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-benzenedicarboxylie 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.