Influence of Phenolic Compounds on the Mechanisms of Pyrazinium

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Influence of Phenolic Compounds on the Mechanisms of Pyrazinium Radical Generation in the Maillard Reaction Qing Bin, Devin G. Peterson, and Ryan J. Elias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf3010838 • Publication Date (Web): 09 May 2012 Downloaded from http://pubs.acs.org on May 15, 2012

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Journal of Agricultural and Food Chemistry

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TITLE: Influence of Phenolic Compounds on the Mechanisms of Pyrazinium Radical

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Generation in the Maillard Reaction

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AUTHORS: Qing Bin§†1, Devin G. Peterson†, Ryan J. Elias§*

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§

Department of Food Science, The Pennsylvania State University, University Park, PA, USA

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†

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, USA

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1

Present address: Department of Food Science and Nutrition, University of Minnesota, St. Paul,

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MN, USA

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TITLE RUNNING HEADER: Phenolic Compounds Affect Pyrazinium Radical Formation in

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the Maillard Reaction

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*CORRESPONDING AUTHOR FOOTNOTE:

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[Fax: (814) 863-6132; email: [email protected]].

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ACS Paragon Plus Environment


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ABSTRACT

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The generation of pyrazinium radical cations during the early stages of the Maillard reaction has

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been previously demonstrated. In this study, the effect of food phenolic compounds [4-

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methylcatechol (4-MeC), (+)-catechin (CAT), and (-)-epigallocatechin-3-gallate (EGCG)] on the

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fate of these intermediates in Maillard model systems was investigated. Aqueous solutions

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containing either glyoxal + alanine (GO-A), or glycolaldehyde + alanine (GA-A), were treated

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with a concentration gradient of each phenolic compound, and quantitative analysis of the

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resulting pyrazinium radicals in these models was performed using electron paramagnetic

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resonance (EPR) spectroscopy. CAT and EGCG were observed to affect pyrazinium radical

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generation rates, in some cases either enhancing or suppressing formation depending on

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concentration, while the simple catechol (4-MeC) had no such effect. A mechanistic study was

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carried out by LC/MS, which suggested that under some conditions, CAT and EGCG react with

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imine intermediates via their A-rings, thus influencing the formation of the enaminol radical

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precursor and, ultimately, pyrazinium radicals. To our knowledge, this is the first study

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demonstrating imine trapping by phenolic compounds under Maillard conditions, and how such

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phenolic quenching reactions can alter pyrazinium radical formation.

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KEYWORDS: Maillard reaction; pyrazinium radical; glyoxal; glycolaldehyde; glycation;

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reactive imine-trapping; catechins; electron paramagnetic resonance

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INTRODUCTION

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The Maillard reaction, often referred to as non-enzymatic browning, refers to the classical

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reaction between a reducing sugar (carbonyl) and an amino acid (amine), and is an important

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reaction mechanism in food and regulatory biology (1). Under food conditions, this reaction is

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well known to alter color and flavor, as well as yielding biologically active compounds. The

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generation of Maillard products in food may be desirable (e.g., the aroma of bakery products,

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coffee) or undesirable (e.g., discoloration, off-flavor development). Because of its importance to

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food quality, this reaction has been the subject of intense study for decades; however, given the

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complexity of this reaction and the difficulty associated with characterizing many Maillard

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products, in particular high molecular weight products, many mechanistic aspects of this reaction

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are unknown. In biological systems, the Maillard reaction has also been studied in relation to

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pathology associated with aging and diabetes (2).

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In recent decades, the presence of radical intermediates species has been confirmed in the early

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stages of the Maillard reaction (3). These species were identified as pyrazinium radical cations

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using EPR, which appear to be key precursors in subsequent Maillard pathways. Namiki and

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Hayashi proposed a mechanism (Figure 1) by which this radical is formed and offered evidence

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that it involved the generation of a glycolaldehyde imine in equilibrium with its enaminol

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tautomer, which is dimerized and oxidized to a resonance stabilized 1,4-dialkylpyrazinium

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radical cation. The authors suggested that the glycolaldehyde imine was formed either from the

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fragmentation of a Schiff base, or by glycolaldehyde–amino acid condensation. Glycolaldehyde

