RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

Annexure II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. NAME OF THE CANDIDATE AND ADDRESS Mr. THIMMAIAH N.V. DEPARTMENT OF PHARMACOLOGY, K. L.E. SOCIETYS COLLEGE OF PHARMACY, J. N. M. C. CAMPUS, NEHRUNAGAR, BELGAUM 590 010.

2.

NAME OF THE INSTITUTION

K. L. E. Ss COLLEGE OF PHARMACY, BELGAUM 590 010.

3.

COURSE OF STUDY AND SUBJECT

MASTER OF PHARMACY IN PHARMACOLOGY

4.

DATE OF ADDMISSION TO COURSE

JUNE 2007

5.

TITLE OF THE TOPIC EFFECT OF CUCURBITA MAXIMA DUCH. SEED EXTRACT ON TESTOSTERONE INDUCED BENIGN PROSTATIC HYPERPLASIA IN RATS.

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BRIEF RESUME OF THE INTENDED WORK 6.1 NEED FOR THE STUDY: Benign prostate hyperplasia (BPH) is a benign and uncontrolled growth of prostate gland; it may leads to bladder out flow obstruction and lower urinary symptoms. BPH is common in men over 50 years of age and frequency increases with age. 1 The etiology of BPH, although not fully elucidated involves hormonal changes in the ageing man. The development and growth of the prostate glands depends on androgen stimulation, mainly by Dihydrotestosterone (DHT) which is formed in the prostate, as in other tissues, through the enzymatic conversion of testosterone into its more active metabolite DHT, catalyzed by prostate 5 alpha-reductase. DHT binds to androgenic receptor and promotes protein synthesis and cellular growth with ageing, the production and accumulation of DHT in the prostate increases, encouraging cell growth and causing hyperplasia. 2 Therefore, anti-androgenic drugs mainly, prostate 5 alpha-reductase inhibitors, are indicators for the treatment of BPH, improving the enlarged prostate and reducing complication.3 BPH also involves augmented adrenergic tone in prostate smooth muscle, regulated through alpha-adrenoceptor. Therefore alpha-adrenoceptor blockers are also used in the treatment of BPH and are particularly effective for relaxing the smooth muscle and improving BPH symptoms. It is reported that many traditional plants like Coconut oil 4, SPLE 5, cucurbita pepo6, Permixon 7, are used in the treatment of BPH because these plants contains fatty acids like oleic acid linoleic acid. These are responsible for 5 alpha-reductase activity. Zinc inhibits the 5alpha-reductase activity,
8, 9

whereas cucurbitacin posses the anti-prostatic

10

by inhibiting the division of glandular prostate cell

and are hence employed in the

treatment of BPH. The phytosterol is also used in the treatment of BPH.11 The Cucurbita maxima Duch. contains fatty acids like oleic acid and linoleic acid, phytosterol like sitosterol,12 15 Zinc and cucurbitacin.10 Literature survey about Cucurbita maxima Duch. it was observed paucty of information regarding its effect on BPH. Therefore the present study is undertaken to investigate the effect of Cucurbita maxima Duch. Seeds extract on testosterone induced BPH in rats

6.2 REVIEW OF LITERATURE Cucurbita maxima Duch.(Field pumpkin) (Fam:Cucurbitaceae) is large climbing herb, annual or perennial, hispid or hairy. Leaves are simple, alternate, nearly orbicular in outline, lobed with deep sinus at the base. Flowers large, yellow, unisexual, solitary, male flowers with three stamens, anthers one single celled, two celled, female flowers with inferior ovary. Fruits are fleshy, seed are ovoid or oblong compressed. The cucurbita maxima Duch. cultivated through out India.16, 17 Cucurbita maxima Duch. was reported to contain phytosterol like avenasterol, spinasterol, sitosterol, campesterol, clerosterol etc. fatty acids like oleic acid, linoleic acid and lineolinic acid. It also contains alpha, beta, and gamma-tocopherol cucurbitaxanthins, cucurbitacins.12 -15 In traditional system of medicine cucurbita maxima is used as anti-prostatic 10, anthelmentic
18

