Synthesis and Structure Identification of Thiol Conjugates of (−)-Epigallocatechin Gallate and Their Urinary Levels in Mice

Abstract

(-)-Epigallocatechin gallate (EGCG), the most abundant and most biologically active compound in tea, has been proposed to have many beneficial health effects. The metabolic fate of EGCG, however, is not well understood. In the present study, we found that EGCG can be oxidized by peroxidase and hydrogen peroxide and then reacted with cysteine or glutathione to form conjugates. The structures of the cysteine and glutathione conjugates of EGCG were identified using 2D NMR and MS. Two thiol conjugates of EGCG (2'-cysteinyl EGCG and 2' '-cysteinyl EGCG) were identified by ESI-LC-MS/MS analysis from the urine samples of mice administered 200 or 400 mg/kg EGCG, i.p. These conjugates were not found in urine samples of mice after receiving EGCG at 50 mg/kg i.p., or 2000 mg/kg i.g., or in human urine following consumption of 3 g of decaffeinated green tea solids (containing 333 mg EGCG). At high doses, EGCG is believed to be oxidized to form EGCG quinone, which can react with glutathione to form the thiol conjugates. These results suggest that detectable amounts of thiol conjugates of EGCG are formed only after rather high doses of EGCG are given to the mice.

Autoxidative quinone formation in vitro and metabolite formation in vivo from tea polyphenol (-)-epigallocatechin-3-gallate: Studied by real-time mass spectrometry combined with tandem mass ion mapping

“…We have previously observed the formation of EGCG-ascorbyl adducts in vitro only in the presence of H 2 O 2 (unpublished data). EGCG-thiol adducts have been found in mouse urine samples after administrated of toxic doses of EGCG [24]. However, EGCG-ascorbyl, EGCG-thiol adducts, and EGCG dimers were not detected in this study with nontoxic doses of EGCG.…”

“…EGCG quinone has been proposed as the key intermediate for the formation of many EGCG oxidation products by us and others [17][18][19], but direct evidence for its existence has been lacking. In the present stud y, we provided for the first time the direct evidence of the formation of EGCG quinone and dimer quinone.…”

N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct

“…These metabolites are hypothesized to arise from cysteine conjugation to the activated carbon in EGCG quinone. We and others have previously reported such reactions between EGCG and thiol-containing compounds such as cysteine and glutathione in cell culture and in vitro systems [14,15].…”

“…It is also interesting to note that the cysteine and glutathione conjugates of EGCG are readily formed in a horseradish peroxidase/H 2 O 2 catalyzed reaction, but the NAC conjugate is produced in only small quantities [14]. Further studies are needed to determine what factors in the present cell culture conditions facilitate the formation of this compound.…”

“…For example, we have observed that daily treatment of xenograft tumor-bearing athymic nude mice with intraperitoneal EGCG results in increased expression of γ-histone 2AX and metallothionein in the tumor and liver [13]. We have also observed that treatment of mice with toxic doses of EGCG results in the excretion of EGCG-2′-cysteine and EGCG-2″-cysteine in the urine [14]. These metabolites are hypothesized to arise from cysteine conjugation to the activated carbon in EGCG quinone.…”