Tea Flavanols Block Advanced Glycation of Lens Crystallins Induced

Dec 1, 2014 - Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina. Research...
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Tea Flavanols Block Advanced Glycation of Lens Crystallins Induced by Dehydroascorbic Acid Yingdong Zhu,† Yantao Zhao,† Pei Wang,† Mohamed Ahmedna,‡ Chi-Tang Ho,§ and Shengmin Sang*,† †

Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States ‡ Department of Health Science, Qatar University, Doha, Qatar § Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, United States

ABSTRACT: Growing evidence has shown that ascorbic acid (ASA) can contribute to protein glycation and the formation of advanced glycation end products (AGEs), especially in the lens. The mechanism by which ascorbic acid can cause protein glycation probably originates from its oxidized form, dehydroascorbic acid (DASA), which is a reactive dicarbonyl species. In the present study, we demonstrated for the first time that four tea flavanols, (−)-epigallocatechin 3-O-gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin 3-O-gallate (ECG), and (−)-epicatechin (EC), could significantly trap DASA and consequently form 6C- or 8C-ascorbyl conjugates. Among these four flavanols, EGCG exerted the strongest trapping efficacy by capturing approximate 80% of DASA within 60 min. We successfully purified and identified seven 6C- or 8C-ascorbyl conjugates of flavanols from the chemical reaction between tea flavanols and DASA under slightly basic conditions. Of which, five ascorbyl conjugates, EGCGDASA-2, EGCDASA-2, ECGDASA-1, ECGDASA-2 and ECDASA-1, were recognized as novel compounds. The NMR data showed that positions 6 and 8 of the ring A of flavanols were the major active sites for trapping DASA. We further demonstrated that tea flavanols could effectively inhibit the formation of DASA-induced AGEs via trapping DASA in the bovine lens crystallin-DASA assay. In this assay, 8C-ascorbyl conjugates of flavanols were detected as the major adducts using LCMS. This study suggests that daily consumption of beverages containing tea flavanols may prevent protein glycation in the lens induced by ascorbic acid and its oxidized products.

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INTRODUCTION Protein glycation takes place through a complicated series of very slow reactions in the body, including the Amadori reaction, Schiff base formation, and the Maillard reaction, giving rise to the formation of advanced glycation end products (AGEs). AGEs have recently received increasing attention as substances that play a significant pathogenic role in the initiation and progression of diabetic complications. The accumulation of AGEs in human has been reported to be more apparent in the elderly and those with diabetic complications.1−4 Particularly, diseases such as retinopathy, nephropathy, atherosclerosis, and cataract formation are considered to be either caused or promoted by AGEs.1 Reactive dicarbonyls are important intermediates of the Maillard reaction and are believed to contribute significantly to intracellular AGE formation. For instance, methylglyoxal (MGO) has been reported to be more active than reducing © XXXX American Chemical Society

sugars as precursor of AGEs and to exert direct toxicity to cells and tissues.5 Ascorbic acid (ASA) levels are markedly high in selected tissues, such as lens, brain, and adrenal gland, especially in the lens, where ASA concentration can reach 3 mM.6 ASA has been shown to participate in glycation reaction7 and to contribute to protein aggregation and cataractogenesis.6,8 This was further supported by a mouse model that selectively overexpresses the human ASA transporter SVCT2 in the lens, thereby increasing the influx of ASA, which led to marked lens browning and AGE accumulation.9 The chemical pathways by which ASA forms glycating agents originate from its oxidized form, dehydroascorbic acid (DASA). Several in vitro studies have reported that DASA can rapidly react with lens proteins via a glycation mechanism and form cross-linked proteins.8,10,11 Received: October 21, 2014

