PREPARATIVE ISOLATION AND PURIFICATION OF FOUR FLAVONOIDS FROM DAPHNE GENKWA SIEB. ET ZUCC. BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY.

Four flavonoids including luteolin, apigenin, 3'-hydroxygenkwanin and genkwanin were isolated and purified from Daphne genkwa Sieb. et Zucc. by high-speed countercurrent chromatography (HSCCC). Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:7:5:5, v/v) was successfully performed by increasing the flow rate of the mobile phase from 1.2 to 2.0 mL/min after 260 min. In a one-step operation, 150 mg of the extracts of D. genkwa was separated to yield 8 mg of luteolin, 25.8 mg of apigenin, 23.6 mg of 3'-hydroxygenkwanin and 35.3 mg genkwanin with the purities of 91.2, 97.4, 94.3 and 95.8%, respectively, analyzed by HPLC using area normalization method. The chemical structures of the four compounds were identified by HPLC, ESI-MS, and (1)H NMR.