Antioxidant Chemistry of Green Tea Catechins. Identification of

In this study, we have isolated and identified for the first time two reaction products of EGCG ..... Figure 3 HMBC correlations (C → H) of the cent...
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Chem. Res. Toxicol. 1999, 12, 382-386

Antioxidant Chemistry of Green Tea Catechins. Identification of Products of the Reaction of (-)-Epigallocatechin Gallate with Peroxyl Radicals Susanne Valcic,† Annette Muders,† Neil E. Jacobsen,‡ Daniel C. Liebler,*,† and Barbara N. Timmermann† Department of Pharmacology & Toxicology, College of Pharmacy, and Department of Chemistry, The University of Arizona, Tucson, Arizona 85721 Received January 12, 1999

(-)-Epigallocatechin gallate (EGCG), isolated from green tea, displays antioxidant properties and is thought to act as an antioxidant in biological systems. However, the specific mechanisms of its antioxidant actions remain unclear. In this study, we have isolated and identified for the first time two reaction products of EGCG derived from its reaction with peroxyl radicals generated by thermolysis of the initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). The products include a seven-membered B-ring anhydride and a novel dimer. The identification of these products provides the first unambiguous proof that the principal site of antioxidant reactions on the EGCG molecule is the trihydroxyphenyl B ring, rather than the 3-galloyl moiety. In contrast to phenoxyl radicals from simple phenolic antioxidants, an initially formed EGCG phenoxyl radical apparently does not form stable addition products with AMVN-derived peroxyl radicals. Characteristic reaction products may provide novel markers for EGCG antioxidant reactions in living systems.

Introduction Green tea, one of the most widely consumed beverages in the world, is an aqueous infusion of dried leaves of Camellia sinensis. It is distinguished by its remarkable content of polyphenols, especially catechins such as (-)epicatechin (EC1), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) (1). Numerous biological activities have been reported for green tea and its constituents, of which the preventive effects against cancer are most notable (2, 3). Many of these activities have been attributed to antioxidant actions of green tea catechins (4-9). For example, Terao et al. (5) showed that EC and ECG are powerful antioxidants against lipid peroxidation when phospholipid bilayers are exposed to aqueous oxygen radicals. Salah et al. (6) reported that green tea catechins and gallic acid inhibit the metmyoglobin-initiated peroxidation of low-density lipoprotein (LDL) lipids and the consumption of LDL R-tocopherol. Previous structureactivity studies (4, 6) suggested that flavonoids with an o-dihydroxy or trihydroxy B ring are the most effective antioxidants. Moreover, catechins containing a gallate ester moiety at the 3-position (ECG and EGCG) have the * To whom correspondence should be addressed. Phone: (520) 6264488. Fax: (520) 626-2466. E-mail: [email protected]. † Department of Pharmacology & Toxicology, College of Pharmacy. ‡ Department of Chemistry. 1 Abbreviations: EGCG, (-)-epigallocatechin gallate; AMVN, 2,2′azobis(2,4-dimethylvaleronitrile); EC, (-)-epicatechin; EGC, (-)-epigallocatechin; ECG, (-)-epicatechin gallate; LDL, low-density lipoprotein; MPLC, medium-pressure liquid chromatography; ESI-MS, electrospray ionization mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; NOE, nuclear Overhauser effect; HMQC, heteronuclear multiple-quantum coherence; HMBC, heteronuclear multiple-bond correlation; DQF-COSY, double-quantum-filtered correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; TMS, tetramethylsilane; DMSO, dimethyl sulfoxide.

Scheme 1. Formation of Catechin Phenoxyl Radical by One-Electron Oxidation

highest activity as antioxidants and are the most effective inhibitors of lipid peroxidation (4, 6). Like other phenolic antioxidants, EGCG probably acts as a chain-breaking antioxidant, which traps peroxyl radicals and thus suppresses radical chain autoxidations. Catechins react with peroxyl radicals via a single electron transfer followed by deprotonation (Scheme 1) (4, 9), but the specific mechanisms of catechin antioxidant reactions remain unclear. The identification of oxidation products formed by reactions of green tea catechins with biologically relevant oxidants could provide insights into the specific mechanisms of antioxidant reactions. In this paper, we report for the first time the structures of two major oxidation products formed by the reaction of EGCG (1) with peroxyl radicals generated by thermolysis of the initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) in oxygenated acetonitrile.

