Scavenging of Acrolein by Food-Grade Antioxidant Propyl Gallate in a Model Reaction System and Cakes

Article abstract of DOI:10.1021/acs.jafc.9b03486

Reactive carbonyl species (RCS), such as acrolein (ACR), glyoxal (GO), and methylglyoxal (MGO), have received extensive attention recently as a result of their high activity and toxicity in vitro and in vivo. In the present study, propyl gallate (PG), a common food antioxidant, was found to effectively trap more ACR than butylated hydroxytoluene and butylated hydroxyanisole through the formation of mono-ACR adducts (PG-ACR) and di-ACR adducts (PG-2ACR). The two adducts were successfully purified, and their structures were elucidated on the basis of their high-resolution mass spectrometry and¹H,¹³C, and two-dimensional nuclear magnetic resonance data. We further identified that PG-ACR had the ability to continue to trap GO and MGO to form PG-ACR-GO and PG-ACR-MGO, respectively, by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we verified that PG could inhibit the production of ACR, GO, and MGO via trapping these RCS simultaneously to form the corresponding adducts in pound cakes using LC-MS/MS.

Full text of DOI:10.1021/acs.jafc.9b03486

Subscriber access provided by GUILFORD COLLEGE
Bioactive Constituents, Metabolites, and Functions
Scavenging of Acrolein by a Food-Grade Antioxidant
Propyl Gallate in model reaction system and in Cakes
Jiaqi Wang, Yongling Lu, Tiesong Zheng, Shengmin Sang, and Lishuang Lv
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03486 • Publication Date (Web): 16 Jul 2019
Downloaded from pubs.acs.org on July 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.

Page 1 of 27
Journal of Agricultural and Food Chemistry
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Scavenging of Acrolein by a Food-Grade Antioxidant Propyl Gallate
in model reaction system and in Cakes
†
†
†
‡
†
Jiaqi Wang , Yongling Lu , Tiesong Zheng , Shengmin Sang , Lishuang Lv *
†Department of Food Science and Technology, School of Food Science and
#
1
1
1
1
1
1
1
1
Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Nanjing,
210023, P. R. China
‡Laboratory for Functional Foods and Human Health, Center for Excellence in
Post-Harvest Technologies, North Carolina Agricultural and Technical State
University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North
Carolina 28081, United States
Corresponding author (Dr. Lishuang Lv, Tel: +86 25 83598286; Fax: +86 25
83598901; E-mail address: lishuanglv@126.com; lulishuang@njnu.edu.cn)
1
1
8
9
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 27
2
2
2
2
2
2
2
2
2
2
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
ABSTRACT
Reactive carbonyl species (RCS), such as acrolein (ACR), glyoxal (GO), and
methylglyoxal (MGO) have received extensive attention recently due to their high
activity and toxicity in vitro and in vivo. In the present study, propyl gallate (PG), a
common food antioxidant, was found to effectively trap more ACR than butylated
hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) through the formation of
mono-ACR adducts (PG-ACR) and di-ACR adducts (PG-2ACR). The two adducts
were successfully purified, and their structures were elucidated based on their HRMS,
1
13
H-, C-, and 2D-NMR data. We further identified that PG-ACR had the ability to
continue to trap GO and MGO to form PG-ACR-GO and PG-ACR-MGO,
respectively, by LC-MS/MS. Furthermore, we verified that PG could inhibit the
production of ACR, GO and MGO via trapping these RCS simultaneously to form the
corresponding adducts in pound cakes using LC-MS/MS.
KEY WORDS: propyl gallate; acrolein; glyoxal; methylglyoxal; pound cakes
3
3
4
5
2
ACS Paragon Plus Environment

