Quercetin Inhibits Advanced Glycation End Product Formation by

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Quercetin Inhibits Advanced Glycation End Product Formation by Trapping Methylglyoxal and Glyoxal Xiaoming Li, Tiesong Zheng, Shengmin Sang, and Lishuang Lv J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504132x • Publication Date (Web): 20 Nov 2014 Downloaded from http://pubs.acs.org on November 23, 2014

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Quercetin Inhibits Advanced Glycation End Product Formation by Trapping

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Methylglyoxal and Glyoxal

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Xiaoming Li‡, Tiesong Zheng‡, Shengmin Sang§, Lishuang Lv‡*

4 5 ‡

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University, 122# Ninghai Road, Nanjing, 210097, P. R. China

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Department of Food Science and Technology, Ginling College, Nanjing Normal

§

Center for Excellence in Post-harvest Technologies, North Carolina Agricultural and

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Technical State University, North Carolina Research Campus, 500 Laureate Way,

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Suite 4222, Kannapolis, North Carolina, 28081, USA

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Corresponding author: Lishuang Lv

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Tel.: +86 25 83598286; Fax: +86 25 83707623.

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E-mail address: [email protected]; [email protected]

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ABSTRACT

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Methylglyoxal (MGO) and glyoxal (GO) are not only endogenous metabolites, but

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also exist in exogenous resources, such as food, beverages, urban atmosphere, and

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cigarette smoke. They have been identified as reactive dicarbonyl precursors of

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advanced glycation end products (AGEs) which have been associated with

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diabetes-related long-term complications. In this study, quercetin, a natural flavonol

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found in fruits, vegetables, leaves, and grains, could effectively inhibit the formation

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of AGEs in a dose-dependent manner via trapping reactive dicarbonyl compounds.

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More than 50.5% of GO and 80.1% of MGO were trapped at the same time by

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quercetin within 1 h under physiological conditions. Quercetin and MGO (or GO)

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were combined at different ratios and the products generated from this reaction were

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analyzed with LC/MS. Both mono-MGO and di-MGO adducts of quercetin were

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detected in this assay using LC/MS, but only tiny amount of mono-GO adducts of

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quercetin were found. Additionally, di-MGO adducts were observed as the dominated

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product with prolonging incubation time. We also revealed that in the bovine serum

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albumin (BSA)-MGO/GO system, quercetin traps MGO and GO directly then

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significantly inhibit the formation of AGEs.

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KEYWORDS: quercetin; methylglyoxal; glyoxal; advanced glycation end products

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(AGEs); diabetic complications.

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Introduction

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Hyperglycemia is considered to be a critical factor to diabetic complications in

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epidemiological studies, especially in type 2 diabetes mellitus.1 Accumulating studies

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have shown that the formation of advanced glycation end products (AGEs) induced

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by hyperglycemia can cause or promote many diseases, such as cataract generation,

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retinopathy, atherosclerosis, and nephropathy.2 This is mainly due to the accumulation

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of AGEs in the tissues, which can modify protein half-life, alter enzyme activity, and

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change protein immunogenicity.3

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AGEs are generated from the reaction of the carbonyl groups of the reducing sugars

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and the free amino groups of the proteins,4 followed by an Amadori rearrangement to

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form the stable Amadori product. Then the product can generate various reactive

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dicarbonyl species, such as deoxyglucosones, glyoxal (GO) and methylglyoxal

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(MGO).5,

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produce reactive dicarbonyl species.7,

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intermediates to form AGEs in vivo.9

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In addition, lipid peroxidation and autoxidation of glucose can also 8

Both MGO and GO are the crucial

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Due to the reactive carbonyl group, MGO and GO can effectively modify proteins

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through reacting with their arginine, lysine, and cysteine residues,9-13 and can also

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exhibit a potential cellular toxicity to DNA.14 Any reaction that increases MGO or GO

