Simultaneous separation and purification of calycosin and formononetin from crude extract of Astragalus membranaceus Bge. var. mongholicus (Bge.) using high-speed counter-current chromatography

A simple and effective high-speed counter-current chromatography ( HSCCC ) method for simultaneous separation and purification of calycosin and formononetin from the ethanolic crude extract of Astragalus membranaceus is described. HSCCC separation was performed using a two-phase solvent system composed of n -hexane-ethyl acetate-methanol-water (2.5: 2.5: 2: 3, v/v). A total of 7.5 mg calycosin and 11.2 mg formononetin was obtained from 500 mg crude extract of Astragalus membranaceus with purities of 98.5 and 98.3%, respectively, determined using HPLC. Chemical structures were confirmed by MS, 1 HNMR.