A

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standard. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and br (broad). The 13C NMR spectra are proton-decoupled. HPLC Analysis. The level of DASA-quinoxaline was analyzed using an HPLC system, consisting of an ESA model 584 HPLC pump, an ESA model 542 autosampler, an ESA organizer, and an ESA 526 UV detector. A Gemini C18 column (150 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA) was used for chromatographic analysis at a flow rate of 1.0 mL/min. The HPLC was performed under binary gradient elution, and mobile phases A (30 mM sodium phosphate buffer containing 1.75% acetonitrile and 0.125% tetrahydrofuran, pH 3.35) and B (15 mM sodium phosphate buffer containing 58.5% acetonitrile and 12.5% tetrahydrofuran, pH 3.45) were used. The gradient elution had the following profile: 5% B from 0 to 3 min; 5 to 20% B from 3 to 10 min; 20 to 30% B from 10 to 15 min; 30 to 100% B from 15 to 20 min; 100% B from 20 to 25 min; and 5% B from 25 to 30 min. The injection volume of the samples was 10 μL. The wavelength of UV detector was set at 318 nm, with ∼100 ng/mL as the limit of detection and ∼1 μg/mL as the limit of quantification for DASA-quinoxaline. LC-MS Analysis. LC-MS analysis was carried out with a ThermoFinnigan Spectra System consisting of an Accela high-speed MS pump, an Accela refrigerated autosampler, and an LCQ Fleet ion trap mass detector (Thermo Electron, San Jose, CA) incorporated with an electrospray ionization (ESI) interface. A Gemini-NX C18 column (150 mm × 4.6 mm i.d., 5 μm, Phenomenex) was used to analyze DASA adducts of flavanols with a flow rate of 0.3 mL/min. The binary mobile phase system consisted of 95% water with 0.2% formic acid (FA) as phase A and 95% methanol with 0.2% FA as phase B. The column was eluted by a gradient progress (0% B from 0 to 5 min; 0 to 20% B from 5 to 35 min; 20−30% B from 35 to 45 min; 30−100% B from 45 to 55 min; 100% B from 55 to 60 min, and then 0% B from 60 to 65 min). The injection volume was 10 μL for each sample. 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 46 arb units and as the auxiliary gas at 15 arb units. The collision-induced dissociation (CID) was conducted with an isolation width of 2 Da and normalized collision energy of 35 for MS/MS analysis. The mass range was measured from 50 to 800 m/ z. Data acquisition was performed with Xcalibur version 2.0 (Thermo Electron, San Jose, CA). Synthesis of DASA-quinoxaline. DASA-quinoxaline was prepared as previously described with some modifications.12 In detail, a mixture of DASA (174 mg, 1.0 mmol) and OPD (216 mg, 2.0 mmol) in 0.5 N HCl (3 mL) was incubated at 37 °C for 5 h. The brown solution was freeze-dried. The dark brown solid was reconstituted in MeOH (2 mL) and purified by preparative HPLC, eluting with a gradient solvent system (0.1% TFA in H2O, solvent A; 0.1% TFA in methanol, solvent B; 10% B, 0−5 min; from 10 to 30% B, 5−15 min; from 30 to 45% B, 15−25 min; from 45 to 80% B, 25−35 min; from 80 to 100% B, 35−40 min; 100% B, 40−45 min; flow rate, 20 mL/ min). The fractions containing the target molecule were pooled, partially evaporated in vacuo, and then freeze-dried, giving the title compound (80 mg, yield 33%) as a light yellow powder. 1H NMR (600 MHz, CD3OD) δ 8.28 (1H, d, J = 8.3 Hz, H-5), 8.00 (1H, t, J = 8.3 Hz, H-6), 8.06 (1H, t, J = 8.3 Hz, H-7), 8.33 (1H, d, J = 8.3 Hz, H8), 5.96 (1H, d, J = 1.7 Hz, H-11), 4.38 (1H, dt, J = 7.7, 1.7 Hz, H-12), 3.89 (1H, dd, J = 11.0, 7.7 Hz, H-13a), and 3.83 (1H, dd, J = 11.0, 6.3 Hz, H-13b); 13C NMR (150 MHz, CD3OD) δ 145.5 (s, C-2), 160.0 (s, C-3), 145.0 (s, C-4a), 132.6 (d, C-5), 130.5 (d, C-6), 131.8 (d, C7), 134.6 (d, C-8), 168.0 (s, C-9), 81.7 (d, C-11), 72.7 (d, C-12), and 63.5 (t, C-13); negative ESI-MS: m/z 245 [M − H]−. Kinetic Study of the Trapping of DASA by Tea Flavanols. DASA (2.0 mM) was incubated with 6 mM flavanols (EGCG, EGC, ECG, or EC) in phosphate buffer solution (100 μL, pH 7.4, 100 mM). The mixed solutions were incubated at 37 °C and shaken at 40 rpm for 0, 5, 10, 15, 20, 30, 40, and 60 min. Next, to each triplicate vial at each time point was added 20 μL of 0.5 N hydrochloric acid (HCl) to stop the reaction and followed by an additional 60 μL of PBS and 20 μL of 100 mM 1,2-diaminobenzene (OPD). The mixture was further

Additionally, an in vivo study revealed that ASA or DASA could be degraded into more reactive forms, such as 2,3-diketogulonic acid and 3-deoxythreosone, in the human lens, which can inflict substantial protein damage via AGE formation.12 Therapeutic options to reduce the morbidity caused by AGEs would be tremendously useful. A therapeutic strategy to prevent and ameliorate diabetic complications is to use antiAGE agents. Many AGE inhibitors, such as aminoguanidine,13,14 tenilsetam,15 carnosine,16 metformin,17,18 and pyridoxamine,19 have been investigated for inhibiting the formation of AGEs and the development of diabetic complications by trapping reactive dicarbonyl species. Unfortunately, all of these pharmaceuticals have safety concerns and serious side effects. Thus, it is pivotal to develop effective and safe therapeutic agents targeting AGEs to prevent diabetics from developing AGE-associated complications. We have previously demonstrated that dietary flavonoids, such as (−)-epigallocatechin 3-O-gallate (EGCG) from tea, phloridzin and phloretin from apples, and genistein from soybeans, could effectively inhibit the formation of AGEs by trapping MGO.20−23 A recent in vivo study showed that oligonol, a new dietary phenolic product derived from lychee fruit polyphenols containing catechin-type monomers and lowmolecular-weight oligomers, attenuated diabetes-induced renal damage through the AGEs-related pathway in db/db mice.24 Very recently, (+)-catechin was reported to be able to ameliorate renal dysfunction in diabetic mice as a consequence of inhibiting AGE formation and cutting off the inflammatory pathway via MGO trapping.25 It has also been reported that tea flavanols, EGCG, (−)-epigallocatechin (EGC), (−)-epicatechin 3-O-gallate (ECG), and (−)-epicatechin (EC), could inhibit the formation of AGEs by trapping MGO at individual stages of protein glycation in vitro.26 Whether tea flavanols could efficiently inhibit the formation of AGEs by trapping DASA and thereby reduce the risk of DASA-induced diabetic complications remains unknown. In the present study, we measured the DASA-trapping ability of four tea flavanols, EGCG, EGC, ECG, and EC, under simulated physiological conditions, and, in particular, we investigated the formation of DASA adducts of flavanols generated from the chemical reaction between DASA and flavanols using NMR experiments and LC-MS techniques. We also tested the potential of these four tea flavanols for inhibiting the formation of AGEs by trapping DASA in a crystallin-DASA assay. The plausible mechanism of inhibition by the tested flavanols is discussed as well.

