HPLC analysis of naturally occurring methylated catechins, 3' '- and 4' '-methyl-epigallocatechin gallate, in various fresh tea leaves and commercial teas and their potent inhibitory effects on inducible nitric oxide synthase in macrophages.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, undergoes substantial biotransformation to species that includes the methylated compounds. Recent studies have demonstrated that the methylated EGCG has many biological activities. In this study, we have investigated the composition of the three O-methylated EGCG derivatives, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (3' '-Me-EGCG), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (4' '-Me-EGCG) and (-)-4'-methyl epigallocatechin-3-O-(4-O-methyl)gallate (4',4' '-di-Me-EGCG) in tea leaves which were picked from various species and at various seasons, ages of leaves, locations, and fermentation levels. Higher levels of 3' '-Me-EGCG and 4' '-Me-EGCG were detected in Chinshin-Kanzai (a species of Camellia sinensis) cultivated in the mountain area of Sansia, Taipei County, Taiwan. Also, these O-methylated EGCG levels were found to be higher in autumn and winter than in spring and summer. The young leaves were found to be richer in the O-methylated compounds than old leaves and the amount of O-methylated EGCG was higher in unfermented longjin green tea than in semifermented oolong tea. However, the fermented black tea and puerh tea did not contain these compounds. 4',4' '-diMe-EGCG could not be detected in either fresh tea leaves or commercial tea leaves. We also found that 3' '-Me-EGCG has a higher inhibitory effect on the nitric oxide generation and inducible nitric oxide synthase (iNOS) expression as compared with EGCG, while 4' '-Me-EGCG and 4',4' '-di-Me-EGCG were less effective.