Effect of Withania somnifera root extract on the sexual behaviour of male rats I. Ilayperuma1, W.D. Ratnasooriya2, T.R. Weerasooriya1 1Department of Anatomy, Faculty of Medicine, University of Ruhuna, Sri Lanka 2Department of Zoology, Faculty of Science, University of Colombo, Sri Lanka Asian J Androl 2002 Dec; 4 : 295- 298 Keywords : Withania somnifera; male sexual competence; aphrodiasiac; libido; antimasculine; male sexual behaviour Abstract Aim: To determine the effect of a methanolic extract of Withania somnifera (L.) Dunal roots on sexual competence of male rats. Methods: Male rats were orally administered 3000 mg.kg-1.day-1 of root extract for 7 days. Their sexual behaviour was evaluated 7 days prior to treatment, day 3 and 7 of treatment, and day 7, 14 and 30 post-treatment by pairing each male with a receptive female. Results: The root extract induced a marked impairment in libido, sexual performance, sexual vigour, and penile erectile dysfunction. These effects were partly reversible on cessation of treatment. These antimasculine effects are not due to changes in testosterone levels or toxicity but may be attributed to hyperprolactinemic, GABAergic, serotonergic or sedative activities of the extract. Conclusion: Use of W. somnifera roots may be detrimental to male sexual competence. 1 Introduction Withania somnifera (L.) Dunal (Family Solanaceae) is a perennial or semiwoody shrub growing in several tropical countries including Sri Lanka [1]. According to traditional medicine of Sri Lanka, India and Nepal, the roots of this plant possess male sexual stimulant/aphrodiasiac activity [1]. However, the activity has not been scientifically documented. On the other hand, in animal models, an alcoholic extract of the root of this plant is shown to possess anticonvulsive, barbiturate potentiation, central nervous system depressant, membrane stabilizing, sedative, hypotensive and gamma amino butyric (GABA) activities[1]. Phytochemically, the roots contain a number of alkaloids, ergostane steroids and amino acids, including tryptophan [1]. Since central nervous system inhibitors [2], centrally acting hypotensives [2], GABA agonists [3], serotonin agonists [4] and a number of alkaloids [5] disrupt libido and sexual performance, it may be well possible that W. somnifera roots could inhibit male sexual function rather than stimulate as claimed in traditional medicine. The aim of this study was therefore to investigate the effects of W. somnifera roots on male sexual competence using laboratory rats and a methanolic extract. 2 Materials and methods 2.1 Plant and preparation W. somnifera roots were purchased from the market and authenticated by Dr. H. M. A. Tissera, Department of Materia Medica, Institute of Indigenous medicine, University of Colombo, Rajagiriya, Sri Lanka. The roots were cut into 1~2 cm pieces and shade dried inside the laboratory for 24 h at room temperature (28 ~30 ). These were finely powdered using an electrical grinder and soaked in 80 % methanol (3 L/kg of root) at room temperature for 14 days. The mixture was then filtered and the filtrate concentrated under negative pressure at 28 2 to obtain the crude extract (a blackish green syrupy mass). Any remaining trace of the solvent was further removed by placing the crude extract in vacuum overnight (the final yield: 2.9 %, w/w). The crude extract was dissolved in absolute ethanol and mixed with an equal volume of an ethanolic solution of polyvinylpyrolidone ( PVP ) ( Sigma Chemical, St. Louis, MO, USA) (1:1.5, crude extract: PVP by weight). The resulting mixture was gradually evaporated under negative pressure at 28 2 to dryness to obtain a co-precipitate. Traces of remaining solvents were removed from the co-precipitate by placing it overnight in vacuum and the latter was stored at 4 until use. The co-precipitate was dissolved in distilled water to a concentration of 3000 mg/mL before use, which is the maximum possible concentration with this method of preparation. 