Large-scale separation of alkaloids from Gelsemium elegans by pH-zone-refining counter-current chromatography with a new solvent system screening method

• Developed a rapid method to optimize solvent system of pH-zone-refining counter-current chromatography.
• Needed fewer steps in the K determination.
• Six oxindole alkaloids preparative separated in one step separation with high purity.

A rapid and simple solvent system screening method was developed for pH-zone-refining counter-current chromatography (CCC) separation, which was much easier to find a suitable solvent system than the traditional method. Using this method, an optimal solvent system composed of hexane–ethyl acetate–methanol–water (3:7:1:9, v/v) was selected for pH-zone-refining CCC separation of alkaloids from the stems of Gelsemium elegans, where 10 mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10 mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter. As a result, six oxindole alkaloids, 420 mg 19-xo-gelsenicine, 456 mg gelsemine, 723 mg koumine, 379 mg 11-methoxygelsemamide, 342 mg gelsenicine and 318 mg humantenine were successfully purified in one step from 4.5 g crude extract with the purity of over 95%, respectively. The structures of the oxindole alkaloids were identified by ESI-MS, 1H NMR and 13C NMR.