PHYTOCHEMICAL INVESTIGATION RESULTS AND DISCUSSION Anticancer activity-guided fractionation of Trigonella stellata extract Mohamed M. Radwan1,2, Safa M. Shams Eldin1,2 Amira S. Wanas1, Abdel-Azim Habib2, Fahima F. Kassem 2, Hala M. Hammoda2 and Mahmoud A. ElSohly1,3 1National Center for Natural Products Research, 2Department of Pharmaceutics and Drug Delivery , School of Pharmacy, The University of Mississippi, University, MS 38677, USA. 2Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt. 3Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, MS 38677. INTRODUCTION Cancer is one of the most serious diseases in humans and nowadays there is a considerable scientific discovery of new anticancer agents from natural products (1). It is estimated that plant derived compounds constitute more than 50% of anticancer agents (2,3). The major aim of analyzing anticancer activity of the crude plant extracts are either to isolate bioactive agents for direct use as anticancer drugs or to identify bioactive compounds that can be used as lead substance in the preparation of semi synthetic drugs to treat cancer (4). Fabaceae plants, particularly of the genus Trigonella are rich in flavonoids and saponins and some species were used in folk medicine as antitumour agents (5). This study aims at evaluating the invitro anticancer activity of the extract of Trigonella stellata using 3 different human cell lines. The most sensitive cell line giving the highest potency of anticancer activity will then be used in testing the different fractions of Trigonella stellata for determining the most active extracts to be used as a platform for phytochemical –based drug discovery. The anticancer activity of the isolated compounds will be further investigated. IN VITRO ANTICANCER ACTIVITY PHYTOCHEMICAL INVESTIGATION OCH3 H2β H3 H2α H1’’ H3’ H5 H6' H4 H5’ H8 H6 EXTRACTION AND ISOLATION RESULTS AND DISCUSSION Dried alcoholic extract of aerial parts of Trigonella stellata (160 g) Hydroalcoholic fractionation Petroleum ether Fraction (64 gm) Methylenechloride Fraction (12gm) Ethylacetate Fraction (23gm) Butanol Fraction (35 gm) Compound 1 Compound 2 Compound 3 The tested alcoholic extract exhibited different potency of cytotoxic activity using the three different cancer cell lines. The highest potency of the tested extract was recorded using the HEPG2 cancer cell lines (IC50 = 4.7 μg mL-1). Accordingly, the four fractions were tested for their anticancer activity using HEPG2 cancer cell line and the butanol fraction was the most active fraction with IC50 value of 1.5μg mL-1 compared to Doxorubicin (0.4 μg mL-1). Three known compounds (1-3) were isolated and chemically identified as 2-hydroxyphenyl D-glucopyranoside (1), Quercetin 3,7-O-α-L-dirhamnoside (2) and soyasaponin I (3) from the butanol extract. The ethylacetate fraction had a strong cytotoxic activity (IC50 =2.9 μg mL-1) from which two known compounds were isolated and identified as p-hydroxybenzoic acid (4) and 4'-7-dihydroxyflavone (5). In addition, one new compound (6) was isolated for the first time from nature and was chemically identified as 2',4'-dihydroxy-7-methoxyisoflavanone-4'-glucupyranoside. While the petroleum ether and chloroform extracts displayed weaker activity with IC50 values of 4.8 and 10.5 μg mL-1 respectively. Compound 4 Compound 5 Compound 6 (New cpd) Silica gel Sephadex ODS References (1)Cragg GM, Newman DJ. Plants as a source of anticancer agents. J Ethnopharmacol. 2005;100:72-79. (2)Gonzales GF, Valerio LG. Medicinal plants from Peru: a review of plants as potential agents against cancer. Anticancer Agents Med Chem. 2006;6:429-444. (3)Bertuccio P, Edefonti V, Bravi F. Nutrient dietary patterns and gastric cancer risk in Italy. Cancer Epidemiology Biomarkers. 2009;18:2882-2886. (4)journal of pharmacy r e s e a r c h 6 ( 2 0 1 3 ) 5 6 5 -5 6 8. Anticancer activity of methanol extract of the seeds of Borreria hispida and Momordica dioica S. Rupachandra, D.V.L. Sarada. (5)Yuldashev, M. P. Flavonoids of the aerial part of Trigonella grandiflora. Chemistry of Natural Compounds (2002), 38(3), 291-29. Trigonella stellata