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.

Keywords:

• fenugreek
• high-speed countercurrent chromatography
• isovitexin
• polyphenol compounds
• response surface methodology
• rhaponticin

Notes

*Coded value in bracket and actual value out of bracket.

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljlc.