INSTITUTE OF PHARMACY

BUNDELKHAND 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 PHARMACY
BUNDELKHAND UNIVERSITY,
JHANSI

CERTIFICATE

This is to certify that present dissertation work entitled

Phytochemical Screening and Pharmacological investigation
on the the leaves of Clitoria ternateaSubmitted 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:
Guide

Place: Jhansi

Dr. Raghuveer Irchhaiya

Reader
Institute of Pharmacy
Bundelkhand University,
Jhansi, (U.P.)

INSTITUTE OF PHARMACY
BUNDELKHAND 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:
Place: Jhansi

Ram kumar

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,
Mr. Tularam

my father

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

fiance

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 villages 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)

CONTENTS
Certificate Head
Certificate Guide
Declaration
Acknowledgement
Abbreviations
List of Tables
List of Figures
S.No.

PAGE NO.
INTRODUCTION
1.1 General

1.

1.2 Plant Profile

1-32

1.3 Disease Profile

2.

REVIEW OF LITERATURE

3.

PLAN OF WORK

4.

COLLECTION AND EXTRACTION

40-42

5.

PHYTOCHEMICAL SCREENING

43-65

5.1 Qualitative Chemical Analysis

5.2 Thin Layer Chromatography
5.3 High Performance Thin Layer

33-38
39

Chromatography
5.4 Column Chromatography
5.5 Characterization Of Isolated Compound
PHARMACOLOGICAL INVESTIGATION
6.

6.1 Evaluation of Antidiabetic Activity

66-77

6.2 Evaluation of Anti-Inflammatory Activity

RESULT AND DISCUSSION

7.1 General
7.

7.2 Phytochemical Screening

78-80

7.3 Evaluation of Antidiabetic Activity

7.4 Evaluation of Anti-Inflammatory Activity

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

19

treatment of Diabetes
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

70

Extract on Alloxan induced Diabetic rats

6.2

The

Antihyperglycemic

Extract
On Glucose Loaded rats

effect

of

Methanolic

71

6.3

The

Extract On

Anti-inflammatory

effect

of

Methanolic

76

Carrageenin-induced rat

LIST OF FIGURES
FIGURE NUMBER AND FIGURE TITLE

PAGE NO.

1.1 Progress of inflammation

4.1

The

Extraction

procedure

25
in

schematic

41

5.1 HPTLC peaks and HPTLC Chromatogram

57

manner

with Rf values
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

70

Extract on Alloxan induced Diabetic rats

6.2 The Antihyperglycemic Effect of Methanolic

72

extract on oral glucose tolerance test

6.3

Anti-inflammatory

effect

of

Methanolic

77

Extract on Carrageenan-induced rat

LIST OF PHOTOGRAPHS
PHOTO NUMBER AND PHOTO TITLE

PAGE NO.

1.Leaves and flower of Clitoria ternatea

2. TLC of Clitoria ternatea extract

54

Abbreviation
Abbreviation
used

Meaning of Abbreviation

ANOVA

Analysis of variance

WHO

World Health Organization

Rf

Resolution Factor

TLC

Thin Layer Chromatography

FA

Formic Acid

EA

Ethyl acetate

CDRI

Central Drug Research Institute

NC

Normal Contral

DC

Diabetic Contral

DM

Diabetic Mellitus

OGTT

Oral Glucose Tolerance Test

BGL

Blood Glucose Level

PG

Plasma Glucose

HPTLC

High Performance Thin Layer Chromatography

FPG

Fasting Plasma Glucose

Hour

CDA

Canadian Diabetes Association

ADA
CTLE
Conc
Dil
S.E.M
IP
IGT
NIDDM
ADM
IDDM
NMR
MS
IR
CC
TMMM
IHP
QC
IDMA
NISCAIR

American Diabetes Association

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 centurys 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 physicians 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

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 blueflowered 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, subcoriaceous, 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 diseases, ulcers of the cornea, tuberculosis glands, elephantiasis, head
ache, cures, tridosha leucoderma burning sensation, pains, biliousness, 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 3monoglucoside,
glucoside,