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MATERIALS AND METHODS

Materials. EGCG, EGC, ECG, EC, DASA, and 1,2-diaminobenzene (OPD) were purchased from Sigma (St. Louis, MO). HPLCgrade solvents and other reagents were obtained from Thermo Fisher Scientific (Waltham, MA). Analytical (250 μm thickness, 2−25 μm particle size) and preparative TLC plates (2000 μm thickness, 2−25 μm particle size) were purchased from Sigma (St. Louis, MO) and Sorbent Technologies (Atlanta, GA), respectively. Sephadex LH-20 (Sigma, St. Louis, MO) was used in open column chromatography (CC) fractionations. Medium-pressure column chromatography was carried out over ODS (60 Å, Sigma, St. Louis, MO) equipped with a LabAlliance series I pump and Spectra/chrom fraction collector CF-2. Bovine lens crystallins were obtained from Sigma (St. Louis, MO). NMR Analysis. 1H, 13C, and two-dimensional (2D) NMR spectra were recorded on a Bruker AVANCE 600 MHz spectrometer (Bruker, Inc., Silberstreifen, Rheinstetten, Germany). Ascorbyl conjugates and DASA-quinoxaline were analyzed in CD3OD, with TMS as an internal B

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incubated in the dark at 37 °C for 9 h. The resulting solution was diluted 10 times, and 80 μL was analyzed by HPLC. The level of DASA-quinoxaline was determined using the HPLC method described above, which was used to reflect the content of the remaining DASA (%) in PBS (Figure 1A). The recovery of DASA-quinoxaline was more than 95% (data not shown). Inhibitory Effects of Tea Flavanols on the Formation of AGEs. In triplicate vials, bovine lens crystallins (2 mg/mL) and DTPA chelating agent (1 mM) were incubated with DASA (5 mM) in the presence or absence of tea flavanols (EGCG, EGC, ECG, or EC) (15 mM) in PBS buffer (pH 7.4, 100 mM) (500 μL) at 37 °C for 7 days. A mixed solution of streptomycin and penicillin was added before incubation to prevente bacterial growth. The AGEs levels were quantified using fluorescence at 360 nm excitation and 460 nm emission wavelengths by a multimode microplate reader (Synergy 2, Biotek) on day 7. Fluorescence intensity was expressed in arbitrary units (AU), and the fluorescence was normalized to that of crystallins incubated without DASA (Figure 1B). Synthesis and Purification of the Major DASA Adducts of Flavanols. Flavanols (EGCG, EGC, ECG, or EC) (1.0 equiv) and DASA (3.0 equiv) were dissolved in PBS (pH 8.0, 100 mM) (30 mL) and then incubated at 37 °C for 3 h (Figure 2). The reaction mixture was subjected to column chromatography over Sephadex LH-20 (70% ethanol as eluent) followed by reverse-phase C18 silica gel mediumpressure column chromatography (CC) (10% methanol as eluent), thereby affording DASA adducts of flavanols, EGCGDASA-1, EGCGDASA-2, EGCDASA-1, EGCDASA-2, ECGDASA-1, ECGDASA-2, and ECDASA-1, as solids (Figure 2). Investigation of the Formation of DASA Adducts of Flavanols in the Crystallin-DASA System via LC-MS. To medium from 7 day (500 μL) harvested from the crystallin-DASA assay was added 500 μL of methanol. The mixture was vortexed and then sonicated for 1 min. The suspension was diluted 10 times and centrifuged at 17 000g for 10 min. The supernatant (80 μL) was transferred into vials for LC-MS analysis according to the method described above. EGCGDASA-1, EGCGDASA-2, EGCDASA-1, EGCDASA-2, ECGDASA-1, ECGDASA-2, and ECDASA-1 in methanol (10 μM for each) were used as authentic references. Statistical Analysis. Statistical analysis was conducted using GraphPad Prism software (version 5). For simple comparisons between two groups, two-tailed Student’s t test was used. A p value of less than 0.05 was considered to be statistically significant in all tests.

