Toxicologically Relevant Aldehydes Produced during the Frying

Jun 20, 2016 - Figure 1. Chemical structures of the compounds synthesized in this ... 2-(3,4-Dihydroxyphenyl)-3,5,8-trihydroxy-10-methyl-9 ... HRMS, m...
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Toxicologically Relevant Aldehydes Produced during Frying Process are Trapped by Food Phenolics Rosario Zamora, Isabel Aguilar, Michael Granvogl, and Francisco J. Hidalgo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02165 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Toxicologically Relevant Aldehydes Produced during Frying Process

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are Trapped by Food Phenolics

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Rosario Zamora, Isabel Aguilar, Michael Granvogl, and Francisco J. Hidalgo*,

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Utrera km 1, Campus Universitario – Edificio 46, 41013-Seville, Spain

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Straβe 34, D-85354 Freising, Germany

Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Carretera de

Lehrstuhl für Lebensmittelchemie, Techniche Universität München, Lise-Meitner-

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*Corresponding author:

Francisco J. Hidalgo

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Phone:

+34954611550

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Fax:

+34954616790

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e-mail:

[email protected]

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ABSTRACT

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The lipid-derived carbonyl trapping ability of phenolic compounds under common food

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processing conditions was studied by determining the presence of carbonyl-phenol

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adducts in both onions fried in the laboratory and commercially crispy fried onions.

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Four

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crotonaldehyde, or (E)-2-pentenal were prepared and characterized by 1H and

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nuclear magnetic resonance (NMR) spectroscopy and high performance liquid

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chromatography coupled to high resolution mass spectrometry (HPLC-HRMS). The

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synthesized compounds were 2-(3,4-dihydroxyphenyl)-3,5,8-trihydroxy-9,10-dihydro-

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4H,8H-pyrano[2,3-f]chromen-4-one (4), 2-(3,4-dihydroxyphenyl)-3,5,8-trihydroxy-10-

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methyl-9,10-dihydro-4H,8H-pyrano[2,3-f]chromen-4-one (5), 2-(3,4-dihydroxyphenyl)-

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3,5-dihydroxy-8-methyl-4H,8H-pyrano[2,3-f]chromen-4-one

and

2-(3,4-

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dihydroxyphenyl)-8-ethyl-3,5-dihydroxy-4H,8H-pyrano[2,3-f]chromen-4-one

(10).

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When onions were fried in fresh rapeseed oil spiked with acrolein, crotonaldehyde, and

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(E)-2-pentenal (2.7 µmol/g of oil), adduct 10 was the major compound produced and

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trace amounts of adducts 4 and 5, but not of adduct 9, were also detected. In contrast,

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compound 4 was the major adduct present in commercially crispy fried onions.

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Compound 10 was also present to a lower extent, and trace amounts of compound 5, but

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not of compound 9, were also detected. These data suggested that lipid-derived

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carbonyl-phenol adducts are formed in food products under standard cooking

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conditions. They also pointed out to a possible protective role of food polyphenols,

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which might contribute to the removal of toxicologically relevant aldehydes produced

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during deep-frying, assuming that the formed products are stable during food

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consumption in the human organism.

carbonyl-phenol

adducts

produced

between

quercetin

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(9),

and

acrolein, 13

C

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KEYWORDS: Aldehydes, Carbonyl-phenol reactions, Deep-frying, Fried onions,

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Lipid oxidation, Quercetin, Reactive carbonyls

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INTRODUCTION

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Lipid oxidation is a major concern during food production because it causes potential

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safety problems and consumer rejection as a consequence of the changes in flavor,

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texture, appearance, and nutritional quality of food products.1–3 All these changes are

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based on numerous complex interrelated reactions induced by oxygen in the presence of

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initiators.4 Although these reactions also occur at low temperatures, reaction rates

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increase with reaction times and temperatures. For that reason, the nature and

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concentrations of the compounds present in frying oils as a consequence of its thermal

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degradation during heating have been a matter of great interest for a long time, as some

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of these compounds are toxicologically relevant and can be ingested directly from the

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degraded oil or via the fried food.5,6 To this respect, recent studies of Granvogl et al.

