ANTIANDROGENIC PROPERTIES OF NEEM SEED OIL (AZADIRACHTA INDICA) IN MALE RAT AND RABBI

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1 Ancient Science of Life, Vol No. VII No. 1 July 1987, ANTIANDROGENIC PROPERTIES OF NEEM SEED OIL (AZADIRACHTA INDICA) IN MALE RAT AND RABBI J. D. SHARMA, R. K. JHA, IRA GUPTA, PRABHA JAIN & V.P. DIXIT Reproductive Physiology Unit, Department of Zooloogy, University of Rajasthan, Jaipur , India Received: 13 June 1986 Accepted: 19 December, 1986 ABSTRACT: Azadirachta indica (Neem) seed oil brings about a significant diminution in spermatozoan motility and density. It leads to reduction of fertility rate in rats and rabbits. The body weight of the animals remain unaffected but the weights of reproductive oranges declined. Reduction of cauda epididymal protein, sialic acid, acid phosphatase and seminal vesicular fructose concentration in rats and rabbits could bedue to antiandrogenic action of the seed oil as confirmed with the help of bioassay techniques. Histopathology of testis revealed arrest of spermatogenesis and sever degenerative changes in the cauda epididymis. INTRODUCTION The population control is a major task of our country since last two decades and the research activities represent the largest concerted efforts to develop non-toxic, inexpensive and effective antifertility agent. The plant kingdom has abundant chemical compounds that elicit pronounced effects in animals and humans. A number of these compounds have become pharmacological tools. Although plant derived contraceptive agents have not been investigated extensively and no such agents have found general acceptance for male fertility regulation 1. Neem seed oil is reputed to have many medicinal properties in traditional system of medicine. Two of its derivatives viz., sodium nimbinate 2 and Sodium nimbidinate 3 were found to possess weak spermicidal action in vitro. Neem oil as a vaginal contraceptive has been developed by Sinha and his associates 4. There is a paucity of literature regarding neem oil affecting male reproductive organs present investigations were under taken to elucidate the properties of neem seed oil in male rats and rabbits. MATERIALS AND METHODS Healthy, adult male albino rats and rabbits of inbred colony housed in air-conditioned animal house at 24 o C ± 2 o C with 14 hour light. Water and food was given ad libitum. Neem seeds were collected from the nearby area of the University campus in the month of June and July. These were shade dried, powdered and the oil extracted in petroleum ether was used for the study. The animals were fed with neem seed oil orally as per experimental design given below. The animals were screened for fertility test and autopsied for detailed pathophysiological study. Reproductive

2 organs were excised, blotted free of blood, weighed and fixed in Bouin s fluid for histological study. The sperm motility and density of cauda epididymal spermatozoa was assessed by the method of Prasad et al 5. The protein 6, sialic acid 7, and fructose 8 were determined. The enzyme activity of acid phosphates was estimated by the method of Fiske and Subbarao 9. In the fertility test, treated males were kept over night with normal cycling estrus / proestrus females in 1: 3 ratios. The vaginal smear was checked next morning. The presence of spermatozoa in vaginal smear or a vaginal plug confirmed the positive mating. The females were checked for implantation site on day 16 of pregnancy. The results were analyzed with the help of Students t test. The animals were sacrificed and epididymis, Seminal vesicle, ventral prostrate and adrenal glands were dissected out, cleared of fats and weighed. The weights of epididymis, seminal vesicle and ventral prostate were increased after T. P. treatment, whereas combined treatment caused little increase in sex accessory organ weights (Table 1). Group Treatment Animal used Autopay day (1) (2) (3) (4) I Control rat (Olive oil) st II Azadirachta indica (neem) seed oil (50 mg/day/rat oral, 60 days) 8 61 st III Neem seed oil (60 mg/day/rat oral, 60 days) st IV Neem seed oil (75 mg/day/rat oral, 60 days) st V Neem seed oil (100 mg/day/rat oral, 60 days) st VI Control rabbits (olive oil) 7 71 st VII Neem seed oil (500 mg/day/rabbit oral, 60 days) 9 71 st

