Characterization of Carbonyl-Phenol Adducts Produced by Food Phenolic Trapping of 4-Hydroxy-2-hexenal and 4-Hydroxy-2-nonenal

Article abstract of DOI:10.1021/acs.jafc.8b07091

4-Hydroxy-2-alkenals disappear in the presence of food phenolics (i.e., cathechin or quercetin), and the corresponding carbonyl-phenol adducts are produced. In an attempt to identify structure(s) of formed adducts, the reactions between model phenolics (resorcinol, 2-methylresorcinol, orcinol, and 2,5-dimethylresorcinol) and hydroxyalkenals (4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal) were studied and the produced adducts were isolated by column chromatography and unambiguously characterized by one- A nd two-dimensional nuclear magnetic resonance and mass spectrometry as dihydrobenzofuranols (1), chromane-2,7-diols (2), and 2H-chromen-7-ols (3). These compounds were mainly produced at slightly basic pH values and moderate temperatures. Their activation energies (E_a) of formation were a25 kJ mol^-1 for adducts 1, a32 kJ mol^-1 for adducts 2, and a38 kJ mol^-1 for adducts 3. A reaction pathway that explains their formation is proposed. All of these results confirm that, analogously to other lipid-derived carbonyl compounds, phenolics can trap 4-hydroxy-2-alkenals in an efficient way. Obtained results provide the basis for the potential detection of carbonyl-phenol adducts derived from hydroxyalkenals in food products.

Full text of DOI:10.1021/acs.jafc.8b07091

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Food and Beverage Chemistry/Biochemistry
Characterization of Carbonyl-Phenol Adducts Produced by Food Phenolic
Trapping of 4-Hydroxy-2-hexenal (HHE) and 4-Hydroxy-2-nonenal (HNE)
Francisco J. Hidalgo, and Rosario Zamora
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07091 • Publication Date (Web): 31 Jan 2019
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Page 1 of 41
Journal of Agricultural and Food Chemistry
Characterization of Carbonyl-Phenol Adducts Produced by Food
Phenolic Trapping of 4-Hydroxy-2-hexenal (HHE) and 4-Hydroxy-2-
nonenal (HNE)
Francisco J. Hidalgo and Rosario Zamora*
Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Carretera de
Utrera km 1, Campus Universitario – Edificio 46, 41013-Seville, Spain
*Corresponding author:
Rosario Zamora
+34954611550
+34954616790
rzamora@ig.csic.es
Phone:
Fax:
e-mail:
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ABSTRACT
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4-Hydroxy-2-alkenals disappear in the presence of food phenolics (i.e. cathechin or
quercetin) and the corresponding carbonyl-phenol adducts are produced. In an attempt
to identify structure(s) of formed adducts, the reactions between model phenolics
(resorcinol, 2-methylresorcinol, orcinol, and 2,5-dimethylresorcinol) and
hydroxyalkenals (4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal) were studied and the
produced adducts were isolated by column chromatography and unambiguously
characterized by 1D and 2D NMR and MS as dihydrobenzofuranols (1), chromane-2,7-
diols (2), and 2H-chromen-7-ols (3). These compounds were mainly produced at
slightly basic pH values and moderate temperatures. Their activation energies (Ea) of
formation were 25 kJ·mol–1 for adducts 1, 32 kJ·mol–1 for adducts 2, and 38 kJ·mol–
1 for adducts 3. A reaction pathway that explains their formation is proposed. All these
results confirm that, analogously to other lipid-derived carbonyl compounds, phenolics
can trap 4-hydroxy-2-alkenals in an efficient way. Obtained results provide the basis for
the potential detection of carbonyl-phenol adducts derived from hydroxyalkenals in
food products.
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KEYWORDS:
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Carbonyl-phenol reactions; 4-hydroxy-2-alkenals; lipid oxidation; Maillard reaction;
phenolics; reactive carbonyls
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INTRODUCTION
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Food lipid profiles change during processing and storage because of the oxidation of
fatty acyl chains. This process is responsible for both changes in food quality and
formation of undesirable compounds.1,2 Among the produced compounds, the formation
of short-chain carbonyl compounds has long attracted the attention of researchers
because of their role in the formation of food flavors and off-flavors3,4 and their
potential toxicity.5,6 In particular, 4-hydroxy-2-alkenals have been specially studied in
this sense. They have been shown to be produced both during food processing7 and food
digestion,8 to contribute to the generation of food flavors,9 and to play a major role in
food safety and body health and disease.10
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Toxicity of all these compounds has been related to their ability to react with the
nucleophiles (mostly amino compounds) present in the nearby and to produce the
corresponding carbonyl-amine adducts. Consequently, the modification of important
biomolecules, such as nucleic acids, proteins and amino phospholipids, by short-chain
aldehydes and other lipid-derived reactive carbonyls has been described in many
studies.11–13
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A possibility for mitigating these reactions, and avoiding the destruction of important
biomolecules, is the use of alternative nucleophiles that can trap the produced carbonyl
compounds. To this respect, recent studies have shown that certain phenolic
compounds14 can effectively trap a wide range of lipid-derived reactive carbonyls and
protect, in that way, amino compounds from degradation.15 This carbonyl-trapping
occurs under common cooking conditions,16 and this ability constitutes an additional
protective function of phenolics for avoiding lipid oxidation consequences.17 Thus, the
reactions of phenolics with alkanals, 2-alkenals, 2,4-alkadienals, 4-oxo-2-alkenals, and
4,5-epoxy-2-alkenals have been studied so far, and the corresponding carbonyl-phenol
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adducts produced have been isolated and characterized.18 In addition, formation of these
carbonyl-phenol adducts has been related to the protective function of some phenolics
on the formation of heterocyclic aromatic amines and biogenic amines by carbonyl-
amine reactions,19,20 and the beneficial properties of polyphenolic-rich foods such as tea
or coffee.21
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Although carbonyl-phenol reactions with different lipid-derived reactive carbonyls
have been described,22–27 the ability of phenolic compounds to trap and produce
carbonyl-phenol adducts with 4-hydroxyalkenals has not been described. To the best of
our knowledge, although several studies have shown that 4-hydroxy-2-nonenal (HNE)
seems to be effectively trapped by phenolics,28,29 neither the produced reaction has been
described in detail nor the structure(s) of the formed adduct(s) has been unequivocally
characterized. In addition, the formation of carbonyl-phenol adducts between 4-
hydroxy-2-hexenal (HHE) and phenolics has not been investigated so far.