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found significant amounts of short chain toxicologically relevant aldehydes, namely 2-

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propenal and (E)-2-butenal (acrolein and crotonaldehyde, respectively) in both frying

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oils and fried foods. Acrolein was present to a higher extent compared to

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crotonaldehyde and the amounts of these compounds were much higher in the frying

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oils than in the fried foods.7–9 Thus, for example, in rapeseed oil heated at 180 ºC, up to

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63.2 mg of acrolein/kg of oil and 27.5 mg of crotonaldehyde/kg of oil were found,

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whereas the amounts of acrolein and crotonaldehyde present in potato chips ranged

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from 23 to 26 µg/kg and from 12 to 25 µg/kg, respectively, depending on the oil used.8

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The fact that the amount of lipid-derived aldehydes in fried foods is reduced

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compared to the content of these aldehydes in the frying oils might be the consequence

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of a limited absorption of these compounds by the foods, but also of a reaction of the

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generated aldehydes with other food components. Among these components, the

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formation of carbonyl-phenol adducts has recently been suggested,10 which would

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imply a mostly unknown protective role of phenolic compounds in the elimination of

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these toxicologically relevant aldehydes, assuming the stability of the formed adducts.

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In an attempt to investigate whether the formed aldehydes in frying oils are

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incorporated into food products in the form of carbonyl-phenol adducts, this study

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aimed at the identification of this kind of adducts in onions submitted to frying in

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rapeseed oil containing acrolein, crotonaldehyde, and (E)-2-pentenal as well as in

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commercially crispy fried onions. These aldehydes were selected because of both their

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confirmed formation in oils submitted to frying7–9 and the fact that the reaction

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mechanism of (E)-2-alkenals with phenolic compounds is now well understood.10 In

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addition, onion was chosen as a model food because its flavonoid composition and

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content is well known.11–15 Thus, total flavonoids in red onions are about 140 mg/100 g

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of fresh weight from which quercetin derivatives are about 85% of total flavonoids. The

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main quercetin derivative is quercetin 3,4’-O-diglucoside (77.1 mg/100 g) followed by

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quercetin 4’-O-glucoside (33.8 mg/100 g), whereas the quercetin content is much lower

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(about 1.31 mg/100 g). However, diglucosides can be hydrolyzed upon maceration,

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among other processes, and the amount of monoglucosides and free quercetin increases.

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During the frying process, the quercetin content is reduced by about 20%.12,13

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MATERIALS AND METHODS

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Safety. Acrolein and crotonaldehyde are hazardous and should be handled carefully.

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Food Samples. Rapeseed oil, red onions (Allium cepa L.), and commercially crispy

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fried onions were purchased at local supermarkets.

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Chemicals. Acrolein, crotonaldehyde, (E)-2-pentenal, and quercetin were obtained

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from Sigma-Aldrich (St. Louis, MO) and were of the highest available quality.

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Sephadex LH-20 was obtained from GE Healthcare Europe (Freiburg, Germany). All

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other chemicals were purchased from Sigma-Aldrich, Fluka (Buchs, Switzerland), or

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Merck (Darmstadt, Germany).

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Preparation of Aldehyde-Quercetin Adducts as Reference Compounds. Keeping

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in mind that quercetin is the major flavonoid present in onions,12,13 the corresponding

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adducts between quercetin and acrolein, crotonaldehyde, or (E)-2-pentenal were

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synthesized and characterized by 1H and

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spectroscopy and high performance liquid chromatography coupled to high resolution

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mass spectrometry (HPLC-HRMS). The chemical structures of the isolated compounds

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are illustrated in Figure 1. These compounds were prepared by dissolving quercetin

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(200 µmol) and the corresponding aldehyde (400 µmol) in methanol (2 mL; containing

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290 µmol of triethylamine). The solution was heated under nitrogen at 100 ºC for 3.5 h

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(acrolein), 10 h (crotonaldehyde), or 96 h ((E)-2-pentenal), respectively. At the end of

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the heating time, the reaction mixture was fractionated on a Sephadex LH-20 column

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using methanol/water (80/20, v/v) as eluent at a flow rate of 15 mL/h. Eluted products

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were detected by mass spectrometry using direct injection.

