Using Response Surface Methodology to Optimize Countercurrent Chromatographic Separation of Polyphenol Compounds from Fenugreek (Trigonella foenum-graecum L.) Seeds

摘要

In this study, we used high-speed countercurrent chromatography (HSCCC) to separate two bioactive polyphenols, rhaponticin and isovitexin, present in fenugreek (Trigonella foenum-graecum L.) seeds. The HSCCC process employed a two-phase solvent system composed of chloroform, methanol, water, and n-butanol in a 4:3:2:1.5 ratio (v:v:v:v). The HSCCC separation conditions were optimized using response surface methodology (RSM). The optimal separation conditions were as follows: separation temperature, 26 degrees C; revolution speed, 874rpm; flow rate, 1.3mL/min. Under these conditions, 21.6mg of rhaponticin and 12.8mg of isovitexin (>96.0% purity) were obtained from 100mg of the crude fenugreek seed extract. The purities and structures of the separated compounds were determined by high-performance liquid chromatography (HPLC), and both proton and carbon nuclear magnetic resonance spectroscopy (H-1 NMR and C-13 NMR, respectively). The results demonstrate that (i) HSCCC is an efficient technique for separating polyphenol compounds from seed extracts of fenugreek (Trigonella foenum-graecum L.) and (ii) RSM is an effective approach for optimizing HSCCC separation conditions.