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International Organization of Scientific Research (IOSR)
 

International Organization of Scientific Research (IOSR)

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The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications ...

The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.

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    International Organization of Scientific Research (IOSR) International Organization of Scientific Research (IOSR) Document Transcript

    • IOSR Journal Of Pharmacy (e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219 Www.Iosrphr.Org Volume 3, Issue 8 (September 2013), Pp 24-28 24 The Effects of Extracts of Neem (Azadirachta Indica) Leaves, On Sperm Count and Sperm Motility of Wistar Rats. Ndodo N.D 1 , Esomonu U.G3 , Onu J.E4 , Okolo R.U 1 , Onwuchekwa C5 , And Anuka J.A2 Department of Human Anatomy1 , Department of Physiology ,5 College of Health Sciences, and Department of veterinary Anatomy4 Usmanu Danfodiyo University, Sokoto, Nigeria. Department of Clinical Pharmacology2 , Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria. Department of Human Anatomy3 , Faculty of Medicine, Bayero University,Kano. ABSTRACT :The effects of water and methanol extracts of Neem (Azadirachta Indica) leaves were studied in male Wister rats following treatment with 20 % w/w and 30% w/w equivalent of water and methanol extracts of Neem leaves incorporated into rat diet and administered orally for 90 days. The control group had normal diet and water ad libitum. Sperm count and motility were evaluated using caudal epididymal sperms. The sperm counts (106 /ml) for the control group was 38.9 ±1.96 while for the 20% w/w water extract of Neem leaves group and 30% w/w water extract of Neem leaves treated group were 38.2 ±1.96 and 40.0 ±1.96 respectively and those of 20% and 30% w/w equivalent of methanol extract treated groups were 38.2 ±1.96 and 39.8 ±1.96 respectively. The values for the sperm motility (%) was 74 ± 2.66 for the control group and 29 ± 2.66, 21 ±2.66, 15 ±2.66, 10 ±2.66, for the 20% and 30% w/w equivalent of water and methanol extracts of Neem leaves treated groups, respectively. The results show that sperm motility was significantly reduced in the treated groups than the control group (P<0.001) while there was no significant variation between the sperm counts of the control and the treated groups. The results suggest a possible antifertility activity in Neem leaves extracts. KEY WORDS: Neem leaves, Sperm counts, Sperm motility, Anti-fertility I. INTRODUCTION Azadirachta indica (neem) tree is regarded as a “tree for solving global problem (National research council, 1992). Almost all parts of the tree are of immense therapeutic importance. Azadirachta indica is one plant with multifunctional properties. Indian researchers have documented more than 60 medicinal uses of Neem. The immunomodulatory activities of neem have been reported (Labadie et al, 1989). The antimicrobial, anti-inflammatory and antipyretic properties have also received attention (Okpanyi and Ezeukwu, 1981). Lai et al, (1986) observed the anti -fertility effects of the neem oil. Riar et al (1990) recorded the potential spermicidal effect, while Upadhyay et al. (1990, 1992), highlighted the immuno -contraceptive properties of neem. Neem oil acted as a spermicidal agent and inhibited sperm motility (Riar et al, 1990; Sharma et al, 1996) Neem-oil has been shown to have anti-fertility activity and to stimulate cell mediated immune response. Immunocontraceptive properties, anti-implantational as well as abortificient effects of neem are well documented (Upadhay et al, 1992, Riar et al 1984, Sinha et al, 1984a, b). A fraction of Neem oil called Nim-76 was said to group only spermicidal activity which makes it suitable for use as pre-coital antifertility formulation for human use, which has undergone phase one clinical trials (Riar et al, 1991, Sai Ram . et al, 2000) Mukherjee and Talwar, (1996) reported that the neem seed extracts, could completely abrogate pregnancy in rodents at an early post implantation stage. The treatment did not have any residual effects on future fertility of the animals. The effects of the extracts on the implantation were due to a transient increase in cytokines Y- interferon and TNF (Tumour Necrosis Factor) after the treatment. Udeinya et al, (2004) demonstrated in-vitro, the broad spectrum anti-cyto-adhesion activity of fractionated acetone-water extract of Neem leaf, known as IRAB, believed to be beneficial in HIV/AIDS, using malaria-infected erythrocytes and metastatic cancer cells. Later, Mbah et al, (2007) reported that IRAB showed safer medicinal properties in clinical trials. This current study evaluated the effects of the Water and methanol extracts of neem leaves on the male reproductive tracts with respect to semen analysis.
