phytochemical screening and pharmacological investigation on the leaves of clitoria ternatea
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Hypogyccemic effect of clitoria ternatea reported by ram patel in 2009TRANSCRIPT
INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,
JHANSI
CERTIFICATE
This is to certify that present dissertation work entitled
“ Phytochemical Screening and Pharmacological
investigation on the leaves of Clitoria ternatea” submitted
in fulfillment of the requirements for the award of the degree of
Master of Pharmacy in “Pharmacognosy” of Institute of
Pharmacy, Bundelkhand University, Jhansi, is a bonafide
work carried out by Ram kumar (En. No. B.U./03/B-2039), under
the guidance and supervision of
Dr. Raghuveer Irchhaiya, during the academic session 2008-
2009.
Date: Dr. S.K. Prajapati
Place: Jhansi Head & Reader,Institute of Pharmacy
Bundelkhand University
Jhansi (U.P.)
INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,
JHANSI
CERTIFICATE
This is to certify that present dissertation work entitled
“Phytochemical Screening and Pharmacological
investigation on the the leaves of Clitoria
ternatea”Submitted in fulfillment of the requirements for the
award of the degree of Master of Pharmacy in
“Pharmacognosy” of Institute of Pharmacy, Bundelkhand
University, Jhansi, is a bonafide work carried out by Ram
kumar (En. No. B.U./03/B-2039), the guidance and supervision of
Dr. Raghuveer Irchhaiya, during the academic session 2008-
2009.
Date:
Place: Jhansi Guide
Dr. Raghuveer IrchhaiyaReaderInstitute of PharmacyBundelkhand University, Jhansi, (U.P.)
INSTITUTE OF PHARMACYBUNDELKHAND UNIVERSITY,
JHANSI
DECLARATION
I hereby declare that this dissertation entitled
“Phytochemical Screening and Pharmacological
investigation on the leaves of Clitoria ternatea” is prepared
under the genial guidance and supervision of Dr. Raghuveer
Irchhaiya,Institute of pharmacy, Bundelkhand University, Jhansi,
(U.P.).
The same is submitted to Bundelkhand University, Jhansi in
partial fulfillment of the requirement for the degree of Master of
Pharmacy in Pharmacognosy.
I further declare that I have not submitted this dissertation
previously for award of any degree or Diploma to me.
Date: Ram kumar Place: Jhansi
Acknowledgement
I wish to express my sincere thanks to all those who assisted
with the completion of my dissertation work.
It is a matter pleasure to acknowledge my respected guide
Dr. Raghuveer Irchhaiya. It is because of his priceless
intellectual guidance, constructive ideas and for having given me
complete independence, affectionate encouragement to put my
desire and thought which paved the way for the successful
completion of this work. It is indeed privilege to work under him.
I express my special regards to Dr. S. K. Prajapati,( H.O.D)
Institute of Pharmacy, Bundelkhand University, Jhansi,for furnishing
me all the necessary facilities to carryout this work and for their
ever helping attitude during the course of this work.
I also express my special regards to Reader, Dr. S. K.
Jain,Institute of Pharmacy, Bundelkhand University, Jhansi, for
furnishing me all the necessary facilities to carry out this work.
I want to thank my mother Smt. Kishori Devi, my father
Mr. Tularam Niranjan and my elder sister Smt.Tara Devi and
her husband Mr Kamlesh Patel. my sister Smt.Sarita Devi and
her husband Mr .Keshavdas Patel , my younger sister Smt.
Brajesh Devi and her husband Mr. Manoj Patel and my elder
brother Mr.Vinay Nirajan and his wife Pratima Niranjan and my
fiancée Archana Patel for their inspiration and motivation
throughout my project works. I am also thankful to my nieces and
nephews for their love and cheering me during the project work.
I am also thankful to my village’s friend Dilip, Birju, Chhotu,
Jitendra, Pramod, Mahendra, Rinku, Shivraj, Lakshmikant, Aanand
and Mansingh for collecting the leaves of Clitoria ternatea.
I am thankful to Mr. Sunil Kumar Niranjan (Lecturer), Mr.Ramji
Swarnkar (Lecturer), Mr. Man Singh (Lecturer), Mr.Shashi Alok
(Lecturer), Mr. Prasant Mishra (Lecturer), Mr. Nandlal Singh
(Lecturer), Mr Sailendra Singh (Lecturer) & Mr. V. K. Dexit (Lecturer)
Institute of Pharmacy, B.U., Jhansi (U.P.), for their cooperation and
help during the course of this work.
I am also thankful to Mr. Ratan Yadav (Lab Assistant),
Mr.Brijkishore, Mr.Santosh Shivhare Mr. Sailes Soni, Mr. Dipak
Shukla for providing me all the chemicals, glassware, instruments
and laboratory facilities to carry out my complete dissertation work.
I take this opportunity to thank my seniors Mr. Chandrahass
yadav ,Mr.T.K. Vashishtha, Mr. Dharmendra Singh, Mr. Atul Vikram
Patel, Mr. Ramshankar and my classmates Mr.O.P.Goutam,
Mr.Himanshu Gurjar, Mr. Lokesh Meena, Mr.Sushil Saini, Mr. Om Pal
Singh, Mr. Brajesh Singh, Mr.Pawan Dhakar, Mr.Vinod Sahu,
Mr.Manish Pathodiya,Mr. Sanjay Dutt, Mr.K.L.Rathor, Mr. Pinkesh
Tiwari, Mr.Yogesh Maddesiya, Rohit and Anoop Gurjar Mrs.Savita
Varma, Mrs. Smita Khare, Mrs.Pratibha Mishra, Mrs. Tanuja,
Ms.Shweta Sachan, Mr. Raghvendra Mishra, & my junior Mr.
Santosh Kumar, Mr. Hemant, for their help and moral support.
I kindly thank to C.D.R.I..., Lucknow for providing FTIR, NMR,
and Mass Spectroscopy.
I am also thankful to Director, NISCAIR New Delhi for providing
library facilities.
I am also thankful to my heartiest friends Mr.Rajneesh Kumar,
Mr. Naveen Patel, Mr. Nitin Paliwal Mr. Ajay Seth, and Mr.Surendra
Patel for giving moral support and valuable advice during my project
work.
Finally I am indebted to those poor animals that took all the
suffering and gave me an opportunity to carrying out this duty and
all who were involved directly or indirectly to work tenure of mine.
I will be grateful to all these people for ever and always want
to live up to all their expectations.
(RAM KUMAR)
Certificate Head
Certificate Guide
Declaration
Acknowledgement
Abbreviations
List of Tables
List of Figures
S.No.PAGE
NO.
1.
INTRODUCTION
1.1 General
1.2 Plant Profile
1.3 Disease Profile
1-32
2. REVIEW OF LITERATURE 33-38
3. PLAN OF WORK 39
4. COLLECTION AND EXTRACTION 40-42
5. PHYTOCHEMICAL SCREENING
5.1 Qualitative Chemical Analysis
5.2 Thin Layer Chromatography
5.3 High Performance Thin Layer
43-65
CONTENTS
Chromatography
5.4 Column Chromatography
5.5 Characterization Of Isolated Compound
6.
PHARMACOLOGICAL INVESTIGATION
6.1 Evaluation of Antidiabetic Activity
6.2 Evaluation of Anti-Inflammatory Activity
66-77
7.
RESULT AND DISCUSSION
7.1 General
7.2 Phytochemical Screening
7.3 Evaluation of Antidiabetic Activity
7.4 Evaluation of Anti-Inflammatory Activity
78-80
8. SUMMARY AND CONCLUSION 81-83
BIBLIOGRAPHY 83-89
ENCLOSURE
ERRATA
LIST OF TABLES
TABLE NUMBER AND TABLE TITLE PAGE NO.
1.1 Diagnostic Criteria for IGT and IFG 18
1.2 List of Some Medicinal plants used in the
treatment of Diabetes
19
1.3 Cell derived mediators 28
1.4 Plasma derived factors 29
4.1 The characterization of methanolic extract 42
5.1 Qualitative Phytochemical analysis 48
5.2 TLC of Methanolic extract 53
5.3 Rf value of the spots of Methanolic extract 53
5.4 Column Chromatography of Isolated compound 59
5.5 Interpretation of IR Spectroscopy 61
5.6 Mass Spectra of Isolated compound 63
5.7 Interpretation of NMR Spectroscopy 64
6.1 The Antihyperglycemic effect of Methanolic
Extract on Alloxan induced Diabetic rats
70
6.2 The Antihyperglycemic effect of Methanolic
Extract
On Glucose Loaded rats
71
6.3 The Anti-inflammatory effect of Methanolic 76
Extract On Carrageenin-induced rat
LIST OF FIGURES
FIGURE NUMBER AND FIGURE TITLE PAGE NO.
1.1 Progress of inflammation 25
4.1 The Extraction procedure in schematic
manner
41
5.1 HPTLC peaks and HPTLC Chromatogram
with Rf values
57
5.2 IR spectra of compound (R9) 61
5.3 Mass Spectra of Isolated compound (R9) 62
5.4 NMR Spectra of Isolated compound (R9) 63
6.1 The Antihyperglycemic Effect of Methanolic
Extract on Alloxan induced Diabetic rats
70
6.2 The Antihyperglycemic Effect of Methanolic
extract on oral glucose tolerance test
72
6.3 Anti-inflammatory effect of Methanolic
Extract on Carrageenan-induced rat
77
LIST OF PHOTOGRAPHS
PHOTO NUMBER AND PHOTO TITLE PAGE NO.
1.Leaves and flower of Clitoria ternatea 8
2. TLC of Clitoria ternatea extract 54
Abbreviation
Abbreviatio
n usedMeaning of Abbreviation
ANOVA
WHO
Rf
TLC
FA
E A
CDRI
N C
D C
D M
OGTT
BGL
PG
HPTLC
FPG
h
CDA
ADA
CTLE
Analysis of variance
World Health Organization
Resolution Factor
Thin Layer Chromatography
Formic Acid
Ethyl acetate
Central Drug Research Institute
Normal Contral
Diabetic Contral
Diabetic Mellitus
Oral Glucose Tolerance Test
Blood Glucose Level
Plasma Glucose
High Performance Thin Layer
Chromatography
Fasting Plasma Glucose
Hour
Canadian Diabetes Association
American Diabetes Association
Conc
Dil
S.E.M
I P
I G T
NIDDM
ADM
IDDM
NMR
M S
I R
C C
T M M M
I H P
Q C
I D M A
N I S C A I R
Clitoria ternatea Leaves Extract
Concentrate
Dilute
Standard Error Mean
International Pharmacopoeia
Impaired glucose tolerance
Non-Insulin dependent Diabetes Mellitus
Administration
Insulin dependent Diabetes Mellitus
Nuclear Magnetic Resonance
Mass Spectroscopy
Infrared Resonance
Column Chromatography
Traditional Medicine Material medica
Indian herbal Pharmacopoeia
Quality Control
Indian Drug medical association
National Institute of Science Communication
and Information Resources
1. INTRODUCTION
1.1 HISTORY OF AYURVEDA:
1.1.1 History of Herbal Medicine: The history of herbal
medicines is as old as human civilization. The documents many of
which are of great antiquity, revealed that plants were used
medicinally in China, India, Egypt and Greece long before the
beginning of the christian era. One of the most famous surviving
remains it Papyrus Ebers, a scroll some go feet long and a foot wide,
dating back to the sixteenth century before christ, The text of
document is dominated by more than 800 formula and 700 different
drugs. The drugs such as acacia, castor oil and fenel are mentioned
along with apparent references to such compounds as iron oxide,
sodium chloride, sodium carbonate and sulphur.
Most of 6 medicinally active substances identified in the
nineteenth and the twentieth century’s were used in the form of
crude extract. In China, many medicinal plants had been in use
since 500 B.C. the oldest known herbal is pent saw written by
Emperor Shen Nung around 3000 B.C. It contains 365 drugs, one for
each day of the year. Indians also, worked meticulously to examine
and classify the herbals, which they come across, into groups called
Gunas Charaka made fifty groups of ten herbs each which according
to him would suffice an ordinary physician’s need similarly, Sushruta
arranged 760 herbs in 7 distinct sets based on some of their
common properties A large portion of the Indian populations even
today depends on the Indian system of medicine Ayurveda, an
ancient science of life. The well known treatises in Ayurveda are
Charak Samita and Sushuta Samita. (Purohit et. al., 2002)
India has an ancient heritage of traditional medicine; Materia
Medica of India provides lot of information on the folklore practices
and traditional aspects of therapeutically important natural
products. Indian traditional medicine is based on various system
including Ayurveda, Siddha and Unani.
The evaluation of these drugs in mostly based on
phytochemical, pharmacological and allied approaches including
various instrumental techniques like chromatography, microscopy
and others. These traditional system of Indian medicine have their
uniqueness, no doubt but there is a common thread running
through these system in their fundemental principles and
practices with the emerging interest in the world to adopt and
study the traditional system and to exploit their potentials based on
different health care system. The evaluation of the rich heritage of
the traditional medicine is essential. (Mukherjee P.K., 2002)
1.1.2 Importance of Herbal therapies:
Herbal medicines are prepared from a variety of plant
materials, leaves, stem, and root, bark and so on they usually
contain many biological active ingredients and are used primary for
treating mild or chronic ailments. Herbs can be prepared at home in
ways using either fresh or dried ingredients.
In the united states today, herbal remedies are not regulated
and come in unpredictable strengths the amount of active
ingredients varies greatly, depending on whether more than are
species of the herb is used and how and when the herb is gathered
and prepared. Because some herbs can be toxic or carcinogenic, all
herbs should be used under the guidance of a health care
practitioner familiar with herbal medicine.
Across the spectrum of alternative medicine, the use of herbs
is varied: Naturopathic medicine, traditional Chinese medicine, and
Ayurvedic medicine, all differ in how diseases are diagnosed and
which herbal remedies are prescribed.
Plants are considered to be medicinal if they possess
pharmacological activities of possible therapeutic use. These
activities are after known as a result of millennia of trial and error
but they have to be carefully investigated if we wish to develop new
drugs that meet the criteria of modern treatment.
The identification of the active principles of medicinal plant
investigation of the extract in order to ensure that they are safe
effective and of constant activity.
