Additive Capacity of [6]-Shogaol and Epicatechin To Trap

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Additive Capacity of [6]-Shogaol and Epicatechin to Trap Methylglyoxal Qiju Huang, Pei Wang, Yingdong Zhu, Lishuang Lv, and Shengmin Sang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02917 • Publication Date (Web): 03 Sep 2017 Downloaded from http://pubs.acs.org on September 3, 2017

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Journal of Agricultural and Food Chemistry

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Additive Capacity of [6]-Shogaol and Epicatechin to Trap Methylglyoxal

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Qiju Huang†, ‡, Pei Wang‡, Yingdong Zhu‡, Lishuang Lv†, *, and Shengmin Sang‡, *

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†

Department of Food Science and Technology, Nanjing Normal University, 122# 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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*Corresponding Author: (Tel: 704-250-5710; Fax: 704-250-5729; E-mail:

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[email protected])

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Or Alternate Corresponding Author: (Tel.: +86 25 83598286; Fax: +86 25

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83598901; E-mail: [email protected])

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ACS Paragon Plus Environment


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Abstract

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Methylglyoxal (MGO), a reactive dicarbonyl species, is thought to contribute to

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the development of long-term pathological diabetes as a direct toxin or as an active

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precursor of advanced glycation end products (AGEs). Trapping MGO by dietary

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phenols to inhibit the MGO induced AGE formation is an approach for alleviating

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diabetic complications. The present study investigated whether dietary compounds

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with different structures and active sites have the additive capacity to trap MGO.

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Ginger phenolic constituent [6]-shogaol and tea flavonoid (-)-epicatechin were

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selected and tested under simulated physiological conditions, showing that they

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additively trapped about 41% MGO at a concentration of 10 µM within 24 h.

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Furthermore, whether [6]-shogaol and epicatechin can retain their MGO trapping

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efficacy in vivo or a biotransformation limits their MGO trapping capacity remain

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virtually unknown. An acute mouse study was carried out by giving a single dose of

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[6]-shogaol, epicatechin, and the combination of both ([6]-shogaol + epicatechin)

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through oral gavage. A mono-MGO adduct of [6]-shogaol was identified from

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[6]-shogaol and [6]-shogaol + epicatechin treated mice, and mono- and di-MGO

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adducts of epicatechin and its metabolite, 3'-O-methyl epicatichin, were detected in

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urine samples collected from epicatechin and [6]-shogaol + epicatechin treated mice.

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To our knowledge, this is the first study demonstrating the additive MGO trapping

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efficacy of [6]-shogaol and epicatechin, and that [6]-shogaol and epicatechin

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retained their MGO trapping capacity in mice.

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KEYWORDS: [6]-Shogaol; Epicatechin; Methylglyoxal; Trapping; Additive effect

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INTRODUCTON

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Increasing evidence indicates that the accumulation of advanced glycation end

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products (AGEs) are closely linked to the pathogenic pathways between

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hyperglycemia and diabetes related complications.1-3 AGEs are a complex and

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heterogeneous group of compounds derived from nonenzymatic glycation between

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reducing sugars and amino residues of proteins, lipids, and nucleic acids. 4, 5 Among

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the precursors of AGEs, methylglyoxal (MGO) is regarded as a major and highly

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reactive α,β-dicarbonyl intermediate in the glycation process, contributing to the

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formation of both intracellular and extracellular MGO-derived AGEs, such as

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argpyrimidine, N ε -(carboxyethyl)lysine, MGO lysine dimer, and MGO-derived

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hydroimidazolone.6-9

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Scavenging MGO and preventing AGE formation have been investigated as

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therapeutic strategies to attenuate MGO-related diabetes mellitus.10 Several

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pharmacological

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monascin,18, 19 and metformin,20-24 have been reported to inhibit the formation of

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AGEs and prevent the deterioration of diabetic complications by scavenging reactive

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dicarbonyl species, mainly MGO. However, these pharmacological reagents can

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impose adverse side effect on diabetes patients, which makes it necessary to exploit

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effective and safe agents to prevent diabetic complications.

reagents,

such

as

aminoguanidine,11-13

pyridoxamine,14-17

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Discovery of MGO scavenging reagents from natural foods and beverages for

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reasons of safety seems feasible, and indeed, many dietary plants and their

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constituents have been described to have the capacity to alleviate MGO-induced



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glycation, mainly due to their MGO-scavenging and antioxidants properties. In our

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previous studies of a number of compounds, including soy isoflavones,25,

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catechins and theaflavins,27, 28 apple polyphenols,29 ginger gingerols and shogaols,30

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have been tested and determined to have significant MGO scavenging capacities in

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in vitro and/or in vivo models. Recently, our structure-activity relationship (SAR)

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study revealed that the A ring is the critical active site in flavonoids that contributes

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to their MGO trapping efficacy.31 Furthermore, we investigated the potential additive

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effects of different flavonoids under simulated physiological conditions, in which we

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found that phloretin and quercetin can additively trap MGO through the same

