A general ionic liquid pH-zone-refining countercurrent chromatography method for separation of alkaloids from Nelumbo nucifera Gaertn

- A general ionic liquid pH-zone-refining CCC method was established for the first time.
- The six alkaloids in lotus plants were obtained with high purity in one run.
- The established general method was applied to lotus leaves and lotus plumules.

The alkaloids from lotus (Nelumbo nucifera Gaertn) are effective in lowering hyperlipemia and level of cholesterol. However, there is not a general method for their separation. In this work, a general ionic liquid pH-zone-refining countercurrent chromatography method for isolation and purification of six alkaloids from the whole lotus plant was successfully established by using ionic liquids as the modifier of the two-phase solvent system. The conditions of ionic liquid pH-zone-refining countercurrent chromatography, involving solvent systems, concentration of retainer and eluter, types of ionic liquids, the content of ionic liquids as well as ionic liquids posttreatment, were optimized to improve extraction efficiency. Finally, the separation of these six alkaloids was performed with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water-[C4mim][PF6] at a volume ratio of 5:2:2:8:0.1, where 10 mM TEA was added to the organic stationary phase as a retainer and 3 mM HCl was added to the aqueous mobile phase as an eluter. As a result, six alkaloids including N-nornuciferine, liensinine, nuciferine, isoliensinine, roemerine and neferine were successfully separated with the purities of 97.0%, 90.2%, 94.7%, 92.8%, 90.4% and 95.9%, respectively. The established general method has been respectively applied to the crude samples of lotus leaves and lotus plumules. A total of 37.3 mg of liensinine, 57.7 mg of isoliensinine and 179.9 mg of neferine were successfully purified in one run from 1.00 g crude extract of lotus plumule with the purities of 93.2%, 96.5% and 98.8%, respectively. Amount of 45.6 mg N-nornuciferine, 21.6 mg nuciferine and 11.7 mg roemerine was obtained in one step separation from 1.05 g crude extract of lotus leaves with the purity of 96.9%, 95.6% and 91.33%, respectively.