CTLE 200mg/kg dose.

Group 4: Test group receive CTLE 400mg/kg dose.

All above doses are given 10ml/kg for orally and 5ml/kg i.p.

Anti-inflammatory Activity:

Carrageenan induced paw oedema in rat:

The method assayed according to Winter et al. The rats were divided in to the four groups. The drug control group, Diclofenac administered at a dose of 10mg/kg P.O. The same volume of normal saline was administered orally to the vehicle control group of rat while bark extract at a dose of 100 and 200 mg/kg was given orally to the test group of animal. The drugs or vehicle were given to experimental animal once at 0 min. Acute paw oedema was induced by subplantar injection of 0.1 ml of 1% freshly prepared carrageenan suspension in normal saline into the right hind paw of each rat. The left hind paw was injected with 0.1% of normal saline. The paw was measured in mm before (0hr) and at a interval of 1st, 2nd, 3rd and 4th hour after injection using verneir caliper (owalabi et al., 2007). The percent inhibitory activity was calculated by following formula (Winter et al., 1962)

% inhibition = 100 (1- Vt /Vc)

Where Vt = oedema paw size of test and Vc= oedema paw size of control

Table: 6.3 The Anti-inflammatory effect of Methanolic Extract On Carrageenan-induced rat

GPDose

Percentage inhibition

1 hr%

Inhibition2 hr

%

Inhibition3 hr

%

Inhibition4 hr

%

Inhibition

I N.C 0.62±0.06 0.78±0.07 0.76±0.05 0.74±0.05

II Std. 0.52±0.05 16.12% 0.59±0.06** 24.35%0.55±0.05*

*27.63%

0.47±0.04*

*36.48%

IIICTLE

(200mg/kg)0.57±0.09 8.06% 0.60±0.10* 23.07% 0.62±0.11* 18.42% 0.50±0.10* 32.43%

IVCTLE

(400mg/kg)0.58±0.08 6.45% 0.61±0.08* 21.79% 0.67±0.09* 11.84% 0.54±0.10* 27.02%

N=6, CTLE= Clitoria ternatea Leaves Extract

The percent inhibition for each group was calculated by comparison with the control group. Values indicate mean± S.E.M (ANOVA test followed by Dunnett’s t-test).Significance variation against control at **P < 0.01

Result:

The control group at 1st, 2nd, 3rd and 4th hour showed oedema volume in ml 0.62±0.06, 0.78±0.07, 0.76±0.05 and 0.74±0.05 respectively. The corresponding mean volume on Diclofenac (10mg/kg) treated group was 0.52±0.05, 0.59±0.06, 0.55±0.05 and 0.47±0.04 respectively, indicating significantly anti-inflammatory activity of Diclofenac from 0 hour onwards when compared to control. The extract in the doses i.e. 200 mg/kg and 400 mg/kg had produced significant inhibition in mean oedema volumes in dose dependent manner from 1 to 4th hour.

Fig:6.3 Anti-inflammatory effect of Methanolic Extract On

Carrageenan-induced rat

7. RESULT & DISCUSSION

7.1 GENERAL:

Clitoria ternatea belongs to the group of herbs having the

family fabaceae formed is cultivated as a perennial herbs all most

though out individually only in the three several significant amount

and chemical constituents like carbohydrates phenolic acid

flavanoids and alkaloids are present in this herb many of them have

been already reported, which results in many ethno medicinal

application on the herb like Antiulcer, Anti-inflammatory,

Cytoprotective Anorexia, dyspepsia etc.

Due to the wide pharmacological properties and the rich

hentaqge owned by the plant, it creates a desire to more widely

explore the plant hence the work has done on this plant and the

results are discussed below.

7.2 PHYTOCHEMICAL SCREENING:

7.2.1 TLC of Methanolic Extract:

The qualitative chromatographic profiles of the extract were

established. The different solvent systems were tried for extract

and the best solvent system was found are as follows Benzene:

Methanol: Formic acid (8.5:1:0.5)

In methanolic extract, five spots were observed with different Rf

value.0.12, 0.24, 0.57, 0.68, 0.92.

7.2.2 HPTLC of Methanolic Extract:

HPTLC of extract show the ten peaks confirming that the ten

compound may be present in the methanolic extract of the leaves of

Clitoria ternatea.

7.2.3 Cloumn Chromatography and characterization of

Methanolic Extract:

After Thin Layer Chromatography and HPTLC of methanolic

extract, the isolation of the constituent of methanolic extract was

carried out by column chromatography and then the compound (R-

9) obtained was analyzed by different analytical technique like IR,

NMR and Mass spectroscopy.

The IR spectra of isolated compound (R-9) shows different

functional group at different wave number shown in Table 5.5 and

Mass spectra of isolated compound (R-9) shows fragmentation

pattern as follows m/z 81, 95, 109, 154.

The NMR spectra of isolated compound (R-9) shows number of

proton and functional group. So the isolated compound may be the

3-neohesperidoside.