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mechanism.31

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tea

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In contrast to the flavonoids, the MGO trapping mechanisms of ginger

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phenolics were different. Trapping of MGO by ginger phenolics ([6]-shogaol and

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[6]-gingerol) involves a classic aldol condensation reaction because of the active

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protons of the α-carbonyl group in their side chains.30 This fact naturally leads to the

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hypothesis that different types of components can additively trap MGO. The daily

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intake of individual dietary phenols may not be high enough to significantly decrease

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the levels of reactive MGO in humans. However, the total intake of phenols from a

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variety of foods can reach the effective doses to trap a significant amount of

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exogenous and endogenous reactive dicarbonyls. In the present study, we

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investigated the additive effects of trapping MGO by ginger phenolic constituent

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[6]-shogaol and tea flavonoid (-)-epicatechin (Figure 1) under an in vitro system.

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Additionally, whether [6]-shogaol, epicatechin, and their major metabolites


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preserved their MGO trapping efficacy was also investigated in an in vivo model.

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MATERIALS AND METHODS

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Materials. [6]-shogaol was purified from ginger root extract according to our

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previous published methods.32 (-)-Epicatechin was purchased from Tokyo Food

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Techno Co., Ltd. (Shizuoka, Japan). MGO (40% in water), DMSO, and

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1,2-diaminobenzene (OPD) were purchased from Sigma (St. Louis, MO). LC-MS

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grade solvents and other reagents were obtained from Thermo Fisher Scientific

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(Waltham, MA).

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Trapping of MGO by [6]-shogaol, epicatechin, and the combination of

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[6]-shogaol and epicatechin. MGO (60 µM) was incubated with [6]-shogaol (10

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µM) and epicatechin (10 µM), or [6]-shogaol + epicatechin (10 µM each) in 100 mM

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potassium phosphate buffer (PBS), pH 7.4, and shaken for 4 and 24 h at 37 °C. Then,

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the reacted mixture

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The solution incubated for 24 h was used for derivatization of the remaining MGO

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by adding 50 mM OPD based on our previous method,27 and the solution incubated

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for 4 h was used for LC-MS analysis.

(500 µL) was added 2 µL of acetic acid to stop the reaction.

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Animal Studies. All mouse experiments conformed to a protocol approved by

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the Institutional Animal Care and Use Committee of the North Carolina Research

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Campus (No: 16-016). Female CF-1 mice were purchased from Charles River

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Laboratories, Inc. (Wilmington, MA) and acclimated for at least 1 week before being

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randomly allocated to different experimental groups. The mice were housed (5



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mice/cage) and kept in air-conditioned quarters with a room temperature of 20 ±

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2 °C, relative humidity of 50 ± 10%, and a light−dark cycle of 12:12 h (7 am to 7

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pm). The mice were allowed free access to water and were fed an AIN-93G diet.

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Twenty mice were divided into a control group and three MGO and

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compound-treated groups (5 mice/group), including MGO and [6]-shogaol, MGO

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and epicatechin, and MGO and [6]-shogaol + epicatechin treated groups, which were

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kept in metabolic cages. For the MGO and compound-treated groups, each mouse

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received a single dose of [6]-shogaol, epicatechin, or a combination of [6]-shogaol

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(200 mg/kg body weight in DMSO) and epicatechin (200 mg/kg body weight in

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DMSO) via oral gavage. Ten min later, MGO solution (1.0 g/kg body weight in

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water) was administrated to the mice via oral gavage. The mice in the control group

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received DMSO followed by water. Twenty-four hour mouse urine and fecal samples

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were collected and stored at −80 °C before analysis.

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Sample preparation.

For the preparation of the urine samples, enzymatic

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deconjugation of the samples was processed as described previously.25, 33 Briefly,

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200 µL urine sample from each group was treated with β-glucuronidase (250 U) and

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sulfatase (3 U) for 3 h at 37 °C and extracted three times with ethyl acetate. The

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ethyl acetate fractions were combined and dried under a gentle stream of nitrogen

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gas and then reconstituted with 200 µL of 80% aqueous methanol with 0.1% acetic

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acid for further analysis. For the preparation of the fecal samples, 100 mg feces from

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each group was mixed with 1 mL of 80% aqueous methanol with 0.1% acetic acid,

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then samples were homogenized for 90 s by a Bead Ruptor Homogenizer (Omni


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International, Kennesaw, GA, USA) and then centrifuged at 14,000 rcf for 10 min.

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The supernatant (200 µL) was collected and diluted 10 times for analysis.

