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Seven compounds from Hibiscus mutabilis L. leaves were first successfully   achieved by two-step high-speed counter-current chromatography with two-phase   solvent system composed of n-butanol-ethyl acetate-water (1:6:9, v/v/v) and   n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v/v/v/). The critical   experimental parameters of first-step separation were optimized with response   surface methodology as follows: flow rate was 1.1 mL/min, revolution speed   was 800 rpm and temperature was 30°C. Under the optimal conditions, around   5.0 mg of salicylic acid, 13.6 mg of rutin, 5.5 mg of genistein were obtained   in 100 mg crude sample. Then, 9.2 mg of potengriffioside A, 4.7 mg of   kaempferol 3-O-rutinoside, 3.0 mg of steppogenin and 2.5 mg of emodin were   obtained by second-step separation. The purities of the seven compounds   determined by UPLC were 96.2%, 93.8%, 95.4%, 94.3%, 98.0%, 94.1% and 90.8%,   respectively. Their chemical structures were identified by electron spray   ionization mass spectroscopy (ESI-MS) and 1H, 13C nuclear magnetic resonance   (NMR). Furthermore, compound steppogenin and genistein were first reported   from Hibiscus mutabilis L. The purification method was simple, efficient and   evaded tedious separation process

Preparative isolation and purification of seven compounds from Hibiscus mutabilis L. leaves by two-step high-speed counter-current chromatography