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levels in tissues or plasma can ultimately lead to the pathology of diabetic

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complications.15 Previous studies have focused mainly on scavenging these reactive

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intermediates with some pharmaceutical agents, such as aminoguanidine,16,

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tenilsetam,18,

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metformin,20,

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and pyridoxamine.22 However, the side effects of 3

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these AGEs inhibitors prompt serious health concerns. For example, aminoguanidine,

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an effective AGEs-inhibitor in both in vitro and in vivo studies, has a high toxicity for

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diabetic patients.5

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Flavonoids are dietary constituents contained in vegetables, fruits, soybean,

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grains, and other plant-derived food. Recent studies show that flavonoids could inhibit

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the oxidative stress and facilitate the detoxification of dicarbonyl species.23, 24 Our

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previous studies have found that genistein exhibits a significant trapping effect on

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MGO to form mono- and di-MGO adducts.25 Several studies have also demonstrated

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the trapping capacity of reactive dicarbonyl compounds by other food-derived

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flavonoids, such as phloretin from apples, (-)-epigallocatechin 3-gallate (EGCG) from

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tea, and proanthocyanidins and anthocyanin from berries.26-28

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Quercetin is a flavonol that exists in many plants, flowers, leaves, and fruits,

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mostly in the form of glycosides, whose A ring is the same as EGCG, phloretion, and

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genistein. It has been reported that quercetin can efficiently inhibit the glycation of

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DNA,

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However, the underlying mechanism of the antiglycation effect of quercetin is still

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largely unknown. In this study, we investigated the efficacy of trapping MGO (and

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GO) and inhibition of the formation of AGEs with quercetin.

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Materials and Methods

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as well as suppress α-dicarbonyl compounds-induced protein glycation.30, 31

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Materials Quercetin was obtained from Nanjing Guangrun Biological Products Co.,

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Ltd (Nanjing, Jiangsu, China). Methylglyoxal (MGO, 40% in water), glyoxal (GO, 40%

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in water), 1,2-diaminobenzene (DB), 2,3-butanedione were purchased from 4

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Sigma-Aldrich (St. Louis, MO, USA). Bovine serum albumin (BSA), DMSO,

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streptomycin and penicillin mixed solution were purchased from Shanghai Sangon

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Biological Engineering Technology Co., Ltd (Shanghai, China). HPLC-grade solvents

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and other reagents were obtained from Shanghai Sinopharm Chemical Reagent Co.,

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Ltd (Shanghai, China). HPLC-grade water was prepared using a Millipore Milli-Q

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purification system (Bedford, MA, USA).

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Determination of the MGO and GO Trapping Capacity of Quercetin by GC.

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MGO (0.5 mM) and GO (0.5 mM) were incubated with quercetin (0.25, 0.5, 1.5, 2.5

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mM) in PBS buffer (pH 7.4, 100 mM) at 37 °C for 0, 10, 30, 60, 120, or 240 min. At

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each time point, the reaction was stopped by adding 10 µL of acetic acid. The samples

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were then stored at -80 °C for further use.

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Preparation of samples: One mL of 100 mM 1,2-diaminobenzene was added to 1

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mL of sample, and then mixed with 0.5 mL of 2,3-butanedione (internal standard) at 1

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mM. The reaction mixture was kept at 60 °C for 10 min. Then, 1 mL of 1 M

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acetaldehyde was added and incubated at 60 °C for 15 min to remove the unreacted

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1,2-diaminobenzene. The mixture was extracted twice with 2 mL of methylene

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chloride. The organic phase was combined and concentrated to 0.5 mL. One µL of this

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sample was injected directly into GC. The remaining percentage of MGO and GO was

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calculated using the equation: remaining (%) = amount of MGO (GO) in test

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compound) (as quinoxalines)/amount of MGO (GO) in control (as quinoxalines) ×

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100.

