The comparative effects of methanol extract of Phyllanthus amarus leaves and Vitamin E on the Sperm parameters of Male guinea pigs

The effects of the methanol extracts of the leaves of Phyllanthus amarus on the sperm parameters of male Guinea pigs were investigated. The effects of Vitamin E on the sperm parameters were also investigated and compared to that of Phyllanthus amarus. The phytochemical screening of the leaves of Phyllanthus amarus was also carried out. The methanol extract of the Phyllanthus amarus leaf (50-200mg/kg) caused a decrease in the sperm count and motility, from 65.0± 2.80 and 66.7±0.33 to 46.0± 2.10 and 37.7± 1.50 respectively. However, this effect was less pronounced at higher doses of Phyllanthus amarus (400 and 800mg/kg). At 400 and 800mg/kg, P. amarus caused a decrease in sperm motility and count from 65.0±2.50 and 66.7± 1.5 to 52.7± 0.50 and 56.7± 0.80 : to 43.3±1.70 and 48.3±1.70 respectively . These effects were dosedependent and comparable to the observed effects of Vitamin E (500IU) on sperm parameters of male guinea pigs. These were significant at P < 0.05 (ANOVA). In timedependent study, the observed effect of P. amarus (800mg/kg) and Vitamin E on the values of sperm count and motility at 28 day were almost the same. These values are 56.7± 3.30 and 48.3± 1.70 for vitamin E; 47.5±2.50 and 47.5± 2.50 for P.amarus respectively for sperm motility and count. These effects were timedependent and statistically significant at P < 0.05 (ANOVA). Finally, the phytochemical screening of the leaves of Phyllanthus amarus revealed the presence of flavonoids, tannins, alkaloids, terpenoids, steroids, saponins and cardiac glycosides. This shows that the leaves of P .amarus do not largely contribute direct to the claims on the use of the aerial part of this plant by traditional medicine practitioners to increase/improve libido in men but it may have some indirect beneficial effects on this claim (like antioxidant activity) that mimic Vitamin E in both action and mechanism of action. However, further studies need to be done to investigate the contribution of the seeds of this plant in the improvement of libido in men and to isolate and characterize the active principles in the extracts of this plant. @ JASEM Phyllanthus amarus Schum (Family Euphorbiaceae) is a widely distributed small erect, tropical annual herbal shrub whose stem has green capsule, and and grows upto10-50cm high and blooms with flowers with 5 white sepals and apical acute anther. The fruit has green capsules,and smooth and fruiting pedicels. The seeds are longitudinally rugose. It is locally called Iyin-olobe (Yoruba, southwest Nigeria) (Adeneye et al, 2006) or kidney stone plant. In Traditional medicine, it is used for its hepatoprotective , antidiabetic, antihypertensive, analgesic, anti-inflammatory and antimicrobial properties (Adeneye et al., 2006).The plant is also used in the treatment of stomach disorders, skin diseases and cold (Kokwaro, 1976, Iwu,1993). It has antidiarrheal effect (Odetola and Akojenu 2000). Its antiviral activity against hepatitis B virus has been established (Thyagarajan et al., 1988; Meixa et al 1995); anticarcinogenic(Joy and kutten, 2000) and antimutagenic activities (Joy and Kutten, 1998). It also has antinociceptive and antiinflammatory activities (kassuya et al, 2003), antidiabetic and antilipidemic potentials (Adeneye et al ,2006). So far, there is no report on the effects of Phyllanthus amarus on sperm parameters, such as sperm count, motility and morphology. Also there is claim on the use of aerial part of Phyllanthus amarus to improve libido or fertility in men, by traditional medicine practitioners. Based on this claim, this study came up to establish, for the first time, a scientific information on the effectiveness of Phyllanthus amarus as fertility agent by its effects on the sperm parameters of male guinea pigs. And also to establish the contribution of the leaves of Phyllanthus amarus to this effect. MATERIALS AND METHODS All the chemicals used were of analar grade. Plant Material The leaves of Phyllanthus amarus were collected from the local garden within the premises of University of Port Harcourt in June 2008. The plant was identified and authenticated by Edwin Nwosu of department of Botany herbarium, University of Port Hacurt .Voucher specimen was maintained at the Herbarium. The fresh leaves collected were airdried for 10days, until a constant weight was attained. Preparation of Extract The dried leaves of Phyllanthus amarus were pulverized (100g). The crude drug was extracted with methanol using Soxhlet extraction method. The solid residue obtained was kept in a capped container in a refrigerator. Different concentrations of the extract were reconstituted from this stock. The comparative effects of methanol extract of Phyllanthus amarus leaves and Vitamin E on the Sperm parameters of .............. * Corresponding author: Obianime, A.W. 38 Experimental animals The male guinea pigs were collected from the animal house of University of Port Harcourt. The weight of the animals ranges from 300-600g. The animals were allowed to acclimatize with the new environment (7) before the experiment. They were housed in a cage of five animals per cage and were adequately feed throughout the experiment. METHOD Phytochemical screening Chemical tests were carried out on the methanolic extracts and on the powdered specimens using standard procedures to identify the constituents (Trease and Evans, 1989; Harborne, 1973) by characteristic colour changes as described by Sofowara, (1993); Odebedy and Sofowara, (1978). Sperm count Semen was diluted 1: 2 using micropipette in test tube with diluting fluid to immobilize and preserve the sperm. The Neubauer chamber (Heamocytometer) was prepared and charged with the diluted seminal fluid and allowed to stand in a moist chamber for 15-20 minutes to allow spermatozoa to sediment to the grid of the counting chamber and counted with 40x objective of light microscope. Motility A drop of undiluted liquefied and well mixed semen was placed on a clean slide and covered with a cover slide and then examined under a light microscope to stimulate both quantitative and qualitative motility. Sperm motility was determined by counting all motile and immotile spermatozoa, in randomly chosen fields using a 40x objective of light microscope. Morphology Smears of semen suspension were made on clean slide and quickly fixed with 95% alcohol. The smear was then stained with dilute (1 : 20) carbol. Fuchsin and examined under the oil immersion objective. 100-300 cells were counted and the v/v of abnormal forms were noted. Semen was also examined for the presence of particulate debris. Excessive contamination of the seminal fluid sample by epithelial cells, red blood cell, white blood cells and immature germ cells were observed and quantified. RESULTS AND DISCUSSION Fig.1: The dose-dependent comparative effects of P. amarus and Vitamin E on sperm motility of male guinea pigs. 0 10 20 30 40 50 60 70 80 sp er m m ot ili ty control value for motility