Figure 1. Trapping of DASA and inhibition of DASA-induced AGEs formation by tea flavanols. (A) Kinetic curves of trapping DASA by EGCG, EGC, ECG, and EC in phosphate buffer (pH 7.4, 37 °C). DASA (2 mM) was incubated with 6 mM flavanols (EGCG, EGC, ECG, or EC) in pH 7.4 phosphate buffer solutions at 37 °C for 0, 5, 10, 15, 20, 30, 40, and 60 min. Results are mean ± SD (n = 3). Bar, standard error. (B) Inhibitory effect of tea flavanols on DASA-induced AGEs formation in a bovine lens crystallin-DASA assay. Crystallin (Cry) was used as a negative control. Cry + DASA was used as a positive control. Results are mean ± SD (n = 3). Bar, standard error; o, not significant; *, p < 0.05; **, p < 0.005. All statistical tests are unpaired Student’s t tests, two-tailed, compared to positive control or EGCG-treated group.

(p < 0.007 for both EGCG and EC versus EGC and ECG) (Figure 1B). Formation and Identification of the Major DASA Adducts of Flavanols. We found that all four tea flavanols could rapidly react with DASA to generate related DASA adducts under slightly basic conditions. Seven DASA adducts, EGCGDASA-1, EGCGDASA-2, EGCDASA-1, EGCDASA-2, ECGDASA-1, ECGDASA-2, and ECDASA-1, were purified from the reaction between flavanols (EGCG, EGC, ECG, or EC) and DASA at a ratio of 1:3 (Figure 2). Their structures were established by analyzing their 1H, 13C, and 2D NMR (DEPT, HSQC, and HMBC) spectra as well as their tandem MS spectra. EGCGDASA-1, synthesized from EGCG and DASA, had a molecular formula of C28H24O17 based on negative ESI-MS at m/z 631 [M − H]− and NMR data. The 1H and 13C NMR spectra of EGCGDASA-1 were highly similar to those of 8-C-ascorbyl EGCG.28 HMBC correlations between H-4 (δH 3.01 and 2.90) and C-4a (δC 102.0), H-6 (δH 6.02), and C-4a (δC 102.0) as well C-5 (δC 161.2) (Figure 3) demonstrated the presence of H-6 (δH 6.02) in ring A of the EGCG moiety, indicating that the DASA residue is attached to the EGCG moiety at the C-8 position. In addition, the molecular weight of EGCGDASA-1 was 174 (MW of DASA, 174) mass units higher than that of EGCG. These spectral features supported that EGCGDASA-1 was 8-C-ascorbyl EGCG, as reported previously from oolong tea.28 EGCGDASA-2, a minor product synthesized from EGCG and DASA, had the same molecular formula of C28H24O17 as that of EGCGDASA-1, based on negative ESI-MS at m/z 631 [M − H]− and NMR data. The 1H and 13C NMR spectra of

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RESULTS Kinetic Study on the Trapping of DASA by Tea Flavanols. Trapping ability of four tea flavanols (EGCG, EGC, ECG, and EC) was evaluated in PBS buffer (pH 7.4, 100 mM) at 37 °C according to the method described above. The kinetic curve of the trapping of DASA by flavanols during the course of 60 min is presented in Figure 1A. Our data showed that DASA in PBS was captured by flavanols gradually and that only 20− 40% of DASA remained in flavanol-treated medium after incubation for 60 min. These observations demonstrated that tea flavanols could efficiently trap DASA. Among them, EGCG exerted the best DASA-trapping activity among the four tested flavanols, followed by EGC, ECG, and EC. This is in agreement with our previous observation in the kinetic study of these four tea flavanols on trapping MGO.27 Inhibitory Effects of Tea Flavanols on the Formation of AGEs. AGEs levels generated in the crystallin-DASA system were monitored by a multimode microplate reader. Four tea flavanols, EGCG, EGC, ECG, and EC, showed significant inhibitory effects on the formation of AGEs compared to the positive control, Cry + DASA (p < 0.02 for all) (Figure 1B). Of which, EGCG and EC displayed a comparable inhibitory effect (p = 0.52) and were much stronger than that of EGC and ECG C

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Figure 2. Synthesis of DASA adducts of flavanols, EGCGDASA-1, EGCGDASA-2, EGCDASA-1, EGCDASA-2, ECGDASA-1, ECGDASA-2, and ECDASA-1. Reagents and conditions: PBS (pH 8.0, 100 mM), 37 °C, 3 h.

Figure 3. Main HMBC correlations (↷) of DASA adducts of flavanols: EGCGDASA-1, EGCGDASA-2, and ECGDASA-1.

Table 1. δH (600 MHz) NMR Spectra Data of DASA Adducts of Tea Flavanolsa no. 2 3 4 6 8 2′ 5′ 6′ 4″ 5″ 6″

