Tea Polyphenol (−)-Epigallocatechin-3-Gallate: A New Trapping

Rutgers University, 164 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, Department ...... Baynes , J. W., and Thorpe , S. R. 1999 Role of o...
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Chem. Res. Toxicol. 2007, 20, 1862–1870

Tea Polyphenol (-)-Epigallocatechin-3-Gallate: A New Trapping Agent of Reactive Dicarbonyl Species Shengmin Sang,*,† Xi Shao,†,‡ Naisheng Bai,§ Chih-Yu Lo,‡ Chung S. Yang,† and Chi-Tang Ho‡ Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers UniVersity, 164 Frelinghuysen Road, Piscataway, New Jersey 08854-8020, Department of Food Science, Rutgers UniVersity, 65 Dudley Road, New Brunswick, New Jersey 08901-8520, and Naturex, 375 Huyler Street, South Hackensack, New Jersey 07606 ReceiVed May 30, 2007

Previous studies have demonstrated that reactive dicarbonyl compounds [e.g., methylglyoxal (MGO) and glyoxal (GO)] irreversibly and progressively modify proteins over time and yield advanced glycation end products (AGEs), which are thought to contribute to the development of diabetes mellitus and its subsequent complications. Thus, decreasing the levels of MGO and GO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. In our studies to find nontoxic trapping agents of reactive dicarbonyl species from dietary sources, we found that (-)epigallocatechin-3-gallate (EGCG), the major bioactive green tea polyphenol, could efficiently trap reactive dicarbonyl compounds (MGO or GO) to form mono- and di-MGO or GO adducts under physiological conditions (pH 7.4, 37 °C). The products formed from EGCG and MGO (or GO), combined at different ratios, were analyzed using LC/MS. We also developed a method to purify the two major mono-MGO adducts of EGCG without derivatization, and their structures were identified as stereoisomers of monoMGO adducts of EGCG based on their 1D and 2D NMR spectra. Our LC/MS and NMR data showed that positions 6 and 8 of the EGCG A-ring were the major active sites for trapping reactive dicarbonyl compounds. We also found that EGCG lost its trapping efficacy under acidic conditions (pH e 4), suggesting a base-catalyzed trapping reaction. The purified mono-MGO adducts of EGCG in this study can be used as standards for further in vivo studies on the possible trapping of reactive dicarbonyl species by EGCG. Introduction Epidemiological studies, including large prospective studies, have indicated that hyperglycemia is the most important factor in the onset and progress of diabetic complications, especially in type 2 (noninsulin-dependent) diabetes mellitus (1–3). Increasing evidence identifies the formation of advanced glycation end products (AGEs)1 as a major pathogenic link between hyperglycemia and diabetes-related complications (4). Nonenzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins, lipids, and DNA. As a first step of AGEs formation, proteins in the tissues are modified by reducing sugars (e.g., glucose) through the reaction between a carbonyl group of the sugar and a free amino group of the proteins, leading to the formation of fructosamines via a Schiff base by Amadori rearrangement (4). Both the Schiff base and the Amadori product further undergo a series of reactions to form new reactive dicarbonyl intermediates, for example, methylglyoxal (MGO) and glyoxal (GO), that can modify proteins to form AGEs of various chemical structures (4, 5). In addition, the autoxidation of glucose and lipid peroxidation are * To whom correspondence should be addressed. Tel: 732-445-3400ext. 300. Fax: 732-445-0687. E-mail: [email protected]. † Department of Chemical Biology, Rutgers University. ‡ Department of Food Science, Rutgers University. § Naturex. 1 Abbreviations: AGEs, advanced glycation end products; EC, (-)epicatechin; ECG, (-)-epicatechin gallate; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin gallate; GO, glyoxal; HMQC, heteronuclear multiple quantum correlation; HMBC, heteronuclear multiple band correlation; MGO, methylglyoxal.