The isolation of these active principles and the determination of
their structure in order that they may be synthesized structurally
modified or simple extracted more efficiently.
The methodology of research into medicinal plants must be
rigorous. Often simple technical errors undermine the value of
research on natural products. There are many who believe that a
little rapid research is sufficient to confirm the reputation of a plant
and who then attempt to proceed from there towards lucrative
industrial production. (Mukherjee P.K., 2002)
1.1.3 Efficacy of Herbal Medicinal Products:
Phytomedicines consist of many chemical constituents with
complex pharmacological effects on the body. They are used
continuously for many decades or centuries, often in ways that
differ from these conventional medical prescribing researches.
Development in phytotherapy has suffered through lack of patent
protection, and the diversity and relatively small scale of the indus-
tries involved compared to the rest of the pharmaceutical industry,
established guidelines for assessing the efficacy and safety of
phytomedicines. The differing regional uses of traditional herbal
remedies present extra difficulties for the harmonization of quality
procedure around the world. Therapeutic efficacy and clinical trials
are two most important criteria for the development of herbal drugs.
Although preliminary assessments of effect can be obtain
through the results of in vitro costing and experiments on animals,
authorities licensing new medicine for public use require evidence of
their effects on human beings. Only carefully planned clinical trials
that minimize experimental bias are able to satisfy these
requirements. Most herbal remedies can call on a tradition of
popular use, which has in practice, allowed manufacturers to submit
relevant bibliographic evidence in reviewing their earlier licenses of
right. (Mukherjee P.K., 2002)
1.1.4 Safety in Herbal Drugs:
Major differences in the assessment of quality, safety and
efficacy would hinder free circulation of herbal medicinal products
may represent risk for consumers.
The complexity of herbal drug preparations and the
interpretation of bibliographic data on safety and efficacy reflecting
the experience gathered during long term use are best addressed
by involving specific expertise and experience. Safety and efficacy
of complex biological products such as herbal medicinal products
are directly linked to Pharmaceutical details such as the way of
production and the specification of extracts.
1.1.5 World Situation on Herbal Medicine:
Traditional medicine is a very important part of health care.
Most population in the developing countries still relies mainly on
indigenous traditional medicine for satisfying their primary health
care needs. Traditional medicine has not however been
incorporated in most national health systems and the potential of
services provided by traditional practitioners is far from being fully
utilized herbal medicines are of great important to the health
individuals and communities but their quality assurance need to be
developed. During last decade, in many developed countries, there
has also been a growing interest in herbal medicine, acupuncture
and alternative systems of medicine. Consequently, an increase in
international trade in herbal medicines and other types of traditional
medicines has occurred proper use of these different types of
medicine has therefore become concern. (Mukherjee P.K., 2002)
1.1.6 The utility of Plants in Current Therapy:
Despite the enormous availability of medicines and, above all
of pharmaceutical specialties, plants have a place in current therapy
as can be justified at least by the four following reasons. There is
renewed interest in using plants in therapy. Such is the case of
Artemisia (Klayman, 1985), a source of quinine. Behind the
therapeutic success of chloroquine and its synthetic derivatives in
the treatment of malaria, the use of quinine passed into a chapter in
the history of medicine. Though a biological phenomenon, that is
now well studied, even at the molecular level, bacteria and
parasites can develop resistance to chemotherapeutics that is, they
undergo selection that results in resistance to a particular chemical
compound. This process has occurred in part, with species of
plasmodium, the causative agent of malaria, to a point that
synthetic antimalarial drugs have lost such a significant part of their
efficiency in the last quarter of the twentieth century that it has
often been necessary to return to the use of quinine.
Currently, there is such a great demand for the plant alkaloids
that extraction laboratories cannot satisfy the growing demand,
maximized now that malaria has again become a great health risk in
topical area. The demand has been accentuated further by the
assistance of the insect vector, to identify their main active
chemical compounds. That sample, even through or partial one
reveals the enormous empirical traditional knowledge about
medicinal plants. Most of this knowledge is verbal and only
incompletely incorporated in historical and folklore work. The
aboriginal knowledge is the fruit of centuries and in capacity of
chemists to modify a molecular structure is almost unlimited the
capacity to invent or create new structures.
Phytochemical investigation carried out during the 1970s
and 1980s have discovered a number of alkaloids and other
pharmacologically active substances that are currently being
studies and that can possible serve as models for new synthetic
compounds. (Barz and Ellis, 1980)
Traditional medicine depends on a number of plants that are
currently used in scientific medicine although they have not yet
been improved upon. Such is the case of digitals purpurea L. and D.
lanata Ehrh. Many other drugs exist to which therapeutic effect
have been attributed As is well known, synthetic chemistry has until
now had little success in obtaining drugs effective in the treatment
of various viral disease, even though immunotherapy has achieved
great successful. We still do not have vaccines for all viral diseases.
It is possible that plants may be useful to treat this disease. An
example from Ecuator is laniqua (Marggricarpis seto-sus Ruiz and
Pavon), the roots of which, in infusion are used in the symptomatic
treatment of measles. Furthermore, numerous plants are known for
certain antineoplastics effects. (Cassady and Douros, 1980)
1.1.7 Differences between Herbs and Other Drugs:
Herbs are different in several respects from the type of
purified therapeutics agents we have become accustomed to call
drugs in the last half of the twentieth the century. In the first place,
they are more dilute than the concentrated chemicals that are
familiar use in the form of aspirin tablets or tetracycline capsules. A
simple example will illustrate the difference. One can take caffeine
for its stimulatory effects on the central nervous system. The usual
dose is 200mg contained in one or two small tablets, depending on
their strength or it is possible to get the some effect by drinking a
caffeine containing beverage such as coffee or tea. Dilution is not
the only difference that just be considered in utilizing medicinal
herbs. In addition to physiologically inert substances such as
cellulose and starch, herbs often contain addition active principles
that may be closely related both chemically and therapeutically to
the active constituents primary responsible for its effects. (James E.
et. al., 2002)
1.1.8 Indian Trade in Medicinal Plants:
The exports of medicinal plants and herbs from India has
been quite substantial in the last few years., India has been the
major supplier of medicinal plants in the world market till 1976
when it was relegated to the second position by South Korea. With
exports worth only Rs. 15 crores during 1978-79. The quantum of
export has dropped to almost half of what it was in 1976-77, when
India exported medicinal plants worth of Rs. 29 crores. During 1988-
89, India exported crude drugs alone to the tune of about 62 crores.
The items of export value are opium, psyllium husks and seeds,
Vinca rosea, Kuth roots, Nux-vomica, Galanga and Senna leaves and
pods. India is the second largest producer of castor seed in the
world. Producing about 1,25,000 tonnes per annum.
With development of phytochemical industry in India,
domestic requirement for various medicinal plants of grew
considerably. Consequently, the Government of India has adopted
restrictive export policy in respect of those crude drugs which were
indiscriminately exploited in the forest.
In accordance with the policy the exports of rauwalfia,
podophyllum, Indian rhubarb, dioscorea, saussurea etc. from India
were restricted. The export of these drugs is, however, permitted by
firms obtaining certificates from the chief conservator of forests or
officer autherized by him that the material is of plantation as
nursery origin.
Apart from requirements of medicinal plants for internal
consumption, India exports crude drugs mainly to developed
countries viz, USA, Germany, France, Switzerland, U.K. and Japan.
Who share between them 75 to 80 percent of the total export of
crude drugs? From India, the principal herbal drugs that have been
finding a good market in foreign countries are Aconite, Aloe,
Bellodena Acorus, Cinchona, Cassia tora, Dioscorea, Digitals,
Ephedra, Plantago (Isabgol), Cassia (Senna) etc.
The total value of export of crude drugs, Ayurvedic put up for
retail have increase from Rs. 394 crores in 1996-97 to Rs. 446
crores in 1998-99. (Purohit et. al., 2002)
1.2 PLANT PROFILE:
Photograph 1: leaves and flower of Clitoria ternatea:
1.2.1 Taxonomical Hierarchy:
Botanical Name : Clitoria ternatea L.
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order : Fabales
Family : Fabaceae
Subfamily : Faboideae
Tribe : Cicereae
Genus : Clitoria
Species : C. ternetea.
1.2.2 Vernacular Name:
Sanskrit : Aparajita, Girikarnika
English : Clitoria, Butterfly pea.
Hindi : Aparajita
Tamil : Kannikkoti
Telgu : Gilagarnika
Kan : Karnike
Malyalam : Samkhupuspam, Aral, Malaya Mukki
1.2.3 Description:
A rambling, pretty, indigenous climber up to 2-3m in height,
extensively grown in gardens for its flowers and also found
commonly as an escape in hedges and thickets throughout India,
up to an altitude of 1500m and in the Andaman Islands. Stem
scandent; leaves pinnately 5-foliolate, 6-13 cm long; leaflets ovate
or oblong, 2-5 cm long flowers papilionaceous, white or bright blue
with yellow or orange centre pods flat, beaked, seeds yellowish
brown, subglobose.
Though a hardly perennial, the climber is grown in gardens as
an annual and trained on bowers or trellises. The white and blue-
flowered types cross naturally resulting in a variety of colors and
single and double forms. (Wealth of India, 2001)
1.2.4 Habitat: Throughout India are hedges and thickets, also
cultivated in gardens.
1.2.5 Propagation: By Seeds
1.2.6 Part Used: Roots, leaves seeds.
Leaves: A good-looking perennial twining herb with terete stems
and branches, leaves compound, imparipinnate, leaflets 5-7, sub-
coriaceous, elliptic- oblong, obtuse.The shoots, leaves and tender
pods are eaten as vegetable in Kerala, and in the Philippines.
Flower Variety: The flower has an almond taste, cooling, acrid,
laxative, alexiteric, anthelminitic tonic to the brain, good for eyes
dise-ases, ulcers of the cornea, tuberculosis glands, elephantiasis,
head ache, cures, tridosha leucoderma burning sensation, pains,
bilious-ness, inflammation, ulcers, “Kapha” snake bites.
Blue Flowered Variety: The root is bitter and has all the
properties of that of the white flowered variety; in addition, it is
aphrodisiac; was density severe bronchitis asthma consumption
useful in as cites and abdominal enlargement (Ayurveda) the roots
purgative and diuretic useful in as cites (Unani).
1.2.7 Cultivation:
The climber yields green fodder throughout the year,
particularly during dry period. It can be grown as a forage legume
either alone or with perennial fodder grasses in Punjab, Rajasthan,
Uttar Pradesh, Gujarat, Maharashtra, Madhya Pradesh, Andhra
Pradesh, Tamil Nadu and Karnataka, and has been recommended
as a forage legume in the Andamans., It is being introduced a
drought resistant pasture in arid and semi-arid regions. The plant is
also suitable as a green manure and cover-crop. Besides
suppressing many perennial weeds, it enriches the soil by fixing
nitrogen.
The seeds are sown in August in rows 30 cm. apart. The crop
starts yielding green fodder (dry matter 21.8%) in 60 days, the first
cutting yielding 20-24 tonnes/ha. Protection from frost and drought
is necessary to ensure fodder supply throughout the year. The green
fodder can also be made into hay, each cutting yielding C. 5.4
tonnes/ha. The hay is used as good quality maintenance feed for
both growing and adult stock and is relished by sheep and goats.
1.2.8 Chemical Constituents:
The high calcium concentration in the plant showed that it can
be exploited as a significant source of calcium brewed as herbal
drink. The presence of stigmast-4-ene-3, 6, diene is reported from
the plant. The roots contain taraxerol and teraxerone. The leaves
contain 3- monoglucoside, 3-rutinoside, 3-neohesperidoside, 3-o-
rhamnosyl- glucoside, 3-o-rhamnosyl galactoside of Kaempferol,
besides kaempferol 3-o- rhamnosyl-o-rhamnosyl- glucoside.It also
contain aparajitin and β- sitosterol. The blue flowers contain
delphinidin-3,5-diglucoside delphinidin-3-β glucoside and its 3-
methyl derivative, malvidin-3β- glucoside, Kaempferol and cyanin
chloride ; the white flowers yield only kaempferol, other substances
present in the seeds are: p-hydroxycinnamic acid, flavonol-3-
glycoside, ethyl-α-D galactopyranoside, adenosine, 3,5,7,4- tetra
hydroxyflavone-3- rhamnoglucoside, a polypeptide, hexacosanol, β-
sitosterol and an anthoxanthin glucoside the seeds also contain
oligosaccharides or flatulene.
1.2.9 Uses:
The leaves are useful in ophthalmopathy, tubercular glands,
amentia, hemicrania, burning sensation, strangury, helminthiasis,
leprosy, leucoderma, elephantiasis, inflammation, vitiated
conditions of pitta, bronchitis, asthma, pulmonary tuberculosis,
ascites, ulcers, visceromegaly and fevers. The roots are bitter,
refrigerant, ophthalmic, laxative, intellect promoting, alexeteric,
diuretic, anthelmintic, depurative, aphrodisiac and tonic. The leaves
are also useful in otalgia hepatopathy and eruptions. The seeds are
cathartic and are useful in visceralgia.
1.3 DISEASE PROFILE:
1.3.1 INTRODUCTION OF DIABETES:
The knowledge regarding diabetes existed since Vedic period
and treatment of diabetes has been mentioned in “Sushruta
Samhita” in 200 B.C. Diabetes mellitus was known to ancient Indian
physicians as ‘Madhumeha’. This term Diabetes mellitus hails its
origin from a Greek word “diabainess” which means “to pass
through” and a Latin word “mellitus” meaning “sweetened” with
honey. Many herbal products including several metals and minerals
have been described for the care of Diabetes mellitus in ancient
literature. A medicinal plant, Galega officinalis, led to discovery and
synthesis of metformin. The modern medicine extensively used to
control blood glucose level. The earliest mention of the medicinal
use of plants is available in the ‘Rig Veda, which was written
between 4500 and 1600 B.C. In the ‘Atharva Veda’ (1500 B.C.). We
find more varied use of drugs. It is Ayurveda which is considered as
an ‘Upa Veda’ in which definite properties of drugs and their uses
have been given in great detail. (Kumar et .al., 2005)
Many oral hypoglycemic agents, such as biguanides and
sulfonylurea are available along with insulin for the treatment of
diabetes mellitus, but these synthetic agents can produce serious
side effects, and in addition, they are not suitable for use during
pregnancy. Since ancient times, diabetes has been treated orally
with several medicinal plants or their extracts based on folklore
medicine. These herbal remedies are apparently effective, produce
minimal or no side effects in clinical experience and are of relatively
low costs as compared to oral synthetic hypoglycemic agents. There
is a growing tendency all over the world to shift from synthetic to
natural products including medicinal plants. Today more than 25%
prescriptions issued in most of the developed countries of Europe
and the United States contains one or more plant drugs. Several
species of plants have been described as having ant diabetic
property. In Indian Materia Medica, 42 medicinal plants have been
recorded for the treatment of diabetes. (Indian Materia Medica,
1954)
According to the National Bureau of plant Genetic Resources,
the world Health Organization has listed more than 21,000 plant
species used around the world for medicinal purposes of which 8000
species of medicinal plants exists in this country. In Africa up to 80%
of the population uses traditional medicine for primary health care.
