Influence of Quercetin and Its Methylglyoxal Adducts on the Formation

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Influence of quercetin and its methylglyoxal adducts on the formation of #-dicarbonyl compounds in lysine and glucose model system Guimei Liu, Qiuqin Xia, Yongling Lu, Tiesong Zheng, Shengmin Sang, and Lishuang Lv J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05811 • Publication Date (Web): 24 Feb 2017 Downloaded from http://pubs.acs.org on February 26, 2017

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Journal of Agricultural and Food Chemistry

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Influence of quercetin and its methylglyoxal adducts on the

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formation of α-dicarbonyl compounds in lysine and glucose model

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system

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Guimei Liu†1, Qiuqin Xia†1, Yongling Lu†, Tiesong Zheng†, Shengmin Sang‡,

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Lishuang Lv†*

6 †

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Department of Food Science and Technology, Nanjing Normal University, 122#

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Ninghai Road, Nanjing, 210097, P. R. China ‡

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Laboratory for Functional Foods and Human Health, Center for Excellence in

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Post-Harvest Technologies, North Carolina Agricultural and Technical State

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University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North

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Carolina 28081, United States

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1

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Corresponding author: Dr. Lishuang Lv

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

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Mailing address: Ninghai Road 122#, Nanjing Normal University, Nanjing, 210097,

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China

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Tel.: +86 25 83598286; fax: +86 25 83598901.

These authors contributed equally to this study.

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ACS Paragon Plus Environment


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Abstract:

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Increasing evidence has identified α-dicarbonyl compounds, the reactive intermediates

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generated during Maillard reaction, as the potential factors to cause protein glycation

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and the development of chronic diseases. Therefore, there is an urgent need to

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decrease the levels of reactive dicarbonyl compounds in foods. In this study, we

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investigated the inhibitory effect of quercetin, a major dietary flavonoid, and its major

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mono- and di-MGO adducts on the formation of dicarbonyl compounds, such as

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methylglyoxal (MGO) and glyoxal (GO) in lysine/glucose aqueous system, a model

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system to reflect the Maillard reaction in food process. Our result indicated

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that quercetin could efficiently inhibit the formation of MGO and GO in a

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time-dependent manner. Further mechanistic study was conducted by monitoring the

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formation of quercetin oxidation and conjugation products using LC/MS. Quercetin

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MGO adducts, quercetin quinones, and the quinones of quercetin MGO adducts were

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detected in the system indicating quercetin plays a due role in inhibiting the formation

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of MGO and GO by scavenging free radicals generated in the system and trapping of

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MGO and GO to form MGO adducts. In addition, we prepared the mono- and

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di-MGO quercetin adducts, and investigated their antioxidant activity and trapping

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capacity of MGO and GO. Our results indicated that both mono- and di-MGO

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quercetin adducts could scavenge DPPH radical in a dose-dependent manner with 2


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more than 40% DPPH were scavenged by the MGO adducts at 10 µM, and di-MGO

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quercetin adduct could further trap MGO to generate tri-MGO adducts. Therefore, we

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demonstrate for the first time that quercetin MGO adducts retain its antioxidant

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activity and its trapping capacity of reactive dicarbonyl species.

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Keywords: quercetin; MGO-quercetin adduct; methylglyoxal (MGO); glyoxal (GO);

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lysine/glucose system

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INTRODUCTION

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The Maillard reaction plays an important role in food processing, contributing to

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the formation of not only aromas, color, flavor, and palatability, but also advanced

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glycation end products (AGEs) when reducing sugars react with amino acids, peptides

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and protein.1 α-Dicarbonyl compounds, such as methylglyoxal (MGO) and glyoxal

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(GO), the important precursors of AGEs, are active intermediates generating under

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different conditions in the early stage of Maillard reaction and non-enzymatic

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glycation of protein.2, 3 As potent glycating agents, α-dicarbonyl compounds shown

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200–50,000-fold more reactive than glucose,4 they can lead to protein modifications,5

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cell toxicity,6 and DNA lesions.7 In vivo, α-dicarbonyl compounds caused the

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formation of irreversibly modified protein tissue and the accumulation of AGEs in

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body led to many diseases such as aging, atherosclerosis, diabetes, and Alzheimer's

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disease.8 MGO and GO are generated during food processing, such as heating,9

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frying,10 and baking,11 or food storage at room temperature.12 Due to the dietary

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exposure of α-dicarbonyl compounds and their-related health disorders, food scientists

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and pharmaceutical researchers paid more attention to investigate scavengers of

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α-dicarbonyl compounds. Recently various pharmacological compounds13 and dietary

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flavonoids14-16 have been evaluated for their inhibitory effects and mechanism.

