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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Journal of Agricultural and Food Chemistry

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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‡*

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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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1

ACS Paragon Plus Environment


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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

160

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 -

163

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

186

(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,

9



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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)

195

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).

204 205

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

227

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.

236

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

237

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

238

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

240

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

244

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

245

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

246

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

247

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

248

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

250

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

252

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

-

12


(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,

260

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

262

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

263

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

264

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

265

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

266

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

267

to disease.

40

It has been reported that dietary flavonoids

268 269

Acknowledgements

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

271

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

273

of China (Project LY12C15001).

274 275

Compliance with Ethics Requirements

276

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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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

16


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384

Figure legends

385

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

387

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

388

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

389

± SD of three replications.

390

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.

392

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

393

(B-D) of quercetin.

394

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

396

replications.

397

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

399

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

400

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

401

of quercetin.

402

17



403

Supplemental Figure 1. LC chromatograms of quercetin after incubation with

404

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

405

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

406

(A and B).

407

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

409

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

410

18


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254x190mm (96 x 96 DPI)



254x190mm (96 x 96 DPI)


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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)



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)



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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TOC graphic 85x47mm (231 x 231 DPI)



254x190mm (96 x 96 DPI)


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Quercetin Inhibits Advanced Glycation End Product Formation by

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