Evaluation of Antidiabetic Activity of Seed Extract of Sesbania grandiflora.

M. PHARM DISSERTATION PROTOCOLSUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE

BYA. S. VENKATESH B.Pharm.

UNDER THE GUIDANCE OF

DR. SHIVAKUMAR SWAMY M.Pharm., Ph.D.H.O.D & PRINCIPAL

MALLIGE COLLEGE OF PHARMACY, BANGALORE

MALLIGE COLLEGE OF PHARMACY

#71 SILVEPURA,BANGALORE 90.

Rajiv Gandhi University of Health Sciences,

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Karnataka, Bangalore.Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 Name and Address of the Candidate

Mr. A. S. VENKATESH

S/o A.V Sathyanarayana

#874, Main road, Amruthur, Kunigal (T), Tumkur: 572111.

02 Name of the Institution Mallige College Of Pharmacy#71 Silvepura,Post : ChikkabanavaraBangalore 90

03 Course of the Study Branch M.Pharm (Pharmacology)

04 Date of Admission to course 14/07/2012

05 Title of the TopicEvaluation of antidiabetic activity of seed extract

of Sesbania grandiflora.

06

Brief resume of the intended work

6.1. Need for the Study

Enclosure – I

6.2. Review of the Literature Enclosure – II

6.3. Objective of the Study Enclosure – III

07

Materials and Methods

7.1. Source of data Enclosure – IV

7.2. Methods of collection of data Enclosure – V

7.3. Does the study require any Investigations on animals? If yes give details

Enclosure – VI

7.4. Has ethical clearance been obtained from your institution in case of 7.3.

Yes

2

08 List of References Enclosure – VII

09 Signature of the Candidate( A S Venkatesh)

10 Remarks of the Guide

The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by A S Venkatesh under my supervision.

11

Name and Designation of (in Block Letters)11.1. Guide

11.2.Signature

11.3.Co-Guide (if any)

11.4.Signature

11.5. Head of the Department

11.6.Signature

Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D.Principal & HODMallige College Of Pharmacy,Bangalore, Karnataka.

Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D.Principal & HODMallige College Of Pharmacy,Bangalore, Karnataka

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Remarks of the Principal

12.1. Signature

The present study is permitted to perform in the Pharmacology laboratory of our institution and the study protocol has been approved by IAEC.

(Dr. Shivakumar Swamy)

3

Enclosure: I

06. Brief resume of the intended work:

6.1. Need for the study:

Introduction:

Carbohydrates from the diet are the primary exogenous source of glucose. Glucose is the

main fuel for energy requirement of the body. Therefore, a continuous supply of glucose is

necessary to ensure proper function and survival of all organs. The impairment in glucose

metabolism therefore, may lead to physiological imbalance and warrants proper management.

Any variation therefore, in normal glucose metabolic pathway may lead to the impaired glucose

metabolism, the onset of hyperglycemia and subsequently diabetes mellitus1

In the last few years there has been an exponential growth in the field of herbal medicine

and these drugs are gaining popularity both in developing and developed countries because of

their natural origin and less side effects. In Indian systems of medicine most practitioners

formulate and dispense their own recipes2. The World Health Organization (WHO) has listed

21,000 plants, which are used for medicinal purposes around the world. Among these 2500

species are in India, out of which 150 species are used commercially on a fairly large scale. India

is the largest producer of medicinal herbs and is called as botanical garden of the world3. The

current review focuses on herbal drug preparations and plants used in the treatment of diabetes

mellitus, a major crippling disease in the world leading to huge economic losses.

Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from

defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is

associated with long-term damage, dysfunction, and failure of different organs, especially the

eyes, kidneys, nerves, heart, and blood vessels.

Symptoms of marked hyperglycemia include polyuria, polydipsia, weight loss,

sometimes with polyphagia, and blurred vision. Impairment of growth and susceptibility to

certain infections may also accompany chronic hyperglycemia. Acute, life-threatening

consequences of uncontrolled diabetes are hyperglycemia with ketoacidosis or the nonketotic

hyperosmolar syndrome.