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LC-MS Analysis. LC-MS analysis was performed using a Spectra system

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consisting of an Ultimate 3000 degasser, an Ultimate 3000 RS pump, an Ultimate

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3000 RS autosampler, an Ultimate 3000 RS column compartment, and an LTQ Velos

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Pro ion trap mass spectrometer (Thermo Electron, San Jose, CA, USA) equipped

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with an electrospray ionization (ESI) interface. Chromatographic separation was

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performed using a 150 mm × 3.0 mm i.d., 5 µm, Gemini C18 column,

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(Phenomenex, Torrance, CA). The mobile phase consisted of 5% aqueous methanol

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with 0.1% formic acid (mobile phase A) and 95% aqueous methanol with 0.1%

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formic acid (mobile phase B). For the determination of the level of remaining MGO,

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the gradient elution was performed for 15 min at a flow rate of 0.2 mL/min using the

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following gradient: 0 to 2.5 min, 50-90% B, 2.5 to 6 min 90-100% B, and 6 to 12

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min held at 100% from, and then the column was re-equilibrated with 50% B for 3

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min. The analysis of [6]-shogaol and its main MGO adduct was identical to our

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previous study.30 For the analysis of epicatechin and its main MGO adducts, the

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gradient was initiated at 10% B and held constant for 5 min, followed by a linear

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increase to 55% from 5 to 25 min, to 100% from 25 to 30 min, and then held for 5

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min. The column was then re-equilibrated with 10% B for 5 min, the flow rate was

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increased to 0.3 mL/min and the injection volume was 10 µL. The negative ion

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polarity mode was set for an ESI ion source with the voltage on the ESI interface

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maintained at approximately 3.6 kV. Nitrogen gas was used as the sheath gas at a



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flow rate of 34 AU and the auxiliary gas at 10 AU. The collision-induced

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dissociation was conducted with an isolation width of 1.0 Da and a normalized

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collision energy of 35 for MS/MS analysis. Data were acquired with Xcalibur

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version 2.0 (Thermo Electron)

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RESULTS AND DISCUSSION Additive MGO-Trapping Effects by [6]-shogaol and epicatechin. Our

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previous investigations demonstrated that different kinds of natural products, such as

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flavonoids including (-)-epigallocatechin 3-gallate, genistein, phloretin, and

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quercetin, or [6]-shogaol and [6]-gingerol derived from ginger can effectively trap

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MGO under physiological conditions (pH 7.4 and 37 °C). Given that these natural

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products usually coexist in our diet like vegetables and fruits, it is necessary to

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evaluate the additive MGO-trapping effect of them. Our laboratory found that

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different flavonoids (phloretein and quercetin) could additively trap MGO, which

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was attributed to the A ring of flavonoids; MGO can react with the two unsubstituted

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carbons in the A ring to form mono- and di-MGO adducts.34 However, there have

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been no studies reporting the additive effect of dietary compounds with different

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active sites in trapping MGO. Therefore, this study focused on the additive effect of

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[6]-shogaol and epicatechin. The formation of MGO adducts of [6]-shogaol is

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believed to involve a classic aldol condensation, due to the α-carbonyl group in its

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structure,35 while epicatechin is a kind of flavonoid that C-6 and C-8 of its A ring can

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be conjugated with MGO.31


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MGO trapping capability of [6]-shogaol, epicatechin, and [6]-shogaol +

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epicatechin was investigated at a concentration of 10 µM with a ratio between each

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compound and MGO at 1:6 under pH 7.4 at 37 °C. Our results indicated that

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[6]-shogaol and epicatechin trapped 11 and 30% MGO, respectively, and 41% MGO

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was trapped by [6]-shogaol + epicatechin over 24 h (Figure 2). Our results indicated

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that ginger shogaol [6]-shogaol and tea flavonoid epicatechin, which had different

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MGO trapping mechanisms, can additively trap MGO.

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Formation of MGO adducts of [6]-shogaol and epicatechin in physiological To determine the underlying mechanism that [6]-shogaol and

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condition.

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epicatechin additively inhibit MGO, the adducts formed in the [6]-shogaol,

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epicatechin, or [6]-shogaol + epicatechin treated reaction were investigated using

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LC-MS analysis in selective ion monitoring (SIM) mode (Figure 3). The reaction

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mixtures of [6]-shogaol and [6]-shogaol + epicatechin with MGO at different ratios

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(1:6 and 1:1:6, respectively) were collected after being incubated for 4 h. Figure 3A

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showed one major peak (tR = 10.2 min, m/z 349 [M+H]+) in both mixtures, the

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retention time and MS/MS spectrum of this peak were identical to that of the

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authentic [6]-shogaol-MGO that we synthesized and purified from the reaction

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mixture of [6]-shogaol and MGO,30 demonstrating that the formation of

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[6]-shogaol-MGO (Figure 3A) in [6]-shogaol and epicatechin + [6]-shogaol treated

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mixtures underwent the same mechanism and formed the same adduct.

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With the same ratios, the reaction mixtures of epicatechin and [6]-shogaol +

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epicatechin with MGO were also collected after 4 h of incubation. As indicated in



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Figure 3B and C, three major peaks (tR = 12.0, 13.0, and 14.9 min) presented 72 Da

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(one molecule MGO) higher than that of epicatechin (m/z 289 [M-H]-), indicating

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that these peaks were mono MGO adducts of epicatechin (Figure 3B), while the

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peaks with the molecular ion at m/z 433 [M-H]- were 144 Da (two molecules of

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MGO) higher than that of epicatechin, which demonstrated that these peaks were

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di-MGO adducts of epicatechin (Figure 3B). In addition, the retention times and

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MS/MS spectrum of those peaks in epicatechin and [6]-shogaol + epicatechin treated

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mixtures were almost identical. All the evidence above further verified that mono

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and di-MGO adducts of epicatechin were produced in both mixtures.

