SEPTEMBER 2000 VOLUME 13, NUMBER 9 © Copyright 2000 by the American Chemical Society
Articles Antioxidant Chemistry of Green Tea Catechins. New Oxidation Products of (-)-Epigallocatechin Gallate and (-)-Epigallocatechin from Their Reactions with Peroxyl Radicals Susanne Valcic, Jeanne A. Burr, Barbara N. Timmermann, and Daniel C. Liebler* Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721 Received April 10, 2000
The green tea catechins (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) react with peroxyl radicals generated by thermolysis of the azo initiator 2,2′-azobis(2,4dimethylvaleronitrile) (AMVN) to produce several oxidation products. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. We isolated and identified a previously unreported reaction product of EGCG and three reaction products of EGC. In the EGCG product, the B-ring was transformed into a ring-opened unsaturated dicarboxylic acid moiety. The EGC products include a seven-membered B-ring anhydride and a symmetrical EGC dimer, both analogues of previously described EGCG oxidation products. The third EGC product was an unsymmetrical dimer. In all identified products, changes occurred solely in the B-ring of EGCG or EGC. This confirmed our previous observation that the principal site of antioxidant reactions in EGCG and EGC is the trihydroxyphenyl B-ring, regardless of the presence of a 3-galloyl moiety. A stoichiometric factor n of 4.16 ( 0.51 was measured for EGCG, whereas factors of 2.20 ( 0.26 was found for EGC and 2.33 ( 0.18 measured for methyl gallate. These values represent the net peroxyl radical trapping per catechin molecule by several competing reactions. EGCG and EGC oxidation involves addition of oxygen, which is not derived from water, but most likely from atmospheric oxygen via peroxyl radicals. Characteristic oxidation products may be useful markers for antioxidant actions in living systems.
Introduction Green tea, a popular beverage brewed from dried leaves of the tea bush (Camellia sinensis), is distin* To whom correspondence should be addressed. Phone: (520) 6264488. Fax: (520) 626-2466. E-mail:
[email protected].
guished by the presence of a group of polyphenols called flavanols or catechins (1). In recent years, the principal green tea catechins, i.e., EC,1 EGC (5), ECG, and EGCG (1), have been recognized to be effective protectants against certain forms of cancer (2, 3). Although the human epidemiology remains inconclusive, catechins display remarkable cancer preventive effects in several
10.1021/tx000080k CCC: $19.00 © 2000 American Chemical Society Published on Web 08/11/2000
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animal models (4, 5). Unlike most flavonoids, catechins exist in tea as aglycones, and despite metabolism by methylation, sulfation, and glucuronidation, catechins are found in blood and tissues following oral ingestion (6-10). The cancer preventive effects often have been attributed to antioxidant actions (11-19). Previous structure-activity studies indicated that the presence of a galloyl ring in the 3-position and a trihydroxyphenyl B-ring are most important for the antioxidant activities of catechins (13, 19, 20). We recently reported the structure of two major oxidation products (3 and 4) of EGCG (1) formed by reactions with peroxyl radicals generated from the azo initiator AMVN (21). The identification of these structures provided the first unambiguous evidence that antioxidant reactions of EGCG (1) with peroxyl radicals involve the trihydroxyphenyl B-ring, rather than the 3-galloyl moiety. In further work on antioxidant chemistry of catechins, we have identified an additional stable oxidation product (2) of EGCG (1) and have extended our studies to include EGC (5) and its oxidation products (68). We also have performed initial studies aimed at elucidating the mechanisms of formation of two major oxidation products of EGCG. We also compared the antioxidant stoichiometries of both catechins and the structurally related methyl gallate in peroxyl radical scavenging reactions.
Experimental Procedures Chemicals and Instrumentation. Green tea was provided by the Royal Estates Tea Co., Division Thomas Lipton. EGCG and EGC were isolated as described previously (22). AMVN was purchased from Polysciences, Inc. (Warrington, PA). H218O was from Isotec, Inc. (Miamisburg, OH). CD3OD, DMSO-d6, and TMS were from Aldrich Chemical Co. (Milwaukee, WI). Gallic acid methyl ester was purchased from Sigma Chemical Co. (St. Louis, MO). BHT was from Aldrich, and methyl linoleate was from NuChek-Prep (Elysian, MN). CH3CN (0.0002% H2O) was from EM Science (Gibbstown, NJ), and all other HPLC grade CH3CN was from Sigma Chemical Co. The polarimeter and the MPLC and HPLC systems used for this study are described in ref 21. The reversed phase HPLC column was an Alltech Econosphere C18 model (10 µm, 10 mm × 250 mm). Ratios of solvent mixtures are volume to volume. The three-dimensional structure model that was used was HGS Molecular Structure Models Type B Set from Maruzen Company Ltd. (Tokyo, Japan). NMR Spectroscopy. NMR samples were dissolved in 0.6 mL of deuterated solvent (Tables 1 and 2), and all spectra were referenced to internal TMS at 0 ppm (1H and 13C). 1H and 13C, HMQC, HMBC, DQF-COSY, and NOESY spectra (23, 24) of 6 were acquired on a Varian Unity-300 (1H, 300 MHz; 13C, 75 MHz) spectrometer at 25 °C. For compounds 7 and 8, 1H NMR and two-dimensional NMR spectra were acquired on a Bruker DRX-600 instrument, and for compound 2, a Bruker DRX-500 spectrometer was used. 13C NMR spectra of 2, 7, and 8 were acquired on a Bruker AMX-500 spectrometer (13C, 125 MHz). The mixing time for the ROESY spectrum of 2 was 200 ms, for 1 Abbreviations: AMVN, 2,2′-azobis(2,4-dimethylvaleronitrile); BHT, 2,6-di-tert-butyl-4-methylphenol; DMSO, dimethyl sulfoxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DQF-COSY, double-quantum-filtered correlation spectroscopy; EC, (-)-epicatechin; ECG, (-)-epicatechin gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin gallate; ESI-MS, electrospray ionization mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; HMBC, heteronuclear multiplebond correlation; HMQC, heteronuclear multiple-quantum coherence; HSQC, heteronuclear single-quantum coherence; MPLC, mediumpressure liquid chromatography; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TMS, tetramethylsilane.