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.Compared with synthetic drugs, drugs derived from the plants are frequently considered

to be less toxic with fewer side effects5. Despite the introduction of hypoglycemic agents from

natural and synthetic sources, diabetes and its secondary complications continue to be a major

medical problem in the world population. Many indigenous Indian medicinal plants have been

found to be useful to successfully manage diabetes with fewer side effects.

World Health Organization (WHO) has suggested the evaluation of the potential of plants as

effective therapeutic agents, especially in areas in which we lack safe modern drugs. Therefore,

the search for the more effective and safer antihyperglycemic agent becomes an area of active

research6

Sesbania grandiflora is an Indian medicinal plant which is extensively used in Ayurveda

and other alternative system of medicine.

Keeping these facts in view, the present study is undertaken to creat a scientific base for the “Evaluation of Antidiabetic activity of Seed Extract of Sesbania grandiflora”.

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ENCLOSURE: II

6.2. REVIEW OF LITERATURE

The plant Sesbania grandiflora (L.) Pers is reported to have great medicinal value in

Indian medicine. Sesbania grandiflora, commonly known as "Sesbania" and "agathi," is widely

used in Indian traditional medicine for the treatment of a broad spectrum of diseases. Sesbania

grandiflora is a fast-growing tree with a typical adult height of between 3 and 5 m. The leaves

are regular and rounded and the flowers are white and large, very characteristic. The fruits look

like flat, long and thin green beans, belongs to the family Fabaceae under the subfamily

Faboideae. It is believed to have originated either in India or Southeast Asia and grows primarily

in hot and humid tropical areas of the world7.

Chemical Composition

Leaf contains- Thiamine, Riboflavin, Niacin, Ascorbic acid.

Flower contain- Protein, Fat, Carbohydrate, β-carotene, Niacin, Ascorbic acid Thiamine,

Riboflavin.

Seed oil contains- Palmitic acid, Stearic acid, Oleic acid, Linoleic acid, Glactomannans.8

Medicinal uses:

Sesbania grandiflora is used for the treatment of a broad spectrum of diseases like gout,

rheumatism, cancer and liver disorders. All parts of Sesbania grandiflora are utilized for

medicine in South eastern Asia and India including preparations derived from the root bark, gum,

leaves, flowers, and fruit. In Ayurvedic medicine the root is applied as a poultice (wet substance

applied to inflammation or sore spots) for application to inflammation and fever and the leaves

are utilized for the treatment of epileptic fits and to cure night blindness9. The juices of the

flowers have a special ability to improve vision and the crushed leaves are applied to sprains and

bruises of all kinds. A tea made from the leaves is believed to have antibiotic, anthelmintic,

antitumor and contraceptive properties. The bark is considered as a tonic and an antipyretic, and

a remedy for gastric troubles and diabetes.

And the tree also has its medicinal effect due to its astringency property; hence it is used

against inflammation, venom and other poisons, bacterial infections and tumors10-12. Sesbania

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grandiflora has been used as an important dietary nutritive source in South East Asian

countries.13

So the all parts of the plant, Sesbania grandiflora are utilized for medicine such as

diuretic, emetic, headaches, smallpox, anemia, bronchitis, anxiolytic, hepatoprotective and have

the potent antidote for tobacco and smoking-related diseases and the flowers are used as

emollient, bronchitis, gout and pain, and the fruits are used for anemia, bronchitis, fever, tumors,

pain and thirst14.

Pharmacological Studies

Recently,Sesbania grandiflora (antianxiety) anxiolytic, hepatoprotective, cardio,

antiurolithiatic and antioxidant activities were reported.

Several reports suggested that the ethanolic extract of the bark of S.grandiflora prevented

acute gastric injury in rats, the leaf juice of S.grandiflora showed antiurolithiatic, antidiabetic

and antioxidant properties15.

