An Efficient Isolation and Purification of Broad Partition Coefficient Range of Anthraquinones From Seeds of Cassia obtusifolia L. by Linear Gradient Counter‐Current Chromatography Coupled With Inner‐Recycling Mode

ABSTRACT Anthraquinones are the main constituents of seeds of Cassia obtusifolia L., which have been used extensively in traditional medicine around the world to cure a variety of illnesses. This work used linear gradient counter‐current chromatography to purify anthraquinones successfully. Two gradient mobile phases were employed: the lower phases of the solvent system combination of n‐ hexane/ethyl acetate/methanol/water ( n ‐Hex/EtOAc/MeOH/H 2 O) (5:5:5:5, v/v) and (5:5:8:2, v/v). The solvent systems employed for the inner‐recycling separation were n ‐Hex/EtOAc/MeOH/H 2 O (5:5:5.5:4.5, v/v) and (5:5:7:3, v/v). Additionally, n‐ hexane/ethanol/water (1:1:0.2, v/v) was utilized as the solvent system to separate the fraction using the offline two‐dimensional separation mode. Lastly, high‐performance liquid chromatography revealed that nine anthraquinones, including aurantio‐obtusin ( 1 ), chryso‐obtusin ( 2 ), obtusin ( 3 ), btusifolin ( 4 ), emodin ( 5 ), rubrofusarin ( 6 ), toralactone ( 7 ), chrysophanol ( 8 ) and physcion ( 9 ) were effectively obtained with purities over 98%. The established technique was used to isolate a series of wide K D ‐value anthraquinones from seeds of C. obtusifolia L. The developed approach demonstrated significant potential for the isolation of complex components with broad polarities from other natural products.