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Flavonoids are the main chemical constituents ofSmilax chinaL. and thought to be responsible for the anti-inflammatory activity ofSmilax chinaL. In this study, a suitable HSCCC method was developed to separate the three main flavonoids in a one-separation from the anti-chronic pelvic inflammation disease (CPID) effective fraction ofSmilax chinaL. with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v). 42 milligrams of astilbin, 10 mg neoisoastilbin, and 6 mg quercetin-3-O-α-L-rhamnoside were separated from 200 mg of the anti-CPID effective fraction ofSmilax chinaL. with purity of 98.3%, 98.7%, 98.5%, respectively, by HPLC analysis. Orthogonal test L9(34) was also applied to investigate the optimum extracting conditions of the three flavonoids. HSCCC was successfully used for the isolation and purification of the three flavonoids from the anti-CPID effective fraction ofSmilax chinaL. and the neoisoastilbin was first separated fromSmilax chinaL. [ABSTRACT FROM AUTHOR]