Identification of Oxidation Products of (-)-Epigallocatechin Gallate and (-)-Epigallocatechin with H 2 O 2 Nanqun Zhu, † Tzou-Chi Huang, ‡ Younong Yu, | Edmond J. LaVoie, | Chung S. Yang, ⊥ and Chi-Tang Ho* ,† Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901-8520, Department of Food Science and Technology, National Pingtung University of Science and Technology, 912, Pingtung, Taiwan, Department of Pharmaceutical Chemistry and Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08854 (-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H 2 O 2 . These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins. Keywords: Catechins; EGCG; EGC; antioxidants; H 2 O 2 INTRODUCTION Tea (Camellia sinensis) is one of the most widely consumed beverages in the world. During the past decade numerous in vitro and in vivo studies have suggested the possible beneficial effects of tea and tea polyphenols in cancer and cardiovascular disease de- velopment (Yang and Wang, 1993; Dreosti et al., 1997; Tijburg et al., 1997; Wiseman et al., 1997). The benefi- cial effects of tea are believed to be mainly due to the antioxidative activity of polyphenolic compounds in green and black tea (Huang et al., 1992; Koketsu, 1997; Wiseman et al., 1997; Yang et al., 1998a,b). The major polyphenolic compounds in tea are catechins, which include (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG). Because the growing body of evidence suggests that tea catechins may act as potent antioxi- dants or modulate key biological pathways in vivo in mammals (Lunder, 1992; Wiseman, 1997), detailed studies of the antioxidation of catechins are of scientific and commercial interest. It is generally accepted that the major pigments of black tea, theaflavins and thearubigins, are produced by enzymatic or chemical oxidation of catechins of green tea (Balentine, 1992; Finger et al., 1992). The first chemical analysis of the products formed from the free radical-initiated oxidation of catechin was reported by Hirose et al. (1991). The major step in the formation of products is the radical-initiated oxidative cleavage of C-3′ and C-4′ of the B-ring. Later, when studying the chemical oxidation of ECG with potassium ferricyanide, Wan et al. (1997) isolated and elucidated a new type of tea pigment. This compound was found to have a novel benzotropolone skeleton formed between the B-ring of one ECG molecule and the galloyl ester group of another. Most recently, the B-ring cleavage products of EGCG were identified when EGCG was reacted with peroxyl radicals (Valcic et al., 1999). All reactions reported so far suggested that the most likely site of oxidation for catechins occurs at the B-ring or gallate ester moiety. As an important oxidant, hydrogen peroxide (H 2 O 2 ), can be produced in vivo in a number of ways. For example, H 2 O 2 is generated during NADH oxidation by cell wall peroxidase, a process that can be stimulated by monophenolic compounds. H 2 O 2 is also generated during autoxidation and enzymic oxidation of catechins (Jiang and Miles, 1993). On the other hand, catechins exhibited a strong capacity for scavenging • OH radicals (Hanasaki et al., 1994) and suppressed cytotoxicity induced by H 2 O 2 (Nakayama, 1994). However, the specific mechanism of catechins oxidized by H 2 O 2 re- mains unclear. In this paper, we report the oxidation products formed by EGC and EGCG with H 2 O 2 . EXPERIMENTAL PROCEDURES Materials. Silica gel F254 TLC plates (259 μm thickness, 2-25 μm particle size) and silica gel (130-270 mesh) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used for chromatography. All solvents and H 2O2 (50%) were of analytical grade quality and purchased from Fisher Scien- tific (Springfield, NJ). NMR and FAB-MS. 1 H NMR (Table 1) and 13 C NMR (Table 2) spectra were obtained on a Varian Gemini-200 instrument (Varian Inc., Melboune, Australia) at 200 and 50 MHz, respectively. 1 H- 1 H COSY, NOESY, HMQC, and HMBC were performed on a U-500 instrument (Varian Inc.). Methanol-d4 was used as solvent, and chemical shifts were expressed in parts per million (δ) using TMS as internal standard. FAB mass spectra were recorded on a Finnigan MAT-90 instrument (Finnigan Corp., Bremen, Germany). All TLC spots were visualized under UV (254 and 365 nm) and with 10% H 2SO4 in EtOH followed by heating. * Corresponding author [fax (732) 932-8004; e-mail ho@ aesop.rutgers.edu]. † Department of Food Science, Rutgers University. ‡ National Pingtung University of Science and Technology. | Department of Pharmaceutical Chemistry, Rutgers Uni- versity. ⊥ Laboratory for Cancer Research, Rutgers University. 979 J. Agric. Food Chem. 2000, 48, 979-981 10.1021/jf991188c CCC: $19.00 © 2000 American Chemical Society Published on Web 03/09/2000