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In an attempt to fill this gap, this manuscript describes the carbonyl-trapping of HNE
by catechin and quercetin. In addition, it unambiguously characterizes the formed
adducts by studying the reactions of HNE and HHE with simple phenolics. For these
characterization studies, resorcinol, 2-methylresorcinol, orcinol, and 2,5-
dimethylresorcinol were employed as model phenolics because of both their high
trapping potential,14 similar to that of food phenolics,15 and their low molecular weight
that facilitated the isolation and characterization of the produced adducts.
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MATERIALS AND METHODS
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Materials. HNE was prepared according to the procedure described by Gardner et
al.30 (1992), who epoxidated 3(Z)-nonenol with 3-chloroperoxybenzoic acid and later
oxidized the 3,4-epoxynonanol obtained with periodinane. This procedure was also
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applied to the synthesis of HHE, although it had to be modified. Briefly, a solution 3(Z)-
hexenol (50 mmol) in 140 mL of dichloromethane were treated slowly with 3-
chloroperoxybenzoic acid (9.3 g) and the obtained mixture was stirred for 1 h at room
temperature. After this time, 10% potassium bicarbonate (140 mL) was added, and the
mixture was stirred for 2 min. The organic layer was then collected and dried over
sodium sulfate. The obtained solution, which contained the produced 3,4-epoxyhexanol,
was added slowly to a solution of periodinane (16 g in 150 mL of dichloromethane) and
the obtained mixture was stirred under dark for 30 min at room temperature. Then, 500
mL of diethyl ether and 230 mL of 1.3 M NaOH were added. The solution was
vigorously stirred for 2 min and the organic layer was collected, dried over sodium
sulfate, and taken to dryness. The residue was fractionated by column chromatography
on silica gel using mixtures of hexane and diethyl ether as eluent. Identity and purity of
obtained HHE were confirmed by means of 1D and 2D NMR and GC-MS.
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Spectroscopic and spectrometric data of this compound are collected in the
Supplementary Material. Synthesized hydroxyalkenals were stored at –30 ºC under
nitrogen. They were stable for long time periods under these conditions.
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Periodinane, unsaturated alcohols, phenolics, and all other chemicals employed in
these studies were purchased from Sigma-Aldrich (St. Louis, MO), Merck (Darmstadt,
Germany) or Fluka (Buchs, Switzerland), and were of the highest available grade.
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Disappearance of the Hydroxyalkenal and Formation of Carbonyl-Phenol
Adducts in the Reaction of HNE with Catechin or Quercetin. A mixture of the
phenolic (30 mol in 170 L of methanol), HNE (30 mol in 30 L of methanol) and
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0.3 M sodium phosphate buffer (300 L), pH 8.0, were heated under nitrogen for
different times at 80 ºC. At the end of the heating, samples were cooled at room
temperature and 50 L of internal standard (54.8 mg of methyl heptanoate in 25 mL of
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methanol) was added. One hundred microliters of the obtained solution was diluted with
acetonitrile (100 L), centrifuged at 2000 g for 5 min, and, finally, studied for HNE
disappearance by GC-MS and for carbonyl-phenol adduct formation by MS.
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Determination of HNE. HNE disappearance was determined directly by GC-MS.
The ions monitored for quantitation of HNE and the internal standard were m/z 86 and
74, respectively. GC-MS analyses were conducted with an Agilent 7820 gas
chromatograph coupled with an Agilent 5977B mass selective detector (MSD),
quadrupole type. Separations were carried out on a fused-silica DB-5MS UI capillary
column (30 m  0.25 mm i.d; coating thickness, 0.25 m), and 1 L of sample was
injected in the pulsed splitless mode. Working conditions were: carried gas, helium (0.8
mL/min); injector, 250 ºC; oven temperature programmed from 40 ºC (3 min) to 200 ºC
at 12 ºC/min and then 1 min at 200 ºC; transfer line to MSD, 280 ºC; ionization EI, 70
eV; ion source temperature, 230 ºC; mass range 50-550 amu.
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The quantitation of HNE was carried out by preparing standard curves of this
compound and using the same procedure described above. HNE concentration was
directly proportional to the adduct/internal standard area ratio (r > 0.99, p < 0.001).
RSD was < 10%.