Experimental Procedures Chemicals and Instrumentation. AMVN was purchased from Polysciences, Inc. (Warrington, PA). EGCG (1) was isolated from green tea provided by the Royal Estates Tea Company, Division of Thomas J. Lipton, as described previously (10). The medium-pressure liquid chromatography (MPLC) system used

10.1021/tx990003t CCC: $18.00 © 1999 American Chemical Society Published on Web 03/24/1999

Oxidation of (-)-Epigallocatechin Gallate Table 1. 1H and 1 (EGCG) (DMSO-d6) position 2 3 4

δC

δH

Chem. Res. Toxicol., Vol. 12, No. 4, 1999 383 13C

NMR and HMBC Data of 1 (EGCG), 2, and 3 3a [CD3CN/D2O (10:1 v/v)]

2 (DMSO-d6) δC

δH

4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1′′ 2′′/6′′

76.6 4.99 (br s) 68.2 5.39 (br s) 25.9 2.96 (dd) (J ) 4, 17 Hz) 2.68 (dd) (J ) 3, 17 Hz) 97.6 155.8 94.5 5.86 (d) (J ) 2 Hz) 156.7 95.7 5.96 (d) (J ) 2 Hz) 156.7 128.8 105.7 6.44 (s) 145.6 132.5 145.6 105.7 6.44 (s) 119.5 108.8 6.85 (s)

74.4 5.18 (br s) 67.6 5.34 (br s) 24.4 2.93 (dd) (J ) 4, 18 Hz) 2.78 (dd) (J ) 3, 18 Hz) 96.6 154.0 94.0 5.88 (d) (J ) 2 Hz) 156.7 95.9 5.97 (d) (J ) 2 Hz) 156.3 155.9 105.5 7.17 (d) (J ) 1 Hz) 157.9 160.9 161.8 111.2 6.31 (br s) 118.6 108.5 6.90 (s)

3′′/5′′ 4′′ COO

145.8 138.7 165.4

145.5 138.5 165.2

HMBC 1′, 2′, 6′ 4a

4a, 5, 7, 8 4a, 6, 7 2, 3′, 4′, 6′

2, 2′, 5′ 1′′, 3′′/5′′, 4′′, 6′′/2′′, COO

δC 76.9 65.0 26.0 98.8 155.4b 95.7c 157.4 97.0c 157.2b 156.6 59.1 105.0 85.8 197.0 127.5 121.0 110.2

δH

HMBC

4.51 (br s) 1′, 2′, 6′ 5.66 (br t) (J ) 3 Hz) 4a, COO 2.92 (dd) (J ) 4, 17 Hz) 2, 3, 4a, 5, 8a 2.88 (d)d (J ) 2, 17 Hz) 5.90d (d) (J ) 2 Hz)

4a, 5, 8, 7

6.00d (d) (J ) 2 Hz)

4a, 6, 7

3.23 (br s)

2, 1′, 3′, 4′, 5′, 6′

6.44 (d) (J ) 1 Hz)

2, 1′, 2′, 4′

6.88 (s)

1′′, 3′′/5′′, 4′′, 6′′/2′′, COO

145.8 139.2 166.7

a Due to the C symmetry of 3, a distinction of the H and C assignments of the two monomeric units could not be made. b Assignments 2 interchangeable. c Assignments interchangeable. d Assignments interchangeable.

