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(−)-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma
BBRC Biochemical and Biophysical Research Communications 302 (2003) 409–414 www.elsevier.com/locate/ybbrc
())-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasmaq Giancarlo Aldini,a Kyung-Jin Yeum,b,* Marina Carini,a Norman I. Krinsky,b,c and Robert M. Russellb b
a Istituto Chimico Farmaceutico Tossicologico, University of Milan, Milan, Italy Jean Mayer USDA Human Nutrition Research Center on Aging, 711 Washington St., Boston, MA 02111, USA c Department of Biochemistry, School of Medicine at Tufts University, Boston, MA, USA
Received 17 January 2003
Abstract When human plasma was exposed to the hydrophilic radical initiator, AAPH, ())-epigallocatechin-(3)-gallate (EGCG) dosedependently inhibited the aqueous compartment oxidation (IC50 ¼ 0:72 lM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, a-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared a-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC50 ¼ 4:37 lM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled a-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: ())-Epigallocatechin-(3)-gallate; Antioxidant; Carotenoids; a-Tocopherol; Aqueous and lipid plasma compartments
Epidemiological and experimental studies suggest that tea intake is associated with a reduced risk of cardiovascular diseases [1–3]. Catechins, which belong to the flavonoid family, are the main components of tea and may be responsible for the alleged protective effect [4]. In particular, the dry extract of a regular green tea (GT) infusion contains 27% of catechin derivatives including (+)-catechin (C), ())-epicatechin (EC), ())-epicatechin gallate (ECG), epigallocatechin (EGC), and ())-epigallocatechin gallate (EGCG) [5], which is the most abunq Abbreviations: A , ascorbyl radical; AH , ascorbate; Aq , aqueous radical; AqH, aqueous hydrogen donor; a-TOC-O , atocopheroxyl radical; a-TOC-OH, a-tocopherol; CAR, carotenoids; CAR , carotenoid alkyl radical; CAR-OO , carotenoid peroxyl radical; CAR-OOH, carotenoid hydroperoxide; EGCG-O , epigallocatechin gallate phenoxyl radical; EGCG, EGCG-OH, epigallocatechin gallate; GT, green tea; LOO , lipid peroxyl radical; LOOH, lipid hydroperoxide; UA, uric acid; UA , uric acid radical. * Corresponding author. Fax: +617-556-3344. E-mail address: [email protected] (K.-J. Yeum).
dant (50% of the total catechins) and most active as an antioxidant [6]. Several mechanisms for the possible protective activity of tea catechins have been proposed such as hypocholesterolemic, anti-inflammatory activity, inhibition of platelet aggregation, and effects on body weight and fat by decreasing nutrient absorption and increasing energy expenditure [7]. However, the most widely proposed mechanism is an antioxidant effect and, in particular, the inhibition of LDL oxidation [8], which is thought to be involved in the development and progression of cardiovascular disease (CVD) [9]. The antioxidant activity of GT catechins in plasma and lipoproteins has been studied in several in vitro and ex vivo models, most of them using AAPH or transition metal ions as hydrophilic radical inducers, and either TBARS, conjugated dienes, or lipid hydroperoxides as markers of the lipid peroxidation process [10–13]. Although these methods are suitable for studying the efficacy of GT catechins in protecting plasma and lipoproteins from oxidative damage, they do not provide
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00192-X
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any information about the mechanism and the site of the antioxidant action. GT catechins could act in several ways: (1) in the aqueous domain or at the aqueous/lipid interface trapping hydrophilic radicals or chelating transition metal ions, (2) in the lipid phase inhibiting the lipid peroxidation cascade through a chain-breaking mechanism, and (3) by sparing/recycling endogenous lipophilic antioxidants such as a-tocopherol and b-carotene [13,5]. Terao et al. [14], using phospholipid liposomes, hypothesized that catechins might be localized near the membrane surface to scavenge aqueous radicals, thereby preventing the consumption of a-tocopherol, which mainly acts as a scavenger of chainpropagating lipid peroxyl radicals. However, to our knowledge no data exist regarding the antioxidant mechanism of GT catechins in human plasma, whose antioxidant composition is similar to that of human interstitial fluid, where lipoprotein oxidation is thought to take place [15]. The aims of this study were to determine (1) in which plasma compartment EGCG acts as an antioxidant and (2) the possible interplay between EGCG and other hydrophilic and lipophilic antioxidants (e.g., ascorbic acid, a-tocopherol, and uric acid). To do this we used a selective method able to measure the oxidizability of the aqueous and lipid compartments of human plasma [16] and aqueous and lipid radical inducers to study a possible sparing/recycling effect of EGCG on other plasma antioxidants.