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Quercetin, a dietary antioxidant flavonoid, exists widely in many fruits, vegetables, 4


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and grains. It exhibits a variety of pharmacological activities17 and also used as a

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supplement or food additive.18 Our previous studies proved that quercetin could

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efficiently inhibit MGO and GO via trapping MGO/GO to form mono- or

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di-MGO-quercetin adducts in C-6 or C-8 position in A- ring under physiological

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conditions19, 20 and in mice.21 Other researchers reported that flavonoid could inhibit

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the formation of α-dicarbonyl compounds by scavenging the free radical.22, 23 As is

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well known, the radical scavenging activity depends on the substituents of the

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heterocyclic (2,3- double bond in C ring) and the number of hydroxyl groups in B

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rings,24,

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important.25, 26 The mono- and di-MGO adducts of quercetin have the same B-ring

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structure as quercetin. It is still unknown whether they are as capable as quercetin to

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scavenge free radicals. It is also unclear whether they have the capacity to further trap

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reactive dicarbonyl species.

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while hydroxyl groups in A ring or 3-OH in C ring seem to be less

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In the present study, we investigated the effects of quercetin on the formation of

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MGO and GO in lysine/glucose model systems using LC/MS. The mono- and

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di-MGO adducts of quercetin were prepared. Their antioxidant activity and inhibitory

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activities on MGO formation in Maillard reaction were elucidated for the first time.

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MATERIALS AND METHODS

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Materials. Lysine and ribose were purchased from Shanghai Sangon Biological

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Engineering Technology Co., Ltd (Shanghai, China). Glucose, fructose and galactose

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were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd (Shanghai,

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China). 1,2-Diaminobenzene (DB), 2,3-butyl diketone, methylglyoxal (MGO, 40% in

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water), glyoxal (GO, 40% in water) and quercetin (98%, HPLC) were purchased from

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Sigma-Aldrich

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(NaH2PO4•2H2O), disodium hydrogen phosphate (Na2HPO4•12H2O), and other

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chemicals were of analytical grade.

(St

Louis,

MO,

USA).

Sodium

dihydrogen

phosphate

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Effect of the Factors on the Formation of MGO and GO in Lys-reducing

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Sugar Systems. In this section, we planned to determine how the type of reducing

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sugar, pH, and temperature affect the formation of MGO and GO in the model

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Maillard reaction system. The experimental conditions were selected based on method

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reported in the literature with slight modification.27 In brief, equimolar solutions of

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reducing sugar (glucose, fructose, ribose and galactose) and lysine (60 mM) were

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prepared in phosphate buffer (6 mL, 0.2 M, pH 7.4), heated at 121 °C for 0, 5, 15, 30,

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40, and 60 min in an oil bath, in screw-capped glass tubes (Schott, 16×160 mm).

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To determine the impact of pH on the formation of MGO and GO, equimolar

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solutions of glucose and lysine (60 mM) were prepared in phosphate buffer with

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different pH (6 mL, 0.2 M, pH 4.0, 6.5, 7.4 and 9.2), heated at 121 °C for 0, 5, 15, 30,

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40, and 60 min in an oil bath, in screw-capped glass tubes.

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To determine the impact of temperature on the formation of MGO and GO,

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equimolar solutions of glucose and lysine (60 mM) were prepared in phosphate buffer

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(6 mL, 0.2 M, pH 7.4), heated at 100, 121, 135, 150 and 170 °C for 0, 5, 15, 30, 40,

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and 60 min in an oil bath, in screw-capped glass tubes.