Valcic et al. Table 1. 1H NMR,
13C
NMR, and HMBC Data of Product 2 in DMSO-d6 2 (DMSO-d6)
position 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 1′′ 2′′/6′′ 3′′/5′′ 4′′ COO
HMBC
δC
δH
73.7 64.9 25.0
4.96 (br s) 5.48 (br s) 2.87 (dd, 4, 17) 2.70 (br d, 17)
1′, 2′, 3′, 4 4a, COO 4a, 5, 8a 2, 3, 4a, 5, 8a
5.96 (d, 2)
4a, 7, 8
5.86 (d, 2)
4a, 6, 7, 8a
6.31 (d, 1.5)
2, 2′, 4′
6.83 (s)
1′′, 3′′/5′′, 4′′, 6′′/2′′, COO
97.0 156.1 95.7 156.3 94.1 154.3 166.3 141.5 125.0 166.1 119.0 108.5 145.1 138.3 164.9
the NOESY spectra of 6 and 8 400 ms, and for the NOESY spectrum of 7 800 ms. Data were processed using Felix 95 (Molecular Simulations, Inc.) on a Silicon Graphics Indy workstation. Mass Spectrometry. LC/MS analyses were performed on a Finnigan MAT TSQ7000 instrument equipped with an HP-1050 HPLC system and an atmospheric pressure ionization source for both positive and negative ESI and APCI. For high-resolution FAB-MS, a JEOL HX 110 instrument equipped with a FAB (Xe) ionization gun was used. Oxidation of EGCG and EGC and Isolation of Reaction Products 2 and 6-8. The oxidation of EGCG (20 × 25 mg) with peroxyl radicals generated from AMVN by thermolysis at 50 °C in oxygenated CH3CN and the workup of the reaction mixture were described in detail previously (21). The EGCG oxidation products were initially separated by reversed phase MPLC using a 0.15% aqueous HCOOH/CH3CN/ethyl acetate mixture (86:12:2) as the mobile phase, yielding crude 2-4. The purification of compounds 3 (12.5 mg) and 4 (17 mg) was described in ref 21. Compound 2 was further purified on Sephadex LH-20 (Pharmacia Biotech) using 100% CH3OH as the eluent followed by HPLC with a 0.15% aqueous HCOOH/ CH3CN mixture (90:10) as the mobile phase to yield 19.4 mg of 3. For the oxidation of EGC (24 × 25 mg), the same procedures were used as described for EGCG (21). EGC oxidation products 6-8 were separated by reversed phase HPLC with a CH3CN/ 0.15% aqueous HCOOH gradient starting with 8% CH3CN for 3 min, and then the mobile phase was programmed to 11% CH3CN over 8 min, held at 11% for 3 min, programmed to 15% CH3CN over 5 min, held at 15% CH3CN for 5 min, and then followed by a final 100% CH3CN wash. Compound 7 was further purified on HPLC with an eluent program consisting of H2O/ CH3CN (92:8) for 3 min followed by a gradient to 11% CH3CN over 8 min, then held at 11% CH3CN for 2 min, and followed by a 100% CH3CN wash. Compound 8 was rechromatographed via HPLC with a CH3CN/0.15% aqueous HCOOH gradient starting with 8% CH3CN for 3 min, followed by a gradient to 11% CH3CN over the course of 17 min. Compounds 6-8 were further purified on Sephadex LH-20 with 100% CH3OH as the eluent to yield 21 mg of 6, 7 mg of 7, and 11 mg of 8. EGCG (1) (see ref 21). Compound 2: [R]25D -85.1° (c 0.2, CH3OH); UV (CH3OH) λmax (log ) 276 nm (3.87). For 1H and 13C NMR (DMSO-d , 500 and 125 MHz) data, see Table 1. EGC 6 (5): [R]25D -57.5° (c 0.3, CH3OH). For 1H and 13C NMR (CD3OD, 300 and 75 MHz) data, see Table 2. Compound 6: [R]25D -8.6° (c 0.7, CH3OH); UV (CH3OH) λmax (log ) 269 nm (sh) (3.79). For 1H and 13C NMR (CD3OD, 300 and 75 MHz) data, see Table 2. Compound 7: [R]25D -61.8° (c 0.2, CH3OH); UV (CH3OH) λmax (log ) 274 nm (sh) (3.75). For 1H NMR (CD3OD, 600 MHz)
Reactions of EGCG and EGC with Peroxyl Radicals Table 2. 1H NMR, EGC (5) (CD3OD)
13C
NMR, and HMBC Data of EGC (5) and 6-8
6 (CD3OD)
7 (CD3OD)
position
δC
2 2′′′ 3 3′′′ 4
79.9
4.74 (br s)
78.7 4.32 (br s)
1′, 2′, 6′
67.5
4.15 (m)
63.2 4.37 (m)
4a
29.2
2.83 (dd, 4, 17) 2.71 (dd, 3, 17)
29.5 2.84-2.72 (m) 4a, 5
δH
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 803
δC
δH
HMBC
4′′′
δC
δH
80.4 79.8 63.6 63.1 28.7
4.65 (br s) 4.38 (br s) 4.69 (m) 4.37 (m) 2.89 (dd, 5, 17) 2.84 (dd, 3, 17) 2.78 (dd, 3, 17)
29.4
8 (DMSO-d6) HMBC
8a, 1′, 2′, 6′ 1′′, 6′′ 4a 4a′′′ 2, 3, 4a, 5, 8a
δC
HMBC
δH
76.2 4.87 (br s)
8a, 1′, 2′, 6′
63.6 4.20 (m)
4a
27.8 2.71 (dd, 4,17) 3, 4a, 5, 8a 2.53 (dd, 2,17) 2, 3, 4a, 5, 8a
2′′′, 3′′′, 4a′′′, 5′′′, 8a′′′
2.75 (dd, 4, 17) 4a 4a′′′ 5 5′′′ 6 6′′′ 7 7′′′ 8 8′′′ 8a 8a′′′ 1′ 1′′ 2′
100.0
99.7
157.9
156.2
96.3a 5.92 (d, 2) 157.9
95.9 5.94 (d, 2) 157.7
95.8a 5.90 (d, 2)
96.9 5.95 (d, 2)
157.6
157.8
131.5
158.2
106.9
5, 7, 8
6.51 (s)
61.0 3.26 (s)
4a, 6, 7
2, 1′, 3′, 4′, 5′, 6′
2′′ 3′ 3′′ 4′ 4′′ 5′ 5′′ 6′ 6′′ a
146.7
105.2
132.5
86.2
146.7
197.7
106.9
6.50 (s)
128.1 6.44 (d, 2)
2, 1′, 4′