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Detection of Carbonyl-Phenol Adducts in the Reaction Between HNE and
Catechin or Quercetin. Detection of carbonyl-phenol adducts was carried out by direct
injection on a triple quadrupole API 2000 mass spectrometer (Applied Biosystems,
Foster City, CA) using an electrospray ionization interface in the negative ionization
mode (ESI–). The nebulizer gas and the curtain gas were set at 19 and 10 (arbitrary
units), respectively. The electrospray capillary voltage was set to –4.5 kV, the
declustering potential was –50 V, the focusing potential was –400 V, and the entrance
potential was –10 V.
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Formation of Carbonyl-Phenol Adducts in the Reaction of 4-Hydroxy-2-
Alkenals and Simple Phenolic Compounds. Determination of conditions that favored
the formation of carbonyl-phenol adducts and the characterization of structures of the
produced adducts was carried out with simple phenolic compounds and by using two
procedures: one for analytical purposes and another for preparative purposes. Both
procedures included the acetylation of the formed adducts to get their stabilization.
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For analytical purposes, a mixture of the phenolic (30 mol in 170 L of water), the
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hydroxyalkenal (30 mol in 30 L of methanol) and 0.3 M buffer (300 L) were heated
under nitrogen at the indicated times and temperatures. At the end of the incubation
time, samples were cooled at room temperature, treated with ethanol (1.2 mL) and taken
to dryness under nitrogen. Then, 30 L of internal standard (36.64 mg of 3(Z)-nonenol
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in 5 mL of dried pyridine), 1 mL of dry pyridine, and 500 L of acetic anhydride were
added, and the mixture was allowed to react under dark for 20 h at room temperature.
After that time, 2 mL of dichlorometane and 2 mL of water were added. The mixture
was stirred for 1 min and the organic layer was collected, and washed 4 times with 2
mL of 5% HCl and once with 2 mL of water. Layers were separated by centrifugation
(2000 g for 5 min) and the organic layer was studied by GC-MS.
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For preparative purposes, reactions were carried out analogously but the phenolic
(2.4 mmol in 13.6 mL of water) and the hydroxyalkenal (2.4 mmol in 2.4 mL of
methanol) were dissolved in 24 mL of 0.3 M sodium phosphate and heated under
nitrogen at 100 ºC for 18 h. After cooling, 120 mL of ethanol were added and the
reaction mixture was taken to dryness. The acetylation was carried out by adding 80 mL
of pyridine and 40 mL of acetic anhydride. After 20 h, 160 mL of dichloromethane and
160 mL of water were added, and the solution was washed four times with 160 mL of
5% HCl and once with 160 mL of water. The organic layer was taken to dryness and the
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residue fractionated by column chromatography on silica gel using mixtures of hexane
and diethyl ether as eluent. Separation was controlled by GC-MS using the conditions
described below.
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Characterization of the Carbonyl-Phenol Adducts Formed in the Reaction of 4-
Hydroxy-2-alkenals and Phenolic Compounds. The reaction between
hydroxyalkenals and phenolics is complex and different carbonyl-phenol adducts were
produced. Figure 1 shows the portion of the total ion chromatogram where carbonyl-
phenol adducts appeared. This figure shows the chromatograms obtained in the
reactions of HHE with resorcinol (Figure 1A), 2-methylresorcinol (Figure 1C), orcinol
(Figure 1E), and 2,4-dimethylresorcinol (Figure 1G), and the chromatograms obtained
in the reactions of HNE with the same phenolics (Figs. 1B, 1D, 1F, and 1H,
respectively). As discussed below, and independently of the phenolic involved, three
types of adducts were always produced (compounds 1–3). Structures for these adducts
are collected in Figure 2. Adducts 1 were isolated and characterized from the reaction of
HHE and 2-methylresorcinol, and adducts 2 and 3 were isolated and characterized from
the reaction of HNE and 2-methylresorcinol. The adducts isolated and characterized
were (Z)-(6-acetoxy-2-(1-acetoxybutyl)-7-methylbenzofuran-3(2H)-ylidene)methyl
acetate (as the pair of diastereomers 1e and 1f), 2-hydroxy-4-(1-hydroxyhexyl)-8-
methylchroman-7-yl acetate (as the pair of diastereomers 2g and 2h) and 1-(7-acetoxy-
8-methyl-2H-chromen-2-yl)hexyl acetate (3g). Spectroscopic and spectrometric data of
all these compounds, as well as of other adducts identified in the assayed reactions, are
collected in Figures S-1, S-2 and S-3, and in Tables S-1, S-2, and S-3 of the
Supplementary Material.
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Phenolic-Hydroxyalkenal Adduct Determination. To study the formation of
carbonyl-phenol adducts and the effect of reaction conditions, formed adducts were
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determined by GC-MS. GC-MS were conducted using the equipment described above
and chromatographic and spectrometric conditions were the same with the exception of
the oven temperature program. Oven temperature was programmed from 100 ºC (1 min)
to 300 ºC at 15 ºC/min and then 5 min at 300 ºC. Carbonyl-phenol adducts were
quantified by preparing standard curves of the isolated adducts (1e and 1f as models of
adducts 1, 2g and 2h as models of adducts 2, and 3g as model of adduct 3). Six
concentration levels were used. Adduct content was directly proportional to
adduct/internal standard area ratio (r > 0.99, p < 0.001). RSD was always < 10%.