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To determine the impact of quercetin on the formation of MGO and GO, equimolar

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solutions of glucose, lysine and quercetin (100 µM) were prepared in phosphate

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buffer (6 mL, 0.2 M, pH 7.4), heated at 170 °C for 0, 5, 15, 30, 40, and 60 min in an

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oil bath, in screw-capped glass tubes. Quercetin was dissolved in DMSO and then

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diluted with phosphate buffer to reach 100 µM.

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At each time point, 1 mL sample was taken and cooled in ice water, then stored at

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-80 °C . MGO and GO levels were analyzed based on our published method.19 In brief,

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1,2-diaminobenzene was used as the derivatization agent and 2,3-butanedione was

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used as the internal standard. Samples were mixed with 1,2-diaminobenzene and kept

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at 60 °C for 10 min, and acetaldehyde was added to react with the rest of

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1,2-diaminobenzene. The mixture was extracted with methylene chloride for GC

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analysis.19

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Preparation of the Major Adducts of Quercetin. Quercetin (0.05 M, 302 mg)

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and MGO (0.5 M) were dissolved in 10.0 mL phosphate buffer (0.2 M, pH 7.4) and

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keep at 40 °C for 48 h. The reaction mixture was loaded onto a Sephadex LH-20

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column and eluted with ethanol to obtain mono-MGO adduct (MM-1, 45 mg) and di-

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MGO adduct (DM-2, 82 mg). The NMR data of mono-MGO and di- MGO adducts

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are shown in the supplementary file.

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Scavenging of MGO/GO by Quercetin and Its MGO Adducts in Lys/glucose

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System. Equimolar solutions of glucose and lysine (60 mM) were prepared in 6.0 mL

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phosphate buffer (0.2 M, pH 7.4), heated at 170 °C in an oil bath, in the presence of 0,

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2.5, 10.0, 50.0, or 100.0 µM quercetin (MM-1 or DM-2) in Lys/glucose system. Then,

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1.0 mL of sample was collected at different time points (0, 5, 15, 30, 40, and 60 min)

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and stored at -80 °C. For time-dependent effect, 100.0 µM of quercetin (MM-1 or

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DM-2) was used. For dose-dependent effect, 30 min was used. The levels of Lys,28

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glucose29 and MGO/GO19 were measured by HPLC or GC.

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LC-MS/MS Analysis. LC/MS analysis was carried out with an Agilent Masshunter

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

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G4212A Diode array detector, and a 6460 QQQ mass detector (Agilent, Santa Clara,

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CA, USA) incorporated with an electrospray ionization (ESI) interface. A 250 × 4.6

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mm i.d., 5 µm ZORBAX Eclipse XDB-C18 column (Agilent, Santa Clara, CA, USA) 8


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was used for separation at a flow rate of 0.6 mL/min. The mobile phase consisted of

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water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid

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(mobile phase B). The gradient was initiated at 10 % B and held constant for 5 min,

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followed by a linear increase to 70% from 5 to 40 min and 90% from 40 to 50 min.

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The column was then re-equilibrated with 10% B for 5 min. The LC eluent was

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introduced into the ESI interface. The negative ion polarity mode was set for ESI ion

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source with the voltage on the ESI interface maintained at approximately 5 kV.

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Nitrogen gas was used as the sheath gas at a flow rate of 45 arb units and the auxiliary

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gas at 5 arb units, respectively. The structural information of quercetin, the major

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MGO adducts of quercetin, and the major MGO adducts of DM-2 was obtained by

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tandem mass spectrometry (MS/MS) through collision-induced dissociation (CID)

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

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

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Effect of MM-1 and DM-2 on Scavenging DPPH Radical. Different

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concentrations of quercetin, MM-1 and DM-2 (0, 0.01, 0.05, 0.1, and 0.5 mM) were

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mixed with DPPH (3.0 mL, 2 × 10-4 mM) in methanol, respectively. Absorbance at

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517 nm was determined after incubation in the dark at room temperature for 30 min.

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All determinations were performed in triplicate.