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GC Analysis. The levels of methylquinoxaline and quinoxaline were analyzed with 5

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an Agilent Gas Chromatograph (7820 Series, Agilent Technologies, Palo Alto, CA,

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USA) equipped with a flame ionization detector (FID). The column was HP-5 MS

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(5%-phenyl)-methylpolysiloxane silica capillary (30 m × 0.32 mm id, film thickness

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0.25 µm, Agilent, Wilmington, DE, USA). The injector temperature was 250 °C and

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the detector temperature was 280 °C with hydrogen, air, and nitrogen flow rates at

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30.0, 300, and 5.0 mL/min, respectively. The injector was in 1:1 split mode. Set flow

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rate of constant carrier gas (nitrogen) to be 2.0 mL/min. The GC oven temperature

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was programmed as follows: the initial oven temperature 40 °C was held for 1 min

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and increased to 140 °C at a rate of 5 °C/min and held for 1 min then increased to

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250 °C at a rate of 50 °C/min and held for 1 min. The total run time was 25.2 min. All

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of the solvents were filtered with 0.2 µm Nylaflo membrane filter. The injection

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volume was 1 µL for each sample solution.

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Inhibitory Effects of Quercetin on the Formation of AGEs. BSA (1.5 mg/mL)

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was incubated with MGO (500 µM) or GO (500 µM) in PBS buffer, pH 7.4, in the

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presence or absence of quercetin (1.5 mM) at 37 °C. Streptomycin and penicillin (0.3

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mL) was added to the solution to prevent bacterial growth. The reaction mixture (500

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µL) was collected and frozen at designated time points (0, 4, 8, 12, 72, 144, 288, 432,

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and 720 h). The amount of AGEs was determined using fluorescence at an

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excitation/emission wavelength of 370/440 nm.

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LC/MS Analysis. LC/MS analysis was performed using an Agilent Masshunter

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System consisting of an 1290 G4220A BinPump, an 1290 G4226A Wellplate sampler,

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an G4212A Diode array detector, and an 6460 QQQ mass detector (Agilent, Santa 6

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Clara, CA, USA) incorporated with electrospray ionization (ESI) interfaces. A 250 ×

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4.6 mm i.d., 5 µm ZORBAX Eclipse XDB-C18 column (Agilent, Santa Clara, CA,

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USA) was used for separation at a flow rate of 0.5 mL/min. The column was eluted

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with 90% solvent B (water with 0.1% formic acid) for 5 min, followed by linear

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increases in A (acetonitrile with 0.1% formic acid) to 60% from 5 to 40 min, to 10%

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from 40 to 41 min, and then with 90% B from 41 to 46 min. The column was then

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re-equilibrated with 90% B for 5 min. The LC eluent was introduced into the ESI

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interface. The negative ion polarity mode was set for ESI ion source. The typical

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operating parameters were as follows: spray needle voltage, 5 kV; nitrogen sheath gas,

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45 (arbitrary units); auxiliary gas, 5 (arbitrary units). The structural information of

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quercetin and the major MGO and GO adducts was obtained by tandem mass

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spectrometry (MS/MS) through collision-induced dissociation (CID) with a relative

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collision energy setting of 35%. Data acquisition was performed with Qualitative

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Analysis of Masshunter (Agilent, Santa Clara, CA, USA).

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LC/MS Analysis of MGO or GO Adducts of Quercetin in the BSA-MGO (or

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GO) System. The procedure of BSA incubation with MGO/GO and quercetin is the

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same as the inhibition effect method described above. Samples were collected at

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designated time points (0, 1, 2, 4, and 8 h) from the quercetin treated BSA-MGO (or

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GO) system and the mono- and di-MGO conjugated quercetin were detected using the

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LC/MS method described above.

