Preparative separation and purification of five anthraquinones from Cassia tora L. by high-speed counter-current chromatography

Five anthraquinones including aurantio-obtusin, 1-desmethylaurantio-obtusin, chryso-obtusin, obtusin, and 1-desmethylchryso-obtusin were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (HPLC) from Cassia tora L. Preparative HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (11:9:10:10, v/v/v/v) was successfully performed by increasing the flow rate of the mobile phase from 1.5 to 2.5 ml/min after 220 min. Consequently, 35.8 mg aurantio-obtusin, 21.6 mg obtusin, 9.3 mg 1-desmethylchryso-obtusin and one partially purified peak fraction (80.6 mg, containing chryso-obtusin and 1-desmethylaurantio-obtusin) were obtained from 300 mg of the crude extract. Then the partially purified fraction was further separated by reversed-phase semi-preparative HPLC, which recovered 24.6 mg 1-desmethylaurantio-obtusin and 51.9 mg chryso-obtusin. The purity of aurantio-obtusin, 1-desmethylaurantio-obtusin, chryso-obtusin, obtusin, and 1-desmethylchryso-obtusin was 98.3, 98.7, 99.5, 97.4 and 99.6%, respectively, as determined by HPLC. The structures of five anthraquinones were identified by UV, MS, 1H NMR.