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Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP
Food Chemistry 317 (2020) 126447
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Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP
T
⁎
Nana Zhanga,b,d,1, Yanfang Chena,e,1, Yueliang Zhaob,e, Daming Fana,c,d, , Lijie Lia,d, Bowen Yana,d, Guanjun Taoa, Jianxin Zhaoa,c,d, Hao Zhanga,c,d, Mingfu Wangb a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region c National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, China d School of Food Science and Technology, Jiangnan University, Wuxi, China e School of Food Science and Technology, Shanghai Ocean University, Shanghai, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: PhIP Caffeic acid 4-Vinylcatechol Microwave heating Dielectric loss
The inhibitory effect of caffeic acid on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was investigated in chemical model systems under microwave heating (MW). A mechanistic study was subsequently carried out to identify the inhibitory mechanism. The results showed that both for conductive heating (CV) and MW, the inhibition of PhIP increased with the concentration of caffeic acid but decreased with the prolongation of heating time. The inhibition on PhIP under MW was always higher than under CV, which were dominated by the difference in dielectric loss (ε″). UPLC-MS analysis showed that caffeic acid releases a CO2 molecule to produce 4-vinylcatechol which can form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. The structure of adduct was characterized as 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. The findings indicate that trapping of phenylacetaldehyde by 4vinylcatechol is a key mechanism of caffeic acid in inhibiting PhIP formation.
1. Introduction Heterocyclic amines (HAs) are a carcinogenic and mutagenic class of polycyclic aromatic compounds produced in protein-rich foods during high temperature processing (Dundar, Sarıçoban, & Yılmaz, 2012; Layton et al., 1995; Wakabayashi, Nagao, Esumi, & Sugimura, 1992). Among them, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is the most frequent and abundant in foods, especially in meat products (Warzecha et al., 2004; Iwasaki et al., 2010). Studies have found that PhIP can cause tumors in animal models (Shirai et al., 1997), and long-term exposure to PhIP increased the risk of mammary carcinogenesis in the second generation through a transplacental route or its secretion into the milk. (Ito et al., 1997). In addition, epidemiological studies have shown that there is a positive correlation between PhIP intake (via meat consumption) and the risks of cancers in different tissues such as breast and colon (Sinha et al., 2000; Sinha et al., 2005). It is reported that the formation of HAs can be inhibited by changing
the processing methods (Gibis, Kruwinnus, & Weiss, 2015; Johansson, Fredholm, Bjerne, & Jägerstad, 1995) or adding antioxidant compounds, especially polyphenols (Janoszka, 2010; Murkovic, Steinberger, & Pfannhauser, 1998; Oguri, Suda, Totsuka, Sugimura, & Wakabayashi, 1998; Quelhas et al., 2010). Studies have found that some specific polyphenols can effectively inhibit the formation of PhIP via trapping of the intermediate, phenylacetaldehyde (Cheng, Chen, & Wang et al., 2008). However, these polyphenols such as naringenin and quercetin usually have hydroxyl groups at the meta-position while the hydroxyl group on the phenyl ring of caffeic acid is at the ortho-position, and the carboxyl group is separated from the phenyl ring by a carbon-carbon double bond. Previous studies found that the inhibitory effects of polyphenols on PhIP formation fell in the order of resorcinol > catechol > hydroquinone (Salazar, Arámbula-Villa, Hidalgo, & Zamora, 2014), which indicates that the inhibitory effect may associate with the position of hydroxyl group. In addition, the degradation of caffeic acid at high temperatures may occur to varying
Abbreviations: HAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine; MW, microwave heating; CV, conductive heating; RMSE, root mean square error; EGCG, epigallocatechin gallate ⁎ Corresponding author at: School of Food Science and Technology, Jiangnan University, Wuxi, China. E-mail address: [email protected] (D. Fan). 1 These authors contribute equally. https://doi.org/10.1016/j.foodchem.2020.126447 Received 9 October 2019; Received in revised form 16 January 2020; Accepted 18 February 2020 Available online 19 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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lasting for 0.5–2 h.
degrees, resulting in a range of phenolic products such as 4-ethylcatechol and catechol. Considering the unique chemical structure and the thermal stability, it is reasonable to assume that the roles of caffeic acid in trapping of phenylacetaldehyde may different from those polyphenols with hydroxyl groups at the meta-position, which implies that the inhibition of PhIP by caffeic acid may be achieved by capturing phenylacetaldehyde by itself, or by its degradation products. Microwave heating (MW) is a green and efficient thermal processing method and has been widely used in food processing. Studies have shown that meats pretreated with microwaves or subjected to direct microwave curing significantly reduced PhIP content (Felton, Fultz, Dolbeare, & Knize, 1994; Knize, Dolbeare, Carroll, Moore, & Felton, 1994). The polyhydroxy structure of caffeic acid may lead to specific response under a microwave field. Reflection loss (RL) is a comprehensive index to evaluate microwave absorption ability while dielectric loss (ε″) is an indicator of the ability to convert microwave energy into heat. Both RL and ε″ have the potential to indirectly affect the PhIP formation under a microwave field (Zhang et al., 2017). However, the relationship between electromagnetic characteristics (RL and ε″) of caffeic acid and its inhibitory effect on PhIP formation remains unclear. In the current study, the effect of caffeic acid on PhIP formation under a microwave field was investigated and the relationship with electromagnetic characteristics was further clarified. A mechanistic study was carried out to identify the inhibitory mechanism.
2.4. Sample preparation for PhIP determination Samples were prepared referring to the method of Fan with slight modifications (Fan et al., 2018). A 40 mL aliquot of the reaction solution was placed in a glass dish for freeze-drying (79480–30, CONCO, USA). Then, 3 mL of 2 mol/L NaOH and 7.5 mL of ethyl acetate were added to the freeze-dried samples obtained from 10 mL aliquots of the reaction solution. The samples were vortexed and ultrasonically extracted for 2–12 min, followed by centrifugation at 6500 r/min for 3 min. The supernatant was dried with nitrogen, dissolved in 0.2 mL of methanol and filtered (0.22 μm) before UPLC-MS analysis. The inhibition rate (%) of caffeic acid against PhIP formation was calculated as follows:
Inhibition rate (%) = (Y1 − Y2)/Y1 × 100
where Y1 is the PhIP content of the blank and Y2 is the PhIP content with the addition of caffeic acid. 2.5. UPLC-MS analysis of PhIP All filtrates were analyzed on a UPLC-MS instrument equipped with a tandem quadrupole mass spectrometer (Waters, USA). Separation of products was carried out on a Waters BEH phenyl column (50 × 2.10 mm, 1.70 μm). The LC conditions were as follows: flow, 0.30 mL/min; column temperature, 35 °C; and injection volume, 10 μL. The mobile phase was composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 10% A; 8 min, 70% A; 9 min, 70% A; 9.5 min, 10% A. Prior to the next injection, 10% A was held for 5 min to equilibrate the column. MS conditions: ion source, ESI+; multi-response monitoring mode; source block temperature, 120 °C; capillary voltage, 2.5 kV; cone voltage, 35 V; desolvation temperature, 400 °C; cone gas flow, 50 L/h; and collision gas (argon) flow, 0.3 mL/min.