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Results 7

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Trapping of MGO and GO Simultaneously by Quercetin under Physiological

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Conditions. The results shown in Figure 1 suggest that quercetin can trap both MGO

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and GO simultaneously and efficiently under physiological conditions. More than

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26.0% of GO and 69.0% of MGO were trapped within 60 min by 0.5 mM quercetin,

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and the trapping efficiency could be up to 50.5% and 80.1%, respectively, when using

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2.5 mM quercetin. Quercetin appeared to trap MGO much more efficient than GO

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when both GO and MGO occurred in the same system.

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Analyzing the Formation of MGO or GO Adducts of Quercetin Using LC/MS.

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We further identified the quercetin-MGO and quercetin-GO adducts using LC/MS

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after incubation of quercetin with MGO or GO at four different ratios (3:1, 1:1, 1:3,

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and 1:10) (Figure 2 and Supplemental Figure 1). Under selective ion monitoring (SIM)

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mode, the structural information of these products was achieved using tandem mass

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analysis (Figure 3 and Supplemental Figure 2). After 4 h of incubation of quercetin

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with MGO (ratio at 1:1), one product peak (tR 23.91 min) appeared in LC

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chromatogram as shown in Figure 2A. This peak had the molecular ion m/z 373 [M -

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H] - and fragment ion m/z 301 [M -72- H] -, indicating the loss of one MGO (m/z 72)

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molecule, suggesting this product was a mono-MGO conjugated quercetin (MM)

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(Figure 3A). When quercetin and MGO at a 1:3 ratio were incubated for 4 h,another

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peak appeared at 21.32 min. This peak had the molecular ion m/z 445 [M - H]-, which

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was 142 mass units heavier than that of the quercetin, indicating that this peak was a

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di-MGO adduct of quercetin (DM-1) (Figure 3B). The third new peak appeared at

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22.68 min (Figure 2A), with the addition of MGO at the ratio of 1:10 for 4 h, which 8

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had the same molecular ion as DM-1, indicating that this peak was also a di-MGO

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adduct of quercetin (DM-2) (Figure 3C). Under the ratio of 1:3, both mono- and

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di-MGO adducts were the major products, and that the amount of unreacted quercetin

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dramatically reduced to 12.28% (Table 1). Whereas, when quercetin and MGO at a

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1:10 ratio were incubated for 24 h, di-MGO adducts became the dominant products

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(Table 1), and one additional peak corresponding to di-MGO adduct (DM-3) was

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observed (Figure 2B). This peak could not be separated fully from the peak of DM-2

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(Figure 2B). When quercetin and GO were incubated at a 1:10 ratio for 24 h, two new

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peaks were observed at 22.02 and 23.98 min (Supplemental Figure 1). They had the

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same molecular ion m/z 359 [M - H]-, which was 58 mass units heavier than that of

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quercetin, indicating both of them are mono-GO adducts of quercetin (Supplemental

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Figure 2A and 2B). However, only tiny amounts of mono-GO adducts were detected

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even when quercetin and GO were incubated at a 1:10 ratio for 24 h, and di-GO

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adducts were not detectable (Supplemental Figure 1A and 1B).

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Inhibitory Effects of Quercetin on the Formation of AGEs. We found that

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quercetin significantly inhibited the formation of AGEs in the BSA-MGO/GO system

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(Figure 4). This result is consistent to our observation on the trapping efficacy of

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MGO/GO by quercetin. When quercetin (0.25 mM) was present in the incubation

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mixture, the inhibition efficiency could be up to 91.0% at 24 h in the BSA-MGO

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system (Figure 4A). While in the quercetin-BSA-GO system, the inhibitory effect of

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quercetin on the formation of AGEs seems less than that of the BSA-MGO system,

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only 76.8% within 30 days (Figure 4B). It may be related to the fact that the formation

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of AGEs by GO was slow.

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Determining the Adduct Formation of Quercetin in the BSA-MGO or

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BSA-GO System by LC/MS. In order to clarify whether trapping of MGO (or GO)

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by quercetin is the major mechanism to prevent the formation of AGEs, LC/MS was

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used to determine the existence of the mono- and di-MGO/GO conjugated quercetin

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in the samples, which were collected after incubation of quercetin with BSA and

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MGO or quercetin with BSA and GO.