99.8 99.9 158.1 158.04 96.93 96.86 158.05 157.9 95.78 95.97
98.3 156.6 5.99 (d, 2) 5.96 (d, 2)
95.9 5.94 (d, 2)
4a, 5/7, 8
156.6 5.98 (d, 2) 5.94 (d, 2)
156.1 156.4 158.2 158.5 54.6
3.71 (br s)
49.9
3.16 (br s)
199.7 97.8 85.4 84.3 111.7 192.7 125.8 124.9
4a, 7, 8 4a′′′, 5′′′, 8′′′
6, 8a 4a′′′, 6′′′, 7′′′, 8a′′′
94.2 5.80 (d, 2)
4a, 6, 7, 8a
154.5 156.6 2, 1′, 4′, 5′, 108.2 7.16 (br s) 6′, 2′′, 3′′, 4′′ 3′, 4′, 5′, 1′′, 3′′, 4′′, 6′′, 2′′′ 156.7
2, 4′, 6′
161.0 160.9 6.67 (br s) 6.58 (d, 1)
2, 2′, 4′ 2′′, 3′′, 4′′, 2′′′
114.2 6.42 (br s)
2, 1′, 2′, 5′
Assignments interchangeable.
and 13C NMR (CD3OD, 125 MHz), see Table 2. Compound 8: [R]25D -33.4° (c 0.5, CH3OH); UV (CH3OH) λmax (log ) 278 (sh) (3.59), 301 nm (3.67). For 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d , 125 MHz) data, see Table 2. 6 Influence of Increasing H2O Concentrations in the Reaction Solvent on the Oxidation of EGCG. EGCG (11 µmol) and AMVN (110 µmol) were dissolved in 9 mL of CH3CN (0.0002% H2O)/H2O mixtures (100:0, 99:1, 95:5, 90:10, and 80: 20) and heated in 10 mL screw-cap vials to 50 °C for 1 h. The reactions were terminated by placing the reaction mixtures on ice. Acetonitrile and water were evaporated in vacuo. The residues were treated with hexane to dissolve excess AMVN which was then separated from the hexane insoluble product mixtures by membrane filtration (0.2 µm Nylon 47 Filter Membranes, Alltech Associates, Inc., Deerfield, IL). The reaction mixtures were rinsed from the membranes with CH3CN, dried, and redissolved in a 500 µL HPLC mobile phase (0.15% aqueous HCOOH/CH3CN mixture), of which 50 µL was injected. Oxidation of EGCG in Acetonitrile and H218O. EGCG (11 µmol) and AMVN (110 µmol) were dissolved in 9 mL of a mixture of CH3CN and H218O (99:1) and heated in a 10 mL screw-cap vial to 50 °C for 1 h. The CH3CN used to prepare these solutions contained 0.0002% residual H2O. The reaction mixture was placed on ice to terminate the reaction, and excess AMVN was extracted with hexane. Simultaneously, control oxidations were conducted in a mixture of CH3CN and H2O (99:1) under the same conditions described above. Both reaction mixtures were dissolved in the mobile phase consisting of H2O and CH3CN (85: 15) and analyzed by reversed phase LC/MS using negative ESI. Effect of the Peroxyl Radical Generation Rate on EGCG Product Distribution. EGCG (11 µmol) and AMVN in five different molar concentration ratios (1:1, 1:2.5, 1:5, 1:10, and 1:20) were dissolved in 5 mL of CH3CN and the mixtures
heated to 50 °C in screw-cap vials. Samples (200 µL) were taken after 15, 30, 45, 60, 90, and 120 min and placed on ice prior to further workup. After evaporation of CH3CN under N2, excess AMVN was extracted from the samples with hexane by membrane filtration as described above. The hexane insoluble reaction products were rinsed from the membranes with CH3CN, dried, and redissolved in a 200 µL HPLC mobile phase (0.15% aqueous HCOOH/CH3CN mixture), of which 20 µL was analyzed. Antioxidant Stoichiometry Measurements. The antioxidant stoichiometry of radical trapping was measured by the kinetic methods previously described by Boozer et al. (25) and modified by Liebler and Burr (26). The accumulation of lipidconjugated dienes, rather than the consumption of oxygen, was used to monitor oxidation of a lipid substrate and its inhibition by antioxidants. Methyl linoleate, antioxidant (BHT, EGCG, EGC, or methyl gallate), and AMVN were dissolved in acetonitrile preheated to 50 °C to start the reaction. Reaction mixtures contained 200 µmol of methyl linoleate, 20 µmol of AMVN, 0.4 µmol of BHT (or 200 nmol of EGCG, 300 nmol of EGC, and 300 nmol of methyl gallate) in a volume of 3 mL. To monitor substrate oxidation, 10 µL samples were removed periodically from the reaction mixture and diluted with 1 mL of acetonitrile, and the absorbance of the sample at 236 nm was measured. Plots of conjugated diene absorbance versus time were used to determine the length of induction period τ. Four experiments were performed for the antioxidant standard BHT, from which the rate of chain initiation Ri was calculated (0.0082 mM min-1). Then, seven to nine experiments were carried out in which EGCG, EGC, or methyl gallate was substituted for the antioxidant standard and their stoichiometric factors for peroxyl trapping n were calculated from eq 1 (see Results).
