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In this paper, an efficient method was successfully established by the combination of macroporous resin (MR) and high-speed counter-current chromatography (HSCCC) for rapid enrichment and separation of aloe-emodin 8-O-β-D-glucoside, emodin 1-O-β-D-glucoside, emodin 8-O-β-D-glucoside and piceatannol 4'-O-β-D-(6″-O-gallate)-glucoside. Six kinds of macroporous resins were investigated in the first step and X-5 macroporous resin was selected for the enrichment of the target compounds. The recoveries of the target compounds reached 89.0, 85.9, 82.3 and 84.9% respectively after 40% ethanol elution. In the second step, the target compounds were separated by HSCCC with a two-phase solvent system composed of chloroform/ethyl acetate/methanol/water (8:1:6:5, v/v). The established method will be helpful for further characterization and utilization of Rheum tanguticum. The results demonstrate that MR coupled with HSCCC is a powerful technique for separation of bioactive compounds from natural products.