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(GA) was reported to be the most effective reactant in yielding pyrazinium radicals. More

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recently, Hofmann and coworkers conducted a series of time-course studies that investigated



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color changes accompanying pyrazinium radical formation during the early stage of the Maillard

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reaction (4). It was demonstrated that glyoxal (GO) is generated in abundance in advance of

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radical formation. Furthermore, GA concentration was seen to increase as a function of

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reductone concentration, leading to increases in observed radical yields, which was presumably

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due to enhanced reduction of GO to GA. In the same study, the role of the pyrazinium radical

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cations in the context of color formation was investigated through the use of a 1,4-

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diethylpyrazinium diquaternary salt. The authors proposed a mechanism involving

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transformation of the pyrazinium radical cations into the hydroxylated 1,4-dialkyl-1,4-

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dihydropyrazines via those diquat intermediates, leading to non-enzymatic browning (4).

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Despite the important role of pyrazinium radical cations as browning precursors, the factors

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affecting their generation and fate in foods are largely unknown. In particular, little attention has

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been given to the effect that other food components (e.g., phenolic compounds) have on

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pyrazinium radical generation. Recent studies by Peterson and Totlani demonstrated that

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Maillard pathways can be affected by (-)-epicatechin (5). The generation of several volatile

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compounds (e.g. pyrazines, furans) in both model systems and bakery products was inhibited in

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the presence of this compound. It was proposed that (-)-epicatechin behaves as a trapping agent

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of carbonyl-containing sugar fragments under such conditions. Isotopic (C13) labeling studies

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provided evidence of the trapping of C2 (e.g., glyoxal), C3 (e.g., methylglyoxal, glyceraldehyde),

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and C4 (e.g., erythrose) sugar fragments by (-)-epicatechin, and the formation of these EC-sugar

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fragment adducts was reported (6). The structure of the EC-methylglyoxal (MGO) adduct was

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elucidated by 2D nuclear magnetic resonance (NMR), revealing that the mechanism of trapping

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was a classical electrophilic aromatic substitution reaction occurring at the highly activated A


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ring (Figure 2) (7). Trapping of reactive dicarbonyls by catechins has also been studied under

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physiological conditions. In a subsequent study, Sang and coworkers reported trapping of MGO

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and GO by EGCG under model physiological conditions, and that this trapping was also

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proceeded by electrophilic aromatic substitution (8). Furthermore, trapping of aldehydes (e.g.,

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glyoxylic acid, acetaldehyde, benzylaldehyde) by (-)-epicatechin in wine has also been reported

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(9,10).

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It was hypothesized that carbonyl-trapping reactions by food phenolic compounds affect

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pyrazinium radical formation in the early stages of the Maillard reaction. GO and GA are thought

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to be the key C2 fragments involved in pyrazinium radical formation (Figure 1), and, as such,

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have been studied extensively as important carbonyls connecting many pathways of the Maillard

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reaction. In the present study, aqueous GO–A and GA–A Maillard models were used to

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investigate how phenolics affect the mechanism of pyrazinium radical formation.

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MATERIALS AND METHODS

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Chemicals. Glyoxal (40 wt. % solution in water), glycolaldehyde dimer, D-glucose, L-alanine,

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(-)-epigallocatechin, (+)-catechin hydrate, 4-methylcatechol, and (-)-epigallocatechin gallate

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were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). All other chemicals and solvents

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were of reagent or HPLC grade. Water was purified through a Barnstead Nanopure Diamond

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water purification system (Thermo Scientific, Dubuque, IA, USA).

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Detection of Pyrazinium Radical Cations in Maillard Models with EPR. Solutions consisting

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of glyoxal (0.2 M) and L-alanine (0.5 M phosphate), or glycolaldehyde (0.1 M) and L-alanine



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(0.5 M) in phosphate buffer (0.1 M, pH 7.0) were treated with varying concentrations of 4-MeC,

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CAT or EGCG. Solutions were deoxygenated with nitrogen gas in their respective reaction

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vessels, and sample aliquots (50 µL) were transferred to, and incubated (25 °C) in, sealed glass

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micropipettes (Brand GmbH, Wertheim, Germany). The EPR spectra of pyrazinium radicals

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were recorded at regular intervals over the course of 200 h on a Bruker eScan X-band

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spectrometer (Bruker BioSpin, Billerica, MA, USA). The experimental parameters were as

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follows: modulation amplitude: 0.69 G; sweep time: 2.62 s; frequency: 9.78 GHz; gain: 1 × 103.