also important sources of reactive dicarbonyl compounds (6–8). The increase in reactive dicarbonyl intermediates, referred to as “carbonyl stress”, has been shown to be the consequence of hyperglycemia in diabetes (9–11). Carbonyl stress compounds such as MGO and GO, the two major R-dicarbonyl compounds found in humans, have recently been given much attention because of their possible clinical significance in chronic and age-related diseases. They accumulate in body fluids and tissues mainly due to accelerated oxidative stress, and they modify proteins, DNAs, and phospholipids to form AGEs (9–11). It has been shown that dicarbonyl compounds are more reactive in the glycation reaction than reducing sugars and play an important role for cross-linking proteins in the glycation process (12–14). It has been reported that in diabetes, the concentration of MGO increases in the lens, blood, and kidney (11, 15, 16). In patients with insulin-dependent and noninsulin-dependent diabetes, the plasma concentration of MGO was 2–6-fold higher than those of healthy individuals (17, 18). It has been well-documented that AGEs progressively accumulate in the tissues and organs, which develop chronic complications of diabetes mellitus, such as retinopathy, nephropathy, neuropathy, and macrovascular disease (5, 14, 19, 20). Trapping of reactive dicarbonyl compounds by several pharmaceutical agents, such as aminoguanidine, metformin, and pyridoxamine, has been shown to be a useful strategy for inhibiting the formation of AGEs, and thus inhibiting or delaying diabetic complications (21–26).

10.1021/tx700190s CCC: $37.00  2007 American Chemical Society Published on Web 11/15/2007

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Figure 1. Structures of EGCG and its two major mono-MGO adducts, EGCGMGO-1 and EGCGMGO-2.

Many studies in humans, animal models, and cell lines suggest potential health benefits from the consumption of tea, including the prevention of cancer, heart disease, obesity, and diabetes (27–29). These effects have been attributed mostly to the polyphenol compounds in tea, in which the most abundant and biologically active polyphenol is (-)-epigallocatechin-3gallate (EGCG) (Figure 1). In addition to its health benefits, as a widely consumed beverage, tea is not known to be toxic. A series of three recent publications on safety studies on EGCG preparations (Teavigo > 90% EGCG) (30–32) concluded that (i) Teavigo was not genotoxic, (ii) the no observed effect level for rats and dogs was 500 mg EGCG preparation/kg/day, and (iii) feeding pregnant rats diet supplemented up to 14000 ppm during organogenesis was nontoxic to dams or fetuses. For humans, the highest repeated dose administered to date is 800 mg EGCG per day for 28 days (33), and no abnormal liver or kidney function was noted in this study. In our previous studies to find nontoxic trapping agents for reactive dicarbonyl species from dietary sources, we compared the trapping effect of MGO by four major catechins in green tea and three major theaflavins in black tea under physiological conditions (pH 7.4, 37 °C). Our results indicated that all of the test compounds could efficiently trap MGO (34). To further understand the mechanisms by which tea catechins trap reactive dicarbonyl compounds, we studied the trapping kinetic of MGO and GO by EGCG. The products formed from the reaction of EGCG and MGO or GO at different ratios were analyzed using LC/MS. We also developed a method to purify the two major mono-MGO adducts of EGCG without derivatization, and their structures were identified as stereoisomers of mono-MGO adducts of EGCG based on their 1D and 2D NMR spectra.

Materials and Methods Materials. EGCG (97% pure with ∼3% ECG) was provided by Mitsui Norin Co. Ltd. (Shizuoka, Japan). RP C-18 silica gel, Sephadex LH-20 gel, TLC plates (250 µm thickness, 2–25 µm particle size), methylglyoxal, glyoxal, quinoxaline, 2-methylquinoxaline, 1,2-diaminobenzene, aminoguanidine, lysine, arginine, and

CD3OD were purchased from Sigma (St. Louis, MO). HPLC-grade solvents and other reagents were obtained from VWR Scientific (South Plainfield, NJ). HPLC-grade water was prepared using a Millipore Milli-Q purification system (Bedford, MA). NMR. 1H (400 MHz), 13C (100 MHz), and all 2D NMR spectra were acquired on a Varian 400 instrument (Varian Inc., Palo Alto, CA). Compounds were analyzed in CD3OD, with TMS as an internal standard. 1H-13C HMQC (heteronuclear multiple quantum correlation) and HMBC (heteronuclear multiple band correlation) experiments were performed as described previously (35). HPLC Analysis. The level of methylquinoxaline and quinoxaline was analyzed using an HPLC system, consisting of a Waters 717 refrigerated autosampler, a Waters associates SP2A2 chromatography pump, and a UV–vis detector (Waters 490E programmable multiwavelength detector). A Supelcosil C18 reversed-phase column (150 mm × 4.6 mm inner diameter; Supelco Co., Bellefonte, PA) was used. The HPLC was performed under binary gradient elution, and mobile phases A (95% acetonitrile and 5% water) and B (5% water and 95% acetonitrile) were used. All of the solvents were filtered with 0.2 µm Nylaflo membrane filter and then degassed. The flow rate was maintained at 1 mL/min, and the mobile phase began with 100% A. It was followed by progressive, linear increases in B to 40% at 35 min, 60% at 45 min, and 100% at 46 min. The mobile phase was then re-equilibrated to 100% at 52 min for 8 min. The injection volume was 50 µL for each sample solution. The wavelength of UV detector was set at 280 nm for methylquinoxaline and 313 nm for quinoxaline. The limit of detection was ∼100 ng/mL, and the limit of quantification was 1 µg/mL for both methylquinoxaline and quinoxaline. Derivatization of MGO or GO with 1,2-Diaminobenzene (DB). The time course of the derivatization of MGO or GO (0.73 mmol) with DB (0.73, 3.65, 7.3, and 14.6 mmol) was studied in phosphate buffer solution (50 mmol/L, pH 7.4) at 37 °C, respectively. The amount of methylquinoxaline or quinoxaline was monitored over a course of 4 h by analyzing small units (10 µL) of the incubation mixtures at different time points (0, 5, 10, 30, 60, 120, and 240 min) using the HPLC method detailed above. The recovery of methylquinoxaline or quinoxaline was more than 95%. Effect of pH on the Derivatization of MGO with DB. The time course of the derivatization of MGO (0.73 mmol) with DB (0.73 and 14.6 mmol) was studied in two different phosphate buffer

1864 Chem. Res. Toxicol., Vol. 20, No. 12, 2007 solutions (50 mmol/L, pH 7.4 and 4.0) at 37 °C, respectively. The amount of methylquinoxaline at different time points (0, 5, 10, 30, 60, 120, and 240 min) was quantified using the method detailed above. Effect of pH on the Trapping of MGO by EGCG. MGO (2.0 mM) was incubated with 6 mM EGCG in two different phosphate buffer solutions (50 mmol/L, pH 7.4 and 4.0) at 37 °C and shaken at 40 rpm speed for 5, 10, 15, 20, 30, 40, and 60 min. Then, to each triplicated vial at each time point, 1 µL of acetic acid was added to stop the reaction and 100 mM DB was added next to derivatize the remaining MGO using the method detailed above. Trapping of MGO or GO by EGCG, Lysine, Arginine, and Aminoguanidine under Physiological Conditions. MGO (2.0 mM) or GO (2.0 mM) was incubated with 6 mM EGCG (or lysine, arginine, and aminoguanidine, respectively) in a pH 7.4 phosphate buffer solution at 37 °C and shaken at 40 rpm speed for 5, 10, 15, 20, 30, 40, and 60 min. Then, to each triplicated vial at each time point, 1 µL of acetic acid was added to stop the reaction and 100 mM DB was added next to derivatize the remaining MGO or GO using the method detailed above. LC/ESI-MS Method. LC/MS analysis was carried out with a Finnigan Spectra System, which consisted of a Surveyou MS pump plus, a Surveyou refrigerated autosampler plus, and a LTQ linear ion trap mass detector (ThermoFinigan, San Jose, CA) incorporated with an electrospray ionization (ESI) interface. A 50 mm × 2.0 mm i.d., 3 µm Gemini C18 column (Phenomenex) with a flow rate of 0.2 mL/min was used for separation. The column elution started with 3 min of isocratic phase of 100% solvent A (5% aqueous methanol with 0.2% acetic acid), followed by progressive, linear increases in solvent B (95% aqueous methanol with 0.2% acetic acid) to 20% at 33 min and 100% at 45 min for 5 min. The mobile phase was then re-equilibrated to 100% A at 51 min for 10 min. The LC elute was introduced into the ESI interface. The negative ion polarity mode was set for an ESI ion source with the voltage on the ESI interface maintained at approximately 5 kV. Nitrogen gas was used as the sheath gas at a flow rate of 30 arb units and the auxiliary gas at 5 arb units, respectively. The structural information of EGCG and the major MGO or GO adducts was obtained by tandem mass spectrometry (MS/MS) through collisioninduced dissociation (CID) with a relative collision energy setting of 30%. Studying the Formation of MGO or GO Adducts of EGCG Using LC/MS. EGCG (14.6 mM) was incubated with different concentrations of MGO or GO (4.87, 14.6, and 43.8 mM) in a pH 7.4 phosphate buffer solution at 37 °C for 60 min, respectively. Next, 10 µL samples were taken and transferred to vials already containing 190 µL of a solution containing 0.2% ascorbic acid and 0.05% EDTA, to stabilize EGCG and the MGO or GO adducts of EGCG. These samples were immediately analyzed or stored at -80 °C before analyzing with LC/MS. Purification of the Major Mono-MGO Adducts of EGCG. EGCG (1.0 g, 2.2 mmol) and MGO (0.131 g, 0.73 mmol) were dissolved in 150 mL of phosphate buffer (50 mmol/L, pH 7.4) and then kept at 37 °C for 60 min. After extraction with ethyl acetate and the evaporation of the solvent in vacuo, the residue was loaded into a Sephadex LH-20 column eluted with ethanol to remove the remaining EGCG and then into a RP C-18 column eluted with 20% aqueous methanol to obtain EGCGMGO-1 (51.2 mg) and EGCGMGO-2 (45.8 mg). 1H and 13C NMR data of EGCGMGO-1 and EGCGMGO-2 were listed in Table 1, and negative ESI-MS of both compounds were found to be m/z 529 [M - H]-.