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NMR Spectroscopy. 1D and 2D NMR spectra were obtained in a Bruker Advance
III spectrometer operating at 500 MHz for protons. Experiments were performed at 24
ºC and acquisition parameters were described previously.16
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Statistical Analysis. All quantitative data are mean  SD values of, at least, three
independent experiments. Analysis of variance was employed to compare different
groups. When F values were significantly different, group differences were evaluated
by the Tukey test.31 Statistical comparisons were carried out using Origin v. 7.0
(OriginLab Corporation, Northampton, MA). The significance level is p < 0.05 unless
otherwise indicated.
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RESULTS
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Disappearance of HNE and Formation of Carbonyl-Phenol Adducts in the
Reaction of HNE with Catechin or Quercetin. Analogously to other lipid oxidation
products,32 HNE is relatively unstable upon heating. Therefore, it disappeared slowly
when heated in sodium phosphate buffer, pH 8 (Figure 3). This loss was linear (r = –
0.975, p = 0.005) during the first 10 h. Nevertheless, when catechin or quercetin were
also present, the hydroxyalkenal disappeared more rapidly (Figure 3). This difference
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was a consequence of the carbonyl-trapping ability of phenolic compounds. Thus, when
reaction mixtures were studied by MS, the appearance of the molecular ions
corresponding to carbonyl-phenol adducts were observed (Figure S-4, Supplementary
material). Thus, the mixture of HNE and catechin exhibited the M+ – 1 molecular ion of
the adducts at m/z 445 and the mixture of HNE and quercetin exhibited the M+ – 1
molecular ion of the adduct at m/z 457. However, these adducts resulted unstable and
they could not be isolated and characterized. To carry out this characterization, the
corresponding adducts with simpler phenolics were prepared.
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Characterization of the Adducts Produced in the Reaction Between
Hydroxyalkenals and Phenolic Compounds. When the reaction between
hydroxyalkenals and simple phenolics was studied, the formation of different carbonyl-
phenol adducts was observed by GC-MS after acetylation (Figure 1). Thus, when the
reaction was carried out with HHE, two main adducts (adducts 1) were always observed
in the different reactions assayed (Figs. 1A, 1C, 1E, and 1G). This kind of adducts were
also produced in the reaction of HNE and phenolics (Figs. 1B, 1D, 1F, and 1H). They
had a molecular weight that corresponded to the addition of the molecular weights of
the carbonyl compound, the phenolic, and three acetyl groups, and the loss of one
molecule of hydrogen. All of them had identical fragmentation pattern (Table S-1,
Supplementary Material), and they were identified as 2,3-dihydrobenzofuranols on the
basis of the 1D and 2D NMR spectra of adducts 1e and 1f, which were isolated and
characterized. Adducts 1e and 1f were produced in the reaction of HHE and 2-
methylresorcinol, but analogous adducts were produced in all assayed reactions.
Spectroscopic and spectrometric data of adducts 1 are collected in the Supplementary
Material.
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Adducts 1e and 1f had very similar NMR spectra. The only difference was the
coupling constants of protons H2 and H1”. This suggested that both adducts were a pair
of diastereomers and carbons C2 and C1” were chiral carbons with different
configuration for both isomers. 1D NMR showed the presence of one isolated olefinic
proton, the existence of two saturated carbons bonded to oxygen, and the occurrence of
the unchanged ethyl group. In addition, most of the initial phenolic molecule remained
unchanged with the exception of one of the hydroxyl groups and its contiguous aromatic
carbon. The study of HMBC and HSQC spectra allowed to determine a structure of 2,3-
dihydrobenzofuranol for the produced adducts (complete chemical structure is shown in
Figure 2). This compound has two chiral carbons (C2 and C1”) and one double bond
between carbons C3 and C1’. The two distereomers that appeared in the chromatograms
are likely the pairs of stereoisomers R,R and S,S on one hand and R,S and S,R, on the
other.
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Differently to adducts 1, adducts 2 and 3 only appeared to a certain extent in
reactions involving HNE. For that reason, they were isolated and characterized from the
reaction of HNE and 2-methylresorcinol. Adducts 2 always appeared to a higher extent
than adducts 3 under the assayed reaction conditions, and, analogously to adducts 1,
they were always produced as a mixture of two diastereomers.
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The molecular ion of adducts 2 was not observed in the mass spectra. This is not
strange because of the presence of free hydroxylic groups observed by NMR. Thus, the
ion with the highest m/z ratio observed by MS was a dehydrated ion (Table S-2,
Supplementary Material). This ion suffered a new dehydration, which suggested the
existence of two free hydroxylic groups in the molecule. Having this into account, the
molecular weight of these adducts corresponded to the addition of the molecular
weights of the carbonyl compound, the phenolic, and only one acetyl group. All adducts
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2 had identical fragmentation pattern, although fragment intensities were different for
the two diastereomers of each adduct (Table S-2, Supplementary Material). They were
identified as chromane-2,7-diols on the basis of the 1D and 2D NMR spectra of adducts
2g and 2h, which were isolated and characterized. Adducts 2g and 2h were produced in
the reaction of HNE and 2-methylresorcinol, but analogous adducts were produced in
all assayed reactions involving HNE. Spectroscopic and spectrometric data of adducts 2
are collected in the Supplementary Material.