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As shown in Figure 5, mono-MGO adduct could be detected after 1 h incubation,

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and di-MGO adduct could be detected after 2 h incubation. They had identical

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retention times and MS/MS spectra to those of the mono- and di-MGO adducts in the

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reaction mixture between quercetin and MGO. But in the BSA-GO system, only

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mono-GO adducts were detectable with very low levels (Supplemental Figure 3).

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Discussion

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Our results revealed that quercetin can rapidly trap MGO and then inhibit the

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formation of AGEs through forming of mono- and di-MGO adducts under neutral and

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alkaline conditions in vitro. In this assay, both mono- and di-MGO adducts of

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quercetin were detected, and the di-MGO adducts became the dominant products

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during prolonged incubation. While only a tiny amount of mono-GO adducts of

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quercetin were detectable in the reaction mixtures of quercetin and GO.

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α-Dicarbonyl compounds are known as important precursors of AGEs, which can 10

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be generated endogenously through either degradation of glucose or early glycation

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products. Rising levels of dietary fructose in the Western diet has been associated

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with an increase of serum MGO and GO.32 In our study, quercetin efficiently trapped

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MGO and GO simultaneously under physiological conditions, as MGO and GO

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appear in the same system. Furthermore, we found that quercetin preferred trapping of

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MGO to that of GO. The potential reason may be that in aqueous solution GO exists

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mainly as the hydrated monomer, dimer, or trimer, whose conversion to free GO is

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slowed down the trapping reaction between quercetin and GO.33 Similar results have

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been reported for EGCG26 and phloretin,27 both of them appeared to trap for GO

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much more slowly than that for MGO.

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To further study whether quercetin can inhibit the formation of AGEs via

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trapping MGO/GO, we analyzed the inhibitory effect of quercetin on the formation of

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AGEs in BSA-MGO/GO system, and then determined the formation of MGO (GO)

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adducts of quercetin by LC-MS. We demonstrated that quercetin can inhibit the

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formation of AGEs via trapping MGO and GO. The amount of AGEs rose

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dramatically with time during incubation with MGO until reaching a plateau. While

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no plateau appeared during incubation with GO, the parameter increased

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progressively with time all along. Results of this study are consistent with previous

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reports that MGO rather than GO is the main dicarbonyl compound responsible for

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albumin glycation.34 Moreover, the inhibitory activity on the formation of AGEs by

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quercetin was much higher in the BSA-MGO system than in the BSA-GO system, one

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of the major causes was that the glycation was dragged down by the conversion 11

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among hydrated monomer, dimer, and trimer to free GO.

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Flavonoids, consisting of two hydroxy substituted aromatic rings joined by a

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three carbon link (a C6-C3-C6 configuration), can be categorized into flavones,

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flavanones, isoflavones, chalcones, anthocyanidins, and flavonols according to

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different C-ring chemical structures. It has been reported that the mechanism of

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inhibition of AGEs is done via trapping MGO for EGCG26 (flavanols), genistein25

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(isoflavones), phloretin27 (chalcones), and proanthocyanidins35 (oligomers of

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flavanols). In view of this fact, we investigated quercetin (flavonols) inhibition of the

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formation of AGEs via trapping MGO and GO. With respect of the structure of these

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flavonoids, EGCG, genistein, phloretin, and quercetin have the same A ring. The same

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mechanism for trapping reactive dicarbonyl species and forming mono- and di-MGO

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adducts at the A ring may be predicted. After quercetin and MGO (1:3) were

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incubated for 24 h, the MS/MS spectrum of the most abundant daughter ion m/z 373

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of the mono-MGO (tR 27.34 min) adduct had the typical breakdown of the C ring (m/z

249

150) and a loss of one H2O to generate the fragment ion 205 [M- H2O -150] - (Figure

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3A, Figure 6), which indicates that the MGO conjugates at the A ring of quercetin.