In Germany, 90% of the population has used a natural remedy at
some point of their life.
Diabetes mellitus (DM) is a widespread disorder, which has long
been recognized in the history of medicine, before the advent of
insulin and oral hypoglycemic drugs, the major form of treatment
involved the use of paints. More than 400 plants are known to have
been recommended ad recent investigations have affirmed the
potential value of some of these treatments. (Baily and Day, 1989)
Diabetes is not new to the medical world. It has been known
since antiquity, almost from 1500 BC. According to Charaka (2nd
century B.C.,) It is called PRAMEHA, i.e. the causative significant of
heredity, obesity and lack of physical activity; clinical features such
as thirst and dryness of mouth, peculiar odour, burning sensation or
lack of sensation in the hands and feet and onset of boils. (Reddy,
2005)
Diabetes mellitus is a group of endocrine syndromes
characterized by hyperglycemia; altered metabolism of lipids, carb-
ohydrates, and proteins, and an increased risk of complications from
vascular disease. Most patients can be classified clinically as having
either type I diabetes mellitus (type I DM formerly known as insulin-
dependent diabetes of IDDM) or type II diabetes mellitus (type II DM
formerly known as non-insulin dependent diabetes of NIDDM).
(Goodman and Gilman, 2001)
1.3.1.1 Type of Diabetes, causes and their treatment:
A. Type I Diabetes:
(Insulin dependent diabetes mellitus IDDM) Insulin dependent
diabetes most commonly afflicts juveniles, but it can also occur in
adults. The disease is characterized by an absolute deficiency of
insulin caused by massive β-cell lesions or necrosis. Loss of β-cell
function may be due to invasion by viruses, the action of chemical
toxin or usually through the actions of autoimmune antibodies
directed against the β-cells. As a result of the destruction of β-cells,
the pancreas fails to responsed to ingestion of glucose and the type
1 diabetes shows classic symptoms of insulin deficiency (Polydipsia,
Polyphagia and Polyuria). Type I diabetes requires exogenous insulin
to avoid hyperglycemia and life threatening ketoacidosis.
a) Cause of Type I Diabetes:
A burst of insulin secretion normally occurs after ingestion of a
meal in response to transient increase in the levels of circulating
glucose and amino acids. In the absorptive period. Low basal levels
of circulating insulin are maintained through β-cells secretion.
However, the type I diabetes has virtually to functional β-cells and
can neither respond o variation in circulating fuels nor maintain
even a basal nephropathy and retinopathy are directly related to the
extent of glycemic control.
b) Treatment of Type I Diabetes:
Type I diabetes requires exogenous (injected) insulin in order
to control hyperglycemia, maintain acceptable levels of glycosylated
haemoglobin and avoid ketoacidosis. The goal of administering
insulin to type I diabetes is to maintain blood glucose concentration
as close to possible and to avoid wide swings in blood glucose levels
that may contribute to long term complications. The use of portable
blood glucose analyzers facilitates close self-monitoring and
treatment.
B. Type II Diabetes:
(Non-Insulin dependent diabetes, mellitus, NIDDM) Most
diabetic are in this category, genetic factors, rather than viruses or
autoimmune antibodies are apparently casua1. The metabolic
alterations observed are milder than those described for IDDM (for
example, NIDDM patients typically are not ketosis) but the long term
clinical consequences can be just as devasting (for example,
vascular complications and subsequent infection can lead to
amputation of the lower limbs.)
a) Cause of Type II diabetes:
In NIDDM the pancreas retains some β-cells function, resulting
in variable insulin levels that are insufficient to maintain
homeostasis patients with type II diabetes are often obese. Type II
diabetes is frequently accompanied by target organ insulin
resistance that limits responsiveness to both endogenous and
exogenous insulin. In some cases, insulin resistance is due to a
decreased number or mutations of insulin receptors.
b) Treatment of Type II diabetes:
The goal in treating type II diabetes is to maintain blood gluc-
ose concentration within normal limits and to prevent the
development of long term complication of disease weight reduction.
Exercise and dietary modification decrease insulin resistance and
correct the hyperglycemia of Type II diabetes in some patients
however most are dependent on pharmacological intervention with
oral hypoglycemic agents. Insulin therapy may be required to
achieve satisfactory serum glucose levels sulphonylureas and
bigunide are two most commonly prescribed oral treatment option.
(Mary et. al., 2000)
C) Type III Gestational Diabetdes:
Gestational diabetes mellitus is an operational classification
(rather than a pathophysiologic condition) identifying women who
develop diabetes mellitus during gestation (Women with diabetes
mellitus before pregnancy are said to have “pregestational
diabetes” and are not included in this group). Women who develop
type I diabetes mellitus during pregnancy and women with
undiagnosed asymptomatic type II diabetes mellitus that is
discovered during pregnancy are classified with gestational diabetes
mellitus. However most women classified with gestational diabetes
mellitus have normal glucose homeostasis during the first half of the
pregnancy and develop a relative insulin deficiency during the last
half of the pregnancy leading to hyperglycemia. The hyperglycemia
resolves in most women after delivery but places them at increase
risk of developing type II diabetes mellitus later in life. (National
diabetes data group, 1995)
1.3.1.2 Characterization of Diabetes Mellitus:
It is metabolic disorder characterized by hyperglycaemia,
glycosuria, hyperlipemia, negative nitrogen balance and sometimes
ketonemia, A wide spread pathologic change is thickening of
capillary basement membrane, increase in vessel wall matrix and
cellular proliferation early atherosclerosis, sclerosis of glomerular
capillaries, retinopathy, neuropathy and peripheral vascular
insufficiency. (Tripathi K.D., 1985)
A. Atherosclerosis:
The diabetic has a two to three fold higher risk of dying
prematurely of atherosclerosis that a non diabetic individual
Foremost is the reduction of LDL-cholesterol levels and triglyceride
while increasing HDL- cholesterol levels. [Mohan Harsh, 2004]
B. Diabetic Neuropathy:
Diabetic neuropathies are among the most frequent
complications of long term diabetes. Loss of peripheral nerve
function. Tingling sensations, number loss of pain, and muscle
weakness may occur as a result of diabetic retinopathies. In rats
increasing the sorbitol concentration in the sciatic nerve is directly
related to decreasing nerve conduction velocity, possible as a result
of decreeased myoinostiol concentration.
C. Diabetic Retinopathy:
World Diabetes Day is celebrated every year on 14 November,
which is incidentally the birthday of Frederick Banting, who together
with Charles Best discovered insulin in the year 1921. This year’s
theme of world diabetes Day was to address retinopathy one of the
complications of DM. Your Eyes and Diabetes: Don’t Lost sight of the
Risks. Diabetic retinopathy is a serious eye disease that can result in
blindness. The retinopathic lesions are divided into background or
“simple” retinopathy (consisting of microaneurysms, haemorrhages,
exudates and retinal edema) and proliferative or malignant (with
newly formed vessels, scaring retinitis proliferens vitreous
haemorrhage and retinal detachement).
D. Diabetic Nephropathy:
This is a common complication and a leading cause of death in
DM. Four types of possible overlapping lesions develop: glomerulo
sclerosis arteriosclerosis of the efferent and afferent arteriosclerosis
of the renal artery and its intrarenal branches; and peritubular
deposits of glycogen fat and mucopolysaccharides. Periodic
monitoring of diabetic patients kidney function (uric acid, creatinine
and creatininme clearance) is important.
E. Diabetic Foot Ulcers:
Ischemia and peripheral neuropathy are the key factors in the
development of diabetic foot ulcers. However foot ulcers are largely
preventable through proper foot care, the avoidance of injury and
tobacco, in any form and employing methods to improve local
circulation. Tobacco constricts the peripheral blood vessels and can
cause Burger’s disease. (Yue et. al., 1984)
1.3.1.3 Diagnosis:
A number of tests for hyperglycemia have been assessed as
diagno-stic tools for DM, namely fasting plasma glucose (FPG),
casual plasma glucose (PG), 2- hour plasma glucose in a 75 gram
oral glucose tolerance test (OGTT) and haemoglobin Ale (Ale). At the
present time, well established diagnostic criteria for DM exist
utilizing all of the above, except Ale. These criteria, which are
supported by the Canadian Diabetes Association (CDA), America
Diabetes Association (ADA) and the World Health Organization, in
the absence of unequivocal hyperglycemia, the diagnosis needs to
be confirmed by repeat testing on a separate day. The diagnostic
criteria for DM are useful to identify patients at risk for developing
the micro vascular complications of diabetes. However there has
been increasing recognition that even lesser degrees of
abnormalities of glucose metabolism are associated with an
increased risk of developing cardio vascular complications and of
course, diabetes. Thus the concept of impaired glucose (IFG) was
introduced. The diagnostic criteria for IFG and IGT have evolved
over time with the emergence of data suggesting a further
lowering of the threshold fasting glucose value from 6.1 to 5.6
mmol/L by the ADA in 2002, However, discrepancies remain
between organization and the CDA’s current definition of IFG is
fasting plasma glucose of 6.1-6.9 mmol/L and IGT is defined as a
2-hour value based on the 75 g OGTT of 7.8-11.0 mmol/L.
Diagnostic criteria for diabetes mellitus according to the Canadian
Diabetes Association, American Diabetes Association and World
Health Organization.
FPG = 7.0 mmol/L
Fasting = no. caloric intake for at least 8 hours.
OR
Casual PG= 11.1 mmol/L + symptoms of diabetes
OR
2hPG in a 75g OGTT = 11.1 mmol/L.
A confirmatory laboratory glucose test must be done in all cases
on another day in the absence of unequivocal hyperglycemia
accomp-anied by acute metabolic decomposition symptoms of
diabetes as polyuria, polydipsia and unexplained weight loss.
Table: 1.1 represents the Diagnostic criteria for impaired
glucose tolerance (IGT) and impaired fasting glucose (IFG) according
to recommendation from the Canadian Diabetes Association (CDA),
American Diabetes Association (ADA) and The World Health
Organization (WHO).
CDA ADA WHO
IGT2hPH OGTT7.8-11.0
2hPG OGTT 7.8-11.0
FPG < 7.0 AND 2hPG OGTT 7.8-11.0
IFG FPG 6.1-6.9 FPG 5.6-6.9FPG 6. 1-6.9 AND 2hPG < 7.8
IFG and IGTBoth of the above criteria
Both of the above criteria
N/A
FPG = fasting plasma glucose
2hPG OGTT = 2 hour plasma glucose in a 75 g oral glucose
tolerance test – all values are in mmol/L.
PG = plasma glucose
OGTT = oral glucose tolerance test
1.3.1.4 Traditional approach of diabetes therapy using
plants:
History of medicine dates back practically to the existence of
human civilization. Natural products, including plants, animals, and
minerals have been the basis of treatment of human disease.
According to the World Health Organization more than70% of the
world population must use traditional medicine to satisfy their
principal health needs. The current accepted modern medicine or
allopathy has gradually developed over the years by scientific and
observational efforts of scientists. However, the basis of its
development remains rooted in traditional medicine and therapies.
Numerous drugs have entered in “International Pharmacopoeia”
structures, which may be used as templates for the development of
new drugs. Natural products are prescribed widely because of their
effectiveness, less side effects and relatively low cost. A great
numbers of medicinal plants used in the control of diabetes mellitus
have been reported.
Table.1.2: List of some Medicinal plants used in the
treatment of Diabetes: (Khan et. al., 2005)
S.No
Plant Name FamilyUseful Part
1. Abroma augusta L.f. Sterculiaceae Bark, Flower
2. Annona squamosa L. Annonaceae Leaves
3. Barleria cristata L. Acanthaceae Roots
4. Beta vulgaris L. Betulaceae Bark
5. Calamug rotang L. Arecaceae Bark
6. Cannabis sativa L. CannabinacaeResin & Leaves
7. Desmodium gyrans L. Papilionaceae Roots
8. Dioscorea alata L. Dioscoreaceae Rhizome
9. Eryngium foelidum L. ApiaceaeWhole
plant
10. Ficus fistulosa L. Moraceae Fruit
11. Gymnema sylvestrisAsclepiadacea
eLeaves
12. Hordeum Vulgare L. Poaceae Seed
13. Ipomaea balatus L. ConvolvulaceaeTuberus Roots
14. Juslicia adhatoda L. Acanthaceae Leaves
15. Kyllianga bulbosa CyperaceaeWhole
plant
16. Lysium barbala L. Solanaceae Fruits
17. Momordica charanlia Cucurbitaceae Fruit
18. Nepeta cataria L. LamiaceaeLeaves & Flowering
19. Oplopanax horridum Umbelliferae Root
20. Picrorhiza kurrooa Scrophulariaceae Herb
21. Quercus lineala Blume Fagaceae Stem bark
22. Rotula aquatica Lour. Boraginaceae Root
23. Swertia chirata Gentianaceae Whole Plant
24.Trigonellafoenum
graecum L.Papilionaceae Seed
Some steroidal plants used for the purpose are barks of various
species of ficus, the roots of ginseng, fenugreek, and the fruit and
seed of various Cucurbitaceae families. It also includes famous
Momordica charantia or Kerala fruit. (Ansari, 2005)
Other plants which are most effective and most commonly
studied in relation to diabetes and their complication are Allium
cepa, Allium sativum, Aloe Vera, Cajannus Cajan, Gymnema Sylve-
stris, Ocimum Sanctum and Tinospora Cordifolia. (Grover et. al.,
2002)
1.3.1.5 Statistics of Diabetes:
It is alarming that India is fast assuming the mantle of being
the diabetic capital of the world. We have the largest number of
diabetics in the world and the number of new cases has increased
from 1% before 1970 to 8% in the 80’s and is still growing as per
ICMR studies. (Reddy, 2005). India is expected to have 40 million
people with diabetes by the year 2010 and 57.2 million by 2025.