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EPR calibration was performed using 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (5 µM).

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All EPR experiments were performed in duplicate.

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Sample Preparation for LC/MS Analysis. Glyoxal (0.2 M) and L-alanine (0.5 M), or

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glycolaldehyde (0.1 M) and L-alanine (0.5 M) solutions were prepared in deoxygenated

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phosphate buffer (0.1 M, pH 7.0) and treated with EGCG (10 or 100 mM). After 1.5 h of

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incubation (at 25 °C), an aliquot (0.5 mL) of the reaction mixture was loaded on a 500 mg C18

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cartridge (Supelco, Bellefonte, PA, USA), which was preconditioned with 5 mL of methanol and

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then 5 mL of water. The cartridge was washed with 2 mL of water, and then eluted with 2 mL of

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methanol. The isolate was subsequently filtered through a 0.20 µm Nylon syringe filter (Millex,

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Billerica, CA, USA) using a 1 mL syringe (Millipore, Bedford, MA, USA) prior to LC/MS

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analysis.

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LC/MS Analysis. Sample analysis was performed using an Agilent 1100 HPLC system (Agilent

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Technologies, Palo Alto, CA, USA) interfaced with a Micromass Q-TOF Micro mass

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spectrometer (Waters, Milford, MA, USA). The HPLC system consisted of a binary pumping


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system (G1312A), a degasser (G1322A), an autosampler (G1313A), a column compartment

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(G1316A), and a diode array UV-vis detector (G1315A). An injection volume of 10 µL was used,

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and samples were separated on an Ascentis Express C18 column (2.1 mm × 150 mm, 2.7 µm)

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(Supelco, Bellefonte, PA, USA) at 35 ºC. The mobile phase was maintained at a flow rate of 250

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µL/min using a binary solvent system of 0.1% formic acid in water (A) and 0.1% formic acid in

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methanol (B). The elution gradient started at 10% B (0-2 min), linearly increased to 50% B (2-23

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min), linearly increased to 100% B (23-30 min), was maintained at 100% B (30-32 min),

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decreased to 10% B (32-35 min), and was finally maintained at 10% B (35-50 min). Analytes

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were detected using an inline Q-TOF Micro mass spectrometer equipped with a LockSpray dual

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exact mass ionization source inlet system, consisting of two electrospray probes for the co-

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introduction of analyte and lock mass reference compound. The source was operated at ES- mode

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with the following conditions: source temperature, 100°C; desolvation temperature, 300°C;

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capillary voltage, 3.0 kV; cone voltage, 30V. An internal reference, reserpine (100 mg/L

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delivered at 50 µL/min), was used to obtain exact mass measurements. The reference spray was

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sampled at an interval of 10 s. Accurate mass measurement was conducted in MS mode (ranged

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50 – 1100 Da) with a scan time of 1.0 s. Structural information of analytes was obtained in

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MS/MS mode with the collision energy set between 15-20 V.

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RESULTS AND DISCUSSION

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Effect of 4-MeC, CAT, and EGCG on Pyrazinium Radical Formation in GO-A Model

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The effect of the three phenolic compounds 4-MeC, CAT, and EGCG (Figure 3) on the

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generation rate of pyrazinium radicals in a GO-A model system was studied by EPR. GO is a

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simple dicarbonyl that is known to be generated during the early stages of the Maillard reaction,

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and is a known precursor to advanced browning products (11). The influence of EGCG on

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radical formation in the GO-A model is shown in Figure 4A. In the absence of EGCG, an EPR

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spectrum characteristic of the pyrazinium radical cation was observed almost immediately, with

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observed hyperfine coupling constants in agreement with previous studies (3). Overall, radical

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intensity remained low throughout the course of the experiment (200 h), which was anticipated

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due to the fact that GO was previously observed not to yield high levels of pyrazinium radicals

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(3); however, high radical yields were observed once GO was converted to GA by reductones (4).