Materials and methods Chemicals. The radical initiators 2,20 -azobis(2,4-amidinopropane) dihydrochloride (AAPH) and 2,20 -azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) were from Wako Chemicals (Richmond, VA, USA). The fatty acid analogue C11-BODIPY 581/591 (BODIPY) and 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Molecular Probes (Eugene, OR, USA). ())-Epigallocatechin-(3)-gallate (EGCG) and a-tocopherol were purchased from Sigma (St. Louis, MO, USA). 2,2-diphenyl-1-picrylhydrazyl (DPPH) was from Fluka (Milwaukee, WI). All-trans-b-carotene (type II) and lycopene were purchased from Sigma Chemical (St. Louis, MO). Lutein was purchased from Kemin Industries (Des Moines, IA). Zeaxanthin, cryptoxanthin, and echinenone were gifts from Hoffmann–La Roche (Nutley, NJ). All the other reagents were of analytical grade. Human plasma oxidation induced by water- and lipid-soluble radical inducers. After an overnight fast (10–12 h), blood from two healthy donors (32 and 35 years old) was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes. In order to reduce the variability of different donors, blood samples from these two subjects were collected and pooled weekly for the duration of the experiment. Immediately after collection, the samples were placed on ice and protected from light. Plasma was obtained by centrifugation at 800g for 20 min at 4 °C and immediately used for the in vitro studies. Aqueous and lipid plasma oxidation was induced by two azo-initiators: AAPH as a water-soluble peroxyl radical generating system and MeO-AMVN as lipid-soluble peroxyl radical initiator. AAPH was prepared in phosphate-buffered saline (50 mM, pH 7.4,
PBS) and stored at )20 °C, while MeO-AMVN was dissolved in CH3 CN immediately before use. In order to obtain homogeneous incorporation, MeO-AMVN was slowly added to the samples with a micro-syringe (10 ll) while stirring. The samples were then gently mixed for 10 s and incubated at 37 °C under aerobic conditions.The amount of free radicals generated by AAPH and MeO-AMVN was kept constant by adjusting the concentration of the two azo-initiators. In the presence of 20 mM AAPH, the flux of aqueous radicals calculated on the basis of the known rate of free radical generation from AAPH at 37 °C (Ri ¼ 1:36 106 [AAPH] mol/l/s) [17] was 2:72 108 mol/l/s. Since the rate of peroxyl radical formation from MeO-AMVN is 14:2 106 [MeO-AMVN] mol/l/s at 37 °C (calculated in micelles) [18], the concentration of the lipophilic azo-initiator was reduced by 10-fold (2 mM) to reach the same order of free radical flux. Measurement of plasma oxidation. Plasma oxidation was measured fluorometrically using two different fluorescent probes (DCFH and BODIPY), as already reported [16]. The final plasma dilution with PBS was 1:5 (v/v). Briefly, DCFH was prepared from DCFH-DA by basic hydrolysis, and aqueous plasma oxidation was measured by monitoring the two-electron oxidation of DCFH to the highly fluorescent compound 20 ,70 -dichlorofluorescein (DCF). The excitation wavelength (kex ) was set at 502 nm (slit 5 nm) and emission (kem ) at 520 nm (slit 5 nm). BODIPY was incorporated into the lipid plasma compartment at a final concentration of 2 lM. Lipid plasma oxidation was determined by monitoring the green fluorescence increase (kex ¼ 500 nm, kem ¼ 520 nm) of the oxidation product of BODIPY. The fluorescence measurements were carried out using a Perkin–Elmer spectrofluorometer (model 650–10 s) with 1 cm path length fluorescence cuvettes. EGCG was prepared in cold PBS immediately before use and added to samples at final concentrations of 0.25, 1, 5, and 10 lM. By considering the final dilution of plasma (1:5, v/v), these concentrations of EGCG were equivalent to: 1.25, 5, 25, and 50 lM. The temporal sequence of adding chemicals to plasma samples was as follows: fluorescence probes, azo-compound, and EGCG. The azo-compounds were added after the fluorescent probes to avoid thermal decomposition during incorporation of BODIPY into the lipid plasma compartment. EGCG was added last to avoid any interference of the fluorescent probe or azo-compound incorporation into plasma compartments. Determination of hydrophilic and lipophilic plasma antioxidants. Plasma:PBS (1:5, by vol) was incubated at 37 °C in the presence and absence of the hydrophilic radical generator, AAPH (10 and 20 mM) or the hydrophobic radical generator, MeO-AMVN (2 mM). After 60 min of incubation in aerobic conditions, the fat-soluble antioxidant nutrients, such as a-tocopherol, b-carotene, lycopene, cryptoxanthin, zeaxanthin, and lutein were extracted and measured using the HPLC method described earlier [19]. Briefly, to a 100 ll aliquot of the reaction mixture, echinenone was added as an internal standard and the reaction mixture was extracted with CHCl3 :CH3 OH (2:1, by vol) containing 0.2% BHT and hexane containing 0.1% BHT; the organic layer was then transferred to another tube, dried under nitrogen, redissolved in ethanol, and injected into an HPLC system with a C30 column (3 lm, 150 4:6 mm, YMC, Wilmington, NC). A Waters 994 programmable photodiode array detector was set at 450 nm for carotenoids and 292 nm for a-tocopherol analyses. The major water-soluble antioxidants (ascorbic acid and uric acid) were measured at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h. For water-soluble antioxidant measurement, the mixtures were immediately deproteinized with perchloric acid (250 mM). Ascorbic acid and uric acid in plasma were analysed by HPLC using an electrochemical detector (Bioanalytical System, N. Lafayette, IN), as described earlier [20]. Results are expressed as percentages with respect to control samples prepared without the azo-compounds. Electron spin resonance experiments. Tocopheroxyl radicals (aTOC-O ) were generated by reaction of a-tocopherol and DPPH according to 1 as described by Rousseau-Richard [21]
G. Aldini et al. / Biochemical and Biophysical Research Communications 302 (2003) 409–414 a-TOC-OH þ DPPH ! a-TOC-O þ DPPHH
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ð1Þ
For sample preparation, a-tocopherol (900 lM) and DPPH (600 lM) in an ethanol solution were mixed for 20 s and 50 ll of the reaction mixture was transferred into a capillary ESR tube. EGCG was added to the mixture immediately after DPPH decolorization (30 s after DPPH addition). After exactly 60 s from the start of the reaction, the ESR spectra were recorded at room temperature with a Bruker EMX spectrometer at 9.5 GHz (X band) equipped with a cylindrical cavity (ER4119HS; Bruker) and under the following instrumental conditions: microwave frequency, 9.316 GHz; microwave power, 15 mW; modulation 2 G; number of scans, 1; and resolution, 1024 points. The spectra were recorded and doubly integrated using a Bruker WINEPR system (version 2.11). Statistical analysis. Results are expressed as means SEM. Statistical analysis was performed with a one-way analysis (ANOVA) followed by DunnettÕs post-test. GraphPad Prism (version 2.01) (GraphPad Software) was used for all analyses. A p value less than or equal to 0.05 was considered significant.