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Effect of DM-2 on Trapping MGO. Equimolar solutions of DM-2 and MGO (0.5 9



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mM) were prepared in 4.0 mL phosphate buffer (0.2 M, pH 7.4), incubated at 100 °C

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for 5 min. The samples were than stored at -80 °C for LC/MS analysis.

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Statistical analysis. Data are expressed as mean ± standard deviation. Statistical

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analyses were performed using the GraphPad Prism. Significant differences among

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treatments were compared using Tukey's test. Each sample was performed in triplicate.

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p < 0.05 were considered significant.

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RESULTS AND DISCUSSION

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Effect of the Factors on the Formation of MGO and GO. Our results indicated

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that all four reducing sugars could react with lysine to generate MGO and GO with

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glucose being the most reactive one (Figure 1A). The amount of MGO and GO were

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all increased in a time-dependent manner with the level of MGO was about two folds

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higher than that of GO in the same system. At 0-30 min, the amount of MGO

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increased sharply.

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Then we selected glucose to further study the impact of pH and temperature on the

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formation of MGO and GO. There was no MGO and GO detected in pH 4.0 in the

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Lys-glucose model system (data not shown). The amount of MGO and GO increased

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sharply from pH 6.5 to 9.2 (p < 0.05) (Figure 1B). As the pH increased from 7.4 to

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9.2, there were about a 5-fold increase of MGO and a 4.5-fold increase of GO after 30

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min incubation of lysine and glucose (Figure 1B). 10


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With increasing temperature from 100 to 170 °C, the level of MGO and GO

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significantly elevated in Lys-glucose system (p < 0.05) (Figure 1C and 1D).

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100 °C, the amount of MGO (0.143 mM) was 5-fold greater than that of GO (0.024

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mM), while there were similar amount of MGO (0.42 mM) and GO (0.39 mM) at

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170 °C.

At

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In view of the above results and considering the conditions used in most of the food

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processing, we chose the Lys-glucose system, pH 7.4, and 170 °C to study the effects

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of quercetin and its MGO adducts on the formation of MGO and GO.

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Effect of Quercetin on the Formation of MGO and GO.

As shown in Figure 2,

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both MGO and GO were rapidly generated during the first 30 mins, and quercetin

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significantly decreased the formation of MGO and GO in a time-dependent manner.

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At 30 min, more than half of the MGO and GO generated in this system were

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diminished by quercetin. In addition, it is interesting to observe that the amount of

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lysine decreased more rapidly than that of glucose even equimolar lysine and glucose

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were used in this system and lysine has two reactive sites for glycation, the

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alpha-amino and the epsilon-amino group. This may due to the generated MGO and

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GO would react with lysine.

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In order to explicate the pathway of eliminating MGO by quercetin in Lys-glucose

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system, LC-MS/MS was used to determine the products of quercetin. As shown in 11



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Figure 3A, two new peaks (QQ-A: RT 25.90 min and QQ-B: RT 28.05 min) were

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appeared after 5 min of incubation of quercetin in Lys-glucose system. The peak at

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25.90 min had the molecular ion m/z 300 [M - H] -, which was one mass unit lower

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than that of quercetin (m/z 301) indicating it was a quercetin semi-quinone (the

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oxidation product of quercetin). The peak at 28.05 min had the molecular ion m/z 299

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[M - H] -, which was 2 mass units lower than that of quercetin (m/z 301) indicating

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that this peak was the ortho-quinone quercetin.30, 31

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As the incubation proceeds to 10 min, there were three additional new peaks

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(MM-1, MM-2, and MMQ-1) appeared (Figure 3B). MM-1 and MM-2 had the same

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molecular ion (373 [M-H] -), which was 72 mass units higher than that of quercetin

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(m/z 301), and similar MS/MS fragments (Table 1) but different retention times (RT,

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23.66 and 23.90 min), suggesting that these products were mono-MGO conjugated

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quercetin. The other new peak (MMQ-1) had molecular ion m/z 371 [M - H] -, which

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was 2 mass units lower than that of mono-MGO conjugated quercetin (m/z 373)

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indicating that this peak is the ortho-quinone of the mono-MGO conjugated quercetin.

