An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gels emium elegans ( G. elegans ) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/ n -hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine ( 1 , 12.1 mg), sempervirine ( 2 , 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine ( 3 , 10.1 mg), koumine ( 4 , 50.5 mg), gelsemine ( 5 , 32.2 mg), gelselvirine ( 6 , 50.5 mg), and 11-hydroxyhumanmantenine ( 7 , 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans , holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.