was equipped with a Bu¨chi 688 pump, a D-Star Instruments DFV-20 fixed wavelength detector, a Linseis L200E recorder, and a Bu¨chi borosilicate 3.3 column (15 mm × 460 mm) filled with Polygoprep 100-30 C18 (25-40 µm, Machery & Nagel, Du¨ren, Germany). The mobile phase was 0.15% aqueous HCOOH/ CH3CN/ethyl acetate (86:12:2). The HPLC system that was used was equipped with a Varian 9012 gradient pump, a Varian 9065 Polychrom detector, and an Alltech Econosphere C18 HPLC column (10 µm, 10 mm × 250 mm). Compounds were eluted with 15% CH3CN in 0.15% aqueous HCOOH at a flow rate of 1 mL/min. Optical rotations were measured on a Jasco P-1020 polarimeter; UV spectra were recorded on a Beckman DU 640 spectrophotometer, and IR spectra of KBr disks were obtained with a Buck Scientific model 500 spectrophotometer. NMR Spectroscopy. NMR samples were dissolved in 0.6 mL of deuterated solvent (Table 1), and all spectra were referenced to internal tetramethylsilane (TMS) at 0 ppm (1H and 13C). 1H and 13C, {1H,13C} heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), double-quantum-filtered correlation spectroscopy (DQFCOSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra (11, 12) of 1-3 were acquired on a Varian Unity-300 (300 MHz for 1H and 75 MHz for 13C) spectrometer at 25 °C. One-dimensional data were acquired with a four-nucleus direct 5 mm probe, and two-dimensional data were acquired with an inverse broadband 5 mm probe. Additional 1H and 13C NMR spectra of 3 were acquired on a Bruker AMX-500 (500 MHz for 1H and 125 MHz for 13C) instrument. The mixing time for recording NOESY spectra was 400 ms. Data were processed using Felix 95 (Molecular Simulations, Inc.) on a Silicon Graphics Indy workstation. All two-dimensional data sets were acquired in the States mode with 2048 complex data points in t2 and 375 (zero-filled to 1024) complex data points in t1. A skewed, 45°-shifted sine-bell weighting function was used in all cases except for in NOESY, where a 90°-shifted sine-bell function was employed. Mass Spectrometry. Spectra generated with negative electrospray mass spectrometry (ESI-MS) and positive atmospheric pressure chemical ionization mass spectrometry (APCI-MS) were recorded with a Finnigan MAT TSQ7000 instrument. Spectra generated with high-resolution fast atom bombardment mass spectrometry (FAB-MS) were recorded with a JEOL HX 110 instrument equipped with a FAB (Xe) ionization gun. Oxidation of 1 (EGCG) and Isolation of Reaction Products 2 and 3. Twenty batches of 25 mg of EGCG and AMVN in

a 10-fold molar excess were dissolved in 20 mL of CH3CN and heated to 50 °C for 2.5 h. The oxidation was terminated after 2.5 h by placing the reaction mixture on ice. Groups of four batches were then combined. After evaporation of the solvent in vacuo, excess AMVN was extracted with hexane. The residue was first applied to reversed-phase MPLC using 0.15% aqueous HCOOH/CH3CN/ethyl acetate (86:12:2) as the mobile phase, yielding crude 2 and 3. Both compounds were purified by reversed-phase HPLC with 0.15% HCOOH/CH3CN (85:15) as the mobile phase to yield 17 mg of 3. Compound 2 was further purified by column chromatography on Sephadex LH-20 (Pharmacia Biotech) with CH3OH as the eluent to yield 12.5 mg of 2. EGCG (1) was isolated from green tea as a white powder as described previously (10): [R]25D -170.4 (c 0.3, CH3OH); UV (CH3OH) λmax (log ) 276 nm (4.14); IR (KBr) νmax 3370 (br), 1690, 1624, 1612, 1516, 1464, 1446, 1366, 1340, 1232, 1142, 1090, 1032, 868, 849, 814, 760, 732 cm-1; 1H NMR [dimethyl sulfoxided6 (DMSO-d6), 300 MHz] see Table 1; 13C NMR (DMSO-d6, 75 MHz) see Table 1; APCI-MS m/z 459. 1H and 13C NMR spectral data measured in acetone-d6 were reported previously (13). Compound 2 was isolated as a colorless amorphous substance: [R]25D -101.9 (c 0.16, CH3OH); UV (CH3OH) λmax (log ) 279 nm (3.96), 299 nm (sh) (3.87); IR (KBr) νmax 3430 (br), 3220, 1704 (br), 1616, 1516, 1440, 1372, 1216, 1142, 1104, 1032, 966, 860, 814, 760, 710 cm-1; 1H NMR (DMSO-d6, 300 MHz) see Table 1; 13C NMR (DMSO-d6, 75 MHz) see Table 1; negative ESI-MS m/z 471; negative high-resolution FAB-MS for C22H15O12 [M - H]- calcd 471.0564, found 471.0569. Compound 3 was isolated as a colorless amorphous substance: [R]25D -44.2 (c 0.21, CH3OH); UV (CH3OH) λmax (log ) 275 nm (4.35); IR (KBr) νmax 3450 (br), 1692 (br), 1680, 1675, 1628, 1612, 1516, 1472, 1460, 1360, 1336, 1228, 1140, 1104, 1096, 1036, 946, 886, 822, 764 cm-1; 1H NMR [CD3CN/D2O (10:1 v/v), 300 MHz] see Table 1; 13C NMR [CD3CN/D2O (10:1 v/v), 75 MHz] see Table 1; negative ESI-MS m/z 929; positive highresolution FAB-MS for C44H35O23 [M + H]+ calcd 931.1569, found 931.1620.

