A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of four prenylflavanones from the crud extract after microbial biotransformation of kurarinone was developed successfully using a stepwise elution with two-phase solvent system composed n-hexane-ethyl acetate-methanol-water at the volume ratios of 1:1:0.7:1 (v/v) and 1:1:1.2:1 (v/v). A total of 7 mg of 4a,5a-dihydroxy norkurarinone (1), 11 mg of 7-methoxyl-4a,5a-dihydroxy norkurarinone (2), 14 mg of 6a-hydroxykurarinone (3) and 10 mg of norkurarinone (4) were obtained in one-step separation from 520 mg of the crude biotransformation sample with purities of 94.0%, 97.8%, 99.5% and 98.7%, respectively. Among them, compounds 1-3 are novel compounds, and their chemical structures were identified on the basis of the extensive spectral methods such as UV, IR, HR-MS and NMR. (C) 2010 Published by Elsevier B.V.