Page 3 of 27
Journal of Agricultural and Food Chemistry
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
INTRODUCTION
Acrolein (ACR), one of the major reactive carbonyl species (RCS), is more toxic
than most of the other species, such as glyoxal (GO) and methylglyoxal (MGO).1-3
ACR was listed in group 3 (“not classifiable as to its carcinogenicity to humans”) by
the International Agency for Research on Cancer (IARC), and it was a precursor of
acrylamide (IARC, group 2A “possibly carcinogenic to humans”).4-6 WHO
7
established a tolerable oral ACR intake of 7.5 mg/kg body weight/day. On the
cellular level, ACR has been reported as one of the causing factors for the
development of diabetic mellitus, Alzheimer's disease, cardiovascular and
cerebrovascular diseases, aging, and cancer8-12 via reacting with the thiol or amino
group or other nucleophilic groups.13-15
ACR can be produced during food processing and storage from lipids,
carbohydrates, and amino acids via oil oxidation reaction, Maillard reaction and
Strecker degradation.16-17 High amounts of ACR were detected in a variety of heated
vegetable oils, frying fats, and fish oil,2, 18-19 for example, perilla oil produced 4160
2
0
ppb ACR after heating at 220 °C for 30 min, and 14.45 ppm ACR was formed in
2
1
fish oil after incubating at 50 °C in dark for 8 h. It has been proven that the carbon
backbone of unsaturated fatty acids, not the glycerol backbone, played a key role to
form ACR using isotope labeled fatty acids, glycerol and synthesized
2
2
triacylglycerides, respectively. There was a positive correlation between the level of
2
3
ACR and linoleic acid in the oils. Even the deep-fried and baked foods, such as
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 27
2
4
7
25
5
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
French fries , bread , and donuts contained ACR that ranged from 0.001 to 0.9
2
6
mg/kg. In addition, ACR was also detected in alcoholic beverages such as wine ,
2
7
28
rums , and whiskies . Therefore, it is urgent to find a strategy to inhibit the
formation of ACR in processed food.
Propyl gallate (PG) is one of the most well-known synthetic phenolic antioxidants
commonly used in food processing to scavenge free-radicals and block lipid
peroxidation.29-30 In the Code of Federal Regulations (CFR), PG was listed as a
“generally recognized as safe” (GRAS) substance (21CFR 184.1660), and it could be
used in fat and oils, baked goods, meat products, snack foods, nut products, and so
3
1
on. The European Food Additives Directive stipulates that the maximum allowable
dose of PG for fat and oils used in the specialized production of heat-treated foods is
3
2
200 mg/kg. In our previous studies, PG have demonstrated effective GO and MGO
scavenging capacities.33-34 Whether PG could also scavenge ACR or trap two different
RCS, considering that PG has two free protons on the benzene ring at the positions
C-2 and C-6, has not been clarified till now. This present study would investigate the
effect and pathway of PG on trapping ACR in model reaction and in pound cakes.
MATERIALS AND METHODS
Materials. ACR, GO (40% in water), MGO (40% in water), 1,2-diaminobenzene
(DB), and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma-Aldrich
(St Louis, MO, USA). PG, BHT, BHA, HPLC grade solvents, and other reagents were
obtained from Shanghai Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
4
ACS Paragon Plus Environment

Page 5 of 27
Journal of Agricultural and Food Chemistry
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Determination of the ACR Trapping Capacity of PG, BHT, and BHA by
HPLC. ACR (0.5 mM) was incubated with PG (0.5 mM), BHT (0.5 mM), and BHA
(0.5 mM) in phosphate buffer (0.2 M, pH 7.0) under a ratio of 1:1 at 100 °C for 10,
30, 60, and 120 min, respectively. At each time point, the reaction was stopped by
adding 2 μL of acetic acid, and 500 μL samples were taken for derivatization.
DNPH derivatization solution (6.3 mM) was prepared by dissolving 62.5 mg of
3
5
DNPH in 50 mL of acetonitrile containing 3 mL HCl (1 mM), and DNPH solution
(300 μL) was combined with the samples (500 μL) and shaken for 2 h at 37 °C for the
derivatization of the remaining ACR. The level of ACR-DNPH was analyzed by an
Agilent HPLC (1260 series, Agilent Technologies, Palo Alto, CA, USA) equipped
with a Diode array detector. A Kromasil 100-5 C column (250 × 4.6 mm i.d.; 5 μm;
1
8
AkzoNobel, Bohus, Sweden) was used. The mobile phase was composed of 0.1%
formic acid in Milli-Q water (solvent A) and acetonitrile (solvent B). Isocratic elution
(1.0 mL/min) was used with 30% A and 70% B, and the detection wavelength was set
at 372 nm. All analyses were performed in triplicate.
Purification of the Major ACR adducts of PG. PG (0.507 g, 0.4 M) was
incubated with 0.471 mL ACR (1.2 M, 1:3) in 5.529 mL phosphate buffer (0.2 M, pH
7.0) at 80 °C for 2 h. The reaction mixture was loaded into a reverse phase silica gel
C (ODS-A-HG, 50 μm, YMC Co., Ltd., Kyoto, Japan) column and eluted with 30,
1
8
50, and 60% methanol. The 60% eluate was collected and then loaded into a silica gel
(MB70-40, 40-75 μm, Fuji Silysia Ltd., Japan) column eluted with
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 27
9
9
dichloromethane/methanol (8:1, v/v) to obtain the PG-ACR (41 mg) and PG-2ACR
(36 mg).
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
NMR analyses. A Bucker AVANCE III 400 MHz instrument (Bruker, Inc.,
Silberstreifen, Rheinstetten, Germany) was used in this study. H, 13C, DEPT
(distortionless enhancement by polarization transfer), HMQC (heteronuclear multiple
quantum correlation), and HMBC (heteronuclear multiple bond correlation) spectra
were acquired at 25°C.
1
1H- and 13C-NMR data of PG-ACR and PG-2ACR are listed in Table 1.
PG-ACR was analyzed in CD OD, and PG-2ACR was analyzed in DMSO.
3
High-Resolution Mass Spectrometry Analyses. PG-ACR and PG-2ACR were
dissolved in methanol. High-resolution mass spectrometry (HRMS) was conducted on
an Agilent 6220 Accurate-Mass TOF mass spectrometer with DART ionization
(Agilent Technologies).
Trapping of GO and MGO by PG-ACR in phosphate buffer. PG-ACR (0.4
M) was incubated with GO and MGO (1:10) in a phosphate buffer (0.2 M, pH 7.0) at
100 °C, respectively. Samples were collected at the designated time points 10, 30, and
120 min, respectively, and then analyzed by LC-MS/MS.
Effect of PG on the formation of ACR, GO and MGO in pound cake
systems. Pound cakes were prepared using 100.0 g margarine containing PG (0, 50,
100, and 200 mg/kg). Then 100.0 g of flour, 100.0 g of sugar, 100.0 g of fresh eggs,
1.0 g of salt and 2.0 g of baking powder (sodium bicarbonate and sodium
6
ACS Paragon Plus Environment