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Adducts 2g and 2h had very similar NMR spectra. However, multiplicities and
coupling constants of protons H2, H3, H4, and H1’ were different for both of them. This
suggested that both adducts were a pair of diastereomers. 1D NMR showed the presence
of two carbons bonded to non-acetylated hydroxyl groups, and the disappearance of
both the carbon-carbon double bond and the carbonyl group initially present in the
hydroxyalkenal. On the contrary, the hydroxyl and pentyl groups initially present in the
hydroxyalkenal remained unchanged. In addition, most of the initial phenolic molecule
remained unchanged with the exception of one of the hydroxyl groups and its
contiguous aromatic carbon. The study of the HMBC and HSQC spectra of both
diastereomers allowed to identify their structure as chromane-2,7-diol derivatives
(chemical structure is shown in Figure 2). This compound has three chiral carbons (C2,
C4, and C1’). However, only two of them are fixed (C4 and C1’). This is likely the
reason for the isolation and characterization of only two diastereomers.
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Finally, a third kind of adducts were also produced. These adducts 3 were the
adducts produced to a lower extent under the assayed conditions. They were only
produced to a certain extent in reactions involving HNE and, differently to adducts 1 or
2, only one adduct could be detected for each phenolic.
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Analogously to adducts 2, the molecular ion of adducts 3 was not observed in the
mass spectra. The ions with highest m/z ratio observed corresponded to the loss of either
ketene or acetic acid (Table S-3, Supplementary Material). Having this into account, the
molecular weight of these adducts corresponded to the addition of the molecular
weights of the carbonyl compound, the phenolic, the loss of one molecule of water, and
the incorporation of two acetyl groups. No free hydroxyl groups could be detected in
these adducts, analogously to adducts 1 and differently to adducts 2. All adducts 3 had
identical fragmentation pattern (Table S-3, Supplementary Material). They were
identified as 2H-chromen-7-ols on the basis of the 1D and 2D NMR spectra of adduct
3g, which was isolated and characterized from the reaction between HNE and 2-
methylresorcinol. Spectroscopic and spectrometric data of adducts 3 are collected in the
Supplementary Material.
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Adduct 3g had NMR spectra that were, to a certain extent, similar to NMR spectra of
adducts 2g and 2h, although significant differences were observed. Thus, the spectra
showed that adduct 3g had one carbon-carbon double bond and only two hydroxyl
groups (both of which were acetylated). On the other hand, and analogously to adducts
2, the occurrence of the unchanged hydroxyl and pentyl groups initially present in the
hydroxyalkenal was observed. In addition, most of the initial phenolic molecule
remained unchanged with the exception of one of the hydroxyl groups and its
contiguous aromatic carbon. The study of their HMBC and HSQC spectra allowed to
identify adducts 3 as 2H-chromen-7-ol derivatives (chemical structure is shown in
Figure 2). This compound has two chiral carbons (C2 and C1’). However, only one
diastereomer could be identified and isolated in studied reactions.
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Effect of pH on the Formation of Carbonyl-Phenol Adducts in the Reaction of
HNE and 2-Methylresorcinol. Three adducts were produced in reactions involving
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HNE and only adducts 1 were produced to a certain extent in reactions involving HHE.
In addition, the yields of all of them depended on the reaction conditions. The study of
the effect of reaction conditions on the disappearance of the phenolic and the formation
of carbonyl adducts 1–3 was carried out by employing HNE as model carbonyl
compound and 2-methylresorcinol as model phenolic because their reaction produced
the three adducts. Figure 4 shows the effect of reaction pH on the disappearance of 2-
methylresorcinol (Figure 4A) and the formation of adducts 1g and 1h (Figure 4B),
adducts 2g and 2h (Figure 4C), and adduct 3g (Figure 4D). As observed in Figure 4A,
the remaining 2-methylresorcinol recovered at the end of the heating time (20 h at 100
ºC) decreased as a function of reaction pH. This decrease was produced because of both
the formation of the carbonyl-phenol adducts and the instability of phenolic compounds
at basic pH values.33
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306
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308
Adducts 1 were mostly produced at pH ~8 (Figure 4B). At this pH, the reaction yield
was about 2% for both isomers 1g and 1h. As shown in Figure 4B, the concentration of
both adducts 1g and 1h increased from pH 6 to pH 8 and then decreased significantly (p
< 0.05) at pH 10.
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310
311
312
313
Adducts 2 were also mostly produced at pH ~8 (Figure 4C). Thus, the concentration
of both adducts 2 increased significantly (p < 0.05) from pH 6 to pH 8 and, then,
decreased to higher pH values. Both diastereomers exhibited a similar behavior and
adduct 2h was produced to a slightly higher yield than adduct 2g (5% vs. 4% after 20 h
at 100 ºC).
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317
Differently to adducts 1 and 2, formation of adduct 3g was not so clearly pH-
dependent and was produced approximately with the same yield (~3% after 20 h at 100
ºC) between pH 6 and pH 9 (Figure 4D). However, its concentration decreased
significantly at pH 10.
