High-speed countercurrent chromatography (HSCCC) was applied to preparative separation and purification of punicalin from pomegranate husk by one-step separation. The two-phase system consisted of n-butyl alcohol-ethyl acetate-water (4:1:5, v/v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 2 ml/min and 25 degrees C respectively. About 100 mg amount of crude extract was separated, yielding 13.1 mg of punicalin at a high purity of over 96%. The mass recovery was more than 90%. The product was assessed by analytical HPLC and characterized by MS, H-1 NMR and C-13 NMR. (C) 2010 Published by Elsevier B.V.