There are more than 125 million persons with diabetes in the world
today, and by 2010 this number is expected to approach 220
million. (Amos et. al., 1997) Some investigators expect the
incidence to double by 2035. Types I and II are both increasing
frequently. Diabetics are 25 times more likely to develop heart
attacks and twice as likely to get strokes as compared to non-
diabetics. About one or two million patients have type I the
remaining 80 to 90% of diabetic patients have type II diabetes.
(Goodman and Gillman, 2001)
1.3.2 INTRODUCTION OF INFLAMMATION:
Inflammation is a complex pathophysiological process
mediated by a variety of singling molecules produced by leukocytes,
macrophages and mast cells as well as by the activation of
complement factors, which bring about edema formation as a result
of extravasations of fluid, proteins etc and pain at the site of
inflammation (White, 1999).
The Roman writer Celsus named the famous four Cardinal Signs
of inflammation as:
Rubor (redness)
Tumor (swelling/ edema)
Color (heat)
Dolor (pain)
The fifth sign function lasea (loss of function) was later added
by Virchow.
1.3.2.1 Types of inflammation:
Depending upon the defense capacity of host and duration of
response inflammation can be classified in to following types;
A. Acute inflammation:
In acute inflammation short duration in which PMN are the
main cells for early body reaction leads to accumulation of fluid and
migration of leucocytes and platelets to the affected site followed by
repair.
Pathophysiology of acute inflammation
The earliest response to tissue injury leads to alterations
including hemodynamic changes and changes in vascular
permeability.
1. Transient vasoconstriction
Irrespective of type of injury of arterioles vasoconstriction may
last longer from 3-5 second with mild form of injury to 5 minutes for
severe form of injury.
2. Persistent progressive vasodilatation
Vasodilatation results in increased blood volume in
microvascular bed in arterioles within half an hour of injury due to
Histamine, 5-HT which are responsible for redness and warmth at
site of acute inflammation.
3. Elevation of local hydrostatic pressure
Progressive vasodilatation may lead local hydrostatic pressure
resulting in transudation of fluid in extracellular space which is
responsible for swelling.
4. Stasis of microcirculation
Slowing is attributed to increased permeability of
microvascular that results in increased in the concentration of red
cells, and thus, raised blood viscosity.
5. Leukocyte migration
Leukocyte sticks to the vascular endothelium due to factors
such as selectin, intigrin and ICAM-1. After attaching to the
endothelial wall these secretes collagenase which cause breakdown
of endothelium and basement membrane and escape out towards
the inflammation site this known as emigration. Chemokinenes are
substances which attract leucocytes toward inflammation site are IL-
2, LT-B4, PF-4 MCP-1.
6. Phagocytosis
Phagocytosis is defined as the process of engulfing of the solid
particulate material by the cells. There are two types of phagocytic
cells, these are PMNs and macrophages and tissue macrophages.
B. Chronic inflammation:
Chronic inflammation is a prolonged reaction arising when the
acute response is insufficient to eliminate proinflammatory agents.
It includes a proliferation of fibroblast & infiltration of the
neutrophiles and exudation. Chronic inflammation has two types
such as specific and non specific can be caused by following 3 ways:
Chronic inflammation following acute inflammation
Recurrent attacks of acute inflammation
Chronic inflammation starting de novo
General features of chronic inflammation
Though there may be differences in chronic inflammatory
response depending upon the tissue involved and causative
organisms, these are the general characteristic of chronic
inflammation;
1. Mononuclear cell infiltration:
Chronic inflammatory lesions are infiltrated by mononuclear
inflammatory cells like phagocytes and lymphoid cells. Phagocytes
are represented by circulating monocytes, tissue macrophages,
epithelioid cells and multinucleated giant cells. The macrophages
compromise the most important cells in chronic inflammation. On
activation they release several biological active substances such as;
acid and neutral proteases, oxygen-derived reactive metabolites
and cytokines. These products bring tissue destruction,
neovascularisation and fibrosis.
2. Tissue destruction or necrosis
Tissue destruction brought about by activated macrophages
which release of a variety of factors like protease, elastase,
collagenase, lipase, reactive oxygen radicals, cytokines (IL-1, IL-8
and TNF), nitric oxide, angiogenesis growth factors etc.
3. Proliferative changes
As results of necrosis, proliferation of small blood vessels and
fibroblasts is simulated resulting in the formation of inflammatory
granulation tissue. Eventually, healing by fibrosis and collagen lying
takes place.
Fig-1.1: Progress of inflammation
Neutrophils migrate from blood vessels to the inflamed tissue via
chemotaxis, where they remove pathogens through phagocytosis
and degranulation.
1.3.2.2 Morphological changes in inflammation:
Specific patterns of acute and chronic inflammation are seen
during particular situations that arise in the body as shown below.
1. Granulomatous inflammation
It is characterized by the formation of granulomas, they are
the result of a limited but diverse number of diseases, among which
are tuberculosis, leprosy, and syphilis.
2. Fibrinous inflammation
Inflammation resulting in a large increase in vascular
permeability allows the blood vessels to pass through fibrin. If an
appropriate procoagulative stimulus is present, such as cancer cells,
fibrinous exudate is deposited. This is commonly seen in serous
cavities, where the conversion of fibrinous exudates into a scar can
occur between serous membranes, limiting their function.
3. Purulent inflammation
Infection of pyrogenic bacteria such as staphylococci causes
inflammation resulting in large amount of pus which consists of
neutrophils, dead cells, and fluid. Large, localized collections of pus
enclosed by surrounding tissues are called abscesses.
4. Serous inflammation
It is characterized by the copious effusion of non-viscous
serous fluid, commonly produced by mesothelial cells of serous
membranes, but may which also be derived from blood plasma. Skin
blisters exemplify this pattern of inflammation.
5. Ulcerative inflammation:
Inflammation occurring near an epithelium can result in the
necrotic loss of tissue from the surface, exposing lower layers.
Excavation in the epithelium is known ulcer.
1.3.2.3 Chemical mediators of inflammation:
These are a large number of endogenous compounds which
can enhance vascular permeability. These are broadly classified
into 2 groups:
A. Cell-derived mediators (Table 1.3)
Vasoactive amines (Histamine, 5-HT)
Arachidonic acid metabolites (Eichosanoids)
Lysosomal components
Platelet activating factors
Cytokines (IL-1, TNF-α, TNF-β, IF-γ and chemokines)
Nitric oxide and oxygen metabolites
B. Plasma-derived mediators (plasma protease) (Table
1.4)
The kinin system
The clotting system
The fibrinolytic system
The complement system
5-Hydroxytryptamine
It is present in tissue like chrommaffin cells of GIT, spleen,
nervous tissue, mast cells and platelets. The actions of 5-HT are
similar to histamine but less potent mediators than histamine in
increasing vascular permeability and vasodilatation.
Platelet activating factor (PAF)
It is released from IgE-sensitized basophiles or mast cells,
other leucocytes, endothelium and platelets. Apart from its action
on platelets aggregation and release reaction, the actions of PAF as
mediators of inflammation are increased vascular permeability,
adhesion of leucocytes to endothelium, Chemotaxis and cell-
mediated immunity to the irritant, implying thereby the role of
hypersensitivity in granulomotous inflammation.
Prostaglandins
PGs play a significant role in different phase of inflammatory
reactions. PGs elicit pain by direct stimulation of sensory nerve
ending and also sensitize sensory nerve endings to other pain
provoking stimuli (Campbell et al., 1991). Especially PGE was
reported to act on cell membrane during inflammatory condition
leading to destabilization in lipoprotein structure of cell membrane
(Bhaskar et al., 1987). PGI2 plays an important role in vascular
function because, like nitric oxide, it inhibits platelet adhesion to the
vascular endothelium and is a strong vasodilator. Thromboxanes
and leukotrienes produce vasoconstriction and are important
modulators.
Table: 1.3 Cell derived mediator
Name Type Source Description
Lysosome granules
Enzymes Granulocytes
These cells contain a large variety of enzymes which perform a number of functions. Granules can be classified as either specific or azurophilic depending upon the contents, and are able to break down a number of substances, some of which may be plasma-derived proteins which allow these enzymes to act as inflammatory mediators.
HistamineVasoactive
amine
Mast cells, basophils, platelets
Stored in preformed granules, histamine is released in response to a number of stimuli. It causes arteriole dilation and increased venous permeability.
IFN-γ CytokineT-cells, NK
cells
Antiviral, immunoregulatory, and anti-tumour properties. This interferon was originally called macrophage-activating factor, and is especially important in the maintenance of chronic inflammation.
IL-8 Chemokine Primarily macrophag
e
Activationand chemoattraction of neutrophils, with a weak
effect on monocytes and eosinophils.
Leukotriene B4
Eicosanoid Leukocytes
Able to mediate leukocyte adhesion and activation, allowing them to bind to the endothelium and migrate across it. In neutrophils, it is also a potent chemoattractant, and is able to induce the formation of reactive oxygen species and the release of lysosome enzymes by these cells.
Nitric oxideSoluble
gas
Macrophage endothelial cells, some
neurons
Potent vasodilator, relaxes smooth muscle, reduces platelet aggregation, aids in leukocyte recruitment, direct antimicrobial activity in high concentrations.
Prostaglandins
Eicosanoid Mast cellsA group of lipids which can cause vasodilation, fever, and pain.
Table: 1.4 Plasma derived factors
M Name Type d by Description
Bradykinin Kinin system
A vasoactive protein which is able
to induce vasodilation, increase
vascular permeability, cause
smooth muscle contraction, and
induce pain.
C3 Complement
system
Cleaves to produce C3a and C3b.
C3a stimulates hishistamine
release by mast cells, thereby
producing vasvasodilation. C3b is
able to bind to bacterial cell walls
an act as an opsonin, which marks
the invader as a target for
phagocytosis.
C5aComplement
system
Stimulates histamine release by
mast cells, thereby producing
vasodilation. It is also able to act
as a chemoattractant to direct
cells via chemotaxis to the site of
inflammation.
Factor XII
(Hageman
Factor)
Liver
A protein which circulates
inactively, until activated by
collagen, platelets, or exposed
basement membranes via
conformational change. When
activated, it in turn is able to
activate three plasma systems
involved in inflammation: the kinin
system, fibrinolysis system, and
coagulation system.
Membrane
attack complex
Complement
system
A complex of the complement
proteins C5b, C6, C7, C8, and
multiple units of C9. The
combination and activation of this
range of complement proteins
forms the membrane attack
complex, which is able to insert
into bacterial cell walls and causes
cell lysis with ensuing death.
Plasmin Fibrinolysis Able to break down fibrin clots,
systemcleave complement protein C3,
and activate Factor XII.
ThrombinCoagulation
system
Cleaves the soluble plasma protein
fibrinogen to produce insoluble
fibrin, which aggregates to form a
blood clot. Thrombin can also bind
to cells via the PAR1 receptor to
trigger several other inflammatory
responses, such as production of
chemokines and nitric oxide.
1.3.2.4 Inflammatory associated disorders:
Asthma
Autoimmune diseases
Chronic prostatitis
Glomerulonephritis
Hypersensitivities
Inflammatory bowel diseases
Pelvic inflammatory disease
Rheumatoid arthritis
Transplant rejection
1.3.2.5 Drugs used as analgesic and anti-inflammatory
agents:
1. Opioid used as analgesic: Morphine, Pholcodine, Pethidine,
Fentayl
2. Corticosteroids: Betamethasone, Betanosolone
3. Drugs that act on COX
a. Nonselective COX inhibitors
Salicylates: Aspirin, Diflunisal
Pyrozolone derivatives: Phenylbutazone, Oxyphenbutazone
Indole derivatives: Indomethacin, sulindac
Propionic acid derivatives: Ibuprofen, Naproxen,
Ketoprofen
Anthranilic acid derivative: Mephenamic acid
Aryl-acetic derivatives: Diclofenac
Oxicam derivatives: Piroxicam, Tenoxicam
Pyrollo-pyrrole derivatives: Ketorol
b. COX-2 Inhibitors
Preferential inhibitors: Nimesulide, Meloxicam, Nabumetone
Selective COX-2 inhibotors: Celocoxib, Rofecoxib, Valdecoxib
2. LITERATURE REIVEW
Naeem A., et al., (2007) reported an alteration high yielding
purification method for clitoria ternatea lectin, In our previous
publication we had reported the purification and characterization
of Clitoria ternatea agglutinin from its seeds on fetuin CL agarose
affinity column, designated CTA [A. Neem, S. Haque, R.H. Khan.
Protein J., 2007] Since CTA binds B-D- galactosides, this lectin can
be used as valuable tool for glycobiology studies in biomedical and
cancer research. So an attempt was made for a high yielding
alternative purification method employing the use of asialoftuin CL
agarose column for the above mentioned lectin, designated CTL.
Kogawa K., et al., (2007) reported Biosynthesis of malonylated
flavonoid glycosides on the basis of malonyltransferase activity in
the petals of clitoria ternatea. The crude malonyltransferase from
the petals of Clitoria ternatea was characterized enzymatically to
investigate its role on the biosynthetic pathways of anthocyanins
and flavonol glycosides. In C. ternatea, a blue flower cultivars (DB)
and mauve flower variety (WM) accumulate polyacylated
anthocyanins (ternatins) and delphinidin3-O- (6”-O-malonyl)-β-
glucoside which is one of the precursors of ternatins, respectively
Moreover WM, accumulates minor delphinidin glycosides-3-O-B
glucoside, 3.-O (2” O-a-rhamnosyl)-B- glucoside .
Juma H.K., et al., (2006) reported evaluation of clitoria, Gliricida
and Mucuna as nitrogen supplements to Napier grass basal diet in
relation to the performance of lactating Jersey cows. A study was
carried out at the Kenya Agricultural Research Institute Mywapa in
Coastal lowland Kenya to evaluate the effects of supplementing
Napier Grass variety Bana (Pennisetum Purpureum ) with clitoria
ternatea (Clitoria), Gliricidia sepium (Gilricidia) and Mucuna
Pruriuens (Mucuna) on feed intake, diet, digestibility and milk yield
of lactating Jersey cows.