Fig. 2. Dose-dependent protective effect of EGCG on aqueous (blank bar) and lipid (filled bar) compartment oxidation after 180 min of incubation. Values are means SEM of five independent experiments.
Results EGCG protective effect on human plasma oxidation induced by water- and lipid-soluble radical inducers When AAPH was used as the radical initiator, aqueous oxidation was already apparent after 30 min of incubation as a consequence of AA consumption, and the rate of oxidation increased after 120 min of incubation, due to uric acid depletion [16]. EGCG addition dose-dependently reduced the oxidative process as detected by DCF fluorescence both before and after uric acid depletion (Fig. 1A). At 60 min, when uric acid is depleted only by 30%, EGCG dose-dependently protected DCF oxidation by 13 2:5% at 0.5 lM and 55 3:4% at 5 lM. At 180 min of incubation, EGCG at 0.25 lM reduced DCFH oxidation by 20:25 0:34% and was almost completely protective at 10 lM (93:0 2:0% protection).
Fig. 1. (A) EGCG inhibits the oxidation of the aqueous plasma compartment induced by AAPH (20 mM) and monitored by DCF fluorescence increase. Values for ascorbate and urate are in the absence of added EGCG. (B) EGCG inhibits the oxidation of the lipid plasma compartment induced by MeO-AMVN (2 mM) and monitored by measuring BODIPY green fluorescence (BODIPY GF). Values are means SEM of five independent experiments. Where not shown, the error bars are covered by the symbols. Left axis: (j) Control; EGCG: (s) 0.25 lM, (M) 0.5 lM, ( ) 1 lM, () 5 lM, and (r) 10 lM; Right axis: (N) ascorbic acid and (}) uric acid.
When plasma containing BODIPY was incubated in the presence of 2 mM MeO-AMVN, a time-dependent increase of green fluorescence was observed, whose rate increased at 90 min following the consumption of atocopherol and b-carotene [16]. The protective effect afforded by EGCG in the lipid domain was found to be less effective than that found in the aqueous compartment; after 180 min of incubation, the lowest effective concentration was 0.5 lM (13:01 0:56%) and 10 lM resulted in 68 2:3% protection (Fig. 1B). In Fig. 2 the protective effect of EGCG in aqueous and lipid compartments after 180 min of incubation is compared; the calculated IC50s in aqueous and lipid compartments were, respectively, 0:72 0:02 and 4:37 0:14 lM. EGCG effect on hydrophilic and lipophilic plasma antioxidant consumption When 20 mM AAPH was added to plasma, ascorbic acid, and uric acid were almost totally consumed within 15 and 180 min, respectively (Fig. 1A). EGCG at all the concentrations tested (0.5–10 lM) was found to be ineffective in reducing the consumption of these two hydrophilic endogenous antioxidants (data not shown). AAPH also induced a significant consumption of lipophilic plasma antioxidants. After 120 min of incubation, the order of consumption expressed as percentage remaining was as follows (Table 1 and Fig. 3): a-tocopherol (3:9 0:9); lycopene (8:5 5:2); lutein (12:8 4:8); zeaxanthin (17:5 6:0); cryptoxanthin (18:9 3:9); and b-carotene (28:9 6:1). EGCG addition was found to significantly and dose-dependently reduce the consumption of all carotenoids (Table 1), suggesting its ability to trap aqueous radicals and hence prevent their diffusion into lipoproteins. The sparing effect of EGCG toward a-tocopherol consumption was significant when
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Table 1 Dose-dependent protective effect of EGCG on carotenoid consumption induced by AAPH (20 mM for 120 min) and MeO-AMVN (2 mM for 60 min) in plasma samples Carotenoid
EGCG (lM)
Radical initiator AAPH
MeO-AMVN
Residual amount (%) b-Carotene
0 (control) 0.5 1 5 10
28:9 6:2 41:5 6:6 54:4 4:9a 58:6 3:3a 64:3 6:4b
50:3 2:3 51:9 1:7 50:1 3:3 57:7 0:5 59:1 3:7
Lycopene
0 (control) 0.5 1 5 10
8:5 5:2 9:3 5:2 18:1 2:4 25:3 1:0a 32:3 4:2b
20:1 4:9 20:1 3:2 19:7 2:6 23:6 1:8 26:7 0:9
Cryptoxanthin
0 (control) 0.5 1 5 10
18:9 3:4 28:5 3:3 36:2 1:7a 48:9 2:1b 56:4 5:0b
41:8 4:2 54:4 2:7 52:4 0:8 45:1 3:1 41:8 1:6
Zeaxanthin
0 (control) 0.5 1 5 10
17:5 5:9 25:6 6:5 48:8 4:8a 58:9 5:1b 62:4 11:9b
40:0 3:1 47:7 5:8 49:6 2:1 44:3 0:9 42:7 2:0
Lutein
0 (control) 0.5 1 5 10
12:8 4:8 26:0 4:3 40:6 5:3a 56:0 4:4b 63:1 8:1b
31:0 5:4 32:6 4:1 33:7 3:9 44:6 5:5 40:4 4:0
10 mM AAPH was used, but not effective at 20 mM AAPH (Fig. 3). When MeO-AMVN was used to induce selective oxidation of the lipid compartment, a significant consumption of a-tocopherol and carotenoids was also observed. EGCG addition was found to be ineffective in sparing carotenoids at all the concentrations tested (0.5– 10 lM) (Table 1) but dose-dependently reduced a-tocopherol consumption (Fig. 3); this effect was significant at 1 lM and total protection was observed at 10 lM (% a-tocopherol remaining: 96:71 1:46 vs. 16:43 1:72 in controls; p < 0:001). EGCG regenerates a-tocopherol via reduction of its phenoxyl radical: ESR experiments Sixty seconds after mixing a-tocopherol with DPPH, the ESR spectrum of DPPH disappeared completely (due to the scavenging activity of a-tocopherol), and the typical spectrum of the a-tocopheroxyl free radical (aTOC-O ) was observed. Consecutive spectra (at 30 s time intervals for 3 min) showing the self-decay of a-TOC-O are shown in Fig. 4A. This decay has been described as a second-order reaction, as reported earlier [21]. EGCG addition dose-dependently accelerated the decay rate of a-TOC-O (Fig. 4B). The quenching effect (calculated 60 s after the beginning of the reaction) was already significant at 2 lM (% inhibition of ESR signal ¼ 8 1:3%) to reach an almost complete disappearance at 25 lM (IC50 ¼ 12:1 1:3 lM). Ascor-
The basal content of carotenoids was as follows: b-Carotene (3:58 0:18 lM); lycopene (2:10 0:23 lM); cryptoxanthin (1:77
0:10 lM); lutein (0:72 0:02 lM); and zeaxanthin (0:34 0:01 lM). a p < 0:05 vs. control. b p < 0:01 vs. control (ANOVA followed by DunnettÕs test).
