PREPARATIVE ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM Hibiscus mutabilis L. LEAVES BY TWO-STEP HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY

Seven compounds from Hibiscus mutabffis L. leaves were successfully achieved by two-step high-speed counter-current chromatography with a twophase solvent system composed of n-butanol-ethyl acetate-water (1:6:9 volume ratio) and n-hexane-ethyl acetate-methanol-water (3:5.3:5 volume ratio). 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 degrees 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 potengriffloside 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, 943, 98.0, 94.1 and 90.8%, respectively. Their chemical structures were identified by electron spray ionization mass spectroscopy (ES/-MS) and H-1, C-13 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.