B ioassay‐guided isolation of antioxidants from A stragalus altaicus by combination of chromatographic techniques

An efficient method for bioassay‐guided preparative isolation of antioxidants from the n ‐butanol extract of A stragalus altaicus Bunge was ingeniously developed by combination of silica gel column chromatography and high‐speed counter‐current chromatography. Under the bioassay‐guidance of antioxidant activities, the antioxidants were gradually separated from the crude sample of A stragalus altaicus Bunge by silica gel column chromatography and high‐speed counter‐current chromatography. Silica gel column chromatography separation was performed with chloroform, chloroform–methanol (100:1∼5:1, v/v) and chloroform–methanol–water (5:1:0.1∼2:1:0.1, v/v/v). High‐speed counter‐current chromatography separation was performed with a two‐phase solvent system composed of ethyl acetate– n ‐butanol–water (2:1:6, v/v/v), which was successfully selected by thin layer chromatography analysis, at a flow rate of 1.5 mL/min. As a result, isorhamnetin‐3‐gentiobioside (20.8 mg), rutin (82.0 mg), and narcissin (12.8 mg) were obtained for the first time from 200 mg of the crude sample, ABS ‐5 of A stragalus altaicus Bunge. The purities were all at over 95% by high‐performance liquid chromatography analysis, and their structures were unambiguously identified by mass spectroscopy, 1 H , and 13 C nuclear magnetic resonance spectroscopy. Antioxidant activities of the three compounds were also assayed by in vitro ABTS [2,2′‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulphonic acid) diamonium salt] radical cation scavenging activity. Among them, rutin possessed the highest antioxidant capacity with SC 50 value of 22.15 μg/m L .