Rajiv gandhi university of health sciences bangalore, karnataka




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ANTIDIABETIC ACTIVITY OF SPINACIA OLERACEA LINN. ON EXPERIMENTAL ANIMALS
Protocol of Dissertation Submitted
By
Mr. KIRAN. M. CHOUDHARI
To
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

Under the guidance of
Mr. NANU. R. RATHOD

Asst. Professor

Department of Pharmacology

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY

BAGALKOT- 587101, KARNATAKA

(2009-2010)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA-BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION




1.

Name of the Candidate and Address


KIRAN M CHOUDHARI

DEPARTMENT OF PHARMACOLOGY

H.S.K.COLLEGE OF PHARMACY

B.V.V.S. CAMPUS

BAGALKOT-587101, KARNATAKA.


2.

Name of the Institution


H.S.K.COLLEGE OF PHARMACY

B.V.V.S CAMPUS

BAGALKOT-587101, KARNATAKA.



3.

Course of Study and Subject



MASTER OF PHARMACY IN PHARMACOLOGY



4.

Date of admission to Course





11-06-2009


5.

Title of the topic:


Antidiabetic activity of Spinacia oleracea Linn. on experimental animals


6.

Brief resume of the intended work



6.1 Need for the study:

Diabetes is a condition in which the body either does not produce enough or does not properly respond to insulin a hormone produced in the pancreas1. Diabetes is the world’s largest endocrine disease with deranged carbohydrate, fats and protein metabolism. As per WHO report, approximately 150 million people have diabetes mellitus world wide and this number may well double by the year 2025. The statistical projection suggests that the number of diabetics will rise from 15 million in the year 1995 to 57 million in 2025, making India apart the country with the highest number of diabetics in the world. Although many drugs and interventions are available to manage diabetics, these are expensive for a developing country, like India apart from their inherent adverse effects. Therefore, it is necessary to look for new avenues to manage this major health problem. As part of the pathogenesis of type II diabetes mellitus, skeletal muscle, liver and adipose tissues become resistance to the hormonal effect of insulin, which in turn leads to decreased insulin-mediated glucose disposal, hepatic glucose overproduction and a marked increase in lipolysis. An addition to the above, hyperinsulinemia is a central pathophysiological feature of type II diabetes mellitus and has been shown to play a key role in the disease evaluation and macrovascular complication. The plants kingdom has become a target for the search by multinational drugs and biological active compound. Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes2.

Hence, the present study was under-taken to explore antidiabetic activity of spinacia oleracea of different extracts on normal and alloxan induced diabetic rats.



6.2 Review of literature :

Plant profile:

Title of plant : Spinacia oleracea Linn.

Family : Amaranthaceae

Synonyms : Basalesoppu, Palak, Spinach

Parts used : Leaves

Habitat : India



Chemical constituents: The plant is a rich source of vitamins, carotene and amino acids. It also contains acetylcholine, sterols both free and esterified (β-spinasterol, 7-stigmasterol, cholesterol), patuletin, quercetin, 6-hydroxymethyl-lumazine, rutin, saponin, catechol, hexadeca- 7, 10, 13-trienoic acid and lipids, flavonoids in particular spinacetin and patuletin, phenolics, sterol and essential amino acids are reported in seed3,4,5,6.

Medicinal action and uses: According to traditional medicinal practitioners, the plant is used for diabetic, urinary calculi, nutritious, febrile affections, inflammation of lungs, bowels and antibacterial activity. Seeds are laxative, used in breathing difficulty, jaundice and inflammation of the liver7.

Pharmacological actions: Antibacterial, antioxidant and CNS depressant. The plant have been reported protective effect in radiation induced oxidative stress and biochemical changes in mouse liver and testis8,9,10 .

6.3 Objective of the study:

Traditionally well known leaves extract of Spinacia oleracea, will be used for the present study. The dose will be selected based on acute toxicity studies11.


1. Evaluation of hypoglycemic activity of extract in rats.




7.