Results Derivatization of MGO or GO with DB. The commonly used assays to determine the levels of MGO and GO involve the derivatization of MGO or GO with DB and its derivatives, followed by the quantification of the resulting quinoxalines by HPLC with ultraviolet, spectrophotometric, fluorescence, or mass detection (17, 36–38). In our study, we modified the

Sang et al. Table 1. δH (400 MHz) and δC (100 MHz) NMR Spectra Data of EGCGMGO-1 and EGCGMGO-2 (CD3OD) (δ in ppm) EGCGMGO-1

EGCGMGO-2

position

δH

δC

δH

δC

2 3 4

5.02 s 5.53 m 2.99 dd 4.0, 17.2 2.88 brd 17.2

78.9 d 69.6 d 27.0 t

5.05 s 5.51 m 2.99 dd 4.0, 17.2 2.88 brd 17.2

79.1 d 69.6 d 26.9 t

5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

6.06 s

5.61 s 2.13 s 6.51 s

6.51 s 6.91 s

6.91 s

158.3 s 96.4 d 156.9 s 105.3 s 154.8 s 99.6 s 72.7 d 211.0 s 25.6 t 130.3 s 106.6 d 146.6 s 133.7 s 146.6 s 106.6 d 121.4 s 110.2 d 146.2 s 139.7 s 146.2 s 110.2 d 167.5 s

6.06 s

5.56 s 2.15 s 6.47 s

6.47 s 6.92 s

6.92 s

158.3 s 96.3 d 156.7 s 105.5 s 155.1 s 99.6 s 72.6 d 212.1 s 25.9 t 130.3 s 106.8 d 146.6 s 133.8 s 146.6 s 106.8 d 121.4 s 110.2 d 146.3 s 139.7 s 146.3 s 110.2 d 167.6 s

existing HPLC-UV method to quantify MGO or GO using DB as the derivatization agent (Figure 2). The effects of reaction time, the pH of the reaction medium, and the concentration of DB on the formation of quinoxalines were studied. Our data showed that DB could effectively react with MGO and GO within 10 min, and a concentration of DB, which was 10–20 fold greater than that of MGO or GO, was optimal for the

Figure 2. (A) Trapping of MGO by EGCG, lysine, arginine, and aminoguanidine and GO by EGCG and arginine in phosphate buffer (pH 7.4, 37 °C). MGO (2.0 mM) or GO (2.0 mM) was incubated with 6 mM EGCG (or lysine, arginine, and aminoguanidine, respectively) in pH 7.4 phosphate buffer solutions at 37 °C for 5, 10, 15, 20, 30, 40, and 60 min, respectively. (B) The effect of pH on the trapping of MGO by EGCG. MGO (2.0 mM) was incubated with 6 mM EGCG in two different phosphate buffer solutions (50 mmol/L, pH 7.4 and 4.0) at 37 °C for 5, 10, 15, 20, 30, 40, and 60 min, respectively. Each value represents the mean ( SD (n ) 3).