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When quercetin was incubated in Lys-glucose system for 30 min, a number of

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new peaks were observed (Figure 3C). Besides the intensity of peaks MM-1 and

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MM-2 increased, a group of new peaks (DM-1, DM-2, DM-3 and DM-4) appeared at

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20.30, 21.15, 21.55 and 22.08 min. These peaks had the molecular ion m/z 445 [M − 12


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H] −, which was 144 mass units higher than that of quercetin (m/z 301), indicating

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they were the di-MGO adducts of quercetin. The appearance of these peaks suggested

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that quercetin suppressed the levels of MGO through trapping MGO to form adducts

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of mono-MGO and di-MGO, which is in accordance with our previous study.19 In

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addition, the intensity of MMQ-1 increased and two new peaks MMQ-2 and MMQ-3

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appeared. Both MMQ-2 and -3 had the molecular ion m/z 371 [M - H] -, which was

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the same as MMQ-1, indicating that they were mono-MGO-quercetin-quinones. This

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is further supported by the observation that all three mono-MGO adducts (MMQ-1,

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MMQ-2, MMQ-3) had the same fragment pattern to generate the daughter ion m/z

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221 [M − 150] – 19, 32, 33 (Table 1). Furthermore, a new peak DMQ-A1 (RT 25.20 min)

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had the molecular ion m/z 443 [M - H] -, which was 2 mass units lower than that of

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di-MGO-quercetin adduct (m/z 445), suggesting this new product was the

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di-MGO-quercetin-quinone. Another new peak DMQ-B1 (25.70 min) had the

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molecular ion m/z 441 [M - H] -, which was 2 mass units lower than that of DMQ-A1

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(m/z 443), suggesting it was a further oxidation product of DMQ-A1. Additionally, a

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small peak TM-1 was observed at 19.26 min, that peak had the molecular ion m/z 517

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[M - H] -, which was 216 mass units higher than that of quercetin (m/z 301),

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suggesting it was a tri-MGO-quercetin adduct. This result indicated that the

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di-MGO-quercetin adduct could have the capacity to further trap MGO. 13



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In this study, a mono-GO-quercetin-quionone was detected at 26.14 min (MGQ-1).

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It had the molecular ion m/z 357 [M - H] -, which was 58 (MW of GO) mass units

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higher than that of quercetin quinone (m/z 299), indicating that it was a

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mono-GO-quercetin-quinone.

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Effect of Quercetin, MM-1 and DM-2 on Scavenging DPPH Radical. We have

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purified the major mono- and di-MGO adducts of quercetin, MM-1 and DM-2

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(Supplementary data). It has been reported that the B-ring of quercetin is the active

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site of its antioxidant activity to scavenging free radicals.24, 25, 34 Since both MM-1 and

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DM-2 have the same B-ring structure as that of quercetin, we hypothesized that both

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MM-1 and DM-2 have the capacity of scavenging free radicals. To test this

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hypothesis, we used DPPH assay as an example to compare the antioxidant activities

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of MM-1, DM-2, and quercetin. Our results indicated that both MM-1 and DM-2

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could scavenge DPPH radical in a dose-dependent manner (Figure 4). More than 40%

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DPPH were scavenged by MM-1 and DM-2 at 10 µM. They showed almost identical

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scavenging activity to that of quercetin at 0.5 mM concentration with up to 90%

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DPPH radical was scavenged by all three compounds.

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Inhibitory Effects of MM-1 and DM-2 on the Formation of MGO/ and GO. As

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shown in Figure 5, both MM-1 and DM-2 decreased the levels of MGO and GO in a

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time- (Figure 5A and 5B) and dose-dependent manner (Figure 5C) in the 14


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lysine/glucose model systems. There were up to 42.4, 46.0, and 50.1% of MGO

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(Figure 5A), 45.6, 54.4, and 59.3% of GO (Figure 5B) were eliminated respectively

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by DM-2, MM-1 and quercetin after 30 min incubation. It suggested that MM-1 or

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DM-2 still have the capacity to suppress the formation of MGO and GO.