2. Antidiabetic activity of extract in alloxan induced diabetic rats.

Material and methods:

7.1 Source of data: The data are collected from the

a) Literature survey, books, websites - Pubmed, Medline, Sciencedirect, Scirus, etc.

b) Publications of journal.

c) Lab based studies.



7.2 Experimental animal groups:

Evaluation of hypoglycemic activity of extract in rats:

Group I - Serve as control receives 0.5% Tween 80

Group II - Low dose of chloroform extract

Group III - Moderate dose of chloroform extract

Group IV - High dose of chloroform extract

Group V - Low dose of methanol extract

Group VI - Moderate dose of methanol extract

Group VII - High dose of methanol extract



Antidiabetic activity of extract in alloxan induced diabetic rats:

Group I - Serve as control receives 0.5% Tween 80

Group II - Glibenclamide (10 mg/kg, body weight)

Group III - Low dose of chloroform extract

Group IV - Moderate dose of chloroform extract

Group V - High dose of chloroform extract

Group VI - Low dose of methanol extract

Group VII - Moderate dose of methanol extract







Group VIII- High dose of methanol extract

7.3 Materials:

Animals : Albino rats either sex

Instruments : Tissue homogenizer, Refrigerated centrifuge, Enzyme assay Kits, Autoanalyzer,

Research centrifuge, etc.

Chemicals : All the chemicals are used in the present study will be AR graded.

7.4 Methods:

7.4.1 Preparation of Spinacia oleracea Linn extract: The leaves are authenticated and collected in the ideal condition and air dried under the shade, powdered of uniform size by passing through the sieve no. # 44 to get a uniform powder. The powders were extracted with petroleum ether to defat, followed by chloroform and methanol. The % of yield of extract will be calculated and dose will be selected based on the toxicity studies.

7.4.2 Evaluation of hypoglycemic activity of extract in rats: The acclimatized animals were fasted for 24 hours with water ad libitum, fasted animals were divided into seven groups. Initial blood sample were drawn at 0 hrs and on interval 1½, 3, 5 hrs after the leaves extracts administration. Blood samples were collected from retro-orbital plexus under anesthesia and were centrifuged at 3000 rpm for 10 minutes to obtained serum and used for estimation of glucose by using glucose kit.

7.4.3 Antidiabetic activity in alloxan induced diabetic rats: Diabetes was induced by a single intraperitoneal injection of freshly prepared alloxan (140 mg/kg, body weight). The rats were maintained on 5 % glucose solution for 24 hrs to prevent hypoglycaemia. After 3 days, rats with hyperglycemic (>200 mg/dl) were used for the experiment. The treatments were continued daily for 28 days, blood samples were collected from retro-orbital plexus under anesthesia. Obtained





serum was used for estimation of glucose using glucose kit, after the 1 hrs of treatment on days

7, 14, 21 and 28 days of treatment12,13.



7.4.4 Evaluation of biochemical parameters:

Serum blood glucose level, glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphate (ALP), cholesterol, triglycerides14 and superoxide dismutase (SOD)15, lipid peroxidations (LPO)16, catalase (CAT)17 and protein in liver homogenate by using spectrophotometric method18.



7.4.5 Statistical analysis:

All the data were expressed in mean ± SEM. The significance of differences in mean between control and treated animals for different parameters were determined by using one way ANOVA and followed by Dunnet’s comparison test and significance difference between groups was evaluated by using student’s t-test.



7.5 Dose the study requires any investigations or interventions to be conducted on patients or other humans/animals? If so describe briefly:

Yes, for the study rats will be used. The effects of preparation on blood serum glucose, liver will be studied by considering physiological, pathological and biochemical parameters using animal models.



7.6 Has ethical clearance been obtained from your institution for performing various tests on animals?

Yes, the study is cleared Institutional Animal Ethics Committee (IAEC) and the copy is enclosed.



8.



REFERENCES:

  1. http://en.wikipedia.org/wiki/Diabetes_mellitus.

  2. Rathod N., Raghuveer I., Chitme H. R., Ramesh Chandra. Antidiabetic activity of Nyctanthes arbortistis. Phcog Mag 2008; 4: 335-339.