2. Materials and methods 2.1. Materials Phenylalanine, anhydrous glucose, creatinine and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PhIP standard and 4-vinylcatechol were from Toronto Research Chemicals (Toronto, Canada). Caffeic acid, formic acid, dodecane and phenylacetaldehyde were from Sigma-Aldrich (St. Louis, MO, USA). SEPHADEX LH-20 glucan gel was from GE Healthcare Biosciences AB (Sweden). DIAION HP20 macroporous resin was from Mitsubishi Corporation (Tokyo, Japan). All solvents used were of analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). An SKM digital display thermostatic heating set was purchased from Runxin Experimental Instrument Co., Ltd. (Shandong, China).
2.6. Determination of dielectric loss A vector network analyzer (E5071C, Agilent, USA) with an openended coaxial line was used to determine the ε″. The high-temperature probe was calibrated with air, a short circuit, and distilled water, respectively. Each treatment was measured in triplicate (Zhang et al., 2019).
2.2. Construction of microwave heating methods
2.7. Calculations of reflection loss
The heating methods was set up referring to Zhang’s method with slight modifications (Zhang et al., 2017). The PhIP chemical model systems (100 mL) were heated in a flexiWAVE microwave synthesizer (Milestone, Italy) (for MW) or a thermostatic heater set at 100 °C for 2 h (for CV). The multi-stage MW program was designed to simulate the heating curve of CV, and the root mean square error (RMSE) was used to evaluate the fitting degree of these two heating curves, as shown in Fig. S1, to ensure that the two methods had similar heating rates. The RMSE value of the two heating curves was 1.75, indicating a high degree of fitting.
RMSE =
1 N
(2)
Computer Simulation Technology (CST) Microwave Studio was used to calculate RL which is an indicator of the overall absorption performance. First, a square reaction system (200 × 200 mm, 4 mm) was designed and the dielectric parameters were then imported. The boundary conditions were set as follows: X and Y (Unit Cell), Z min (Electric Et = 0), and Z max (open (add space)). After that, the simulation was started (Aguilar, Al-Joumayly, Hagness, & Behdad, 2010). 2.8. Sample preparation for the detection of thermal stability of caffeic acid
N
∑ (TMW − TCV
)2
n=1
The caffeic acid solutions (3 mmol/L) were treated with CV or MW at 100 °C for 0, 0.5, 1.0, 1.5 and 2 h, respectively, and subsequently cooled in ice water. Then, 10 mL aliquots of the samples were extracted with 20 mL of ethyl acetate. The upper layer was concentrated on a rotary evaporator under vacuum and then dissolved with 1 mL of methanol before UPLC-MS.
(1)
2.3. Construction of PhIP chemical model system Referring to Fan’s method (Fan et al., 2018), glucose, phenylalanine and creatinine were dissolved in deionized water to ensure final concentrations of 10, 20 and 20 mmol/L, respectively. Then, 0–5 mmol/L of caffeic acid was added to the system. The samples were immediately cooled in ice water after MW or CV treatment, with the treatments
2.9. Separation and purification of caffeic acid degradation products Referring to Cheng’s method (Cheng, Wong et al., 2008), 120 mL of 2
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Fig. 1. Amount of PhIP and percentage of inhibition on PhIP as a function of the concentration of caffeic acid (a) (c) and the heating time (b) (d). Values are mean ± standard deviation, n = 3, *p < 0.05, **p < 0.01.
the caffeic acid solution treated with MW at 100 °C for 2 h was extracted with ethyl acetate and n-hexane at a volume ratio of 1: 2: 1. The upper solution was concentrated on a rotary evaporator under vacuum, dissolved with 2 mL of methanol and separated using a Sephadex LH-20 column (40 × 4 cm). Next, 100 g of Sephadex LH-20 gel was swelled with 70% methanol overnight, stirred and added to a column pre-rinsed with anhydrous methanol. When the liquid level fell to the gel interface, the sample was then added into the column and drained until the liquid level was flush with the gel interface. Elution was carried out with methanol, wherein the eluent was collected until the sample color spread to 4 cm from the lower end of the column, and a 15 mL collection volume was taken for each tube. Collection was stopped when the colored part of the sample was completely diffused out of the gel column. Aliquots of 1.5 mL of all eluates were filtered and analyzed with HPLC. The mobile phase was composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 70% A, 30% B; 50 min, 70% A, 30% B. The components with the same retention time were combined, concentrated by a rotary evaporator (RV3V, IKA, Germany), dissolved with 0.5 mL of methanol, filtered and then subjected to UPLC-MS analysis. The expected isolates were combined and purified again using a Sephadex column. A Nicolet Nexus FTIR spectrometer was used for structural comparison of the main degradation products of caffeic acid.
composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 5% A; 15 min, 20% A; 20 min, 40% A; 25 min, 80% A; 27 min, 100% A; 30 min, 5% A. MS conditions: ion source, ESI-; MSE with both low and high collision energies; source block temperature, 120 °C; capillary voltage, 3.0 kV; cone voltage, 35 V; desolvation temperature, 400 °C; cone gas flow, 600 L/h; and collision gas (argon) flow, 1.5 mL/min.
2.10. UPLC-MS analysis of caffeic acid degradation products
2.12. Statistical analysis
All filtrates were analyzed on a UPLC-MS instrument equipped with a tandem quadrupole time-of-flight (Q-TOF) mass spectrometer (Waters, USA). LC was performed on a Waters Acquity UPLC with a photodiode array (PDA) detector and an autosampler. Referring to Cheng’s method (Cheng, Wong et al., 2008), separation of products was carried out on a Waters BEH C18 column (50 × 2.10 mm, 1.70 μm). The LC conditions were as follows: flow, 0.30 mL/min; column temperature, 45 °C; and injection volume, 10 μL. The mobile phase was
Statistical analyses were performed using GraphPad Prism 7.0 and Microsoft Excel 2010. Origin 8.5 software was used to integrate the areas of time-fluorescence intensity curves. Masslynx V4 was used to analyze the UPLC-MS data. All experiments were performed three times and the data were presented as mean ± SD.