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4 Bioassay Castrated immature male rats were treated as outlined Group Treatment Autopsy day Animal used I Castration + Olive oil 4 th 6 (0.1 ml twice a day, i.m) II Castration + Testosterone propionate (T. P.) ( mg/animal twice a day, i.m) 4 th 6 III Castration + TP + Neem oil (TP = mg/animal twice a day, i.m mg Neem oil / animal twice a day oral) 4 th 6 TABLE I Bioassay for androgenic / antiandrogenic activity of Neem seed oil in 21 day old castrated male rats S. No. I Treatment Castration + olive oil (0.1 ml twice a day, i.m) (6) Initial Body Wt. 25 ± 3.8 Body Wt. (g) (After 7 days castration) 31 ± 4.9 Final Body Wt (g) (After treatment) 32.0 ± 5.5 Epididymis (mg) 18.3 ± 1.5 Seminal Vesicle (mg) 11 ± 0.6 Ventral prostrate (mg) 6.5 ± 1.3 Adrenal (mg) 11.3 ± 1.5 II Castration + TP ( mg/animal twice a day i.m) (6) 25.3 ± ± ± ± ± ± ± 0.3 III Castration + T. P. + Neem oil (6) 26.6 ± ± ± ± ± ± ± 0.3 Figures in parentheses represent the number of animals examined. All figures ± S. E. M.

5 RESULTS: Organ weights Body weights were not affected after Neem seed oil feeding. However the weight of testes were significantly decreased at 75 mg (P<0.001) and 100 mg (P<0.01) dose levels. The reduction in epididymal weights were dose dependent and significant reduction was noted with increased doses. Seminal vesicle and ventral prostate weights were elevated at 50 mg dose but reduced at higher dose levels (Fig. I, II, III & IV).

6 Fertility test The sluggishly motile spermatozoa were unable to fertilize normal cycling females. The test was cent per cent negative throughout the experiment, except with 50 mg dose, where it was 62% negative. The libido remain unchanged as evinced by vaginal plug formation. Spermatozoa motility The spermatozoan motility declined significantly (P<0.001) at various dose levels (Fig. V.) Spermatozoa density Cauda epididymal spermatozoa density declined and was dose dependent (50 mg: P<0.01; 60, 75 and 100 mg : P<.001). Similar changes were observed in rabbit cauda epididymal sperm suspension (Fig. VI). Biochemical analysis The concentration of protein decreased at 75 and 100 mg dose level in rat and 500 mg dose level in rabbit. The sialic acid contents and acid phosphatase enzymes activity in cauda epididymis decreased significantly in comparison to controls (P<0.001). Fructose concentration of seminal vesicle was significantly reduced (P <.001, Fig. VII).

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8 Histology The histology of testes evinced spermatogenic arrest at spermatocyte stage β-phase. Severe degenerative changes were observed in the cadua epididymis, viz. reduction in tubular diameter, infiltration of connective tissue and disintegration of epidymal tubules (Plante I, Fig. 1 6). DISCUSSION The present study revealed that the Neem seed oil treatment in rats and rabbits resulted in dose dependent changes in weights and biochemical parameters of reproductive organs. As these organs depends upon the levels of circulating testosterone for the maintenance of their structural and functional integrity 10, reduction in androgen dependent parameters by the Neem seed oil treatment may cause the so-called androgen deprivation effect to target organs by probably influencing the Leydig cells and/or by affecting testosterone synthesis in the testis 11. This in turn results in alteration in the internal milieu of the epididymis thereby rendering them hostile for sperm maturation and survival. Therefore sluggishly motile spermatozoa were unable to fertilize normal cycling female, although the libido remain unaffected. The 100% negative fertility test in present study may be attributed to lack of foreward progression and reduction in density of spermatozoa and altered biochemical milieu of cauda epididymis. Sharma and Saksena 3 (in vitro study on human spermatozoa) reported a weak