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Figure 1. Reversed phase HPLC chromatograms of reaction products derived from the oxidation of EGCG (A) and EGC (B) by peroxyl radicals generated by thermolysis of AMVN in oxygenated acetonitrile at 50 °C.
Results Oxidation Products of EGC and EGCG. EGC and EGCG were isolated from green tea (22) and oxidized with peroxyl radicals generated by thermolysis of AMVN in oxygenated acetonitrile as described previously for EGCG (21). Figure 1 shows reversed phase HPLC chromatograms of EGCG (1) and EGC (5) reaction products. The structures of the EGCG oxidation products 3 and 4 were reported previously (21). Compound 2 was isolated as a colorless amorphous compound. The negative ESI-MS of 2 showed the molecular ion [M - H]- at m/z 447. Negative high-resolution FAB-MS confirmed the [M - H]- ion at m/z 447.0561 (calcd 447.0563) corresponding to the molecular formula C20H16O12 for 2. Further structural information was obtained from negative ESI-MS/MS analysis of the ion at m/z 447. The presence of the [M - H - 152]- ion at m/z 295 that corresponded to the ion created after cleavage of the gallic acid ester and the occurrence of the galloyloxy ion at m/z 169, typical for galloylated catechins (EGCG and ECG) (27), suggested that the galloyl moiety in 2 remained unchanged when compared to 1. The ions at m/z 429, 251, and 233 reflected the loss of H2O from the [M - H]- ion and the losses of CO2 and H2O from the [M - H - galloyl]- ion. Also, the 1H and 13C NMR data of 2 were similar to those of EGCG in regard to rings A and C as well as the galloyl moiety (21). The HSQC, HMBC, and DQF-COSY correlations of 2 showed that all the connectivities of the A- and C-rings and the gallic acid ester function were the same as for 1, suggesting that, as for 3 and 4 (21), the only changes in the molecule had occurred in ring B. The signal shape of the C-ring protons H-2 and H-3 (both broad singlets) and the observed cross-peak between the two protons in the ROESY spectrum indicated an unchanged stereochemistry in positions 2 and 3 in 2 compared to 1, an observation we previously also reported for oxidation products 3 and 4 (21). For the remaining part of the molecule, the 13C NMR spectrum exhibited the signals of three quaternary and one tertiary carbons at δC 141.5, 166.1, 166.3, and 125.0, respectively. In the HSQC experiment, the tertiary carbon at δC 125.0 correlated with an olefinic proton at δH 6.31 (d, J ) 1.5 Hz) that exhibited a DQF-COSY cross-peak to H-2 of ring C (δH 4.96). Both protons also exhibited HMBC cross-peaks with their respective HSQC-correlated carbons and to the three remaining quaternary carbons at δC 141.5, 166.3, and 166.1. The MS data, the chemical shifts, and the
Valcic et al.
correlations observed in the two-dimensional NMR experiments led us to the conclusion that ring B of 1 had opened to an unsaturated dicarboxylic acid after the loss of two carbons. ROESY cross-peaks between C-ring protons H-2 and H-3 and the olefinic proton at δH 6.31 and between the latter proton and H-2′′ and H-6′′ of the galloyl moiety revealed a Z-configuration of the dicarboxylic acid moiety. On the basis of all spectroscopic data, we assigned 2 the structure shown in Figure 2. From the EGC (5) oxidation mixture, compounds 6-8 were isolated by HPLC as amorphous substances. The 1 H and 13C NMR spectra of 6 were similar to those of the EGCG oxidation product 4 (21), except for the missing signals of the galloyl moiety (Table 2). With negative and positive FAB-MS, no molecular ion could be observed but the negative ESI-MS of 6 revealed a molecular mass of 626 Da, which corresponded to the molecular formula C30H26O15. The difference of 304 Da between the molecular mass of 6 and that of EGCG oxidation product 4 (930 Da) corresponds to the lack of two galloyl moieties (152 Da twice) in 6 when compared to 4. The presence of only 15 carbon signals in the 13C NMR spectrum and only eight proton signals in the 1H NMR spectrum together with the observed molecular mass of 626 Da indicated that 6 was a symmetrical dimeric oxidation product of EGC (5). The 1H and 13C NMR spectra of 6 revealed that, as with the EGCG oxidation products 2-4, the A- and C-rings again remained unchanged when compared to those of 5. For the dimer-connecting B-ring, all one- and two-dimensional NMR data of 6 corresponded to those observed for 4. On the basis of the spectroscopic evidence, we concluded that 6 was the EGC analogue of EGCG oxidation product 4 (Figure 2). The C2 symmetry of the molecule caused the appearance of only half of the carbon signals in the 13C NMR spectrum of 6. As with 4, 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 for 6. The negative ESI mass spectrum of product 7 exhibited molecular ion [M - H]- at m/z 625. This ion [M - H]was also observed by high-resolution negative FAB-MS at m/z 625.1165 (calcd 625.1193), corresponding to the molecular formula C30H26O15 for 7, the same molecular composition as determined for 6. While the 1H and 13C NMR spectra of 6 exhibited signals for only eight protons and for only half of the 30 carbons due to the C2 symmetry of the molecule, the 1H and 13C NMR spectra of 7 revealed signals of 16 protons and of all 30 carbons. The 1H and 13C NMR chemical shifts indicated that 7 was another dimeric EGC oxidation product, similar to 6, but unsymmetrically connected. The 1H NMR data revealed the signals of two sets of A- and C-ring protons. The four doublet signals between δH 5.99 and 5.94 were assigned to H-6 and H-6′′′ and H-8 and H-8′′′ of two A-ring moieties (A1 and A2, Figure 2). The two multiplets at δH 4.69 and 4.37, the two broad singlets at δH 4.65 and 4.38, and the signals at δH 2.89 (dd, J ) 5, 17 Hz), 2.84 (dd, J ) 3, 17 Hz), 2.78 (dd, J ) 3, 17 Hz), and 2.75 (dd, J ) 4, 17 Hz) were assigned to two sets of H-3 and H-3′′′, H-2 and H-2′′′, and H-4a+b and H-4′′′a+b protons, respectively. The HSQC and HMBC connectivities of these protons were similar to those observed for 6, indicating that 7 had two sets of unchanged A- and C-rings (A1 and A2, and C1 and C2) and that oxidation had again occurred solely on the B-ring, as seen with the other oxidation products. When compared to those of 6,
Reactions of EGCG and EGC with Peroxyl Radicals
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 805
Figure 2. Structures of EGCG (1), EGC (5), and their oxidation products 2-4 and 6-8.