2.11. Separation, purification and identification of caffeic acid degradation product – phenylacetaldehyde adducts Caffeic acid and phenylacetaldehyde were dissolved in diethylene glycol in a ratio of 1:5 (v/v) to ensure the final concentration of the two was 25:125 mmol/L. The mixture was heated at 100 °C for 4 h and subsequently cooled in ice water. The mixture was extracted with water, ethyl acetate and n-hexane at the ratio of 1:1:2:1. The supernatant was dried under vacuum and dissolved with 2 mL of methanol. Separation was carried out on a Sephadex LH-20 column (40 × 4 cm). A 2 mg sample of the purified target was dissolved in 1 mL of chloroform and analyzed with 1H NMR (Avance III-400 MHz, Bruker, Germany).
3
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3. Results and discussion
degradation products of caffeic acid. The ion fragments of 4-vinylcatechol are shown in Table S1. Khuwijitjaru and co-workers heated caffeic acid at temperatures ranging from 100 to 250 °C and found that the carboxyl groups on the side chain were the most susceptible to being removed (Khuwijitjaru et al., 2014). Müller reported that 4-ethylcatechol was produced during roasting of coffee beans via pyrolysis of caffeic acid, and the formation pathway may involve 4-vinylcatechol as the reaction intermediate (Jia, Wang, Qi, Hong, & Lee, 2016; Müller et al., 2006). The relative molecular mass of 4-vinylcatechol is 136, and the molecular formula is C8H8O2, consistent with previous results. In combination with previous studies, it is reasonable to speculate that the main degradation product of caffeic acid formed under CV and MW is 4-vinylcatechol. The heattreated caffeic acid standard was separated and purified by a glucan gel chromatography column, and the degradation product of molecular weight 136 was localized by UPLC-MS. The components were concentrated, collected and re-purified, followed by comparison of the FTIR spectrum (Nicolet NcxUs, Thermo Electron, USA) with the 4-vinylcatechol standard. As shown in Fig. 2 (c), the FTIR spectra of the two were basically coincident. Thus, it can be determined that the degradation product of caffeic acid under CV and MW is 4-vinylcatechol, and a proposed pathway is shown in Fig. S2. The principal reaction is the release of CO2 from caffeic acid through thermal decarboxylation to produce 4-vinylcatechol with a molecular weight of 136. This is consistent with the conclusion of Terpinc (Terpinc et al., 2011).
3.1. Effect of caffeic acid and heating time on PhIP formation under microwave heating In this study, 0–5 mmol/L of caffeic acid solutions were added to PhIP-producing systems which were heated at 100℃ for 2 h to investigate the effect of the concentration of caffeic acid on PhIP formation under CV and MW. As shown in Fig. 1 (a), the amount of PhIP decreased with the increasing concentration of caffeic acid, 0.032 and 0.0051 ng/mL at 5 mmol/L under CV and MW, respectively. In fact, it is difficult to compare the results with previous works due to the existence of various variables such as the heating conditions, extraction methods and different systems. While in Fig. 1 (c), both for CV and MW, the inhibition of PhIP increased with the concentration of caffeic acid, and the trend toward greater inhibition gradually slowed as the concentration continued to increase. This finding may be explained by the fact that increased concentration promotes the reaction between the polyphenol (caffeic acid) and the intermediate of PhIP formation (phenylacetaldehyde). However, if the concentration of caffeic acid is increased beyond a certain level, in contrast, its inhibitory effect is weakened, implying that its reaction with phenylacetaldehyde is suppressed. In addition, at the same concentration, the inhibitory effect of caffeic acid against PhIP was always higher under MW than under CV. For example, at 5 mmol/L, the inhibition of PhIP was 93.7% under MW and 73.8% under CV. This difference suggests that at lower concentrations, MW either more strongly promotes the dissolution of caffeic acid or increases its activity, which leads to better inhibitory effects (Bouras et al., 2015; Wang et al., 2008). In addition, caffeic acid (3 mmol/L) was added to the reaction system to investigate its inhibitory effect on PhIP during heating times of 0–2 h. As shown in Fig. 1 (b), PhIP content increased with the heating time, 0.063 and 0.012 ng/mL at 2.0 h under CV (control, 0.128 ng/mL) and MW (control, 0.078 ng/mL), respectively. While in Fig. 1 (d), both under CV and MW, the inhibition of PhIP decreased with prolonged heating time. Moon and co-workers (Moon & Shin, 2013) investigated the inhibitory effect of epigallocatechin gallate (EGCG) on PhIP as a function of heating time at different temperatures. Their results showed that the inhibition of PhIP decreased at all temperatures with the extension of heating time due to the thermal instability of EGCG, the degradation constant of which increased by 180% when heated. Other studies found that decarboxylation or free radical reactions occurred when caffeic acid was heated, accompanied by various degrees of degradation (Khuwijitjaru et al., 2014; Moon & Shibamoto, 2010; Müller, Lang, & Hofmann, 2006). Therefore, we speculate that the decreasing inhibitory effect of caffeic acid on PhIP with time may be caused by thermal degradation during heating. In addition, the inhibition rate under MW always declined less than under CV and remained higher. This indicates that microwave heating not only promotes a high level of PhIP inhibition, but also weakens the decrease of the inhibition rate caused by prolonged heating time. This may be due to the difference in electromagnetic behavior of caffeic acid during MW, which is reflected in the change of microwave absorption ability or dielectric properties.
3.3. Effect of heating time on reflection loss, dielectric loss and caffeic acid degradation products formation In order to show whether the difference of electromagnetic characteristics (RL and ε″) under CV and MW contributes to better inhibitory effects on PhIP formation, firstly, the RL of caffeic acid (3 mmol/L) heated for 0–2 h were evaluated. As shown in Fig. 3 (a), with the extension of heating time, there were no differences of RL under MW and CV, which indicates that the overall microwave absorption abilities of caffeic acid are basically the same. Based on this, the ε″ of caffeic acid was further determined to evaluate whether there are differences in the ability to convert microwave energy into heat. As can be seen from Fig. 3 (b), the ε″ of caffeic acid was higher under MW than under CV, which indicates that the systems under microwave field are able to absorb more microwave energy and further convert it into heat, and finally, indirectly promotes the inhibition on PhIP formation under MW. Additionally, in order to verify that whether the decreasing inhibitory effect of caffeic acid on PhIP with time was caused by thermal degradation during heating, here, the effect of heating time on the formation of main degradation products (4-vinylcatechol) was evaluated. UPLC-MS was carried out to analyze the caffeic acid standards (3 mmol/L) treated with CV or MW at 100℃for 0–2 h. As shown in Fig. 3 (c), under both CV and MW, the amount of 4-vinylcatechol increased with the heating time while the similar trend was not found in the inhibition of PhIP. This phenomenon may attribute to the basically unchanged RL values with heating time, and further indicate that the thermal degradation of caffeic acid may not dominate the inhibitory effect. In addition, at each stage of heat treatment, the concentration of 4-vinylcatechol under MW was always higher than that of CV. When heated for 2 h, the formation of 4-vinylcatechol under MW reached 0.092 ng/mL while that under CV was 0.065 ng/mL. Combined with the results above, the ε″ of caffeic acid under MW was always higher than under CV, it can be concluded that the effect of ε″ on PhIP formation was dominant during the heating times, which may compensate for or even exceed the reduced inhibitory effect caused by the degradation of caffeic acid.