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C nuclear magnetic resonance (NMR)

2-(3,4-Dihydroxyphenyl)-3,5,8-trihydroxy-9,10-dihydro-4H,8H-pyrano[2,3-

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f]chromen-4-one (4). HRMS, m/z 357.06154 (M+ – 1), error 0.2 ppm. 1H NMR (500

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MHz, DMSO-d6): δ 1.97 (br, 2, H9), 2.88 (br, 2, H10), 3.38 (br, OH), 5.58 (br, 1, H8),

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6.18 s (s, 1, H6), 6.90 (d, 1, J = 8.5 Hz, H5’), 7.61 (dd, 1, J = 2.1 Hz, J = 8.5 Hz, H6’),

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7.73 (d, 1, J = 2.1 Hz, H2’), 9.47 (br, OH), 12.30 (br, OH).

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DMSO-d6): δ 14.85 (C10), 26.87 (C9), 93.29 (C8), 98.89 (C6), 101.26 (C13), 104.20

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(C11), 115.32 (C2’), 116.18 (C5’), 120.43 (C6’), 122.64 (C1’), 136.56 (C3), 145.64

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(C3’), 147.26 (C2’), 148.27 (C4’), 153.10 (C14), 158.59 (C12), 158.78 (C5), 176.51

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(C4).

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C NMR (125.7 MHz,

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2-(3,4-Dihydroxyphenyl)-3,5,8-trihydroxy-10-methyl-9,10-dihydro-4H,8H-

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pyrano[2,3-f]chromen-4-one (5). HRMS, m/z 371.07763 (M+ – 1), error 0.9 ppm. 1H

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NMR (500 MHz, DMSO-d6): δ 1.38 (d, 3, J = 7.0 Hz, H1”), 1.94 (br, 2, H9), 3.38 (br,

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OH), 3.41 (br, 1, H10), 5.50 (br, d, 1, J = 7.9 Hz, H8), 6.18 s (s, 1, H6), 6.91 (d, 1, J =

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8.5 Hz, H5’), 7.60 (dd, 1, J = 2.2 Hz, J = 8.5 Hz, H6’), 7.73 (d, 1, J = 2.2 Hz, H2’), 9.52

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(br, OH), 12.38 (br, OH).

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(C1’), 56.49 (C9), 92.76 (C8), 98.64 (C6), 104.38 (C11), 105.74 (C13), 115.27 (C2’),

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116.20 (C5’), 120.25 (C6’), 122.61 (C1’), 136.56 (C3), 145.71 (C3’), 147.31 (C2),

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148.27 (C4’), 153.44 (C14), 158.88 (C12), 159.16 (C5), 176.51 (C4).

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C NMR (125.7 MHz, DMSO-d6): δ 19.02 (C10), 21.74

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2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-8-methyl-4H,8H-pyrano[2,3-f]chromen-4-

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one (9). HRMS, m/z 353.0625 (M+ – 1), error 4.8 ppm. 1H NMR (500 MHz, DMSO-d6):

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1.42 (d, 3, J = 6.6 Hz, H1”), 3.36 (br, OH), 5.15 (m, 1, H8), 5.83 (dd, 1, J = 3.2 Hz, J =

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10.0 Hz, H9), 6.23 s (s, 1, H6), 6.87 (dd, 1, J = 1.1 Hz, J = 10.0 Hz, H10), 6.91 (d, 1, J

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= 8.5 Hz, H5’), 7.62 (dd, 1, J = 2.2 Hz, J = 8.5 Hz, H6’), 7.73 (d, 1, J = 2.2 Hz, H2’),

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9.52 (br, OH), 12.49 (br, OH), 12.65 (br, OH), 12.99 (br, OH). 13C NMR (125.7 MHz,

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DMSO-d6): δ 21.56 (C1”), 72.88 (C8), 98.83 (C6), 101.63 (C13), 104.48 (C11), 115.21

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(C2’), 116.00 (C10), 116.25 (C5’), 120.58 (C6’), 122.36 (C1’), 124.55 (C9), 136.61

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(C3), 145.68 (C3’), 147.57 (C2), 148.39 (C4’), 150.47 (C14), 159.24 (C12), 160.71

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(C5), 176.57 (C4).

