60 u00b0 c the filtered solution was then concentrated to dryness under vacuum at 45 u00b0 c the obtained dry extract was mixed with acidic water ph 2 the supernatant was filtered through a 0 22 u03bcm nylon membrane the filtrate was then extracted by diethyl ether using separating funnel the layer of diethyl ether was then separated and evaporated the obtained gummy residue was then subjected to thin layer chromatography using petroleum ether and ethyl acetate for separating the active compounds two compounds were separated namely hydroxyl p anisaldehyde c8h8o3 0 011 g rf 0 47 and p hydroxyacetophenone c 8 h 8 o 2 0 009 g rf 0 47 the two compounds were identified by gc mass and hplc hplc analysis of separated compounds the hydroxyl p anisaldehyde c 8 h 8 o 3 and p hydroxyacetophenone were quantified by hplc coupled with uv detection hplc uv hplc analysis was performed with an hplc instrument agilent 1100 usa equipped with a quaternary solvent delivery system a column oven and an uv detector agilent 1100 vwd usa twenty microliters of sample solution were injected into the column manually separation was achieved on an hypersil ods2 column 4 6 mm u00d7 250 mm 5 u03bcm from dalian elite analytical instruments co ltd the column temperature was set to 25 u00b0 c and the detection wavelength was set at 280 nm the mobile phase was 25 methanol with isocratic elution at a flow rate of 1 0 ml min fig1 and 2 show a chromatogram for the simultaneous determination of the two compounds respectively biological activity antimicrobial activities of the 3 hydroxyl p anisaldehyde c 8 h.