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

Abstract

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°C; revolution speed, 874 rpm; flow rate, 1.3 mL/min. Under these conditions, 21.6 mg of rhaponticin and 12.8 mg of isovitexin (>96.0% purity) were obtained from 100 mg 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 (1H NMR and 13C 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.