3.2. Identification of main degradation products of caffeic acid Considering that caffeic acid degrades easily when heated, here, the main degradation products was first identified for further analysis. The caffeic acid standard was treated under CV or MW at 100 °C for 2 h. The mass spectrum of the main degradation products with low collision energy is shown in Fig. 2 (a), wherein the m/z of the parent ion was 135.0436 and the relative molecular weight was 136.0514. Further analysis of the mass spectrum with high collision energy, as shown in Fig. 2 (b), shows that there were ion fragments with m/z of 135.0456, 117.0345 and 107.0526, which were consistent with previous studies (Terpinc et al., 2011) and further used to qualitatively identify the main 4
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Fig. 2. The mass spectra with low collision energy (a) and high collision energy (b), and the FTIR spectra (c) of the main degradation products of caffeic acid and 4vinylcatechol standard.
3.4. Identification of caffeic acid degradation product – phenylacetaldehyde adduct
inhibitory effect of caffeic acid on PhIP formation may be due to the capture of phenylacetaldehyde by its degradation product. To further identify the adducts, the mass spectra of the two target products were analyzed, as shown in Fig. 5. It was found that for the primary mass spectrometry fragments with m/z of 255, the main products had m/z of 135 and 119 (Fig. 5 (a)), which coincide with the fragments of 4-vinylcatechol and phenylacetaldehyde. When increasing the collision energy, the content of the ion with m/z of 255 decreased while that of the above-mentioned fragments increased (Fig. 5 (b)). Therefore, it can be concluded that the parent ion of the compound has m/z of 255, and its molecular weight is 256, making it an adduct of one molecule of 4-vinylcatechol with one molecule of phenylacetaldehyde. Similarly, the compound with m/z of 391 has main products with m/z of 135 and 271 (Fig. 5 (c)), which coincides with the fragments of one molecule and two molecules of 4-vinylcatechol, respectively. When increasing the collision energy, the content of the ion with m/z of 391 decreased, while that of the fragment with m/z of 135 increased (Fig. 5 (d)). This indicated that the parent ion of the compound has m/z of 391, and the molecular weight is 392. This compound is formed by fragmentation of phenylacetaldehyde to form a fragment of 271, together with a dimer structure of 4-vinylcatechol, so the compound is an adduct of two molecules of 4-vinylcatechol with one molecule of phenylacetaldehyde.
Studies have shown that several polyphenols, such as quercetin, naringenin, dihydromyricetin and EGCG, can capture phenylacetaldehyde, an intermediate during PhIP formation, and form new adducts to inhibit PhIP formation (Cheng, Chen et al., 2008; Cheng, Wong et al., 2008; Cheng et al., 2009). Given that caffeic acid differs from other polyphenols both structurally and in terms of thermal stability, its inhibition pathway against PhIP may be unique, which means that the trapping of phenylacetaldehyde may be achieved by caffeic acid or its degradation products. Therefore, to further clarify the role of caffeic acid in trapping phenylacetaldehyde and further inhibiting PhIP formation, a caffeic acid–phenylacetaldehyde mixture was heated under MW, in which the concentrations of the two reactants were 25 and 125 mmol/L, respectively. The mixture was then extracted with ethyl acetate and n-hexane at a ratio of 1: 2: 1, concentrated, and separated by an LH-20 Sephadex column. Each tube of the eluate was 20 mL, and the sequence of elution of the target was determined by HPLC. A total of 24 tubes were collected from the 300 mL reaction system. The parent ion of m/z = 255 was found in the first eluate tube, which was confirmed by selecting ions for which the retention time of the target was 14.21 min, as shown in Fig. 4 (a) and 4 (b). The parent ion of m/ z = 391 was found in the second tube, which was further verified by the fact that the retention time of the target was 15.72 min, as shown in Fig. 4 (c) and 4 (d). Table S2 showed the proposed molecular structure of new adducts, both m/z = 255 and m/z = 391 were formed from 4vinylcatechol and phenylacetaldehyde. The results indicate that the
3.5. Structural characterization of caffeic acid degradation product – phenylacetaldehyde adduct Mass spectral analysis showed that the two targets clearly contained 5
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Fig. 3. Reflection loss (a), dielectric loss (b) and the formation of 4-vinylcatechol (c) as a function of the heating time. Asterisks in Fig. 3 (b) indicate the significant differences between CV and MW treatments. Values are mean ± standard deviation, n = 3, *p < 0.05, **p < 0.01.
inferred. The compound has an aldehyde-based hydrogen at the chemical shift of 9.76, and three hydrogens are derived from a methyl group at the chemical shift of 1.34. This methyl group may be due to 4vinylcatechol undergoing an addition reaction with phenylacetaldehyde, causing the carbon-carbon double bond to become saturated. In Fig. 6, the presence of hydrogen No. 1, 2 and 3 indicates that caffeic acid undergoes a decarboxylation reaction triggered by the absorption of electromagnetic energy under a microwave field and its subsequent conversion into heat. An addition reaction occurs between the unsaturated bond of 4-vinylcatechol and phenylacetaldehyde, which
fragment ions having m/z of 135 and 119. However, the UV absorption of the two targets was not strong, and that of the ion with a molecular weight of 392 was even weaker. Therefore, the target with a molecular weight of 256 was selected for further separation and purification by preparative chromatography. The 1H NMR spectrum is shown in Fig. 6, wherein δ = 9.76 (d, 1H, J = 2.4 Hz), δ = 7.21–7.19 (m, 5H), δ = 7.01–7.00 (m, 1H), δ = 6.62–6.41 (m, 3H), δ = 5.3–4.9 (m, 2H), δ = 3.65 (dd, 1H, J = 2.8, 10.0 Hz), δ = 3.47–3.43 (m, 1H), δ = 1.34 (d, 3H, J = 6.8 Hz). From the 1H NMR spectrum, the following structural details of the compound with a molecular weight of 256 can be
Fig. 4. The TIC spectrum (a) and selective ion spectrum (b) of the parent ion of m/z = 255, and the TIC spectrum (c) and selective ion spectrum (d) of the parent ion of m/z = 392. 6
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Fig. 5. The mass spectra of the parent ion of m/z = 255 with both low (a) and high (b) collision energies, and the mass spectra of the parent ion of m/z = 392 with both low (c) and high (d) collision energies.
