Rajiv Gandhi University of Health Sciences s138

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERATATION

1. /

NAME OF THE CANDIDATE AND ADDRESS

/ SARASVATHI.V
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 / 06/06/2007
5. /

TITLE OF THE TOPIC:

“Evaluation of Anticanceractivity of Strychnos potatorum Linn in Balb/c mice”

6. /

BRIEF RESUME OF THE INTENDED WORK

6.1 Need for the study:
Cancer is a basically a disease of cell characterized by shift in a control mechanism that govern a cell in proliferation and differentiation.1 Cancer is a second leading cause of death behind heart diseases. Heart disease have declined 45% but increasing cancer death still is continue since from 1997 according to the data base of the National Cancer Institute Survival Epidemiology and End {NCISEE} results population data from the senses that 1.36 million new cases of invasive cancers were diagnosed 563700 persons died from cancer i.e. percentage distribution of new cancer and cancer death increases becoming major disease in world 2. Remarked differences can be found in incidence of cancers and depending upon genetic, environmental factors 3.The carcinogenicity by UV rays and chemical agents are the major causes for cancer. Tubacco is used through the cigarette and other means are most common risk factors for the cancer and other associated diseases.4
Treatment of cancer with chemotherapeutic agents, along with radiation and surgery. The chemotherapeutic agents provides a temporary improve of symptoms and signs of cancers and most of the chemotherapeutic agents are given in combination with the radiation therapy and surgery5 and patient has to consume for the long duration and these drugs have more toxic to the patient and economically burden to the patients6. The most of the plant based medicine Strychnos potatorumLinn as shown promising agents for the treatment of the cancer with safe, efficacy and economical
With this object the present study Strychonos potatorum Linn belongs to family Loganiaceae has wide spread through out south India. It is used as a traditional medicine for the treatment of pain, ulcer and various ailments 7 .The photochemical studies are reported that it contains tannins, flavanoids, alkaloids etc. from this object, the present study was designed to evaluate the anticancer activity of Strychonos potatorum Linn by in vitro and in vivo model of Balb/c mice.
6.2 Review of literature:-
Plant Profile:
Title of plant: Strychnos potatorum Linn
Family : Loganiaceae
Synonyms : Clearing nut tree, Nirmali ,
Parts used : Dried seeds
Chemical constituents: Moisture-8.26%, Nitrogen-1.33%, Total alkaloids-0.17%,Ash-1.34%,Sucrose-1-2%, Loganin 7,Flavanoids,Tannins etc
Medicinal action and uses: -
Traditionally fresh Strychnos potatorum Linn seeds are indicated in tumors and pains.8
Leucorrhoea, excessive heat, ulcers, anemia, menorrhagia, asthma, and the pulp is the good for the treatment of bronchits9.
Pharmacological activity: -
Aqueous extract of Strychnos potatorum Linn seeds have hepatoprotective, antioxidant activity , Methanolic extract is reported that has an antidiarrheol activity, anthalmentic, and antiallergic and mast cell membrane stabilizing activity10 .
6.3Objectives of the study: -
Traditionally well known seed extract of Strychonos potatorum Linn, will be used for the present study and objectives of the present study are to evaluate
1. Anticancer activity of the extracts.
2.Effect of the extracts on physiological parameters associated with anticancer activity
3. Effect of the extracts on hematological parameters.
4. Effect of the extracts on Biochemical parameters.
7. / METHODS AND MATERIALS:
7.1 Source of data: All the data will be collected from the experimental animal model and the animals will be subdivided into following groups:
a) Group I Normal / positive control (n=6)
b) Group II Tumor control (n=6)
c) Group III Cyclophosphamide (n=6)
d) Group IV Low dose of chloroform extract (n=6)
e) Group V Moderate dose of chloroform extract (n=6)
f) GroupVI High dose of chloroform extract (n=6)
g) GroupVII Low dose of Methanolic extract (n=6)
h) Group VIII Moderate dose of Methanolic extract (n=6)
i) Group IX High dose of Methanolic extract (n=6)
7.2 Materials :
Animals : Female Balb/c mice
Chemicals : All the chemicals are AR grade
Instruments : Hemocytometer, Binocular microscope, Centrifuge machine etc
7.3 Methods:
