RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE

M. PHARM SYNOPSIS

DECEMBER- 2011-12

“Evaluation of Anti-obesity activities of Crotalaria juncea L. in

albino rats.” BY

Mr. SREEDHAR K.S

DEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Dr. B.M. Vrushabendra Swamy M. Pharm, Ph. D, FICCP.

Director/Professor

Department of Pharmacology

GAUTHAM COLLEGE OF PHARMACY

SULTHANPALYA, R.T. NAGAR, BANGALORE-560032

KARNATAKA

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKAANNEXURE-II

1. Name of The Candidate and

Address

Mr. Sreedhar K.S

Permanent Address:Sreedhar K.S. S/O Sriramreddy. Kodirampura Rantavalalu Post, Madhugiri taluk,Tumkur district.572132

Postal Address:Gautham college of Pharmacy,Sultan palya , RT Nagar post,Bengaluru - 560032.

2. Name of the Institution GAUTHAM COLLEGE OF PHARMACYSulthan Palya, RT Nagar post.Bengaluru- 560032.

3. Course of study and Subject Master of Pharmacy in Pharmacology

4. Date of admission 19/12/2011

5. Title of the topic:

“Evaluation of Anti-obesity activities of Crotalaria juncea L. in albino rats”.

6. BRIEF REVIEW OF THE INTENDED WORK :

6.1 INTRODUCTION:

Health is defined as soundless of physical, mental or moral condition,

especially freedom from bodily pain or disease, but true health is more than that. It

includes the joy of living, the power and ability to lead a satisfying and purposeful

life.

Obesity and related disorders are the leading causes of illness and mortality in

the developed World [1].

Effects of excess weight on morbidity and mortality have been known since

the time of Hippocrates, who said,” sudden death is more common in those who are

naturally fat than in the lean”[2].

The ultimate cause of obesity is an imbalance between calorie intake and

energy expenditure, but the pathologic mechanisms, which lead to this imbalance are

still not understood. Obesity is believed to be of both genetic and environmental

origin, involving excess calorie intake, decreased physical activity, social and

economic forces and metabolism and endocrine abnormalities[3].

The World Health Organization has estimated that perhaps 80% of earths 6

billion inhabitance rely upon traditional medicine for their primary health care needs,

and a major part of this therapy involves the use of plant extracts or their active

principles.

Crotalaria juncea L. known as Sunn or Sunn hemp, is a tropical Asian plant of

the legume family (Fabaceae).Grown as a source of green manure, fodder and the

lignified fiber obtained from its stem, it bears yellow flowers and elongate, alternate

leaves. Sunn Hemp is also being looked at as a possible bio-fuel Annual, c. 60–

250 cm tall.

Many ascending branches, pubescent. Leaf simple, 2.5-10.5 cm long, 6–

20 mm broad, linear or oblong, obtuse or subacute, apiculate, pubescent on both sides,

hairs appressed, silky. Petiole 1.2-2.5 mm long; stipules almost absent. Inflorescence

an erect terminal and lateral raceme, up to 30 cm long, 12-20-flowered. Pedicel c. 3–

7 mm long. Bract minute; bracteoles 2, below the calyx, 1.8-2.0 cm long, pubescent,

teeth linear-lanceolate. Corolla bright yellow. Vexillum ovate-oblong, slightly

exserted. Fruit; 2.5-3.2 cm long, sessile, pubescent, 10-15-seeded [4].

6.2 NEED FOR THE STUDY

Obesity is abnormal or excessive fat accumulation that presents risk to health.

The body mass index (BMI) of a person is 25-30 kg/m2 indicates overweight and

above 30 kg/m2 represents obesity. World Health Organization (WHO) assigns obesity

as global epidemic. WHO’s latest study indicate that globally in 2005, approximately

1.6 billion adults (15+) were overweight and at least 400 million adults were obese.

Further WHO projects that by 2015 approximately 2.3 billion people will be

overweight and more than 700 million will be obese. Once it was considered that

obesity was only in high income countries. But now a day, it has spread dramatically

in medium and low income countries [5].

