Preparative isolation and purification of flavonoids from the Chinese medicinal herb Belamcanda by high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) was applied for separation and purification of flavonoids from the extract of belamcanda. High efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-ethyl acetate-methanol-water (4:5:5:5, v/v) by eluting the lower mobile phase at a flow-rate of 1.2mL/min and a revolution speed of 800 rpm. Three well-separated peaks were obtained in the HSCCC chromatogram and their purities were determined by HPLC-UV absorption spectrometry. These peaks were characterized by ESI-MS(n) and NMR, and the data compared with the reference standards where three peaks were identified as isorhamnetin, irigenin and hispidulin. The purities of each peak were 94, 95 and 90% respectively. In HSCCC experiment, 100 mg of the crude extract were separated yielding 10 mg of isorhamnetin, 8 mg of irigenin and 7 mg of hispidulin. HSCCC thus provides a cost-effective alternative to preparative scale HPLC for the semi-preparative scale separation and purification of flavonoids from Belamcanda.