Page 7 of 27
Journal of Agricultural and Food Chemistry
1
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
pyrophosphate in a 4:3 ratio) were weighted and mixed. The mixing and baking
3
6
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
methods were those described by Wilderjans et al. The pound cakes were baked in a
YXP 101-2 oven (Shanghai Zaomiao Food Co., Ltd., Shanghai, China) at 175 °C for
45 min. After cooling, the pound cakes were finely grounded and stored at -80 °C for
further analysis.
Pound cakes samples (0.5 g) were extracted with a mixture of methanol and
phosphate buffer (0.2 M, pH 7.4) in a 1:1 ratio and the supernatant was collected,37
ACR was quantified using the method described above, and GO and MGO were
3
8
derivatived with DB and quantified according to our published method. All analyses
were performed in triplicate. The samples of pound cake containing 200 mg/kg PG
were analyzed by LC-MS/MS.
LC-MS/MS Analysis. The same Agilent Masshunter System described in our
previous publication was used for this study.33
Negative ESI ion mode was used, and tandem mass spectrometry (MS/MS) with
collision-induced dissociation (CID, 15% relative collision energy) was used to obtain
the structural information of the major RCS adducts of PG.
For the analysis of the mixture of PG-ACR incubated with GO or MGO, the
mobile phases consisted of phase A (0.1% formic acid in water) and phase B
(acetonitrile). The gradient elution was performed using the following gradient: 20%
B for 3 min, 3 to 15 min, 20-46% B, 15 to 18 min, 46-70% B, and 18 to 19 min 70%
B. The column was then re-equilibrated with the initial phase for 3 min.
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 27
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
For the analysis of pound cakes samples, the mobile phases consisted of phase A
(0.1% formic acid in water) and phase B (methanol). The gradient elution was
performed using the following gradient: 0-3 min, 30% B, 3 to 20 min, 30-70% B,
20-25 min, 70-80% B, and 25 - 26 min, 80% B. The column was then re-equilibrated
with the initial phase for 5 min.
Statistical analysis. The assays and experiments were performed in triplicate
and all data were expressed as mean and standard deviation. Statistical analyses were
conducted using Tukey's test. A p value < 0.05 was considered statistically different.
SPSS 13.0 statistical software was applied to analysis the comparisons (IBM SPSS
Software, New York, USA).
RESULTS AND DISCUSSION
Scavenging capacity of PG, BHT, and BHA on ACR. Our results demonstrated
that PG could efficiently trap ACR in a time-dependent manner, and it had a much
stronger trapping capacity on ACR than BHT and BHA. Almost half of the ACR
(48.40%) was scavenged after incubation with PG for 10 min, and up to 72.80% of
ACR was eliminated at 60 min, whereas only 16.75 and 16.97% of ACR was
scavenged by BHT and BHA, respectively (Figure 1), although the anti-lipid-oxidant
capability of BHA was higher than that of PG.39-40
Purification and structure elucidation of PG-ACR and PG-2ACR. In this
study, we did observe a temperature dependent trapping of ACR by PG (data not
shown). At 80°C, PG-ACR and PG-2ACR are the major products, and PG-2ACR is
8
ACS Paragon Plus Environment

