PREPARATIVE ISOLATION AND PURIFICATION OF FIVE FLAVONOIDS FROM POGOSTEMON CABLIN BENTH BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND PREPARATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

High-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of pogostone and four flavonoids from Pogostemon cablin (Blanco) Benth. An efficient HSCCC separation was achieved on a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (11: 5: 11: 5, v/v/v/v). Three well-separated peaks were obtained in the HSCCC chromatogram. The first and the second fractions each contained two flavonoids that were further separated by preparative HPLC. Consequently, the separation yielded 11.5 mg of 4',5-Dihydroxy-3',7-dimethoxyflavanone at a purity of 99%, 20.3 mg of 5-Hydroxy-7,3',4'-trimethoxyflavanone at a purity of 98%, 18 mg of 5,4'-Dihydroxy-3,7,3'-trimethoxyflavone at a purity of 96%, and 8 mg of 5-Hydroxy-3,7,4'-tetramethoxyflavone at a purity of 98%. The third HSCCC fraction yielded 18.5 mg of pogostone at a purity of 95%. The chemical structures of these compounds were identified by ESI-MSn, H-1-NMR, and C-13-NMR.