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Effect of Phenolic and Hydroxyalkenal Concentrations on the Formation of
Carbonyl-Phenol Adducts in the Reaction of HNE and 2-Methylresorcinol. Adduct
formation depended on the concentration of both the phenolic and the hydroxyalkenal
present. Figure 5 shows the remaining 2-methylresorcinol (Figure 5A) and the formed
adducts 1 (Figure 5B), 2 (Figure 5C), and 3 (Figure 5D), as a function of the
concentration of the phenolic (0–50 mol) in the reaction between 2-methylresorcinol
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and 30 mol of HNE after 20 h at 100 ºC. Because increasing amounts of 2-
methylresorcinol were added, the amount of remaining 2-methylresorcinol increased as
a function of the amount of 2-methylresorcinol added (Figure 5A). However, this
increase achieved a maximum when 30 mol of 2-methylresorcinol was added,
328
329
330
although only 30 mol of HNE was available for the reaction with the phenolic.
Formation of polymeric structures at high concentrations of the phenolic might be
hypothesized.
331
332
333
334
The effect of phenolic concentration on adduct 1 formation was also quite surprising
(Figure 5B). These adducts were produced to a high extent at low concentrations of the
phenolic and the presence of higher amounts of 2-methylresorcinol decreased linearly
the concentration of adducts 1. Thus, reaction yield of adduct 1g was 3% when 10 mol
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336
337
of 2-methylresorcinol was added, and this yield decreased to 1.7% when 50 mol of 2-
methylresorcinol was added. Something similar occurred with adduct 1h, and its
formation yield decreased from 2.9% to 1.4%.
338
339
340
341
Differently to adducts 1, adducts 2 exhibited a much more expectable behavior and a
linear increase in their concentrations was observed when the amount of 2-
methylresorcinol increased from 0 to 30 mol (Figure 5C). Thus, yield of adduct 2g
increased from 0 to 3.4% when 2-methylresorcinol concentration increased from 0 to 30
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mol and then remained approximately constant. Analogously, adduct 2h increased
from 0 to 4.2% when 2-methylresorcinol increased from 0 to 30 mol and then
increased slightly to 5.3% when 50 mol of 2-methylresorcinol was added.
345
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347
The behavior of adduct 3g was analogous to that of adducts 2, and its yield increased
from 0 to 2.2% when 2-methylresorcinol increased from 0 to 30 mol and, then,
decreased slightly at higher concentrations of 2-methylresorcinol.
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Figure 6 shows the remaining 2-methylresorcinol (Figure 6A) and the formed
adducts 1 (Figure 6B), 2 (Figure 6C), and 3 (Figure 6D), as a function of the
concentration of the hydroxyalkenal (0–50 mol) in the reaction between 30 mol of 2-
methylresorcinol and HNE after 20 h at 100 ºC. As expected, increasing amounts of
hydroxyalkenal produced a decrease in the remaining 2-methylresorcinol (Figure 6A).
This decrease was linear between 0 and 30 mol of HNE and higher amounts of the
aldehyde did not produce a higher decrease in the remaining 2-methylresorcinol.
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357
358
Differently to that observed for increasing amounts of 2-methylresorcinol, the yield
of adducts 1 increased linearly between 0 and 40 mol of HNE (Figure 6B). Thus, the
yield of adduct 1g increased to 3.3% when 40 mol of HNE were added and the yield of
adduct 1h was 2.6% when the same amount of HNE was added.
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361
A similar behavior was observed for adducts 2, although adduct 2g only increased
linearly between 0 and 20 mol of HNE, and adduct 2h increased linearly between 0
and 50 mol of HNE (Figure 6C).
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364
Finally, adduct 3g also increased linearly between 0 and 40 mol of HNE (Figure
6D). However, and differently to other adducts, the yield obtained with 50 mol of
HNE was significantly much higher than that expected for a linear increase.
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Effect of Time and Temperature on the Formation of Carbonyl-Phenol Adducts
in the Reaction of HNE and 2-Methylresorcinol. Time courses of adducts formation
in the reaction between 2-methylresorcinol and HNE are collected in Figures 7, 8, and 9
for adducts 1, 2, and 3, respectively. Concentration of all adducts increased linearly as a
function of time at all assayed temperatures (from 60 to 140 ºC). Formation rates were
determined for all adducts at the different assayed temperatures by using the equation:
371
[adduct] = k·t
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375
where k is the rate constant and t is the time. Determined rate constants were then used
in an Arrhenius plot to calculate the activation energies (Ea) of formation of the
different adducts (Figure 10). The Ea were determined from the slopes of the lines of
best fit. The Ea determined for the formation of the different adducts were: 25.0  2.1
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377
kJ·mol–1 for adduct 1g, 25.8  2.1 kJ·mol–1 for adduct 1h, 33.2  1.7 kJ·mol–1 for
adduct 2g, 31.9 1.2 kJ·mol–1 for adduct 2h, and 38.4  3.8 kJ·mol–1 for adduct 3g.
378
DISCUSSION
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387
Previous studies have shown that the lipid-derived carbonyl trapping by phenolic
compounds is a complex reaction in which the different nucleophilic groups of the
phenolics (the hydroxyl groups and their contiguous aromatic carbons in m-diphenols)
react with the reactive carbons of the carbonyl compound.18 Because there is a large
variety of lipid-derived carbonyl structures and because some of these carbonyls are
easily degraded to produce new lipid carbonyls,32 the variety of structures of the
initially produced carbonyl-phenol adducts is also considerable.18 Furthermore, many of
these adducts still have reactive groups and further reactions, including polymerizations,
are usually produced.