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Similarly, the MS/MS spectrum of the three di-MGO adducts had the same fragment

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patent to generate the daughter ion 277 [M- H2O -150]

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Therefore, we could determine that the positions 6 and 8 on the A ring of quercetin

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were the major active sites for trapping MGO (Figure 7), that comprise the essential

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structural requirement for flavonoids to scavenge methylglyoxal.36 Quercetin is the

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major representative of the flavonoid subclass flavonols.37 It is widely distributed in

-

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(Figures 3B-3D and 6).

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nature, existing in foods such as apples, onions, teas, berries, red wine, and some

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herbs. Quercetin is a strong antioxidant,38 and also possesses antihistamine and

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anti-inflammatory properties.39,

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including luteolin, rutin, EGCG, and quercetin show effective inhibition on MGO

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mediated AGEs formation by 82.2, 77.7, 69.1, and 65.3%, respectively.41 Our results

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demonstrate the reaction kinetics for quercetin trapping MGO and GO simultaneously,

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as well as the mechanism of quercetin inhibiting the formation of AGEs in the

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BSA-MGO/GO system. In conclusion, using the food-borne flavonoid quercetin

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intervention to scavenge these dicarbonyl compounds is likely to be an effective

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strategy to inhibit the formation of AGEs and prevent AGE-mediated processes linked

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to disease.

40

It has been reported that dietary flavonoids

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Acknowledgements

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This work was supported by NSF of Jiangsu province of China (Project BK2012850),

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Program of Natural Science Research of Jiangsu Higher Education Institution of

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China (Project 12KJB5500005) and Natural Science Foundation of Zhejiang province

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of China (Project LY12C15001).

274 275

Compliance with Ethics Requirements

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There is no conflict of interest for all authors.

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This article does not contain any studies with human or animal subjects.

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metabolism and diabetic complications. Biochemical Society Transactions 2003, 31, 1358-1363. 10. Lo, T.; Westwood, M. E.; McLellan, A. C.; Selwood, T.; Thornalley, P. J., Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. Journal of Biological Chemistry 1994, 269, 32299-32305. 11. Shamsi, F. A.; Partal, A.; Sady, C.; Glomb, M. A.; Nagaraj, R. H., Immunological Evidence for Methylglyoxal-derived Modifications in Vivo DETERMINATION OF ANTIGENIC EPITOPES. Journal of Biological Chemistry 1998, 273, 6928-6936. 12. Klöpfer, A.; Spanneberg, R.; Glomb, M. A., Formation of Arginine Modifications in a Model System of N α-tert-Butoxycarbonyl (Boc)-Arginine with Methylglyoxal. Journal of agricultural and food chemistry 2010, 59, 394-401. 13. Gao, Y.; Wang, Y., Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal. Biochemistry 2006, 45, 15654-15660. 14. Kang, J. H., Oxidative damage of DNA induced by methylglyoxal in vitro. Toxicology letters 2003, 145, 181-187. 15. Kilhovd, B. K.; Juutilainen, A.; Lehto, S.; Rönnemaa, T.; Torjesen, P. A.; Hanssen, K. F.; Laakso, M., Increased serum levels of methylglyoxal-derived hydroimidazolone-AGE are associated with increased cardiovascular disease mortality in nondiabetic women. Atherosclerosis 2009, 205, 590-594. 16. Thomas, M.; Baynes, J.; Thorpe, S.; Cooper, M., The role of AGEs and AGE inhibitors in diabetic cardiovascular disease. Current drug targets 2005, 6, 453-474. 17. Cervantes-Laurean, D.; Schramm, D. D.; Jacobson, E. L.; Halaweish, I.; Bruckner, G. G.; Boissonneault, G. A., Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites. The Journal of nutritional biochemistry 2006, 17, 531-540. 14

