SEPARATION AND PURIFICATION OF THREE FLAVONOIDS FROM DAPHNE GENKWA SIEB. ET ZUCC.: COMPARISON IN PERFORMANCE BETWEEN MEDIUM-PRESSURE LIQUID CHROMATOGRAPHY AND HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

Abstract

The two methods of medium-pressure liquid chromatography (MPLC) and high-speed countercurrent chromatography (HSCCC) are compared in the separation and purification of crude extract from Daphne genkwa Sieb. et Zucc. The MPLC method was performed with the mobile phase composed of methanol-0.1% aqueous acetic acid in the gradient elution which yielded apigenin, 3′-hydroxygenkwanin, and genkwanin at high purities of 95.3%, 94.0%, and 91.5%, respectively, for 230 min. The same three components were isolated by HSCCC also at high purities of 93.4%, 94.3%, and 92.8%, respectively, using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:7:5:5, v/v) for 400 min. Although both methods produced effective separations of Daphne genkwa Sieb. et Zucc., MPLC gave much shorter separation time, whereas HSCCC yielded substantially greater amounts of target compounds due to avoiding irreversible sample loss onto the solid support with less consumption of the solvent. The chemical structures of purified three compounds were identified by HPLC, ESI-MS, and 1H NMR.