The epidemic of obesity is on the rise and this is probably due to increasingly

sedentary lifestyles combined with easy availability of palatable, high fat foods. Its

prevalence has shown a startling Increase in all age groups and in all the countries of

the world during past 19 years. In 1980, a Department of health Survey in U.K.

showed that 6% of men and 8% of women were obese, as defined by a BMI >30

kg/m2.In 1995, 15% of men and 16.5% of women were obese and more than half of

the adult population is now either overweight or obese. It has been predicted that if

this trend continues to the year 2005, the prevalence of obesity among men and

women would by then be 18 and 24% - exactly three times the target figure. There has

also been an increase in comorbid risk factors like coronary heart disease,

hypertension, various cancers (e.g., endometrium, ovaries, breast, colon), gall bladder

disease, NIDDM, arthritis, respiratory disease, pregnancy complications, menstrual

difficulties, varicose veins, psychological problems (poor self-esteem, depression,

poor employment prospects)[6,7].

The successful management of obesity is possible through lifestyle changes in

diet and physical activity. But this requires extreme efforts. The market for safe and

efficacious drug is therefore potentially huge, but currently available therapies are not

having potential and these are associated with lot of side effects. Currently FDA

approved only two drugs in US for long-term treatment for obesity. These are Orlistat

and Sibutramine [8].

In severe cases, surgery is performed or an intra gastric balloon is placed to

reduce stomach volume and/or bowel length, leading to earlier satiation and reduced

ability to absorb nutrients from food [9].

Dietary obesity can be induced readily in laboratory rodents by giving high fat

diets or cafeteria diets. Obesity also occurs in rodents given a palatable sugar solution

in additional to laboratory chow. These animals consume only about half of as much

chow as animals not given sugar, additional calories from sugar solution generally

results in greater total dietary energy intake and development of profound obesity [10].

Recently, there has been increasing interest in the use of medicinal plants. The

use of medicinal plants in modern medicine suffers from the fact that though hundreds

of plants are used in the world to prevent or to cure diseases. Recently search for

appropriate anti-obesity agent has been focused on plants used in traditional medicine

because of leads provided by natural products that may be better treatment than

currently used drugs.

There are reports that Crotalaria juncea L. is used traditionally as anti-obesity

plant, so, present study selected leaves of Crotalaria juncea L. plant for evaluation of

anti-obesity activity by using high fat diet induced, monosodium glutamate induced

obesity and fructose induced obesity in rats.

6.3 REVIEW OF LITERATURE:

Botanical name: Crotalaria juncea L

Synonym: Crotalaria benghalensis Lam

Family: Fabaceae

Vernacular names: English: Indian hemp, Bombay hemp, Sun hemp.

Hindi: kharif, sannai sun

Tamil: sanal, sannappu

Bengali: ghore sun, shon, shonpat[11].

Telugu: Sanamachettu, Gilaka, Gilligintha[12].

Distribution & Description: Native: Australia, India, although generally considered a

tropical or subtropical crop, sunn hemp is drought resistant and is widely adaptable to

different soil types. Its tolerance to salt and frost is low [11].

Folk uses: The seeds are said to purify the blood and are used to treat impetigo and

psoriasis [11], roots are used for fever, amenorrhea, and dysentery. Leafs are used

anorexia, blood disorders, amenorrhoea , obesity[12].

Fiber: The major significance of sunn hemp lies in its valuable fiber, which is

extracted from the bark and used to make twine, rug yarn, cigarette and tissue papers,

fishnets, sacking, canvas and cordage. Fiber is stronger when wet; it is fairly resistant

to mildew, moisture and microorganisms in salt water[11].

Sunn hemp fiber has greater tensile strength and is more durable under

exposure than jute. It is not as strong as hemp, sunn hemp an excellent candidate for

paper-making[11].

Reported activities:

- Antifertility activity of various extracts of Crotalaria juncea L. seeds in male mice[13].

- Anti-inflammatory and anti-ulcerogenic effect of Crotalaria juncea L. in albino rats[14].

- Evaluation of activity of methanolic extract of anti-diarrhoeal Crotalaria juncea L. in albino wistar rats[15].

- Antibacterial activity of seed and flower parts of Crotalaria juncea L [16].

Chemical constituents: An Abundance of mucilage, linoleic acid (62.36%)[17],

steroids, flavonoids , phenols, glycosides and triterpinoides apart from that it contains

some of the interesting compounds which include monocrotaline, riddelline,

seneciphylline, senecionine, trichodesmine, chodesmine, galactose specific lectin and

cardiogenin 3-O-[OH]-d-xylopyranosid[18].

6.4 OBJECTIVES OF THE STUDY:

The main objective of the proposed work is to induce obesity by using high fat

diet[19-21], Monosodium glutamate[22], fructose induced[23] obesity in rats and screening

of Crotalaria juncea L. Leaves extract in rats.