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18. Peyroux, J.; Sternberg, M., Advanced glycation endproducts (AGEs): pharmacological inhibition in diabetes. Pathologie Biologie 2006, 54, 405-419. 19. Webster, J.; Urban, C.; Berbaum, K.; Loske, C.; Alpar, A.; GÄrtner, U.; De Arriba, S. G.; Arendt, T.; MÜnch, G., The carbonyl scavengers aminoguanidine and tenilsetam protect against the neurotoxic effects of methylglyoxal. Neurotoxicity research 2005, 7, 95-101. 20. Schurman, L.; McCarthy, A.; Sedlinsky, C.; Gangoiti, M.; Arnol, V.; Bruzzone, L.; Cortizo, A., Metformin reverts deleterious effects of advanced glycation end-products (AGEs) on osteoblastic cells. Experimental and clinical endocrinology & diabetes 2008, 116, 333-340. 21. Beisswenger, P.; Ruggiero-Lopez, D., Metformin inhibition of glycation processes. Diabetes & metabolism 2003, 29, 6S95-6S103. 22. Price, D. L.; Rhett, P. M.; Thorpe, S. R.; Baynes, J. W., Chelating activity of advanced glycation end-product inhibitors. Journal of Biological Chemistry 2001, 276, 48967-48972. 23. Romagnolo, D. F.; Selmin, O. I., Flavonoids and cancer prevention: a review of the evidence. Journal of nutrition in gerontology and geriatrics 2012, 31, 206-238. 24. Morimitsu, Y.; Yoshida, K.; Esaki, S.; Hirota, A., Protein glycation inhibitors from thyme (Thymus vulgaris). Biosci Biotechnol Biochem 1995, 59, 2018-2021. 25. Lv, L.; Shao, X.; Chen, H.; Ho, C.-T.; Sang, S., Genistein Inhibits Advanced Glycation End Product Formation by Trapping Methylglyoxal. Chemical Research in Toxicology 2011, 24, 579-586. 26. 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. Chemical research in toxicology 2007, 20, 1862-1870. 27. Shao, X.; Bai, N.; He, K.; Ho, C.-T.; Yang, C. S.; Sang, S., Apple polyphenols, phloretin and phloridzin: new trapping agents of reactive dicarbonyl species. Chemical research in toxicology 2008, 21, 2042-2050. 28. Wang, W.; Yagiz, Y.; Buran, T. J.; Nunes, C. d. N.; Gu, L., Phytochemicals from berries and grapes inhibited the formation of advanced glycation end‐products by scavenging reactive carbonyls. Food Research International 2011, 44, 2666-2673. 29. Sengupta, B.; Uematsu, T.; Jacobsson, P.; Swenson, J., Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: a calorimetric and spectroscopic study. Biochem Biophys Res Commun 2006, 339, 355-361. 30. Wang, Y.; Ho, C. T., Flavour chemistry of methylglyoxal and glyoxal. Chem Soc Rev 2012, 41, 4140-4149. 31. Hsieh, C.-L.; Lin, Y.-C.; Yen, G.-C.; Chen, H.-Y., Preventive effects of guava (Psidium guajava L.) leaves and its active compounds against α-dicarbonyl compounds-induced blood coagulation. Food Chemistry 2007, 103, 528-535. 32. Lee, O.; Bruce, W. R.; Dong, Q.; Bruce, J.; Mehta, R.; O’Brien, P. J., Fructose and carbonyl metabolites as endogenous toxins. Chemico-Biological Interactions 2009, 178, 332-339. 33. Commission, E., Scientific Committee on Consumer Products (SCCP) Opinion on Glyoxal, the 4th plenary of June 2005. http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_023.pdf. 34. Sadowska-Bartosz, I.; Galiniak, S.; Bartosz, G., Kinetics of glycoxidation of bovine serum albumin by methylglyoxal and glyoxal and its prevention by various compounds. Molecules 2014, 19, 4880-4896. 35. Peng, X.; Cheng, K.-W.; Ma, J.; Chen, B.; Ho, C.-T.; Lo, C.; Chen, F.; Wang, M., Cinnamon bark proanthocyanidins as reactive carbonyl scavengers to prevent the formation of advanced glycation 15