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Identification of MGO adducts of [6]-shogaol and epicatechin in mice. The

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in vivo environment is quite different from in vitro, and many factors may influence

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this trapping reaction, such as pH, oxygen, pressure, and the presence of other

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endogenous and exogenous compounds. Thus, whether [6]-shogaol, epicatechin, or

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their combination could trap MGO in vivo remained unknown. To answer this

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question, we analyzed the formation of mono- and di-MGO adducts of [6]-shogaol

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and epicatechin in urine and fecal samples collected from [6]-shogaol, epicatechin,

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and [6]-shogaol plus epicatechin treated mice under the same LC-MS conditions

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used in our in vitro studies.

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In the SIM chromatogram of m/z 349 [M+H]+ (molecular ion peak of the

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mono-MGO adduct of [6]-shogaol), consistent with our in vitro study, only one peak

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(tR = 10.2 min) was detected in the mouse fecal samples collected from the acute

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study (Figure 4A). This peak shared the same retention time and MS/MS spectrum


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with those of the authentic mono-MGO-[6]-shogaol, which established that this peak

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is the mono-MGO adduct of [6]-shogaol. The mono- and di-MGO adducts of

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epicatechin were detected in the urine samples collected in the acute study (Figure

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4B and C). Both in epicatechin and [6]-shogaol + epicatechin treated mouse urine,

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three peaks (tR = 11.7, 12.8 and 14.7 min) were observed correspond to the

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mono-MGO adducts of epicatechin (molecular ion peak at m/z 361 [M-H]-), all

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peaks were 72 Da higher than that of epicatechin (m/z 289 [M-H]-), which indicated

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that they were mono-MGO adducts of epicatechin. This finding was further

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confirmed by the observation that these peaks included a fragment ion that lost one

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MGO unit (m/z 72), and the tandem

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(m/z 289 (MS3 289/361)) was almost identical to that of epicatechin (MS2 289)

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(Figure 4B). When selecting m/z 433 as monitoring ion, seven peaks with the same

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molecular weight with different retention times were detected as shown in Figure 4B

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and C, those peaks also shared similar tandem mass spectra, including a fragment

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ion at m/z 361 (MS3 361/433) which was identical to that of mono-MGO-epicatechin

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(MS2 361) (Figure 4C), showing that these peaks were di-MGO adducts of

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epicatechin, which was further supported by a fragment ion (m/z 289 (MS4

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289/361/433)) lost two MGO units (m/z 144) and was nearly coincident to that of

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epicatechin (MS2 289) (Figure 4B and C).

mass spectrum

of

this fragment ion

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Identification of the metabolites of [6]-shogaol and epicatechin, and their

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MGO adducts in mice. Since both [6]-shogaol and epicatechin are extensively

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metabolized in mice, we searched the metabolites of [6]-shogaol and epicatechin,



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and the MGO adducts of these metabolites in mouse urine samples. We did not find

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mono- and/or di-MGO adducts of [6]-shogaol metabolites in mouse urine, or at least

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their quantities were not sufficient enough to be detected under our LC-MS

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conditions. Therefore, whether the metabolites of [6]-shogaol can trap the MGO

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effectively in vivo requires future research.

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Both in epicatechin and [6]-shogaol + epicatechin treated mouse urine samples,

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we observed two peaks (m/z 303 [M-H]-) that were 14 mass units higher than that of

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epicatechin (m/z 289 [M-H]-), which preliminarily indicated that they were

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methylated metabolites of epicatechin (Figure 5). Previous studies reported that the

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retention time of 3'-O-methyl epicatechin was prior to that of 4'-O-methyl

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epicatechin (with a 4:1 ratio) in the synthesis of 3'-O-methyl epicatechhin using

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catechol-O-methyl- transferase (COMT) (Figure 1B).36 Therefore, we identified that

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the major peak (tR = 19.2 min) as 3'-O-methyl epicatechin, while the minor peak (tR

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= 20.5 min) was 4'-O-methyl epicatechin. Both peaks had similar tandem mass

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spectra of fragment ions.

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It was noticeable that the methylation occurred on the B-ring of epicatechin,

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and C-6 and C-8 of A ring are the active sites to trap MGO, so the trapping efficacy

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of epicatechin metabolites should be preserved. To investigate whether metabolites

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of epicatechin still have the capability of trapping MGO, epicatechin, and

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[6]-shogaol + epicatechin treated mouse urine samples were analyzed using LC-MS.

