Preparative Isolation and Purification of Anthraquinones from Cassia Seed by High-Speed Countercurrent Chromatography

Abstract

A high-speed countercurrent chromatography (HSCCC) technique in a semi‐preparative 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‐methyl‐anthraquinone (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‐6,7,8‐tri‐methoxy‐3‐methylanthraquinone (3 mg) from 100 mg of the sample. The purities 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.