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Radical intensity was observed to increase as a function of EGCG concentration (5 – 20 mM),

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with maximum radical yield observed at 20 mM ([GO]:[EGCG] = 10:1). However, the intensity

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of the pyrazinium radical signal began to decrease in the presence of EGCG at concentrations

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equal to, and exceeding, 50 mM. Figure 4D depicts the EPR spectrum of the pyrazinium

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radical observed in the system described above and related dose response. Substituting CAT for

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EGCG resulted in a similar trend (Figure 4B), with radical intensity increasing with CAT

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concentrations up to, and including, 20 mM CAT, and signal suppression observed at 50 mM

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(100 mM CAT was not included in the present study due to solubility restrictions in the model

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system). However, this trend was not seen for 4-MeC (Figure 4C), suggesting that catechol or

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gallate groups were not responsible for this effect. Furthermore, given the similarity between the

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CAT and EGCG treatments with respect to observed radical intensities, it was hypothesized that


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the A-rings of these two compounds were the reactive sites for carbonyl trapping, as previously

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reported by Noda and Peterson (12).

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Identification of Key Intermediates in GO-A-EGCG Model

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To investigate the effect of phenolic compounds in our model systems on pyrazinium radical

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formation, a link between the precursors of pyrazinium radicals (GO and GA) in our models and

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the previously reported ability of the A-ring of catechins to covalently trap these sugar fragments

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(7,8) by electrophilic aromatic substitution reactions was explored. LC/MS analysis of related

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phenolic-sugar fragment products in the GO–A Maillard model containing low and high

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concentrations of EGCG (10 and 100 mM) was conducted. When EGCG concentration was held

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at 10 mM, several major pseudomolecular ions were observed by LC/MS. Based on the observed

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masses, reaction product structures were predicted, which were further supported by MS/MS

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fragmentation patterns. Figure 5A and 5B illustrate the pseudomolecular ions 515 [M-H]- and

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573 [M-H]- detected and supported as mono-GO and di-GO adducts of EGCG, respectively.

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These reaction products were in agreement with other studies wherein the same products were

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observed in both food systems (7) and under biological conditions (8).

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In addition to GO adducts, glyoxal imine adducts of EGCG were also reported (Figure 6) and

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supported by the fragmentation ions of its sibling scan: fragments 434 [M-152-H]- and 416 [M-

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170-H]- indicate the neutral loss of one galloyl group (MW 152) and one gallic acid group (MW

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170) on the gallate ring, respectively; fragment 497 [M-89-H]- indicates the loss of an alanine

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molecule (MW 89); fragment 305 [M-129-152-H]- indicates the loss of a glyoxal imine molecule

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(MW 129) and a galloyl group (MW 152). The predicted structure of this analyte was further



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supported by accurate mass measurement with a reported molecular weight of 586.1199 Da and

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a predicted formula of [C27H24NO14]- (0.3 ppm). Based on the product structure, two possible

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mechanisms are proposed for the formation of this adduct, illustrated in Figure 7. In mechanism

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1, this product was generated by the reactive intermediate, glyoxal imine, formed from the initial

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condensation between glyoxal and alanine. EGCG appears to trap this intermediate through

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electrophilic aromatic substitution on the A ring to form the MW 587 adduct (Ia). In mechanism

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2, GO is more reactive towards EGCG and forms EGCG-GO adduct, which further reacts with

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amino group of alanine using the aldehyde group to form adduct Ib. Both adduct Ia and Ib

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provide a basis to explain the enhancement of pyrazinium radical formation by EGCG in GO-A

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model (Figure 8). Both structures have the trigonal planar conformation for the N-C-C moiety

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(i.e., the moiety forming the pyrazine ring structure) that freely tautomerize and easily dimerize

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and, subsequently oxidize, to generate the same pyrazinium radical cations consisting of a stable

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π-system. EGCG is predicted to be liberated from the radical’s pyrazine ring and, once liberated,

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is available to serve as a catalyst for further reactions. The hyperfine structure of the pyrazinium

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radical from the EPR spectra did not change with EGCG addition, and the enhancement of

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radical formation increased with EGCG concentration up to 1:10 (EGCG:GO). Other than MW

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587 adduct, a di-substituted EGCG-imine adduct (6 and 8 positions substituted by a glyoxal

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imine and a GO) was also observed as a minor product.