Results and Discussion EGCG (1) was isolated from green tea as described previously (10) and oxidized with peroxyl radicals by thermolysis (50 °C) of AMVN in oxygenated acetonitrile.

384 Chem. Res. Toxicol., Vol. 12, No. 4, 1999

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Figure 1. Reversed-phase HPLC chromatogram of reaction products derived from the oxidation of EGCG by peroxyl radicals generated by thermolysis of AMVN in oxygenated acetonitrile.

AMVN generates peroxyl radicals by thermolysis and oxygen addition:

RNdNR f 2R• + N2

(1)

2R• + 2O2 f 2ROO•

(2)

Analysis by reversed-phase HPLC with diode array UV detection (Figure 1) showed residual 1 and two major oxidation products, 2 and 3, in the reaction mixture. Compounds 2 and 3 were isolated via MPLC and HPLC and identified on the basis of their spectroscopic data. Compound 2 was isolated as a colorless, amorphous substance. The negative ESI-MS of 2 showed the molecular ion [M - H]- at m/z 471. Negative high-resolution FAB-MS showed an ion at m/z 471.0569 which corresponds to a molecular formula C22H16O12 for 2 (calcd for [M - H]- 471.0564). The UV spectrum had a maximum at λ ) 279 nm (log  ) 3.96) and a shoulder at 299 nm. The optical rotation [R]25D was -101.9 (c 0.16, CH3OH). The 1H and 13C NMR spectra of 2 were similar to those of 1 (EGCG) (Table 1). The two signals at δH ) 5.97 and 5.88 ppm (both d, J ) 2 Hz) were assigned to the two meta-coupled aromatic protons of ring A (Figure 2). The two broad singlets at δH ) 5.34 and 5.18 ppm and the signals at δH ) 2.93 ppm (dd, J ) 4, 18 Hz) and δH ) 2.78 ppm (dd, J ) 3, 18 Hz) were similar to protons H-3, H-2, H-4a, and H-4b, respectively, of ring C of 1. Proton H-2 in 2 showed a downfield shift of 0.2 ppm when compared with H-2 in 1 (Table 1). While in catechins, in which H-2 and H-3 are in the trans configuration, H-2 appears as a 7-8 Hz doublet, the signal of H-2 in 2 was a broad singlet typical for epicatechins in which H-2 and H-3 are in the cis configuration (13). Supported by the observed nuclear Overhauser effets (NOE) between H-2 and H-3 in 2, the signal of H-2 indicated an unchanged stereochemistry in positions 2 and 3 in 2 when compared to those in 1. Therefore, 1 and 2 belong to the (-)epicatechin group, for which a 2R,3R absolute configuration was proven with the crystal structure (14). The singlet at δH ) 6.90 ppm (2H) was assigned to H-2′′ and H-6′′ of the galloyl moiety. All HMQC, HMBC, and DQFCOSY correlations of the protons mentioned above were identical to those observed for 1, suggesting that rings A and C and the galloyl moiety of 1 are still intact in 2

Figure 2. Structures of 1 (EGCG), oxidation products 2 and 3, 4 (EGC), and 5 (methyl gallate).

Figure 3. HMBC correlations (C f H) of the central part of 2.

and that the changes in the molecule had only occurred in ring B of 1. For the remaining unidentified part of molecule 2, the 1 H and 13C NMR data together with the high-resolution FAB-MS data indicated two olefinic carbons with their correlated protons, four quaternary carbons, and four oxygens, one of which is a hydroxyl. To connect these atoms with each other in the modified B ring and to ring C, five degrees of unsaturation were available. For the modified B ring of 2, the1H NMR spectrum exhibited two signals at δH ) 7.17 ppm (d, J ) 1 Hz, H-2′) and δH ) 6.31 ppm (br s, H-6′) which had HMQC correlations to carbons at δC ) 105.5 ppm (C-2′) and δC ) 111.2 ppm (C-6′), respectively. To the proton H-2′ (δH ) 7.17 ppm), HMBC cross-peaks were observed from two quaternary carbons at δC ) 157.9 ppm (C-3′) and δC ) 160.9 ppm (C-4′), from C-2 of the C ring (δC ) 74.4 ppm) and from the olefinic carbon at δC ) 111.2 ppm (C-6′) (Figure 3). Proton H-6′ (δH ) 6.31 ppm) had HMBC correlations with C-2 of the C ring, with the tertiary carbon at δC ) 105.5 ppm (C-2′), and with a quaternary carbon at δC ) 161.8 ppm (C-5′). In addition, HMBC correlations were observed between H-2 and a quaternary carbon at δC ) 155.9 ppm (C-1′). The DQF-COSY experiment revealed allylic couplings between H-6′ and H-2′ and between H-6′ and H-2. In a NOESY experiment, cross-peaks from both B-ring protons H-2′ and H-6′ to