2.2 Animal and treatment Adult male Wistar rats of proven fertility were used. Rats were housed under standardized animal house conditions with free access to food and tap water. The animals were assigned randomly into 4 groups (Treated 1, Treated 2, Control 1 and Control 2) of 10 rats each. Rats of the two treated groups were orally administered 3000 mg.kg-1.day-1 (1mL) of the crude extract for 7 days. This dose is equivalent to fifteen times the human dose [6]. The Control animals were dosed with 1 mL of the vehicle (PVP) in a similar manner. All animals were observed daily for mortality, overt signs of toxicity, stress, aversive behaviour and non-sexual behaviour [5]. Food and water intake was recorded. On day 1 post-treatment, rats from Treated 1 and Control 1 groups were weighed and killed. Blood was collected and serum prepared and SGOT, SGPT and urea nitrogen levels were determined using Randox enzyme kits and a spectrophotometer (Jasco V500, Jasco Corporation, Tokyo, Japan). The gross morphology of the testes, epididymides, seminal vesicles, coagulating glands, central prostrate, liver, adrenal glands and kidneys were observed. The wet weights of these organs were recorded and expressed as a percentage of body weight. Seminal vesicular fluid was squeezed out and the pH determined. Female rats were brought into estrous by sequential treatment with estradiole benzoate (Sigma Chemical, St Louis, MO, USA) and progesterone (Sigma Chemical, St Louis, MO, USA). Rats of the Treated 2 and Control 2 groups were individually placed in a wire cage with a 10 min adaptation period. An oestrous female was placed into each cage 7 days prior to treatment, and at days 3 and 7 of treatment and days 7, 14 and 30 post-treatment. The masculine sexual behaviour parameters were monitored as described elsewhere [5]. 2.3 Statistical analysis Data are expressed as meanSEM. Statistical evaluations were made using Mann-Whitney U-test and McNemer test as appropriate. Significance was set at P<0.05. 3 Results There were no treatment related deaths, overt signs of toxicity, stress, aversive behaviours or non-sexual behaviour. Food and water intake remained unaltered. The results are summarised in Table 1. As shown, significant changes in all the parameters monitored or computed were evident on day 7 of treatment and day 7 post-treatment. None of the treated rats ejaculated during treatment period and up to day 7 post-treatment. Complete abolition of intromission was also evident on day 7 of treatment and day 7 post-treatment. In contrast, at least 2~7 rats attempted to mount during this period. Further, none of the rats that mounted exhibited aberrant mounting. Table 1. Effect of Withania somnifera root extract on sexual behaviour of male rats (n=10). bP <0.05, cP <0.01, compared with controls (Maun-Whitney U- test and McNermer test). Pre-treatment Treatment Post -treatment Day 7 Day 3 Day 7 Day 7 Day 14 Day 30 % mounted Vehicle Extract 100 100 100 50 100 20c 100 60 100 70 100 90 % intromitted Vehicle Extract 100 100 100 20c 100 0c 100 0c 100 40 100 70 % ejaculated Vehicle Extract 100 100 100 0c 100 0c 100 0c 100 40b 100 70 Number of mounts Vehicle Extract 13.8 1.6 13.3 1.7 13.7 1.5 2.5 1.1c 14.9 1.3 1.0 0.7c 14.5 1.4 3.2 1.1c 13.6 1.9 4.6 1.1c 13.7 1.5 5.6 1.2c Number of intromissions Vehicle Extract 8.2 1.1 8.6 1.2 10.2 1.4 0.3 0.2c 12.7 1.1 0c 11.3 1.3 0c 10.7 1.8 1.0 0.5c 8.0 1.0 2.3 0.8c Mount latency (s) Vehicle Extract 58.4 14.0 50.3 15.7 60.7 12.3 538.3132.3 59.2 11.5 722.3 118.5b 61.5 9.2 521.0 113.1 c 59.4 5.4 372.7 117.9c 60.1 8.4 218.1 90.5 Intromission latency (s) Vehicle Extract 58.6 14.1 51.1 15.5 61.2 12.1 811.066.8c 59.8 11.4 900.0 0.0c 61.9 9.2 900.0 0.0c 60.6 5.3 579.9 131.3c 64.7 9.3 330.0 126.4 Ejaculation latency (s) Vehicle Extract 1458.0 75.9 1440.0107.7 1464.0100.1 1800.00.0c 1416.0110.7 1800.00.0c 1398.0 104.7 1800.0 0.0 c 1494.0 91.0 1740.0 40.0b 1440.0 85.8 1554.0 96.1 Copulatory efficiency Vehicle Extract 0.6 0.1 0.7 0.1 0.7 0.1 0.1 0.1c 0.9 0.0 0c 0.8 0.0 0c 0.8 0.1 0.2 0.1c 0.6 0.0 0.3 0.1b Intercopulatory interval (s) Vehicle Extract 179.1 20.8 173.1 25.6 157.1 28.0 270.0 192.1b 111.1 14.3 0c 120.3 10.9 0c 187.2 51.3 208.5 91.8 175.3 18.7 291.2 80.4 Intromission ratio Vehicle Extract 0.4 0.0 0.4 0.0 0.4 0.0 0.1 0.0c 0.5 0.0 0c 0.4 0.0 0c 0.4 0.0 0.1 0.0c 0.4 0.0 0.2 0.1b In the treated rats, the time required to mount and intromit was significantly increased from day 3 of treatment to day 14 post-treatment; the frequencies of mounting and intromission, the copulatory efficiency and intromission ratio were also significantly suppressed throughout the study period; the intercopulatory interval was significantly altered during treatment and on day 7 post-treatment. The root extract treatment had no significant effect on the serum SGOT, SGPT, and the urea nitrogen levels, as well as the gross appearance, wet weight of the organs and the pH of the seminal vesicular fluid (data not shown). 