Preparative isolation and purification of anthraquinones from Cassia seed by high-speed countercurrent chromatography

A high-speed countercurrent chromatography (HSCCC) technique in a semipreparative scale has been applied to separate and purify anthraquinones from the extract of Cassia seeds. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-ethyl acetate-methanol-water (4:1:3:2, v/v/ v/v) by eluting the lower mobile phase at a flow rate of 1.5 mL/min under a revolution speed of 750 rpm. A total of five well separated peaks were obtained in the HSCCC chromatogram, and their purities were determined by HPLC-UV absorption spectrometry. These peaks were characterized by ESI-MSn and the data compared with the reference standards. Five peaks were identified as 1,2,6-trihydroxy-7, 8-dimethoxy-3- methylanthraquinone (7 mg), 1,2,6,8-tetrahydroxy-7-methoxy-3-methylanthraquinone (4 mg), 2-hydroxy- 1,6,7,8-teramethoxy-3-methylanthraquinone (9 mg), 6-dihydroxy-1,7,8-trimethoxy-3-methylanthraquinone (2 mg), and 1,2-dihydroxy-6,7,8tri-methoxy-3-methylanthraquinone (3 mg) from 100 mg of the sample. The purifies of obtained fractions were 98, 95, 96, 95, and 96%, respectively. HSCCC, thus, provides a cost effective alternative to preparative scale HPLC for the semi-preparative-scale separation and purification of anthraquinones from Cassia seeds. With appropriate modifications, the technique can also be applicable to other herbs in general.