Page 9 of 27
Journal of Agricultural and Food Chemistry
1
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
the major product at 100°C. In order to purify both PG-ACR and PG-2ACR, we chose
80°C instead of 100°C. The PG-ACR and PG-2ACR were purified from the reaction
mixtures. Their structures were determined after analyzing the HRMS, MS/MS, and
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
13
NMR ( H, C, DEPT, HMQC, and HMBC) data.
-
PG-ACR had a [M-H] peak at m/z 267.0879 with the molecular formula of
C H O from the high-resolution mass spectrum (Supplementary Figure. S1). The
1
3
16
6
-
theoretical m/z value of [M-H] for this molecule is 267.0874. The molecular weight
of the PG-ACR was 56 mass units (MWACR, 56) higher than that of PG, suggesting
that PG-ACR was the mono-ACR adduct of PG. This is also supported by the
-
observation of the fragment ion at m/z 211 [M-H] (loss of one ACR) in the tandem
mass spectrum of PG-ACR (Supplementary Figure. S2). The PG-ACR NMR data
1
13
were shown in the Supplementary Figures. S3-S6. The H- and C-NMR spectra
suggested the PG-ACR carbon skeleton was similar to PG from C-1 to C-10 (Table
1
1). In the H-NMR spectrum of PG-ACR, the singlet signal at δ 7.13 represented
H
only one proton instead of two protons (δ 7.08, s) for PG, suggesting that ACR
H
conjugated with PG at the C-2 position of the benzene ring. In the HMBC spectrum,
correlations between the proton at δ 3.10 (H-11) and the carbons at δ 119.88 (C-1),
H
C
118.68 (C-2), 142.47 (C-3), 28.67 (C-12), and 93.05 (C-13) were observed, indicating
that this proton was H-11 and that the ACR group was connected at the C-2 position
of the benzene ring. In addition, the proton at δ 5.57 (H-13) was correlated with δ
H
C
141.11 (C-3), which suggested that ACR formed a cyclic semi-acetal structure.
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 27
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
PG-ACR has one chiral center at C-13 position. However, we only observed one set
1
13
of H- and C-NMR signals for the PG-ACR we purified from the reaction mixture.
Therefore, we tentatively identified the planar structure of PG-ACR as shown in
Figure 2.
-
The high-resolution mass spectrum of PG-2ACR gave a [M-H] peak at m/z
325.1139 with a molecular formula of C H O (Supplementary Figure. S7). The
1
6
20
7
-
theoretical m/z value of [M-H] for this molecule is 323.1136. The molecular weight
of PG-2ACR was 56 mass units (MWACR, 56) higher than that of PG-ACR and 112
mass units higher than that of PG, indicating that it was the di-ACR adduct of PG.
This is further supported by the observation of the fragment ion losing one ACR unit
-
-
at m/z 267 [M-H] and the fragment ion losing two ACR units at m/z 211 [M-H] in
the MS/MS spectrum of PG-2ACR (Supplementary Figure. S8). The PG-2ACR
1
NMR data were shown in the Supplementary Figures. S9-S13. The H-NMR
spectrum of PG-2ACR was similar to that of PG, except that the two proton signals at
1
δ 7.08 (H-2 and H-6) in the H-NMR spectrum of PG disappeared, indicating that
ACR conjugated with PG at both positions C-2 and C-6 of the benzene ring. In
addition, there were two sets of proton signals for the ACR group (δ 2.63 m, 4H;
H
1
3
1.78 s, 4H and 5.49 d, 2H). Its C-NMR spectrum also showed two sets of carbon
signals for the ACR group (δ 18.96, 2C; 27.88, 2C, and 91.59, 2C). In addition, the
C
HMBC spectrum showed the proton at δ 5.49 (H-13 or H-16) was correlated with δ
H
C
141.11 (C-3 or C-5), it suggested that two cyclic semi-acetal structures were formed
1
0
ACS Paragon Plus Environment

Page 11 of 27
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
04
on the benzene ring. Therefore, the PG-2ACR has two chiral carbons at C-13 and
1
3
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
C-16. In theory, PG-2ACR should have four stereoisomers. The C-NMR spectrum
of the purified PG-2ACR has two sets of almost identical signals, suggesting it is a
mixture of stereoisomers. However, we were not able to separate them. Therefore, we
tentatively identified the planar structure of PG-2ACR as shown in Figure 2.
Trapping of GO and MGO by PG-ACR in phosphate buffer. Due to the
PG-ACR still having one free proton at the C-6 position on the benzene ring, and
demonstrating that it could further trap another ACR to form PG-2ACR (Figure 2),
we hypothesized that PG-ACR might have a similar capacity of further trapping GO
and MGO in the same system. To prove this hypothesis, LC-MS/MS was used to
determine the products of PG-ACR incubated with GO and MGO in phosphate buffer,
respectively.
As shown in Figure 3A, three major new peaks (PG-ACR-GO-1,
PG-ACR-GO-2, and PG-ACR-GO-3) appeared after 10 min of incubation of
-
PG-ACR with GO, and they had the same molecular ion (m/z 325 [M-H] ), being 58
-
2
mass units higher than PG-ACR (m/z 267 [M-H] ), and similar MS fragments (Table
2) but different retention times (RT 11.31, 10.76 and 9.53 min), indicated that these
compounds were mono-GO-conjugated PG-ACR adducts. As the incubation
proceeded from 30 to 120 min, one additional new peak (PG-ACR-GO-4) appeared,
-
and this peak (RT 7.87 min) also had the molecular ion m/z 325 [M - H] and similar
MS/MS fragments to the first three peaks, indicated it was also the mono-GO product
1
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 27
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
of PG-ACR. Therefore, our results indicated that PG-ACR, the product of PG
capturing ACR, could further capture GO to form four isomers of mono-GO adduct.
As shown in Figure 3B, one major new peak (PG-ACR-MGO, RT 12.51 min)
appeared after 10 min of incubation of PG-ACR with MGO in phosphate buffer. It
-
had a molecular ion at m/z 339 [M-H] , which was 72 mass units higher than that of
-
2
PG-ACR (m/z 267 [M-H] ), and its MS spectrum displayed fragment ions at m/z 267
-
-
[M-H] (loss of one MGO) and m/z 211 [M-H] (loss of one MGO and one ACR),
suggesting that the product was mono-MGO-conjugated PG-ACR. With time
increasing from 10 to 120 min, the level of PG-ACR-MGO significantly increased
until only one product of PG-ACR-MGO was found in the incubation of PG-ACR
with MGO.
Effects of PG on the formation of ACR, GO, and MGO in pound cakes. In
order to understand whether PG could simultaneously scavenge ACR, GO, and MGO
generated during food processing, we investigated the inhibitory effects of PG on the
formation of ACR, GO, and MGO in an oil-rich food, the pound cakes. It has been
reported that PG was stable up to 180 °C and decomposed in two steps. The first step
occurred at the 180-365°C interval (63.09%), the second step occurred at the
365-650°C interval (36.91%).41-42 There PG is a good candidate compound to be used
in making pound cakes, which requires a temperature of 175 °C. As shown in Figure
4, PG could inhibit the accumulation of ACR in a dose-dependent manner, up to
60.25 % of ACR was scavenged by 200 mg/kg PG after baking at 175 °C for 45 min
1
2
ACS Paragon Plus Environment

