Efficient Protocol for Isolation of Rhaponticin and Rhapontigenin with Consecutive Sample Injection from Fenugreek (Trigonella foenum-graecum L.) by HSCCC.

High efficiency and less solvent consumption are the essential requirements of high-speed countercurrent chromatography (HSCCC), especially for the large-scale preparation. In this study, an efficient HSCCC strategy with consecutive sample injection was successfully developed to rapidly separate and purify rhaponticin and rhapontigenin from the seeds of the Chinese medicinal herb fenugreek (Trigonella foenum-graecum L.). The effective separation was achieved using n-hexane-ethyl acetate-methanol-water (1:4:2:6, v/v/v/v) as the two-phase solvent system, in which the mobile phase was eluted at an optimized flow rate of 2.2 mL/min and a revolution speed of 850 rpm. After consecutively loading four identical fenugreek samples, each containing 120 mg, HSCCC separation yielded 146.4 mg of rhaponticin and 174.8 mg of rhapontigenin with purities of 98.6 and 99.1%, respectively, as determined by high-performance liquid chromatography at 320 nm. Their chemical structures were identified using UV spectroscopy, (1)H-NMR and (13)C-NMR. The HSCCC method with consecutive sample injection allowed faster separation and produced less solvent waste, suggesting that it is an efficient way to rapidly separate and purify natural products on a large scale.