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An efï¬cient method for the isolation and puriï¬cation of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis by high speed counter-current chromatography (HSCCC) was established in this paper. The ether extracts of Radix Eupatorii Chinensis were puriï¬ed by HSCCC with a solvent system of hexyl hydrideâethyl acetateâmethanolâwater (1:2:1:2, v/v/v/v). The upper phase was used as the stationary phase and the lower phase as the mobile phase. About 8.4 mg of 12,13-dihydroxyeuparin was obtained from 200 mg of ether extracts from Radix Eupatorii Chinensis in one-step HSCCC separation, with the purity of 96.71%, as determined by HPLC. After methanolâwater recrystallization, the purity of 12,13-dihydroxyeuparin reached 99.83%. Such a simple and effective method was fairly useful to prepare pure compound as reference substances for related study on Radix Eupatorii Chinensis. Keywords: Radix Eupatorii Chinensis, High speed counter-current chromatography (HSCCC), 12,13-dihydroxyeuparin