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In the SIM chromatogram of m/z 375 [M-H]- (molecular ion peak of the mono-MGO

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adduct of 3’-O-methyl epicatechin), three peaks (tR = 15.2, 15.9, and 17.3 min) were


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observed with a fragment ion of m/z 303. The tandem mass spectrum with a

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fragment ion (MS3 303/375) was almost identical to that of 3'-O-methyl epicatechin

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(Figure 6), which demonstrated that these three peaks were the mono-MGO adducts

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of 3'-O-methyl epicatechin.

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According to a similar approach, di-MGO adducts of 3'-methyl epicatechin

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were also confirmed by observing the fragment ions that lost one MGO molecular

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(m/z 375 (MS3 375/477)) and two MGO molecules (m/z 303 (MS4 303/375/447))

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which correspondended to those of mono-MGO-3'-methyl epicatechin and 3'-methyl

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epicatechin, respectively (Figure 6). Our results clearly demonstrated that the

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metabolites of epicatechin still possess the MGO-trapping efficacy.

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In summary, this investigation revealed the additive MGO-trapping effect of

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dietary compounds with different structures, which provides meaningful suggestions

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about a healthy diet for people to prevent the development of diabetic complications.

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Additionally, this is the first report to illustrate that epicatechin and its metabolites

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are still competent to scavenge MGO in vivo, this discovery was consistent with our

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in vitro results and further confirmed our previous findings that the A ring of

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flavonoids is the active site mainly responsible for trapping reactive dicarbonyl

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species.

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ACKNOWLEDGEMENT

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The authors are grateful for financial supports from National Natural Science

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Foundation of China grant 31571783 to L. Lv. and USDA-NIFA grant



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2012-67017-30175 to S. Sang.


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C.; Gonçalves, L.; Seiça, R., Pyridoxamine Reverts Methylglyoxal‐induced Impairment of Survival Pathways During Heart Ischemia. Cardiovascular therapeutics 2013, 31, e79-e85. 16. Nagaraj, R. H.; Sarkar, P.; Mally, A.; Biemel, K. M.; Lederer, M. O.; Padayatti, P. S., Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal. Arch. Biochem. Biophys. 2002, 402, 110-119. 17. Voziyan, P. A.; Metz, T. O.; Baynes, J. W.; Hudson, B. G., A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation. J. Biol. Chem. 2002, 277, 3397-3403. 18. Shi, Y.-C.; Liao, V. H.-C.; Pan, T.-M., Monascin from red mold dioscorea as a novel antidiabetic and antioxidative stress agent in rats and Caenorhabditis elegans. Free Radical Biology and Medicine 2012, 52, 109-117. 19. Hsu, W.-H.; Lee, B.-H.; Chang, Y.-Y.; Hsu, Y.-W.; Pan, T.-M., A novel natural Nrf2 activator with PPARγ-agonist (monascin) attenuates the toxicity of methylglyoxal and hyperglycemia. Toxicology and applied pharmacology 2013, 272, 842-851. 20. Kinsky, O. R.; Hargraves, T. L.; Anumol, T.; Jacobsen, N. E.; Dai, J.; Snyder, S. A.; Monks, T. J.; Lau, S. S., Metformin scavenges methylglyoxal to form a novel imidazolinone metabolite in humans. Chemical research in toxicology 2016, 29, 227-234. 21. Kender, Z.; Fleming, T.; Kopf, S.; Torzsa, P.; Grolmusz, V.; Herzig, S.; Schleicher, E.; Racz, K.; Reismann, P.; Nawroth, P., Effect of metformin on methylglyoxal metabolism in patients with type 2 diabetes. Experimental and Clinical Endocrinology & Diabetes 2014, 122, 316-319. 22. Lu, J.; Ji, J.; Meng, H.; Wang, D.; Jiang, B.; Liu, L.; Randell, E.; Adeli, K.; Meng, Q. H., The protective effect and underlying mechanism of metformin on neointima formation in fructose-induced insulin resistant rats. Cardiovascular diabetology 2013, 12, 58. 23. Beisswenger, P.; Ruggiero-Lopez, D., Metformin inhibition of glycation processes. Diabetes Metab. 2003, 29, 6S95-6S103. 24. Beisswenger, P. J.; Howell, S. K.; Touchette, A. D.; Lal, S.; Szwergold, B. S., Metformin reduces systemic methylglyoxal levels in type 2 diabetes. Diabetes 1999, 48, 198-202. 25. Wang, P.; Chen, H.; Sang, S., Trapping Methylglyoxal by Genistein and Its Metabolites in Mice. Chem. Res. Toxicol. 2016, 29, 406-414. 26. 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. 27. Sang, S.; Shao, X.; Bai, N.; Lo, C.-Y.; Yang, C. S.; Ho, C.-T., Tea polyphenol (−)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol. 2007, 20, 1862-1870.