ECDASA-1

EGCDASA-1

5.00 4.25 2.88 2.80 6.01

brs brs dd 16.8 4.2 dd 16.8 1.8 s

4.93 4.21 2.86 2.78 5.99

7.05 6.80 6.93 4.24 4.12 3.86 3.69

d 1.8 d 8.4 dd 8.4 1.8 d 6.6 q 4.8 dd 10.8 4.2 dd 10.8 6.0

6.59 s

6.09 s 6.50 s

6.59 4.23 4.10 3.85 3.66

6.50 4.31 4.11 3.70 3.61

2‴ 6‴ a

brs brs dd 16.8 4.8 dd 16.8 2.4 s

EGCDASA-2

s d 6.6 q 6.6 dd 11.0 4.8 dd 11.0 6.0

4.95 4.23 2.92 2.80

brs brs m m

s d 5.0 m m m

ECGDASA-1 5.21 5.55 3.02 2.91 6.02

brs brs dd 17.4 4.8 dd 17.4 2.4 s

7.03 6.72 6.94 4.36 4.13 3.86 3.69 6.86 6.86

d 2.0 d 8.4 dd 8.4 2.0 d 6.0 q 5.4 dd 11.4 5.4 dd 11.4 6.0 s s

ECGDASA-2 5.09 5.54 3.08 2.80

brs brs dd 17.3 4.5 d 17.3

6.10 6.91 6.68 6.77 4.28 4.07 3.76 3.60 6.90 6.90

s s d 8.3 d 8.3 d 5.2 q 5.5 dd 11.2 5.3 dd 11.2 6.2 s s

EGCGDASA-1

EGCGDASA-2

5.15 5.55 3.01 2.90 6.02

5.02 5.55 3.06 2.80

brs brs dd 17.4 4.8 dd 17.4 1.8 s

brs brs dd 16.8 4.2 dd 16.8 2.4

6.61 s

6.11 s 6.48 s

6.61 4.38 4.13 3.85 3.68 6.86 6.86

6.48 4.30 4.08 3.76 3.60 6.92 6.92

s d 6.0 q 5.4 dd 10.8 4.8 dd 10.8 6.0 s s

s d 5.4 q 6.0 dd 10.8 4.8 dd 10.8 6.0 s s

CD3OD, δ in ppm and J in Hz.

the EGCG moiety at the C-6 position. A hemiketal group (δC 112.4) at C-3″ in the structure of the DASA residue was established by HMBC cross-peaks between C-3″ (δC 112.4) and H-4″ (δH 4.30) as well H-5″ (δH 4.08) (Figure 3), indicating that the hydroxyl group at either C-5 or C-7 is substituted. However, the resonance at δC 158.5 (C-5) was

EGCGDASA-2 were very similar to those of EGCGDASA-1 (Tables 1 and 2), suggesting it is an EGCG-DASA adduct as well. HMBC correlations between H-4 (δH 3.06 and 2.80) and C-8a (δC 159.9), H-8 (δH 6.11), and C-8a (δC 159.9) as well as C-7 (δC 156.1) (Figure 3) showed the presence of H-8 (δH 6.11) in ring A, suggesting that the DASA residue is linked to D

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Table 2. δC (150 MHz) NMR Spectra Data of DASA Adducts of Tea Flavanolsa

a

no.

ECDASA-1

EGCDASA-1

EGCDASA-2

ECGDASA-1

ECGDASA-2

EGCGDASA-1

EGCGDASA-2

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

79.8 d 67.1 d 29.2 t 161.4 s 91.5 d 158.7 s 102.7 s 102.6 s 155.0 s 131.8 s 116.0 d 145.8 s 145.5 s 115.0 d 119.2 d 174.8 s 80.1 s 112.1 s 84.0 d 71.4 d 63.4 t

79.9 d 67.1 d 29.2 t 161.4 d 91.4 d 158.7 s 102.7 s 102.6 s 155.0 s 131.0 s 106.7 d 146.6 s 133.4 s 146.6 s 106.7 d 175.1 s 80.1 s 112.1 s 84.1 d 71.3 d 63.4 t

80.5 d 66.3 d 27.8 t 158.8 s 104.0 s 156.0 s 98.2 d 96.7 s 160.1 s 130.4 s 106.9 d 146.7 s 133.9 s 146.7 s 106.9 d 175.1 s 81.2 s 112.4 s 84.6 d 71.5 d 63.3 t

78.6 d 69.6 d 26.7 t 161.2 s 91.5 d 158.9 s 102.8 s 101.9 s 154.9 s 130.9 s 116.0 d 145.8 s 145.7 s 114.8 d 119.2 d 175.0 s 80.1 s 112.2 s 84.2 d 71.3 d 63.3 t 121.3 s 110.2 d 146.2 s 139.8 s 167.6 s

79.2 d 68.9 d 26.4 t 158.6 s 104.1 s 156.2 s 98.3 d 96.0 s 160.0 s 130.8 s 116.0 d 146.1 s 146.0 s 115.0 d 119.3 d 174.9 s 80.1 s 112.5 s 84.6 d 71.3 d 63.2 t 121.2 s 110.2 d 146.3 s 139.9 s 167.3 s

78.6 d 69.6 d 26.7 t 161.2 s 91.5 d 158.9 s 108.8 s 102.0 s 154.9 s 130.3 s 106.7 d 146.5 s 133.5 s 146.5 s 106.7 d 175.2 s 80.1 s 112.2 s 84.0 d 71.3 d 63.3 t 121.3 s 110.2 d 146.2 s 139.7 s 167.6 s

79.7 d 68.8 d 26.3 t 158.5 s 104.0 s 156.1 s 98.3 d 96.0 s 159.9 s 130.1 s 106.8 d 146.6 s 133.8 s 146.6 s 106.8 d 174.9 s 80.1 s 112.4 s 84.6 d 71.2 d 63.2 t 121.2 s 110.2 d 146.2 s 139.8 s 167.3 s

