This paper reports a way to increase the production yield of counter-current chromatography by using two-phase solvent systems in sample pretreatment. This strategy succeeds in the case of anti-EV71 saponin preparation from Anemarrhena asphodeloides. Briefly, 15 g of water extract was refined by solvent partition with ethyl acetate/1-butanol/water (1:4:5, v/v) to yield 4.17 g of the upper part, which in turns yielded 635 mg of timosaponin B-II (purity 96.5%) and 672 mg of anemarsaponin B (97.8%) after gradient CCC separation with 1-butanol/water (containing 0.01 mol/L CuSO in water, 1:1, v/v) and ethyl acetate/1-butanol/water (3:17:20, v/v/v). In a similar way, the ethanol extract (8 g) was partitioned between the diphase solvent system of ethyl acetate/1-butanol/water (4:1:5) to get 2.16 g of refined fraction and yielded 358 mg of timosaponin A-III (96.4%) by CCC elution using ethyl acetate-methanol-water (4:1:5, v/v/v). To remove mangiferin, the salt CuSO was added; it formed a complex with mangiferin, thereby enlarging the separation coefficient between mangiferin and timosaponin B-II.