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

Honglei Xu*a, Tingting Zhou*a, Jun Wena, Guorong Fana* & Yutian Wua a Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University , Shanghai, P.R. China * Second Military Medical University, School of Pharmacy, 325 Guohe Road, Shanghai 200433, P.R. China E-mail: Guorfan@yahoo.com.cn Abstract 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 500 mg partially purified extract of the hawthorn leaves, 188.4 mg 2″-O-rhamnosylvitexin, 28.5 mg 4″′-O-rhamnosylrutin, and 55.3 mg 4″-O-glucosylvitexin with purities of over 97%, respectively, were obtained. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.