The whole study is divided into two phase

Phase I:

- Collection and authentication of plant material. Collected plant material

subject to extraction with 70% v/v alcohol using Soxhlet apparatus with

hydro alcoholic (70%v/v).

- To investigate preliminary phytochemical constituents present in the

extract.

Phase II:

To evaluate Anti-Obesity activity of Crotalaria juncea L. leaves extracts

by using the experimental animal models like:

High fat diet induced obesity.

Monosodium glutamate induced obesity.

Fructose induced obesity

Parameters to be studied.

- Body weight

- Body temperature

- Lipid profile(HDL, LDL, VLDL, TG, Total Cholesterol).

- Serum glucose

- SGOT

- SGPT

- Parametrial adipose tissue weight

7.0 MATERIALS AND METHODS:

7.1 Source of Data:

Whole work is planned to generate data from laboratory studies i.e.

experiments are performed as described in references. Experimental studies in journals

and in text books available with college and various institutions.

1. Standard Books:

Goodman and Gilmann’s: The Pharmacological basis of

Therapeutics.

H.Gerhard Vogel’s: Drug Discovery and Evaluation.

Katzung’s : Basic and clinical pharmacology.

Rang and Dale’s: Pharmacology.

Tortora: Human Anotomy and Physiology.

Guyton and Hall’s: A Text Book of Medical Physiology.

Wealth of India.

Materiamedica.

Glossary of Indian medicinal plants.

2. Internet source:

Google

Pub med

www.sciencedirect.com

Wikipedia

SCOUPS

Helinet

Ovid

Open J-Gate

DOAJ

Chemical Abstracts

CABI

International Pharmaceutical Abstract

3. Journal sources:

Indian Journal of Pharmacology.

Journal of Pharmacology and experimental Therapeutics.

Journal of Ethano pharmacology.

American Journal of Pharmacology and Toxicology.

Phytochemistry.

7.2ANTI-OBESITY ACTIVITY:

A. Experimental animals: Adult albino rats weighing approximately 150-200

g will be used for anti-obesity activity. And rats are used for the acute

toxicological studies. The animals will be fed with standard diet and will be

given water ad libitum.

B. Preparation of extract[15]: About 250 g of powdered shade dried leaves of

Crotalaria juncea L. are subjected to successive Soxhlet extraction by using

Hydro alcoholic. The prepared extracts are concentrated to lesser volume

under reduced pressure and evaporate to dryness. The prepared extract is

suspended in distilled water containing 2%v/v tween 80 (suspending agent).

C. Dose: A dose of Hydro alcoholic extract will be taken as per the toxicity

studies.

D. Grouping of animals: The animals are divided into 6 groups of six rats in

each group and the treatment given once.

7.3 GROUPING OF ANIMALS[25]:

In high fat diet induced obesity animals:Experimental Animals

Albino wistar rats weighing 160-220g were divided into six groups of six in

each group.

High Fat Composition:

Commercially available edible dalda (vanaspathy) and culinary grade coconut oil

were obtained from local market. The high fat diet (HFD) was prepared by

homogenously mixing dalda and coconut oil in the ratio of 3:2 w/w.

Induction of Obesity

Group I animals were administered with 10ml distilled water per kg body

weight orally once daily for a period of four weeks by oral gavaging technique and

served as negative control. For the Group II, III, IV, V, and VI in addition to normal

diet and water prepared high fat diet was administered by gavaging to induce obesity.

HFD was gavaged at the dose rate of 10ml per kg body weight to each animal orally

daily for a period of four weeks.

Treatment Protocol

Once the Obesity was induced between 0 to 4th week of the experiment, from

the beginning of fifth week to the end of the eighth week, the Hydro alcoholic extract

of Crotalaria junceac L (HECJ) treatment was carried out.

Group-I: Distilled water was administered and served as negative control.

Group-II: Distilled water was administered and served as positive control

Group-III: Standard drug (Fenofibrate 200mg/kg, p.o) was administered.

Group-IV: HECJ was administered at a dose rate of 200mg/kg, p.o body

weight.

Group-V: HECJ was administered at a dose rate of 400mg/kg, p.o body

weight.

Group-VI: HECJ was administered at a dose rate of 600mg/kg, p.o body

weight.

After the completion of eighth week i.e., 56 days, on 57th day blood was

collected for the estimation of biochemical parameters. Before collection of blood the

animals were kept overnight fasting.