Fig. 3. Dose-dependent effect of EGCG on a-tocopherol depletion induced by AAPH (10 and 20 mM) and MeO-AMVN (2 mM). The basal content of a-tocopherol was 42:08 1:28 lM. Values are means SEM of three independent experiments. *p < 0:05 vs. control; **p < 0:01 vs. control (ANOVA followed by DunnettÕs test).
Fig. 4. ESR spectra relative to the time-course of a-TOC-O decay in the absence (A) and presence (B) of EGCG (15 lM). a-TOC-O was induced by the stable radical DPPH.
G. Aldini et al. / Biochemical and Biophysical Research Communications 302 (2003) 409–414
bic acid, the physiological recycling agent of a-tocopherol, showed an IC50 ¼ 14:2 1:2 lM (data not shown).
Discussion The aim of the present investigation was to add to our understanding of the antioxidant mechanism of EGCG in human plasma. By using a selective fluorescent method able to induce and monitor the oxidative process in both the aqueous and lipid phases, we found that EGCG dose-dependently protected both compartments. However, the antioxidant efficiency of EGCG was six times greater in the aqueous vs. the lipid domain. The IC50s of EGCG, calculated after 180 min of incubation, in the aqueous and lipid plasma compartments were 0.72 and 4.37 lM, respectively. EGCG dose-dependently reduced AAPH-induced consumption of lipophilic antioxidants such as a-tocopherol and the carotenoids, suggesting that EGCG, by acting as a radical-scavenger in the aqueous compartment, limits the diffusion of the radical species to the lipid domain, thereby preventing the lipid-oxidation cascade and, as a consequence, lipophilic antioxidant depletion. By contrast, EGCG was ineffective (up to 10 lM) in sparing ascorbic acid, as previously described [22,13]. As reported by Lotito and Fraga [13], ascorbic acid may be able to prevent catechin depletion, and in view of the redox potentials [E(EGCG-O , Hþ /EGCGOH) ¼ 0.43 V]; E(A , H+/AH ) ¼ 0.28 V], it is thermodynamically feasible for ascorbic acid to regenerate EGCG from its aroxyl radical, EGCG-O . These interactions are depicted in Scheme 1, which includes the ability of a-tocopherol to prevent carotenoid oxidation as previously reported by Yeum et al. [23]. Although EGCG had a lower activity in the lipophilic than in the aqueous compartment, it was still found to dose-dependently inhibit oxidative damage in the lipid compartment induced by MeO-AMVN. The protective effect could be ascribed to one of the following
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mechanisms among others: (1) EGCG diffuses into lipoproteins where it acts as a chain-breaking antioxidant; (2) EGCG binds to the surface of lipoproteins where it recycles a-tocopherol from the tocopheroxyl radical. To understand whether EGCG is able to diffuse inside lipoproteins or remains localized on the outer surface of LDL, we studied the sparing effect of EGCG towards a-tocopherol and both polar and apolar carotenoids, using lipophilic peroxyl radicals generated by MeO-AMVN. EGCG at all concentrations tested (1– 10 lM) failed to prevent the depletion of either polar or apolar carotenoids, located, respectively, in the shell and core of the lipoproteins [24], while dose-dependently maintaining a-tocopherol, which resides at or near the surface of the lipoprotein particles [25]. These results indicate that EGCG is unable to diffuse into the shell/ core of the lipoprotein particles, but significantly binds to the outer surface of lipoproteins where the sparing/ recycling effect on a-tocopherol can occur. To demonstrate the direct reaction of EGCG with the tocopheroxyl radical, a direct ESR technique was used. EGCG was found to quench the tocopheroxyl radical with a potency similar to that of ascorbic acid, supporting the idea that EGCG regenerates a-tocopherol through a Htransfer mechanism. In addition, Zhu et al. [26] found that the a-tocopherol level in isolated LDL was partially restored when GT catechins were added during AAPHinduced depletion. Taken together, these data provide direct and unequivocal evidence for the regeneration of a-tocopherol via reduction of its phenoxyl radical by EGCG in LDL particles. Acknowledgments This research has been supported in part by the US Department of Agriculture, under agreement number 1950-51000-048-01A. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. We thank B. Gindelsky and E. Seyoum at Nutrition Evaluation Laboratory (HNRC) for the HPLC analyses of uric acid and ascorbic acid.