Figure 3. HMBC correlations (C f H) of the central part of 7.
the spectra of 7 not only exhibited two sets of A- and C-rings but also exhibited twice as many signals for the connecting (B1 and B2 ring) moiety. Four protons were observed for the connecting moiety of 7. Two olefinic protons were at δH 6.67 (br s, H-6′) and δH 6.58 (d, J ) 1 Hz, H-6′′) with HSQC correlations to carbons at δC 125.8 and 124.9, respectively. The protons at δH 3.71 (br s, H-2′) and δH 3.16 (br s, H-2′′) were correlated to carbons at δC 54.6 and 49.9. HMBC correlations (Figure 3) between H-2 and H-2′′′ of each C-ring moiety and the above-mentioned carbons at δC 125.8/124.9 and δC 54.6/49.9 indicated that the protons at δH 3.71 and δH 3.16 and those at δH 6.67 and δH 6.58 were located in positions 2′ and 2′′and 6′ and 6′′, similar to H-2′ and H-6′ in 6. However, the DQFCOSY cross-peak between both protons at δH 3.71 (H-2′) and δH 3.16 (H-2′′) and HMBC correlation between H-2′ (δH 3.71) and carbon at δC 49.9 (C-2′′) indicated a vicinal relationship between the protons at H-2′ and H-2′′ in 7, which was not the case for the H-2′ protons (δH 3.26) of both monomer moieties in 6. The small coupling constant between H-2′ and H-2′′ in 7 (both signals are broad singlets) is due to an almost 90° dihedral angle between the two protons. Additionally, the 13C NMR spectrum of 7 revealed the presence of two keto groups (δC 199.7 and 192.7) and six quaternary carbons at δC 158.2, 158.5, 85.4, 84.3, 111.7, and 97.8. On the basis of HMBC correlations (Figure 3), we could assign the carbons at δC 158.2 (C-1′) and δC 158.5 (C-1′′) as those that connect the B-rings to the C-rings. In addition to the HMBC correlation mentioned above, H-2′ and H-2′′ both showed cross-peaks with carbons at δC 111.7, 97.8, 85.4, and 84.3.
Figure 4. Selected NOESY correlations of 7.
But the proton at δH 3.16 (H-2′′) also showed an additional HMBC correlation to the carbonyl carbon at δC 199.7. For the olefinic protons at δH 6.67 (H-6′) and δH 6.58 (H-6′′), additional HMBC cross-peaks were observed between H-6′ and the carbon at δC 85.4 and between H-6′′ and carbons at δH 84.3 and 97.8. When compared to the C-2′ (δC 61.0) in 6, the two corresponding carbons in 7, C-2′ (δC 54.6) and C-2′′ (δC 49.9), had experienced upfield shifts of 6.4 and 11.1 ppm, respectively, due to the lack of vicinal hydroxyl groups for C-2′ and C-2′′ in 7. A smaller upfield shift for C-2′ than for C-2′′ is expected because of the neighboring carbonyl function (C-3′) for C-2′. A 13C NMR chemical shift of δC 199.7 seems unusually low for a saturated keto group; however, similar resonances have been observed for saturated carbonyl functions especially with oxygen substituents in their vicinity (28, 29). The downfield shift of C-5′ (δC 111.7) in 7 can be explained by its allylic character compared to the corresponding C-3′ in 6. On the basis of the spectroscopic evidence, we assigned 7 the structure shown in Figure 2. As with the symmetrical molecule 6, the chiral centers at positions 2′ and 2′′ in 7 can be either both R- or both S-configured. Because of the cage structure of the two connected B-ring moieties in 7, R/Sor S/R-configurations at positions 2′ and 2′′ are not possible. In the NOESY spectrum of 7 (Figure 4), H-2′ gave a cross-peak with H-6′, but H-2′′ had no cross-peak with H-6′′, which had its only cross-peak with H-2′′′. This observation can be explained assuming that the sixmembered ring of the B2 moiety is more planar than the
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six-membered ring of the B1 moiety, as suggested by a three-dimensional structure model. In addition to the cross-peak with H-2′, H-6′ showed an additional crosspeak with H-3 and a smaller cross-peak with H-2. These NOE effects would appear to be consistent with both possible diastereomers. However, in the three-dimensional structure model of the S/S-configured isomer, the H-3 proton appeared to be closer to H-6′ than H-2 did. This observation is consistent with the NOESY spectrum of 7, in which the cross-peak between H-2 and H-6′ is smaller than that of H-3 with H-6′. In a model of the R/Rconfigured isomer, H-3 seems to be farther and H-2 seems to be closer to H-6′ when compared to the other isomer. Thus, for the R/R-configured diastereomer, H-6′ should have a smaller NOESY cross-peak with H-3 than with H-2. On the basis of the NOESY spectral data and this reasoning, we conclude that in 7 the two chiral centers at positions 2′ and 2′′ are most likely both S-configured. For product 8, negative high-resolution FAB-MS gave molecular ion [M - H]- at m/z 319.0446 (calcd 319.0454), which corresponded to the molecular formula C15H12O8 for 8. The UV/vis spectrum of 8 had a λmax at 301 nm (log ) 3.67) and a shoulder at 278 nm (log ) 3.59) similar to that of 3, in which λmax was at 279 nm (log ) 3.96) and the shoulder at 299 nm (log ) 3.87) (21). The lack of the gallic acid ester function in 8 probably contributes to the difference between the UV/vis spectra of 3 and 8. Both UV/vis spectra are distinguishably different from those of EGCG (1), EGC (5), and the other oxidation products (2, 4, 6, and 7), where λmax ranges between 269 and 276 nm. The molecular mass difference of 152 Da between 8 ([M - H]- at m/z 319) and 3 ([M H]- at m/z 471) corresponded to the lack of the galloyl moiety in 8. Also, the 1H and 13C NMR data of 8 indicated that EGC was again oxidized solely on the B-ring and that A- and C-rings in 8 were unchanged when compared