Page 13 of 27
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
46
in pound cakes, while the GO and MGO trapping efficiency of PG was 41.81% and
49.17%, respectively, lower than that of ACR.
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Studies the inhibitory mechanism of PG on the formation of ACR, GO and
MGO in pound cake by LC-MS/MS. To understand if PG can inhibit ACR, GO,
and MGO in baked foods via trapping or by antioxidant ability, a LC-MS/MS was
used to detect the products of PG in pound cakes. As shown in Figure 5, there were
three peaks (PG-ACR, RT 21.61 min; PG-MGO, RT 15.93 min; PG-GO, RT 14.02
-
-
-
min) that had molecular ion at m/z 267 [M-H] , m/z 283 [M-H] , and m/z 269 [M-H] ,
respectively, in pound cakes with 200 mg/kg PG, which were 56, 72, and 58 mass
units (MWACR, 56; MWMGO, 72; MW , 58) higher than that of PG, suggesting that
GO
PG-ACR was the mono-ACR adduct of PG, PG-MGO was the mono-MGO adduct of
PG, and PG-GO was the mono-GO adduct of PG. This was also supported by the
-
2
formation of the fragment ion PG (m/z 211 [M-H] ) in the MS spectra of PG-ACR,
PG-GO, and PG-MGO (Figure 5). However, with current limits of detection we
failed to detect the PG-ACR-GO and PG-ACR-MGO adducts in pound cakes system,
which may due to there are not enough RCS to be further trapped by the mono RCS
adducts of PG.
In conclusion, our results suggested that PG, as a popular food antioxidant, has
an effective inhibitory ability on ACR through the formation of mono- and di-ACR
adducts. Firstly, we purified and identified the structure of mono- and di-ACR adducts
of PG by NMR data. Moreover, this is also the first study to demonstrate that
1
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 27
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
PG-ACR could further capture different RCS (MGO and GO) at the C-6 position,
after capturing one molecule of ACR in the same system. Our data supports that PG is
far more than an antioxidant, it also has the potential to suppress the generation of
toxic RCS, such as ACR, MGO and GO, simultaneously during food processing.
ABBREVIATIONS USED
ACR, acrolein; GO, glyoxal; MGO, methylglyoxal; RCS, reactive carbonyl species;
PG, propyl gallate; PG-ACR, mono-ACR adduct of PG; PG-2ACR, di-ACR adduct of
PG; PG-ACR-GO, mono-GO adduct of PG-ACR; PG-ACR-MGO, mono-MGO
adduct of PG-ACR; PG-MGO, mono-MGO adduct of PG; PG-GO, mono-GO adduct
of PG
FUNDING SOURCES.
This work was supported by the National Natural Science Foundation of China (Grant
31571783).
CONFLICT OF INTEREST
The authors declare no competing financial interest.
SUPPORTING INFORMATION DESCRIPTION
Figure S1. High resolution mass spectrometry of PG-ACR
Figure S2. Mass spectrum (MS/MS) of PG-ACR
Figure S3. 1H-NMR spectrum of PG-ACR
Figure S4. 13C-NMR spectrum of PG-ACR
1
4
ACS Paragon Plus Environment