References
Scheme 1. Antioxidant mechanism of EGCG in human plasma.
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())-Epigallocatechin-(3)-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasmaq Giancarlo Aldini,a Kyung-Jin Yeum,b,* Marina Carini,a Norman I. Krinsky,b,c and Robert M. Russellb b
a Istituto Chimico Farmaceutico Tossicologico, University of Milan, Milan, Italy Jean Mayer USDA Human Nutrition Research Center on Aging, 711 Washington St., Boston, MA 02111, USA c Department of Biochemistry, School of Medicine at Tufts University, Boston, MA, USA
Received 17 January 2003
Abstract When human plasma was exposed to the hydrophilic radical initiator, AAPH, ())-epigallocatechin-(3)-gallate (EGCG) dosedependently inhibited the aqueous compartment oxidation (IC50 ¼ 0:72 lM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, a-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared a-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC50 ¼ 4:37 lM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled a-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: ())-Epigallocatechin-(3)-gallate; Antioxidant; Carotenoids; a-Tocopherol; Aqueous and lipid plasma compartments
Epidemiological and experimental studies suggest that tea intake is associated with a reduced risk of cardiovascular diseases [1–3]. Catechins, which belong to the flavonoid family, are the main components of tea and may be responsible for the alleged protective effect [4]. In particular, the dry extract of a regular green tea (GT) infusion contains 27% of catechin derivatives including (+)-catechin (C), ())-epicatechin (EC), ())-epicatechin gallate (ECG), epigallocatechin (EGC), and ())-epigallocatechin gallate (EGCG) [5], which is the most abunq Abbreviations: A , ascorbyl radical; AH , ascorbate; Aq , aqueous radical; AqH, aqueous hydrogen donor; a-TOC-O , atocopheroxyl radical; a-TOC-OH, a-tocopherol; CAR, carotenoids; CAR , carotenoid alkyl radical; CAR-OO , carotenoid peroxyl radical; CAR-OOH, carotenoid hydroperoxide; EGCG-O , epigallocatechin gallate phenoxyl radical; EGCG, EGCG-OH, epigallocatechin gallate; GT, green tea; LOO , lipid peroxyl radical; LOOH, lipid hydroperoxide; UA, uric acid; UA , uric acid radical. * Corresponding author. Fax: +617-556-3344. E-mail address: [email protected] (K.-J. Yeum).
dant (50% of the total catechins) and most active as an antioxidant [6]. Several mechanisms for the possible protective activity of tea catechins have been proposed such as hypocholesterolemic, anti-inflammatory activity, inhibition of platelet aggregation, and effects on body weight and fat by decreasing nutrient absorption and increasing energy expenditure [7]. However, the most widely proposed mechanism is an antioxidant effect and, in particular, the inhibition of LDL oxidation [8], which is thought to be involved in the development and progression of cardiovascular disease (CVD) [9]. The antioxidant activity of GT catechins in plasma and lipoproteins has been studied in several in vitro and ex vivo models, most of them using AAPH or transition metal ions as hydrophilic radical inducers, and either TBARS, conjugated dienes, or lipid hydroperoxides as markers of the lipid peroxidation process [10–13]. Although these methods are suitable for studying the efficacy of GT catechins in protecting plasma and lipoproteins from oxidative damage, they do not provide
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00192-X
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any information about the mechanism and the site of the antioxidant action. GT catechins could act in several ways: (1) in the aqueous domain or at the aqueous/lipid interface trapping hydrophilic radicals or chelating transition metal ions, (2) in the lipid phase inhibiting the lipid peroxidation cascade through a chain-breaking mechanism, and (3) by sparing/recycling endogenous lipophilic antioxidants such as a-tocopherol and b-carotene [13,5]. Terao et al. [14], using phospholipid liposomes, hypothesized that catechins might be localized near the membrane surface to scavenge aqueous radicals, thereby preventing the consumption of a-tocopherol, which mainly acts as a scavenger of chainpropagating lipid peroxyl radicals. However, to our knowledge no data exist regarding the antioxidant mechanism of GT catechins in human plasma, whose antioxidant composition is similar to that of human interstitial fluid, where lipoprotein oxidation is thought to take place [15]. The aims of this study were to determine (1) in which plasma compartment EGCG acts as an antioxidant and (2) the possible interplay between EGCG and other hydrophilic and lipophilic antioxidants (e.g., ascorbic acid, a-tocopherol, and uric acid). To do this we used a selective method able to measure the oxidizability of the aqueous and lipid compartments of human plasma [16] and aqueous and lipid radical inducers to study a possible sparing/recycling effect of EGCG on other plasma antioxidants.
