Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl3-MeOH-(0.1 M; 0.2 M; 0.3 M) HCl (4: 1.5: 2; 4: 2: 2; 4: 3: 2, v/v), CHCl3-MeOH- 0.2M HCl (4: 2: 2, v/v) and CHCl3-MeOH-0.3M HCl (4: 3: 2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94-99% as determined by HPLC. Their chemical structures were characterized on the basis of H-1-NMR, C-13-NMR, and LC-ESI-Q-TOF-MS/MS analyses.