    • The Effects Of Extracts Of Neem… 25 II. MATERIALS AND METHODS 2.1.Extraction of Plant Material Water Extraction 1000g of powdered Neem leaves were dissolved in distilled water in a soxhlet apparatus; the liquid extract was further concentrated in a rotary evaporator to yield a solid aqueous extract weighing 30g. 2.2.Methanol Extraction Using Soxhlet extractor, 1000g of powdered Neem leaves were extracted in methanol to yield 20g of dry, concentrated extract with the aid of rotary evaporator. 2.3.Animal Treatment Procedure Twenty-five male Wistar rats of proven fertility weighing between 200g-250g, were used. They were housed in a Perspex cage with stainless steel-mesh tops, kept in the animal house of the Human Anatomy department, ABU. The rats had free access to food and water and were maintained in standard environmental conditions. The rats were fed growers mash and provided with water ad libitum. The rats were divided into 5 groups, A, B1, B2, C2 and C2. Group A is the control group while groups B to C were treatment groups. Group A: this is the control group consisting of 5 male rats. This group received normal diet and water. Group B: this group was treated orally with Water extract of Neem leaves. B1 received 20% w/w equivalent of water extract of Neem leaves mixed with the feed; for 12 weeks while B2, received 30% w/w equivalent of the aqueous extract mixed with the feed for 12 weeks. Group C: This group was treated orally with methanol extract of Neem leaves. C1 group received 20% w/w equivalent of methanol extract of Neem leaves mixed feed for 12 weeks. C2 group: received 30% w/w equivalent of methanol extract of Neem leaves mixed with feed for 12 weeks. Each group consisted of 5 wistar rats. 2.4.Estimation of Cauda epididymal sperm count The caudal epididymis is removed from the testis and teased inside 1ml normal saline in a Petri dish as described by Kempinas and Lamano cavalho (1988). 2.5.Estimation of Percentage Sperm Motility A drop of the incubated cells was placed on a clean slide using a Pasteur pipette and then covered with cover-slip, the condenser Iris sufficiently closed to give good contrast. Several microscopic fields were examined. The approximate percentages of the sperm motilities were recorded. 2.6.Estimation of Morphology A thin smear of the incubated semen on a slide was fixed with 95% v/v ethanol for 5-10 minutes, and allowed to air-dry. The smear was washed with sodium bicarbonate-formalin solution, to remove any mucus which may be present. The smear was rinsed with several changes of distilled water. It was then covered with dilute (1 in 20) carbol fuchsin and allowed to stain for 3 minutes. The stain was washed off with distilled water, and counter stained by covering the smear with dilute (1: 20) loeffer‟s methylene blue for 2 minutes. The stain was washed off with distilled water, drained and allowed to air-dry. The viewing was under X40 magnification after smearing the slide with oil. III. RESULTS 3.1.Neem on Sperm Count The effect of Water and Methanol extracts of neem leaves on sperm counts of Wistar rats is shown by fig.1 Analysis shows that there was no significant variation between control and treated group, at p<0.05
    • The Effects Of Extracts Of Neem… 26 Fig.1 shows the effect of water and methanol extracts of Neem leaves on sperm count of wistar rats, following oral administration for 90days.There was no significant difference between the control and treated group. P>0.05 36 38 40 42 44 A B1 B2 C1 C2 Groups Sperm count (109 /L) s pm .count Sperm motility The effect of Neem leaf extracts on sperm motility is shown in Fig.2. Results show that there was highly significant variation between control and neem treated groups at P< 0.001. There was no significant difference between means of treated groups. The effect was markedly pronounced in the C2 group (30% w/w equivalent methanol extract group) Fig. 2 shows the effect of water and methanol extracts of Neem leaves on sperm motility of wistar rats.The control group is highly significantly higher than the treated groups at p<0.0001 0 20 40 60 80 100 A(Control) B1 B2 C1 C2 Groups Sperm Motility(%)
