Counter-current chromatographic method for preparative scale isolation of picrosides from traditional Chinese medicine Picrorhiza scrophulariiflora

Apocynin, androsin, together with picroside I, II and III from crude extracts of Picrorhiza scrophulariiflora were isolated by means of high-speed counter-current chromatography (CCC) combining elution-extrusion (EE) and cycling-elution (CE) approach. The EECCC took full advantages of the liquid nature of the stationary phase for a complete sample recovery and extended the solute hydrophobicity window, while CECCC showed its unique advantage in achieving effective separation of special compounds through preventing stationary phase loss. In the present work, the biphasic liquid system composed of n-hexane/ethyl acetate/methanol/water (1:2:12, v/v/v/v) was used for separation of apocynin and androsin, ethyl acetate/n-butanol/water/formic acid (4:1:5:0.005, v/v/v/v) for picroside I, II and III. However, due to the extremely similar K values (K-1/K-2 approximate to. 1.2), picroside I and III were always eluted together by several biphasic solvent systems. In this case, the CECCC exhibited great superiority and baseline separated in the sixth cycle using ethyl acetate/water (1:1, v/v) as biphasic liquid system. Each fraction was analyzed by UPLC-UV and ESI-MS analysis, and identified by comparing with the data of reference substances. Compared with classical elution, the combination of EE and CE approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.