Department of Biochemical Engineering, School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
Molecules. 2013 Dec 12;18(12):15490-500. doi: 10.3390/molecules181215490.
pH-Zone refining counter-current chromatography was successfully applied to the preparative isolation and purification of six alkaloids from the ethanol extracts of Uncaria macrophylla Wall. Because of the low content of alkaloids (about 0.2%, w/w) in U. macrophylla Wall, the target compounds were enriched by pH-zone refining counter-current chromatography using a two-phase solvent system composed of petroleum ether-ethyl acetate-isopropanol-water (2:6:3:9, v/v), adding 10 mM triethylamine in organic stationary phase and 5 mM hydrochloric acid in aqueous mobile phase. Then pH-zone refining counter-current chromatography using the other two-phase solvent system was used for final purification. Six target compounds were finally isolated and purified by following two-phase solvent system composed of methyl tert-butyl ether (MTBE)-acetonitrile-water (4:0.5:5, v/v), adding triethylamine (TEA) (10 mM) to the organic phase and HCl (5 mM) to aqueous mobile phase. The separation of 2.8 g enriched total alkaloids yielded 36 mg hirsutine, 48 mg hirsuteine, 82 mg uncarine C, 73 mg uncarine E, 163 mg rhynchophylline, and 149 mg corynoxeine, all with purities above 96% as verified by HPLC Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and 1H-NMR spectroscopy.
pH 区带精制逆流色谱法成功地应用于从钩藤乙醇提取物中分离和纯化 6 种生物碱。由于钩藤中生物碱含量较低（约 0.2%，w/w），因此通过 pH 区带精制逆流色谱法，使用石油醚-乙酸乙酯-异丙醇-水（2:6:3:9，v/v）组成的两相溶剂系统，在有机固定相中添加 10 mM 三乙胺，在水相移动相中添加 5 mM 盐酸，对目标化合物进行富集。然后使用另一种两相溶剂系统进行 pH 区带精制逆流色谱法，进行最终的纯化。最后，通过以下两相溶剂系统（甲基叔丁基醚（MTBE）-乙腈-水（4:0.5:5，v/v）），在有机相中添加三乙胺（TEA）（10 mM），在水相移动相中添加 HCl（5 mM），从 2.8 g 富集的总生物碱中分离出 36 mg 毛钩藤碱、48 mg 毛钩藤碱、82 mg 育亨宾 C、73 mg 育亨宾 E、163 mg 钩藤碱和 149 mg 考来烯胺，所有产物的纯度均超过 96%，通过高效液相色谱法（HPLC）验证。它们的结构通过电喷雾电离质谱（ESI-MS）和 1H-NMR 光谱鉴定。
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