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28. Lo, C. Y.; Li, S.; Tan, D.; Pan, M. H.; Sang, S.; Ho, C. T., Trapping reactions of reactive carbonyl species with tea polyphenols in simulated physiological conditions. Molecular nutrition & food research 2006, 50, 1118-1128. 29. 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. Chem. Res. Toxicol. 2008, 21, 2042-2050. 30. Zhu, Y.; Zhao, Y.; Wang, P.; Ahmedna, M.; Sang, S., Bioactive ginger constituents alleviate protein glycation by trapping methylglyoxal. Chem. Res. Toxicol. 2015, 28, 1842-1849. 31. Totlani, V. M.; Peterson, D. G., Epicatechin carbonyl-trapping reactions in aqueous Maillard systems: identification and structural elucidation. J. Agric. Food Chem. 2006, 54, 7311-7318. 32. Sang, S.; Hong, J.; Wu, H.; Liu, J.; Yang, C. S.; Pan, M. H.; Badmaev, V.; Ho, C. T., Increased growth inhibitory effects on human cancer cells and anti-inflammatory potency of shogaols from Zingiber officinale relative to gingerols. J. Agric. Food Chem. 2009, 57, 10645-50. 33. Chen, H.; Lv, L.; Soroka, D.; Warin, R. F.; Parks, T. A.; Hu, Y.; Zhu, Y.; Chen, X.; Sang, S., Metabolism of [6]-shogaol in mice and in cancer cells. Drug Metabolism and Disposition 2012, 40, 742-753. 34. Shao, X.; Chen, H.; Zhu, Y.; Sedighi, R.; Ho, C.-T.; Sang, S., Essential structural requirements and additive effects for flavonoids to scavenge methylglyoxal. J. Agric. Food Chem. 2014, 62, 3202-3210. 35. Zhu, Y.; Zhao, Y.; Wang, P.; Ahmedna, M.; Sang, S., Bioactive ginger constituents alleviate protein glycation by trapping methylglyoxal. Chem. Res. Toxicol. 2015, 28, 1842-9. 36. Yang, C. S.; Maliakal, P.; Meng, X., Inhibition of Carcinogenesis by Tea. Annu. Rev. Pharmacol. Toxicol. 2002, 42, 25-54.



Figure legends Figure 1. (A) Structures of [6]-shogaol (6S) and its MGO adduct, MGO-[6]-shogaol (MGO-6S); (B) Structures of epicatechin (EC) and its methylated metabolites, and their MGO adducts .

Figure 2. Trapping of MGO (60 µM) by [6]-shogaol (6S), epicatechin (EC), and the combination of [6]-shogaol and epicatechin (6S + EC, equal molar combination) at 10 µM in phosphate buffer (pH 7.4 and 37 °C) at 24 h. Data are presented as the means ± standard deviation (SD) of three replications.

Figure 3. (A) SIM chromatograms of mono-MGO-[6]-shogaol (MGO-6S) adduct in [6]-shogaol:MGO (6S:MGO; 1:6) and [6]-shogaol:epicatechin:MGO (6S:EC:MGO; 1:1:6) incubation systems, as well as MGO-6S standard; (B) LC chromatograms of epicatechin (EC) after incubation with MGO (1:6 molar ratio) for 4 h; (C) LC chromatograms of [6]-shogaol (6S) and epicatechin (EC) after incubation with MGO (1:1: 6 molar ratio) for 4 h. The chromatograms of epicatechin (EC) , mono, and di-MGO adducts of epicatechin (mono-MGO-EC and Di-MGO-EC) were obtained using SIM mode.

Figure 4. (A) SIM chromatograms and ESI-MSn (n=2; negative ion) spectra of mono-MGO-[6]-shogaol (MGO-6S) adduct in [6]-shogaol (6S) and [6]-shogaol (6S) + epicatechin (EC) treated mouse feces, as well as mono-MGO-[6]-shogaol (MGO-6S) standard; (B) SIM chromatogram and ESI-MSn (n=2-3; negative ion) spectra of mono-MGO-epicatechin (mono-MGO-EC) in epicatechin (EC) and [6]-shogaol (6S) + epicatechin (EC) treated mouse urine; (C) SIM chromatogram and ESI-MSn (n=2-4;


Page 18 of 26

Page 19 of 26


negative ion) spectra of di-MGO-epicatechin (di-MGO-EC) in epicatechin (EC) and [6]-shogaol (6S) + epicatechin (EC) treated mouse urine.

Figure 5. (A) SIM chromatogram of 3'-methyl epicatechin (3'-MeEC) and 4'-methyl epicatechin (4'-MeEC) in epicatechin (EC) and [6]-shogaol (6S) + epicatechin (EC), and vehicle treated mouse urine samples. (B) ESI-MSn (n=2; negative ion) spectra of 3'-methyl epicatechin (3'-MeEC) and 4'-methyl epicatechin (4'-MeEC)..

Figure 6. (A) SIM chromatogram and ESI-MSn (n=2-3; negative ion) spectra of mono-MGO-3'-methyl epicatechin (mono-MGO-3'-MeEC) in epicatechin (EC) and [6]-shogaol (6S) + epicatechin (EC) treated mouse urine; (B) SIM chromatogram and ESI-MSn

(n=2-4;

negative

ion)

spectra

of

di-MGO-3'-methyl

epicatechin

(di-MGO-3'-MeEC) in epicatechin (EC) and [6]-shogaol (6S) + epicatechin (EC) treated mouse urine.