In vivo studies using ascites and solid tumor models strongly support in vitro findings as

SF2(Sesbania Fraction 2) administration may serve as a potential anticancer drug, the triterpene

containing fraction of S.grandiflora exhibits anticonvulsant and anxiolytic activity16.

The oral administration of an ethanolic extract of S.grandiflora leaves (200 mg/kg/day)

shows significant hepatoprotective17, anti-microbial18, analgesic and antipyretic activity19,

In vitro studies using methanol and aqueous extract of S.grandiflora bark exhibits

antihelmintic activity20, ethanol extract of S.grandiflora bark shows anti-ulcer activity21, and

methanol extract of S.grandiflora shows wound healing property22.

The aqueous suspension of S.grandiflora diminishes oxidative stress and ameliorates

antioxidant capacity in liver and kidney of rats exposed to cigarette smoke.23

In vitro study using petroleum ether extract of S.grandiflora seeds exhibits antihelmintic

property.24

Ethanol extract of S.grandiflora against ehrlich ascites carcinoma in albino mice exhibits

anticancer activity.25

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The effect of salinity on chlorophyll and carbohydrate contents of S.grandiflora is

evaluated,the elevated levels of the total soluble and insoluble carbohydrate in the shoot and root

are considered to play an important role in the osmotic adjustment.26

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ENCLOSURE: III

6.3. OBJECTIVES OF THE STUDY

The present research work is an attempt to evaluate the possible Antidiabetic efficacy

using extract of Seed of Sesbania grandiflora in rats with the following objectives.

Collection, authentication and extraction of Seed of Sesbania grandiflora

Qualitative estimation of phytoconstituents.

Standardization of the therapeutic dose and confirmation of any acute toxicity

Evaluation of Antidiabetic activity in Alloxan induced experimental model.

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ENCLOSURE: IV

07. MATERIALS AND METHODS

7.1. SOURCE OF DATA:

The work is aimed to generate data from experiments to be conducted at pharmacology

laboratory of our institution. Albino rats and mice will be used for this purpose.

Animals:

Adult male albino rats, and albino rabbits will be used for this purpose. The animal will be

obtained from animal house of Mallige College of Pharmacy. Animal clearance will be obtained

from institutional animal ethical committee for experimental purpose. They will be maintained

under laboratory condition with controlled environment of temperature, humidity as per

committee for the purpose of control and supervision of experiments on animal (CPCSEA)

guidelines. They will be provided with standard diet and water ad libitum.

Drugs:

Glibanclamide will be received as a gift sample from manufacturers will be used in this

study, all other chemicals used will be of analytical grade.

The experiments involve the following steps:

1. Collection and authentication of the Seed of Sesbania grandiflora.

2. Extraction of Seed of Sesbania grandiflora

3. Qualitative analysis of phytoconstituents

4. Evaluation of the extract of flower for the Antidiabetic activity in rats.

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ENCLOSURE-V

7.2. METHOD OF COLLECTION OF DATA:

1. Collection of raw material:

Seed of Sesbania grandiflora will be collected from the surrounding gardens of

Bangalore, Karnataka. The sample will be identified and authenticated by the botanist. Fresh

leaves and flowers will be cleaned and shade dried at room temperature.

2. Extraction of Sesbania grandiflora:

The powdered materials will be extracted with 70% alcohol by Soxhlet’s extraction

method27. The extracts will be concentrated using rotary flash evaporator and percentage yield of

the same will be recorded. Finally the extract will be used for qualitative phytochemical analysis

and to evaluate Antidiabetic activity.

3. Qualitative Phytochemical Analysis:

The crude extracts thus obtained will be subjected for preliminary phytochemical analysis using standard procedures described in the literature.