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To further understand how mono- and di-MGO adducts of quercetin inhibit the

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formation of MGO and GO, using DM-2 as an example, we analyzed the products of

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DM-2 generated during the incubation of DM-2 with MGO using LC/MS. As shown

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in Figure 6, Three new peaks (DMQ-A2, DMQ-A3, and DMQ-B2) appeared at 22.77,

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23.80, and 23.41 min. DMQ-A2 and DMQ-A3 had the molecular ion m/z 443 [M -

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H]-, which was 2 mass units lower than that of DM-2 (m/z 445), suggesting they were

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the quinones of DM-2. DMQ-B2 had the molecular ion m/z 441 [M - H] -, which was

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2 mass units lower than that of DMQ-A2 or -A3 (m/z 443), suggesting it was a further

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oxidation product of DMQ-A2 or -A3. In addition, two tri-MGO-quercetin adducts,

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TM-1 and TM-2, were generated. TM-2 (RT 20.29 min) had the same molecular ion

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m/z 517 [M - H]- as that of TM-1, which was 216 mass units higher than that of

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quercetin (m/z 301) and both peaks had similar MS/MS fragments 373/445 (MS2)

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(Figure 6), suggesting they were tri-MGO-quercetin adducts. Thus, our data

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demonstrated for the first time that quercetin could still trap MGO in the position of

15



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B-ring to form tri-MGO adducts when the positions of the A-ring were occupied by

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MGO.

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In conclusion, this study reported that quercetin could efficiently inhibit the

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formation of MGO and GO in lysine/glucose model systems, indicating it can be used

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in food system to lower the levels of reactive dicarbonyl species. Our results further

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supported the literature finding that quercetin showed inhibition against formation of

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both reactive carbonyl species and total fluorescent AGEs in a cookie model.35 One

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potential limitation of adding quercetin to food system is it may affect the color of

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foods fortified with quercetin, which depends on the amount of quercetin added to the

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food system. A more recent study reported that the addition of quercetin increased the

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color of biscuits.36

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The observation of the formation of quinones of quercetin and its MGO adducts

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suggests that quercetin may serve as an antioxidant to scavenge free radicals

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generated during Maillard reaction. The detection of the MGO and GO adducts of

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quercetin indicates that quercetin could inhibit the formation of reactive dicarbonyl

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species via trapping them during food processing. Furthermore, this is the first study

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to demonstrate that the MGO adducts of quercetin retain quercetin’s antioxidant

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activity and trapping capacity of reactive dicarbonyl species. This is also the first

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report to show that the B-ring of quercetin has the capacity to trap MGO. Due to the 16


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instability of the quinones of quercetin and its MGO adducts and the tri-MGO adducts

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of quercetin, we failed to purify them and confirm their structures using NMR. It is

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worthwhile to apply quercetin as well as other dietary flavonoids into processed foods

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to inhibit the formation of toxic dicarbonyl compounds and AGEs.

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ABBREVIATIONS USED

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MGO, methylglyoxal; GO, glyoxal; MM, mono-MGO-quercetin adduct; DM,

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di-MGO-quercetin adduct; TM, tri-MGO-quercetin adduct; MG, Mono-GO-querctin

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adduct; MMQ, mono-MGO-quercetin quinone; DMQ, di-MGO-quercetin quinone;

300

MGQ, mono-MGO-quercetin quinone.

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FUNDING SOURCES. This work was supported by National Natural Science

302

Foundation of China (Grant No. 31571783)

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CONFLICT OF INTEREST

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There is no conflict of interest of all authors.

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406

Figure legends

407

Figure 1. Effect of the type of reducing sugar, pH, and temperature on the formation

408

of MGO and GO in Lysine-reducing sugar systems. (A) Impact of the type of

409

reducing sugar on the formation of MGO and GO. Lysine (60 mM) and reducing

410

sugar (glucose, fructose, ribose, and galactose, 60 mM) was incubated at 121 °C in

411

phosphate buffer (6 mL, 0.2 M, pH 7.4) for 0, 5, 15, 30, 40, and 60 min, respectively.