Page 15 of 27
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
88
Figure S5. HMBC-NMR spectrum of PG-ACR
89
90
91
92
93
94
95
Figure S6. HMQC-NMR spectrum of PG-ACR
Figure S7. High resolution mass spectrometry of PG-2ACR
Figure S8. Mass spectrum (MS/MS) of PG-2ACR
Figure S9. 1H-NMR spectrum of PG-2ACR
Figure S10. 13C-NMR spectrum of PG-2ACR
Figure S11. HMBC-NMR spectrum of PG-2ACR
Figure S12. HMQC-NMR spectrum of PG-2ACR
2
2
96
97
Figure S13. DEPT-NMR spectrum of PG-2ACR
References
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
1. Conklin, D. J.; Barski, O. A.; Lesgards, J. F.; Juvan, P.; Rezen, T.; Rozman, D.; Prough, R. A.;
Vladykovskaya, E.; Liu, S.; Srivastava, S.; Bhatnagar, A., Acrolein consumption induces systemic
dyslipidemia and lipoprotein modification. Toxicol. Appl. Pharmacol. 2010, 243 (1), 1-12.
2. Suh, J. H.; Niu, Y. S.; Hung, W. L.; Ho, C. T.; Wang, Y., Lipidomic analysis for carbonyl species
derived from fish oil using liquid chromatography-tandem mass spectrometry. Talanta 2017, 168,
31-42.
3. Wang, Y.; Cui, P., Reactive Carbonyl Species Derived from Omega-3 and Omega-6 Fatty Acids.
J. Agric. Food Chem. 2015, 63 (28), 6293-6296.
4. International Agency for Research on Cancer. Dry cleaning, some chlorinated solvents and other
industrial chemicals. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 1995, 63,
337-372.
5. International Agency for Research on Cancer. Some industrialchemicals. IARC Monographs on
the Evaluation of Carcinogenic Risks to Humans 1994, 60, 389-433.
6. Duda-Chodak, A.; Wajda, Å.; Tarko, T.; Sroka, P.; Satora, P., A review of the interactions
between acrylamide, microorganisms and food components. Food Funct. 2016, 7 (3), 1282-1295.
7. Abraham, K.; Andres, S.; Palavinskas, R.; Berg, K.; Appel, K. E.; Lampen, A., Toxicology and
risk assessment of acrolein in food. Mol. Nutr. Food Res. 2011, 55 (9), 1277-1290.
8. Moghe, A.; Ghare, S.; Lamoreau, B.; Mohammad, M.; Barve, S.; McClain, C.; Joshi-Barve, S.,
Molecular mechanisms of acrolein toxicity: relevance to human disease. Toxicol. Sci. 2015, 143 (2),
242-255.
9. Burcham, P. C., Acrolein and human disease: untangling the knotty exposure scenarios
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 27
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
accompanying several diverse disorders. Chem. Res. Toxicol. 2017, 30 (1), 145-161.
10. Kim, B. G.; Lee, P. H.; Lee, S. H.; Hong, J.; Jang, A. S., Claudins, VEGF, Nrf2, Keap1, and
nonspecific airway hyper-reactivity are increased in mice co-exposed to allergen and acrolein. Chem.
Res. Toxicol. 2019, 32 (1), 139-145.
11. Wang, H. T.; Tsou, H. H.; Hu, C. H.; Liu, J. H.; Liu, C. J.; Lee, C. H.; Liu, T. Y., Acrolein is
involved in the synergistic potential of cigarette smoking- and betel quid chewing-related human oral
cancer. Cancer Epidemiol. Biomarkers Prev. 2019, 28 (5), 954-962.
12. Zhu, Q.; Zhuang, X.; Lu, J., Neuroprotective effects of baicalein in animal models of Parkinson's
disease: A systematic review of experimental studies. Phytomedicine 2019, 55, 302-309.
13. Igarashi, K.; Uemura, T.; Kashiwagi, K., Acrolein toxicity at advanced age: present and future.
Amino Acids 2018, 50 (2), 217-228.
14. Guillen, M. D.; Goicoechea, E., Toxic oxygenated alpha,beta-unsaturated aldehydes and their
study in foods: a review. Crit. Rev. Food Sci. Nutr. 2008, 48 (2), 119-136.
15. Jiang, C.; Jiang, L.; Li, Q.; Liu, X.; Zhang, T.; Dong, L.; Liu, T.; Liu, L.; Hu, G.; Sun, X.; Jiang,
L., Acrolein induces NLRP3 inflammasome-mediated pyroptosis and suppresses migration via
ROS-dependent autophagy in vascular endothelial cells. Toxicology 2018, 410, 26-40.
16. Thurer, A.; Granvogl, M., Generation of desired aroma-active as well as undesired toxicologically
relevant compounds during deep-frying of potatoes with different edible vegetable fats and oils. J.
Agric. Food Chem. 2016, 64 (47), 9107-9115.
17. Stevens, J. F.; Maier, C. S., Acrolein: sources, metabolism, and biomolecular interactions relevant
to human health and disease. Mol. Nutr. Food Res. 2008, 52 (1), 7-25.
18. Ben Hammouda, I.; Freitas, F.; Ammar, S.; Da Silva, M.; Bouaziz, M., Comparison and
characterization of volatile compounds as markers of oils stability during frying by HS-SPME-GC/MS
and Chemometric analysis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2017, 1068-1069,
322-334.
19. Fujisaki, M.; Endo, Y.; Fujimoto, K., Retardation of volatile aldehyde formation in the exhaust of
frying oil by heating under low oxygen atmospheres. J. Am. Oil Chem. Soc. 2002, 79 (9), 909-914.
20. Endo, Y.; Hayashi, C.; Yamanaka, T.; Takayose, K.; Yamaoka, M.; Tsuno, T.; Nakajima, S.,
Linolenic acid as the main source of acrolein formed during heating of vegetable oils. J. Am. Oil Chem.
Soc. 2013, 90 (7), 959-964.
21. Shibata, A.; Uemura, M.; Hosokawa, M.; Miyashita, K., Acrolein as a major volatile in the early
stages of fish oil TAG oxidation. J. Oleo Sci. 2018, 67 (5), 515-524.
22. Ewert, A.; Granvogl, M.; Schieberle, P., Isotope-labeling studies on the formation pathway of
acrolein during heat processing of oils. J. Agric. Food Chem. 2014, 62 (33), 8524-8529.
23. Molina-Garcia, L.; Santos, C. S. P.; Cunha, S. C.; Casal, S.; Fernandes, J. O., Comparative
fingerprint changes of toxic volatiles in low PUFA vegetable oils under deep-Frying. J. Am. Oil Chem.
Soc. 2017, 94 (2), 271-284.
24. Osorio, V. M.; de Lourdes Cardeal, Z., Determination of acrolein in french fries by solid-phase
microextraction gas chromatography and mass spectrometry. J. Chromatogr. A 2011, 1218 (21),
3332-3336.
25. Lane, R. H.; Smathers, J. L., Monitoring aldehyde production during frying by reversed-phase
1
6
ACS Paragon Plus Environment