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Figure 3. (A) LC chromatograms of EGCG after incubation with different ratios of MGO (3:1, 1:1, and 1:3) for 60 min. MM and DM stand for mono- and di-MGO, respectively. (B) LC chromatogram of EGCG after incubation with an equal molar ratio of MGO (1:1) for 60 min. The chromatograms of EGCG, mono-, and di-MGO adducts of EGCG were obtained using SIM mode.

derivatization. In addition, we found that the pH of the reaction medium did not influence the derivatization. Trapping of MGO or GO by EGCG, Lysine, Arginine, and Aminoguanidine under Physiological Conditions. We found that EGCG could efficiently trap MGO or GO under physiological conditions (pH 7.4, 37 °C). More than 90% MGO was trapped within 5 min, and 70% GO was trapped within 1 h (Figure 2). Our data also demonstrated that EGCG reacted more effectively with MGO or GO, as opposed to lysine, arginine, or aminoguanidine (Figure 2). We quantified the levels of MGO or GO by derivatization with DB to form methylquinoxaline and quinoxaline, respectively, and the products were quantified by HPLC with an UV detector. In addition, we found that EGCG could not trap reactive dicarbonyl compounds under acidic conditions (pH 4) (Figure 2). Studying the Formation of MGO or GO Adducts of EGCG Using LC/MS. To understand the mechanism by which EGCG traps MGO or GO, we studied the formation of carbonyl adducts under different ratios of EGCG to MGO or GO (3:1, 1:1, and 1:3) using LC/MS (Figures 3 and 4). The structural information of these products was obtained using LC/MS/MS analysis under selective ion monitoring (SIM) mode (Figures 5 and 6). At a 3:1 ratio, we found that the mono-MGO and monoGO adducts were the major products, repectively. During the incubation of EGCG with MGO (3:1 ratio), three major new peaks appeared in the LC chromatogram (Figures 3 and 5A). All three new peaks had the same molecular ion (529 [M H]-) and MS/MS fragments but different retention times. All three compounds had the fragment 457 ([M - 72 - H]-), showing that they all lost one MGO (m/z 72) molecule (Figure 5), thus indicating that they were mono-MGO adducts of EGCG. Furthermore, all three compounds had the fragments 359 ([M - 170 - H]-) and 377 ([M - 152 - H]-), exhibiting the typical

loss of one gallic acid group (M - 170) and one galloyl group (M - 152), respectively. Therefore, the conjugation did not occur on the gallate ring of EGCG. There were six major new peaks that appeared in the LC chromatogram after incubation of EGCG and GO (3:1) (Figure 6A). All six new peaks had the same molecular ion (515 [M - H]-) and similar MS/MS fragments but different retention times. Five of them had the fragment 457 ([M - 58 - H]-), meaning that they lost one GO (m/z 58) molecule, and all six of them had the fragments 345 ([M - 170 - H]-) and 363 ([M - 152 - H]-), signifying the typical loss of one gallic acid group (M - 170) and one galloyl group (M - 152), respectively. This loss again indicated that the conjugation did not occur on the gallate ring of EGCG. When EGCG and MGO (or GO) at a 1:1 ratio were incubated at 37 °C, it was observed that the amounts of mono-MGO or mono-GO adducts formed were higher than those from the reaction at a 3:1 ratio, and di-MGO or di-GO adducts were formed as minor products (Figures 3 and 4A). At a 1:3 ratio, we found that di-MGO adducts and mono- and di-GO adducts were the major products (Figures 3 and 4A). During the incubation of EGCG and MGO (1:3), five major new peaks appeared in the LC chromatogram (Figures 3A and Figure 5B). All five new peaks had the same molecular ion (601 [M - H]-) and MS/MS fragments but had different retention times. All five of the compounds had the fragment 529 ([M -72 - H]-), meaning that one MGO (m/z 72) molecule was lost (Figure 5B). This fragment had almost identical MS/MS fragments as those from the mono-MGO adduct of EGCG. All of these observations indicated that they were di-MGO adducts of EGCG. In addition, all of these compounds had the fragments 431 ([M - 170 H]-) and 449 ([M - 152 - H]-), which represented the typical loss of one gallic acid group (M - 170) and one galloyl group (M - 152), respectively, indicating that the conjugation did not

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Figure 4. (A) LC chromatograms of EGCG after incubation with different ratios of GO (3:1, 1:1, and 1:3) for 60 min. MG and DG stand for monoand di-GO, respectively. (B) LC chromatogram of EGCG after incubation with an equal molar ratio of GO (1:1) for 60 min. The chromatograms of EGCG, mono-, and di-GO adducts of EGCG were obtained using SIM mode.