3-rutinoside,

3-o-rhamnosyl

3-neohesperidoside,

galactoside

of

3-o-rhamnosyl-

Kaempferol,

besides

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

aparajitin and - sitosterol. The blue flowers contain delphinidin-3,5diglucoside delphinidin-3- glucoside and its 3-methyl derivative,
malvidin-3-

glucoside, Kaempferol and cyanin chloride ; the white

flowers yield only kaempferol, other substances present in the seeds

are:

p-hydroxycinnamic

galactopyranoside,

acid,

adenosine,

flavonol-3-glycoside,
3,5,7,4-

tetra

ethyl--D

hydroxyflavone-3-

rhamnoglucoside, a polypeptide, hexacosanol, -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 (2 nd
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

group

of

endocrine

syndromes

characterized by hyperglycemia; altered metabolism of lipids, carbohydrates, 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 insulindependent 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 -cell lesions or necrosis. Loss of -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 -cells. As a result of the destruction of -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 -cells secretion. However,
the type I diabetes has virtually to functional -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 -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 glucose

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 years theme of
world

diabetes

Day

was

to

address

retinopathy

one

of

the

complications of DM. Your Eyes and Diabetes: Dont 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 Burgers disease. (Yue et. al., 1984)
1.3.1.3 Diagnosis:
A number of tests for hyperglycemia have been assessed as diagnostic 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 CDAs current definition of IFG
is fasting plasma glucose of 6.1-6.9 mmol/L and IGT is defined
2-hour

value based

as a

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 accompanied 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

Organization (WHO).

Association

(ADA)

and

The

World

Health

CDA
2hPH OGTT
7.8-11.0

ADA
2hPG OGTT
7.8-11.0

IFG

FPG 6.1-6.9

FPG 5.6-6.9

IFG and IGT

Both of the above

criteria

Both of the
above
criteria

IGT

WHO
FPG < 7.0 AND 2hPG
OGTT 7.8-11.0
FPG 6. 1-6.9 AND
2hPG < 7.8
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

1.

Abroma augusta L.f.

2.

Annona squamosa L.

3.

Barleria cristata L.

4.

Family
Sterculiaceae
Annonaceae

Useful Part
Bark, Flower
Leaves

Acanthaceae

Roots

Beta vulgaris L.

Betulaceae

Bark

5.

Calamug rotang L.

Arecaceae

Bark

6.

Cannabis sativa L.

Cannabinacae

7.

Desmodium gyrans L.

Papilionaceae

Roots

8.

Dioscorea alata L.

Dioscoreaceae

Rhizome

9.

Eryngium foelidum L.

Apiaceae

Whole plant

10.

Ficus fistulosa L.

Moraceae

Fruit

11.

Gymnema sylvestris

Asclepiadaceae

Leaves

12.

Hordeum Vulgare L.

Poaceae

Seed

13.

Ipomaea balatus L.

Convolvulaceae

Tuberus
Roots

14.

Juslicia adhatoda L.

Acanthaceae

Leaves

15.

Kyllianga bulbosa

Cyperaceae

Whole plant

16.

Lysium barbala L.

Solanaceae

Fruits

17.

Momordica charanlia

Cucurbitaceae

Fruit

18.

Nepeta cataria L.

Lamiaceae

19.

Oplopanax horridum

Umbelliferae

Resin & Leaves

Leaves &
Flowering
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 80s 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

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 IL2, 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
organisms,

depending
these

upon

are

the

the

tissue

general

involved

and

characteristic

of

causative
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

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

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-, TNF-, IF- 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

strong

vasodilator.

Thromboxanes

and

leukotrienes produce vasoconstriction and are important modulators.

Table: 1.3 Cell derived mediator
Name

Type

Source

Description
These cells contain a large
variety

of

enzymes

perform

which

number

of

functions. Granules can be

classified as either specific or
Lysosome
granules

azurophilic
Enzymes

depending

upon

Granulocytes the contents, and are able to

break

down

substances,
may

some

be

proteins

number
of

of

which

plasma-derived

which

enzymes

allow

these

act

as

to

inflammatory mediators.
Histamine

Vasoactive

Mast cells,

Stored in preformed granules,

amine

basophils,

histamine is

platelets

response

to

released
a

number

stimuli. It causes

in
of

arteriole

dilation and increased venous

permeability.
Antiviral,
and
IFN-

Cytokine

T-cells, NK
cells

immunoregulatory,

anti-tumour

properties.