Parimaladeve B. et al., (2004) reported evaluation of antipyretic
potential of clitoria ternatea L. extract in rats. The methanol extract
of Clitoria ternatea L. root (MECTR) blue flowered variety (family:
faba-ceae), was evaluated for its anti-pyretic potential on normal
body temperature and yeast induced pyrexia in albino rats. Yeast
suspension (10ml/kg body wt.) increased rectal temperature after
19 hours of subcutaneous injection. The extract at doses of 200,300
and 400 mg/kg. Body wt., p.o., produced significant reduction in
normal body temperature and yeast provoked elevated temperature
in a dose dependent manner. The effect extended up to 5 hours
after the drug administration. The anti- pyretic effect of the extract
was comparable to that of paracetamol (150 mg/Kg. body wt., p.o.,)
a standard anti-pyretic agent.
Nataraja K., et al., (2005) reported screening of antibacterial
activity in the extract of clitoria ternatea. Hexane methanol and
water extracts of leaf, stem and roots of white flowered variety of
clitoria ternatea (Linn.) (Febaceae) used by Indian traditional healers
for treating ulcer, eye infections, bronchitis, tuberculosis and/or anti-
inflammatory properties were screened for in vitro antibacterial
activities.
Kelemu S., et al., (2005) reported evaluation of antipyretic
potential of clitoria ternatea L. extract in rats. The tropical forage
legume clitoria ternatea (L.) has important agronomic traits such as
adaptation to a wide range of soil conditions and resistance to
drought. It is resistant to a number of pathogens and pests. These
import-ant traits gave reasons to look more closely at the plant. A
highly basic small protein was purified from seeds of C. ternatea to
homogeneity by using ultrafiltration with Centricon-3 membrane
tubes and preparative granulated- bed isoelectric focusing (IEF). A
single protein band was obtained on both sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and IEF gels.
Shroff S.K., et al., (2003) reported clitoria ternatea and the CNS.
The present investigation was aimed at determining the spectrum of
activity of the methanolic extact of Clitoria ternatia (CT) on the CNS.
The CT was studied for its effect on cognitive behavior, anxiety,
depression, stress and convulsions induced by pentylenetetrazol
(PTZ) and maximum electroshock (MES) to explain these effects the
effect of CT was also studied on behavior mediated by dopamine
(DA), noradrenaline, serotonin and acetylcholine. The extract
decreased time requ-ired to occupy the central platform (transfer
latency TL) in the elevated plus maze (EPM) and increased
discrimination index in the object recognition test, indicating no
tropic activity.
Nagappa A.N. et al., (2003) studied antidiabetic activity of
Terminalia catappa Linn. Fruits. In view of alleged antidiabetic
potential, effect of the petroleum ether, methanol, and aqueous
extracts of Terminalia catappa Linn (combretaceae) fruit, on fasting
blood sugar levels and serum biochemical analysis in alloxan
induced diabetic rats were investigated. All the three extracts of
Terminalia catappa produced a significant antidiabetic activity at
dose levels 1/5 of their lethal doses. Concurrent histological studies
of the pancreas of these animals showed comparable regeneration
by methanolic and aqueous extracts which were earlier, necrosed
by alloxan.
Babu V. et al., (2003) reported antidiabetic activity of ethanol
extract of Cassia kleinii leaf in streptozotocin induced of diabetic
rats and isolation of an active fraction and toxicity evaluation of the
extract.
Kazuma K. et al., (2003) reported malonylated flavonal
glycosides from the petals of clitoria ternatea. Three flavonol
glycosides kaempf-erol 3-O-(2”-O-a-rhamnosyl-6-O-malonyl)-β-
glucoside, querce-tin 3-O-(2---O-a rhamnosyl-6”-O-malonyl-β-
glucoside and myricetin 3-O- (2”-6-di-O-a-rhamnosyl)-β-glucoside
were isolated from the petals of Clitoria ternatea cv., Double Blue,
Together with eleven known flavonol glycosides. Their structures
were identified using UV, MS and NMR spectroscopy.
Boominathan R. et al., (2003) reported Anti- Inflammatory,
analgesic and antipyretic properties of Clitoria ternatea root. Clitoria
ternatea roots methanol extract when given by oral route to rats
was found to inhibit both the rat paw oedema caused by carrageen
in and vascular permeability induced by acetic acid in rats.
Moreover the extract exhibited a significant inhibition in yeast
induced pyrexia in rats. In the acetic acid- induced writhing
response, the extract markedly reduced the number of writhings at
doses of 200 and 400 mg/kg. (p.o.,) in mice.
Kazuma K. et al., (2003) reported flavonoid composition related
to petal color in different lines of Clitoria ternatea. Flavonoids in the
petals of several Clitoria ternatea lines with different petal colors
were investigated with LC/MS/Ms. Delphinidin 3-O-(2”O-a-rhamnosyl-
6-O-malonyl)-B- glucoside was newly isolated from the petals of a
mauve line (wm) together with three known anthocyanins. They
were identified structurally using UV, Ms, and NMR spectroscopy.
Although ternatins a group of 15 (poly) acylated delphinidin
glucosides were identified in all the blue petal lines Double Blue and
Albiflora WM, accumulated delphinidin glucosides were identified in
all the blue petals lines.
Rai K.S. et al., (2002) reported clitoria ternatea root extract
enhances a cetylcholine content in rat hippocampus. Treatment with
100 mg/Kg. of clitoria ternatea aqueous root extract (CTR,) for 30
days in neonatal and young adult age groups of rat, significantly
increased acetylcholine (ACh) content in their hippocampi as
compared to age matched controls. Increase in ACh content in their
hippocampus may be the neurochemical basis for their improved
learning and memory.
Chaudhari U.S. and Hutke V., (2002) reported Ethano-medico-
botanical information on some plants used by melghat tribes of
Amravati district, Maharashtra. The paper deals with ethnobotanical
uses of 14 plant species among the Korian and Gond tribes living in
Melghat forests of Amravati district. The study comprises
information on traditional formulations, modes of administration and
the ailments for which they are effective. Use of root of clitoria
ternatea mixed with hen blood and honey in chronic cough is found
to be a unique method of cure. Chlorophytum borivilianum and
Plumbago are preferred as medicines, moreover leaves are
generally uses food.
Terahara N. et al., (1996) Reported Five new anthocyanins,
Ternatins A3, B4, B3B2, and D2 from clitoria ternatea flowers.: Five
new ternatins 1-5 have been isolated from Clitoria ternatea flowers,
and the structures have been determined by chemical spectroscopic
methods as delphinidin 3-GCG-5-GCG-3-GCG-5CG3’- and 3’-GCGC-
side chains respectively in which G is D-glucose and C is a coumaric
acid. Pigment 1 had symmetric 3’5- side chains. Compounds 3 and 4
are structural isomers. These trernatins were shown to form an intra
molecular stacking between the aglycon ring and the 3’5-‘side
chains in solution.
Bavaliya N.K., (1993) reported poisonous lagurnes of Rajasthan 5
The paper deals with 33 poisonous leguminous species which are
toxic to men, animals, fishes and livestock or other living things
while enumerating the species are arranged alphabetically, with
their habit common/local name (s) toxic part of the plant toxic to
which living organism and their distribution in state of Rajasthan in
a tabular form.
Terahara N. et al., (1990) Reported acylated anthocyanims of
clitoria ternatia flowers and their acyl moieties. Two acyl moieties
prepared by alkaline deacylation or H2O2 oxidation of ternatin
mixture from Clitoria ternatea flowers, were determined as E-4-O-B-
D glucopyranoisyl p-coumaric acid and 6-O-malonylD-
glucopyranose respectively through FABMS and NMR. Furthermore
six ternatins A1, A2, B1, B2, D1 and D2 in C. ternmatea flowers
were isolated by reversed phase HPLC and their structures were
partly characterized as highly acylated delphinidin derivatives.
Venkatesh S. et. al., (1969) reported antidiabetic activity of
helicteres isora root. The different extracts of the roots of helicteres
isora (Family Sterculiaceae) were tested for antidiabetic activity by
glucose tolerance test in normal rats and alloxan induced diabetic
rats. alloxan diabetic rats the maximum reduction in blood glucose
was observed after 3h at a dose level 250 mg/kg of body weight.
3. PLAN OF WORK
1- Literature survey of selected medicinal plant.
2 - Collection and Authentication of Clitoria ternatea leaves.
3 - Phytochemical Investigation:
A. Extraction of drug powder
B. Phytochemical test.
C. Thin layer chromatography, HPTLC and Column
Chromatography.
D. Identification and characterization of constituent by H-
NMR, IR and Mass spectroscopy.
4 - Assesment of antidiabetic activity.
5 - Assesment of anti-inflammatory activity.
6 - Statistical Analysis.
4. COLLECTION & EXTRACTION
4.1 COLLECTION AND AUTHENTICATION OF CRUDE DRUG
The fresh leaves of Clitoria ternatea was collected during the
month of September 2008, from my village Kailiya and gandoli
(Distt-Jalaun), the Kush Nursury, Gwalior Road, Jhansi and from the
Institute of Pharmacy, Bundelkhand University, Jhansi The plant
materials was taxonomically identified and authenticated by Dr.
Gaurav Nigam, Botany Department, Bundelkhand University, Jhansi.
Herbarium and Museum Division with ref. no. BU/BOT /376/24-01-
2009.
4.2 EXTRACTION:
The leaves of Clitoria ternatea were shaded dried until
cracking sound was observed during breakage, and then these are
made into coarsely powdered from using dry grinder. The powdered
leaves of the plant (600 gm.) was packed in soxhlet apparatus and
continuously extracted with petroleum ether (40-600C) till complete
extraction, after completion of extraction the solvent was
removed by distillation and then concentrated extract obtained was
dried under reduced pressure using rotatory evaporator at
temperature not exceeding 400C and then give moderate heating
on water bath. A pale green extract approximate 18 gm. was
obtained. From the drug, petroleum ether was removed and the
defatted drug was extracted with methanol till complete extraction,
after completion of extraction the solvent was removed by
distillation and then concentrated extract obtained dried under
reduced pressure at temperature not exceeding 400C and then give
moderate heating on water bath. The methanolic extract obtained
was greenish black in colour, weighed about 40 gm. The both
petroleum ether and methanolic extract was kept in petridish and it
was stored in desiccator at cool place (Mukherjee, 2002).
Powdered Crude Drug
Extracted with Petroleum Ether
(40-600C)
Defatted Powdered Drug Petroleum Ether Extract
Dried in Hot Air Oven Below 500 C
Extracted with Methanol Solvent removed by Distillation
Extracted Drug (Discarded) Methanolic extract
Traces of solvent removed under
Reduced pressure
Transferred remaining to a tarred dish
and dried to constant weight
Extract of Drug was collected and stored
in a Dessicator at room temperature
Fig.4.1 The Extraction procedure in schematic manner.
Table:4.1 The characteristics of methanolic extract.
S.
No.Characteristics
Methanolic
extract
Pet. Ether
extract
1. Extractive Value (%) 6.66 % 3%
2. Physical appearance Semisolid mass Semisolid mass
3. Colour Greenish black Yellowish green
4. Odour Odourless Odourless
5. Taste bitter bitter
5. PHYTOCHEMICAL SCREENING
The systematic phytochemical investigations not only help in
revealing the active components but also help in the synthesis of
better and newer analogues and congeners of higher therapeutics
activities or the various active principals isolated from plants. The
products of the investigations sometimes prove to be significant
than the ordinary plant constituent.
It is desirable not only for the discovery of new therapeutic
agents but also because such information may lead to the new
source of economically useful material and intermediates for the
synthesis of complex chemical substances. Again isolation of the
compound which is not necessarily of any intrinsic value in itself
but has a novel chemical structure may be stimulate the chemist
to modify the molecule to obtain semi synthetic substances having
medicinal and other useful properties.
Modern pharmacognosy has been developed rapidly to the
improvement made in the technology of isolation process which
includes the development techniques such as column, paper, thin
layer, gas, liquid, high performance liquid and droplet counter-
current chromatographic procedure. These methods have allowed
the rapid isolation of compounds, which are previously difficult to
obtain by classical procedures. The most important factor has been
the development of new spectroscopic techniques which are used to
identify structures of the isolated compounds, by which it become
easy to develop the new molecules and it is beneficial for the
research point of view, and now-a-days every research laboratory
having the latest techniques which help in the development of new
compounds.
PLANT PHYTOCHEMICAL SCREENING:
1. Qualitative chemical analysis.
2. Thin Layer Chromatography
3. High Performance Thin Layer Chromatography
4. Isolation of active constituent.
a) Column Chromatography
5. Characterization by
a) IR Spectroscopy
b) Mass Spectroscopy
c) Proton NMR
5.1 QUALITATIVE CHEMICAL ANALYSIS:
The plant extracts were subjected to preliminary
phytochemical screening for the detection of various plant
constituents present in the leaves of Clitoria ternatea.
a)Test for alkaloids:
Stirrer a small portion of the methanolic extract with a
few drops of dilute hydrochloric acid and filter. The filtrate were
tested with various alkaloid reagents such as Mayer’s reagent
(cream precipitate) Dragendroff’s reagent (orange brown
precipitate) and Wagner reagent (reddish brown precipitate).
Mayer’s reagent: Few drops of Mayer’s reagent were added
in each of the extract and observed formation of the white or cream
colored precipitates.
Dragendorff’s reagent: Few drops of dragendorff’s reagent
were added in each of the extract and observed formation of the
orange yellow or brown colored precipitates.
Wagner’s reagent: Few drops of Wagner reagent were
added in each of the extract and observed formation of the reddish
brown precipitates.
b) Test for Carbohydrates: Dissolve small quantities of
methanolic extract in 4 ml of distilled water and filter. The filtrate
may be subjected to Molisch’s test to detect the presence of
carbohydrates.
Molisch’s test: To small quantity of extract few drops of α-
napthol (20% in ethyl alcohol) were added. Then about 1 ml of
concentrated sulphuric acid was added along the side of the
tube. Reddish violet ring appeared at the junction of two
layers. It indicates the presence of carbohydrates.