Figure 5. LC/MS/MS spectra of (A) mono-MGO and (B) di-MGO adducts of EGCG.

occur on the gallate ring of EGCG. There were four major new peaks that appeared in the LC chromatogram after incubation of EGCG and GO (1:3) (Figures 4A and 6B). Two of them had

the same molecular ion (515 [M - H]-), MS/MS fragments, and retention times as those observed from the 3:1 ratio. The other two peaks had the molecular ion 573 [M - H]-), which

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Figure 6. LC/MS/MS spectra of (A) mono-GO and (B) di-GO adducts of EGCG.

were 116 (58 × 2) mass units higher than that of EGCG, indicating that they were di-GO adducts of EGCG. Both of them had the fragments, 403 ([M - 170 - H]-) and 421 ([M - 152 - H]-), which signaled the typical loss of one gallic acid group (M - 170) and one galloyl group (M - 152), respectively, indicating that the conjugation did not occur on the gallate ring of EGCG (Figure 6B). Purification and Structure Elucidation of the Major Mono-MGO Adducts of EGCG. We purified the acetylated derivatives of two mono-MGO adducts of EGCG using a chiral column and determined their structures after analyzing the NMR data of the acetylated derivatives (34). In this study, we successfully purified two major mono-MGO adducts of EGCG, EGCGMGO-1 and EGCGMGO-2, without derivatization (Figure 1). EGCGMGO-1 was assigned the molecular formula C25H22O13 based on negative-ion ESI-MS ([M - H]- at m/z 529) and 13C NMR data. Its 1H and 13C NMR spectra showed very similar patterns as compared to the spectra of EGCG (Table 1) (39). The 1H NMR spectrum of EGCGMGO-1 exhibited signals for the B-ring (H-2′, 6′ at δH 6.51 s), C-ring (H-2 at δH 5.02 s, H-3 at δH 5.53 m, and H-4 at δH 2.99 dd 4.0, 17.2, and 2.88 brd 17.2), and D-ring (H-2″, 6″ at δH 6.91 s), which were similar to those of EGCG, indicating that the B-, C-, and D-rings of EGCGMGO-1 did not undergo any changes. In comparison with the 1H NMR spectrum of EGCG, EGCGMGO-1 only had one singlet signal for one proton of the A-ring (δH 6.06 s), instead of the one singlet signal for two protons of EGCG (δH 5.96 s 2H). There were also two additional proton signals for the MGO group (δH 5.61 s, 1H and 2.13 s, 3H) in the 1H NMR spectrum of EGCGMGO-1. The major differences in the 13C spectra of EGCGMGO-1 and EGCG were (i) the presence of three additional carbons at δC 25.6 t, 72.7 d, and 211.0 s for MGO group in EGCGMGO-1 and (ii) the presence of a

Figure 7. Significant HMBC (HfC) correlations of EGCGMGO-1 and EGCGMGO-2.

quaternary carbon at δC 105.3 in lieu of an unsubstituted aromatic carbon from the A-ring of EGCG. In addition, the molecular weight of EGCGMGO-1 was 72 (M.W. of MGO, 72) mass units higher than that of EGCG. All of these spectral features supported the presence of a MGO group in EGCGMGO-1 at either the C-8 or the C-6 position of the A-ring. The HMBC spectrum showed correlations between δC 154.8 and H-2 (δH 5.02 ppm), δC 154.8 and H-4 (δH 2.99 and 2.88 ppm), and δC 158.3 and H-4, thus establishing the signal at δC 154.8 ppm as that of C-9 and δC 158.3 ppm as that of C-5 (Figure 7). This carbon nucleus resonating at δC 154.8 ppm showed no coupling to the A-ring proton (δ 6.06 ppm), whereas the carbon at δC 158.3 ppm had a cross-peak with the A-ring proton in the HMBC spectrum. Therefore, the MGO group was located at the C-8 position of the A-ring. This was further confirmed by the cross-peaks in the HMBC spectrum (Figure 7) between δC

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Figure 8. Formation pathway of mono- and di-MGO adducts of EGCG under neutral or base conditions.