Oxidation of (-)-Epigallocatechin Gallate

the two galloyl protons H-2′′ and H-6′′ and to H-2 were observed. An additional cross-peak was observed between H-6′ and H-3. On the basis of this evidence, we assigned 2 the structure of the anhydride shown in Figure 2. Unsaturated seven-membered ring anhydrides or muconic acid anhydrides are known to be stable oxidation products of o-benzoquinones (15) or catechols (16), and comparison of NMR data of 2 with those reported for muconic acid anhydrides supports the structure of 2. In the IR spectrum of 2, the typical carbonyl bands of muconic acid anhydrides at around 1780 and 1740 cm-1 were not observed. When compared with the IR spectrum of 1, which showed the carbonyl absorption of the galloyl ester as a sharp medium strong band at 1698 cm-1, the IR spectrum of 2 showed an intensified and broadened carbonyl band with maxima at 1724 and 1704 cm-1. This shift to lower frequencies of the anhydride carbonyl bands could be due to the hydroxy group in the ortho position with respect to the carbonyl, which is known to cause 30-60 cm-1 shifts to lower frequencies in hydroxypyrones (17, 18) or hydroxynaphthoquinones (19). Compound 3 was isolated as a colorless, amorphous substance with a UV maximum at λ ) 275 nm (log  ) 4.35) and an optical rotation [R]25D of -44.2 (c 0.21, CH3OH). As with compound 2, the NMR spectral data indicated that rings A and C as well as the galloyl moiety of 1 are still intact in 3 and that all changes in the molecule again occurred solely in ring B (Table 1). The stereochemistry of positions 2 and 3 of ring C in 3 was apparently unchanged when compared to that of 1 on the basis of the signal shape of H-2 and similar NOEs between protons H-2 and H-3 in the cis configuration in 3. The 13C NMR spectrum of 3 showed a total of 22 signals. The molecular formula of 3 was determined to be C44H34O23 by both negative ESI-MS and positive highresolution FAB-MS (m/z calcd for [M + H]+ 931.1569, found 931.1620), indicating that 3 is a symmetrical dimer, whose monomer units are joined at their B rings. Examination of the spectroscopic data of the central part of 3, which includes the connection between the two monomer units formed from their B rings, identified a symmetrical C12H8O7 moiety to which the two unchanged galloyl-substituted C and A rings of the starting material are connected. Eight unsaturation equivalents were available for this central partial structure. For the central moiety of 3, the 1H NMR spectrum showed only two signals at δH ) 6.44 ppm (d, J ) 1 Hz, H-6′) and at δH ) 3.23 ppm (br s, H-2′), which had HMQC correlations to carbons at δC ) 127.5 ppm (C-6′) and δC ) 59.1 ppm (C-2′), respectively. Both H-2′ and H-6′ showed cross-peaks to H-2 in a DQF-COSY experiment and cross-peaks to H-2 and H-3 in a NOESY experiment. In addition, NOE correlations were observed between H-6′ and H-2′′ and H-6′′ of the galloyl moiety. In the HMBC spectrum, H-6′ showed cross-peaks with C-2 and C-2′, and with a quaternary carbon at δC ) 85.8 ppm (C4′) whose chemical shift indicated a single oxygen substituent (Figure 4). Proton H-2′ showed correlations with C-6′, C-2, and C-4′, with a carbonyl at δC ) 197.0 ppm (C-5′), and with a quaternary carbon at δC ) 105.0 ppm (C-3′), whose chemical shift was an indication of two oxygen substituents. In addition, HMBC correlations were observed between H-2 and C-2′, between H-2 and C-6′, and between H-2 and a quaternary carbon at δC )

Chem. Res. Toxicol., Vol. 12, No. 4, 1999 385

Figure 4. HMBC correlations (C f H) of the central part of 3.

