Application of High‐speed Countercurrent Chromatography–Evaporative Light Scattering Detection for the Separation of Seven Steroidal Saponins from Dioscorea villosa

Abstract

Steroidal saponins in Dioscorea species are chemically characterised as spirostanol and furostanol saponins, and have been used as standard marker compounds due to their chemotaxonomical significance and their important biological activities. To design a simple, rapid and efficient method for the separation of steroidal saponins with a high degree of purity using high-speed countercurrent chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD). In the first step, reversed-phase mode HSCCC (flow rate: 1.5 mL/min; revolution speed: 800 rpm) using n-hexane:n-butanol:water [3:7:10 (v/v/v)] was employed to separate furostanol saponins from n-butanol soluble extracts of Dioscorea villosa. After the first HSCCC run, spirostanol saponins retained in the stationary phase were subjected to the second HSCCC (normal-phase mode; flow rate: 2.0 mL/min; revolution speed: 800 rpm). A two-phase solvent system composed of chloroform:methanol:isopropanol:water [10:6:1:4 (v/v/v/v)] was employed in the second HSCCC. The structures of isolates were elucidated by (1) H-NMR, (13) C-NMR, ESI-MS and HPLC analysis. Three furostanol saponins, parvifloside (27.3 mg), methyl protodeltonin (67.1 mg) and trigofoenoside A-1 (18.5 mg) were isolated from the n-butanol soluble extract of D. villosa by the first HSCCC run. Subsquent normal-phase HSCCC of the spirostanol-rich extract led to the separation of four spirostanol saponins: zingiberensis saponin I (15.2 mg), deltonin (31.5 mg), dioscin (7.7 mg) and prosapogenin A of dioscin (3.4 mg).