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Effects of Withania Coagulans Extract and Morphine on Spermatogenesis in Rats

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Effects of Withania Coagulans Extract and Morphine on Spermatogenesis in Rats Noori et al Tropical Journal of Pharmaceutic al Research April 201 9 ; 1 8 ( 4 ): 817 - 822 ISSN: 1596 - 5996 (print); 1596 - 9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.or g http://dx.doi.org/10.4314/tjpr.v1 8 i 4 . 19 Original Research Article Effects of Withania coagulans extract and morphine on spermatogenesis in rats Ali Noori 1 *, Leila Amjad 1 , Fereshteh Yazdani 2 1 Department of Biology, 2 Young Researchers and Elite Club, F alavarjan Branch, Islamic Azad University, Isfahan, Iran *For correspondence: Email: [email protected]; Tel: +98 - 913169918; Fax: +98 - 0381 - 3330709 Sent for review : 2 5 September 201 7 Revised accepted: 11 March 201 9 Abstract Purpose : To investig ate the comparative effects of Withania coagolans extract and morphine on spermatogenesis in rats Methods: W. coagolans was collected from Sistan and Baluche stan, Iran and 50 and 100 mg/kg body weight doses of methanol extract and 5, 10 and 15 mg/kg body w eight doses of morphine were administered parenterally to the rats which were divided into groups. Blood sampl es were collected and the level s of luteinizing hormone ( LH ), follicle stimulating hormone (FSH), and testosterone were assayed. The testicular ti ssue was isolated for histopathological examination. Results: No significant changes were observed in levels of LH, FSH and testosterone in treated groups (p < 0.05). However, there was significant difference between the treated groups for extract plus mo rphine groups, in terms of the number of spermatogonium, spermatocytes and spermatide variation. Moreover, the results indicate tissue disorders in all groups relative to control. The extract caused more disturbances in spermatogenesis compared to morphine , and appears to improve param e ters related to s permatogenesis. Conclusion: The results show that the higher dose of Withania coagolans extract (100 mg/kg) exerts varying effects on reproductive parameters. Moreover, the lower dose of Withania coagolans e xtract (50 mg/kg) enhanced spermatogenesis while also protecting against the damaging effects of morphine. Keywords: Morphine, Spermatogenesis, Spermatogonium, Spermatide, Spermatocyte, Withania coagolans This is an Open Access article that uses a fund - ing model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://c reativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestop enaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopu s, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline Internati onal, Open - J - Gate and Pharmacy Abstracts INTRODUCTION hypothalamus, also, their function includes hypophysis secretions in the testis [2]. Morphine Addiction is a reversible chronic disorder that is is used clinically to control severe pain, h owever, spreading among different communities around it has many side effects, such as vomiting, the world [1]. Opium comes from Papaver nausea, constipation, tolerance, and rhoeas , which includes at least 20 alkaloids, one dependence [2]. Moreover, morphine reduces of which i s morphine alkaloids. Opium acts stages of spermatogenesis through inhibiting through drug receptors. These receptors are luteinizing hormone (LH). Male fertility r equires dispersed in limbic system, thalamus and normal spermatogenesis. ---------------------------------------------------------- -------------------------------------------------------------------------------------------817 © 201 9 Noori A, Amjad L, Yazdani F . This work is licensed under the Creative Commons Attribution 4.0 International License Noori et al LH an d follicle - stimulating hormone (FSH) are ratio for preparing concentrations of 50, 100 involved in the endocrine mechanism through act mg/kg body weight. on the testosterone testosterone produced by the leydig cells [3]. Thus, a combination of Preparation of morphine testosterone, LH and FSH activity are needed for stimulating spermatogenesis. LH and FSH are Morphine was prepared in normal saline at doses glycoprotein hormones, called gonadotropins, of 5, 10 and 15 mg/kg body weight of rat. that modulate gonadal functions [4]. LH affects on leydig cells and causes to produce testis Animals steroids. On the other hand, FSH is the main motivator for growth of testis seminiferous Adult male Wistar rats (200 - 250 g) were tubules [5]. Moreover, spermatogenesis depends obtained from Iran Pasteur Institute and divided