Hyphenated HSCCC-DPPH• for rapid preparative isolation and screening of antioxidants from Selaginella moellendorffii

A method for rapid preparative isolation and screening of antioxidants has been developed by combining preparative HSCCC with on-line radical scavenging detection by use of DPPH center dot (1,1-diphenyl-2-picrylhydrazyl) as model radical. The HSCCC effluent was split into two parts. The major part was collected and the rest was introduced, through a switch valve, directly into a reaction coil for on-line evaluation of antioxidant activity. Each of the antioxidants separated by HSCCC reacted postcolumn with the DPPH center dot solution in methanol and was observed as a negative peak at 517 nm of size corresponding to its antioxidant activity. The on-line method was successfully used for preparative isolation and screening of antioxidant components from an ethyl acetate extract of the traditional Chinese medicine Selaginella moellendorffii. A total of 114.7 mg amentoflavone, 12.4 mg hinokiflavone, 7.2 mg podocarpusflavone A, and 1.1 mg ginkgetin, identified as the main radical scavengers, were obtained with purity of 99.4, 98.6, 98.9, and 99.2%, respectively, as determined by LC, from 1.0 g ethyl acetate extract. Their chemical structures were identified by MS and by UV and NMR spectroscopy.