, diuretic, urinary anti-inflammatory and sedative. It was also used in

treatment of haemorrhoids, renal insuffiency, nephritis or glomerulonephritis, edema and renal calculi.10, 16, 17 Benign prostate hyperplasia (BPH) is a benign and uncontrolled growth of prostate gland; it may leads to bladder out flow obstruction and lower urinary symptoms. BPH is present in more than 50% men aged over 60 years. Between 15% and 30% of these men have lower urinary tract symptoms like smooth muscle dysfunction, urinary retention etc. In England and Wales, up to 25%of men undergo prostatectomy because of acute urinary retention, which doubles the risk of death and morbidity compared with elective surgery. More over, chronic urinary retention can lead to renal failure, and is responsible for 15% of prostatectomies. Other complications include recurrent urinary tract infection, bladder calculi, and haematuria 19. So, the present study is planned to evaluate the effects of Cucurbita maxima Duch. on BPH. 6.3 OBJECTIVES OF STUDY The objectives of the proposed study are: 1. Effect of Cucurbita maxima seed Duch. Extract on prostate to body weight ratio. 2. Effect of Cucurbita maxima Duch. Seed extract on alpha adrenergic activity in rats using vasdeferens. 7. 3. Effect of Cucurbita maxima Duch. Seed extract on 5 alpha-reductase activity. MATERIALS AND METHODS 3

7.1 SOURCE OF DATA: The sources of data will be from the laboratory experiments, which involves extraction of the seeds and their pharmacological activities on experimental animals, such as Adult Wistar male rats. 7.2 METHOD OF COLLECTION OF DATA: (Including sampling procedure, if any) MATERIALS: 1. Alcoholic extract of Cucurbita maxima Duch. Seeds by using Soxhlets extractor. 2. Standard drug a) Finasteride b) Norepinephrine c) Testosterone 3. Chemicalsa) Olive oil b) Dithiothretiol (DTT) c) Ethylene diamine tetraacetic acid (EDTA) d) Reduced nicotinamide adenine dinucleotide phosphate (NADPH) e) Ethyl acetate f) Nitrogen gas (IOLAR-1) METHODS: The following methods will be employed to study the effects of cucurbita maxima Duch. on BPH. 1. In vitro study of 5 alpha-reductase activity. 20 1.1Enzyme preparation. Albino Wistar male Rats will be anesthetized with anesthetic ether and the prostates will be dissected and homogenated in phosphoric acid buffer solution at pH 6.5 contains sucrose, DTT and EDTA. The homogenate will be centrifuge and supernatant is taken as a source of enzyme. Testosterone will be dissolved in ethanol. A NADPH will be prepared in potassium phosphate buffer at pH 6.5.

1.2 Estimation of 5 alpha-reductase activity. 4

Various concentrations of Cucurbita maxima Duch. Seed Extract prepared in potassium phosphate buffer, Testosterone, NADPH and enzyme solution will be incubated at specific temperature for 3hr and reaction terminated by the addition of ethyl acetate. The ethyl acetate layer will be separated and taken for the estimation of DHT. 1.3 Estimation of DHT by gas chromatography. DHT will be estimated by using gas chromatography attached with flame ionization detector. The instrument is equipped with a stainless steel column packed with 3% OV17. Nitrogen will be used as a carrier gas at a flow rate of 30mL/min. Ethyl acetate layer will be injected in to gas chromatographic column and peak area of the DHT will be calculated. The formation of DHT is taken as a measure of 5 alpha-reductase activity. 2. In vitro study on the alpha-adrenergic antagonistic activity.20 Male rats will be anesthetized with ether and the vasdeferens will be dissected and freed from extraneous tissues and suspended in organ bath containing Tyrode solution gassed with 95% O2 and 5% CO2 at 37oC. The contraction of the tissue will be recorded using Norepinephrine in different concentration with or without Cucurbita maxima Duch. Seed extract. 3. In vivo study on the activity against experimental prostatic Hyperplasia.20 The male rats will be selected and randomized in to five groups. Groups I II III IV V Treatment Olive oil Testosterone(3mg/kg) Testosterone(3mg/kg)+Cucurbita maxima Duch.seed extract (250mg/kg) Testosterone(3mg/kg)+Cucurbita maxima Duch.seed extract (500mg/kg) Testosterone(3mg/kg)+Cucurbita maxima Duch. seed extract (750mg/kg)

On the 22nd day rats will be anesthetized using anesthetic ether. The prostate glands will be collected and the prostatic to body weight ratio will be recorded.

7.3

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 techniques on Albino Wistar male rats. 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3? The Institutional Animal Ethical Committee has approved the proposed work.

