Elucidation of the Partial Structure of Polymeric Thearubigins from Black Tea by Chemical Degradation

Abstract

Butanol-soluble neutral and acidic thearubigins were prepared by a combination of solvent extraction, fractional precipitation, and Toyopearl column chromatography. These pigments showed similar properties to those of Roberts' thearubigins on the paper chromatogram, and a convex broad band with several peaks on reversed-phase HPLC. The thearubigins were methylated, degallated, and chemically degraded with KMnO₄ under alkaline conditions. From the degradation products, methyl esters of 4-methoxy benzoic acid, 3, 4-dimethoxy benzoic acid, 3, 4, 5-trimethoxy benzoic acid, 3, 4-dimethoxy-1, 2-benzenedicarboxylic acid, 3, 4-dimethoxy-1, 5-benzenedicarboxylic acid, 3, 4-dimethoxy-1, 6-benzenedicarboxylic acid, 3, 4, 5-trimethoxy-1, 2-benzenedicarboxylic acid, 4, 5, 6-trimethoxy-1, 2, 3-benzenetricarboxylic acid, 4, 5, 4', 5'-tetramethoxy-2, 2'-diphenic acid, and 3, 4, 5, 3', 4', 5'-hexamethoxy-2, 2'-diphenic acid were detected by using GC-MS and GC-SIM. The butanol-soluble neutral thearubigins, which had been purified by Toyopearl column chromatography, were hydrolyzed by treating with HCl-BuOH. Cyanidin and delphinidin were identified in the reaction mixture. These results suggest that thearubigins are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates which have a bond at C4, C6, C8, C2', C5', and C6' in the flavan-3-ol units. In addition to the C4-C8 or C6 interflavanoid linkage, a C6'-C6' linkage was also found, and a possible partial structure for thearubigin is proposed.