on the function of testosterone that is required for into 6 groups of eight animals each (48 rats). initiating spermatogenesis at maturity and the They were maintained under controlled maintenance of this process in the adult [6 ]. temperature (22°±1°C), with 12 h light/12 h dark conditions for 1 week before the start of the With regard to the harmful effects of chemical ex periments for adaptation to laboratory drugs for the treatment of a ddiction, the use of conditions. The procedures in this study were herbal medicines is considered [1]. Withania carried out in a accordance with the institution's coagulans Dual. belongs to family Solanaceae. scientific procedures for animals and was This plant is a general medicinal plant in many approved by the Institutional Animal Care and parts of Indai and Pakistan. Phytochemical Use Committee (IACUC) [8] . This study was research of the W. coagulans aqueous and approved by the Medical Research Ethics methanol fru its extracts showed the presence of Committee of the Islamic Azad University of alkaloids, steroids, phenolic compounds, tannins, Falavarjan Branch (approval no. saponins, carbohydrates, proteins, amino acids IR.IAU.FALA.REC.1397.025). and organic acids [7]. This plant is rich in withanolide that is a group of alkaloids. The The animals were randomly divided into 6 groups investigation on W. coagulans re vealed that it (n= 10 per group) and received intraperitoneally known as to treatment of ulcers, rheumatism injec tion. These groups include control group that bronchitis, nervous exhaustion, disability, received normal saline, morphine treated group insomnia, failure in children thrive and impotence (5, 10 and 15 mg/kg), morphine (5, 10 and 15 [7]. With regard to the harmful effects of mg/kg) plus extract (50 mg/kg), morphine (5, 10 morphine on spermatogenesis the aim of this and 15 mg/kg) plus extract (100 mg/kg), extract study was there fore to compare the effects of W. treated groups (50 and 100 mg/kg), respectively. coagulans extract and morphine on The animals were treated with morphine (groups spermatogenesis in rats. 2, 3, 4) once daily for 8 days (the first 2 days with concentration of 5, the second 2 days with EXPERIMENTAL concentration of 10 and the third 4 days with concentration of 15 mg/kg) then, 2 h af ter Collection of plants treatment with morphine they were treated with extract (groups 3, 4) once daily for 6 days to The roots of Withania coagulans were collected evaluate the therapeutic effect of the extract on in Iranshahr County, Sistan and Baluchestan of morphine disorders [9]. province, Iran, in May 2013. A v oucher specimen was deposited in a herbarium of the Research - The animals were anesthetized with ketamine Institute of Isfahan Fores ts and Rangelands by injections and blood samples were taken directly Dr. Feizi. from the heart one day after the last injection of extract. The hormones LH, FSH, testosterone Preparation of extract were assayed using autoanalyzer (902 Hitachi Automatic Analyzer, Roche, India). T he rats were The roots of W. coagulans were air - dried under sacrificed by cervical dislocation and their testes shade and ground to fine powder using electric were rapidly removed and testicular tissue was blender, then, 60 gr of root powder were fixed in 10 % formalin, dehydrated in ethanol, extracted with 200 mL methanol by soxhlet cleared in xylene and embedded in paraffin. extraction for 12 h. The residue was vaporized Sections were prepared and then stained with using a rotary evaporator. The dried extracts (8 Hematoxylin - Eosin (H&E) for observe of gr) were stored stored at 4 °c until used. The testicular tissue disorders and countin g of extracts were dissolved in phosphate buffered different cells (spermatogonium, spermatocyte I s aline and tween and tween 80, at a 1:4 weight and spermatide) (Olympus, Japan). To determine Trop J Pharm Res, April 2019; 18(4): 818 Noori et al the number of sperms in the epididymis, the tissue, cell apoptosis, incomplete cell division epididymis tail was removed. Then, for the and the abnormal cells. These changes were release of the sperms, the epididymis tail was cut more in the groups treated with morphine, into 2 ml of 37 °C physiological serum and morphine plus extract 100 mg/kg and extract 100 sperms were diluted to (1:20) with normal saline. mg/kg compared to other groups (Figure 1). Finally, 10 μl was placed on a neobar lam, and the total number of sperms as well as the DISCUSSION percentage of sperm motility was counted. According to the results of this study, the levels Statistical
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