to those in EGC. In the 1H NMR spectrum of 8, two broad singlets were observed for the B-ring at δH 7.16 (H-2′) and δH 6.42 (H-6′) that corresponded to the carbons at δC 108.2 and 114.2, respectively, in the HSQC spectrum. The 13C NMR spectrum revealed four additional signals for the B-ring moiety at δC 156.6, 156.7, 160.9, and 161.0. In the HMBC experiments, cross-peaks were observed between H-2 and the carbons at δC 156.6 (C-1′), 108.2 (C-2′), and 114.2 (C-6′). Proton H-2′ (δH 7.16) showed 3J connectivities to C-2, C-4′ (δC 161.0), and C-6′ (δC 114.2). The proton at δH 6.42 (H-6′) showed cross-peaks to C-2, C-1′, C-2′, and C-5′ (δC 160.9). The DQF-COSY experiment revealed allylic couplings between H-6′ and H-2, between H-6′ and H-2′, and between H-2 and H-2′. On the basis of the spectroscopic data and their comparison with those of 3 (21), we assigned 8 the structure shown in Figure 2. Role of H2O in the Oxidation of EGCG with Peroxyl Radicals. As demonstrated with the identification of oxidation products 2-4 and 6-8, the reaction pathway of oxidations of EGCG and EGC with peroxyl radicals involves the addition of oxygen. Water is a possible source of the additional oxygen atom. To explore the influence of water on the EGCG oxidation with peroxyl radicals, two experiments were carried out. First, we examined the influence of increasing H2O concentrations in the reaction solvent on the distribution of EGCG oxidation products. EGCG was oxidized with AMVN (10fold molar excess) in acetonitrile alone and in various acetonitrile/H2O mixtures with increasing H2O concen-
Valcic et al.
trations (0, 1, 5, 10, and 20% H2O) for 1 h at 50 °C. After reaction for 1 h, the HPLC analysis showed that EGCG (1) was consumed and that products 3 and 4 had been formed at the same rate regardless of the H2O concentration (data not shown). Second, we investigated the incorporation of 18O into EGCG products formed by oxidations in acetonitrile and H218O. The previous experiment showed that the formation of oxidation products 3 and 4 was not influenced by increased concentrations of H2O in the reaction solvent. To determine whether the additional oxygen in the B-rings of 3 and 4 could still originate from water, we conducted oxidation experiments of EGCG with AMVN in acetonitrile/water mixtures (99:1) using labeled (H218O) versus unlabeled (H2O) water. If the additional oxygen in products 3 and 4 originated from H2O, it should be possible to observe an incorporation of an 18O atom from H218O into molecules 3 and 4. After reaction for 1 h, the reaction mixtures were analyzed by LC/MS (data not shown). The use of the negative ESI-MS data required a modified HPLC mobile phase that consisted of an 85:15 H2O/acetonitrile mixture. The lack of formic acid in the mobile phase had almost no effect on the retention times of EGCG (1) and product 4, but the retention time of 3 shifted from 7.4 min (with acid) to 2.4 min (without acid), which is probably due the deprotonation of the R-hydroxy group in 3 in the absence of formic acid (data not shown). The negative ESI-MS data of compounds 3 and 4 showed the same molecular ions [M - H]- at m/z 471 and 929, respectively, regardless of whether the oxidations had been carried out in the presence of 18O-labeled or unlabeled water. The experiment with labeled water demonstrated that the additional oxygen incorporated in 3 and 4 was not derived from water, but most likely from molecular oxygen. Time Course Studies of EGCG Oxidation. To further characterize the effect of reaction conditions on competing antioxidant reactions of EGCG, we studied the effect of oxidation rate on EGCG depletion and product distribution. EGCG was oxidized by AMVN-derived peroxyl radicals at 50 °C in acetonitrile at EGCG:AMVN molar ratios of 1:1, 1:2.5, 1:5, 1:10, and 1:20. The oxidation of EGCG accellerated with increasing AMVN concentrations, reflecting the increased rate of peroxyl radical generation. However, increasing AMVN concentrations produced no significant changes in the relative yields of the anhydride 3 and symmetrical dimer 4 (data not shown). Formation of product 2 was not assessed in these experiments. Antioxidant Stoichiometry of Reactions with EGCG, EGC, and Methyl Gallate. To further evaluate the mechanisms of antioxidant reactions of EGCG, EGC, and methyl gallate, the antioxidant stoichiometry of radical trapping was measured by the kinetic method originally described by Boozer et al. (25) and modified by Liebler and Burr (26). The stoichiometric factor for peroxyl trapping, n, is calculated from eq 1
n ) Riτ/[ArOH]
(1)
where Ri is the rate of chain initiation, [ArOH] is the concentration (millimolar) of the antioxidant, and τ is the length of the induction period during which substrate autoxidation is suppressed by the inhibitor. Methyl linoleate was used as the oxidizable substrate, and AMVN was used to generate peroxyl radicals. Substrate oxidation was monitored by spectrophotometric measure-
Reactions of EGCG and EGC with Peroxyl Radicals Table 3. Antioxidant Kinetic Parameters and n Values for Antioxidant Reactions of EGCG, EGC, and Methyl Gallate in Acetonitrile EGCG (1) EGC (5) methyl gallate
[ArOH]a
τb
nc
0.0667 0.100 0.100
33.8 ( 4.2 (7) 26.9 ( 3.2 (7) 28.4 ( 2.2 (8)
4.16 ( 0.51 2.20 ( 0.26 2.33 ( 0.18
a Antioxidant concentration (millimolar). b τ is the length of the induction period, during which substrate autoxidation is suppressed by the inhibitor. c n is the stoichiometric factor for peroxyl trapping.
