Isolation and Purification of Three Flavonoids from the Hawthorn Leaves by High Speed Countercurrent Chromatography, Combined with Isocratic Preparative Reversed-Phase High Performance Liquid Chromatography

Three flavonoids including 4'''-O-rhamnosylrutin, 2 ''-O-rhamnosylvitexin, and 4 ''-O-glucosylvitexin were isolated and purified from the hawthorn leaves by high speed countercurrent chromatography (HSCCC), combined together with isocratic preparative reversed-phase high performance liquid chromatography. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-butanol-water (1:1, v/v). After two useful fractions were obtained by HSCCC using the optimal solvent system, one of them, fraction II, was further purified by reversed-phase semipreparative high performance liquid chromatography. From 500mg partially purified extract of the hawthorn leaves, 188.4mg 2 ''-O-rhamnosylvitexin, 28.5mg 4'''-O-rhamnosylrutin, and 55.3mg 4 ''-O-glucosylvitexin with purities of over 97%, respectively, were obtained. The structure identification of all pure fractions was carried out by UV, MS, H-1 NMR and C-13 NMR.