Fig. 6. The 1H NMR spectrum of the compound with a molecular weight of 256.
finally produces 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. In summary, the inhibition pathway of caffeic acid against phenylacetaldehyde formation under a microwave field is shown in Fig. S3. The inhibition mechanism of caffeic acid against PhIP under a microwave field is different from that of meta-dihydroxy polyphenols. The decarboxylation product of caffeic acid under MW forms an adduct with the intermediate of PhIP formation, phenylacetaldehyde, thus
inhibiting PhIP formation by blocking the interaction of phenylacetaldehyde with creatinine. 4. Conclusion The inhibition of PhIP formation by caffeic acid increased with the caffeic acid concentration but decreased with the prolongation of heating time. The inhibitory effect of caffeic acid on PhIP formation was 7
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stronger under MW than under CV, which was dominated by ε″. The effect of ε″ may compensate for or even exceed the reduced inhibitory effect caused by the degradation of caffeic acid. The inhibitory effect of caffeic acid on PhIP under a microwave field involves capturing phenylacetaldehyde, a key PhIP intermediate, which is also the case for inhibition by meta-dihydroxy polyphenols. The difference is that caffeic acid is first degraded to form 4-vinylcatechol, which then forms an adduct with phenylacetaldehyde, finally leading to significant inhibition of PhIP formation.
the formation of heterocyclic aromatic amines and sensory quality in fried bacon. Food Chemistry, 168, 383–389. Ito, N., Hasegawa, R., Imaida, K., Tamano, S., Hagiwara, A., Hirose, M., & Shirai, T. (1997). Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in the rat. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 376(1–2), 107–114. Iwasaki, M., Kataoka, H., Ishihara, J., Takachi, R., Hamada, G. S., Sharma, S., & Tsugane, S. (2010). Heterocyclic amines content of meat and fish cooked by Brazilian methods. Journal of Food Composition and analysis, 23(1), 61–69. Janoszka, B. (2010). Heterocyclic amines and azaarenes in pan-fried meat and its gravy fried without additives and in the presence of onion and garlic. Food Chemistry, 120(2), 463–473. Jia, C., Wang, X., Qi, J., Hong, S., & Lee, K. (2016). Antioxidant properties of caffeic acid phenethyl ester and 4-vinylcatechol in stripped soybean oil. Journal of Food Science, 81(1), C35–C41. Johansson, M., Fredholm, L., Bjerne, I., & Jägerstad, M. (1995). Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues. Food and Chemical Toxicology, 33(12), 993–1004. Khuwijitjaru, P., Plernjit, J., Suaylam, B., Samuhaseneetoo, S., Pongsawatmanit, R., & Adachi, S. (2014). Degradation kinetics of some phenolic compounds in subcritical water and radical scavenging activity of their degradation products. Canadian Journal of Chemical Engineering, 92(5), 810–815. Knize, M., Dolbeare, F., Carroll, K., Moore, D., II, & Felton, J. (1994). Effect of cooking time and temperature on the heterocyclic amine content of fried beef patties. Food and Chemical Toxicology, 32(7), 595–603. Layton, D. W., Bogen, K. T., Knize, M. G., Hatch, F. T., Johnson, V. M., & Felton, J. (1995). Cancer risk of heterocyclic amines in cooked foods: An analysis and implications for research. Carcinogenesis, 16(1), 39–52. Moon, J.-K., & Shibamoto, T. (2010). Formation of volatile chemicals from thermal degradation of less volatile coffee components: Quinic acid, caffeic acid, and chlorogenic acid. Journal of Agricultural and Food Chemistry, 58(9), 5465–5470. Moon, S.-E., & Shin, H.-S. (2013). Inhibition of mutagenic 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) formation using various food ingredients in a model systems. Food Science and Biotechnology, 22(2), 323–329. Müller, C., Lang, R., & Hofmann, T. (2006). Quantitative precursor studies on di- and trihydroxybenzene formation during coffee roasting using “in bean” model experiments and stable isotope dilution analysis. Journal of Agricultural and Food Chemistry, 54(26), 10086–10091. Murkovic, M., Steinberger, D., & Pfannhauser, W. (1998). Antioxidant spices reduce the formation of heterocyclic amines in fried meat. Z Lebensm Unters Forsch, 207(6), 477–480. Oguri, A., Suda, M., Totsuka, Y., Sugimura, T., & Wakabayashi, K. (1998). Inhibitory effects of antioxidants on formation of heterocyclic amines. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis, 402(1), 237–245. Quelhas, I., Petisca, C., Viegas, O., Melo, A., Pinho, O., & Ferreira, I. (2010). Effect of green tea marinades on the formation of heterocyclic aromatic amines and sensory quality of pan-fried beef. Food Chemistry, 122(1), 98–104. Salazar, R., Arámbula-Villa, G., Hidalgo, F. J., & Zamora, R. (2014). Structural characteristics that determine the inhibitory role of phenolic compounds on 2-amino-1methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) formation. Food Chemistry, 151, 480–486. Shirai, T., Sano, M., Tamano, S., Takahashi, S., Hirose, M., Futakuchi, M., ... Ito, N. (1997). The prostate: A target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) derived from cooked foods. Cancer Research, 57(2), 195–198. Sinha, R., Gustafson, D. R., Kulldorff, M., Wen, W. Q., Cerhan, J. R., & Zheng, W. (2000). 2-Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine, a carcinogen in high-temperature-cooked meat, and breast cancer risk. Journal of the National Cancer Institute, 92(16), 1352–1354. Sinha, R., Peters, U., Cross, A. J., Kulldorff, M., Weissfeld, J. L., Pinsky, P. F., ... Hayes, R. B. (2005). Meat, meat cooking methods and preservation, and risk for colorectal adenoma. Cancer Research, 65(17), 8034–8041. Terpinc, P., Polak, T., Šegatin, N., Hanzlowsky, A., Ulrih, N. P., & Abramovič, H. (2011). Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food Chemistry, 128(1), 62–69. Wakabayashi, K., Nagao, M., Esumi, H., & Sugimura, T. (1992). Food-derived mutagens and carcinogens. Cancer Research, 52(7 Suppl), 2092s–2098s. Wang, Y., You, J., Yu, Y., Qu, C., Zhang, H., Ding, L., & Li, X. (2008). Analysis of ginsenosides in Panax ginseng in high pressure microwave-assisted extraction. Food Chemistry, 110(1), 161–167. Warzecha, L., Janoszka, B., Błaszczyk, U., Stróżyk, M., Bodzek, D., & Dobosz, C. (2004). Determination of heterocyclic aromatic amines (HAs) content in samples of household-prepared meat dishes. Journal of Chromatography B, 802(1), 95–106. Zhang, N., Fan, D., Chen, M., Chen, Y., Huang, J., Zhou, W., & Chen, W. (2017). Concentration-related microwave heating processes: Electromagnetic interference of Maillard reaction substrates (glucose and lysine). RSC Advances, 7(39), 24382–24386. Zhang, N., Fan, D., Zhao, Y., Wu, Y., Yan, B., Zhao, J., ... Zhang, H. (2019). Dielectric loss mediated promotion of microwave heating in the Maillard reaction. LWT, 101, 559–566.
