Separation and purification of water-soluble iridoid glucosides by high speed counter-current chromatography combined with macroporous resin column separation

•HSCCC isolates iridoid glucosides from an herbal medicinal plant for the first time.•A suitable two-phase solvent system was optimized for preparative HSCCC.•Four iridoid glucosides were obtained with preparative HSCCC by one-step separation.

Four iridoid glucosides, shanzhiside methyl ester, phloyoside II, chlorotuberside, and penstemonoside, were isolated and purified from an herbal medicinal plant for the first time by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system composed of ethyl acetate–n-butanol–water (5:14:12, v/v/v). A total of 37 mg of shanzhiside methyl ester, 29 mg of phloyoside II, 27 mg of chlorotuberside, and 21 mg of penstemonoside with the purity of 99.2%, 98.5%, 97.3%, and 99.3%, respectively, were obtained in one-step separation within 4 h from 150 mg of crude extract. To the best of our knowledge, this is the first report of separation and purification of iridoid glucosides from natural sources by HSCCC. The chemical structures of all the four compounds were identified by ESI-MS, 1H NMR, and 13C NMR.