Page 17 of 27
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
60
liquid chromatography. J. Assoc. Off. Anal. Chem. 1991, 74 (6), 957-960.
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
26. Bauer, R.; Cowan, D. A.; Crouch, A., Acrolein in wine: importance of 3-hydroxypropionaldehyde
and derivatives in production and detection. J. Agric. Food Chem. 2010, 58 (6), 3243-3250.
27. Loquet, C.; Toussaint, G.; Letalaer, J. Y., Studies on mutagenic constituents of apple brandy and
various alcoholic beverages collected in western France, a high incidence area for oesophageal cancer.
Mutat. Res. 1981, 88 (2), 155-164.
28. Miller, B. E.; Danielson, N. D., Derivatization of vinyl aldehydes with anthrone prior to
high-performance liquid chromatography with fluorometric detection. Anal. Chem. 1988, 60 (7),
622-626.
29. Additives, E. P. O. F., Scientific Opinion on the re-evaluation of propyl gallate (E 310) as a food
additive. EFSA Journal 2014, 12 (4), 3642-3646.
30. Garrido, J.; Garrido, E. M.; Borges, F., Studies on the food additive propyl gallate: synthesis,
structural characterization, and evaluation of the antioxidant activity. J. Chem. Educ. 2011, 89 (1),
130-133.
31. Final report on the amended safety assessment of propyl gallate. Int. J. Toxicol. 2007, 26 Suppl 3,
89-118.
32. EU, European Parliament and Council Directie No.95/2/ECof 20 February 1995 on food
additives other than colours and sweeteners. 1995; Vol. 61, p 23-24.
33. Cui, H.; Tao, F.; Hou, Y.; Lu, Y.; Zheng, T.; Sang, S.; Lv, L., Dual effects of propyl gallate and
its methylglyoxal adduct on carbonyl stress and oxidative stress. Food Chem. 2018, 265, 227-232.
34. Hou, Y.; Xie, Z.; Cui, H.; Lu, Y.; Zheng, T.; Sang, S.; Lv, L., Trapping of glyoxal by propyl,
octyl and dodecyl gallates and their mono-glyoxal adducts. Food Chem. 2018, 269, 396-403.
35. Wang, W.; Qi, Y.; Rocca, J. R.; Sarnoski, P. J.; Jia, A.; Gu, L., Scavenging of toxic acrolein by
resveratrol and hesperetin and identification of adducts. J. Agric. Food Chem. 2015, 63 (43),
9488-9495.
36. Wilderjans, E.; Pareyt, B.; Goesaert, H.; Brijs, K.; Delcour, J. A., The role of gluten in a pound
cake system: A model approach based on gluten-starch blends. Food Chem. 2008, 110 (4), 909-915.
37. Arribas-Lorenzo, G.; Morales, F. J., Analysis, distribution, and dietary exposure of glyoxal and
methylglyoxal in cookies and their relationship with other heat-induced contaminants. J. Agric. Food
Chem. 2010, 58 (5), 2966-2972.
38. Li, X.; Zheng, T.; Sang, S.; Lv, L., Quercetin inhibits advanced glycation end product formation
by trapping methylglyoxal and glyoxal. J. Agric. Food Chem. 2014, 62 (50), 12152-12158.
39. Murcia, M. A.; MartínezTomé, M., Antioxidant activity of resveratrol compared with common
food additives. J. Food Prot. 2001, 64 (3), 379-384.
40. Zhang, Y. L.; Shen, Y. X.; Zhu, Y. C.; Xu, Z. M., Assessment of the correlations between
reducing power, scavenging DPPH activity and anti-lipid-oxidation capability of phenolic antioxidants.
LWT-Food Sci. Technol. 2015, 63 (1), 569-574.
41. Galico, D. A.; Nova, C. V.; Guerra, R. B.; Bannach, G., Thermal and spectroscopic studies of the
antioxidant food additive propyl gallate. Food Chem. 2015, 182, 89-94.
42. Reda, S. Y., Evaluation of antioxidants stability by thermal analysis and its protective effect in
heated edible vegetable oil. Ciencia Tecnol. Aliment. 2011, 31 (2), 475-480.
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 27
4
4
01
02
1
8
ACS Paragon Plus Environment