Materials and methods Chemicals. The radical initiators 2,20 -azobis(2,4-amidinopropane) dihydrochloride (AAPH) and 2,20 -azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) were from Wako Chemicals (Richmond, VA, USA). The fatty acid analogue C11-BODIPY 581/591 (BODIPY) and 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Molecular Probes (Eugene, OR, USA). ())-Epigallocatechin-(3)-gallate (EGCG) and a-tocopherol were purchased from Sigma (St. Louis, MO, USA). 2,2-diphenyl-1-picrylhydrazyl (DPPH) was from Fluka (Milwaukee, WI). All-trans-b-carotene (type II) and lycopene were purchased from Sigma Chemical (St. Louis, MO). Lutein was purchased from Kemin Industries (Des Moines, IA). Zeaxanthin, cryptoxanthin, and echinenone were gifts from Hoffmann–La Roche (Nutley, NJ). All the other reagents were of analytical grade. Human plasma oxidation induced by water- and lipid-soluble radical inducers. After an overnight fast (10–12 h), blood from two healthy donors (32 and 35 years old) was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes. In order to reduce the variability of different donors, blood samples from these two subjects were collected and pooled weekly for the duration of the experiment. Immediately after collection, the samples were placed on ice and protected from light. Plasma was obtained by centrifugation at 800g for 20 min at 4 °C and immediately used for the in vitro studies. Aqueous and lipid plasma oxidation was induced by two azo-initiators: AAPH as a water-soluble peroxyl radical generating system and MeO-AMVN as lipid-soluble peroxyl radical initiator. AAPH was prepared in phosphate-buffered saline (50 mM, pH 7.4,
PBS) and stored at )20 °C, while MeO-AMVN was dissolved in CH3 CN immediately before use. In order to obtain homogeneous incorporation, MeO-AMVN was slowly added to the samples with a micro-syringe (10 ll) while stirring. The samples were then gently mixed for 10 s and incubated at 37 °C under aerobic conditions.The amount of free radicals generated by AAPH and MeO-AMVN was kept constant by adjusting the concentration of the two azo-initiators. In the presence of 20 mM AAPH, the flux of aqueous radicals calculated on the basis of the known rate of free radical generation from AAPH at 37 °C (Ri ¼ 1:36 106 [AAPH] mol/l/s) [17] was 2:72 108 mol/l/s. Since the rate of peroxyl radical formation from MeO-AMVN is 14:2 106 [MeO-AMVN] mol/l/s at 37 °C (calculated in micelles) [18], the concentration of the lipophilic azo-initiator was reduced by 10-fold (2 mM) to reach the same order of free radical flux. Measurement of plasma oxidation. Plasma oxidation was measured fluorometrically using two different fluorescent probes (DCFH and BODIPY), as already reported [16]. The final plasma dilution with PBS was 1:5 (v/v). Briefly, DCFH was prepared from DCFH-DA by basic hydrolysis, and aqueous plasma oxidation was measured by monitoring the two-electron oxidation of DCFH to the highly fluorescent compound 20 ,70 -dichlorofluorescein (DCF). The excitation wavelength (kex ) was set at 502 nm (slit 5 nm) and emission (kem ) at 520 nm (slit 5 nm). BODIPY was incorporated into the lipid plasma compartment at a final concentration of 2 lM. Lipid plasma oxidation was determined by monitoring the green fluorescence increase (kex ¼ 500 nm, kem ¼ 520 nm) of the oxidation product of BODIPY. The fluorescence measurements were carried out using a Perkin–Elmer spectrofluorometer (model 650–10 s) with 1 cm path length fluorescence cuvettes. EGCG was prepared in cold PBS immediately before use and added to samples at final concentrations of 0.25, 1, 5, and 10 lM. By considering the final dilution of plasma (1:5, v/v), these concentrations of EGCG were equivalent to: 1.25, 5, 25, and 50 lM. The temporal sequence of adding chemicals to plasma samples was as follows: fluorescence probes, azo-compound, and EGCG. The azo-compounds were added after the fluorescent probes to avoid thermal decomposition during incorporation of BODIPY into the lipid plasma compartment. EGCG was added last to avoid any interference of the fluorescent probe or azo-compound incorporation into plasma compartments. Determination of hydrophilic and lipophilic plasma antioxidants. Plasma:PBS (1:5, by vol) was incubated at 37 °C in the presence and absence of the hydrophilic radical generator, AAPH (10 and 20 mM) or the hydrophobic radical generator, MeO-AMVN (2 mM). After 60 min of incubation in aerobic conditions, the fat-soluble antioxidant nutrients, such as a-tocopherol, b-carotene, lycopene, cryptoxanthin, zeaxanthin, and lutein were extracted and measured using the HPLC method described earlier [19]. Briefly, to a 100 ll aliquot of the reaction mixture, echinenone was added as an internal standard and the reaction mixture was extracted with CHCl3 :CH3 OH (2:1, by vol) containing 0.2% BHT and hexane containing 0.1% BHT; the organic layer was then transferred to another tube, dried under nitrogen, redissolved in ethanol, and injected into an HPLC system with a C30 column (3 lm, 150 4:6 mm, YMC, Wilmington, NC). A Waters 994 programmable photodiode array detector was set at 450 nm for carotenoids and 292 nm for a-tocopherol analyses. The major water-soluble antioxidants (ascorbic acid and uric acid) were measured at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h. For water-soluble antioxidant measurement, the mixtures were immediately deproteinized with perchloric acid (250 mM). Ascorbic acid and uric acid in plasma were analysed by HPLC using an electrochemical detector (Bioanalytical System, N. Lafayette, IN), as described earlier [20]. Results are expressed as percentages with respect to control samples prepared without the azo-compounds. Electron spin resonance experiments. Tocopheroxyl radicals (aTOC-O ) were generated by reaction of a-tocopherol and DPPH according to 1 as described by Rousseau-Richard [21]
G. Aldini et al. / Biochemical and Biophysical Research Communications 302 (2003) 409–414 a-TOC-OH þ DPPH ! a-TOC-O þ DPPHH
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ð1Þ
For sample preparation, a-tocopherol (900 lM) and DPPH (600 lM) in an ethanol solution were mixed for 20 s and 50 ll of the reaction mixture was transferred into a capillary ESR tube. EGCG was added to the mixture immediately after DPPH decolorization (30 s after DPPH addition). After exactly 60 s from the start of the reaction, the ESR spectra were recorded at room temperature with a Bruker EMX spectrometer at 9.5 GHz (X band) equipped with a cylindrical cavity (ER4119HS; Bruker) and under the following instrumental conditions: microwave frequency, 9.316 GHz; microwave power, 15 mW; modulation 2 G; number of scans, 1; and resolution, 1024 points. The spectra were recorded and doubly integrated using a Bruker WINEPR system (version 2.11). Statistical analysis. Results are expressed as means SEM. Statistical analysis was performed with a one-way analysis (ANOVA) followed by DunnettÕs post-test. GraphPad Prism (version 2.01) (GraphPad Software) was used for all analyses. A p value less than or equal to 0.05 was considered significant.
