Isolation and structure identification of C-19 diterpenoid alkaloids from Aconitum carmichaeli by high-speed counter-current chromatography

Three C-19 diterpenoid alkaloids were isolated and purified from the lateral roots of Aconitum carmichaeli Debx. (Fuzi in Chinese) by high-speed counter-current chromatography (HSCCC). A mixture of n-hexane-ethyl acetate-methanol-water (3:5:4:5, v/v/v/v) was used as the two phase solvent system. The lower phase was used as the mobile phase and was operated at a flow rate of 2.0 mL/min, while the apparatus was rotated at 850 r/min, and the detection wavelength was at 235 nm. Under these conditions, 15.3 mg of beiwutine, 35.1 mg of mesaconitine and 22.7 mg of hypaconitine were obtained from 90 mg of crude extract in one-step separation with the purities of 97.9%, 96.2% and 99.2%, respectively, determined by high performance liquid chromatography. The structures of these three compounds were identified by electrospray ionization mass spectrometry (ESI-MS), H-1-nuclear magnetic resonance (H-1-NMR) and C-13-NMR. The results indicate that HSCCC is a powerful technique for the purification of diterpenoid alkaloids from the lateral roots of Aconitum carmichaeli Debx.