Preparative isolation of caffeoylquinic acid isomers from Kuding tea by salt‐containing aqueous two‐phase extraction and purification by high‐speed countercurrent chromatography

Caffeoylquinic acid and its derivatives are important groups of natural plant phenols with antioxidant and neuroprotective properties. In this study, a salt‐containing aqueous system consisting of ethanol/ammonium sulfate/water 32:21:47 wt% was used to enrich these polar phenols from crude Kuding tea extract. In a one‐step extraction, the yields of 1‐ O ‐caffeoylquinic acid and 3‐ O ‐caffeoylquinic acid can be up to three times that obtained by using a 1‐butanol/water system. To purify polar caffeoylquinic acid, a two‐step high‐speed countercurrent chromatography method with a conventional organic/water system and a salt‐containing aqueous system was introduced. In the first step, dicaffeoylquinic acids and their derivatives were separated from the crude extract using n‐hexane/ethyl acetate/methanol/water (0.05% formic acid) 1:5:2.5:5 v/v. And caffeoylquinic acids and polar 1,3‐ O ‐dicaffeoylquinic acid were collected as a mixed fraction. In the second step, the mixture was futher purified using a 1‐butanol/ethanol/saturated ammonium sulfate/water (9:1:6:4, v/v) solvent system. 1‐ O ‐Caffeoylquinic acid, 3‐ O ‐caffeoylquinic acid, chlorogenic acid, and 1,3‐ O ‐dicaffeoylquinic acid were well separated with high purities of 90%. The chemical structures of the isolated compounds were identified by liquid chromatography with diode array detection and electrospray ionization mass spectrometry using a standard reference.