STUDIES ON COUMARIN COMPOUNDS ISOLATED FROM MELILOTUS INDICA AND ITS EFFECT ON SOME PATHOGENIC BACTERIA AND HELA CELLLINE

Abstract

The active compounds in crude extracts have been separated by Liquid –Liquid extraction using diethyl ether. The separated compounds from two phases were studied by TLC. The ether phase of extracts prepared by solvents were contained compounds with Rf values (0.65,0.5,0.4,0.27, 0.2), but the aqueous phase was free of any compound belong to coumarins, while the ether phase of extracts prepared by buffer solutions were contained compounds with Rf values (0.65 and 0.4) , but the aqueous phase was free of any compound belong to coumarins. The concentrations of coumarin in crude extracts have been determined, the greater concentration were 1.501 and 2.734 mg/gm plant in acetate buffer extracts for the first and second times, respectively, and lowest concentration were determined in tris buffer extracts.The identified studies by high performance liquid chromatography (HPLC) was revealed the presence of one peak with retention time(RT) 8.53min. which is the equal to RT of standard coumarin. The antibacterial activity of acetate buffer, ethanol 80% extracts and purified coumarin have been determined against pathogenic bacteria by determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The MIC range of acetate extract were 16.2-19.7mg/ml against pathogenic isolates while the MIC range of ethanol 80% extract were 3.3-5.4mg/ml.Ps. aeruginosa was distinguished as most sensitive against extracts. The MIC values of purified coumarin were 0.62-1.37mg/ml and the most sensitive isolate against compound was E.coli. Cytotoxicity of standard and purified coumarin were tested against cell line (HeLa cell) by using four concentrations (25,50,100 and 200μg/ml) for exposure time 48hrs. The results revealed that standard and purified coumarin had cytotoxic effect with increasing effectiveness with concentration under our study.