Application of high-speed counter-current chromatography and preparative high-performance liquid chromatography mode for rapid isolation of anthraquinones from Morinda officinalis How.

Five anthraquinones including alizarin-1-methylether, 1,2-dimethoxy-3-hydroxyanthraquinone, 1-hydroxy-3-hydroxymethylanthraquinone, rubiadin-1-methylether and anthragallol-2-methylether were isolated and purified by high-speed counter-current chromatography (HSCCC) and followed by preparative high-performance liquid chromatography (prep-HPLC) mode from Morinda officinalis How. n-Hexane–ethyl acetate–methanol–water (6:4:5:5, v/v/v/v) was employed as the two-phase solvent system in HSCCC. Consequently, partially purified fraction “1” (90.6 mg, containing alizarin-1-methylether and 1,2-dimethoxy-3-hydroxyanthraquinone), partially purified fraction “2” (52.5 mg, containing 1-hydroxy-3-hydroxymethylanthraquinone and rubiadin-1-methylether) and 19.8 mg anthragallol-2-methylether were obtained from 300 mg of the crude extract. Then the partially purified fractions were further separated by prep-HPLC, which recovered 54.9 mg alizarin-1-methylether, 10.2 mg 1,2-dimethoxy-3-hydroxyanthraquinone, 16.4 mg 1-hydroxy-3-hydroxymethylanthraquinone and 18.2 mg rubiadin-1-methylether. The purity of each compound was over 96%, as determined by HPLC. The structures of five anthraquinones were identified by MS and 1H NMR. It is the first report of discovering 1,2-dimethoxy-3-hydroxyanthraquinone from the plant of M. officinalis How. The results demonstrated that this separation mode can integrate the advantages of HSCCC and prep-HPLC to improve separation efficiency, and adopted method was a feasible, economical and efficient technique for rapid preparative isolation of complicated natural products.