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

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–H(2)O (4 : 1 : 5, v/v/v) with 10 mM TFA added to the organic phase as the stationary phase and 10 mM NH(3)·H(2)O added to the aqueous phase as the mobile phase. The first fraction was separated using EtOAc–H(2)O (1 : 1, v/v) (10 mM TFA was added in the upper phase and 20 mM NH(3)·H(2)O 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.