Separation and purification of two new and two known alkaloids from leaves of Nitraria sibirica by pH-zone-refining counter-current chromatography

The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica. The separation was performed with a solvent system of MtBE-n-BuOH-H2O (2:2:5, v/v) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica. The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n-hexane-n-BuOH-H2O (1.5:3.5:5, v/v) and (2:3:5, v/v), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC-ELSD, and their structures were identified by ESI-MS, 1D and 2D NMR.