Fig. 2. Dose-dependent protective effect of EGCG on aqueous (blank bar) and lipid (filled bar) compartment oxidation after 180 min of incubation. Values are means SEM of five independent experiments.
Results EGCG protective effect on human plasma oxidation induced by water- and lipid-soluble radical inducers When AAPH was used as the radical initiator, aqueous oxidation was already apparent after 30 min of incubation as a consequence of AA consumption, and the rate of oxidation increased after 120 min of incubation, due to uric acid depletion [16]. EGCG addition dose-dependently reduced the oxidative process as detected by DCF fluorescence both before and after uric acid depletion (Fig. 1A). At 60 min, when uric acid is depleted only by 30%, EGCG dose-dependently protected DCF oxidation by 13 2:5% at 0.5 lM and 55 3:4% at 5 lM. At 180 min of incubation, EGCG at 0.25 lM reduced DCFH oxidation by 20:25 0:34% and was almost completely protective at 10 lM (93:0 2:0% protection).
Fig. 1. (A) EGCG inhibits the oxidation of the aqueous plasma compartment induced by AAPH (20 mM) and monitored by DCF fluorescence increase. Values for ascorbate and urate are in the absence of added EGCG. (B) EGCG inhibits the oxidation of the lipid plasma compartment induced by MeO-AMVN (2 mM) and monitored by measuring BODIPY green fluorescence (BODIPY GF). Values are means SEM of five independent experiments. Where not shown, the error bars are covered by the symbols. Left axis: (j) Control; EGCG: (s) 0.25 lM, (M) 0.5 lM, ( ) 1 lM, () 5 lM, and (r) 10 lM; Right axis: (N) ascorbic acid and (}) uric acid.
When plasma containing BODIPY was incubated in the presence of 2 mM MeO-AMVN, a time-dependent increase of green fluorescence was observed, whose rate increased at 90 min following the consumption of atocopherol and b-carotene [16]. The protective effect afforded by EGCG in the lipid domain was found to be less effective than that found in the aqueous compartment; after 180 min of incubation, the lowest effective concentration was 0.5 lM (13:01 0:56%) and 10 lM resulted in 68 2:3% protection (Fig. 1B). In Fig. 2 the protective effect of EGCG in aqueous and lipid compartments after 180 min of incubation is compared; the calculated IC50s in aqueous and lipid compartments were, respectively, 0:72 0:02 and 4:37 0:14 lM. EGCG effect on hydrophilic and lipophilic plasma antioxidant consumption When 20 mM AAPH was added to plasma, ascorbic acid, and uric acid were almost totally consumed within 15 and 180 min, respectively (Fig. 1A). EGCG at all the concentrations tested (0.5–10 lM) was found to be ineffective in reducing the consumption of these two hydrophilic endogenous antioxidants (data not shown). AAPH also induced a significant consumption of lipophilic plasma antioxidants. After 120 min of incubation, the order of consumption expressed as percentage remaining was as follows (Table 1 and Fig. 3): a-tocopherol (3:9 0:9); lycopene (8:5 5:2); lutein (12:8 4:8); zeaxanthin (17:5 6:0); cryptoxanthin (18:9 3:9); and b-carotene (28:9 6:1). EGCG addition was found to significantly and dose-dependently reduce the consumption of all carotenoids (Table 1), suggesting its ability to trap aqueous radicals and hence prevent their diffusion into lipoproteins. The sparing effect of EGCG toward a-tocopherol consumption was significant when
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Table 1 Dose-dependent protective effect of EGCG on carotenoid consumption induced by AAPH (20 mM for 120 min) and MeO-AMVN (2 mM for 60 min) in plasma samples Carotenoid
EGCG (lM)
Radical initiator AAPH
MeO-AMVN
Residual amount (%) b-Carotene
0 (control) 0.5 1 5 10
28:9 6:2 41:5 6:6 54:4 4:9a 58:6 3:3a 64:3 6:4b
50:3 2:3 51:9 1:7 50:1 3:3 57:7 0:5 59:1 3:7
Lycopene
0 (control) 0.5 1 5 10
8:5 5:2 9:3 5:2 18:1 2:4 25:3 1:0a 32:3 4:2b
20:1 4:9 20:1 3:2 19:7 2:6 23:6 1:8 26:7 0:9
Cryptoxanthin
0 (control) 0.5 1 5 10
18:9 3:4 28:5 3:3 36:2 1:7a 48:9 2:1b 56:4 5:0b
41:8 4:2 54:4 2:7 52:4 0:8 45:1 3:1 41:8 1:6
Zeaxanthin
0 (control) 0.5 1 5 10
17:5 5:9 25:6 6:5 48:8 4:8a 58:9 5:1b 62:4 11:9b
40:0 3:1 47:7 5:8 49:6 2:1 44:3 0:9 42:7 2:0
Lutein
0 (control) 0.5 1 5 10
12:8 4:8 26:0 4:3 40:6 5:3a 56:0 4:4b 63:1 8:1b
31:0 5:4 32:6 4:1 33:7 3:9 44:6 5:5 40:4 4:0
10 mM AAPH was used, but not effective at 20 mM AAPH (Fig. 3). When MeO-AMVN was used to induce selective oxidation of the lipid compartment, a significant consumption of a-tocopherol and carotenoids was also observed. EGCG addition was found to be ineffective in sparing carotenoids at all the concentrations tested (0.5– 10 lM) (Table 1) but dose-dependently reduced a-tocopherol consumption (Fig. 3); this effect was significant at 1 lM and total protection was observed at 10 lM (% a-tocopherol remaining: 96:71 1:46 vs. 16:43 1:72 in controls; p < 0:001). EGCG regenerates a-tocopherol via reduction of its phenoxyl radical: ESR experiments Sixty seconds after mixing a-tocopherol with DPPH, the ESR spectrum of DPPH disappeared completely (due to the scavenging activity of a-tocopherol), and the typical spectrum of the a-tocopheroxyl free radical (aTOC-O ) was observed. Consecutive spectra (at 30 s time intervals for 3 min) showing the self-decay of a-TOC-O are shown in Fig. 4A. This decay has been described as a second-order reaction, as reported earlier [21]. EGCG addition dose-dependently accelerated the decay rate of a-TOC-O (Fig. 4B). The quenching effect (calculated 60 s after the beginning of the reaction) was already significant at 2 lM (% inhibition of ESR signal ¼ 8 1:3%) to reach an almost complete disappearance at 25 lM (IC50 ¼ 12:1 1:3 lM). Ascor-
The basal content of carotenoids was as follows: b-Carotene (3:58 0:18 lM); lycopene (2:10 0:23 lM); cryptoxanthin (1:77
0:10 lM); lutein (0:72 0:02 lM); and zeaxanthin (0:34 0:01 lM). a p < 0:05 vs. control. b p < 0:01 vs. control (ANOVA followed by DunnettÕs test).