CD3OD, δ in ppm.

shifted downfield as compared to the signal at δC 156.1 (C-7) in EGCGDASA-2, demonstrating that the substituted hydroxyl group is at the C-5 position, thus establishing the O-bridge of C(3″)−O−C(5). In addition, the molecular weight of EGCGDASA-2 was 174 (MW of DASA, 174) mass units higher than that of EGCG. All of these spectral features support EGCGDASA-2 as being 6-C-ascorbyl EGCG, as shown in Figure 2. EGCDASA-1, a major product synthesized from EGC and DASA, had a molecular formula of C21H20O13 based on negative ESI-MS at m/z 479 [M − H]− and NMR data. The 1H and 13C NMR spectra of EGCDASA-1 were the same as those of 8-C-ascorbyl EGC. Particularly, signals observed at δH 5.99 (H-6)/δC 91.4 (C-6) in ring A of EGCDASA-1 indicated attachment of the DASA residue to the EGC moiety at the C-8 position. Additionally, the molecular weight of EGCDASA-1 was 174 mass units higher than that of EGC, corresponding to a DASA residue. Thus, EGCDASA-1 was identified as being 8C-ascorbyl EGC, as reported previously from oolong tea.28 EGCDASA-2, a minor product synthesized from EGC and DASA, had the same molecular formula of C21H20O13 as that of EGCDASA-1, based on negative ESI-MS at m/z 479 [M − H]− and NMR data. The 1H and 13C NMR spectra of EGCDASA-2 were similar to those of EGCGDASA-2 (Tables 1 and 2). The main difference between these two compounds in their 1H NMR spectra was that the protons at δH 6.92 (2H, s) of a gallate group in EGCGDASA-2 disappeared in EGCDASA-2. Moreover, the signal at δH 4.23 (H-3) in the 1H NMR spectrum of EGCDASA-2 was vastly shifted upfield compared to the proton at δH 5.55 (H-3) in EGCGDASA-2 (Table 1), indicating that a gallate group at C-3 in EGCDASA-2 is missing. In particular, signals observed at δH 6.09 (H-8)/δC

98.2 (C-8) in ring A of the EGC moiety strongly agreed with the attachment of the DASA residue to the EGC moiety being at the C-6 position. In addition, the molecular weight of EGCDASA-2 was 152 mass units less than that of EGCGDASA-2, also corresponding to the loss of a gallate group. Thus, EGCDASA-2 was identified as being 6-C-ascorbyl EGC, as shown in Figure 2. ECGDASA-1, a major product synthesized from ECG and DASA, had a molecular formula of C28H24O16 based on negative ESI-MS at m/z 615 [M − H]− and NMR data. The 1H and 13C NMR spectra of ECGDASA-1 were very similar to those of EGCGDASA-1. The major difference between these two compounds in their 1H NMR spectra was the appearance of a 1,3,5-trisubstituted benzene ring [δH 7.03 (1H, d, J = 2.0 Hz), δH 6.72 (1H, d, J = 8.4 Hz), and δH 6.94 (1H, dd, J = 8.4, 2.0 Hz)] in ECGDASA-1 instead of a 1,3,4,5-tetrasubstituted benzene ring [δH 6.86 (2H, s)] in EGCGDASA-1 (Table 1). Resonances at δC 130.9 (s), 116.0 (d), 145.8 (s), 145.7 (s), 114.8 (d), and 119.2 (d) in the 13C NMR spectrum of ECGDASA-1 also supported the existence of a 1,3,5trisubstituted benzene ring. HMBC correlations between H-4 (δH 3.02 and 2.91) and C-4a (δC 101.9), H-6 (δH 6.02), and C4a (δC 101.9) as well as C-5 (δC 161.2) (Figure 3) indicated that the DASA residue is attached to the ECG moiety at the C8 position. In addition, the molecular weight of ECGDASA-1 was 16 mass units less than that of EGCGDASA-1. All of these spectral features established that ECGDASA-1 is 8-C-ascorbyl ECG, as shown in Figure 2. ECGDASA-2, a minor product synthesized from ECG and DASA, had a molecular formula of C28H24O16 based on negative ESI-MS at m/z 615 [M − H]− and NMR data. The 1H and 13C NMR spectra of ECGDASA-2 were very similar to those of EGCGDASA-2. The major E

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Figure 4. LC chromatograms (TIC and SIM) and MS2 (MS/MS) spectra of EGCGDASA-1 (A), ECGDASA-1 (B), EGCDASA-1 (C), and ECDASA-1 (D) in the respective flavanol (EGCG, ECG, EGC, or EC)-treated samples after incubation with bovine lens crystallins at 37 °C for 7 days as well as corresponding authentic standards obtained by negative ESI/MS interface. TIC, total ion current; SIM, selected ion monitoring; ESI, electrospray ionization.