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In the presence of higher EGCG concentrations, the reverse trend was observed with a decrease

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in radical intensity observed at concentrations above 1:2 (EGCG:GO) (Figure 4A). We

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hypothesize that when EGCG concentrations are relatively high, MW 587 adduct is more likely

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to form novel reaction products (e.g., the GO-EGCG-EGCG adduct) at the expense of forming


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pyrazinium radical compounds. To further investigate this possibility, the GO–A model was

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incubated in the presence of 100 mM of EGCG and analyzed by LC/MS. A comparable amount

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of molecular ion 1044 [M-H]- was detected, which was not observed (or only observed at low

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levels) in the low EGCG system. MS/MS analysis of this 1044 [M-H]- ion (Figure 9) revealed

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the major fragmentation ions and structural information as follows: fragment of 586 [M-458-H]-

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indicates the loss of an EGCG molecule (MW 458); fragment 955 [M-89-H]- indicates the loss of

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an alanine molecule (MW 89); fragment 785 [M-89-170-H]- indicates the loss of a gallic acid

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molecule (MW 170) and an alanine molecule (MW 89); fragment 497 [M-458-89-H]- indicates

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the loss of an EGCG (MW 458) and an alanine molecule (MW 89). This compound was

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predicted to be a glyoxal imine di-EGCG adduct (II), which was further supported by accurate

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mass measurement giving a molecular weight of 1044.2094 Da and a predicted formula of

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[C49H42NO25]- (4.1 ppm). The proposed mechanism for the formation of adduct II is shown in

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Figure 10. At higher EGCG concentrations, MW 587 adduct (Ia or Ib) is predicted to react

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more readily with other EGCG molecules to form adduct II. Once bound to another EGCG

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molecule, it is proposed that MW 587 adduct loses its reactivity and its sp2 planar conformation

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required to form pyrazinium radicals. Therefore, at high concentration of EGCG, the phenolic

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quenches the radical precursors by disarming their chemical reactivity (i.e., conformation

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required for the production of pyrazinium radicals). This proposed mechanism explains the

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suppression of pyrazinium radical intensity observed when concentrations of EGCG/CAT are

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high relative to GO.

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Effect of 4-MeC, CAT, and EGCG on Pyrazinium Radical Formation in GA-A Model



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The effect of the model phenolic compounds 4-MeC, CAT, and EGCG on the generation rate of

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pyrazinium radicals in a GA-A model was also investigated by EPR. Glycolaldehyde (GA) is the

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smallest hydroxyl carbonyl that is known to be generated from sugar fragmentation during the

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early stages of the Maillard reaction (3), and been reported to efficiently generate pyrazinium

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radicals via condensation with amino acids, yielding the direct radical precursor, enaminol

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(Figure 1). The GA-A model was treated with the test phenolic compounds at concentrations

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ranging from 0 to 100 mM (Figure 11A). It was observed that both EGCG and CAT

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significantly inhibited pyrazinium radical formation in the GA-A model, and that the radical

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intensity declined as a function of EGCG/CAT concentration. 4-MeC, again, showed no such

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effect under these conditions.

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Identification of Key Intermediates in GA-A-EGCG Model

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LC/MS analysis of the GA-A Maillard model containing EGCG was performed, which revealed

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a pseudomolecular ion of 588 [M-H]- (Figure 12). Fragmentation ions and structural information

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obtained from MS/MS were as follows: fragment 499 [M-89-H]- indicates the loss of an alanine

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molecule (MW 89); fragment 347 [M-89-152-H]-, indicating the loss of an alanine (MW 89) and

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a galloyl group (MW 152). The structure of this compound was predicted to be the

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glycolaldehyde imine adduct of EGCG (III) that was further supported by accurate MS analysis

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with a reported molecular weight of 588.1371 Da and the predicted formula [C27H26NO14]- (3.1

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ppm). In addition to adduct III, the di-substituted EGCG-imine adducts (6 and 8 positions

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substituted by two glycolaldehyde imine molecules or by imine + GA) were also observed as

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minor products in the GA-A-EGCG model. Similar to adduct I, the formation of adduct III

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(Figure 13) was proposed occur via the reactive intermediate, glycolaldehyde imine (by


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condensation between GA and alanine), which is further trapped by EGCG to give adduct III.