412

(B) Effect of pH on the formation of MGO and GO. Lysine (60 mM) and glucose

413

were incubated at 121 °C in phosphate buffer with different pH (6 mL, 0.2 M, pH 4.0,

414

6.5, 7.4, and 9.2) for 0, 5, 15, 30, 40, and 60 min, respectively. (C and D) Effect of

415

temperature on the formation of MGO (C) and GO (D). Lysine (60 mM) and glucose

416

was incubated at different temperatures (100, 121, 135, 150 and 170 °C) for 0, 5, 15,

417

30, 40 and 60 min, respectively.

418

Figure 2. Inhibitory effects of quercetin on the formation of MGO/GO in

419

Lysine-glucose system. Lysine (Lys) (60 mM) incubated with glucose (60 mM) in

420

phosphate buffer (0.2 M, pH 7.4) at 170 °C in the presence or absence of quercetin

421

(100.0 µM).

422

Figure 3. Total ion chromatograms of the oxidation and conjugation products of

423

quercetin (100.0 µM) after incubating with lysine (Lys) and glucose (60 mM, 1:1) for

424

5 (A), 10 (B), and 30 (C) min at pH 7.4, 170 °C. 20


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425

Figure 4. Effect of quercetin, mono-MGO (MM)- and di-MGO (DM) quercetin

426

adducts (10.0, 50.0, 100.0, and 500.0 µM) on scavenging DPPH radical at room

427

temperature.

428

Figure 5. Mono-MGO (MM)-, and di-MGO (DM) quercetin adducts and quercetin

429

inhibited the formation of MGO and GO in a time- (A and B) and dose-dependent (C)

430

manner.

431

Figure 6. Total ion chromatogram of di-MGO (DM) quercetin adduct incubated with

432

MGO (1:1) for 5 min at 100 °C.

433

21



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434

Table 1. ESI-MS and ESI-MS2 fragment ions of the quercetin, its quinone, and their

435

corresponding MGO adducts* Time (min)

5

10

30

Compound

RT (min)

MW

MS1 (m/z)

MS2 (m/z)

QQ-A

25.90

301

300[M-H]-

/

QQ-B

28.05

300

299[M-H]-

271/226/147

Quercetin

27.41

302

301[M-H]-

273/227/149

MM-1

23.66

374

373[M-H]-

301/255/233/205

MM-2

23.90

374

373[M-H]-

327/301/223/205/179

QQ-A

25.99

301

300[M-H]-

/

QQ-B

28.21

300

299[M-H]-

271/226/147

MMQ-1

29.09

372

371[M-H]-

325/301/221/177

MM-1

23.13

374

373[M-H]-

301/255/223/205

MM-2

23.71

374

373[M-H]-

327/301/223/205/179

DM-1

20.30

446

445[M-H]-

417/355/301

DM-2

21.14

446

445[M-H]-

427/373/357/301/277/251

DM-3

21.55

446

445[M-H]-

327/301/277/205

DM-4

22.08

446

445[M-H]-

357/301/251/151

TM-1

19.25

518

517[M-H]-

445/427/373

MMQ-1

28.94

372

371[M-H]-

325/301/221/177

MMQ-2

25.90

372

371[M-H]-

301/221/177

MMQ-3

28.50

372

371[M-H]-

271/221/177

DMQ-A1

25.20

444

443[M-H]-

425/371/355/327/275

DMQ-B1

25.70

442

441[M-H]-

423/371/343/327/273/247

MGQ-1

26.13

358

357[M-H]-

207/149

436

* Abbreviation used: QQ, quercetin quinone; MM, mono-MGO-quercetin adduct; DM,

437

di-MGO-quercetin

438

mono-MGO-quercetin

439

mono-GO-quercetin quinone.

adduct; quinone;

TM,

tri-MGO-quercetin

DMQ,

di-MGO-quercetin

22


adduct;

MMQ,

quinone;

MGQ,

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440

Figure 1

441

442 443

23



444

Figure 2

445

24


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446

Figure 3

447 448

25



449 450

Figure 4

451 452

26


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453

Figure 5

454

27



455

Figure 6

456 457

28


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458

Graphic Abstract

459

29


Influence of Quercetin and Its Methylglyoxal Adducts on the Formation

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