Parameters studied for this test were body temperature, bodyweights, average

feed intake, weights of liver, kidneys, spleen, paramatrial adipose tissue, blood

glucose, total cholesterol, HDL, LDL, VLDL, triglycerides, atherogenic index.

Parameters to be studied.

Body weight: The body weight (g) will be recorded on day 1 and then on alternate

days for 40 days in each group.

Body temperature: The body temperature will be recorded on day 39 using rectal

telethermo meter before and after drug administration at 30, 60, 90, 120 and 180min

with a contact time of 1 minute.

Biochemical parameters: Serum glucose, SGOT, SGPT, Lipid profile (HDL, LDL,

VLDL, TG, Total Cholesterol) will be measured on the 40th day in each group.

Parametrial adipose tissue weight: On the 40thday of the experiment rats will be

sacrificed, parametrial adipose tissue will be dissected and weighed.

In Monosodium glutamate induced obesity:Experimental animals:

Albino wistar rats weighing 120-150gms were divided into 6 groups of six in

each group.

Induction of obesity:

Group I animals were administered with distilled water orally once daily by oral

gavaging technique and served as negative control. For the Group II, III, IV, V and VI

in addition to normal diet and water prepared Monosodium glutamate solution was

administered by gavaging to induce obesity. Monosodium glutamate was gavaged at

the dose rate of 8mg/kg body weight to each animal orally daily up to 7days.

Treatment Protocol:

Once the obesity was induced between 1st and 7th day of the experiment, from 8th

to 48th days of Hydro alcoholic extract of Crotalaria junceac L (HECJ) treatment was

carried out.

Group-I: Distilled water will be supplied and serve as negative control.

Group-II: Only Monosodium glutamate will be supplied and served as positive

control.

Group-III: standard drug (Fenofibrate 200mg/kg) will be supplied orally.

Group-IV: HECJ was administered at a dose rate of 200mg/kg, p.o body

weight.

Group-V: HECJ was administered at a dose rate of 400mg/kg, p.o body

Weight.

Group-VI: HECJ was administered at a dose rate of 600mg/kg, p.o body

weight.

After completion of 48th day, on 49th day blood is collected for the estimation

of biochemical parameters. Before collection of blood, the animals were kept

overnight fasting.

Parameters studied for the test are body weights, weights of liver, kidneys,

spleen, parametrial adipose tissue, blood glucose, total cholesterol, HDL, VDL,

VLDL, triglycerides, SGOT, SGPT, Atherogenic index.

Parameters to be studied.

Body weight: The body weight (g) will be recorded on day 1 and then on alternate

days for three weeks in each group.

Body temperature: The body temperature will be recorded on day 20 th day using

rectal telethermo meter before and after drug administration at 30, 60, 90, 120 and

180min with a contact time of 1 minute.

Biochemical parameters: Serum glucose, SGOT, SGPT, Lipid profile (HDL, LDL,

VLDL, TG, Total Cholesterol) will be measured on the21st day in each group.

Parametrial adipose tissue weight: On the 21stday of the experiment rats will be

sacrificed, parametrial adipose tissue will be dissected and weighed.

In Fructose load test

Experimental Animals:

Albino wistar rats weighing 160-220g were divided into six groups of six in

each group.

Experimental Design

Group-I: Distilled water was administered and served as negative

control.

Group-II: Distilled water was administered and served as positive control

Group-III: Standard drug (Fenofibrate 200mg/kg, p.o) was administered

Group-IV: HECJ was administered at a dose rate of 200mg/kg, p.o body

weight

Group-V: HECJ was administered at a dose rate of 400mg/kg, p.o body

weight.

Group-VI: HECJ was administered at a dose rate of 600mg/kg, p.o body

weight.

This treatment was continued for 3 days, once daily. The time to the last

administration of the doses were at 5 h prior to the oral administration of fructose.

Following that Group-I animals were received distilled water at a dose of 0.3ml/100g

body weight, and Group- II, III, IV, V, and VI were supplied with 60% w/v fructose

solution by gavage at a dose of 0.3 ml/100 g p.o body weight at the start and then had

free access to the fructose solution for 6 h after supplying neither diet nor water

overnight. Then the blood was collected from animals for the estimation of

triglycerides.

Parameter studied for this test is triglyceride levels.

