Preparative separation of alkaloids from Nelumbo nucifera Gaertn by pH-zone-refining counter-current chromatography

Preparative separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn has been successfully performed for the first time using pH-zone-refining counter-current chromatography. Two kinds of two-phase solvent systems were applied to preparative CCC isolation. The first was the system composed of MtBE-water (1:1, v/v), 10 mM triethylamine in organic stationary phase and 5 mM HCl in aqueous mobile phase, which could only separate neferine partly from the crude extract. The second was the system composed of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v), 10 mM triethylamine in organic stationary phase and 5 mM HCl in aqueous mobile phase, which was the optimum for large-scale CCC isolation. 2.5 g of the crude alkaloid was purified in one-step separation of 7 h, yielding 151 mg of liensinine, 118 mg of isoliensinine and 572 mg of neferine with the purities of 93.0, 95.1 and 97.0%, respectively.