in ECDASA-1 is missing. Particularly, signals observed at δH 6.01 (H-6)/δC 91.5 (C-6) in ring A of the EC moiety also are in agreement with the attachment of the DASA residue to the EC moiety being at the C-8 position. In addition, the molecular weight of ECDASA-1 was 152 mass units less than that of ECGDASA-1, corresponding to a gallate group. These spectral features suggested that ECDASA-1 is 8-C-ascorbyl EC, as shown in Figure 2. Determining the Formation of DASA Adducts of Flavanols in the Crystallin-DASA System Using LC-MS. In order to further understand whether the inhibition of AGEs formation was due to the trapping of DASA by tea flavanols (EGCG, EGC, ECG or EC), we determined the existence of the DASA adducts of flavanols in the samples collected after incubating the respective flavanols with crystallins and DASA using LC-MS (Figure 4). In Figure 4A, the peak (tR 42.2 min) corresponding to the molecular ion of the mono-DASA adduct of EGCG in the EGCG-treated crystallin-DASA sample had an almost identical tR and MS/MS spectrum as those of the authentic EGCGDASA-1 that we synthesized and purified from the reaction between EGCG and DASA. This clearly demonstrates the presence of EGCGDASA-1 in the EGCGtreated crystallin-DASA sample. In the same way, EGCDASA-1, ECGDASA-1, and ECDASA-1 were identified in the EGC-, ECG-, and EC-treated samples, respectively, by comparison of their retention times and MS/MS spectra with those of

difference between these two compounds in their 1H NMR spectra was the appearance of a 1,3,5-trisubstituted benzene ring [δH 6.91 (1H, s), δH 6.68 (1H, d, J = 8.3 Hz), and δH 6.77 (1H, d, J = 8.3 Hz)] in ECGDASA-2 instead of a tetrasubstituted benzene ring [δH 6.92 (2H, s)] in EGCGDASA-1 (Table 1). Resonances at δC 130.8 (s), 116.0 (d), 146.1 (s), 146.0 (s), 115.0 (d), and 119.3 (d) in the 13C NMR spectrum of ECGDASA-2 also agreed with the existence of a 1,3,5-trisubstituted benzene ring. Particularly, signals observed at δH 6.10 (H-8)/δC 98.3 (C-8) in ring A of the ECG moiety are in agreement with the attachment of the DASA residue to the ECG moiety at the C-6 position. In addition, the molecular weight of ECGDASA-2 was 16 mass units less than that of EGCGDASA-2. Thus, ECGDASA-2 was deduced as being 6-Cascorbyl ECG, as shown in Figure 2. ECDASA-1, a major product synthesized from EC and DASA, had a molecular formula of C21H20O12 based on negative ESI-MS at m/z 463 [M − H]− and NMR data. The 1H and 13C NMR spectra of ECDASA-1 were very similar to those of ECGDASA-1 (Tables 1 and 2). The main difference between these two compounds in their 1H NMR spectra was that protons at δH 6.86 (2H, s) of the gallate group in ECGDASA-1 disappeared in ECDASA-1. Moreover, the signal at δH 4.25 (H3) in the 1H NMR spectrum of ECDASA-1 was vastly shifted upfield as compared to the proton at δH 5.55 (H-3) in ECGDASA-1 (Tables 1), indicating that a gallate group at C-3 F

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Figure 5. Formation pathway of 8C-ascorbyl conjugates (major) or 6C-ascorbyl conjugates (minor) of flavanols under simulated physiological conditions.

structure, more or less, facilitates the trapping efficacy. In addition, it is worth emphasizing that the derivatization of DASA by OPD under acidic conditions (e.g., 0.5 N HCl) is much more efficient than that under literature-reported neutral conditions (e.g., methanol).12 In fact, we observed that the reaction of OPD with DASA was not completed until 3 days using the literature-reported conditions (data not shown). Furthermore, we investigated the inhibitory effects of these four tea flavanols against the formation of AGEs by trapping DASA in a crystallin-DASA assay (Figure 1B). It is the first report of tea flavanols inhibiting the formation of DASAinduced AGEs in vitro. Our results showed that all four tea flavanols could significantly inhibit the formation of DASAinduced AGEs, compared to positive control (Cry + DASA). As expected, EGCG had the best ability to inhibit the formation of DASA-related AGEs. Interestingly, EC was found to show a comparably high inhibition to that of EGCG in this assay, which is different from it having the lowest DASA-trapping efficacy in the kinetic study above. This may be due to various factors. Further study, therefore, is warranted in the future. In order to gain insight into the plausible mechanism of inhibition by flavanols, we investigated the formation of DASA adducts of tea flavanols by treating DASA with individual flavanols. We found that the reaction of flavanols with DASA proceeded readily under slightly basic conditions, forming the obvious DASA conjugates in 3 h. As a consequence, seven conjugates were purified and identified as 8C-ascorbyl conjugates (EGCGDASA-1, EGCDASA-1, ECGDASA-1, and ECDASA-1) and 6C-ascorbyl conjugates (EGCGDASA-2, EGCDASA-2, and ECGDASA-2), as shown in Figure 2. Of which, five ascorbyl conjugates, ECGDASA-1, ECDASA-1,

authentic standards (Figure 4B−D). In addition, EGCGDASA2, EGCDASA-2, and ECGDASA-2 were detected as minor conjugates in the EGCG-, EGC-, or ECG-treated medium as well (data not shown).