Fehling’s Test: The 1 ml of Fehling’s reagent (copper
sulphate in alkaline conditions) was added to the filtrate of the
extract in distilled water and heated in a steam bath. Brick red
precipitates appeared which confirm the presence of
carbohydrates.
C) Test for Glycosides:
Hydrolyse small portion of the extract with dilute hydrochloric
acid for a few hours in water bath and subjected to hydrolysate to
Liebermann Burchard’s, Keller Killiani, Sodium nitrosoprusside and
Borntrager’s tests to detect the presence of different glycoside.
Small quantity of the extract was taken separately and subjected to
the following tests.
Keller Killiani Test: The 1 ml of glacial acetic acid containing
traces of FeCl3 and 1 ml of concentrated H2SO4 was added to
the extract carefully.
Bluish green colour appeared which confirm the presence of
glycosides in the extract.
Sodium nitrosoprusside test: The extract was made
alkaline with few drops of 10% sodium hydroxide and then
freshly prepared sodium nitrosoprusside solution was added
to it. Blue colour confirms the presence of glycosides in the
extract.
Borntrager’s test: The 1ml of benzene and 0.5 ml of dilute
amonia solution were added to the extract. A reddish pink
colour was obtained which show the presence of glycosides in
the extract.
D) Test for phenolic compounds and tannins:
Take small quantities of alcoholic extract in water and test for
the presence of phenolic compounds and tannins with dilute
ferric chloride solution (5%) and lead acetate test.
Ferric chloride test: On addition of ferric chloride solution
(5%) green or blue colour was observed, due to the presence
of phenolic compounds and tannins. No colour appeared which
shows the absence of phenolic compounds.
Lead Acetate test: Few drops of lead acetate solution (5%)
were added to the alcoholic extract. white precipitate was
appeared which confirm the presence of phenolic compounds.
E) Test for flavonoids:
Ammonia test: Filter paper strip were dipped in the alcoholic
solutions of the extract and ammoniated. The filter paper
changed its colour to yellow which indicates the presence of
flavonoids.
Pew test for flavonoids: To the small portion of the extract,
a piece of metallic magnesium/zinc was added followed by
addition of 2 drops of concentrated hydrochloric acids. A
brownish colour confirmed the presence of flavonoids in all
the extract.
F) Test for proteins and free amino acids:
Add small portion of alcoholic extract in a few ml of distilled
water and subjected the solution to million’s, Biuret and
Ninhydrin tests.
Million’s test: To the small portion of extract 5-6 drops of
million’s reagent (solution of mercury nitrate and nitrous acid)
were added. A red colour precipitate appeared which confirms
the presence of proteins and free amino acids.
Ninhydrin test: To the extract, lead acetate solution was
added to precipitate tannins and filtered. The filtrate was
spotted on a paper chromatogram, sprayed with ninhydrin
reagent and dried at 1100C for 5 minutes. Violet spots were
seen which confirm the presence of proteins and free amino
acids.
Biuret test: 1 ml of 40% sodium hydroxide solution and
2dropsof 1% copper sulphate solution was added to the
extract (1 ml.). The formation of violet color indicates the
presence of proteins.
Xanthoprotein test: 1ml of concentrated nitric acid was
added to the extract. Boil the white precipitate, if any, and
cool. Added 20% of sodium hydroxide or ammonia solution.
Orange color indicates the presence of aromatic amino acids.
G) Test for saponin:
Dilute small portion of alcoholic extract with distilled water to 20
ml and shake in a graduated cylinder for 15 minutes. A one -
centimeter layer of foam indicates the presence of saponin.
H) Test for Steroids:
Lieberman Burchard’s test: Dissolved the extract in 2 ml of
chloroform in a dry test tube. Added 10 drops of acetic
anhydride and 2 drops of concentrated sulphuric acid. The
solution becomes red, then blue and finally bluish green,
indicating the presence of steroids.
Salkowski test: Dissolved the extract in chloroform and
added equal volumes of concentrated sulphuric acid. The
formation of bluish red to cherry red color in chloroform layer
and green fluorescence in the acid layer represents the
steroid components in the tested extract.
Table: 5.1 Qualitative Phytochemical Analysis:
S.No
.Tests
Methanolic
extract
Pet. Ether
extract
1.
Alkaloids
Dragendorff’s test
Wagner’s test
Mayer’s test
Hager’s test
--
--
--
--
--
--
--
--
2.Carbohydrates
Molisch’s test
Fehling’s test
+
--
+
--
3.Proteins
Biuret test
Xanthoprotein test
+
--
+
--
4. Amino acid
Ninhydrin test+ --
5. Flavonoids
Shinoda test + --
6.Phenolic
compounds-- --
7.Glycoside
Keller Killiani test
Borntrager’s test
+
+
--
--
5.2 THIN LAYER CHROMATOGRAPHY:
5.2.1 General:
The technique thin layer chromatography was first introduced by
Izmailov and Shraiber in 1938. They used this technique for
separating plant extract on 2 mm thick and firm adhesive layer of
alumina set on glass plate. Condon, Gorden and Martin (1944)
started using filter papers. Williams carried out chromatograph on
adsorbent layer sandwiched between two glass plates; one of them
has a small hole through which solutions and developing solvents
were applied to the layer.
In 1958, Stahl demonstrated applications of TLC, a method
based on adsorption chromatography which is at important
analytical tool for qualitative and quantitative analysis of a number
of phytochemical substances. TLC is the first important step for
phytochemical screening. It also serves as a pilot technique for
column chromatography. (Chatwal, 2004)
TLC is the method mainly uses to investigate the presence of
chemical constituent qualitatively and quantitatively in the plant
extract. It is used to investigate alkaloids, glycosides, isoprenoids,
lipid component, sugar and their derivates etc. This component can
also run with standards for the investigation. It is an easy, versatile
and reliable method to establish authenticity, identity and purity.
(Kokate et. al., 2000)
5.2.2 Basic Principles of TLC:
Separation by TLC is effected by the application of the
mixture or extract as a spot or thin line on to a sorbent that has
applied to a backing plate. Analytical TLC plates (thickness 0.1-0.2
mm) are commercially available; e.g., the commonest analytical
silica gel plate is the 20x20 cm. Plastic or aluminum backed
Kieselgel 60 F254 plate, which has a 0.2 mm thickness of silica
sorbent. The plate is then placed into a tank with sufficient suitable
solvent to just wet the lower edge of the plate-sorbent but not
enough to wet the part of the plate where the spots were applied.
The solvent from then migrates up the plates through the sorbent
by the capillary action. A process known as development
The information provided a finished chromatography includes
the migrating behavior of the separated substances. It is given in
the form of the Rf value (relative to front)
Distance travelled by spot
Rf=
Distance travelled by solvent front
(Mukherjee P. K., 2002)
5.2.3 Methods & equipments of Thin Layer
Chromatography Apparatus:
The apparatus employed consisted of the following components:
1. Rectangular glass chambers (30 x 15 x 8 cm) with ground
glass rim on which a glass lid was placed. Grease was
applied on the rim of the chamber to make the glass jar
airtight.
2. Glass Sheets (20 x 5 cm) used for the preparation of thin
layer plates.
3. Sprayer for detection.
5.2.4 Preparation of Plates:
Silica gel G was used as the adsorbent. Slurry of it was prepared
with distilled water in a glass pestle mortar. The slurry was poured
on the clean and dry glass plates and spread on the plate as a
uniform coating using a glass rod. These plates were then placed on
a leveled surface in the horizontal position and allowed to air dry for
20-25 minutes.
5.2.5 Activation of Plates:
When the plates were dried they were placed in an oven.
Maintained at 1100 C for 30 minutes. The prepared plates were
stored in a closed desiccated cabinet and removed only when
required for use.
5.2.6 Preparation of Samples:
About 100 mg of test material was dissolved in 10 ml of the
respective solvent and was used for the TLC studies.
5.2.7 Application of Spots:
The spots were applied on the activated plate at a distance of 2
cm from one end of the plate and 3cm from each other with the help
of a fine capillary tube or diameter less than 1mm. The solvent was
removed from spot by air drying. The position of the origin was
marked.
5.2.8 Saturation of TLC chamber:
The inner wall of the chamber was lined with filter paper from all
side except the front face to maintain a solvent saturated
atmosphere. The solvent system was poured into the chamber up to
a height of 1 cm from the base. The mouth of the chamber was then
closed with a rectangular glass plate and made airtight with grease.
The chamber was then allowed to stand till the filter paper became
completely wetted with solvent vapors.
5.2.9 Development of Chromatograms:
Chromatograms were developed by one way ascending TLC. The
plate carrying spots was placed squarely in the developing chamber
and the lid was replaced as quickly as possible to minimize
disturbance of the solvent saturated atmosphere. The developing
solvent was allowed to travel up the plate until it reached the
desired level (10 to 15 cm). The plate was then removed from the
chamber; solvent front was marked and dried in air at room
temperature.
5.2.10 Detection of Spots:
The number and position of the various constituents present in
the mixtures was determined by spraying the plate with the 1%
vanalin in sulphuric acid and the plate was heated at 1100C for 10
minutes and the spots were marked. Rf value was calculated for well
defined spots.
5.2.11 Advantages of TLC:
a) TLC is an elegantly simple procedure for chromatography in
all kinds of solid-liquid and liquid- liquid systems.
b) TLC can be performed on an analytical as well as on a
preparative scale.
c) It may be applied to almost the entire spectrum of chemical
compounds.
d) TLC can be used for uncovering adulterations of food as well
as decomposition of foods and drugs caused by improper
storage or incorrect use, because of its great resolving power.
e) TLC is of great help in chemical taxonomy.
f) In chemical laboratory, morphological appearance of tissues
can be associated with their chemical composition as detected
by chromatographic patterns of tissue extracts.
g) The great advantages of TLC are often most profitably
exploited when TLC is employed in conjugation with other
methods of analysis. (Sethi, 2005)
5.2.12 Thin Layer Chromatography of Methanolic Extract:
100 mg of methanolic extract was weighted and dissolved in 10
ml of methanol and filtered. Filterate was taken as sample for TLC.
Table 5.2: TLC of Methanolic extract:
S. No Solvent systemNumber of spots
Resolution
1Chloroform: Ethylacetate
( 6:4 )4
spotGood
2Benzene: Diethylether
(3:7)4
spotGood
3Ethylacetate: : Methanol: Water
(7:2:1)4
spotGood
4Benzene: Ethylacetate
(8.5:1.5)4 spot Good
5Benzene: Methanol: Formic Acid
(8.5:1:0.5)5
spotExcellent
Adsorbent – Activated Silicagel-G
Detecting agent – Iodine
According to combination tried above it was found that Benzene:
Methanol: Formic Acid (8.5:1:0.5) may be the best solvent.
Table 5.3: Rf value of the spots of methanolic extract:
S. No. Rf value Developed in iodine
1. 0.12 Brownish
2. 0.24 Brownish
3. 0.57 Brownish
4. 0.68 Brownish
5. 0.92 Brownish
Photo: 2 TLC of Clitoria ternatea extract
Detecting agent – Iodine
5.3 High Performance Thin Layer Chromatography:
5.3.1 General:
Standardized manufacturing procedures and suitable analytical
tools are required to establish the necessary framework for quality
control in herbals. Among those tools separation techniques
including high performance liquid chromatography (HPLC), high
performance thin layer chromatography (HPTLC) and capillary
electrophoresis are the most widely used to establish reference
fingerprints of herbs, against which raw materials can be evaluated
and finished products can be assayed.
High performance thin layer chromatography also known under
the synonym planar chromatography which is a modern, powerful
analytical technique with separation power and reproducibility
superior to TLC.
5.3.2 Advantages of HPTLC:
Unsurpassed flexibility by design and being an off line
technique, HPTLC is extremely flexible with following advantages.
Choice of Detection.
Cost and time efficiency
User friendliness and Result Presentation
One time use of the TLC plate.
5.3.3 Requirements for HPTLC standardization:
For the analysis of herbals, HPTLC offers a number of
advantages. This technique is especially suitable for comparison of
samples on scanning densitometry or video technology. It has
become a cost and time effective alternative to HPLC.
Fingerprint analysis by HPTLC or HPLC is one of the most
powerful tools to link the botanical identify to the chemical
constituent profile of the plant, in combination with microscopic
investigations the fingerprint provides for a convenient identity
check. It can also be used to detect adulterations in raw materials.
This technique further describes the quality of the herb and the
herbal preparation. High performance thin layer chromatography
can also be employed for quantitative determination of such marker
compounds.
The production of most herbals preparations includes some
extraction process. It is essential for quality assurance that this
extraction is standardized, the quantity of marker compounds of
their relative abundance assayed by HPTLC or HPLC which are the
principle methods of monitoring. When choosing marker compounds
for a particular herb or herbal preparation. It is of critical
importance that chemically well characterized standards are
available for their qualification. It is often impossible to separate all
components of a plant extract completely.
A useful method for this purpose could be adopting the following
parameters as standard.
Place HPTLC plate 10 x 10 cm or 20 x 10 cm.
Sample application: application of bands (5-10 mm in length)
using the spray on technique.
Chamber saturated twin-trough or flat bottom chamber.
Developing distance 50-60 mm
Derivatization by immersion
Fig: 5.1 HPTLC of Clitoria ternatea extract
5.3.4 Result:
HPTLC of extract show the ten peaks confirming that the ten
compound may be present in the methanolic extract of the leaves of
Clitoria ternatea.
5.4 COLUMN CHROMATOGRAPHY:
It is used for separation and isolation of different constituents of
the methanolic extract of Clitoria ternatea.
5.4.1 Apparatus:
A glass column of 60 cm. length and 2.9 m diameter was taken
it was thoroughly cleaned, dried and checked for any type of
leakage. At the lower end of the column about one inch bed of glass
wool was placed for collection of elutes. Clean and dry beaker (100
ml) was uses.
5.4.2 Adsorbent:
Silica gel (60-120 Mesh, Merck) was used for column
Chromatography. The powder was activated at 1100C for an hour in
hot air oven prior to use in the column.
