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 flavanoids 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 800rpm. 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-MSn 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, 100mg of the crude extract were separated yielding 10mg of isorhamnetin, 8mg of irigenin and 7mg 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.