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DISCUSSION Dietary flavonoids have been reported to effectively inhibit the formation of AGEs by trapping MGO.20−23,26 Little is known about whether dietary flavonoids, especially dietary flavanols, could reduce or inhibit the formation of DASA-induced AGEs. Flavanols are one of the main subclasses of dietary flavonoids and highly present in commonly consumed beverages, such as green tea.29 A study revealed that a 500 mL bottle of a green tea beverage contained a total of 240 mg of flavanols, principally in the form of 105 mg of EGCG, 77 mg of EGC, 22 mg of ECG, and 17 mg of EC.30 Such high contents of flavanols in green tea beverages may offer chances for flavanols to inhibit the formation of DASA-induced AGEs, thereby reducing or preventing the progression of DASA-induced diabetic complications. In the present study, we evaluated the DASA-trapping ability of four tea flavanols, EGCG, EGC, ECG, and EC, under simulated physiological conditions using an OPD derivatization assay.12 As a consequence, we revealed for the first time that all four tea flavanols could efficiently trap DASA in PBS (pH 7.4, 37 °C) (Figure 1A). Of which, EGCG exerted the best trapping ability followed by EGC, and the latter had slightly better activity than that of ECG and EC. These observations suggested that (1) two free hydroxyl groups at C-5 and C-7 in ring A in the structure of tea flavanols are critical to their activity and (2) an additional hydroxyl group in ring B in the G

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EGCGDASA-2, EGCDASA-2, and ECGDASA-2, were reported as new compounds for the first time. In addition, 8Cascorbyl conjugates were the major adducts and 6C-ascorbyl conjugates were the minor adducts in their respective treatments. These results suggested that tea flavanols capture DASA in the system via the formation of 6C- or 8C-ascorbyl conjugates, but mainly in the form of 8C-ascorbyl conjugates. The chemical pathway for the generation of ascorbyl conjugates of flavanols is considered to successively undergo two-step nucleophilic additions at active sites in ring A according to our previous hypothesis from the study of MGO adducts,20,21 as shown in Figure 5. First, deprotonation of OH-5 in flavanol by a weak base generates a carbanion species at C-6 or C-8 as a nucleophile, which attacks the carbonyl group at C-2″ in DASA to form the key intermediate 6C- or 8C-adduct, respectively. Next, nucleophilic OH-5 or OH-7 attacks the carbonyl group at C-3″ in the DASA residue to close the furan ring, thereby producing 6C- or 8C-ascorbyl conjugates. The kinetics of this proposed reaction course is a subject for future study. Using the 6C- or 8C-ascorbyl conjugates that were purified from the model reaction between flavanols and DASA, we studied whether flavanols can inhibit the formation of AGEs through the trapping of DASA in a crystallin-DASA assay. 8CAscorbyl conjugates of flavanols, EGCGDASA-1, EGCDASA-1, ECGDASA-1, and ECDASA-1, were unambiguously detected in their respective assays using LC-ESI/MS (Figure 4). Our results demonstrated for the first time that four tea flavanols can inhibit the formation of DASA-induced AGEs by irreversibly converting DASA into ascorbyl conjugates. This further supports our hypothesis that dietary flavonoids can inhibit the formation of AGEs by trapping reactive dicarbonyls. It is well-known that the Age-Related Eye Disease Study (AREDS) formulation that contains high amount of vitamin C has been used for the treatment of age-related macular degeneration (AMD) in clinical trials.31 It is possible that the administration of the AREDS formula will lead to lens AGEs accumulation; however, this remains largely unknown. Recently, an investigation of eye tissues from rats that were fed green tea extract showed that eye structures including choroid-sclera, retina, lens, and cornea absorbed significant amounts of individual flavanols, such as EGC and EGCG.32 This indicates that green tea consumption may be of benefit to the eyes against oxidative and carbonyl stresses. Human studies in China and the U.S. have found that consuming ∼6 cups a day of green tea for 7 days decreases oxidative DNA damage, lipid peroxidation, and free radical generation in both smokers and nonsmokers.33 These findings in combination with the current study suggest that coadministration of vitamin C with green tea or green tea extract may be the best approach to achieve eye health in clinical use. In conclusion, the present study revealed that tea flavanols could efficiently inhibit the formation of DASA-induced AGEs and consequently form 8C-ascorbyl conjugates in vitro in a crystallin-DASA system, indicating that tea flavanols may prevent the development of diabetic complications, particularly diseases such as retinopathy and cataract. Thus, flavanolenriched foods and beverages, such as green tea, as effective dietary strategies, hold great potential for preventing the development of diabetic complications and therefore could reduce the morbidity and mortality of individuals with diabetes. Whether increasing the intake of tea flavanols lowers the risk of diabetic complications, especially for retinopathy and cataract, in humans is an important subject for future study.

Article

AUTHOR INFORMATION

Corresponding Author

*Tel.: 704-250-5710; Fax: 704-250-5709; E-mail: ssang@ncat. edu or [email protected]. Funding

This project was supported by a grant from the Qatar National Research Fund, NPRP no. 5-220-3-063 to S.S. and M.A. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS AGEs, advanced glycation end products; ASA, ascorbic acid; DASA, dehydroascorbic acid; EC, (−)-epicatechin; ECG, (−)-epicatechin 3-O-gallate; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin 3-O-gallate; HMBC, heteronuclear multiple band correlation; MGO, methylglyoxal; SIM, selected-ion monitoring; TIC, total ion current

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