This interferon was originally

called

macrophage-activating

factor,

and

is

especially

important in the maintenance

of chronic inflammation.
Activationand chemoattraction
IL-8

Chemokine

Primarily
macrophage

of neutrophils, with a weak

effect

on

monocytes

and

eosinophils.
Able

to

mediate

leukocyte

and

activation,

adhesion

allowing them to bind to the

endothelium
Leukotriene
B4

and

migrate

across it. In neutrophils, it is

Eicosanoid Leukocytes

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 oxide

Potent

vasodilator,

relaxes

Macrophage

smooth

muscle,

reduces

Soluble

endothelial

platelet aggregation, aids in

gas

cells, some

leukocyte recruitment, direct

neurons

antimicrobial activity in high

concentrations.

Prostagland
ins

A group of lipids which can

Eicosanoid

Mast cells

cause vasodilation, fever, and

pain.

T
able: 1.4 Plasma derived factors
M Name

Type d by

Description
A vasoactive protein which is able to
induce

Bradykinin

Kinin system

vasodilation,

increase

vascular permeability, cause smooth

muscle

contraction,

and

induce

pain.
Cleaves to produce C3a and C3b.
C3a stimulates hishistamine release

C3

Complement
system

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.
Stimulates
mast

C5a

histamine

cells,

release

thereby

by

producing

Complement

vasodilation. It is also able to act as

system

a chemoattractant to direct cells via

chemotaxis

to

the

site

of

inflammation.
Factor XII

Liver

A protein which circulates inactively,

(Hageman

until activated by collagen, platelets,

Factor)

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.
A

complex

of

the

complement

proteins C5b, C6, C7, C8, and

multiple
Membrane

Complement

attack complex

system

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
system

Able to break down fibrin clots,

cleave complement protein C3, and
activate Factor XII.
Cleaves the soluble plasma protein
fibrinogen

to

produce

insoluble

fibrin, which aggregates to form a

Thrombin

Coagulation

blood clot. Thrombin can also bind

system

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.
petals

of

Clitoria

The crude malonyltransferase from the

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)-- 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: fabaceae), 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 antipyretic 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 sulfatepolyacrylamide 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 kaempferol 3-O-(2-O-a-rhamnosyl-6-O-malonyl)--glucoside, querce-tin 3-O(2---O-a rhamnosyl-6-O-malonyl--glucoside and myricetin 3-O- (26-di-O-a-rhamnosyl)--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-(2O-a-rhamnosyl-6-Omalonyl)-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-medicobotanical 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-GCGCside chains respectively in which G is D-glucose and C is a coumaric
acid. Pigment 1 had symmetric 35- 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 35-side chains
in solution.
Bavaliya N.K., (1993) reported poisonous lagurnes of Rajasthan

The paper deals with 33 poisonous leguminous species which are

toxic to men, animals, fishes and livestock
while enumerating the species

or other

living things

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 pcoumaric 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
and their

were isolated by reversed phase HPLC

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,
Chromatography.

HPTLC

and

Column

D. Identification and characterization of constituent by HNMR, 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 (DisttJalaun), 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 /376/24-012009.
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 40 0C

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 40 0C 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

Extracted Drug (Discarded)

Solvent removed by Distillation

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

chemical structure

may be

value

stimulate the

in itself but has a novel

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 Mayers reagent (cream
precipitate) Dragendroffs reagent (orange brown precipitate) and
Wagner reagent (reddish brown precipitate).
Mayers reagent: Few drops of Mayers reagent were added in
each of the extract and observed formation of the white or cream
colored precipitates.

 Dragendorffs reagent: Few drops of dragendorffs reagent were

added in each of the extract and observed formation of the orange
yellow or brown colored precipitates.
Wagners 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 Molischs test to detect the presence of carbohydrates.

Molischs test: To small quantity of extract few drops of 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.

Fehlings Test: The 1 ml of Fehlings 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 Burchards, Keller Killiani, Sodium nitrosoprusside and
Borntragers 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 H 2SO4 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.

Borntragers 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 millions, Biuret and
Ninhydrin tests.

Millions test: To the small portion of extract 5-6 drops of

millions 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 Burchards 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

Pet. Ether

extract

extract

Alkaloids
1.

2.

3.

4.

Dragendorffs test

--

--

Wagners test

--

--

Mayers test

--

--

Hagers test

--

--

Molischs test

Fehlings test

--

--

Carbohydrates

Proteins
Biuret test

Xanthoprotein test

--

--

--

--

--

--

Keller Killiani test

--

Borntragers test

--

Amino acid
Ninhydrin test

5.

