Application of preparative high-speed countercurrent chromatography for the separation and purification of three...

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, ¹ H and ¹³ C NMR data. Furthermore, they were efficient scavengers of 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals in a concentration-dependent manner.