ment of conjugated diene accumulation. With an assigned antioxidant stoichiometric factor n of 2 (26), BHT was used as antioxidant standard to determine the Ri value for autoxidations initiated at 50 °C by AMVN-derived peroxyl radicals in acetonitrile solutions containing methyl linoleate as the autoxidation substrate. Reproducible τ values were estimated from breaks in the oxidation curves (Table 3). The calculated Ri value of 0.0082 mM min-1 for the standard antioxidant BHT then was used to estimate the stoichiometric factors n for EGCG, EGC, and methyl gallate (Table 3). The stoichiometric factor n of EGCG was approximately twice those of EGC and methyl gallate.
Discussion Numerous researchers have demonstrated the antioxidant and free radical scavenging abilities of catechins (11-15, 17-20, 30-32). Various structure-antioxidant activity studies concluded that the presence of the gallate group in the 3-position and a trihydroxy B-ring play the most important roles in the free radical scavenging abilities of the catechins. For example, Guo et al. (19) investigated the free radical scavenging effects of catechins on superoxide anions (O2-), singlet oxygen (1O2), peroxyl radicals, and the DPPH radical by ESR. They demonstrated that galloylated catechins displayed antioxidant effects superior to those of nongalloylated catechins and that the scavenging effects of catechins with a trihydroxyphenyl B-ring were stronger than those with an o-dihydroxyphenyl B-ring. These results were in agreement with the study by Salah et al. (13) which showed that catechins containing a galloyl moiety in the 3-position (EGCG and ECG) have the highest activity as antioxidants and are the most effective inhibitors of lipid peroxidation. Nanjo et al. (20) reported that both the trihydroxyphenyl B-ring and the 3-galloyl moiety of EGCG are involved in reactions with the DPPH radical. Other studies have been focused on elucidating antioxidant mechanisms of catechins by partial structural characterization of reaction products. For example, Sawai and Sakata (33) subjected reaction mixtures of green tea catechins with DPPH radicals to NMR analysis and suggested that in (+)-catechin and (-)-epicatechin the B-ring is changed to an o-quinone structure because of the appearance of two carbonyl signals. In a similar EGC oxidation, no carbonyl signals were observed, suggesting that EGC does not form an o-quinone structure when reacted with DPPH. Kondo et al. (17, 18) investigated the scavenging effects of the four major catechins (EGCG, EGC, ECG, and EC) on peroxyl radicals both in a liposomal and in an aqueous system. They concluded that EGC was the least effective of the four catechins that were studied. Solely on the basis of LC/MS analysis, spectrophotometric data, and semiempirical molecular
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 807
orbital calculations, they concluded that EGC is transformed to a quinone-like compound that might form active oxygen species. They also suggested that EC, ECG, and EGCG were converted to anthocyanin-like compounds with cleavage of the gallate moiety for ECG and EGCG. However, the data did not allow the unambiguous assignment of structures to any of the products that were detected. We have employed a different strategy. Identification of oxidation products formed by reaction of green tea catechins with biologically relevant oxidants can provide insights into the specific mechanisms of antioxidant actions. Our previous studies of antioxidant reactions of the phenolic antioxidants R-tocopherol and EGCG have employed the azo initiator AMVN as a source for peroxyl radicals in acetonitrile. Although this system may seem to lack biological relevance, two considerations justify its use. First, AMVN thermolysis in oxygenated solutions selectivly generates peroxyl radicals without the participation of other oxidants and catalysts (e.g., metal ions), which degrade primary reaction products and complicate interpretation of the chemistry that is involved. Study of peroxyl radical scavenging chemistry is particularly important as peroxyl radicals are the principal species scavenged by chain-breaking antioxidants. Second, previous work on vitamin E oxidation indicated that both the identities and distribution of products formed from AMVN-initiated R-tocopherol oxidations in acetonitrile (34, 35) closely modeled the fate of R-tocopherol in liposomal membranes (36-38), in isolated subcellular fractions (39, 40), in intact organs (41), and in whole animals in vivo (42). Our previous identification of EGCG products 3 and 4 (21) provided the first unambiguous evidence that antioxidant reactions of EGCG (1) with peroxyl radicals involve the trihydroxyphenyl B-ring, rather than the 3-galloyl moiety. With the current study, we substantiate our previous observations with the identification of the third EGCG product (2), again resulting from oxidation of the trihydroxyphenyl B-ring of EGCG. Moreover, we identified EGC oxidation products 6 and 8 that are analogues of 4 and 3, respectively. The fact that catechins with trihydroxyphenyl B-rings form the same products when oxidized with peroxyl radicals, regardless of the presence of a 3-galloyl moiety, emphasizes that the trihydroxyphenyl B-ring is the principal site of antioxidant actions in EGCG and EGC. Indeed, we have not yet detected any products resulting from oxidation of the galloyl moiety. In oxidations of EGC with peroxyl radicals, a third product was identified as an unsymmetrical dimer (7), again resulting from oxidation on the trihydroxyphenyl B-ring. We did not observe the EGCG analogue of EGC product 7. This may reflect steric hindrance caused by the two galloyl rings in 4. Our data provide some new insights into the mechanisms by which catechins scavenge peroxyl radicals. First, phenoxyl radicals derived from EGCG and EGC apparently do not form stable radical addition products with AMVN-derived peroxyl radicals, as do those from simple phenolic antioxidants (e.g., tert-butylhydroxytoluene) (43, 44), R-tocopherol (34, 35, 45), and the soy isoflavone genistein (46). Second, oxygen that is added during catechin oxidation is not incorporated from water in the reaction medium, but instead most probably from atmospheric oxygen via peroxyl radicals. Although atmospheric oxygen could also be incorporated directly by
808
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 Scheme 1. Proposed Mechanism for the Formation of Oxidation Product 3
oxygen addition, this appears less likely because the generation of catechin-derived peroxyl radical intermediates would result in a net peroxyl radical consumption of zero (see Schemes 1-3). Third, all of the oxidation products we have characterized contain oxidative modifications exclusively on the B-ring. Another important aspect of the oxidation of EGCG and EGC is the formation of an apparently polymeric group of oxidation products, which elute with the acetonitrile wash in HPLC analyses of reaction products (Figure 1). We have not characterized these products, but we presume they arise from radical-initiated polymerization reactions. The product yields reported in Experimental Procedures reflect considerable purification and
Valcic et al.
