Summary: Activity of alkaloid extract of caricapryl–99 seeds (Carica papaya Linn seeds) on the serum levels of steroid hormones was studied in adult male albino rats. Three tolerated doses obtained from the graph of percectage toxicity (10, 50 and 150 mg/kg) were separately administered orally, daily for three days to three groups of male rats (n=5) while group four of 5 rats received the vehicle (corn oil) as control. The results showed that 10mg/kg/d caused increase serum levels of FSH and estrogen but decrease in the serum levels of LH and testosterone compared to control; 50mg/kg/d elevated the serum levels of FSH, estrogen but inhibited testosterone; while 150mg/kg/d pretreatments caused a significant decrease (

Agwu, B

James, UW

Okeke, CP

Udoh, FV

Udoh, PB

Umoren, EB

English

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13  Nigerian Journal of Physiological Sciences 24 (1): 13 –15 ©Physiological Society of Nigeria, 2009 Available online/abstracted at http://www.bioline.org.br/np; www.ajol.info/journals.njps; www.cas.org  EFFECT OF CARICAPRYL-99 SEED ALKALOID EXTRACT ON THE SERUM LEVELS OF SEX HORMONES AND PITUITARY GONADOTROPHINS IN MALE ALBINO RATS  P. B. UDOH, F. V. UDOH*, E. B. UMOREN. U. W. JAMES, C. P. OKEKE AND B. AGWU Department of Zoology and Environmental Biology, University of Calabar, Calabar, Cross River State, Nigeria. *Department of Pharmacology,  College of Basic Medical Sciences,  University of Calabar, Calabar, Nigeria  E-mail: pbudoh@yahoo.com Tel: +234 (0) 8035091976, +234 (0) 8037933923     Summary:  Activity of alkaloid extract of caricapryl–99 seeds (Carica papaya Linn seeds) on the serum levels of steroid hormones was studied in adult male albino rats. Three tolerated doses obtained from the graph of percectage toxicity (10, 50 and 150 mg/kg) were separately administered orally, daily for three days to three groups of male rats (n=5) while group four of 5 rats received the vehicle (corn oil) as control. The results showed that 10mg/kg/d caused increase serum levels of FSH and estrogen but decrease in the serum levels of LH and testosterone compared to control; 50mg/kg/d elevated the serum levels of FSH, estrogen but inhibited testosterone; while 150mg/kg/d pretreatments caused a significant decrease (p<0.01) in the serum levels of FSH, LH and testosterone. The results permitted the conclusion that caricapryl-99 treatment inhibited the serum level of the androgen, testosterone which might result in a male infertility. Key words: Alkaloid extracts, Caricapryl, infertility and pituitary gonadotrophins, male rats.   Introduction The need for fertility regulation in men cannot be overemphasized. While chemical methods for fertility regulation in woman are readily available (Chinoy and Padman, 1996; WHO, 1996) the “male pill” remains a distant prospect as effective contraceptive regimens are still toxic and inconvenient. Acceptable male contraceptive methods should reduce the burden traditionally placed almost exclusively on the female partner. Caricarpyl-99 seeds (Caricaceae) have been used in Nigerian herbal medicine for treatment of typhoid fever, malaria and other bacterial infections. However, our previous investigation into the activity of the seed extract in male rats showed that the seed extract treatment induced seminiferous tubules hypertrophy which could lead to male reproductivc dysfunction. (Udoh et al, 2005a) Phytochemistry showed that the seed extract contained alkaloid glycosides, saponins, flavonoids, reducing compound, polyphenol, phlobatanins and hydroxymethl-anthraquinones. (Udoh and Kehinde, 1999)  These observations led to the histological examination of the sections of pituitary gonadotrophs of rats treated with the seed extract. The result showed hypertrophy and hyperplasia of the cell morphology, whereas the gonads (testes) sections showed gradual degeneration of germ cells, Sertoli and Leydig cells. (Udoh et al, 2005c) Further observation of the effect of the extract on the fertility in male and female rats showed that extract treatment prevented ovum fertilization as well as reduced sperm count in male rats. (Udoh and Udoh, 2005b; Udoh et al, 2005a)  Therefore, this study was to investigate the effect of the seed extract on serum levels of testosterone, LH/ICSH, estrogen and FSH in male rats.  Materials and methods  Preparation of plant extracts  The ripe fruits of C. papaya were obtained from the University of Calabar vicinity around the month of June and July. The ripe seeds were removed, washed and oven dried at 800 C for 48h. They were milled into fine powder using electric blender (Osterizer). The powdered sample (100g) of C. papaya was wrapped in a thimble and placed in a 500 cm3 extractor (M&G England). The sample was extracted in absolute ethanol at 600 C. The extract was evaporated to a paste form at 400C for 8h to a paste form. The paste plant extract was preserved in the refrigerator (Thermocool, TEC).  Treatment Twenty adult male albino rats weighing between 190 – 200g were divided into 4 groups (n=5). Group 1 received corn oil (0.5ml) daily as controls, groups 2, 3 and 4 received 10, 50 and 150mg/kg/d for 3 days respectively following the method adopted by Udoh et al, 2005c; Udoh, 2007. 24h after treatment the rats were sacrificed and blood sera were prepared for hormonal assays. 