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2-(3,4-Dihydroxyphenyl)-8-ethyl-3,5-dihydroxy-4H,8H-pyrano[2,3-f]chromen-4-one

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(10). HRMS, m/z 367.0810 (M+ – 1), error 3.6 ppm. 1H NMR (500 MHz, DMSO-d6):

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0.97 (t, 3, J = 7.3 Hz, H2”), 1.74 (qu, 2, J = 7.3 Hz, H1”), 3.36 (br, OH), 4.98 (m, 1,

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H8), 5.83 (dd, 1, J = 3.4 Hz, J = 10.1 Hz, H9), 6.23 s (s, 1, H6), 6.89 (dd, 1, J = 1.1 Hz,

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J = 10.1 Hz, H10), 6.91 (d, 1, J = 8.5 Hz, H5’), 7.61 (dd, 1, J = 2.2 Hz, J = 8.5 Hz,

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H6’), 7.72 (d, 1, J = 2.2 Hz, H2’), 9.52 (br, OH), 12.49 (br, OH), 12.65 (br, OH), 12.99 7 ACS Paragon Plus Environment

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(br, OH).

C NMR (125.7 MHz, DMSO-d6): δ 9.23 (C2”), 28.39 (C1”), 77.49 (C8),

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98.75 (C6), 101.69 (C13), 104.39 (C11), 115.21 (C2’), 116.24 (C5’), 116.40 (C10),

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120.59 (C6’), 122.37 (C1’), 123.20 (C9), 136.52 (C3), 145.66 (C3’), 147.39 (C2),

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148.35 (C4’), 150.42 (C14), 159.47 (C12), 160.69 (C5), 176.52 (C4).

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Deep-Frying Experiments. Deep-frying experiments were carried out on rapeseed

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oil (100 g) containing acrolein, crotonaldehyde, and (E)-2-pentenal (each 2.7 µmol/g of

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oil), which were added before heating the oil. The oil was firstly pre-heated for 9 min to

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achieve 160 ºC and then heated in the presence, or absence, of thin slices of onions (5 g)

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for further 3 min. At the end of the heating time, the heated oils were analyzed for

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aldehyde contents and the fried onions were analyzed to identify aldehyde-quercetin

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adducts.

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Determination of Aldehydes in Heated Oils. Aldehydes were determined by 1H

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NMR spectroscopy analogously to the analysis of oil components developed by

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Sopelana et al.16 Briefly, oil samples (200 mg) were diluted with CDCl3 (400 µL),

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which contained 0.2% of non-deuterated chloroform, and their spectra were obtained by

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a Bruker Advance III spectrometer (Karlsruhe, Germany) operating at 500 MHz.

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Quantitation of the three aldehydes was carried out by considering the area of the CHCl3

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signal (at δ 7.29 ppm) as internal standard. The proton of the aldehyde group appeared

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as a doublet that was independent for each one of the three analyzed aldehydes and

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appeared far from other signals in the spectrum. The signals of the aldehydic proton

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appeared at δ 9.55 (d, J = 7.4 Hz) for acrolein, 9.47 (d, J = 7.9 Hz) for crotonaldehyde,

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and 9.49 (d, J = 7.7 Hz) for (E)-2-pentenal. Four samples were analyzed for each heated

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oil and each sample was measured three times. Therefore, the aldehyde content for each

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analyzed oil was the mean value of twelve determinations.