9 spermicidal action of sodium nimbidinate a derivative of neem oil (spermatozoa killed in 30 min). However, Sinha et al 4 have reported that the spermatozoa (human and monkey) became immotile within 30 sec. of contact with Neem oil. The cellular integrity of epididymis is androgen dependent 12, 13. Martan and Risley 14 have reported that mouse epididymal acid phosphatase is under the control of androgen. In the present finding the various androgen dependent parameters viz. protein, sialic acid and acid phosphatase of cauda epididymis revealed significant decrease, indicate that the Neem seed oil treatment resulted in fall of circulating androgen level, changing Status quo of epididymis. The most important free sugar secreted by the Seminal vesicle is fructose15 and this sugar can serve as a good index to evaluate the secretory activity of the seminal vesicle16. Reduction in the fructose concentration of seminal vesicle after Neem seed oil treatment further support the antiandrogenic nature of the seed oil. Altered histophysiology of the testis and the epididymis is in agreement with antiandrogenic nature of Neem seed oil. The bioassay carried out is castrated immature males support the antiandrogenic nature of Neem seed oil. CONCLUSION In conclusion neem seed oil brings about antifertility effects in male rats and rabbits and they are attributed to the anti androgenecity. Further studies are necessary for its practical applicability as male contraceptive. REFERENCES 1. Farnsworth, N. R., The potential consequence of plant extinction in the United States on the current and future availability of prescription drugs. Paper presented at Symposium on estimating the value of Endangered species : Responsibilities and Role of Scientific Community. Annual Meeting American Assoc. Advan. Sci., Washinington, DC, January 4, 20. (1982). 2. Sharma, V. N. and Saksena, K. P. Spermicidal action of sodium nimbinate. Indian J. Med. Res., Vol. 47, No. 3, (1959). 3. Sharma, V. N. and Saksena, K. P. Sodium nimbidinate. In vitro study of its spermicidal action. Indian J. Med. Sci., Vol. 12, (1959). 4. Sinha, K. C., Riar, S. S., Tiwary, R. S., Dhawan, A. K., Jaya Bardhan, Thomas Pauling Jain, A. K. and Jain, R. K. Neem seed oil as a vaginal contraceptive. Indian J. Med. Res., Vol. 79, (1984). 5. Prasad, M.R. N., Chinoy, N. J and Kadam, K. M. Changes in succinate dehydrogenase levels in the rat epididymis under normal and altered physiological conditions. Fert. Steril. Vol. 23, (1972).

10 6. Lowry, O. H., Rosebrough, M. J., Farr, A. L. and Randall, R. J. Protein measurement with Folin-phenol reagent J. Biol. Chem., Vol. 193, (1951). 7. Waren, L. The thiobarbituric acid assay of sialic acid. J. Biol. Chem Vol ( ). 8. Foreman, D., Gaylor, L., Evans, E. and Trella, C. A. A modification of the Roe procedure for determination of fructose in tissues with increased specificity. Anat. Biochem., (1973) Vol. 56, Fiske, C. H. and Subbarao, Y. Colorimetric determination of phosphorous. J. Biol. Chem., Vol. 66, (1925). 10. Chinoy, N. J., Sheth, K. M., Seethalakshmi, L., Parmar, P.Y., Sanjeevan, A. G., Rao, M. V., Sharma, J. D., Chinoy, M. R., Maithili, R. S. Trivedi, D. G., Ashok Kumar R., and Geetha Ranga Studies on reproductive physiology of animals with special references to fertility control. Comp. Physiol. Ecol. Vol. 7, (1982). 11. Chinoy, N. J., Sheth, K. M., Seethalakshmi, L., Effects of estradiol benzoate on rat testicular and adrenal testosterone production in vitro. Experientia (Communicated). 12. Lubicz-Nawrocki, C. M. The inhibition of fertilizing ability of epididymal spermatozoa by the administration of estradiol benzoate to testosterone maintained hypophysectomized or castrated hamsters. J. Endocr., Vol. 61, (1974). 13. Orgeloin-Cirst, M. C., Danzo, B. J. and Davies, J. Endocrine control of the development and maintenance of sperm fertility ability in the epididymis. In: Handbook of Physiology Edocrinology. Vol. V, ch. 15, (1975). 14. Martan, J. and Risley, P. L. Holocrine secretory cells of the rat epididymis. Anat. Rec. Vol. 146, (1963). 15. Mann, T. The Biochemistry of semen and of the male reproductive tract John Willey and Sons. Inc., New York, (1964). 16. Bandes, D. Male accessory sex organs. Academic Press, New York, (1974).