CRediT authorship contribution statement Nana Zhang: Conceptualization, Methodology, Formal analysis, Writing - review & editing. Yanfang Chen: Data curation, Software, Writing - original draft. Yueliang Zhao: Formal analysis, Investigation. Daming Fan: Conceptualization, Supervision, Funding acquisition, Resources. Lijie Li: Software, Validation. Bowen Yan: Visualization. Guanjun Tao: Data curation. Jianxin Zhao: Visualization. Hao Zhang: Project administration. Mingfu Wang: Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province-Outstanding Youth Foundation [BK20170052]; the National Natural Science Foundation of China [31671821]; the National First-class Discipline Program of Food Science and Technology [JUFSTR20180102]. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.126447. References Aguilar, S. M., Al-Joumayly, M. A., Hagness, S. C., & Behdad, N. (2010). Design of a miniaturized dual-band patch antenna as an array element for microwave breast imaging. Antennas and Propagation Society International Symposium (APSURSI) (pp. 1– 4). IEEE. Bouras, M., Chadni, M., Barba, F. J., Grimi, N., Bals, O., & Vorobiev, E. (2015). Optimization of microwave-assisted extraction of polyphenols from Quercus bark. Industrial Crops & Products, 77, 590–601. Cheng, K.-W., Chen, F., & Wang, M. (2008). Preventive potential and mechanism of dietary phenolics on the formation of mutagenic heterocyclic amines. The 14th world conference of food science and technology (pp. 394–395). . Cheng, K. W., Wong, C. C., Chao, J., Lo, C., Chen, F., Chu, I., & Wang, M. (2009). Inhibition of mutagenic PhIP formation by epigallocatechin gallate via scavenging of phenylacetaldehyde. Molecular Nutrition & Food Research, 53(6), 716–725. Cheng, K.-W., Wong, C. C., Cho, C. K., Chu, I. K., Sze, K. H., Lo, C., & Wang, M. (2008). Trapping of phenylacetaldehyde as a key mechanism responsible for naringenin’s inhibitory activity in mutagenic 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine formation. Chemical Research in Toxicology, 21(10), 2026–2034. Dundar, A., Sarıçoban, C., & Yılmaz, M. T. (2012). Response surface optimization of effects of some processing variables on carcinogenic/mutagenic heterocyclic aromatic amine (HAA) content in cooked patties. Meat Science, 91(3), 325–333. Fan, D., Li, L., Zhang, N., Zhao, Y., Cheng, K.-W., & Yan, B. (2018). A comparison of mutagenic PhIP and beneficial 8-C-(E-phenylethenyl) quercetin and 6-C-(E-phenylethenyl) quercetin formation under microwave and conventional heating. Food & Function, 9(7), 3853–3859. Felton, J., Fultz, E., Dolbeare, F., & Knize, M. (1994). Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties. Food and Chemical Toxicology, 32(10), 897–903. Gibis, M., Kruwinnus, M., & Weiss, J. (2015). Impact of different pan-frying conditions on
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Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP
T
⁎
Nana Zhanga,b,d,1, Yanfang Chena,e,1, Yueliang Zhaob,e, Daming Fana,c,d, , Lijie Lia,d, Bowen Yana,d, Guanjun Taoa, Jianxin Zhaoa,c,d, Hao Zhanga,c,d, Mingfu Wangb a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region c National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, China d School of Food Science and Technology, Jiangnan University, Wuxi, China e School of Food Science and Technology, Shanghai Ocean University, Shanghai, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: PhIP Caffeic acid 4-Vinylcatechol Microwave heating Dielectric loss
The inhibitory effect of caffeic acid on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was investigated in chemical model systems under microwave heating (MW). A mechanistic study was subsequently carried out to identify the inhibitory mechanism. The results showed that both for conductive heating (CV) and MW, the inhibition of PhIP increased with the concentration of caffeic acid but decreased with the prolongation of heating time. The inhibition on PhIP under MW was always higher than under CV, which were dominated by the difference in dielectric loss (ε″). UPLC-MS analysis showed that caffeic acid releases a CO2 molecule to produce 4-vinylcatechol which can form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. The structure of adduct was characterized as 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. The findings indicate that trapping of phenylacetaldehyde by 4vinylcatechol is a key mechanism of caffeic acid in inhibiting PhIP formation.
1. Introduction Heterocyclic amines (HAs) are a carcinogenic and mutagenic class of polycyclic aromatic compounds produced in protein-rich foods during high temperature processing (Dundar, Sarıçoban, & Yılmaz, 2012; Layton et al., 1995; Wakabayashi, Nagao, Esumi, & Sugimura, 1992). Among them, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is the most frequent and abundant in foods, especially in meat products (Warzecha et al., 2004; Iwasaki et al., 2010). Studies have found that PhIP can cause tumors in animal models (Shirai et al., 1997), and long-term exposure to PhIP increased the risk of mammary carcinogenesis in the second generation through a transplacental route or its secretion into the milk. (Ito et al., 1997). In addition, epidemiological studies have shown that there is a positive correlation between PhIP intake (via meat consumption) and the risks of cancers in different tissues such as breast and colon (Sinha et al., 2000; Sinha et al., 2005). It is reported that the formation of HAs can be inhibited by changing
the processing methods (Gibis, Kruwinnus, & Weiss, 2015; Johansson, Fredholm, Bjerne, & Jägerstad, 1995) or adding antioxidant compounds, especially polyphenols (Janoszka, 2010; Murkovic, Steinberger, & Pfannhauser, 1998; Oguri, Suda, Totsuka, Sugimura, & Wakabayashi, 1998; Quelhas et al., 2010). Studies have found that some specific polyphenols can effectively inhibit the formation of PhIP via trapping of the intermediate, phenylacetaldehyde (Cheng, Chen, & Wang et al., 2008). However, these polyphenols such as naringenin and quercetin usually have hydroxyl groups at the meta-position while the hydroxyl group on the phenyl ring of caffeic acid is at the ortho-position, and the carboxyl group is separated from the phenyl ring by a carbon-carbon double bond. Previous studies found that the inhibitory effects of polyphenols on PhIP formation fell in the order of resorcinol > catechol > hydroquinone (Salazar, Arámbula-Villa, Hidalgo, & Zamora, 2014), which indicates that the inhibitory effect may associate with the position of hydroxyl group. In addition, the degradation of caffeic acid at high temperatures may occur to varying
Abbreviations: HAs, heterocyclic amines; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine; MW, microwave heating; CV, conductive heating; RMSE, root mean square error; EGCG, epigallocatechin gallate ⁎ Corresponding author at: School of Food Science and Technology, Jiangnan University, Wuxi, China. E-mail address: [email protected] (D. Fan). 1 These authors contribute equally. https://doi.org/10.1016/j.foodchem.2020.126447 Received 9 October 2019; Received in revised form 16 January 2020; Accepted 18 February 2020 Available online 19 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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lasting for 0.5–2 h.
