Application of preparative high‐speed countercurrent chromatography for the separation and purification of three flavonoids from Oroxylum indicum

An efficient method was established by high‐speed countercurrent chromatography (HSCCC) for the separation and purification of three flavonoids from Oroxylum indicum. Optimized by single‐factor and orthogonal experiments, the optimal extraction conditions were an extraction temperature of 50°C, a solid‐to‐liquid ratio of 1:50 (g/ml), an ethanol concentration of 75% and an extraction time of 45 min. Using a two‐phase solvent system composed of chloroform–methanol–water (6:10:5, v/v/v), the preparative separation was successfully performed by HSCCC in head‐to‐tail elution mode. Totals of 12.63 mg of oroxin A at a purity of 97.61% with 96.46% recovery, 10.96 mg oroxin B at a purity of 98.32% with 98.81% recovery, and 9.34 mg baicalein at a purity of 98.64% with 97.87% recovery were obtained in one‐step separation from 200 mg crude extract. Their chemical structures were confirmed by melting points, HPLC, UV, FTIR, MS, 1H and 13C NMR data. Furthermore, they were efficient scavengers of 1,1‐diphenyl‐2‐picrylhydrazyl and hydroxyl free radicals in a concentration‐dependent manner.