Page 20 of 26

MGO

A [6]-Shogaol (6S)

MGO-[6]-Shogaol (MGO-6S)

B

8-Mono-MGO-EC

6-Mono-MGO-EC

8-Mono-MGO-3'-MeEC

6-Mono-MGO-3'-MeEC

Trapping MGO to form Mono-MGO adducts

Catechol-O-methyl transferase (COMT) 3'-O-methyl epicatechin (3'-MeEC)*

Epicatechin (EC)

Trapping MGO to form Di-MGO adducts

6,8-Di-MGO-EC

Figure 1


6,8-Di-MGO- 3'- MeEC

Page 21 of 26


Figure 2



20

13.76 80

40

5

10

15

20

10.25

100

6S:EC:MGO = 1:1:6 MGO-6S M.W 349 [M+H]+

80 60

Relative Abundance

0

17.04 8.41 5.54 7.80

0

5

20

15.51 16.58

21.06

4.96 8.35 9.54

0 0

5

10

15

20

25

10.25

100

13.04

60 40 20

16.61 18.32 21.48 9.83 5.67 8.43

0 0

5

10

Time (min)

15

20

Relative Abundance

Relative Abundance

MGO-6S standard M.W 349 [M+H]+

15 14.97

20

60 12.00 40 20

18.48 21.29

11.03 2.65 8.91 0 5

10

100 11.06

80

15

20

Di-MGO-EC M.W 433 [M-H]17.05

40

20.28 23.50

20 4.07

0

7.83 8.40 5

10

15

20

100

40 20 1.44 0

7.92 9.40 5

10

40

16.99 23.45

20 8.30 4.15 7.71 0 5

10

15 14.88

12.99

25

Mono-MGO-EC M.W 361 [M-H]-

80 60

20

11.91

40 20 7.71 8.80

0 0

5

18.46

10

15 14.39

100

25

Di-MGO-EC M.W 433 [M-H]-

13.70 10.94

80

21.26 20

60 17.00

40 20 3.10

0 0

23.43

7.69 8.28 5

10

15

20

25

13.38

60

0

60

25

EC M.W 289 [M-H]-

80

14.87

13.72 10.96

0

13.38

100

6S:EC:MGO = 1:1:6 (4 h) TIC 14.40

80

25

14.46 13.75

60

100

25

Mono-MGO-EC M.W 361 [M-H]-

80

0 80

10

100

0 40

20.29 23.49

20

0

9.50 5.65 8.34

14.92

60

15.56 18.22 21.45

0

11.08

Relative Abundance

40

100

Relative Abundance

60

C

EC:MGO = 1:6 (4 h) TIC 14.47

Relative Abundance

Relative Abundance

6S:MGO = 1:6 MGO-6S M.W 349 [M+H]+

80

Relative Abundance

Relative Abundance

100

Relative Abundance

B

10.24

14.96 15

20.07 20

25.80 25

Time (min)

Figure 3


Relative Abundance

A

Page 22 of 26

100

EC M.W 289 [M-H]-

80 60 40 20 1.37

0 0

7.80 9.36 5

10

15.27 15

Time (min)

21.32 20

25


6S-treated mouse feces

80 60

9.49 40

10.59 14.98

6.81 7.97 20 0 6

8

10

14

10.29

100

60

13.47 14.33

9.49

6.72 7.18

20 0 6

8

10

12

80

40

11.72 20.24

20 10.71 0

5

10

13.67 14.80

7.78 8.86 9.60

0 6

8

10

12

14

60

60 40 313.17 348.25

261.15

175.11 0 200

300 273.04

20.18 23.82

20 8.58 10.61 0

100

MS 2 : 349 [M+H]+ MGO-6S standard

80 60 40

331.24 20 139.05

215.07

333.24

0 100

200

300

m/z

80

5

10

15

20

25

289.26

MS 2 : 361 [M-H]Mono-MGO-EC

60 343.18 40 181.80 245.78 161.82

20

400

362.18

0 100

200

MS 3 :

80

300 245.18

80

40

205.16

20 125.38

203.26

247.10

0 100

150

200

100

MS 2 :

250 245.20

300

[M-H]-

289 EC standard

80 60

205.34

40 20

203.57 109.61 165.71

245.96 290.11

0 100

150

200

250

16.87

10.76

20 8.40 0 0 100

5

10

15

20 20.11

6S+EC-treated mouse urine 13.47 14.22 M.W 433 [M-H]-

80 60

25 23.30

16.79

10.59 40 20 7.90

0 0

5

10

15

20

Time (min)