  3. Chatterjee A., Pakrashi S. The Treatise on Indian Medicinal Plants. National Institute of Science Communication. 1st ed. New Delhi 1997; 1: pp. 69-70.

  4. Elke Aehle., Sophie Raynaud-Le Grandic., Robert Ralainirina., Sylvie Baltora-Rosset., Francois Mesnard., Christophe Prouillet., et al. Development and evaluation of an enriched natural antioxidant preparation obtained from aqueous spinach extracts by an adsorption procedure. Food Chemistry 2004; 86: 579-585.

  5. Dawidar M. A., Amer M. A. Sterol content of Spinacia oleracea. Phytochemistry 1973; 12; 1180-1181.

  6. Madhava Chetty K., Sivaji K., Tulasi Rao K. Flowering plants of Chittoor district. 1st ed.

Student offset printers, Tirupathi, India 2008: pp. 296.

  1. Nadkarni K. M., Nadkarni A. K. Indian Materia Medica. 2nd ed. Popular prakashan,

Mumbai, India 2007; 1: pp. 1164-1165.

  1. Das S., Guha D. CNS depressive role of aqueous extract of Spinacia oleracea L. leaves in adult male albino rats. Indian J Exp Biol 2008; 46: 185-190.

  2. Bhatia L. A., Jain M. Spinacia oleracea L. protects against gamma radiations: a study on glutathione and lipid peroxidation in mouse liver. Phytomedicine 2004; 1: 607- 615.

  3. Sisodia R., Yadav K. R., Sharma V. K., Bhatia L. A. Spinacia oleracea modulates radiation induced biochemical changes in mice testis. Indian J Pharm Sci 2008; 70: 320-326.




  1. OECD: guidelines 425 acute oral toxicity-up-and-down-procedure. 2006.

  2. Manonmani G., Bhavapriya V., Kalpana S., Govindasamy S., Apparanantham T. Antioxidant activity of Cassia fistula flowers in alloxan induced diabetic rats. J Ethnopharmacol 2005; 97: 39-42.

  3. Pari L., Amaranath Satheesh. Antidiabetic activity of Boerhaavia diffusa L. effect on hepatic key enzymes in experimental diabetes. J Ethnopharmacol 2004; 91: 109-113.

  4. Achyut N. K., Kesari S., Singh S., Gupta R., Watal G. Studies on the glycemic and lipidemic effect of Murraya koenigii in experimental animals. J Ethnopharmacol 2007; 112: 305-311.

  5. Beauchamp C., Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971; 44: 276-287.

  6. Ohkawa H., Ohishi N., Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acids reaction. Anal Biochem 1979; 95: 351-358.

  7. Sinha A. Calorimetric assay of catalase. Anal Biochem 1972; 47: 389-394.

  8. Lowry O. H., Rosebrough N. J., Farr L., Randall R. J. Protein measurement with the Folin-Phenol reagent. J Biol Chem 1951; 193: 265-272.



9.


SIGNATURE OF CANDIDATE




(KIRAN. M. CHOUDHARI)


10.


REMARKS OF THE GUIDE



This investigation is undertaken to validate traditional claims of spinacia oleracea for the treatment of diabetes mellitus.

11.

NAME AND DESIGNATION OF

THE GUIDE


Mr.NANU. R. RATHOD

Asst. Professor






12.


SIGNATURE





13.


CO-GUIDE



-------------


14.


SIGNATURE



-------------



15.


HEAD OF THE DEPARTMENT



Dr. I. S. MUCHANDI

H.O.D. , Department of Pharmacology

H.S.K.College of Pharmacy

B.V.V.S. Campus

Bagalkot-587101.



16.


SIGNATURE




17.


REMARKS OF THE PRINCIPAL

The above mentioned information is correct and I recommended the same for approval.





18.


NAME OF THE PRINCIPAL



Dr. I. S. MUCHANDI

H.O.D. , Department of Pharmacology

H.S.K.College of Pharmacy

B.V.V.S. Campus

Bagalkot-587101.



19.


