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.

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/H2O) (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/H2O (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 KD-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.