Fig. 3. Dose-dependent effect of EGCG on a-tocopherol depletion induced by AAPH (10 and 20 mM) and MeO-AMVN (2 mM). The basal content of a-tocopherol was 42:08 1:28 lM. Values are means SEM of three independent experiments. *p < 0:05 vs. control; **p < 0:01 vs. control (ANOVA followed by DunnettÕs test).
Fig. 4. ESR spectra relative to the time-course of a-TOC-O decay in the absence (A) and presence (B) of EGCG (15 lM). a-TOC-O was induced by the stable radical DPPH.
G. Aldini et al. / Biochemical and Biophysical Research Communications 302 (2003) 409–414
bic acid, the physiological recycling agent of a-tocopherol, showed an IC50 ¼ 14:2 1:2 lM (data not shown).
Discussion The aim of the present investigation was to add to our understanding of the antioxidant mechanism of EGCG in human plasma. By using a selective fluorescent method able to induce and monitor the oxidative process in both the aqueous and lipid phases, we found that EGCG dose-dependently protected both compartments. However, the antioxidant efficiency of EGCG was six times greater in the aqueous vs. the lipid domain. The IC50s of EGCG, calculated after 180 min of incubation, in the aqueous and lipid plasma compartments were 0.72 and 4.37 lM, respectively. EGCG dose-dependently reduced AAPH-induced consumption of lipophilic antioxidants such as a-tocopherol and the carotenoids, suggesting that EGCG, by acting as a radical-scavenger in the aqueous compartment, limits the diffusion of the radical species to the lipid domain, thereby preventing the lipid-oxidation cascade and, as a consequence, lipophilic antioxidant depletion. By contrast, EGCG was ineffective (up to 10 lM) in sparing ascorbic acid, as previously described [22,13]. As reported by Lotito and Fraga [13], ascorbic acid may be able to prevent catechin depletion, and in view of the redox potentials [E(EGCG-O , Hþ /EGCGOH) ¼ 0.43 V]; E(A , H+/AH ) ¼ 0.28 V], it is thermodynamically feasible for ascorbic acid to regenerate EGCG from its aroxyl radical, EGCG-O . These interactions are depicted in Scheme 1, which includes the ability of a-tocopherol to prevent carotenoid oxidation as previously reported by Yeum et al. [23]. Although EGCG had a lower activity in the lipophilic than in the aqueous compartment, it was still found to dose-dependently inhibit oxidative damage in the lipid compartment induced by MeO-AMVN. The protective effect could be ascribed to one of the following
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mechanisms among others: (1) EGCG diffuses into lipoproteins where it acts as a chain-breaking antioxidant; (2) EGCG binds to the surface of lipoproteins where it recycles a-tocopherol from the tocopheroxyl radical. To understand whether EGCG is able to diffuse inside lipoproteins or remains localized on the outer surface of LDL, we studied the sparing effect of EGCG towards a-tocopherol and both polar and apolar carotenoids, using lipophilic peroxyl radicals generated by MeO-AMVN. EGCG at all concentrations tested (1– 10 lM) failed to prevent the depletion of either polar or apolar carotenoids, located, respectively, in the shell and core of the lipoproteins [24], while dose-dependently maintaining a-tocopherol, which resides at or near the surface of the lipoprotein particles [25]. These results indicate that EGCG is unable to diffuse into the shell/ core of the lipoprotein particles, but significantly binds to the outer surface of lipoproteins where the sparing/ recycling effect on a-tocopherol can occur. To demonstrate the direct reaction of EGCG with the tocopheroxyl radical, a direct ESR technique was used. EGCG was found to quench the tocopheroxyl radical with a potency similar to that of ascorbic acid, supporting the idea that EGCG regenerates a-tocopherol through a Htransfer mechanism. In addition, Zhu et al. [26] found that the a-tocopherol level in isolated LDL was partially restored when GT catechins were added during AAPHinduced depletion. Taken together, these data provide direct and unequivocal evidence for the regeneration of a-tocopherol via reduction of its phenoxyl radical by EGCG in LDL particles. Acknowledgments This research has been supported in part by the US Department of Agriculture, under agreement number 1950-51000-048-01A. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. We thank B. Gindelsky and E. Seyoum at Nutrition Evaluation Laboratory (HNRC) for the HPLC analyses of uric acid and ascorbic acid.
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Scheme 1. Antioxidant mechanism of EGCG in human plasma.
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