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 (0.10 mol/L in the lower phase) was selected. 9.2 mg isoirigenin (1), 46.4 mg irigenin (2) and 1.2 mg 5,7,4′-trihydroxy-6,3′,5′-trimethoxyisoflavone (3) were simultaneously purified from 100 mg 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.