HECJ: Hydro alcoholic extract of Crotalaria junceac L

P.O: Post oral

7.4 COLLECTION OF BLOOD AND ANALYSIS PROCEDURE:

Blood samples will be collected by Retro orbital sinus. Serum glucose, Lipid

profile(HDL, LDL, VLDL, TG, Total Cholesterol) SGOT, SGPT will be measured.

7.5 STATISTICAL ANALYSIS:

All the values that are generated out of this study execution will be expressed as

mean ± SEM from six animals. Statistical difference in mean will be analyzed using

one way ANOVA (Analysis of Variance) followed by Dunnett’s ‘t’ test. P values

less than 0.05 were considered as indicative of significance.

7.6. Does the study require any investigation or intervention to be conducted on

patients or other humans or animals? If so, please mention briefly.

Yes, the above study requires In-vivo screening techiniques on Wistar rats.

7.7. Has ethical clearance been obtained from your institution.

The copy of ethical clearance certificate is enclosed.

8. REFERENCES:

1. Giampiero Muccioli, Tschop Matthias, Papotti Mauro, DeghenghiRomanco,

Heiman Mark, et.al. Neuroendocrine and peripheral activities of ghrelin

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2. Suresha BS. Investigation of the anti-obesity activity of poly herbal

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3. Stunkard Albert J. Current views on obesity Am J Med 1996;100:230-236.

4. http://www.en.wikipedia.org/wiki/Crotalaria _ juncea (Accessed on 17-06-12).

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17-06-12).

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9. Imaz I, Martínez-Cervell C, García-Alvarez EE, Sendra-Gutiérrez JM,

González-Enríquez J. "Safety and effectiveness of the intra gastric balloon for

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SpeciesInfo.asp?SpID=618 (Accessed on 19-06-12).

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district A.P. India, 1sted 2008;85.

13. Vijaykumar B, Sangamma I, Sharanabasappa A, Saraswati Paitl B.

Antifertility Activity of Various Extracts of Crotalaria juncea Linn., Seeds in

Male Mice .Philippine J Sci 2003;132(1):39-46.

14. Purnima ashok, Rajani GP, Arulmozhi S, Basavaraj Hulkoti, Desai BG,

Rajendran R. Anti-inflammatory and Anti-ulcerogenic Effect of Crotalaria

juncea Linn. in Albino Rats. Iranian J Pharmacol Ther 2006 ; (5):141-144.

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diarrhoeal activity of methanolic extract of Crotalaria juncea Linn. in albino

wistar rats. International Journal of Preclinical Research 2011;2(2):66-70.

16. Hemendra S. Chouhan and Sushil K. Singh. Antibacterial Activity of Seed

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(3): 212-215.

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22. Dolnikoff M , Martín-Hidalgo A, Machado UF, Lima FB, Herrera E.

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9 SIGNATURE OF THE CANDIDATE:

10 REMARKS OF THE GUIDE:

“Evaluation of Anti-obesity activities of Crotalaria juncea L. in albino rats.” to be carried out by Mr.Sreedhar K.S of M. Pharm has been discussed and worked out under my directions and supervision as an official guide. The project work envisaged is of great importance in the field of pharmacology. The work can be carried out in pharmacology laboratory of Gautham College of Pharmacy for which facilities are available. Hence the project is viable and is recommended for clearance.

11 NAME AND DESIGNATION OF:

11.1 GUIDE: Dr. B M VrushabendraSwamy M.Pharm,Ph.D, FICCP

Director / Professor & Head,Department of Pharmacology.Gautham College of PharmacyBangalore- 32

11.2 SIGNATURE:

11.3 HEAD OF THE DEPARTMENT: Dr. B M Vrushabendra Swamy M.Pharm, PhD, FICCP.

Director / Professor & Head,Department of Pharmacology.Gautham College of Pharmacy

11.4 SIGNATURE

12 CLEARENCE FROM INSTITUTIONAL ETHICAL COMMITTEE:

The study is cleared from Animal Ethical Committee of the Institution.

(Approval no:491/01/c/CPCSEA)

13 13.1 REMARKS OF THE PRINCIPAL:

The program and research work that Mr. Sreedhar K.S is undertaking have

potential implication in the field of Pharmacology. The work can be carried out in the

Research Laboratories of Pharmacology Department at Gautham college of

Pharmacy.

Hence the project is recommended and requested for clearance and approval.

13.2 SIGNATURE

Prof. ArchanaSwamy. PM.Pharm (Ph.D)

PrincipalGautham College of PharmacyBhuvaneswarinagar,R.T.Nagar Post,Bangalore- 32.