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REFERENCES: 1. Clifford GM, Farmer RD. Medical therapy for benign prostatic hyperplasia: a review of the literature. European Urology 2000; 38(1): 2-19. 2. Bartsch G, Rittmaster RS, Klocker H. Dihydrotestosterone and the Concept of 5 -Reductase Inhibition in Human Benign Prostatic Hyperplasia. European Urology 2000; 37: 367-380. 3. Bartsch G, Rittmaster RS, Klocker H. Dihydrotestosterone and the concept of 5 alpha-reductase inhibition in human benign prostatic hyperplasia. World Journal of Urology 2002; 19: 413-25. 4. Maria de LA, Vivian M, Rosa M, Daisy C, David M. Effects of coconut oil on testosterone-induced prostatic hyperplasia in Sprague-Dawley rats. Journal of Pharmacy and Pharmacology 2007; 59(7): 995-9. 5. Franklin CL, Kurt Dreikorn A, Borkowski J, Braeckman L, Denis. Review of recent placebo-controlled trials utilizing phytotherapeutic agents for treatment of BPH. Prostate 1998; 37: 187-193. 6. Gossell-Williams M, Davis A, OConnor N. Inhibition of testosteroneinduced hyperplasia of the prostate of Sprague-Dawley rats by pumpkin seed oil. J Med Food 2006; 9(2): 284-6. 7. Raynaud JP, Cousse H, Martin PM. Inhibition of type 1 and type 2 5 alpha-reductase activity by free fatty acids, active ingredients of permixon. J Steroid Biochem Mol Biol 2002; 82(2-3): 233-9. 8. Fahim MS, Wang M, Sutcu MF, Fahim Z. Zinc arginine, a 5 alpha-reductase, reduces rat ventral prostate weight and DNA without affecting testicular function. Andrologia 1993; 25(6): 369-375. 9. Emily H, Michelle Yan. Zinc and prostate cancer. Linus Pauling institute spring/summer 2005 research report. 10. Gordon M, Gragg. Encyclopedia of Medicinal plants. 2nd edition, Education and health library publisher 2000; Vol-2, 605-6. 11. Barges RR, Windeler J, Trampisch HJ, Senge TH. Randomized, placebocontrolled double-blind clinical trial of beta-sitosterol in patients with benign prostatic hyperplasia. The lancet 1995; 345(8964): 1529-32. 12. Ram P, Rastogi, Mehrota BN. Compendium of Indian Medicinal plants. 7

Central Drug Research Institute, Lucknow and Information Directorate New Delhi Publishers 1993; Vol-2, 229. 13. Ram P, Rastogi, Mehrota BN . Compendium of Indian Medicinal plants. Central Drug Research Institute, Lucknow and Information Directorate New Delhi Publishers 1993; Vol-4, 238-9. 14. David GS, Fred JE, Liping Wang, Jay-lin Jane. Oil and Tocopherol Content and Composition of Pumpkin seed Oil in 12 Cultivators. Journal of Agricultural and Food chemistry 2007; 55: 4005-13. 15. Abak K, Sari N, Cetiner B. Changes of protein, fat content and fatty acid composition in naked pumpkin seeds influenced by sowing time. ISHS Acta Horticulturae 492. 16. Vaidyaratnam PS. Variers Arya Vaidya Sala - Indian Medicinal Plants: A Compendium of 500 species. Chennai: Orient Longman, 1994 (reprint 2002); Vol-2, 239-240. 17. Kirtikar KR, Basu BD. Indian Medicinal Plants, 2nd Edition, Dehradun: International Book Distributors, Book Sellers and Publishers 1999; Vol.-2, 11554-6. 18. Lohan LC, Khanikor HN, Ahmed N, Gogoi AR. Preliminary pharmacological and anticestodal screening of cucurbita maxima. Indian Journal of Pharmacology 1978; 10(4): 315-17. 19. Thorpe A and Neal D. Benign prostatic hyperplasia. The Lancet 2003; 361(9366): 1359-67. 20. Mitra SK, Sundaram R, Mohan AR, Gopumadhavan MV. Protective effect of prostane in experimental prostatic hyperplasia in rats. Asian Journal of Andrology 1999; 1: 175-79

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SIGNATURE OF THE CANDIDATE REMARKS OF THE GUIDE The above information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance. NAME & DESIGNATION OF (IN BLOCK LETTERS) 11.1 GUIDE Prof. A. D. TARANALLI. M. Pharm. PROFESSOR & HEAD, DEPT. OF PHARMACOLOGY, K.L.E.Ss COLLEGE OF PHARMACY, 11.2 SIGNATURE 11.3 CO-GUIDE (if any) 11.4 SIGNATURE 11.5 HEAD OF THE DEPARTMENT BELGAUM-10. -NA-NA-

11.

Prof. A. D. TARANALLI. M. Pharm. PROFESSOR & HEAD, DEPT. OF PHARMACOLOGY, 11.6 SIGNATURE 12. 12.1 REMARKS OF THE CHAIRMAN & PRINCIPAL K. L. E. Ss COLLEGE OF PHARMACY, BELGAUM-10 The above-mentioned information is correct and I recommend the same for approval.

Dr. F.V. MANVI. M. Pharm. Ph.D. PRINCIPAL K. L. E. Ss COLLEGE OF PHARMACY 11.2 SIGNATURE BELGAUM-590010