Isolation of -Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography.

-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5'''- and 5'''-epigallocatechin gallate-8-C -ethyl-2-pyrrolidinone (-EGCG-cThea and -EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. -hexane-ethyl acetate-methanol-water (1:5:1:5, ///) and 2. -hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, ///) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite values, α, and  . After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of -EGCG-cThea and 2.9 mg of -EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that - and -EGCG-cThea possessed in vitro inhibition effects on -glucosidase with IC of 70.3 and 161.7 μM, respectively.