154.8 (C-9) and H-11 (δH 5.61 s) and δC 105.3 (C-8) and H-11 (δH 5.61 s). Thus, the structure of EGCGMGO-1 was identified as shown in Figure 1. The negative-ion ESI-MS of EGCGMGO-2 displayed a molecular ion peak at m/z [M - H]- 529, allowing us to conclude that the molecular formula was C25H22O13, which was the same as that of EGCGMGO-1. The NMR spectra of EGCGMGO-2 showed signal patterns similar to those of EGCGMGO-1 (Table 1). The 1H NMR spectrum of EGCGMGO-2 displayed signals for the B-ring (H-2′, 6′ at δH 6.47 s), C-ring (H-2 at δH 5.05 s, H-3 at δH 5.51 m, and H-4 at δH 2.99 dd 4.0, 17.2, and 2.88 brd 17.2), and D-ring (H-2″, 6″ at δH 6.92 s). There was only one proton (singlet signal) for the A-ring (δH 6.06 s), and there were two additional proton signals for the MGO group (δH 5.56 s, 1H and 2.15 s, 3H). The 13C NMR spectrum of EGCGMGO-2 displayed 25 carbon signals, 15 of which were assigned to the A-, B- and C-rings of flavan-3-ols, seven of which were the signals for the gallate group and three of which were for the MGO group (δC 25.9 t, 72.6 d, and 212.1 s) (Table 1). Similarly, one quaternary carbon and one unsubstituted aromatic carbon were observed at δC 105.5 and 96.3 ppm instead of two unsubstituted aromatic carbon from the A-ring. The spectral information allowed us to conclude that a MGO group was also located at the A-ring in EGCGMGO-2. The HMBC spectrum showed correlations between δC 155.1 and H-2 (δH 5.05 ppm), δC 155.1 and H-4 (δH 2.99 and 2.88 ppm), and δC 158.3 and H-4, thus establishing the signal at δC 155.1 ppm as that of C-9 and δC 158.3 ppm as that of C-5 (Figure 7). This carbon nucleus resonating at δC 155.1 ppm showed no coupling to the single A-ring proton (δ 6.06 ppm), whereas the carbon at δC 158.3 ppm had a cross-peak with the single A-ring proton in the HMBC spectrum. Therefore, the MGO group was also located at the C-8 position of A-ring. This was further confirmed by the cross-peaks in the HMBC spectrum (Figure 7) between δC 155.1 (C-9) and H-11 (δH 5.61 s) and δC 105.3 (C-8) and H-11 (δH 5.61 s). As a result,

the difference between EGCGMGO-1 and EGCGMGO-2 was the stereochemistry of the newly formed hydroxyl group at position 11; one had the R-configuration and the other had the S-configuration. Full assignments of the 1H and 13C NMR signals of EGCGMGO-1 and EGCGMGO-2 were made by analyzing the signals in HMBC and HMQC spectra (Table 1).

Discussion In this study, we found that EGCG could rapidly trap both MGO and GO under neutral or alkaline conditions. To our surprise, the trapping efficacy of EGCG for GO, which had two aldehyde groups, was much lower than that for MGO, which had one aldehyde and one keto group. It has been reported that the hydrated monomer is the main form of GO in aqueous solution and that the hydrated monomer tends to polymerize into a GO dimer and trimer (6). There is an equilibrium between the monomer, dimer, trimer, and free GO. Therefore, the trapping reaction between EGCG and GO is slowed down by the transformation from the hydrated monomer, dimer, and trimer to free GO. It has been reported that MGO and GO, the two major R-dicarbonyl compounds found in humans, are extremely reactive and readily modify lysine and arginine residues on proteins to form AGEs (26). Our data showed that EGCG was more reactive than lysine and arginine in terms of trapping MGO or GO, indicating that EGCG has the potential to compete with lysine and arginine in vivo and therefore prevent the formation of AGEs. In addition, we also found that EGCG was more reactive at trapping MGO than the pharmaceutical agent, aminoguanidine, which has been shown to inhibit the formation of AGEs by trapping of reactive dicarbonyl compounds in vivo (21, 40, 41). We have purified two major products, EGCGMGO-1 and -2, from the reaction between EGCG and MGO at a 3:1 molar ratio. Their structures were identified as the mono-MGO adducts of