SIGNATURE






OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)

HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY,

BAGALKOT-587101, KARNATAKA
REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE

“BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision)

RULES 1998”

Ref: HSKCP/IAEC, Clear /2009-10/1-8




CERTIFICATE

This is to certify that Mr. KIRAN. M. CHOUDHARI a student of first M. Pharm is permitted to carry out experiments on animals for the dissertation/thesis work entitled as “Antidiabetic activity of Spinacia oleracea Linn. on experimental animals’’ as per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.



Animal house in charge CHAIRMAN


Form B

See rule [6 (a) and 8(a)]
PART A



(1)

Name and address of the Establishment:




HSK College Of Pharmacy, B.V.V.S Campus, Bagalkot.587101,Karnataka

(2)

Date and Registration Number of the Establishment:

821/01/a CPCSEA



(3)

Name, address and Registration NO. of the

breeder from whom acquired and the date of

acquisition:


OFFICE OF CPCSEA,

Ministry of environment and forest

IIIrd seaward road, Valmiki nagar

Thiruvanmiyur, Chennai-600041.

Tamil Nadu



(4)

Place where the animals are presently kept:


Animal House

HSK College Of Pharmacy, B.V.V.S Campus, Bagalkot.587101,Karnataka



(5)

Place where the experiment is to be performed:


PG Lab-II, Department of pharmacology, HSK College Of Pharmacy, B.V.V.S Campus, Bagalkot.587101,Karnataka



(6)

The date on which the experiment is to commence and the duration of the experiment:

15 MAY 2010



The protocol form for the research proposal – PART B in the case of experiments using other than non-human primate animals for ongoing/new projects, PART C for use of non-human primates for new projects and PART D for use of non-human primate for extension of ongoing projects – should be duly filled, singed and annexed with this form.

Signature
Dated: (Name and Designation)
Place:



PART – B
Protocol form for Research Proposal to be submitted to the Committee on use of small animals / Animals other than non human Primate in Biomedical Research for ONGOING / NEW PROJECTS



1.

Project Title :


“Antidiabetic activity of Spinacia oleracea Linn. on experimental animals’’




2.

Investigation (s) :

Designation


Mr.NANU. R. RATHOD

Asst. Professor





3.

Department (s) :



Department of pharmacology, HSK College Of Pharmacy, BVVS Campus Bagalkot.587101,Karnataka


4.





  1. Funding Source (s): if any


------


(b) Are sufficient funds available for purchase and maintenance of the animals


Yes

©


Duration of present project :





  1. Number of months :




6 months

  1. Date of start of the Project :

(Experiment)

15 MAY 2010




  1. Date of termination of the project :



15 DEC 2010




5.


Date by which approval is needed in case the project is to be funded by outside agency (If less than six weeks from the date of admission, please justify below):




--------





6.

Summary of project briefly summarize in laymen’s term the background, the objective and the experiment approach.





  1. Background:




Enclosed

  1. Objectives




Enclosed

  1. Experimental procedure:



Enclosed



7.

(a) Name of species


Wister albino rats

Age


Sex

Weight

4-6 Weeks

Either

150-250 gms


(b)


Rationale for selection




Approximate number of animals required during the first 12 months.

42+ 48=90



Justification of number (define treatment group and number per group)


15 groups, Each group containing six animals.

Number of animals housed per weeks


20


8.

List all invasive Non Surgical Animal Procedures and Potentially Stressful Noninvasive procedures to be used (Example IM injection, foot pad injection, venapunctures).



Invasive non-surgical procedure


Procedure and Approximate Frequency:


Enclosed






9.


10.


Anesthetic and/or Analgesic and Dosage:

Anesthetic ether

Test substance injected and/or applied:

Test substance will given orally


Does the protocol prohibit the use of anaesthetic and analgesic for the conduct of painful procedures?
NO


With surgical procedure/Experimental procedure be performed?
NO



(a) Will the animal be sacrificed after surgery?
NO






(b) Give anticipated post operative survival time:
NO


©


Personal who will perform the surgery


Name

Designation

Qualification











11.

Will hazardous agent such as radioisotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?


No

INVESTIGATOR SIGNATURE



DATE: ____________________







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