Vol 9, Issue 6, 2016 Online - 2455-3891 Print - 0974-2441 EFFECT OF UNSAPONIFIABLE FRACTION OF SEEDS OF HYGROPHILA SPINOSA T. ANDER ON TESTOSTERONE PRODUCTION OF RAT LEYDIG CELLS IN VITRO NIRAJ VYAS*, MANAN RAVAL Department of Pharmacognosy, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Anand - 388 421, Gujarat, India. Email: nirajvyas4me@gmail.com Received: 12 July 2016, Revised and Accepted: 16 July 2016 ABSTRACT Objective: Seeds of Hygrophila spinosa (HS) T. Ander (Acanthaceae) are traditionally used as aphrodisiac and spermatogenic in Indian System of Medicine. Preliminary phytochemical screening of plant revealed the presence of triterpenoids and sterols in seeds. The study was planned to assess the effect of unsaponifiable fraction prepared from seeds of HS on isolated rat Leydig cells for testosterone (T) production using in vitro method. Methods: Leydig cells were isolated from Wistar rats, aseptically, in vitro by collagenase cell dispersion method. Cells (2×10 6 cells/ml) were then incubated with a unsaponifiable fraction of HS (10, 100 and 1000 µg/ml dose levels in triplicate) in an incubator at 37°C under atmosphere of 95% CO 2 condition for 3 hrs in aseptic condition. Dehydroepiandrosterone was used as positive control in the study. The amount of testosterone secreted in culture media was estimated using high performance thin-layer chromatography (TLC). Benzene: Ethyl acetate (5:5% v/v) was employed as mobile phase and silica gel G F 254 aluminum coated TLC plate as the stationary phase. Results: The results indicated dose-dependent increase in testosterone concentration in test groups. Isolated rat Leydig cells treated with the test fraction showed increased amount of testosterone present in culture media as compared to that of control. Conclusion: Unsaponifiable fraction prepared from seeds of HS showed ability to enhance biosynthesis of testosterone in isolated rat Leydig cells. In vitro studies showed that the fraction might act locally in testis on Leydig cells and stimulated testosterone synthesis. Keywords: Aphrodisiac, Spermatogenic, Hygrophila spinosa, Testosterone. INTRODUCTION The plant Kokilaksha is known as H. spinosa (HS) in Sanskrit literature belonging to Acanthaceae family and its seeds have been used in Ayurvedic preparations as Vajikarana Aushadhi, i.e., aphrodisiac and spermatogenic [1-3]. The plant has shown many pharmacological activities such as cooling, tonic, spermatorrhea, aphrodisiac, and spermatogenic [4]. Phytochemical study of this plant showed that seeds contain mucilage, sterols, unidentified alkaloids, fatty acids, minerals, and carbohydrates [5-12]. Literature survey revealed that this plant is not systematically screened for the claimed activity. Hence, unsaponifiable fraction prepared from seeds of HS was screened for testosterone production and release by in vitro method using isolated Leydig cells from male Wistar rats. METHODS Reagents and chemicals All the solvents and chemicals used were of analytical grade and procured from Loba Chemicals, India. Materials and media used for in vitro studies were purchased from Sigma-Aldrich, USA, Acros Organics, India and Hi-media, India. Plant materials Whole plants of HS were collected in August from tribal area of Anand district, Gujarat, India. The plant samples were identified by taxonomist at J and J Science College, Nadiad, Gujarat, India. The specimen of the collected plant material was submitted to the department for future reference with specimen number 2011/NV/HS. Seeds were separated from the plants, dried under shade and milled using laboratory grinder to 70# powder. This powder was used for further extraction process. Preparation of unsaponifiable fraction Dried seed powder (4 kg) was extracted using 5000 ml of hexane (00160, Loba Chemie) in Soxhlet’s extraction apparatus at 60°C for 48 hrs. Hexane extract was filtered and refluxed with sufficient quantity of 10% potassium hydroxide (05378, Loba Chemie) in methanol (00196, Loba Chemie) for saponification purpose. The content was removed and mixed with equal amount of water. The content was then partitioned with solvent ether (001040, Loba Chemie) to separate unsaponified matter. Ethereal extracts were pooled and passed through anhydrous sodium sulfate to remove moisture present. All ethereal portions were mixed together and evaporated to dryness using rotary vacuum evaporator (Heidolph, Germany) at 30°C. The yield of unsaponifiable fraction was determined and found to be 1.2% w/w. The fraction was then subjected to TLC studies for detection of sterols and triterpenoidal compounds present. Detection of sterols and triterpenoidal compounds The unsaponifiable fraction was evaluated for the presence of sterols and triterpenoidal compounds using TLC. Post chromatographic derivatization was performed using Liebermann-Burchard reagent [13]. Optimized mobile phase employed to separate these compounds on silica gel coated TLC plates (silica gel G 60 F 254 , Merck) was hexane:ethyl acetate:methanol:glacial acetic acid (6:4.5:0.5:0.2 v/v/v/v) with saturation time of 10 minutes. Animals Protocols for in vitro studies were approved by the Institutional animal Ethics Committee (IAEC) constituted as per the norms of Committee for the Purpose of Control and Supervision of Experiments on Animals. The protocol numbers assigned were RPCP/IAEC/2013-14/R29, RPCP/IAEC/2011-12/R7 respectively. Healthy male Wistar rats of weight 250-350 g were used in the experiments. Rats were received Research Article © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2016.v9i6.14049