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Nevertheless, the number of mechanisms by which the initial carbonyl-phenol
adducts are produced is relatively limited. Thus, when only an isolated carbonyl group
is present, such as in alkanals, the reaction is an addition of the aromatic carbon of the
phenolic to the carbonyl carbon of the alkanal.22 However, the reaction is more complex
when a conjugated carbon-carbon double bond is present in the carbonyl compound. In
this case, the reaction is always initiated by the addition of either the hydroxyl group or
its contiguous aromatic carbon in the phenolic to the , carbon-carbon double bond of
the lipid carbonyl. After this initial addition, the molecule is then stabilized by blocking
the carbonyl group, usually by formation of a hemiacetalic structure. This occurs in 2-
alkenals,23,24 2,4-alkadienals,25 and 4-oxo-2-alkenals.26 As an exception, in 4,5-epoxy-2-
alkenals, the reaction is initiated by the attack of the hydroxyl group of the phenolic to
the epoxide ring of the lipid carbonyl because of the high reactivity of this three
membered ring.27
401
402
403
404
405
406
407
4-Hydroxy-2-alkenals are basically ,-unsaturated carbonyl compounds. Therefore,
its behavior, at least at the initial steps, should be similar to other unsaturated carbonyl
compounds. In addition, and because of the presence of an additional hydroxylic group
in the carbonyl compound, the involvement of this group in the stabilization of the
formed structure might be expected. This is what has been observed in this study,
although the later stabilization of adducts 1 was different to that previously observed for
other lipid carbonyls.
408
409
410
411
412
Figure 11 collects a possible reaction pathway that explains the formation of the
different produced adducts. Analogously to other unsaturated carbonyl compounds, the
reaction is initiated by addition of either the hydroxyl group or its contiguous aromatic
carbon in the phenol to the carbon-carbon double bond of the aldehyde. This would
produce adducts 5 and 4, respectively.
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Stabilization of adduct 4 occurs simply by forming the corresponding hemiacetal (6),
analogously to that observed in reactions involving 2-alkenals24 or 2,4-alkadienals.25
The later acetylation of adduct 6 stabilizes the structure of the adduct and produce the
adducts 2 isolated and characterized in this study. Curiously, acetylation only occurs in
the phenolic hydroxyl group. An interaction between the other two hydroxyl groups
might be hypothesized to explain that they were not acetylated under standard
acetylation conditions (20 h at 25 ºC).
420
421
422
423
424
425
Stabilization of adduct 5 is more complex. A possibility of stabilization is the
addition of the aromatic carbon contiguous to the hydroxyl group to the carbonyl group
and the formation of the chromane-4,7-diol (7). However, this compound is not stable
and, after dehydration, the most stable 2H-chromen-7-ol (8) is produced. An analogous
dehydration was observed in the reaction between phenolics and 2-alkenals.24 After
acetylation, the corresponding adducts 3 were produced.
426
427
428
429
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431
An additional possibility, not observed previously with other lipid carbonyls, is the
oxidation of adduct 5 to produce the corresponding ,-unsaturated carbonyl compound
9, which would be latter stabilized by addition of the aromatic carbon of the phenol. A
stabilization of this kind was previously observed in 4,5-epoxy-2-alkenals.26 A later
tautomerization would be responsible of the existence of adduct 10 and, after
acetylation, of adduct 1.
432
433
434
435
436
437
The effect of studied reaction conditions is in agreement with the proposed reaction
pathways. Thus, a slight basicity would increase the nucleophilicity of phenolic active
groups and would favor the reaction as observed in Figure 4 for most adducts. In
addition, the reaction involves equimolecular amounts of the phenolic and the carbonyl
compound. Therefore, the concentration of most adducts increased linearly when the
concentration of either 2-methylresorcinol or HNE increased from 0 to 30 mol, which
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454
is the amount present of the other reactant (Figures 5 and 6). The most surprising result
in relation to this conclusion was that observed for adducts 1, because their
concentration decreased when the concentration of 2-methylresorcinol increased (Figure
4). This behavior might be likely related to the required oxidation step from adduct 5 to
adduct 9 (Figure 11) that can be hypothesized to be inhibited because of the antioxidant
activity of the phenolic compound. Thus, the higher amount of phenolic present, the
higher inhibition of the oxidative reaction and the lower yield of adduct 1. Other
possibility is a higher reactivity of adduct 10 in comparison to that of compounds 2 and
8. Thus, compound 10 might react away in the presence of excess of phenolics more
easily than compounds 2 or 8. Finally, adduct concentrations increased linearly as a
function of incubation times and Ea increased in the order adducts 1 < adducts 2 <
adducts 3. For that reason, adducts 1 were the main adducts produced at low
temperature and were produced for the two hydroxyalkenals assayed. However, adduct
stability also played a major role and, at high temperatures and long incubation times,
the main adducts present in hydroxyalkenal/phenolic reaction mixtures were always
adducts 2. This kind of hemiacetalic adducts, although derived from acrolein, has been
detected in commercially crispy fried onions,16 which suggest a certain thermostability.
455
456
457
458
459
460
461
All these results confirm that, analogously to other lipid-derived carbonyl
compounds, phenolics are also able to trap 4-hydroxy-2-alkenals, which is likely
contributing to the mitigation of some adverse consequences described for these
undesirable products of lipid oxidation.34–35 Furthermore, they provide the basis for the
potential determination of carbonyl-phenol adducts derived from hydroxyalkenals in
food products. This determination may help to evaluate the oxidative stress to which
such food has been exposed during processing or storage.
