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Scavenging of Toxic Acrolein by Resveratrol and Hesperetin and Identification of Adducts Weixin Wang, Yajing Qi, James R. Rocca, Paul Sarnoski, Aiqun Jia, and Liwei Gu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03949 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 17, 2015
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Journal of Agricultural and Food Chemistry
Scavenging of Toxic Acrolein by Resveratrol and Hesperetin and Identification of Adducts Weixin Wang†, ‡, Yajing Qi†, #, James R. Rocca§, Paul J. Sarnoski†, Aiqun Jia‡*, Liwei Gu†*
†
Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, Florida, USA 32611; ‡
School of Environmental and Biological Engineering, Nanjing University of Science and
Technology, Nanjing, Jiangsu, China 210094; #
§
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China 214122;
Advanced Magnetic Resonance Imaging & Spectroscopy, McKnight Brain Institute, University of
Florida, Gainesville, Florida, USA 32611
*Corresponding authors (Tel: +1-352-392-1991 ex 210, Fax: +1-352-392-9467, Email:
[email protected] for Liwei Gu; Tel: +86 25 84315512, Fax: +86 25 84315512, Email:
[email protected] for Aiqun Jia)
List of abbreviation: COSY: Correlation spectroscopy HSQC: Heteronuclear single-quantum correlation HMBC: Heteronuclear multiple-bond correlation 1
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ABSTRACT
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The objective of this study was to investigate the ability of resveratrol and hesperetin to
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scavenge acrolein in pH 7.4 at 37 oC. About 6.4% or 5.2% of acrolein remained after reacting
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with resveratrol or hesperetin for 12 hrs at equimolar concentrations. An acrolein-resveratrol
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adduct and two acrolein-hesperetin adducts were isolated. Their structures were elucidated
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using mass and NMR spectroscopy. Acrolein reacted with resveratrol at the C-2 and C-3
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positions through nucleophilic addition and formed an additional heterocyclic ring. Two similar
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monoacrolein-conjugated adducts were identified for hesperetin. Spectroscopic data suggested
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each acrolein-hesperetin adduct was a mixture of four stereoisomers due to the existence of
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two chiral carbon atoms. Yield of adducts was low at pH 5.4 but increased at pH 7.4 and 8.4.
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Higher pH also promoted the formation of di-acrolein adducts. Results suggest that resveratrol
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and hesperetin exert health benefits in part through neutralizing toxic acrolein in vivo.
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KEYWORDS: acrolein, resveratrol, hesperetin, adducts, aldehyde
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INTRODUCTION
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Acrolein (1, Figure 1) is a toxic α, β-unsaturated aldehyde that exists in human body due to lipid
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peroxidation, polyamine catabolism,1 heating or baking of carbohydrate-containing foods, and
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overheating cooking oil. Acrolein is also found in engine exhaust and cigarette smoke,2 a
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growing body of evidence suggests acrolein is associated with and a causative factor for
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Alzheimer’s,3, 4 Parkinson’s,5, 6 diabetes,7 lung carcinogenesis,8, 9 atherosclerosis and
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cardiovascular diseases.10 Like other reactive carbonyl species, acrolein causes cross-linking
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between proteins, DNA,11 and phospholipids in physiological systems after carbonyls react with
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the thiol or amino group or other nucleophilic groups. It reacts with nucleophilic sites on
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transcription factors such as NF-κB and AP-1 to disrupt their functions.12 It causes the formation
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of advanced glycation end products which play a major pathogenic role in diabetes and its
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complications.13, 14 Identifying natural compounds that scavenge reactive carbonyl species
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remains a useful strategy to prevent or mitigate many carbonyl-associated chronic conditions.
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Several natural phenolic compounds have been suggested to be effective in scavenging reactive
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carbonyl species. For example, phloretin was found to scavenge acrolein by forming di-acrolein
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conjugated adducts.15 Resveratrol was shown to protect human retinal pigment epithelial cells
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from acrolein-induced damage.16 Curcumin protected neurons against the toxicity induced by
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acrolein.17 Resveratrol (2, Figure 1) is a stilbenoid found in grapes (0.13−47.6 mg/kg) and red
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wine (0−18.03 mg/L).18 Hesperetin (3, Figure 1) is a flavonoid found exclusively in citrus fruits
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(14.4−33 mg/kg).19 Both compounds are known to be antioxidants and possess health
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promoting properties.20-24 The objective of this study was to investigate the ability of resveratrol
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and hesperetin to scavenge acrolein and to determine the structure of formed adducts. We also
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aimed to elucidate the reaction mechanisms of scavenging acrolein by phenolic compounds.
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MATERIALS AND METHODS
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Reagents. Resveratrol was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ).
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Hesperetin was obtained from MP Biomedical, LLC (Solon, OH). Acrolein was a product from
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Alfa Aesar Company (Ward Hill, MA). Sephadex LH-20 was purchased from Sigma-Aldrich (St.
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Louis, MO). HPLC-grade solvents and other chemicals were obtained from Fisher Scientific Co.
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(Pittsburgh, PA).
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Reaction between acrolein and phenolic compounds. Resveratrol, hesperetin and acrolein
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were dissolved in DMSO:phosphate buffer (pH 7.4) (50:50, v/v) to a concentration of 20 mM.