4 Discussion Other investigators have shown that W. somnifera roots have antistress [1], anabolic and hyperphagic effects [1] and non-toxic even after chronic oral administration [8]. Thus the antilibido effect could be attributed to a selective action of the extract. A lack of any marked change in the gross appearance and wet weight of sexual accessory glands, and pH of seminal vesicular fluid suggest that the extract is not acting via androgen changes. Hyperprolactenemia inhibit male sexual behaviour and libido [2, 9]. Drugs inhibiting or stimulating the central nervous system may change blood prolactin levels [2]. Thus, the antilibido action in the present study, at least partly, may be mediated through prolactin mechanism. It is well established that GABA and serotonin agonists [3, 4] disrupt libido and male sexual behaviour in rats more or less in a similar manner as shown in this study. W. somnifera roots possess GABA mimetic activity [1] and contains serotonin precursors [1]. Hence, a strong possibility exists that the root extract depresses libido via the GABAergic and serotonergic systems. Suppression in libido is frequently associated with sedatives and antihypertensives [2, 10]. Both effects have been reported with the root of this plant [1] and therefore it is also possible that the extract induced antilibido effects through this mechanism. In the treated rats, there is a substantial prolongation in the mounting and intromission latencies and a decrease in the mounting and intromission frequencies and copulatory efficiency, which is suggestive of suppression of sexual arousability/motivation and vigour [5]. On the other hand, none of the treated rats exhibited aberrant mounting behaviour, which indicates that the penile tactile sensations remained uninhibited [11]. The extract treatment also caused a reduction in the intromission ratio indicating an erectile dysfunction [5]. This is possible as root extract of this plant is known to have potent hypotensive, sedative and central nervous system depressant activities [1]; impotence is a major side effect of several western drugs possessing these activities [2,10]. In addition, the root extract caused a prolongation in the intercopulatory interval in rats indicating a disturbed sexual performance [5]. In conclusion, this study shows, for the first time, that oral administration of methanolic root extract of W. somnifera impairs sexual competence of male rats, possibly by a multifactorial mechanism. References [1] Arambewela L, Silva R. In: Withania somnifera. Colombo: Ceylon Institute of Scientific and Industrial Research; 1999. p 1-26. [2] Smith CG. Drug effects on male sexual function. Clin Obstet Gynecol 1982; 25: 525-31. [3] Agmo A, Paredes RG, Sierra L, Garces I. The inhibitory effects on sexual behaviour and ambulatory activity of the mixed GABAa/GABAb agonist progabide are differentially blocked by GABA receptor agonists. Pscyhopharmacol 1997; 129: 27-34. [4] Fernandez-Guasti A, Escalante AL, Ahlenius S, Hillegaart V, Larsson K. Stimulation of 5-HT1A and 5-HT1B receptors in brain regions and its effects on male rat sexual behaviour. Eur J Pharmacol 1992; 210: 121-9. [5] Ratnasooriya WD, Dharmasiri MG. Effects of Terminalia catappa seeds on sexual behaviour and fertility of male rats. Asian J Androl 2000; 2: 213-9. [6] Annonimus. The use of traditional medicine in primary health care: A manual for health workers in South-East Asia. SEARO regional health papers 1990; 19: 96-7. [7] Williams K, Mckinnells S, Saunders PTK, Walker M, Fisher JS, Turner KJ, et al. Neonatal exposure to potent environmental oestrogens and abnormalities of the male reproductive system in the rat: evidence for importance of the androgen-oestrogen balance and assessment of the relevance to man. Hum Reprod Update 2001; 7: 236-47. [8] Sharma S, Dhanukar S, Karandikar SM. Effects of long term administration of the roots of Ashwagandha and Shatavari in rats. Indian Drugs 1985; 23: 133-9. [9] Chambers KC, Phoenix CH. Sexual behaviour and serum level of prolactin, testosterone and oestradiol in young and old Rhesus males. Physiol Behav 1992; 52: 13-6. [10] Horowitz JD, Globle AJ. Drugs and impaired male sexual function. Drugs 1979; 18: 206-17. [11] Lucio RA, Manzo J, Martinez-Gomez M, Sachs BD, Pacheco P. Participation of pelvic nerve branches in male rat copulatory behaviour. Physiol Behav 1994; 55: 241-6. home Correspondence to: Professor W. D. Ratnasooriya, Department of Zoology, University of Colombo, Colombo-03, Sri Lanka. Tel: +94-1-503 399 E-mail: dappvr@sltnet.lk Received 2002-02-25 Accepted 2002-11-07