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Glycolaldehyde imine was reported to be a reactive precursor in the generation of pyrazinium

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radicals, through tautomerization (to enaminol) and dimerization (3). Once bound to EGCG, the

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imine loses its sp2 orbital hybridization to tautomerize to enaminols, and thus its ability to form

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pyrazinium radicals. Hence, as observed in the EPR spectra above, in the presence of EGCG (or

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CAT), pyrazinium radical formation was suppressed in the GA-A Maillard model.

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In summary, catechins were reported to affect the formation rates of pyrazinium radicals in both

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GO–A and GA–A models, presumably through trapping of electrophilic imine intermediates.

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This mechanism is proposed to either catalyze the radical generation, or function as a quenching

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agent of the radical precursor enaminol depending on the sugar fragment involved

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(hydroxycarbonyl vs. dicarbonyl sugar fragment). Carbonyl-trapping by phenolics were, for the

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first time, reported as a mechanism to alter reactive imine intermediates linked to pyrazinium

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radical formation in Maillard systems. As pyrazinium radical formation is linked to non-

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enzymatic browning, these findings provide key insights into the effect that phenolics have on

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the Maillard reaction, which is of major importance to food quality and, potentially, human

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health.

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REFERENCES

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(1)

Nursten, H. E. The Maillard reaction: chemistry, biochemistry, and implications; Royal Society of Chemistry: Cambridge, UK, 2005.

289 290 291

(2)

Baynes, J. W. Monnier, V. Ames, J.; Thorpe, S. The Maillard reaction: chemistry at the interface of nutrition, aging, and disease; 8th International Symposium on the Maillard Reaction. Ann. N. Y. Acad. Sci. 2005, 1043.

292 293

(3)

Namiki, M.; Hayashi, T. A new mechanism of the Maillard reaction involving sugar fragmentation and free radical formation. ACS Symposium Series 1983, 215, 21-46.

294 295 296

(4)

Hofmann, T. Bors, W.; Stettmaier, K. Studies on radical intermediates in the early stage of the nonenzymatic browning reaction of carbohydrates and amino acids. J. Agric. Food Chem. 1999, 47, 379-390.

297 298 299

(5)

Totlani, V. M.; Peterson, D. G. Influence of epicatechin reactions on the mechanisms of Maillard product formation in low moisture model systems. J. Agric. Food Chem. 2007, 55, 414-420.

300 301 302

(6)

Totlani, V. M.; Peterson, D. G. Reactivity of epicatechin in aqueous glycine and glucose maillard reaction models: quenching of C2, C3, and C4 sugar fragments. J. Agric. Food Chem. 2005, 53, 4130-4135.

303 304 305

(7)

Totlani, V. M.; Peterson, D. G. Epicatechin carbonyl-trapping reactions in aqueous maillard systems: Identification and structural elucidation. J. Agric. Food Chem. 2006, 54, 7311-7318.

306 307 308

(8)

Sang, S. Shao, X. Bai, N. Lo, C.-Y. Yang, C. S.; Ho, C.-T. Tea polyphenol (-)epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol. 2007, 20, 1862-1870.

309 310 311

(9)

Es-Safi, N. E. Fulcrand, H. Cheynier, V.; Moutounet, M. Competition between (+)catechin and (-)-epicatechin in acetaldehyde-induced polymerization of flavanols. J. Agric. Food Chem. 1999, 47, 2088-2095.

312 313 314 315

(10)

Es-Safi, N. E. Le Guernevé, C. Cheynier, V.; Moutounet, M. New phenolic compounds formed by evolution of (+)-catechin and glyoxylic acid in hydroalcoholic solution and their implication in color changes of grape-derived foods. J. Agric. Food Chem. 2000, 48, 4233-4240.


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316 317 318

(11)

Hofmann, T. Quantitative studies on the role of browning precursors in the Maillard reaction of pentoses and hexoses with L -alanine. Eur. Food. Res. Technol. 1999, 209, 113-121.

