8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols as the marker compounds of Chinese dark teas formed in the post-fermentation process provide significant antioxidative activity

Phytochemical investigation of the aqueous extract of pu-erh tea afforded eight novel 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols (puerins I-VIII) by 1H, 13C, two-dimensional nuclear magnetic resonance (NMR) and high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS) analysis. Comparative chemical analysis of green tea, black tea and Chinese dark teas confirmed that these compounds were the marker compounds of Chinese dark teas. Furthermore, fungal fermentation was indispensable for the biosynthesis of these novel compounds. Through single fungal fermentation, it was proved that catechins and theanine were the precursors of puerins I-VIII. HPLC-DAD-ESI/MS analysis elucidated the biosynthetic pathway for puerins I-VIII. Puerins I-IV have potential protective effects for the human micro-vascular endothelial cells (HMEC) injury induced by hydrogen dioxide compared to other tea polyphenols. 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols could be used in the quality control and authentication of Chinese dark teas.