Combined chromatographic strategy based on macroporous resin, high-speed counter-current chromatography and preparative HPLC for systematic separation of seven antioxidants from the fruit of Terminalia billerica.

In the present study, combined chromatographic strategy based on macroporous resin, high-speed counter-current chromatography and preparative high-performance liquid chromatography for systematic separation of antioxidants from crude samples guided by high-performance liquid chromatography with 1,1-diphenyl-2-picrylhydrazyl has been successfully established. Based on this strategy, seven antioxidants including isorugosin A, -1,2,3,6-tetragalloyl-D-glucose, chebulinic acid, 1,2,3,4,6-penta-O-galloyl- -D-glucose, chebulagic acid, ethyl gallate, and gallic acid were obtained from the fruit of Terminalia billerica. First, high-performance liquid chromatography with 1,1-diphenyl-2-picrylhydrazyl experiment showed the presence of seven main antioxidants in the crude extract of the fruit of Terminalia billerica. Then, a macroporous resin column chromatography method was developed for the enrichment of these seven antioxidants. Finally, an efficient method based on high-speed counter-current chromatography and preparative high-performance liquid chromatography was developed for the separation of these antioxidants. In the selection of solvent systems, it was found that acetic acid could be a good regulator for modifying the partition coefficient values of tannins. The present study provides a reference for systematic separation of antioxidants from crude samples. Considering the general existence of antioxidants in crude samples, this combined chromatographic strategy might lead to broader application prospects.