Vol 13, Issue 5, 2020 Online - 2455-3891 Print - 0974-2441 EVALUATION OF IN VITRO HEPATIC TOXICITY OF LEAVES OF PTEROSPERMUM ACERIFOLIUM (L.) WILLD. RANA DATTA 1 , SANKHADIP BOSE 2 , SUDIP KUMAR MANDAL 3 * 1 Department of Pharmacology, Gupta College of Technological Sciences, Asansol, West Bengal, India. 2 Department of Pharmacognosy, Bengal School of Technology, Hooghly, West Bengal, India. 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dr. B. C. Roy College of Pharmacy and Allied Health Sciences, Bidhannagar, West Bengal, India. Email: gotosudip79@gmail.com Received: 31 January 2020, Revised and Accepted: 18 March 2020 ABSTRACT Objective: The objective of the study was to determine the in vitro hepatic toxicity profile of methanolic extract of leaves of Pterospermum acerifolium (L.) Willd. (MEPA) using a mammalian hepatic cell line (HepG2). Methods: To assess its in vitro hepatic toxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-2,5-diphenyltetrazolium bromide assay using MEPA at a concentration of 25 µg, 50 µg, 100 µg, 200 µg, and 300 µg was carried out. Sorafenib tosylate was used as the standard agent to assess cytotoxicity. Results: The inhibitory concentration 50 (IC 50 ) value for HepG2 cell lines was determined after 24 h. Thereafter the cytotoxicity study was again performed with the ½ IC 50 , IC 50 , and 2IC 50 doses of MEPA. Experimentally, the IC 50 was found to be 150.42 µg/ml for HepG2 using MEPA. A dose- dependent cytotoxicity and reduction in optical density were also observed with incremental MEPA administration. Conclusion: The cytotoxic potential of MEPA was found to be much less than that of sorafenib tosylate. Thus, MEPA shows in vitro cytotoxicity in mammalian hepatic cells (HepG2) at a concentration as low as 100 µg. Keywords: Pterospermum acerifolium, Cytotoxicity, In vitro, Cell line, Hepatocarcinoma. INTRODUCTION Demand for herbal medicine has increased tremendously in recent years; however, there many issues regarding their safety. Very less (<10%) marketed herbal products are actually standardized. Furthermore, quality control measures are not followed on many occasions [1]. Even in the literature, very little toxicological information is available. Since some plants produce toxic constituents for defense purposes, it becomes absolutely necessary to explore the safety profile of herbal drugs. The plant Pterospermum acerifolium (L.) Willd., belonging to the family Sterculiaceae, is used by the tribals of Chotanagpur, Konkan, and Arunachal Pradesh region of India, for the treatment of different diseases such as wound healing and hemostatic activities [2,3]. However, no toxicological data are available for the plant. Preliminary phytochemical studies showed the presence of alkaloids, flavonoids, and glycosides in the methanolic extract of P. acerifolium (L.) Willd. (MEPA). Conventionally, the presence of alkaloids has been implicated to cytotoxicity; thus, it is worth to elucidate the toxicological profile of MEPA both from therapeutic and toxicological standpoint. Herbal medicines are normally considered safe, but chronic administration may lead to cumulative toxicity. Thus, it is equally important to evaluate P. acerifolium (L.) Willd. for its toxicological profile. In vitro toxicity testing is frequently done on mammalian hepatic cell line HepG2 [4]. HepG2 through a cancer cell line is frequently used to assess in vitro toxicity. It was first derived from a 15-year-old Caucasian American male. In vitro studies offer additional advantages over the traditional animal models on many aspects. It allows a species-specific, simpler, and more detailed analysis [5]. In vitro studies, if designed well, can very well replace whole animal studies in days to come. The present study was undertaken to elucidate the in vitro toxicological implications (if any) on HepG2 cell lines, to establish the safety profile of MEPA. METHODS Preparation of extracts The leaves of P. acerifolium were collected from Asansol, West Bengal, in the month of September 2013 and 2014 at 11 a.m. The plant was identified and authenticated as P. acerifolium (L.) Willd. by the Director, Acharya Jagadish Chandra Bose Indian Botanic Garden, Shibpur, Howrah, India. The leaves of P. acerifolium were dried in the shade of about 30°C and crushed into a coarse powder. Cell culture Hepatocellular carcinoma cell line HepG2 was purchased from National Facility for Animal tissue and cell culture, Pune, India, and supplied from Indian Institute of Chemical Biology for in vitro studies. Hepatoma cells were subcultured after every 2 days at an initial concentration of 1×10 6 cells/ml and maintained in sterile Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum [5]. The culture was maintained at 37°C in a humidified atmosphere containing 5% CO 2 in the air [6]. During subculturing or during the use of HepG2 in experiments, this adherent property has been diminished by adding ×1 Trypsin solution in the cell [7]. In all the experiments, untreated hepatic cells were used as a control group. Preparation and extraction of the tested plant sample Leaves of P. acerifolium (L.) Willd. were air-dried in the shade. The dried leaves were extracted with methanol, were crushed, and then extracted by continuous hot extraction process for 72 h in Soxhlet apparatus, using a reflux condenser. Then, the solvent was removed by filtration. Fresh solvent was added and further extracted for 3 h. The extract was concentrated by vacuum under reduced pressure. Thereafter, the extract was lyophilized for 4 h to produce methanol free extract. It was kept in a container, sealed with parafilm and stored at 4°C in an airtight container, and was designated as MEPA. Stock solution was prepared as 1 mg/ml in phosphate buffer saline (PBS) from which desired doses were tested. © 2020 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.2020.v13i5.36998 Research Article