    • The Effects Of Extracts Of Neem… 27 IV. DISCUSSION This study was designed to ascertain whether water and methanol extracts of Neem leaves had effects on the fertility of male Wistar rats. In view of the abundance of Neem in the northern region of Nigeria, and the massive dependence of the local populace on this same plant, it is expedient that the role of the Nigerian breed of the Neem should be subjected to fertility evaluation on the male. Furthermore, researchers in Asia have ascribed certain antifertility effect on the Neem oil in the female rats (Sharma et al, 1996, 2001). The extracts also produced no effect on the mean organ weights of Testes, Epididymides, Seminal vesicles, and ventral prostate glands, of Wistar rats at P<0.05. This agrees with the findings of Krause and Adami, (1983) The unaffect mean weight of testes could possibly explain the lack of definite testicular damage that could have inhibited active spermatogenesis particularly during meiosis (Purvis and Hansson, 1981). Since there was no alteration of the testicular milieu, the observed active spermatogenesis is in line with the unaffected mean testicular weight (Krause and Adami, 1983).However; this observation is contrary to the result obtained by Melis, (1999) with extracts of Stevia rebaudiana on rats, where he observed decreased testicular, seminal vesicular and Epididymal weights. This finding was interpreted as an indication of reduced plasma testicular androgens. Testicular weight is said to be related to the number of spermatids and spermatozoa present in the testis (Gupta et al, 2000). Since, there was no testicular weight loss, it is consistent that the spermatids and spermatozoa number were not affected as observed in this study. It equally follows that normal spermatogenesis was maintained in both the water and methanol treated Neem groups. The few and dispersed Leydig cells observed might suggest reduction in testicular androgens which in turn may affect fertility. This effect might not be a direct effect but might be mediated via the Hypothalamic- pituitary-Testicular axis. According to Cunningham and Huckins, (1979) low testosterone concentration may be adequate for maintenance of spermatogenesis. However, no hormonal assay was done to ascertain the role of androgens in the observed fertility reduction in the methanol and water extract treated groups. Nevertheless, the role of testosterone in this case, appears persuasive and overwhelming. It is known that Testosterone stimulates the synthesis of specific Epididymal proteins essential for testicular sperm differentiation; as such spermatozoa released from the Seminiferous tubules would not acquire progressive motility and fertilizing ability during their passage through the Epididymis Male rats following Neem leaf extracts, did not cause reduction in caudal Epididymal sperm count at p<0.05 but rather a drastic reduction in caudal Epididymal sperm motility was observed particularly in C2 group as well as in other treated groups than the control. This effect seem to be dose and extraction solvent dependent. This finding does not support impairment of testicular sperm production but rather a post- testicular effect, may be at subcellular level, such as a possibility that the extract could inhibit the Epididymal fluid absorption or concentration mechanism by vasodilatation (Melis, 1999). Another possibility could be by decrease in Testicular androgens. The Thirty percent weight by weight (30%) w/w equivalent of methanol extract of neem leaves (C2) significantly increased the PCV (p<0.001) of the treated rats. This finding underscores little or no toxicity observed in Neem extracts. This result is at variance with Abubakar (1997) who observed decrease in PCV with increase in dose of Neem leaves extract (data not shown). The B2 group surprisingly was significantly lower than the control (P<0.001).This could not also be explained. However, TLC was not affected(data not shown) contrary to Abubakar, (1997). The maintenance of spermatogenesis in this work is in line with Despande et al (1980). Spermatozoa of the control group showed normal morphology and excellent motility. Late spermatids seen in close association with the Sertoli cells show that androgen binding protein was not disturbed. It was also observed that testicular tissues seemed to undergo degeneration in some fields of the treated groups. This effect could not be explained. It might be due to processing error. This study equally established that methanol extract produced more response to the various parameters under study than water extract. Again that the effect were also dose dependent as 30% w/w equivalent groups showed a better response than 20% w/w equivalent groups in both water and methanol extracts of Neem leaves. The fertility index of the C2 group was seriously reduced when compared to control that had 100% results. V. CONCLUSION Finally these findings point to the possible antifertility effect of methanol and water extracts of Neem leaves on male Wistar rats. REFERENCE. [1] Abubakar, G (1997): „Effect of neem leaf on some biochemical parameters,‟ University of Maidugri, Project report. [2] Cunningham G.R and Huckins C.(1979) Persistence of complete Spermatogenesis in the presence of low intratesticular Testosterone, Endocrinology 105:177-187 [3] Despande VY, Mendulkar KN. Sadre N.K.(1980) “Male antifertility activities of Azadirachta indica in mice” J. postgrad. Med, 26:167-170 [4] Gupta, R.S. Pramod Kumar, V.P. Dexit M.P. Dobhal (2000) “Antifertility studies or the root extract of Baleria priovitis linn in male albino rats. Journal of Ethnopharmacol. 70: 111-117