Flavonoids
Shinoda test

6.

7.

Phenolic
compounds
Glycoside

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

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 110 0C for 10
minutes and the spots were marked. R f 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 system
Chloroform: Ethylacetate
( 6:4 )
Benzene: Diethylether
(3:7)
Ethylacetate: : Methanol: Water
(7:2:1)
Benzene: Ethylacetate
(8.5:1.5)
Benzene: Methanol: Formic Acid
(8.5:1:0.5)

Number
of spots

Resolution

4 spot

Good

4 spot

Good

4 spot

Good

4 spot

5 spot

Good

Excellent

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 110 0C 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.
1.

Elute
Hexane: Benzene (95:5)

Volume
collected
(ml)

No of spot

Code

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 R9 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

(ppm)

of

Std. (ppm)

of

Isolated Inference

No.of Protons

Compouund

Compouund

8.91

8.30

1H

6.66

6.68

1H

6.86

6.89

1H

7.74

7.54

2H

3.96

3.94

dd

1H

3.71

3.71

1H

3.47

3.48

1H

3.50

3.48

ddd

1H

3.83

3.83

dd

1H

3.66

3.66

dd

1H

3.30

3.26

1H

3.63

3.66

dd

1H

0.91

0.92

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 3neohesperidoside, 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 diabetogenic activity. The compound turned out to be specifically cytotoxic
to -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 -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 monosodium 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 -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 252 0C relative humidity 5015%
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.

Blood Glucose Level (mg/dl) at hr

Dose

GP

0 hr

1 hr

2 hr

3 hr

N.C

75.75
3.93

75.56
2.20

76.63
1.59

76.06
1.48

II

D.C

343.37
8.04

342.19
6.37

340.52
5.48

333.69
4.57

III

CTLE
(200mg/kg)

340.82
4.51

289.95
3.01***

272.48
3.72***

260.01
4.98***

IV

CTLE
(400mg/kg)

347.52
4.92

293.11
2.76***

271.52
2.48***

256.19
2.50***

Glibenclamide
(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
Blood Glucose Level (mg/dl) at minutes
GP

Dose

0
minutes

30
minutes

60
minutes

120
minutes

Control
(4g/kg)

75.92
2.21

177.50
4.38

151.89
3.54

126.32
3.61

II

CTLE
(200mg/kg)

71.52
1.37

159.50
3.73**

135.68
2.10***

110.37
1.64**

III

CTLE
(400mg/kg)

77.30
3.07

153.40
2.52***

130.73
2.38***

101.74
1.60***

IV

Glibenclamide
(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 60C 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

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 252C 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

GP

Percentage inhibition

Dose
1 hr

%
Inhibition

2 hr

%
Inhibition

%
Inhibition

%
Inhibition

N.C

0.620.06

II

Std.

0.520.05

16.12%

0.590.06**

24.35%

0.550.05**

27.63%

0.470.04**

36.48%

0.570.09

8.06%

0.600.10*

23.07%

0.620.11*

18.42%

0.500.10*

32.43%

0.580.08

6.45%

0.610.08*

21.79%

0.670.09*

11.84%

0.540.10*

27.02%

IV

CTLE
(200mg/kg)
CTLE
(400mg/kg)

0.760.05

4 hr

III

0.780.07

3 hr

0.740.05

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 Dunnetts 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 0.620.06, 0.780.07, 0.760.05 and 0.740.05
respectively. The corresponding mean volume on Diclofenac (10mg/kg)
treated group was 0.520.05, 0.590.06, 0.550.05 and 0.470.04
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.

1.0

NC
Std.
CTLE (200mg/kg)
CTLE (400mg/kg)

Paw oedema

0.8
0.6
0.4
0.2

CT
LE

0m
g/
kg
)
(2
0

CT
LE

(4
00
m
g/
kg
)

.
St
d

NC

0.0

hour (hrs)
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
chemical constituents

significant

amount and

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 R f
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 3neohesperidoside.
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 0.620.06, 0.780.07, 0.760.05 and 0.740.05
respectively. The corresponding mean volume on Diclofenac (10mg/kg)
treated group was 0.520.05, 0.590.06, 0.550.05 and 0.470.04
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 (DisttJalaun), 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 /376/24-012009.
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 40 0C

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 40 0C 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 R f
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 3neohesperidoside.
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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