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382 383

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Figure legends

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Figure 1. Trapping of MGO and GO simultaneously by quercetin under physiological

386

conditions (pH 7.4, 37 ℃). MGO (0.5 mM) and GO (0.5 mM) were incubated with

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quercetin (0.25, 0.5, 1.5, and 2.5 mM) in pH 7.4 phosphate buffer solutions at 37 ℃

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for 10, 30, 60, 120, and 240 min. (A) MGO, (B) GO. Data are presented as the means

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± SD of three replications.

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Figure 2. LC chromatograms of quercetin after incubation with different ratios of

391

MGO (3:1, 1:1, 1:3, and 1:10) for 4 h (A) and 24 h (B), respectively.

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Figure 3. Tandem MS/MS spectra of mono-MGO adducts (A) and di-MGO adducts

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(B-D) of quercetin.

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Figure 4. Inhibitory effect of the formation of AGEs by quercetin in the BSA-MGO

395

(or GO) assay. (A) MGO, (B) GO. Data are presented as the means ± SD of three

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replications.

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Figure 5. LC chromatograms of quercetin and mono- and di-MGO adducts of

398

quercetin after incubation of quercetin in the BSA-MGO assay for 1, 2, 4, and 8 h as

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well as after the incubation of quercetin with MGO at a 1:3 ratio for 4 h.

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Figure 6. Formation pathway of the major fragments of mono- and di-MGO adducts

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of quercetin.

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Supplemental Figure 1. LC chromatograms of quercetin after incubation with

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different ratios of GO (3:1, 1:1, 1:3, and 1:10) for 4 h (A) and 24 h (B), respectively.

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Supplemental Figure 2. Tandem MS/MS spectra of mono-GO adducts of quercetin

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(A and B).

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Supplemental Figure 3. LC chromatograms of quercetin and mono-adducts of

408

quercetin after incubation of quercetin in the BSA-GO assay for 1, 2, 4, and 8 h as

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well as after the incubation of quercetin with GO at a 1:3 ratio.

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Tandem MS/MS spectra of mono-MGO adducts (A) and di-MGO adducts (B-D) of quercetin. 254x190mm (96 x 96 DPI)

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LC chromatograms of quercetin and mono- and di-MGO adducts of quercetin after incubation of quercetin in the BSA-MGO assay for 1, 2, 4 and 8 h as well as after the incubation of quercetin with MGO at a 1:3 ratio. 254x190mm (96 x 96 DPI)

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Formation pathway of the major fragments of mono- and di-MGO adducts of quercetin. 254x190mm (96 x 96 DPI)

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Formation pathway of mono- and di-MGO adducts of quercetin under neutral or base conditions. 254x190mm (96 x 96 DPI)

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Table 1 Percentage of quercetin-MGO products for 4 and 24 h at different ratio. DM-1

Quercetin:MGO

DM-2 + DM-3

MM

Quercetin

Timepoints (h)

4

24

4

24

4

24

4

24

3:1

-

-

-

-

4.59

10.91

95.41

89.09

1:1

12.28

7.87

-

-

11.43

30.38

76.29

61.75

1:3

16.89

45.33

-

10.07

33.87

38.68

49.24

5.92

1:10

54.05

56.17

3.85

12.90

29.82

30.93

12.28

-

“-”, No detected Percent (%) = peak area of each product at 4 h or 24 h at each ratio / total sum of peak areas of products at 4 h or 24 h at each ratio.

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