Abstract
This study employed the online HPLC‐2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonate radical cation (ABTS^+^^·^) bioassay to rapidly determine antioxidant compounds occurring in the licorice extract of Glycyrrhiza uralensis. The negative peaks of the ABTS^+·^ radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS^+^‐based antioxidant activity profile showed that three peaks exhibited antioxidant activity, and then the high‐speed counter‐current chromatography technique of preparative scale was successfully applied to separate the three peaks I‐III in one step from the licorice extract. The high‐speed counter‐current chromatography was performed using a two‐phase solvent system composed of n‐hexane–ethyl acetate–methanol–water (6.5:5.5:6:4, v/v). Yields of the three peaks, dehydroglyasperin C (I, 95.1% purity), dehydroglyasperin D (II, 96.2% purity), and isoangustone A (III, 99.5% purity), obtained were 10.33, 10.43, and 6.7% respectively. Chemical structures of the purified dehydroglyasperin C (I), dehydroglyasperin D (II), and isoangustone A (III) were identified by ESI‐MS and ^1^H‐ and ^13^C‐NMR analysis.