Original Article Contraceptive Efficacy of Citrullus colocynthis Methanolic Extract in Male Rats Aksha Sharma1, Priyanka Sharma2, Mridula Chaturvedi3 and Suresh C. Joshi*1,4 1Reproductive Toxicology Unit, Center for advanced studies, Department of Zoology 2P.G. Department of Zoology, Agrawal College, Jaipur 3BBD College, Chimanpura, Jaipur 4Centre for Converging Technology, University of Rajasthan, Jaipur – 302 055 (India) ABSTRACT To study the effects of Citrullus colocynthis on testicular function and fertility, 70% methanolic extract of C. colocynthis was fed orally to male albino rats at the dose levels of 75 and 150 mg/kg body wt/day for 60 days. Oral administration of C. colocynthis caused a Address for significant decrease in testicular weight and sperm concentration. Correspondence The level of serum testosterone, LH and FSH also declined and spermatogenesis was impaired. The histoarchitecture of the testes in Reproductive the rats receiving C. colocynthis methanolic extract showed Toxicology Unit, degenerative changes and disruption of spermatogenesis and the Center for advanced number of spermatogonia remained significantly decreased. Thus the studies, Department antifertility effects of C. colocynthis seemed to be mediated by of Zoology, University disturbances in structure and testicular function including leydig and of Rajasthan, Jaipur Sertoli cells resulting in an alteration in physio-morphological events (INDIA); of spermatogenesis. E-mail: s_c_joshi2003 @rediffmail.com Keywords: Testicular weight, Sperm concentration, Testosterone, Spermatogenesis, Degenerative changes, Antifertility. INTRODUCTION The quest for the oral contraceptive to design new and effective contraceptives agent that can control human fertility is from compounds of plant origin3. Use of continue since ancient times1. Although a ethnobotanical information in medicinal variety of synthetic contraceptive agents are plant research has gained considerable available, but because of their side effects attention in recent years, especially in these cannot be used continuously2. segments of the scientific community4. The Scientists and researchers throughout the use of herbs is very common in developing world are realising the importance of countries, particularly in rural communities. avoiding the harmful effects of synthetic However, an increase in the use of plants compounds, and thus cooperating in efforts American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org Joshi et al__________________________________________________ ISSN 2321 – 2748 has been observed in metropolitan areas of Animals and animal treatment developed countries during the last decade5. Healthy adult male albino rats (Rattus Citrullus colocynthis (L.) Schrad is norvegicus) of Wistar Strain of an average an important medicinal plant belonging to body weight 150-200 gms with proven the family of Cucurbitaceae. C. colocynthis fertility have been employed for contain large amounts of phenolics and experimentation. The animals were kept in flavonoids that have antioxidant activities6. clean polypropylene cages covered with It has a beneficial effect on improving the chrome plates grills and maintained under glycemic profile without severe adverse controlled environmental conditions (12-h effects in type II diabetic patients7. It was light: 12-h dark). The animals were mostly used as purgative, anthelmentic, antipyretic, maintained on standard pellet diet procured carminative, cures tumours, leucoderma, from Ashirwad Industries, Chandigarh (India) asthma, jaundice, enlargement of spleen, and occasionally on germinated/ sprouted tuberculous glands of the neck, elephantiasis gram and wheat seeds as an alternative feed. and ulcers, also reported that fresh fruit and They were given clean water ad libitum. The seeds are eaten as laxative and for removing guidelines for care and use of animals for kidney stones8. scientific research (INSA, 2000) were strictly The therapeutic efficacy of C. followed throughout the course of colocynthis extract in treatment of human investigation. diseases has been established. Thus the aim of this study was to investigate the effect of Experimental design methanolic extract of C. colocynthis on Doses were freshly prepared by various male fertility parameters to evaluate making a suspension of the methanolic extract its possible contraceptive efficacies in wistar of Citrullus colocynthis was daily made in strain male rats. distilled water for administration and administered forcibly through mouth by pearl MATERIAL AND METHODS point needle during the study duration. Animals were equally distributed into three Plant collection treatment groups, each consisting of 10 The plant material was collected from animals: the university campus and authenticated at the Group I: Animals of this group were given Herbarium, Department of Botany, University sterile distilled water alone orally for 60 days. of Rajasthan, Jaipur in comparison with the This group was serves as vehicle treated pre existing voucher specimen (RUBL control. 