156.6 ppm (C-1′), indicating the connection between ring C and the central part of 3. The carbon chemical shifts for the central part of 3 indicated one carbonyl and one olefinic double bond per monomer. When the eight degrees of unsaturation calculated for the central part were considered, the atoms of the C12H8O7 moiety must be connected within four rings. On the basis of the HMBC correlation observed for 3, we assigned the structure shown in Figure 2. As part of cage structures, 1,4-hydroxylated 1,4-oxygenbridged six-membered rings are known to be stable (2024). The NMR data of the central oxygen-bridged ring of 3 are similar to those of a comparable known synthetic compound (25). Because all structural changes during the oxidation of 1 occurred solely in the B ring, it is reasonable to assume that for the two monomer moieties in 3 the stereochemistry of R-configured positions 2 and 3 in the C rings remains unchanged, which is supported by the similar signal shape of H-2 and the similar NOE correlations between H-2 and H-3 in 3 and 1. Furthermore, 3 cannot possess mirror plane symmetry since it is optically active. The observance of only 22 sharp signals in the 13C NMR spectrum for the 44-carbon dimer can be explained by a C2 symmetry of the molecule. Two diastereomers in which the chiral centers at position 2′ in both monomer units are either both R-configured or both S-configured are possible. However, HPLC and 13C NMR data did not indicate a mixture of diastereomers. The identification of products 2 and 3 provides the first unambiguous proof that the principal site of antioxidant reactions on the EGCG molecule (1) is the trihydroxyphenyl B ring. Determinants of catechin reactivity toward oxidants are the presence of 3′- and 4′-hydroxy groups on the B ring (6, 7). Incorporation of an additional 5′-hydroxy group in the B ring and the ester linkage via the 3-OH to gallic acid further increase reactivity (7). The work of Jovanovic et al. (9) indicated that the phenoxyl radical of epigallocatechin (4) exhibits a one-electron reduction potential of 0.43 V, whereas that of methyl gallate (5) exhibits a one-electron reduction potential of 0.56 V. Our data thus confirm the view that the B ring, rather than the 3-galloyl moiety, is the principal site of antioxidant reactivity in EGCG (1). Nanjo et al. (8) reported that both the trihydroxyphenyl B ring and the 3-galloyl moiety of EGCG (1) were involved in reactions with the diphenylpicrylhydrazyl radical. Although reaction at the 3-galloyl moiety was inferred from the greater effectiveness of EGCG compared to epigallocatechin (4) in quenching the diphenylpicrylhydrazyl electrospin resonance signal, oxidation of the 3-galloyl moiety in EGCG was not confirmed by product analysis. It nevertheless seems likely that oxidation of the 3-galloyl moiety could

386 Chem. Res. Toxicol., Vol. 12, No. 4, 1999

occur in the presence of an excess quantity of the relatively stable diphenylpicrylhydrazyl radical. Indeed, we have observed that methyl gallate reacts with peroxyl radicals under the same conditions we used to oxidize EGCG, albeit at a greatly reduced rate. Although further work is needed to establish the chemistry of EGCG phenoxyl radicals, the structures of products 2 and 3 allow two inferences to be drawn. First, the EGCG phenoxyl radical apparently does not form stable addition products with peroxyl radicals, as has been observed for simple phenolic antioxidants (26, 27) (e.g., butylhydroxytoluene) and R-tocopherol (28-30). Second, competing reactions consume the EGCG phenoxyl radical, as two different products have been identified. These reaction pathways may involve either radical or cationic intermediates and certainly involve the addition of oxygen, either from water or from molecular oxygen. The disproportionation of EGCG radicals or electron transfer from EGCG radicals to peroxyl radicals may lead to the formation of cationic intermediates. Work currently being carried out in our laboratories addresses these mechanistic questions. The identification of product structures provides potential markers for EGCG antioxidant reactions. Although reaction with peroxyl radicals certainly is among the most probable antioxidant reactions of polyphenolic compounds in biological systems, reactions with other oxidants also may occur. Accordingly, we are investigating the reaction of EGCG with other biologically relevant oxidants, including singlet oxygen, peroxynitrite, and hydroxyl radicals. The identification of oxidant-specific products together with the application of sensitive and specific analytical methods may provide analytical approaches to evaluating the antioxidant actions of catechins in biological systems.

Acknowledgment. We thank J. H. Wertheim, Tea Importers, Inc., and the Royal Estates Tea Company, Division of Thomas J. Lipton, for providing green tea leaves, Dr. Arpad Somogyi for the determination of FAB mass spectra, and Dr. Thomas McClure of the Southwest Environmental Health Sciences Core whose help in the acquisition of the ESI-MS data is appreciated. This investigation was supported by NIH grants CA75599 and ES06694.

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