degrees, resulting in a range of phenolic products such as 4-ethylcatechol and catechol. Considering the unique chemical structure and the thermal stability, it is reasonable to assume that the roles of caffeic acid in trapping of phenylacetaldehyde may different from those polyphenols with hydroxyl groups at the meta-position, which implies that the inhibition of PhIP by caffeic acid may be achieved by capturing phenylacetaldehyde by itself, or by its degradation products. Microwave heating (MW) is a green and efficient thermal processing method and has been widely used in food processing. Studies have shown that meats pretreated with microwaves or subjected to direct microwave curing significantly reduced PhIP content (Felton, Fultz, Dolbeare, & Knize, 1994; Knize, Dolbeare, Carroll, Moore, & Felton, 1994). The polyhydroxy structure of caffeic acid may lead to specific response under a microwave field. Reflection loss (RL) is a comprehensive index to evaluate microwave absorption ability while dielectric loss (ε″) is an indicator of the ability to convert microwave energy into heat. Both RL and ε″ have the potential to indirectly affect the PhIP formation under a microwave field (Zhang et al., 2017). However, the relationship between electromagnetic characteristics (RL and ε″) of caffeic acid and its inhibitory effect on PhIP formation remains unclear. In the current study, the effect of caffeic acid on PhIP formation under a microwave field was investigated and the relationship with electromagnetic characteristics was further clarified. A mechanistic study was carried out to identify the inhibitory mechanism.
2.4. Sample preparation for PhIP determination Samples were prepared referring to the method of Fan with slight modifications (Fan et al., 2018). A 40 mL aliquot of the reaction solution was placed in a glass dish for freeze-drying (79480–30, CONCO, USA). Then, 3 mL of 2 mol/L NaOH and 7.5 mL of ethyl acetate were added to the freeze-dried samples obtained from 10 mL aliquots of the reaction solution. The samples were vortexed and ultrasonically extracted for 2–12 min, followed by centrifugation at 6500 r/min for 3 min. The supernatant was dried with nitrogen, dissolved in 0.2 mL of methanol and filtered (0.22 μm) before UPLC-MS analysis. The inhibition rate (%) of caffeic acid against PhIP formation was calculated as follows:
Inhibition rate (%) = (Y1 − Y2)/Y1 × 100
where Y1 is the PhIP content of the blank and Y2 is the PhIP content with the addition of caffeic acid. 2.5. UPLC-MS analysis of PhIP All filtrates were analyzed on a UPLC-MS instrument equipped with a tandem quadrupole mass spectrometer (Waters, USA). Separation of products was carried out on a Waters BEH phenyl column (50 × 2.10 mm, 1.70 μm). The LC conditions were as follows: flow, 0.30 mL/min; column temperature, 35 °C; and injection volume, 10 μL. The mobile phase was composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 10% A; 8 min, 70% A; 9 min, 70% A; 9.5 min, 10% A. Prior to the next injection, 10% A was held for 5 min to equilibrate the column. MS conditions: ion source, ESI+; multi-response monitoring mode; source block temperature, 120 °C; capillary voltage, 2.5 kV; cone voltage, 35 V; desolvation temperature, 400 °C; cone gas flow, 50 L/h; and collision gas (argon) flow, 0.3 mL/min.
2. Materials and methods 2.1. Materials Phenylalanine, anhydrous glucose, creatinine and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PhIP standard and 4-vinylcatechol were from Toronto Research Chemicals (Toronto, Canada). Caffeic acid, formic acid, dodecane and phenylacetaldehyde were from Sigma-Aldrich (St. Louis, MO, USA). SEPHADEX LH-20 glucan gel was from GE Healthcare Biosciences AB (Sweden). DIAION HP20 macroporous resin was from Mitsubishi Corporation (Tokyo, Japan). All solvents used were of analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). An SKM digital display thermostatic heating set was purchased from Runxin Experimental Instrument Co., Ltd. (Shandong, China).
2.6. Determination of dielectric loss A vector network analyzer (E5071C, Agilent, USA) with an openended coaxial line was used to determine the ε″. The high-temperature probe was calibrated with air, a short circuit, and distilled water, respectively. Each treatment was measured in triplicate (Zhang et al., 2019).
2.2. Construction of microwave heating methods
2.7. Calculations of reflection loss
The heating methods was set up referring to Zhang’s method with slight modifications (Zhang et al., 2017). The PhIP chemical model systems (100 mL) were heated in a flexiWAVE microwave synthesizer (Milestone, Italy) (for MW) or a thermostatic heater set at 100 °C for 2 h (for CV). The multi-stage MW program was designed to simulate the heating curve of CV, and the root mean square error (RMSE) was used to evaluate the fitting degree of these two heating curves, as shown in Fig. S1, to ensure that the two methods had similar heating rates. The RMSE value of the two heating curves was 1.75, indicating a high degree of fitting.
RMSE =
1 N
(2)
Computer Simulation Technology (CST) Microwave Studio was used to calculate RL which is an indicator of the overall absorption performance. First, a square reaction system (200 × 200 mm, 4 mm) was designed and the dielectric parameters were then imported. The boundary conditions were set as follows: X and Y (Unit Cell), Z min (Electric Et = 0), and Z max (open (add space)). After that, the simulation was started (Aguilar, Al-Joumayly, Hagness, & Behdad, 2010). 2.8. Sample preparation for the detection of thermal stability of caffeic acid
N
∑ (TMW − TCV
)2
n=1
The caffeic acid solutions (3 mmol/L) were treated with CV or MW at 100 °C for 0, 0.5, 1.0, 1.5 and 2 h, respectively, and subsequently cooled in ice water. Then, 10 mL aliquots of the samples were extracted with 20 mL of ethyl acetate. The upper layer was concentrated on a rotary evaporator under vacuum and then dissolved with 1 mL of methanol before UPLC-MS.