Preparation of Strychnos potatorum Linn extract: -
Strychnos potatorum Linn seeds were purchased from Kholapur (Maharashtra) after authentication by taxonomist of Ayurvedic medical college Bagalkot, 5kg of seeds were powdered to fine texture and passed through sieve no 40 to obtain uniform texture and extracted successively using Soxhelt extractor by Petroleum ether, Chloroform and methanol for 24 hrs cycle, the extract obtained was concentrated using rotatory evaporator at 50oc . Extract was dried in freeze dryer preserved in aseptic condition before the experiment. The percent yield of petroleum ether, chloroform and methanol was
Calculated and found to be 0.22%, 0.154% & 0.506% respectively. The dried extract was reconstituted in 50% DMSO for further study10.
Induction of Ehrlich Ascites Carcinoma
The Ascitic carcinoma bearing mice (donor) was taken 15 days after tumor transplantation .The ascitic fluid is drawn using an 18-gauge needle into sterile syringe. A small amount tested for microbial contamination. Tumor viability was determined by Trypan blue exclusion test and cells were counted using haemocytometer. The ascitic fluid was suitably diluted in normal saline to get a concentration of 10 6 cells/ml of the tumor cell suspension. This was injected intreperitonial route to obtain ascitic tumor. The mice were weighed on the day of tumor inoculation. And then for each three days. Treatment was started 24 hours after tumor inoculation. Cyclophosphamide was injected on two alternate day’s 1st and 3rd day, by intreperitonial route. Extracts were administered till the 9th day by intraperitonial route.11
Evaluation:
I. Physical parameters :
1. Percentage increase in body weight as compared to day-012
2. Median survival time (MST)
3. Percentage increase in life span (%ILS)13.
4. Mean survival time (MEST)
5. Percentage increase in life span (%ILS)
6. Cell viability test (% survivors of malignant cells in ascetic fluid)14
2. Hematological parameters:
Total WBC, DLC, RBC and Hemoglobin content15.
In order to detect the influence of plant extracts on the status of EAC bearing mice comparison was made amongst for groups of mice for each extract on the fourteenth day after transplantation.
These comprised as
1.Normal mice
2.Tumor bearing mice
3.Tumor bearing mice is treated with one dose of cyclophosphamide
4. Tumor bearing mice treated with plant extracts for 5 days.
3. Biochemical parameters:
After the collection of the blood samples the mice were sacrificed and their liver was excised, rinsed in ice-cold normal saline followed by cold 0.15 mol/lit. Tris-Hcl buffer (pH 7.4) blotted dry and weighed 10% w/v homogenate was prepared in 0.15 mol/l Tris-Hcl buffer and a portion utilized for the estimation of lipid peroxidation16 and other portion of the same after precipitating with TCA was used for the estimation of Glutathione.17 The remaining homogenate was centrifuged at 1500rpm for the estimation of Superoxidedismutase, catalase and protein content18-20.
In vitro studies:-
Tryphan Blue Exclusion Method (Cell viability test)
This is one of the methods to assess cytotoxicity of anticancer compounds. It comes under preliminary screening of anticancer compounds. This test is based on the principle that living cell membrane has the ability to prevent the entry of dye. Hence, they remain unstained and can be easily distinguished from dead cells, which take the dye. The percentage of viable cells was determined as follows
Method:
The ascetic fluid was withdrawn from animals which were induced with EAC cell lines, 0.1 ml of ascetic fluid was aspirated, and it was diluted with 2ml of phosphate buffered saline (PBS). To this equal volume of different extracts were mixed & this mixture incubated for3hrs at 370c. Then the mixture was added to 0.5ml of tryphan blue & mixed thoroughly. The diluted suspension was charged into hemocytometer .
The viable cells were (unstained) counted in a WBC chamber under a microscope and the mean numbers of cells in four chambers were calculated as follows
Total number of cells=Mean number of cells Dilution factor (2) ×10 4
Statistical analysis:
Results will be analyzed by one way ANOVA followed by Schefee’s post-hoc test using Graph prism computer package
7.4 Does the study require any investigation or to be conducted on patients or other humans/animal? If so please describe briefly