do not necessarily indicate the product distribution in the reactions. It is clear that a large fraction of the consumed catechins goes to polymeric products, but we are unable to make reliable estimates. The catechins and their products all display largely similar UV spectra, with differences in molar absorptivity apparently not greater than about 2-3-fold at 270 nm. Thus, it would appear from inspection of the chromatograms (Figure 1) that we have identified at least half of the catechin monomer and dimer products. On the basis of the results of our experiments, we propose for the formation of products 3 and 4 the mechanisms shown in Schemes 1 and 2, respectively. An initial one-electron oxidation of EGCG by a peroxyl radical generates a EGCG phenoxyl radical (11, 15). This phenoxyl radical is the first and only common intermediate in the formation of products 3 and 4. For the formation of product 3, the phenoxyl radical reacts with a second peroxyl radical to form an unstable AMVN adduct. Heterolytic cleavage of the peroxide with simultaneous ring enlargement produces product 3. For the formation of 4, the phenoxyl radical attacks a second EGCG molecule to form an EGCG dimer radical. This dimer radical forms an unstable adduct with a second peroxyl radical generated from AMVN. Heterolytic cleavage of the peroxide then is followed by re-
Scheme 2. Proposed Mechanism for the Formation of Oxidation Product 4
Scheme 3. Proposed Mechanism for the Formation of Oxidation Product 7
Reactions of EGCG and EGC with Peroxyl Radicals
arrangement to 4 with loss of an AMVN-derived alcohol fragment. Since compounds 8 and 6 are EGC analogues of 3 and 4, we assumed that their formation follows mechanisms similar to those of 3 and 4 (Schemes 1 and 2). For the formation of product 7, we propose the mechanism shown in Scheme 3, which is a slight variation of the one proposed for 4 and 6. We have not carried out experiments to address the mechanism of formation of the dicarboxylic acid 2. However, hydrolysis of the anhydride 3 followed by oxidative decarboxylation would appear to be a logical route. The view that the B-ring is the exclusive site of catechin oxidation in these experiments appears to conflict with our measured antioxidant stoichiometries of EGCG, EGC, and methyl gallate. EGCG exhibited an antioxidant stoichiometric factor of approximately 4, which is twice that measured for EGC and methyl gallate. EGCG contains both a galloyl moiety and a trihydroxyphenyl B-ring, whereas methyl gallate contains only the galloyl moiety and EGC only the trihydroxyphenyl B-ring. Thus, the antioxidant stoichiometry suggests that the trihydroxyphenyl B-ring and the galloyl moiety both contribute equally to antioxidant reactions. However, we identified no products in which the galloyl moiety was oxidized. The apparent inconsistency between our product data and antioxidant stoichiometry data may be accounted for by three considerations. First, in our product studies, oxidations of EGCG and EGC were not taken to completion, to avoid the generation of secondary oxidation products, which would complicate characterization of the product mixture. In contrast, oxidations in which stoichiometric factors were measured resulted in complete consumption of the catechins. Thus, oxidation of the galloyl moiety may have occurred after more facile oxidation of the trihydroxyphenyl B-ring, but still within the time frame of the measurement of τ in Table 3. Consistent with this view is our observation that, under the conditions we used to oxidize the catechins, methyl gallate reacts with peroxyl radicals, albeit at a greatly reduced rate. Second, some of the products of EGCG and EGC oxidations remain to be characterized, and these could include products of oxidation at the galloyl moiety. Finally, the measured antioxidant stoichiometry for EGCG actually represents the net effect of several product-forming mechanisms, which lead to the dicarboxylic acid 2, the anhydride 3, the dimer 4, and the polymeric products and any other products that have not yet been identified. Each of these pathways may consume different numbers of peroxyl radicals. Our data collectively suggest that B-ring oxidation is the most facile reaction pathway, but perhaps not the only one that contributes to the antioxidant effect. A related issue is the effect of radical flux on the distribution of products of catechin oxidations. We postulate that trapping of catechin radical intermediates is a key step in determining whether monomeric products (e.g., anhydrides) versus dimers are formed. Thus, we expected that varying the rate of radical generation (by changing the concentration of the initiator AMVN) would shift the distribution of products. However, our results indicated that when the AMVN:catechin molar ratio was varied from 1:1 to 20:1, there was no appreciable change in the product distribution. Together with the results of our previous work (21), our data suggest that multiple product-forming reactions
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 809
contribute to peroxyl radical scavenging by catechins. However, it appears most likely that B-ring oxidation products of EGCG and EGC would probably predominate under conditions of partial oxidation likely to occur when these compounds act as antioxidants biologically. These B-ring oxidation products are therefore the most likely potential markers for antioxidant reactions of EGCG and EGC in biological systems. Analyses of these products could thus provide a unique tool for assessing the contribution of antioxidant reactions to the diseasepreventive effects of catechins.
Acknowledgment. We thank Dr. Arpad Somogyi for the determination of FAB mass spectra, Dr. Thomas McClure for assistance in the LC/MS studies, Ms. Annette Muders for assistance in product isolations, and Dr. Neil E. Jacobsen for assistance with NMR instrumentation. This investigation was supported by NIH Grants CA 75599 and ES06694.
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