100 80

25

415.24

MS 2 : 433 [M-H]Di-MGO-EC

60 40

361.34 397.34

20 253.80 281.52

415.94 434.30

0 200

250

300

350 400 289.14

450

100

Mono-MGO-EC

40

23.37

60

400

289/361 [M-H]-

60

400

Relative Abundance

100

25

11.67

40

100

Relative Abundance

MS 2 : 349 [M+H]+ MGO-6S

20

20

6S+EC-treated mouse urine M.W 361 [M-H]-

12.73 80

100

273.00 100 80

15 14.66

20.12

EC-treated mouse urine 14.28 M.W 433 [M-H]-

100

Time (min)

40 20

23.87

0

0

MGO-6S standard

60

EC-treated mouse urine M.W 361 [M-H]-

60

14

Time (min)

Relative Abundance

12.80

10.30

100

Relative Abundance

80

100

6S+EC-treated mouse feces

80

40

12

100

Relative Abundance

100

C 14.75

Relative Abundance Relative Abundance


B 10.28

Relative Abundance

Relative Abundance Relative Abundance Relative Abundance

A


Page 23 of 26

300

m/z

Figure 4


80

MS 3 :

361/433 [M-H]-

Di-MGO-EC

343.15

60 40 181.07 161.13

20

245.17

0 100

200

300 245.08

400

100 80

MS 4 : 289/361/433 [M-H]Di-MGO-EC

60 205.08

40

179.07

20 125.03

271.13

0 100

150

200

m/z

250

300


B

A

100

Relative Abundance

80

3'-MeEC 19.24

60

4'-MeEC

40 20.52

20

Relative Abundance

1.62

7.68 10.90 15.81 16.60

0 100

28.17

22.25

32.61 33.25

19.24

3'-MeEC

6S+EC treated mouse urine M.W 303 [M-H]-

80 60

20.51 28.17

18.62

28.95

40

22.27

3.22

261.20

20

15.81 19.58

27.64

29.36 32.67

165.54

179.78

10

15

250

303 [M-H]4'-MeEC

304.83

20

33.57 37.81

25

30

300

35

244.25

80 60

304.16

259.25 285.20

40 217.41 165.54

20

151.62

202.55

219.34

261.19

187.65

0 150

5

270.22

200

MS2:

25.45

0 0

235.60

204.76 139.70

137.58

Control mouse urine 10.13 10.90

285.21

40

100

32.57

28.94

20

219.50

60

m/z

33.53 37.04

0 100

60

304.16 259.29

4'-MeEC

1.43 4.00 7.68 10.90 16.61

80

244.33

80

150

40 20

MS2: 303 [M-H]3'-MeEC

0

Relative Abundance

EC treated mouse urine M.W 303 [M-H]-

100

Page 24 of 26

40

Time (min)

Figure 5


200

250

m/z

300

15.22 40 22.93 23.64 26.07 21.57

20 7.72 8.86 12.75

0 5 100 80 60

10

15

20

25

15.93

6S+EC-treate d mouse urine M.W 375 [M-H]-

17.32

15.17

40 20

22.88

7.68 9.59 12.65

0

10

15

23.59

20

26.03

80 60

357.29

MS 2 : 375 [M-H]Mono-MGO-3'-MeEC

220.58 303.38 376.21 40 221.57 20 313.37 377.01 244.58 261.73 0 200 250 300 350 400

100

MS 3 : 303/375 [M-H]Mono-MGO-3'-MeEC

450

259.13

219.10 217.14

80

285.10

60 40

288.07 20

139.09 165.11

204.13

150

200

288.67

0

100 80

250

300

259.19

MS 2 : 303 [M-H]3'-MeEC

244.19 219.18 285.16

60 40 20

288.13 139.31 165.27

204.31

150

200

303.19

0 250

EC-treated mouse urine M.W 447 [M-H]-

80 60

16.72 17.09 14.06

22.35 19.07

40 20 7.63 8.95

0 5 100

15 20 16.71 17.04 16.17

6S+EC-treate d mouse urine M.W 447 [M-H]-

80 60

25.41

12.16

10

14.77

25

22.33

25.34

19.12

40 20 7.59 8.88

0 5

Time (min) 100

100

25

Relative Abundance


B

60

5

Relative Abundance

15.99 17.38

Relative Abundance

80

EC-treate d mouse urine M.W 375 [M-H]-

Relative Abundance

A

100




Page 25 of 26

12.16

10

15

20

Time (min)

100

25

429.28

MS 2 : 447 [M-H]Di-MGO-3'-MeEC

80 60

263.43

40

411.34 448.20

281.30

20

317.42 357.33 375.33

448.87

0 250

300

350

400

450

500

357.14

100

MS 3 : 375/447 [M-H]Di-MGO-3'-Me EC

80 60 40

303.13

220.04

20

221.10 261.14

331.21

0 200 100 80

250

300

350

MS 4 : 303/375/447 [M-H]Di-MGO-3'-MeEC

400

219.14 217.16

60

450

259.18

285.17

40 20 0

139.18 163.00

204.08

150

200

300

m/z

Figure 6


288.18 250

m/z

300


GRAPHIC TOC


Page 26 of 26

Additive Capacity of [6]-Shogaol and Epicatechin To Trap

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