Epicatechin Oxidation Dimers Produced by Crude Tea Enzymes: Isolation, Structural Elucidation, and Potential Link to Thearubigin Formation.

Enzymatic oxidation of epicatechin, a major tea catechin, produces a color like that of black tea infusion and a broad liquid chromatography (LC) peak (DhC2-1). Herein, DhC2-1 was isolated and identified as an unstable mixture of an isomer of the epicatechin oxidative dimer DhC2-4 and intermediate IV (DhC2-1b), while this mixture readily degraded to DhC2-1a. Nuclear magnetic resonance (NMR) analysis established DhC2-4 as a γAB-type dehydrodicatechin possessing a unique C-tetrahydropyran-cyclohexenone-tetrahydrofuran-A ring linkage. To explore a possible structural relationship with thearubigins (TRs), a TR fraction TR13-19, purified from Keemun black tea, was acid-hydrolyzed; the resulting hydrolysate (m/z 575.1234 [M-H]-) exhibited LC retention similar to that of DhC2-1 and DhC2-4. Furthermore, characteristic 13C NMR signals of TR13-19 at δC 194.40, 165.06-165.19, 94.41, and 41.29 ppm matched those of DhC2-4, suggesting that γAB-type dehydrodicatechins may be structurally related to TRs.