Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter-current chromatography fractionation and liquid chromatography/mass spectrometry analysis

In this paper, an effective method combing fast elution-extrusion counter-current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three-coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n-hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL-capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were 15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.