Application of complexation high-speed counter-current chromatography in the separation of 5-hydroxyisoflavone isomers from Belamcanda chinensis (L.) DC

A novel separation technique of complexation high-speed counter-current chromatography (HSCCC) using copper ion as a complexation agent was first developed to isolate 5-hydroxyisoflavone isomers from Belamcanda chinensis (L.) DC. According to the partition coefficient and separation factor, the two-phase solvent system composed of light petroleum-ethyl acetate-methanol-water (3:5:3:5, v/v) and copper nitrate (0.10mol/L in the lower phase) was selected. 9.2mg isoirigenin (1), 46.4mg irigenin (2) and 1.2mg 5,7,4'-trihydroxy-6,3',5'-trimethoxyisoflavone (3) were simultaneously purified from 100mg crude extract by HSCCC with the purity of 95.06%, 96.98% and 93.69%, respectively. As evidenced by the results of UV-Vis spectroscopy, the stoichiometries of the copper ion with the three 5-hydroxyisoflavones were all 1:1 and their chelating power was 3>2>1. Those explained the complexation HSCCC behavior. It is the first report that includes the practical application of complexation HSCCC and explanation of its chromatographic behavior.