Page 19 of 27
Journal of Agricultural and Food Chemistry
4
4
03
Table 1. δ (400 MHz) and δ (100 MHz) NMR Spectral Data of PG, Mono-
H
C
04
05
ACR Adduct of PG (PG-ACR; CD OD, δ in ppm and J in Hz), and Di-ACR Adduct
3
4
of PG (PG-2ACR; DMSO, δ in ppm and J in Hz)
No.
PG
PG-ACR
PG-2ACR
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
7
8
9
-
120.46
108.90
144.98
138.29
144.98
108.90
167.48
-
-
-
-
-
118.51
117.31
141.11
138.01
142.49
110.89
167.50
-
-
-
-
-
-
-
122.17/122.13
113.01/112.88
139.89/139.75
135.83/135.77
139.89/139.75
113.01/112.88
168.73/168.68
66.38
7.08 (s)
-
-
-
7.08 (s)
-
7.13 (s)
-
4.19 (t, J = 6.4) 66.18
1.76 (m) 21.76
1.03 (t, J = 7.4) 9.52
4.17 (t, J = 6.4) 65.72
1.77 (m) 21.82
4.15 (tt, J = 6.4, 1.8)
1.67 (m)
22.05
1
1
1
1
1
1
1
0
1
2
3
4
5
6
1.03 (t, J = 7.4) 9.60
3.10 (t, J = 6.4) 19.20
0.94 (t, J = 7.4)
2.63 (m)
11.04/11.03
18.96/18.75
27.88/27.71
91.59/91.24
18.96/18.75
27.88/27.71
91.59/91.24
-
-
-
-
-
-
-
-
-
-
-
-
1.93 (m)
27.29
1.78 (s)
5.57 (s)
91.08
5.49 (ddd, J = 12.6, 6.0, 3.0)
2.63 (m)
-
-
-
-
-
-
1.78 (s)
5.49 (ddd, J = 12.6, 6.0, 3.0)
4
06
1
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 27
2
4
4
07
08
Table 2. ESI-MS and ESI-MS Fragment Ions of PG-ACR, and PG-ACR
Corresponding GO or MGO Adducts
1
2
Compound
RT (min)
MW
MS (m/z)
MS (m/z)
PR-ACR
14.42
11.31
10.76
9.53
268
326
326
326
326
340
267 [M-H]-
325 [M-H]-
325 [M-H]-
325 [M-H]-
325 [M-H]-
339 [M-H]-
124/169/211/223
211/223/267
PG-ACR-GO-1
PG-ACR-GO-2
PG-ACR-GO-3
PG-ACR-GO-4
PG-ACR-MGO
169/211/267
211/223/267/307
211/223/267
7.87
12.51
211/223/267/321
4
09
2
0
ACS Paragon Plus Environment

Page 21 of 27
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
10
Figure legends
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Figure 1. Scavenging of ACR (0.5 mM) by PG, BHT and BHA (0.5 mM) at 100 ˚C
in phosphate buffer (pH 7.0). Each value was presented as the mean ± standard
deviation of three replications. (Different uppercase letters (A and B) indicate
significant (p < 0.05) difference of each sample among the times; different lowercase
letters (a and b) indicate significant (p < 0.05) difference among the samples at each
time-point.)
Figure 2. Chemical structures of ACR, PG, mono-, and di-ACR adducts of PG, and
significant HMBC (H→C) correlations of mono- and di-ACR adducts of PG.
Figure 3. HPLC chromatograms of PG-ACR (0.5 mM) incubation with 0.5 mM GO
(A) and MGO (B) under a ratio of 1:1 at 100 ˚C in phosphate buffer (0.2 M, pH 7.0)
for 10, 30, 120 min, respectively.
Figure 4. Inhibitory effect of PG on the formation of ACR, GO, MGO in pound
cakes. Each value was presented as the mean ± standard deviation of three
replications. (Different uppercase letters indicate significant (p < 0.05) difference of
the samples among the concentration; different lowercase letters indicate significant
(p < 0.05) difference of the sample among the species of RCS at each concentration.)
Figure 5. Total ion chromatogram of the extract of pound cakes in the presence of PG
2
(200 mg/kg) and the ESI-MS and MS fragment ions of PG, PG-ACR, PG-MGO, and
PG-GO.
2
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 27
4
31
4
4
32
33
Figure 1
2
2
ACS Paragon Plus Environment

Page 23 of 27
Journal of Agricultural and Food Chemistry
4
4
4
34
35
36
Figure 2
2
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 27
4
4
4
37
38
39
Figure 3
2
4
ACS Paragon Plus Environment

Page 25 of 27
Journal of Agricultural and Food Chemistry
4
4
40
41
Figure 4
4
4
42
43
2
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 27
4
4
44
45
Figure 5
4
46
2
6
ACS Paragon Plus Environment

Page 27 of 27
Journal of Agricultural and Food Chemistry
4
47
GRAPHIC TOC
4
4
48
49
2
7
ACS Paragon Plus Environment