4. Screening of hypoglycemic activity:

A. Effect of Seed extract of Sesbania grandiflora on blood glucose level on normal albino rats28.

Male albino strain rats weighing (160–200 g), aged 8-14 weeks older are to be equally

divided into four groups of six Albino rats each. Animals belonging to Group I-normal control

group will be administered only vehicle and Group II-standard group will be administered

reference drug Glibenclamide suspended in the vehicle while Group III- will be administered

with plant extract of S.grandiflora (250mg/kg,p.o.) and Group IV will be administered plant

extract of S.grandiflora (500mg/kg,p.o.) respectively for 28 days, and blood samples will be

collected from tail vein prior to dosing (day 0) and then at regular intervals of day 7, 14, 21 and

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28 respectively and to be subjected to fasting blood glucose level. The fasting blood glucose

level will be estimated by glucometer29. The fasting blood glucose level and percentage reduction

will be recorded.

Group I - Normal control - vehicle

Group II - Standard group - Glibenclamide (0.25mg/kg, p.o.)

Group III - Plant extract of S.grandiflora(250mg/kg,p.o.)

Group IV - Plant extract of S.grandiflora (500mg/kg,p.o.)

Data to be recorded:

Effect of extract on Normoglycemia:

Group Treatment % Reduction in blood glucose level

0 min 30 min 60min 90min 120 min

I

II

III

IV

B. Evaluation of Hypoglycemic activity in Alloxan induced experimental albino rats30.

Albino rats of either sex weighing 150 -180 gm are used in this experiment. In this

method, albino rats were fasted in individual cage for 24 hours. Care was taken to avoid

carophagy. Diabetes was induced in the rat by injecting alloxan monohydrate intra peritoneally

in a single dose of 125mg/kg in 0.95 of weight/volume sodium chloride, to the overnight fasted

rat. The rats were kept for next 24 hours on 10% of glucose solution contained in bottle, kept in

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their cages, to prevent hypoglycemia. After 72 hours of injection, fasting blood glucose level will

be measured. The animal whose glucose level should not rise to more than 250mg/dl will be

rejected.

The diabetic rats will be selected and divide into four groups containing 6 animal each,

drug treatment as follows

Group-I- Diabetic control

Group -II- Diabetic rats treat with standard Glibenclamide (0.25 mg/kg; p.o.)

Group -III - Diabetic rats treat with crude plant extract (250mg/kg; p.o.)

Group -IV - Diabetic rats treat with crude plant extract (500mg/kg; p.o.)

The treatment will be continued for a period of 28 days respectively following oral

administration by gastric intubation, using a force-feeding needle to the experimental animals.

Plasma glucose will be estimated by glucometer on withdrawing blood samples from tail vein

prior to dosing (day 0) and then at regular intervals of day 7, 14, 21and 28 respectively all

groups of animals and a data will be recorded. The body weight, food and fluid intake of all

groups of animals will be monitored on a daily basis for 28 days at regular time. Fixed amount of

rat chow and fluid will be given to each rat and replenished the next day.

Data to be recorded:-

Effect of extracts on diabetic rats:

Group Treatment

% Reduction in blood glucose level

0days 7thday 14thday 21stday 28thday

I

II

III

IV

V

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C.Histopathological investigation:-

At the end of 28th day, all the rats will be sacrificed by cervical dislocation and

pancrease will be dissected out and tissues will be placed in 10% formalin (dilute to 10% with

20 mM phosphate buffer pH 7.4) for 1 hr to rectify shrinkage due to high concentration of

formalin and the tissue will be processed for histopathological studies.

4. Statistical Analysis:

The data obtained from the study will be subjected for statistical analysis using One-way

ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical

significance of the results.

5. Work plan details:

Total duration for the completion of proposed research work may be ten months.

1. Collection of plant materials including authentication - One month.

2. Duration of experimentation on animals including - Five months.

preparation of crude extracts

3. Literature collection - Two months.

4. Dissertation writing and communication of research - Two months.

papers.

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Enclosure-VI

7.3 Does the study require any investigation or interventions to be conducted on patients

or other humans and animals? if so please describe briefly.

The proposed study requires the investigation on albino rats of either sex (Wistar Strain)

weighing 150 - 200 gm for the hypoglycemic activity.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

The present study protocol will be approved from Institutional Animal Ethical Committee .

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Enclosure VII

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