462
ASSOCIATED CONTENT
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463
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website
at DOI:
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469
Spectroscopic and spectrometric data of compounds isolated and characterized in this
study. Proposed fragmentation, retention indexes and mass spectra of 2,3-
dihydrobenzofuranols (1), chromane-2,7-diols (2), and 2H-chromen-7-ols (3). Mass
spectra of catechin/HNE and quercetin/HNE reaction mixtures (PDF)
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471
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473
474
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476
AUTHOR INFORMATION
Corresponding author
*R.Z.: tel, +34954611550; fax: +34954616790; e-mail: rzamora@ig.csic.es.
ORCID
Francisco J. Hidalgo: 0000-0003-2098-728X
Rosario Zamora: 0000-0003-3016-2177
Funding
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479
This study was supported in part by the European Union (FEDER funds) and the Plan
Nacional de I + D of the Ministerio de Economía y Competitividad of Spain (project
AGL2015-68186-R).
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Notes
The authors declare no competing financial interest.
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FIGURE CAPTIONS
Figure 1. Total ion chromatograms obtained for the reactions between: A, 4-hydroxy-2-
hexenal (HHE) and resorcinol; B, 4-hydroxy-2-nonenal (HNE) and resorcinol; C, HHE
and 2-methylresorcinol; D, HNE and 2-methylresorcinol; E, HHE and orcinol; F, HNE
and orcinol; G, HHE and 2,5-dimethylresorcinol; and H, HNE and 2,5-
dimethylresorcinol, in sodium phosphate buffer, pH 8, after 20 h at 100 ºC under
nitrogen, and later acetylation.
Figure 2. Adducts identified in this study. Compounds 1e (R = ethyl; R’ = methyl; R” =
H), 1f (R = ethyl; R’ = methyl; R” = H), 2g (R = pentyl; R’ = methyl; R” = H), 2h (R =
pentyl; R’ = methyl; R” = H), and 3g (R = pentyl; R’ = methyl; R” = H) were isolated
and characterized by NMR and MS. Carbon numbering corresponds to that employed in
NMR spectra (spectroscopic and spectrometric data are collected in the Supplementary
Material).
Figure 3. Time-course of 4-hydroxy-2-nonenal (HNE) disappearance at 100 ºC in
sodium phosphate buffer, pH 8, and in the presence of catechin () and quercetin ().
HNE disappearance in the absence of phenolics is also shown for comparison ().
Figure 4. Effect of pH on: A, remaining 2-methylresorcinol (Rem. MeRes); B, adduct 1
formation; C, adduct 2 formation; and D, adduct 3 formation, in the reaction of 2-
methylresorcinol (30 mol) and 4-hydroxy-2-nonenal (30 mol) after 20 h at 100 ºC
under nitrogen. The compounds determined were: 2-methylresorcinol (,), adduct 1g
(,), adduct 1h (,), adduct 2g (,), adduct 2h (,), and adduct 3g (,).
Open symbols correspond to sodium phosphate buffers and closed symbols correspond
to sodium borate buffers.
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Figure 5. Effect of 2-methylresorcinol (MeRes) concentration on: A, remaining 2-
methylresorcinol (Rem. MeRes); B, adduct 1 formation; C, adduct 2 formation; and D,
adduct 3 formation, in the reaction of 2-methylresorcinol and 4-hydroxy-2-nonenal (30
mol) in sodium phosphate buffer, pH 8, after 20 h at 100 ºC under nitrogen. The
compounds determined were: 2-methylresorcinol (), adduct 1g (), adduct 1h (),
adduct 2g (), adduct 2h (), and adduct 3g ().
Figure 6. Effect of 4-hydroxy-2-nonenal (HNE) concentration on: A, remaining 2-
methylresorcinol (Rem. MeRes); B, adduct 1 formation; C, adduct 2 formation; and D,
adduct 3 formation, in the reaction of 2-methylresorcinol (30 mol) and HNE in sodium
phosphate buffer, pH 8, after 20 h at 100 ºC under nitrogen. The compounds determined
were: 2-methylresorcinol (), adduct 1g (), adduct 1h (), adduct 2g (), adduct 2h
(), and adduct 3g ().
Figure 7. Time courses of formation of adducts: A, 1g, and B, 1h, at 60 (), 80 (),
100 (), 120 (), and 140 ºC (). Structures of adducts 1g and 1h are given in Figure
2.
Figure 8. Time courses of formation of adducts: A, 2g, and B, 2h, at 60 (), 80 (),
100 (), 120 (), and 140 ºC (). Structures of adducts 2g and 2h are given in Figure
2.
Figure 9. Time courses of formation of adduct 3g at 60 (), 80 (), 100 (), 120 (),
and 140 ºC (). Structure of adduct 3g is given in Figure 2.
Figure 10. Arrhenius plot of adducts 1g (), 1h (), 2g (), 2h (), and 3g ().
Structures of adducts 1g, 1h, 2g, 2h, and 3g are given in Figure 2.
Figure 11. Proposed reaction pathways for the formation of adducts 1–3 in the reaction
of phenolics and 4-hydroxy-2-alkenals. R can be either ethyl or pentyl groups. R’ and
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Journal of Agricultural and Food Chemistry
R” can be either hydrogen or a methyl group. Ac2O is acetic anhydride and Pyr is
pyridine.
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