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The derivatization agent for acrolein was 2, 4-dinitrophenylhydrazine. 2,
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4-dinitrophenylhydrazine (6 mM) was prepared by dissolving 62.5 mg of 2,
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4-dinitrophenylhydrazine crystal and HCl (1 M, 3 mL) in 50 mL acetonitrile. Equal volumes of
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acrolein and resveratrol buffer solutions were mixed and incubated at 37 oC for 0, 0.25, 0.5, 0.75,
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1, 1.5, 2, 4, 8, 12, 24, and 48 h. Acrolein or resveratrol buffer solutions were also incubated with
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phosphate buffer as controls. Reaction between hesperetin and acrolein was carried out in the
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same manner. The reaction was terminated by mixing 300 μL of 2, 4-dinitrophenylhydrazine
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with 50 μL of reaction media.
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For reaction at different pH, resveratrol, hesperetin or acrolein was dissolved in 4 Environment ACS Paragon Plus
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DMSO:phosphate buffer at pH 5.4, 6.4, 7.4 or 8.4 (50:50, v/v) to a concentration of 20 mM.
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Acrolein was incubated with phosphate buffer, resveratrol or hesperetin solutions at different
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pH at 37 oC for 12h. The final concentrations of all reagents in the mixed solutions were 10 mM.
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HPLC and HPLC-MS analyses of acrolein-conjugated adducts. Detection of the remaining
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resveratrol, hesperetin and acrolein were performed on a HPLC (Model 1200, Agilent
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Technologies, Palo Alto, CA) equipped with a diode array detector and a Zorbax SB-C18 column
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(250 mm × 4.6 mm i.d., 5 μm, Agilent Technologies, Palo Alto, CA). Mobile phases were
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composed of 0.1% formic acid in water (mobile phase A) and methanol (mobile phase B). The
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flow rate was 1 mL/min and the injection volume was 20 μL. The linear gradient for the
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detection of remaining resveratrol, hesperetin and acrolein was: 0-7 min, 60-68% B; 7-7.1 min,
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68-100% B; 7.1-11.5 min, 100-100% B; 11.5-12 min, 100-60% B; followed by 5 min of
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re-equilibration. The linear gradient for detection of acrolein-resveratrol adducts and
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acrolein-hesperetin adducts was: 0-18 min, 53-60% B; 18-19 min, 60-100% B; 19-24 min,
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100-100% B; 24-25 min, 100-53% B; followed by 5 min of re-equilibration. Detection wavelength
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was set at 280 nm.
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A HCT ion trap mass spectrometer (Bruker Daltonics, Billerica, MA) with electrospray ionization
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interface was operated at negative mode using nebulizer 50 psi, drying gas 10 L/min, a drying
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temperature of 350 oC, trap drive level 100%, and compound stability 100%. Mass spectra were
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scanned from m/z 150 to 1000. The ions of possible adducts in the full scan spectrum were
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isolated and their product ion spectra (MS2) were recorded.
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Purification of acrolein-phenolic compound adducts. Resveratrol and acrolein were dissolved in
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DMSO:water (50:50, v/v) to a concentration of 100 and 1000 mM respectively. Resveratrol
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solution (25 mL) was mixed with equal volume of acrolein and incubated at 37 oC for 24 h. The
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reaction mixture was dispersed in 500 mL water and extracted with 100 mL ethyl acetate three
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times. Ethyl acetate was evaporated on a SpeedVac concentrator and the residue was dissolved
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in methanol before it was loaded on a Sephadex LH-20 column (30 cm×2.5 cm). Twelve fractions
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were collected by eluting the column with 400 mL methanol. Each fraction was concentrated on
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a SpeedVac concentrator and analyzed by HPLC-ESI-MSn. Fractions with similar composition
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were combined. The fractions containing possible adducts were re-loaded on Sephadex LH-20
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column to increase their purity. An acrolein-resveratrol adduct was obtained after separation by
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HPLC with a Zorbax SB-C18 column (250 mm × 4.6 mm i.d., 5 μm, Agilent Technologies, Palo Alto,
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CA).
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Hesperetin and acrolein were dissolved in DMSO:phosphate buffer (pH 7.4) (50:50, v/v) to a
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concentration of 40 mM. 20 mL of hesperetin was mixed with an equal volume of acrolein and
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incubated at 37 oC for 1 h. The procedure to purify acrolein-hesperetin adducts was similar to
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that of acrolein-resveratrol adduct. Two acrolein-hesperetin adducts were obtained after HPLC
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separation. These two adducts had same molecular weight but different retention time on the
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basis of HPLC-DAD-MSn analysis.
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NMR analyses. Purified adducts were dissolved in CD3OD. 1H and 13C spectra and 2-Dimensional
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COSY, HSQC, and HMBC spectra were acquired using an Avance II spectrometer (Bruker BioSpin
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GmbH, Rheinstetten, Germany) operating at field strength of 14 tesla (600 MHz) in a 54-mm
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bore or an Avance III spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at
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field strength of 14 tesla (600 MHz) in a 51-mm bore tube.
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High resolution mass spectrometry and polarimetry analyses. High resolution mass
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spectrometry was conducted on an Agilent 6220 Accurate-Mass TOF mass spectrometer with
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DART ionization (Agilent Technologies, Palo Alto, CA). Optical rotations were measured on a
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P-2000 polarimeter (JASCO Corp., Japan) in methanol solution. Wavelength of the light was 589
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nm and room temperature was 22±0.5 oC.
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Statistics. Samples were analyzed in triplicate or duplicate. Duplicate analyses were shown as
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averages and triplicate analyses were expressed as mean ± standard deviation. One-way
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analyses of variance with Tukey-Kramer HSD pair-wise comparison of the means were
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performed using JMP software (Version 9, Cary, NC). Statistical significance was determined by
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p
