Polymerization of EGCG using polyphenol oxidase and the purification of EGCG dimer

Abstract

Tea leaves from Camellia sinensis can be classified into four different types of tea: white, green, oolong and black tea according to degree of fermentation. Black tea is produced by a fermentation process from the green leaves and contains oxidized catechins. These catechins form dimers, oligomers, and polymers in a heterogeneous mixture known as thearubigins. Thearubigins constitute more than 70% of the dry mass of an average black tea aqueous infusion. However, the structures of the catechins in thearubigins are remained poorly characterized. The monomeric catechins are oxidized with polyphenol oxidases(PPO), peroxidases(POD) and catalases which are critical in fermentation process. In this study, epigallocatechin-3-O-gallate(EGCG), the most abundant monomeric catechins, was oxidized to EGCG dimers through radical oxidative reaction using laccase, a member of the tea PPOs family. Electrospray ionization tandem LC-MS (ESI-LC/MS) was applied for the characterization of the dimers, theacitrin(TC), dehydrotheasinensin(DhTS), and theasinensin(TS), which were produced by the radical oxidative reaction. They were also analyzed by HPLC and LC/MS/MS to identify their structures according to their fragmentation patterns. The dimers were dismutated with heat treatment after oxidation step in order to mimic the burning and drying in the last step of fermentation in the laboratory. The result of the heat treatment, theasinensin A(TSA) was the major dimer product and was separated through Sephadex LH-20 and C18 column. Finally, 27.6mg of purified TSA was obtained in 100ml scale, and it is the first time that TSA has been produced by enzymatic reaction and isolated.