    • The Effects Of Extracts Of Neem… 28 [5] Katsuri, M. Nazeer, A.R, Pathan, K.M, Parveen D.S, Munivannan B. (1997): Leaves on the seminal vesicle and ventral prostate in albino rats, Indian J. exp. Biol. 34:582-583-590. [6] Kempinas, and Lamano-Carvalho T.I (1988): “A method for estimating the concentration of spermatozoa in the rat cauda epididymis” Lab. Animal (22), 154-156. [7] Krause, W and Adami, M.(1983): “Extracts of Neem (Azadirachta indica) Seed kernel do not inhibit spermatogenesis in the rat”, Proc., pp483-490, 2nd int. Neem. Conf., Rauischholzhausen. [8] Labadie R.P, Vander Nat, J.M, Simons J.M, .Kroes, B.H (1989) An ethno pharmacognostic approach to search for immunomodulatory plants. Plants med. 55, 339-348 [9] Lai R. Sam Karnaryanan, A., Mathur V.S. Sharma P.L. (1986): “ Antifertility effect of Neem oil in female albino rats by Intra vaginal and oral routes” Indian J. med. Res. 83,89-92 [10] Mbah, A. U., Udeinya, I. J., Shu, E. N., Chijioke, C. P., Nubila, T., Udeinya, F., Muobuike, A., [11] Mmuobieri, A. and Obioma, M. S. (2007). Fractionated neem leaf extract is safe and [12] increases CD4+ cell levels in HIV/AIDS patients. Am. J. Ther. 14(4): 369-374. [13] Melis M.S (1999) Effect of chronic administration of Stevia rebandiana on fertility in rats. J. Ethnopharmacol.167: 157-161. [14] Mukherjee, S and Talwar. G.P (1996): Termination of pregnancy in rodents by oral administration of pranneem, a purified neem seed extract. An. J. Reprod. Immuno. 35,57-56. [15] National Research Council (1992): Neem: A tree for slowing global problems. 1st ed., pp30-55, National Academy press, Washington. [16] Okpanyi, S.N and Ezeukwu, G.C. (1981) Anti-inflamatory and antipyretic activities of A. indica. Planta medica, 43:59-63. [17] Purvis K and Hansson, V (1981): Hormonal regulation of spermatogenesis: regulation of target Cell response, Ind. J. Androl 3: 81-143 [18] Riar, S.S, Bardham, J, Thomas, P., Kain, A.K and Prasad R. (1984): Mechanism of antifertility action of Neem Oil, Ind. J. Med. Res 88:339-342 [19] Riar, S.S, Devakumar, C. (1991): Anti-fertility activity of volatile fraction of Neem oil. Conception, sep, 44 (3): 319-326 [20] Riar; S.S, Devkumar, C, Ilavazhagam, G. And Bardham, J. (1990)” volatile fraction of Neem oil as a spermicide.”Contraception (42) 479-487 [21] SaiRam, M., llavazhagan, G., Sharma, S.K, Dhanraj, S.A, Suresh, B., Parida M.M, Jana, A.M, Kumar, D and selvamurthy, W. (2000): Anti-microbial activity of a new vaginal contraceptive Nim-76 from neem oil(A. indica) J. ethnopharmacol. 71:277-382 [22] Sharma R.K, Agarwal A. (1996): Role of reactive oxygen species in male infertility Urology 48: 835-850. [23] Sharma R.K, Pasqualotto F. F, Nelson D.R, and Thomas A.J, Agarwal A. (2001): Relationship between seminal white blood cell counts oxidative stress in men treated at an infertility clinic. J. Androl 22:575-583 [24] Sharma R.S. Rajalakshmi and Jeyaraj D.A.(2001) “Current status of fertility Control methods in India” J.Biosci, 26:395-399. [25] Sharma S.K, Sai Ram m, llavazhamgan G;Devendra K, Shiraji S.S and Selvamurthy W;(1996) “Mechanism of action of Nim-76: a novel vaginal contraceptive from neem oil;” Contraception (54) 373-378. [26] Sinha K.C, Riar S.S, Tiwary, A.K; P(1984): Neem oil as a vaginal contraceptive. Indian Journal medical Research 79: 131-136. [27] Sinha, K.C, Riar S.S, Bardham, J., Thomas P., Kain, A.K, Jain, R.K, (1984): Anti-implantation effect of neem oil, Indian Journal Medical Research. 80:708-710. [28] Udeinya, I. J., Mbah, A, U., Chijioke, C. P. and Shu, E. N. (2004). An anti-malarial extract from [29] neem leaves is antiretroviral. Trans. Royal Soc. Trop. Med. Hyg. 98:453-437. [30] Upadhay S.N. Kaushik S., and Talwar, G.P (1990). Antifertility effects of Neem (Azadirachita indica) oil by single intrauterine administration: a novel method for contraception. Proc. pp 175-179, R.Soc London. [31] Upadhay, S.N, Dhawan, S., Garga, S. and Talwar G.P (1992)” Immunomodulatory effects of Neem (A. Indica) oil” Int. J. Immunopharmacol. 171: 1187-1193