Trapping of Dicarbonyl Species by EGCG

EGCG with the MGO conjugated at position 8 of the EGCG A-ring for both compounds. The only difference between the two products is the stereochemistry of the newly formed hydroxyl group, one with R-configuration and the other with S-configuration. However, we could not determine which compound had the R-configuration and which compound had the S-configuration using NMR (including 2D ROESY). The LC/MS/MS spectra under SIM mode showed that there were three major new peaks (MM-1, -2, and -3) corresponding to the molecular ion of mono-MGO adducts of EGCG (529 [M H]-). They all had almost identical MS/MS fragments, indicating they had similar structures. All three peaks displayed the typical loss of one gallic acid group or one gallate group, indicating that the MGO group was not conjugated on the gallate ring. On the basis of the structures of two of those three peaks that we have identified, it is most likely that the MGO group is conjugated at position 6 of EGCG in the structure of peak 3 (MM-3). In theory, there should be two isomers for mono-MGO adducts of EGCG at position 6: one with R-configuration and the other with S-configuration. It is possible that one peak overlapped with one of the three peaks that we observed from the LC chromatogram since their structures were so similar. During our purification, we noticed that it was difficult to separate them from each other. We also observed that they were unstable during the purification. Therefore, we were only able to purify the two major products. Our results clearly indicate that the major active site of EGCG is at position 6 and 8 of the A-ring and that the gallate ring does not play an important role in the trapping of reactive dicarbonyl species. This may explain our previous observation that both (-)-epicatechin (EC) and EGC had similar trapping efficacy of MGO as compared to catechin gallate, (-)epicatechin 3-gallate (ECG), and EGCG. Many studies have shown that dietary flavonoids, which are widely distributed in fruits, vegetables, grains, and beverages, such as tea, coffee, and wine, are strong antioxidants and may prevent diabetes and its complications. However, the mechanisms by which dietary flavonoids prevent the development of diabetic complications are unclear. It has been observed in vitro that dietary flavonoids may inhibit the formation of AGEs by trapping reactive dicarbonyl compounds. A recent study investigated the inhibitory effect of dietary flavonoids, including catechin, EC, ECG, EGC, EGCG, kaempferol, luteolin, naringenin, quercetin, and rutin, on different stages of protein glycation, including MGOmediated protein glycation (42). Luteolin, rutin, EGCG, and quercetin exhibited significant inhibitory effects on MGOmediated AGEs formation by 82.2, 77.7, 69.1, and 65.3%, respectively, and the other flavonoids showed 13–54% inhibitory effects at a concentration of 100 µM. Although the authors did not discuss the mechanism involved, it appears that flavonoids can trap MGO and therefore inhibit the formation of AGEs. A more recent study found that flavonoids containing vicinyl dihydroxyl groups, such as quercetin and myricetin, could significantly decrease the level of GO during the autoxidation of glucose in vitro (43). These authors also found that rutin and its metabolite, quercetin, could effectively inhibit GOderived AGEs formation during glycation of collagen I by glucose (43). Because luteolin, rutin, and quercetin have the same A-ring structure as that of EGCG, they may have the same mechanism to trap reactive dicarbonyl species and form A-ring MGO or GO adducts. However, the reaction between dietary flavonoids and reactive dicarbonyl compounds needs to be further characterized. It is also important to know whether

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dietary flavonoids can effectively inhibit the formation of AGEs by trapping reactive dicarbonyl compounds in vivo. On the basis of our observation, a mechanism of EGCG trapping MGO to form mono- and di-MGO adducts is proposed (Figure 8). The slightly alkaline pH can increase the nucleophilicity at positions 6 and 8 of the A-ring of EGCG, facilitating the addition of MGO at these two positions to form mono- and di-MGO adducts. It is unclear why the trapping reaction does not occur on the gallate ring, which has been considered to play an important role in the antioxidative effect of EGCG. The Victrihydroxyl group may make position 2″ or 6″ of the gallate ring less nucleophilic than positions 6 and 8, as well as too sterically hindered to react with MGO or GO. In summary, we found that EGCG was a very efficient trapping agent of reactive dicarbonyl species under in vitro conditions (pH 7.4, 37 °C). It could rapidly react with MGO or GO to form related adducts, and the major active sites were positions 6 and 8 of the A-ring. We also found that EGCG was more reactive than lysine, arginine, and the pharmaceutical agent, aminoguanidine, in terms of trapping reactive dicarbonyl species, MGO or GO. The structural information of the MGO or GO adducts of EGCG obtained from LC/MS and NMR analysis will help us to further understand whether EGCG can decrease the levels of reactive dicarbonyl species in vivo through the same mechanism. Acknowledgment. This work was supported by the NIH Center for Dietary Supplements Research on Botanicals and Metabolic Syndrome, Grant #1-P50 AT002776-01, and Rutgers University.

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