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.