Yes, the above study requires to be carried out in mice. The effects of preparation on cancer will be studied by considering physiological, pathological, biochemical parameters using animal models.
7.5 Has ethical clearance been obtained from your institution for performing various tests on animals?
Yes, the study is cleared from institutional animal’s ethics committee & the copy is enclosed with the protocol.
8. / REFERENCES: -:
1. Otits W. Brawely, Barnett S. Kramer. Prevention & early detection of cancer. Kasper,.Braunwald, Fauchi ,Hauser ,Longo ,Jameson ,Harrison’s principles of Internal medicine-16th ed Vol-I , McGRAW-HILL Medical publishing Divison: Newyork(2005):441-467.
2. Neoplastic disease and immunosuppression, P.N.Bennett, M.J.Brown, Clinical pharmacology Churchill Livingstone, An imprint of Elsevier (2005), 9th ed; 603.
3. Bruce A,Chabner,Philip C.Amrein,Brian J. Dichaelson, Constantine S.Mitsiades, Paul E. Goss, David P.Ryan, Sumant Ramachandra, Paul G. Richardson, Jeffrey G. Supko, Wyndham H. Wilson-Antineoplastic agents,Brunton Lazo parker ,Goodman and Gilman’s The pharmacological Basis of therapeutics. McGRAW-HILL, Medical publishing division Newyork, (2006), 11th ed; 1335.
4. Edward chu, MD, and Alan C. Sartorelli, Phd, Bertram G katzung. Basic and clinical pharmacology McGRAW-HILL, Medical publishing division Newyork,(2004), 9th ed;903.
5. Carol McManus Balmer, Amy WV, and Andrea Iannucci. Cancer treatment and Chemotherpy Joseph T Dipro, Robert L.Talbert, Gary. C.Yee, Gary.R.Matzke, Barbara .G. Wells, L. Michael posey Pharmacotherpy .A pathophysiologic approach McGRAW-HILL Medical publishing division Newyork (1999), 6th Ed; 2280.
6. Kirsten M, Duncan, Gary Ogawa, and Unamarie Clibon, Supportive care therapies for patients with cancer, Eric T.Herfindal. Dick R. Gourley Text book of therapeutics Drug and Disease ManagementLibrary of Congress Cataloging-in-publication data (2000), 7th edition; 2025
7. The wealth of India, Raw material, Volume-x: SP-W.New Deihi: CSIR; 2003.
8. Kirtikar KR, Basu BD. Indian medicinal plants.4th ed. Deharadun;International book Distributors; 1996.
9. Nadkarni KM, Nadkarni AK.Indian Metrica Medica. 3rd ed Mumbai: Popular prakashan; 1998
10.Chitme HR, Patil UJ. Antiallergic and mast cell membrane stabilizing activity of Strychnos
potatorum Linn. Phytomed 2006. (In Pres).
11. Uma Devi P, Rao BSS and Soloman FE “Effect of plumbagin on the radiation induced cytogenic
and cell cycle changes in mouse Ehrlich ascites carcinoma in vivo”. Ind J Exp Biol 1998;
36:891-895.
12. Echardt AE, Malone BN and Goldstein I cancer Res 1982; 42:2977.
13. Umadevi P, Emerson Soloman F, and Sharada AC Indian J Exp Biol 1994; 32:523.
14. Mazumdar UK, Gupta M, Suryyendubikas M and Dilip M. Anti tumor activity of Hygrophila
spinosa on Ehrlich ascites carcinoma and sarcoma-180 induced mice. Ind J Exp Biol 1997:35:473-
477.
15.Naigonkar AV, Burande MD, A manual of medical laboratory technology .2nd ed. Nirali prakashan:
1996.
16.Onkava H, Onishi N, Yagi K. Assay for lipid peroxidation in animal tissue by thiobarbituric acid
reaction. Anal Biochem 1979:95; 351-358.
17.Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophysics 1979:82:70-77.
18.Kakkar p, Dos B, Vishwanathan PN. A modified spectrophotometric assay of superoxide
dismutase. Ind J Biochem Biophys; 1984; 21:130-132.
19.Aebi H. Methods in enzymology. Packer L editor.v1059.New York: Academic press 1974.
20.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol
reagent. Biol. Chem. 1951:193:265-275.
9. / SIGNATURE OF CANDIDATE / (SARASVATHI .V)
10. / REMARKS OF THE GUIDE / The present study has been proposed based on the results earlier study, the results obtained from the study may validate and substantiates the traditional therapeutic claim.
11. / NAME AND DESIGNATION OF
THE GUIDE / Prof.I.S.Muchandi
Principal and H.O.D., Department of Pharmacology
12. / SIGNATURE
13. / CO-GUIDE / ------
14. / SIGNATURE / ------
15. / HEAD OF THE DEPARTMENT / Prof. 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 /

Prof. 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 / 2007-08 / 1-8

CERTIFICATE

This is to certify that Miss.SARASVATHI.V a student of FIRST M.pharm is permitted to carry out experiments on animals for the dissertation / thesis work entitled “Evaluation of Anticancer activity of Strychnos potatorum linn. Seeds extract in mice as per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.