5.4.3 Packing of the Column:
The wet packing method was adopted. Initially the lower end of
the glass column was plugged with glass wool. Methanol was poured
on to the glass wool to release any air bubbles, which might be
trapped with the flat end of a packing rod. A portion of the slurry of
activated silica gel in hexane was poured in to the column.It should
be added continuousily. The side of the column was tapped gently
with a glass rod to even the compaction of the particles as the silica
gel settled. The outlet of the column was then adjusted so that the
eluent was continuously released but a small solvent head was
maintained on the top of the column.
5.4.4 Eluent:
Hexane, Benzene Methanol and Ethanol are generally used for
elution of column with increasing polarity.
5.4.5 Application of Sample:
The sample was prepared in respective solvent and added
slowly by the sides of the glass column without disturbing the
column packing. Then outlet of column was opened until the sample
got absorbed in silica gel in column.
5.4.6 Column Chromatography of Methanolic extract:
5.4.6.1 Preparation of Sample:
Methanolic extract was dried in reduced pressure and dissolve in
minimum quantity of methanol, mixed with silica gel, then dried,
and applied in the column and eluted with Benzene: Methanol
(90:10)
5.4.6.2 Collection of Samples in Volumetric:
First approximately 500ml, solvent Hexane: Benzene was
prepared for eluting column, which is collected in 25 ml. volumetric
flask and TLC was performed for each volumetric flask. The samples
showing the same TLC pattern were mixed as shown in table 5.4
Table 5.4: Column Chromatography of isolated compound
S.No.
EluteVolume
collected (ml)
No of spot
Code
1. Hexane: Benzene (95:5) 1-4 No spot R-1
2. Hexane: Benzene (90:10) 5-10 No spot R-2
3. Hexane: Benzene (85:15) 11-20 No spot R-3
4. Hexane: Benzene (75:25) 21-30 One spot R-4
5. Hexane: Benzene (50:50) 31-40 No spot R-5
6. Benzene (100) 41-50 No spot R-6
7. Benzene: Methanol (95:5) 51-60 No spot R-7
8. Benzene: Methanol (90:10) 61-70 Two spot R-8
9. Benzene: Methanol (85:15) 71-80 One spot R-9
10 Benzene: Methanol (75:25) 81-90 Three spot R-10
11. Benzene: Methanol (50:50) 91-100 No spot R-11
12 Methanol (100) 101-110 No spot R-12
The entire fraction (1to 40) were subjected to TLC using solvent
system Hexane: Benzene and entire fraction (41to 100) were
subjected to TLC using solvent system Benzene: Methanol. Fraction
R-9 showed single spot and was in sufficient quantity for analysis,
hence selected for further characterization. Other fraction R-1, R-2,
R-5 showed no spot and fraction R-4 was not studied due to very
minimum quantity of the isolates so the fractions were not applied
for further isolation.
5.5 CHARACTERIZATION OF ISOLATED COMPOUND:
The compound which is isolated in column chromatography is
characterized by the analytical techniques such as Infrared
spectroscopy, NMR spectroscopy and Mass spectroscopy.
5.5.1 Infrared Spectroscopy
Infrared spectroscopy is generally sensitive to the presence of
functional groups in the samples. The most powerful aspects of
Infrared spectroscopy is that it allows identification of unknown
compound. IR spectroscopy of compound (R-9) was performed in
CDRI, Lucknow. Spectra of compound have shown in figure 5.3. The
interpretation that can be made from spectra has shown in table 5.5
Fig: 5.2 IR spectra of compound (R-9)
Table 5.5: Interpretation of IR Spectroscopy
Wave number (cm-1) Functional Group
3357.3 O-H Stretching alcohol and phenols
2945.2 C-H Stretching Alkane
2833.3 C-H Stretching in aldehyde
1453.4 C-H Bending in Alkane
1114.3 C-N Vibration in Aliphatic
1030.3 C-OH
5.5.2 Mass Spectroscopy:
The mass spectroscopy of compound (R-9) was performed at
C.D.R.I. Lucknow, the mass spectra is used to determine the
possible fragmentation in the compound. The mass spectra of
compound have shown in Fig. 5.4 the spectra exhibited various
peeks suggesting fragmentation pattern.
Fragmentation data of isolated compound (R-9): 81, 95, 109,
154.
Fig: 5.3 Mass Spectra of Isolated compound (R-9)
Table 5.6: Interpretation of Mass Spectroscopy
m/z Relative intencity
95 100
109 85
154 70
81 65
5.5.3 NMR Spectroscopy:
The NMR spectroscopy of compound (R-9) was performed at
C.D.R.I. Lucknow, the mass spectra is used to determine the
possible Proton in the compound. The NMR spectra of compound
have shown in Fig. 5.5
Fig: 5.4 NMR Spectra of Isolated compound (R9)
Table 5.7: Interpretation of NMR Spectroscopy
δ(ppm) of Std.
Compouund
δ(ppm) of Isolated
Compouund
Inference No.of Protons
8.91 8.30 s 1H
6.66 6.68 d 1H
6.86 6.89 d 1H
7.74 7.54 s 2H
3.96 3.94 dd 1H
3.71 3.71 t 1H
3.47 3.48 t 1H
3.50 3.48 ddd 1H
3.83 3.83 dd 1H
3.66 3.66 dd 1H
3.30 3.26 t 1H
3.63 3.66 dd 1H
0.91 0.92 d 3H
Analytical Result of Isolated Compound R-9:
In 3-neohesperidoside, functional group OH, CH, C-OH, hydroxy,
alcohal, Vinylic present and elemental analysis of isolated
compound show C=37.19%, H=12.88%.
According to above study, the isolated compound may be 3-
neohesperidoside, colour has yellowish green and Rf values of
isolated compound 0.49 and elemental analysis of isolated
compound show C=37.19%, H=12.88%.
Chemical name – 3-neohesperidoside
Molecular formula- C30H33O19
6. PHARMACOLOGICAL INVESTIGATIONS
6.1 EVALUATION OF ANTIDIABETIC ACTIVITY:
6.1.1 EXPERIMENTAL METHODS OF DIABETES:
1. Alloxan Induced Diabetes:
A. Purpose and Rational:
It has been described mainly for dogs; rabbits, and rats,
guinea pigs have been found resistant to it. In most species,
triphasic time course is observed a rise of glucose found by a
decrease, probably due to depletion of islets from insulin, again
followed by sustained increase of blood glucose.
B. Procedure:
a) Rabbits: Weighing 2.0 to 3.5 Kg are infused via ear with 150
mg/Kg alloxan monohydrates (5.0 gm/100 ml. pH 4.5) for 10
minutes resulting in 70% of the animals become hyperglycemic and
uricosuric.
b) Rats of wistar or Sprange-Dawley strain: weighing 150-
200 gm are injected subcutaneous with 100-175 mg/Kg alloxan.
c) Male Beagle dogs: Weighing 15-20 Kg are injected
intravenously with 60 mg/Kg alloxan, subsequently animals receive
daily 1000 ml 5 Glucose solution with 10 I.U. regular insulin for one
week and canned food ad libitum.
2. Streptozotocin Induced Diabetes:
A. Purpose and Rational:
The antibiotic streptozotocin is an antidiabetic having diabe-
togenic activity. The compound turned out to be specifically
cytotoxic to β-cells of the pancreas.
B. Procedure:
Male wistar rats weighing 150-220 gm fed with standard diet
were injected with 60 mg/kg streptozotocin intravenously. Six to
eight hour after streptozotocin injection, the serum insulin values
are increased up to 4 times resulting in hypoglycemic phase. This is
followed by persistant hyperglycemia. Severity and onset of
diabetes symptoms depend on the dosage of streptozotocin.
Although 60 mg/kg dosage of streptozotocin cause hyperglycemia in
24-48 hours upto 800 mg % due to β-cells degranulation yet a
steady state is reached in 10 to 14 days allowing the use of animals
for pharmacological test. Other authors have also described the
modification of method in other animals.
3. Hormone Induced Diabetes:
A. Growth Hormone Induced Diabetes:
Pure anterior pituitary growth hormone shows diabetogenic
action in cats. Rats of any age subjected to a similar treatment do
not become diabetic but grow faster and shows striking hypertrophy
of the pancreatic islets.
B. Corticosteroid Induced Diabetes:
Forced fed rats treated with cortisone causes hyperglycemia
and glycosuria. In the guinea pig and rabbit, experimental corticoid
diabetes could be obtained without forced feeding. In the rats, the
adrenal cortex stimulated by corticotrophin has the capacity to
secrete steroids which induced steroids diabetes.
4. Other Diabetogenic Compounds:
Various chelators like dithiazone, gold thioglucose and mono-
sodium glutamate in a single i.v. dose of 40-100 mg/kg to cats,
rabbits, hampster, rats cause a triphasic diabetic state in rabbit.
Initial phase is hyperglycemic and normoglycemic and again
permanent hyperglycemic in 24-72 hours due to complete or partial
degranulation of β-cells.
Many other methods are also described by author. The
method which we have used here is alloxan induction method and
oral glucose tolerance test. (Vogel, 2004)
6.1.2 EXPERIMENTAL WORK:
1. Effect of methanolic extract on alloxan induced diabetic rats.
2. Effect of methanolic extract on glucose loaded rats.
A. Animals:
The adult male albino rats of weight 180-240 gm were
selected for the study. All animals were procured from disease free
animal house, Institute of Pharmacy, Bundelkhand University, Jhansi.
The Institute of Pharmacy is approved by Institutional Animal Ethical
Committee (716/02/a/CPCSEA). The animals were housed in
polypropylene cages, 5 per cage with free access to standard
laboratory diet and water ad libitum. The rats were maintained
under standard laboratory conditions at 25±20C relative humidity
50±15% and normal photo period (12 h dark/ 12h light) were used
for experiment.
B. Drugs:
Alloxan of CDH, New Delhi was used for the inducted of
diabetes and was obtained from Department of Pharmacy and the
standard drug i.e. glibenclamide was send by Sun Pharmaceutical
Industries, J & K.
C. Extraction of Plant:
The powder of leaves of Clitoria ternatea was subjected to
extraction in methanol. The extract was then concentrated at
reduced pressure and used for the experimentation.
D. Preparation of Dose:
The Dose of 200 mg/kg and 400 mg/kg of methanol extract
was selected for the test. All the doses was given orally after
making emulsion in vehicle i.e. 1% acacia gum and the standard
drug i.e. glibenclamide was given orally (10 mg/kg) in the vehicle.
1. Effect of Methanolic extract on alloxan induced
diabeticrats:
A) Induction of experimental diabetes:
Diabetes mellitus was induced by administering
intraparitoneal injection of alloxan monohydrate 120 mg/kg
(Nagappa A. N.,2003) to the overnight fasted rats. Five days after
administration of alloxan, fasting blood glucose of 300 to 450 mg/dl
were included in the study.
B) Sample collection:
Blood sample were collected from tail nipping and glucose
level was determined by an automatic electronic glucometer
(Accuchek comfort). (Vats et al., 2002)
C) Procedure:
After checking the fasting blood glucose in overnight fasted
diabetic rats. They were divided into five groups of five rats each
and one group of non-diabetic rats.
All the doses were given in the following manner
1st Group- normal control group received vehicle.
2nd Group-diabetic control received vehicle.
3rd Group-Received alcoholic extract at dose of 200 mg/Kg
orally.
4th Group- Received alcoholic extract at dose of 400 mg/Kg.
orally.
5th Group- Received standard drug i.e. Glibenclamide (10
mg /Kg. in Vehicle) orally. [Nagappa A.N., 2003]
The treatment was continued for 3 hours. During the period
water was supplied ad libitum. All the doses were administered
orally by the oral feeding needle. The effect of extract on Blood
glucose levels was estimated on overnight fasted rats on 0 hour, 1
hour, 2 hr and 3 hr by the method described before. The general
behaviors of the animals were recorded. The blood glucose level in
(Mean ± SEM) is shown in the Table 6.1.
Table 6.1: The Antihyperglycemic effect of Methanolic
Extract on Alloxan induced Diabetic rats.
GPDose Blood Glucose Level (mg/dl) at hr
0 hr 1 hr 2 hr 3 hr
I N.C75.75±3.93
75.56±2.20
76.63±1.59
76.06±1.48
II D.C343.37±8.04
342.19±6.37
340.52±5.48
333.69±4.57
IIICTLE
(200mg/kg)340.82± 4.51
289.95±3.01***
272.48±3.72***
260.01±4.98***
IVCTLE
(400mg/kg)347.52 ±4.92
293.11±2.76***
271.52±2.48***
256.19±2.50***
VGlibenclamid
e(10mg/kg)
346.35±4.28
287.90±2.51***
253.46 ±2.77***
238.67±2.36***
N.C. = Normal Control ;
D.C. = Diabetic Control
CTLE= Clitoria ternatea Leaves Extract
***P < 0.001 show significant when compare with group II
Fig: 6.1 The Antihyperglycemic effect of Methanolic
extract on alloxan induced diabetic rats.
2. Effect of Methanolic extract on oral glucose tolerance
test:
The hypoglycemic effect of methanolic extract of Clitoria
ternatea leaves was study on glucose loaded rats.
Protocol:
In this glucose tolerance test fasted normal rats were divided
into sifour groups of five animals each, Group I served as control
and received vehicle. Group IV received standard drug
glibenclamide at an oral dose of 10 mg/kg and Group II and III
received methanolic extract orally at a dose of 200 mg/kg and 400
mg/kg respectively. The rats of all the groups were given glucose
(4g/kg), 30min after the extract and drug administration Blood
samples were collected by tail nipping just prior to glucose loading
and blood glucose levels were measured by Accuchek Comfort
glucometer. Basal value is those after which glucose was
administered.
Table: 6.2 The Antihyperglycemic effect of Methanolic Extract On Glucose Loaded
rats
GP
Dose
Blood Glucose Level (mg/dl) at minutes
0minutes
30 minutes
60 minutes
120 minutes
IControl (4g/kg)
75.92±2.21
177.50±4.38
151.89±3.54
126.32±3.61
IICTLE
(200mg/kg)71.52±1.37
159.50±3.73**
135.68 ±2.10***
110.37±1.64**
IIICTLE
(400mg/kg)77.30±3.07
153.40±2.52***
130.73±2.38***
101.74±1.60***
IVGlibenclamide
(10mg/ kg)81.85±2.52
147.01±2.00***
119.81±2.86***
86.97±3.03***
CTLE= Clitoria ternatea Leaves Extract
***P < 0.001 show significant when compare with group I
Fig. 6.2: The Antihyperglycemic effect of Methanolic
extract on Glucose loaded rats.
