Rapid Screening and Preparative Isolation of Antioxidants from Hance Using HSCCC Coupled with DPPH-HPLC Assay and Evaluation of Their Antioxidant Activities.

An efficient method using high-speed counter-current chromatography (HSCCC) coupled with DPPH-HPLC assay has been developed for rapid screening and preparative isolation of antioxidants from ethyl acetate fraction of Hance. Target-guided by DPPH-HPLC assay, two antioxidants, galangin and kaempferide, were targeted and further separated with purities of 99.3% and 98.5% by HSCCC using petroleum ether-ethyl acetate-methanol-water (0.8 : 1 : 1 : 0.8, /) as the solvent system. The antioxidant activities of galangin and kaempferide were further evaluated by measuring their inhibiting effects on superoxide anion radical, hydroxyl radical, and hydrogen peroxide in different luminol chemiluminescence (CL) systems. As a result, galangin and kaempferide both showed potent antioxidant activities. Results of the present study indicated that the combinative method by offline coupling DPPH-HPLC and HSCCC could be widely applied for rapid screening and isolation of antioxidants from complex TCM extract.