20689). Stem and leaves of C. colocynthis Group II: Animals of this group were were shed dried and powdered. administered with methanolic extract of Citrullus colocynthis at the oral dose of 75 Plant crude extract preparation mg/kg/ body wt/day, for 60 days. Shed dried stem and leaves of C. Group III: Animals of this group were colocynthis were powdered and extracted administered with methanolic extract of with 70% methanol for 36-48 hours by Citrullus colocynthis at the oral dose of 150 Soxhlet extraction method. The extract was mg/kg/ body wt/day, for 60 days. filtered and then methanol was separated under reduced pressure to obtain a solid mass. Body and organ weight measurement Various doses of C. colocynthis methanolic After the last day of treatment, the extract were administered orally for 60 days. body weight of each animal was recorded and AJPCT[2][2][2014]229-241 Joshi et al__________________________________________________ ISSN 2321 – 2748 they were killed along with control animals. microtome at 5 μm thickness, and stained Blood was collected in sterile tubes by cardiac with hematoxylin and eosin (H&E) for light puncture. Testes of each animal were excised microscopic examination. from the surrounding tissue and blotted free of blood for weighing. Hormone analysis Serum concentration of total Sperm density determination testosterone, FSH and LH of control and Total number of sperms were counted treated groups were measured by using haemocytometer after further diluting radioimmunoassay (RIA)21. the sperm suspension from testis. The sperm density was calculated in million per ml as Statistical Calculations per dilution9. The data obtained from the above experiments were subjected to statistical Fertility Test analysis. All the values were expressed in The mating exposure test of all the terms of mean ± SEM. The data were animals was performed. They were cohabited analyzed statistically by using Student’s “t” with proesterous females in the ratio 1:3. The test and the significance of differences was set vaginal plug and presence of sperm in the at P < 0.01 and P < 0.001. vaginal smear was checked for positive mating. Females were separated and resultant RESULTS pregnancies were noted, when dam gave birth. The number and size of litters delivered Testicular weight, Sperm Dynamics and were recorded. Fertility was calculated in Fertility control as well as in treated groups. A significant decrease was observed in weight of testes after administration of Serum biochemistry methanolic extract of C. colocynthis at dose Serum was isolated and stored for levels of 75mg (p≤0.01) and 150mg detection of protein content10, total (p≤0.001) in comparison to controls (Table cholesterol11, phospholipids12, triglyceride13, 1). The testicular sperm density also acid phosphatase and alkaline phosphatase14. decreased significantly (P ≤ 0.001) after C. colocynthis administration. Control rats Hematology showed 100% positive fertility in the mating The blood samples were collected exposure test while the rats exposed to 75 mg from the heart and analyzed for blood urea15, and 150 mg/kg b. wt/day dose level showed blood sugar16, hematocrit level17, RBC and 25% and 60% negative fertility respectively WBC18. (Table 1). Tissue Biochemistry Effect of C. colocynthis on Serology of Protein10, Glycogen19, Sialic acid20 treated rats and Cholesterol11 were estimated in testis. No significant changes were observed in serum total protein, phospholipid, Testicular histology triglyceride, total cholesterol, acid Testes of rats of all experimental phosphatase and alkaline phosphatase levels groups were fixed in Bouin’s fixative for at after administration of methanolic extract of least 48 h, processed by paraffin wax C. colocynthis at 75 and 150 mg/kg dose impregnation method, cut using a rotary levels (Table 2). AJPCT[2][2][2014]229-241 Joshi et al__________________________________________________ ISSN 2321 – 2748 Effect of C. colocynthis on Haematology of DISCUSSION treated rats After administration of methanolic In the present study oral extract of C. colocynthis, the levels of total administration of methanolic extract of C. erythrocyte count (TEC), total leukocyte colocynthis at the doses 75 and 150 mg/kg count (TLC), haemoglobin, haematocrit, orally for 60 days produced a dose dependent blood sugar and blood urea revealed a non- adverse effect on fertility of male rats. significant change in 75 and 150 mg/kg b. wt. Reduction in the weight