6.1.3 STATISTICAL ANALYSIS:
The data were statistically evaluated using one way Anova.
expressed as Mean ± SEM followed by Tukey test using the Graph
pad instant Demo (Data set 1.IS) version P. values of 0.05 or less
were considered to be significant.
Result:
The methanolic extract of the drug showed marked effect for
decreasing the blood glucose level and rectifying the problem like
fatigue and irritation associated with the disease. Two concentration
of the extract were used for the investigation i.e. 400 mg/kg and
200 mg/kg against the standard glibenclamide 10 mg/kg dose
showed 23.12 % decrease in blood glucose level, 200mg /kg showed
21.92% decrease and standard drug showed 28.52% decrease
during the study of two week when compare with the standard drug.
400mg/kg dose of methanolic extract was near about as effective as
standard drug (glibenclamide).
When the activity of extract was done by the glucose
tolerance test in glucose loaded rats, the methanolic extract
400mg/kg showed significant effect on the blood glucose level but
extract of 200 mg/kg did not show the significant decrease in blood
glucose level. The value of p is less than 0.001 except in 200 mg/kg
in glucose tolerance test.
6.2 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY:
A. Screening of the acute inflammatory agents:
1. Ultraviolet erythema in guinea pig
After cleaning of back skin they are chemically depilated by a
suspension of barium sulfide. The guinea pigs are placed in a
leather cuff with a hole of 1.5 to 2.5 cm size punched in it, allowing
the ultraviolet radiation to reach only this area. The erythema is
scored 2 and 4 h after exposure (Yawalkar, 1991).
2. Paw edema
After injecting 0.1 ml of 1% solutions of carrageenan into the
plantar side of the left hind paw. The paw volume measured by
plethysmometer after injection, and for a specific time period after
challenge.
B.Screening of the chronic inflammatory agents:
1. Granuloma formation
After sacrificed of animal on 8th day cotton pellet are removed
that was placed on both sides in scapular region on first day. After
drying at 60°C for 24 h, net dry weight is determined (Ismail et al.,
1997).
2. Sponge implantation technique
Standard size and weight (10.0 ±0.02 mg) sponges are inserted
into dorsal cavities by insertion of blunt forceps. For estimation of
the fluid phase of sponge are exudates, e.g. protein content and
enzyme levels are noted.
3. Glass rod granulomas
The glass rods together with the surrounding connective tissue is
removed from sacrificed animal in which rods are placed in caudal
region by blunted forceps for 7 days before under goes
histopathological study (Vogel et al., 1990)
Animals:
Albino adult male rat weighing 220-280 gm were used for
assessment of anti-inflammatory activity. All animals supplied by
Central Drug Research Institute, Lucknow and kept at animal house
B.U. Jhansi. There were maintained standard environmental
condition (R.H. - 55-65%, room temperature 25±2°C and 12 hr light/
dark cycle) and were fed with standard pellet diet and water ad
libitum. Each experiment group constitute of six animal housed in
separate cages. All experiments were carried out with the consent
of Institutional Animal Ethical committee of the institute Approved
with reff, no. (716/02/a/CPCSEA)
Drugs and Chemicals:
Carrageenan (Himedia, Mumbai), Diclofenac (Alfa Remedies,
Ambala), The Methanol, Chloroform and Petroleum ether is provided
by Institute of Pharmacy, Bundelkhand University Jhansi.
Acute toxicity Study:
The limit test for acute toxicity was carried out at 2000 mg/kg
oral dose of CTLE in group of three rats (OECD 423 guidelines). The
rats undergoes for 2 hr behavioral, neurological and autonomic
profiles and morbid state. There also notice mortality rate in
duration 24 hours.
Doses and Treatments:
Rats were divided into different groups (n= 5). Diclofenac
(10mg/kg) was administered orally in mice and rats in acetic acid
induced writhing and carrageenan induced oedema. The control
groups received 0.9% saline. The dose of 1% carrageenan was
taken as 0.1 ml subplantar administered in rats is introduced in
animals as i.p (10ml/kg).
The rats were divided into four groups of five animals each in
a group to receive various treatments as mentioned bellow.
The characteristics of the groups are as follows:
Group 1: Control (Normal) rats given only saline.
Group 2: control standard group receive Indomethacin
Group 3: Test group receive CTLE 200mg/kg dose.
Group 4: Test group receive CTLE 400mg/kg dose.
All above doses are given 10ml/kg for orally and 5ml/kg i.p.
Anti-inflammatory Activity:
Carrageenan induced paw oedema in rat:
The method assayed according to Winter et al. The rats were divided in to the four groups. The drug control group, Diclofenac administered at a dose of 10mg/kg P.O. The same volume of normal saline was administered orally to the vehicle control group of rat while bark extract at a dose of 100 and 200 mg/kg was given orally to the test group of animal. The drugs or vehicle were given to experimental animal once at 0 min. Acute paw oedema was induced by subplantar injection of 0.1 ml of 1% freshly prepared carrageenan suspension in normal saline into the right hind paw of each rat. The left hind paw was injected with 0.1% of normal saline. The paw was measured in mm before (0hr) and at a interval of 1st, 2nd, 3rd and 4th hour after injection using verneir caliper (owalabi et al., 2007). The percent inhibitory activity was calculated by following formula (Winter et al., 1962)
% inhibition = 100 (1- Vt /Vc)
Where Vt = oedema paw size of test and Vc= oedema paw size of control
Table: 6.3 The Anti-inflammatory effect of Methanolic Extract On Carrageenan-induced rat
GPDose
Percentage inhibition
1 hr%
Inhibition2 hr
%
Inhibition3 hr
%
Inhibition4 hr
%
Inhibition
I N.C 0.62±0.06 0.78±0.07 0.76±0.05 0.74±0.05
II Std. 0.52±0.05 16.12% 0.59±0.06** 24.35%0.55±0.05*
*27.63%
0.47±0.04*
*36.48%
IIICTLE
(200mg/kg)0.57±0.09 8.06% 0.60±0.10* 23.07% 0.62±0.11* 18.42% 0.50±0.10* 32.43%
IVCTLE
(400mg/kg)0.58±0.08 6.45% 0.61±0.08* 21.79% 0.67±0.09* 11.84% 0.54±0.10* 27.02%
N=6, CTLE= Clitoria ternatea Leaves Extract
The percent inhibition for each group was calculated by comparison with the control group. Values indicate mean± S.E.M (ANOVA test followed by Dunnett’s t-test).Significance variation against control at **P < 0.01
Result:
The control group at 1st, 2nd, 3rd and 4th hour showed oedema volume in ml 0.62±0.06, 0.78±0.07, 0.76±0.05 and 0.74±0.05 respectively. The corresponding mean volume on Diclofenac (10mg/kg) treated group was 0.52±0.05, 0.59±0.06, 0.55±0.05 and 0.47±0.04 respectively, indicating significantly anti-inflammatory activity of Diclofenac from 0 hour onwards when compared to control. The extract in the doses i.e. 200 mg/kg and 400 mg/kg had produced significant inhibition in mean oedema volumes in dose dependent manner from 1 to 4th hour.
Fig:6.3 Anti-inflammatory effect of Methanolic Extract On
Carrageenan-induced rat
7. RESULT & DISCUSSION
7.1 GENERAL:
Clitoria ternatea belongs to the group of herbs having the
family fabaceae formed is cultivated as a perennial herbs all most
though out individually only in the three several significant amount
and chemical constituents like carbohydrates phenolic acid
flavanoids and alkaloids are present in this herb many of them have
been already reported, which results in many ethno medicinal
application on the herb like Antiulcer, Anti-inflammatory,
Cytoprotective Anorexia, dyspepsia etc.
Due to the wide pharmacological properties and the rich
hentaqge owned by the plant, it creates a desire to more widely
explore the plant hence the work has done on this plant and the
results are discussed below.
7.2 PHYTOCHEMICAL SCREENING:
7.2.1 TLC of Methanolic Extract:
The qualitative chromatographic profiles of the extract were
established. The different solvent systems were tried for extract
and the best solvent system was found are as follows Benzene:
Methanol: Formic acid (8.5:1:0.5)
In methanolic extract, five spots were observed with different Rf
value.0.12, 0.24, 0.57, 0.68, 0.92.
7.2.2 HPTLC of Methanolic Extract:
HPTLC of extract show the ten peaks confirming that the ten
compound may be present in the methanolic extract of the leaves of
Clitoria ternatea.
7.2.3 Cloumn Chromatography and characterization of
Methanolic Extract:
After Thin Layer Chromatography and HPTLC of methanolic
extract, the isolation of the constituent of methanolic extract was
carried out by column chromatography and then the compound (R-
9) obtained was analyzed by different analytical technique like IR,
NMR and Mass spectroscopy.
The IR spectra of isolated compound (R-9) shows different
functional group at different wave number shown in Table 5.5 and
Mass spectra of isolated compound (R-9) shows fragmentation
pattern as follows m/z 81, 95, 109, 154.
The NMR spectra of isolated compound (R-9) shows number of
proton and functional group. So the isolated compound may be the
3-neohesperidoside.
7.3 EVALUATION OF ANTIDIABETIC AND ANTI-
INFLAMMATORY ACTIVITY:
The methanolic extract of the drug showed marked
effect for decreasing the blood glucose level and rectifying the
problem like fatigue and irritation associated with the disease. Two
concentration of the extract were used for the investigation i.e. 400
mg/kg and 200mg/kg against the standard glibenclamide 10 mg/kg.
400mg/kg dose showed 23.12 % decrease in blood glucose level,
200 mg/kg showed 21.92% decrease and standard drug showed
28.52% decrease during the study of two week when compare with
standard drug, 400 mg/kg dose of methanolic extract was near
about as effective as standard drug (glibenclamide).
When the activity of extract was done by the glucose
tolerance test in glucose loaded rats, the extract showed significant
effect on the blood glucose level but extract of 200 mg/kg did not
show the significant decrease in blood glucose level. The value of p
is less than 0.001 except in 200 mg/kg in glucose tolerance test.
The control group at 1st, 2nd, 3rd and 4th hour showed oedema volume in ml 0.62±0.06, 0.78±0.07, 0.76±0.05 and 0.74±0.05 respectively. The corresponding mean volume on Diclofenac (10mg/kg) treated group was 0.52±0.05, 0.59±0.06, 0.55±0.05 and
0.47±0.04 respectively, indicating significantly anti-inflammatory activity of Diclofenac from 0 hour onwards when compared to control. The extract in the doses i.e. 200 mg/kg and 400 mg/kg had produced significant inhibition in mean oedema volumes in dose dependent manner from 1 to 4th hour.
8. SUMMARY AND CONCLUSION
The fresh leaves of Clitoria ternatea was collected during the
month of September 2008, from my village Kailiya and gandoli
(Distt-Jalaun), the Kush Nursury, Gwalior Road, Jhansi and from the
Institute of Pharmacy, Bundelkhand University, Jhansi The plant
materials was taxonomically identified and authenticated by Dr.
Gaurav Nigam, Botany Department, Bundelkhand University, Jhansi.
Herbarium and Museum Division with ref. no. BU/BOT /376/24-01-
2009.
The leaves of Clitoria ternatea were shaded dried until cracking
sound was observed during breakage, and then these are made into
coarsely powdered from using dry grinder. The powdered leaves of
the plant (600 gm.) was packed in soxhlet apparatus and
continuously extracted with petroleum ether (40-600C) till complete
extraction, after completion of extraction the solvent was
removed by distillation and then concentrated extract obtained was
dried under reduced pressure using rotatory evaporator at
temperature not exceeding 400C and then give moderate heating
on water bath. A pale green extract approximate 18 gm. was
obtained. From the drug, petroleum ether was removed and the
defatted drug was extracted with methanol till complete extraction,
after completion of extraction the solvent was removed by
distillation and then concentrated extract obtained dried under
reduced pressure at temperature not exceeding 400C and then give
moderate heating on water bath. The methanolic extract obtained
was greenish black in colour, weighed about 40 gm. The both
petroleum ether and methanolic extract was kept in petridish and it
was stored in desiccator at cool place (Mukherjee, 2002).
The qualitative chromatographic profiles of the extract were
established. The different solvent systems were tried for extract
and the best solvent system was found are as follows Benzene:
Methanol: Formic acid (8.5:1:0.5)
In methanolic extract, five spots were observed with different Rf
value.0.12, 0.24, 0.57, 0.68, 0.92.
HPTLC of extract show the ten peaks confirming that the ten
compound may be present in the methanolic extract of the leaves of
Clitoria ternatea.
After Thin Layer Chromatography and HPTLC of methanolic
extract, the isolation of the constituent of methanolic extract was
carried out by column chromatography and then the compound (R-
9) obtained was analyzed by different analytical technique like IR,
NMR and Mass spectroscopy.
The IR spectra of isolated compound (R-9) shows different
functional group at different wave number shown in Table 5.5 and
Mass spectra of isolated compound (R-9) shows fragmentation
pattern as follows m/z 81, 95, 109, 154.
The NMR spectra of isolated compound (R-9) shows number of
proton and functional group. So the isolated compound may be the
3-neohesperidoside.
In view of the ethanobotanical and traditional claims of Clitoria
ternatea plant used as hypoglycemic agent and wide use of its leaf,
root and flower extract in Ayurvedic practice, it is proposed to
evaluated anti-diabetic activity of methanolic Clitoria ternatea leaf
extract in alloxan induced hyperglycemic rats. In glucose loaded
normal rats, hypoglycemia was observed maximum at 120 minutes
after administration of CTLE. Single dose administration of CTLE
produce significant hypoglycemic effect in alloxan treated
hyperglycemic rats. The methanolic extract of Clitoria ternatea
leaves also show the anti-inflammatory effect in carrageenan
induced rat.
In conclusion, the study indicates that the methanol extract of
leaves posses anti-diabetic and anti-inflammatory properties which
suggest the presence of biologically active components. The extract
might be promoting glucose uptake and metabolism or inhibiting
hepatic gluconeogenesis. Result from the phytochemical analysis of
Clitoria ternatea revealed the presence of flavonoids, which has also
been isolated from the other plant and found to stimulate secretion
or possess an insulin-like effect.
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