Chem. Pharm. Bull. 55(12) 1768—1772 (2007)

duction, and therefore represents one of the most important beverages in the world. Additionally, black tea is rich in polyphenols compared to other beverages, and the various health benefits associated with its consumption, including antioxidative, anticancer and anti-inflammatory activity, have recently been investigated. Black tea is produced by crushing the fresh leaves of Camellia sinensis, and the constituents of the leaves are enzymatically converted to numerous secondary products that contribute to the characteristic color and flavor of black tea. In this process tea catechins, which are the major constituents of fresh tea leaves and mainly comprise ( )-epicatechin, ( )-epigallocatechin and their galloyl esters, are oxidized to yield a complex mixture of secondary polyphenols including theaflavins, theasinensins and oolongtheanins. However, the major components of the secondary polyphenols have not been chemically characterized due to their complexity and the difficulties involved in their separation and purification. In our previous studies concerning the enzymatic oxidation of tea catechins, oxidative coupling reactions of catechin B-rings were demonstrated. On the other hand, 60—80% of the total tea catechins possess galloyl esters located at the C-3 hydroxy group and although oxidation of galloyl groups must be important in the formation of black tea polyphenols, only limited examples of oxidative coupling of galloyl groups has been reported. Our previous in vitro experiments showed that enzymes preferentially oxidize the catechol B-rings of epicatechin and the resulting quinone, which is a potential oxidizing reagent, subsequently oxidizes pyrogallol rings, the redox potential of which is lower than that of the catechol ring. The catechol quinone also undergoes coupling reactions with other aromatic rings. Taking the reaction mechanism into account, ( )-epicatechin 3O-gallate (1), which possesses both the catechol ring and galloyl groups, was used in the present study to examine the oxidation of galloyl groups. In this paper, we describe the structure and mechanism of production of catechin trimer (3) and tetramer (4) produced by the oxidation of galloyl groups. The enzyme source used in our study should be noted here. Given that catechin oxidation in fresh tea leaf is a complex chemical process that acts on individual tea catechins, in vitro model fermentation experiments using pure catechins were employed in this study. However, the tea leaf homogenate or crude enzymes prepared from tea leaf did not display sufficient activity required for performing large scale experiments necessary to supply sufficient amounts of oxidation products needed for isolation and structure determination. Previously, we showed that homogenates of Japanese pear or banana could synthesize typical black tea polyphenols from tea catechins. The reactions proceeded in a manner similar to the oxidation catalyzed by tea leaf enzymes, and the products were consistent with catechin oxidation in fresh tea leaf. Consequently, Japanese pear homogenate was employed as the enzyme source in the present study.