319 320 321

(12)

Noda, Y.; Peterson, D. G. Structure-reactivity relationships of flavan-3-ols on product generation in aqueous glucose/glycine model systems. J. Agric. Food. Chem. 2007, 55, 3686-3691.

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FIGURE CAPTIONS

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Figure 1. Mechanism of pyrazinium radical cation formation in the Maillard reaction (3,4).

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Figure 2. (-)-Epicatechin-carbonyl-trapping reaction and formation of EC-MGO adducts (7).

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Figure 3. Structures of (-)-EGCG, (+)-catechin, and 4-methylcatechol.

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Figure 4. Effects of (A) EGCG, (B) CAT, and (C) 4-MeC on generation of pyrazinium radical in

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GO–A aqueous model at 25°C. The EPR spectra showing the effect of EGCG on radical

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formation in the GO-A model is shown (D).

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Figure 5. LC/MS/MS chromatogram and spectrum of (A) mono-substituted and (B) di-

340

substituted EGCG–GO adducts generated from the GO–A–EGCG model.

341 342

Figure 6. LC/MS/MS chromatogram and spectrum of analyte MW 587 (586 [M-H]-) generated

343

from the GO–A–EGCG model.

344 345

Figure 7. Two possible mechanisms for the formation of analyte MW 587 (Ia and Ib) in the GO–

346

A–EGCG model. Conjugation is predicted also to occur at position 8 of EGCG (not shown).

347


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348

Figure 8. Proposed mechanism for the effect of low EGCG concentration on pyrazinium radical

349

formation in the GO–A model. EGCG conjugation products Ia and Ib are predicted to dimerize

350

to form pyrazinium radicals given by their sp2 orbital hybridization.

351 352


353

from the GO–A–EGCG model.

354 355

Figure 10. Proposed mechanism of the generation of analyte MW 1045 (II) and the effect of high

356

EGCG concentration on pyrazinium radical formation in the GO–A model.

357 358

Figure 11. Effects of (A) EGCG, (B) CAT, and (C) 4-MeC on generation of pyrazinium radical

359

in the GA–A aqueous model at 25°C.

360 361


362

from the GA–A–EGCG model.

363 364

Figure 13. Proposed mechanism of generation of analyte MW 589 (III) and the inhibitory effect

365

of EGCG on pyrazinium radical formation in the GA–A model. Conjugation is predicted also to

366

occur at Position 8 of EGCG (not shown).



367 368

FIGURES

369

Figure 1

370 371


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372 373

Figure 2

374



375 376

Figure 3

377


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378 379

Figure 4a

380 381



382 383

Figure 4b

384 385


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386 387

Figure 4c

388



389 390

Figure 4d

391 392


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393 394

Figure 5a

395 396



397 398

Figure 5b

399 400


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401 402

Figure 6

403 404 405 406



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407 408

Figure 7 Mechanism 1

OH

(-)-EGCG HO

OH O

H

O +

O

H2N

Glyoxal

OH

O OH

O

OH

OH

O

- H2O

N

OH

O NH OH

HO

O

Alanine

OH

O

OH OH

OH

O

HO

OH

O

OH

O

O

OH [Ia]

Glyoxal imine

OH

(MW = 587)

Mechanism 2 OH (-)-EGCG HO

O O OH

OH

O

O O

409

Glyoxal

OH OH

OH

OH HO

O

HO

OH

H

OH

OH OH

OH

- H2O

OH OH O H2N

OH

OH OH OH

Alanine

410


O OH OH

HO O

OH

N

O

O

O

OH

O

O

OH [Ib] (MW = 587)

OH

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411 412

Figure 8

413



414 415

Figure 9

416 417


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418 419

Figure 10 EGCG

O

NH HO

EGCG

O Ia HO

Pyrazinium Radical

NH EGCG

N HO

HO

OH EGCG

O Ib

420

EGCG EGCG

O II

MW 1045

MW 587



421 422

Figure 11a

423 424


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425 426

Figure 11b

427 428



429 430

Figure 11c

431


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432 433

Figure 12

434



435 436 437

Figure 13

438 439


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440


TABLE OF CONTENTS GRAPHIC

441


Influence of Phenolic Compounds on the Mechanisms of Pyrazinium

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