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Determination of Aldehyde-Quercetin Adducts in Fried Onions. Onions fried in

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the laboratory and commercially crispy fried onions (5 g each) were homogenized in

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water (20 mL) and hexane (50 mL). The resulting mixture was centrifuged at 7500 g for

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10 min at room temperature. The organic layer was discarded and other 50 mL of

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hexane were added. The mixture was homogenized again and centrifuged at 7500 g for

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10 min. The organic layer was discarded and methanol (80 mL) was added. The mixture

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was newly homogenized and centrifuged at 7500 g for 10 min. The supernatant was

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collected and the solid was extracted with methanol/water (80/20, v/v; 100 mL). The

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resulting mixture was centrifuged at 7500 g for 10 min and the supernatant was

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collected. Both supernatants were combined, concentrated to about 4 mL using a

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rotatory evaporator (35 ºC, 16 mbar) and fractionated on a Sephadex LH-20 column

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using methanol/water (80/20, v/v) as eluent at a flow rate of 15 mL/h. Fractions

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obtained were analyzed by HPLC-HRMS.

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NMR Spectroscopy. All NMR spectra were obtained by a Bruker Advance III

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spectrometer operating at 500 MHz for protons. For 1H spectra, acquisition parameters

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were: spectral width 10000 Hz, relaxation delay 1 s, number of scans 16, acquisition

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time 3.277 s, and pulse width 90º, with a total acquisition time of 1 min 17 s. For 13C

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spectra, acquisition parameters were: spectral width 27500 Hz, relaxation delay 2 s,

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acquisition time 1.188 s, and number of pulses depended on the concentration of the

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sample. All experiments were performed at 23 ºC. For structural determinations, COSY,

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HMQC and HMBC experiments were carried out.

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Low Resolution MS. To control carbonyl-phenol reactions and the eluates from the

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LH-20 column during the isolation of reference compounds, a triple quadrupole API

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2000 mass spectrometer (Applied Biosystems, Foster City, CA) was employed by using

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an electrospray ionization interface in the negative ionization mode (ESI–). The 9 ACS Paragon Plus Environment

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nebulizer gas and the curtain gas were set at 19 and 10 (arbitrary units), respectively.

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The electrospray capillary voltage was set to –4.5 kV, the declustering potential was –

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50 V, the focusing potential was –400 V and the entrance potential was –10 V.

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HPLC-HRMS. The HPLC-ESI-MS system consisted of a Dionex Ultimate 3000RS

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U-HPLC (Thermo Fisher Scientific, Waltham, MA) coupled to a micrOTOF-QII ultra

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high resolution time-of-flight mass spectrometer (UHR-TOF) with q-TOF geometry

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(Bruker Daltonics, Bremen, Germany). Chromatographic separation was performed on a

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Zorbax Eclipse XDB-C18 column (15 cm × 0.46 cm i. d., 5 µm) from Agilent (Santa

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Clara, CA). As eluent A, a mixture of acetonitrile containing 0.2% formic acid and 4

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mM ammonium formiate (30/70, v/v) was used. As eluent B, acetonitrile containing

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0.2% formic acid was employed. The flow rate was 0.5 mL/min in linear gradient mode:

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0-13 min 7% B, 13-20 min from 7 to 60% B, 20-30 min 60% B, 30-32 min from 60 to

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90% B, 32-42 min 90% B, 42-45 min from 90 to 7% B. A split post-column with a flow

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rate of 0.25 mL/min was inserted directly into the mass spectrometer ESI source. The

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scan range applied was m/z 50-1500 and mass resolving power was always over 18,000

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(m/∆m). The instrument was operated in the negative ion mode. Mass spectra and data

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were obtained by broadband Collision Induced Dissociation (bbCID) mode, providing

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MS and MS/MS spectra simultaneously. Collision energy was estimated dynamically

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based on appropriate values for the mass and stepped across a ±10% magnitude range to

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ensure good quality fragmentation spectra. The instrument control was performed using

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Compass 1.3 for micrOTOF-Q II + Focus Option Version 3.0 (Bruker Daltonics).

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Statistical Analysis. Statistical differences among the amounts of aldehydes

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remaining in the oils after heating in the presence or in the absence of onions were

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evaluated by the Student t-test.17 These comparisons were carried out using Origin,

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version 7.0 (OriginLab Corporation, Northampton, MA). Significance level was p