14  Hormonal Assays Serum samples were assayed for the following; Follicle stimulating hormones (FSH), Luteinizing/intestitial cell stimulating hormone (LH/ICSH), testosterone and estrogen (estradiol). The method used was microwell enzyme-linked immunoassay (ELISA) using analytical grade reagents (Syntron Bioresearch Inc. USA)  Statistical Analysis All data for control and experimental groups were subjected to statistical evaluation, using student t-test and analysis of variance (ANOVA) for significant differences between the controls and experimental groups at p<0.05.  Results Testosterone serum level Caricapryl-99 seeds extract (10, 50 and 150mg/kg/d) repeated treatment daily for 3 days caused a significant (p<0.05) decrease in the serum level of testosterone in male rats. The level of decrease was dose related (Table 1).     Luteinizing hormone (LH) serum level  Repeated treatment of male rats with the alkaloid extract for 3 days caused a dose related decrease in the serum level of LH (table 1). The level of decrease was statistically significant (p<0.05).  Estrogen serum level The alkaloid extract of (10, 50 and 150mg/kg/d) caused an increase in the serum level of estrogen in male rats. Doses of 10 and 50 mg/kg/d administered daily for 3 days caused a sharp rise in the serum level of estrogen (Table 1). Whereas the highest dose of 150 mg/kg/d induced gradual increase (Table 1).   Follicle-stimulating hormone (FSH) serum level Pretreatment with the alkaloid extract daily for 3 days caused an increase in the serum level of FSH in males compared to control (Table 1). The increase in the serum level of FSH in male rats was statistically significant (p<0.05).  Discussion  The results of the study revealed a decrease in the serum level of testosterone. This observation was similar to the earlier findings of Udoh et al, 2005c; Udoh and Kehinde, 1999; Udoh and Ekpenyong, 2001. In their report dietary intake of the seed extract caused reduction in body weight gain compared to control, testicular atrophy, spermatogenesis arrest and degeneration of spermatozoa. The reduction in the serum level of testosterone could probably be due to the decrease of serum levels of LH/ICSH observed in this investigation. Leydig cells secrete testosterone by the stimulatory effect of LH (Udoh and Udoh, 2005b; Udoh et al, 2005a; Udoh et al, 2005c). In males, reduction of testosterone level might impaire spermatogenesis and cause male infertility. This study further observed a dose dependent increase in the estrogen serum level. This increase might probably be due to the conversion of testosterone to estrogen (Carr and Blackwell, 1993; Chinoy and Padman, 1996).      TABLE 1: Effect of Caricapryl-99 Seeds extract on Sex hormones levels and pituitary gonadotrophins in male albino rats  Groups Testosterone (ng/ml) LH/ICSH (µIu/ml) Estrogen (pg/ml) FSH (µIu/ml) 10mg/kg (5) 1.68 + 0.86 0.60 +0.01 81.6 +0.02* 1.73 +0.03* 50mg/kg (5) 1.33 +0.16* 0.43 +0.01* 60.0 +0.01* 2.00 +0.7* 150mg/kg (5) 0.96 +0.3* 0.33 +0.07* 43.33 +0.05* 0.46 +0.03* Controls (5) 2.53 + 0.39 1.27 +0.03 26.6 +0.10 0.96 +0.03 Values are expressed as mean + SEM. Number in parenthesis represents number of animals for treatment group, *p<0.05, t-test.  References Chinoy, R. J. and Padman, P. (1996). Antifertility investigation and benzene extract or Carica papaya seeds in male albino rats. J Med. Aromatic Plant Sci. 18 (3): 489 – 494. Carr, B. R. and Blackwell, R. E. (1993). Neuroendocrinology of reproduction in Bruce R. Carr and Richard E. Blackwell (eds), Textbook Reproductive Medicine (pp.157). Norwalk: Appleton & Lange. Udoh, P., Essien, I. and Udoh, F. (2005a). Effect of Carica papaya (paw-paw) seeds extract on the morphology of pituitary-gonadal axis of male albino rats. Phytotherapy Res. 19: 1065-1068. P. B. Udoh et al 14  Udoh, F. V. and Udoh, P. B. (2005b). Hepatotoxicity of the methanol extract of C. papaya seeds in Wistar Rats. Pharmaceutical Biol. 43(40): 349-352. Udoh, F. V., Udoh, P. B. and Umoh, E. E. (2005c). Activity of alkaloid extract of carica papaya seeds on reproductive functions in male wistar rats. Pharmaceutical Biol. 43(6): 563-367. Udoh, F. V. (2007). The effects of Gnetum africanum leaf extracts on some biochemical indices and endocrine functions in albino rats. A Ph.D. Thesis, University of Calabar, Calabar, Nigeria.  Udoh, P. and Ekpeyong J. (2001). Effects of Mucuna urens (Horse eye bean) on the gonads of male Guinea pigs. Phytopher. Res. 15: 99-102. Udoh, P. and Kehinde, A. (1999). Studies on Antifertility effects of pawpaw seeds (Carica papaya) on the gonads of male Albino rats. Phytother. Res. 13. 226-228. United Nation, Department of Int. Econ. & Soc. Affairs (1989) World Population prospects 1988. Population studies No. 106 UN New York of Neem: A tree for solving global problem. National Academy: Washington D. C. 1992. WHO (1996) Task Force on methods for the regulation of male Contraceptive efficacy of testosterone and oligospermia in normal Men. Fertility and Sterility. Int. J. Androl. 65 821-29.        Caricapryl-99 seed extract and male sex hormones 15 

Effect Of CARICAPRYL-99 Seed Alkaloid Extract On The Serum Levels Of Sex Hormones And Pituitary Gonadotrophins In Male Albino Rats

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Effect Of CARICAPRYL-99 Seed Alkaloid Extract On The Serum Levels Of Sex Hormones And Pituitary Gonadotrophins In Male Albino Rats

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