7.3 EVALUATION OF ANTIDIABETIC AND ANTI-

INFLAMMATORY ACTIVITY:

The methanolic extract of the drug showed marked

effect for decreasing the blood glucose level and rectifying the

problem like fatigue and irritation associated with the disease. Two

concentration of the extract were used for the investigation i.e. 400

mg/kg and 200mg/kg against the standard glibenclamide 10 mg/kg.

400mg/kg dose showed 23.12 % decrease in blood glucose level,

200 mg/kg showed 21.92% decrease and standard drug showed

28.52% decrease during the study of two week when compare with

standard drug, 400 mg/kg dose of methanolic extract was near

about as effective as standard drug (glibenclamide).

When the activity of extract was done by the glucose

tolerance test in glucose loaded rats, the extract showed significant

effect on the blood glucose level but extract of 200 mg/kg did not

show the significant decrease in blood glucose level. The value of p

is less than 0.001 except in 200 mg/kg in glucose tolerance test.

The control group at 1st, 2nd, 3rd and 4th hour showed oedema volume in ml 0.62±0.06, 0.78±0.07, 0.76±0.05 and 0.74±0.05 respectively. The corresponding mean volume on Diclofenac (10mg/kg) treated group was 0.52±0.05, 0.59±0.06, 0.55±0.05 and

0.47±0.04 respectively, indicating significantly anti-inflammatory activity of Diclofenac from 0 hour onwards when compared to control. The extract in the doses i.e. 200 mg/kg and 400 mg/kg had produced significant inhibition in mean oedema volumes in dose dependent manner from 1 to 4th hour.

8. SUMMARY AND CONCLUSION

The fresh leaves of Clitoria ternatea was collected during the

month of September 2008, from my village Kailiya and gandoli

(Distt-Jalaun), the Kush Nursury, Gwalior Road, Jhansi and from the

Institute of Pharmacy, Bundelkhand University, Jhansi The plant

materials was taxonomically identified and authenticated by Dr.

Gaurav Nigam, Botany Department, Bundelkhand University, Jhansi.

Herbarium and Museum Division with ref. no. BU/BOT /376/24-01-

2009.

The leaves of Clitoria ternatea were shaded dried until cracking

sound was observed during breakage, and then these are made into

coarsely powdered from using dry grinder. The powdered leaves of

the plant (600 gm.) was packed in soxhlet apparatus and

continuously extracted with petroleum ether (40-600C) till complete

extraction, after completion of extraction the solvent was

removed by distillation and then concentrated extract obtained was

dried under reduced pressure using rotatory evaporator at

temperature not exceeding 400C and then give moderate heating

on water bath. A pale green extract approximate 18 gm. was

obtained. From the drug, petroleum ether was removed and the

defatted drug was extracted with methanol till complete extraction,

after completion of extraction the solvent was removed by

distillation and then concentrated extract obtained dried under

reduced pressure at temperature not exceeding 400C and then give

moderate heating on water bath. The methanolic extract obtained

was greenish black in colour, weighed about 40 gm. The both

petroleum ether and methanolic extract was kept in petridish and it

was stored in desiccator at cool place (Mukherjee, 2002).

The qualitative chromatographic profiles of the extract were

established. The different solvent systems were tried for extract

and the best solvent system was found are as follows Benzene:

Methanol: Formic acid (8.5:1:0.5)

In methanolic extract, five spots were observed with different Rf

value.0.12, 0.24, 0.57, 0.68, 0.92.

HPTLC of extract show the ten peaks confirming that the ten

compound may be present in the methanolic extract of the leaves of

Clitoria ternatea.

After Thin Layer Chromatography and HPTLC of methanolic

extract, the isolation of the constituent of methanolic extract was

carried out by column chromatography and then the compound (R-

9) obtained was analyzed by different analytical technique like IR,

NMR and Mass spectroscopy.

The IR spectra of isolated compound (R-9) shows different

functional group at different wave number shown in Table 5.5 and

Mass spectra of isolated compound (R-9) shows fragmentation

pattern as follows m/z 81, 95, 109, 154.

The NMR spectra of isolated compound (R-9) shows number of

proton and functional group. So the isolated compound may be the

3-neohesperidoside.

In view of the ethanobotanical and traditional claims of Clitoria

ternatea plant used as hypoglycemic agent and wide use of its leaf,

root and flower extract in Ayurvedic practice, it is proposed to

evaluated anti-diabetic activity of methanolic Clitoria ternatea leaf

extract in alloxan induced hyperglycemic rats. In glucose loaded

normal rats, hypoglycemia was observed maximum at 120 minutes

after administration of CTLE. Single dose administration of CTLE

produce significant hypoglycemic effect in alloxan treated

hyperglycemic rats. The methanolic extract of Clitoria ternatea

leaves also show the anti-inflammatory effect in carrageenan

induced rat.

In conclusion, the study indicates that the methanol extract of

leaves posses anti-diabetic and anti-inflammatory properties which

suggest the presence of biologically active components. The extract

might be promoting glucose uptake and metabolism or inhibiting

hepatic gluconeogenesis. Result from the phytochemical analysis of

Clitoria ternatea revealed the presence of flavonoids, which has also

been isolated from the other plant and found to stimulate secretion

or possess an insulin-like effect.

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