Preparative separation of seven phenolic acids from Xanthii Fructus using pH-zone-refining counter-current chromatography combined with semi-preparative high performance liquid chromatography

Abstract

Phenolic acids represented by caffeoylquinic acids in Xanthii Fructus have various pharmacological activities such as anti-inflammatory, anti-nociceptive, anti-oxidative and anti-allergic effects. In this study, pH-zone-refining counter-current chromatography was successfully applied in the segmentation of crude samples and further separation of phenolic acids from Xanthii Fructus. We initially segmented 1.6 g of the crude sample to yield three sample fractions using a two-phase solvent system composed of EtOAc–ACN–H₂O (4 : 1 : 5, v/v/v) with 10 mM TFA added to the organic phase as the stationary phase and 10 mM NH₃·H₂O added to the aqueous phase as the mobile phase. The first fraction was separated using EtOAc–H₂O (1 : 1, v/v) (10 mM TFA was added in the upper phase and 20 mM NH₃·H₂O was added in the lower phase) solvent system, the second fraction containing low-content compounds was separated using semi-preparative high performance liquid chromatography, and the third fraction contained one pure compound. As a result, seven phenolic acids including six caffeoylquinic acid isomers (3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid) and caffeic acid were successfully isolated from Xanthii Fructus with purities above 90%. This study demonstrated that pH-ZRCCC is an efficient preparative separation method for phenolic acids, especially isomeric caffeoylquinic acids, from natural products.