(1)
2.3. Construction of PhIP chemical model system Referring to Fan’s method (Fan et al., 2018), glucose, phenylalanine and creatinine were dissolved in deionized water to ensure final concentrations of 10, 20 and 20 mmol/L, respectively. Then, 0–5 mmol/L of caffeic acid was added to the system. The samples were immediately cooled in ice water after MW or CV treatment, with the treatments
2.9. Separation and purification of caffeic acid degradation products Referring to Cheng’s method (Cheng, Wong et al., 2008), 120 mL of 2
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Fig. 1. Amount of PhIP and percentage of inhibition on PhIP as a function of the concentration of caffeic acid (a) (c) and the heating time (b) (d). Values are mean ± standard deviation, n = 3, *p < 0.05, **p < 0.01.
the caffeic acid solution treated with MW at 100 °C for 2 h was extracted with ethyl acetate and n-hexane at a volume ratio of 1: 2: 1. The upper solution was concentrated on a rotary evaporator under vacuum, dissolved with 2 mL of methanol and separated using a Sephadex LH-20 column (40 × 4 cm). Next, 100 g of Sephadex LH-20 gel was swelled with 70% methanol overnight, stirred and added to a column pre-rinsed with anhydrous methanol. When the liquid level fell to the gel interface, the sample was then added into the column and drained until the liquid level was flush with the gel interface. Elution was carried out with methanol, wherein the eluent was collected until the sample color spread to 4 cm from the lower end of the column, and a 15 mL collection volume was taken for each tube. Collection was stopped when the colored part of the sample was completely diffused out of the gel column. Aliquots of 1.5 mL of all eluates were filtered and analyzed with HPLC. The mobile phase was composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 70% A, 30% B; 50 min, 70% A, 30% B. The components with the same retention time were combined, concentrated by a rotary evaporator (RV3V, IKA, Germany), dissolved with 0.5 mL of methanol, filtered and then subjected to UPLC-MS analysis. The expected isolates were combined and purified again using a Sephadex column. A Nicolet Nexus FTIR spectrometer was used for structural comparison of the main degradation products of caffeic acid.
composed of acetonitrile (A) and 0.1% aqueous formic acid (B) of the following gradients: 0 min, 5% A; 15 min, 20% A; 20 min, 40% A; 25 min, 80% A; 27 min, 100% A; 30 min, 5% A. MS conditions: ion source, ESI-; MSE with both low and high collision energies; source block temperature, 120 °C; capillary voltage, 3.0 kV; cone voltage, 35 V; desolvation temperature, 400 °C; cone gas flow, 600 L/h; and collision gas (argon) flow, 1.5 mL/min.
2.10. UPLC-MS analysis of caffeic acid degradation products
2.12. Statistical analysis
All filtrates were analyzed on a UPLC-MS instrument equipped with a tandem quadrupole time-of-flight (Q-TOF) mass spectrometer (Waters, USA). LC was performed on a Waters Acquity UPLC with a photodiode array (PDA) detector and an autosampler. Referring to Cheng’s method (Cheng, Wong et al., 2008), separation of products was carried out on a Waters BEH C18 column (50 × 2.10 mm, 1.70 μm). The LC conditions were as follows: flow, 0.30 mL/min; column temperature, 45 °C; and injection volume, 10 μL. The mobile phase was
Statistical analyses were performed using GraphPad Prism 7.0 and Microsoft Excel 2010. Origin 8.5 software was used to integrate the areas of time-fluorescence intensity curves. Masslynx V4 was used to analyze the UPLC-MS data. All experiments were performed three times and the data were presented as mean ± SD.
2.11. Separation, purification and identification of caffeic acid degradation product – phenylacetaldehyde adducts Caffeic acid and phenylacetaldehyde were dissolved in diethylene glycol in a ratio of 1:5 (v/v) to ensure the final concentration of the two was 25:125 mmol/L. The mixture was heated at 100 °C for 4 h and subsequently cooled in ice water. The mixture was extracted with water, ethyl acetate and n-hexane at the ratio of 1:1:2:1. The supernatant was dried under vacuum and dissolved with 2 mL of methanol. Separation was carried out on a Sephadex LH-20 column (40 × 4 cm). A 2 mg sample of the purified target was dissolved in 1 mL of chloroform and analyzed with 1H NMR (Avance III-400 MHz, Bruker, Germany).
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3. Results and discussion
degradation products of caffeic acid. The ion fragments of 4-vinylcatechol are shown in Table S1. Khuwijitjaru and co-workers heated caffeic acid at temperatures ranging from 100 to 250 °C and found that the carboxyl groups on the side chain were the most susceptible to being removed (Khuwijitjaru et al., 2014). Müller reported that 4-ethylcatechol was produced during roasting of coffee beans via pyrolysis of caffeic acid, and the formation pathway may involve 4-vinylcatechol as the reaction intermediate (Jia, Wang, Qi, Hong, & Lee, 2016; Müller et al., 2006). The relative molecular mass of 4-vinylcatechol is 136, and the molecular formula is C8H8O2, consistent with previous results. In combination with previous studies, it is reasonable to speculate that the main degradation product of caffeic acid formed under CV and MW is 4-vinylcatechol. The heattreated caffeic acid standard was separated and purified by a glucan gel chromatography column, and the degradation product of molecular weight 136 was localized by UPLC-MS. The components were concentrated, collected and re-purified, followed by comparison of the FTIR spectrum (Nicolet NcxUs, Thermo Electron, USA) with the 4-vinylcatechol standard. As shown in Fig. 2 (c), the FTIR spectra of the two were basically coincident. Thus, it can be determined that the degradation product of caffeic acid under CV and MW is 4-vinylcatechol, and a proposed pathway is shown in Fig. S2. The principal reaction is the release of CO2 from caffeic acid through thermal decarboxylation to produce 4-vinylcatechol with a molecular weight of 136. This is consistent with the conclusion of Terpinc (Terpinc et al., 2011).
3.1. Effect of caffeic acid and heating time on PhIP formation under microwave heating In this study, 0–5 mmol/L of caffeic acid solutions were added to PhIP-producing systems which were heated at 100℃ for 2 h to investigate the effect of the concentration of caffeic acid on PhIP formation under CV and MW. As shown in Fig. 1 (a), the amount of PhIP decreased with the increasing concentration of caffeic acid, 0.032 and 0.0051 ng/mL at 5 mmol/L under CV and MW, respectively. In fact, it is difficult to compare the results with previous works due to the existence of various variables such as the heating conditions, extraction methods and different systems. While in Fig. 1 (c), both for CV and MW, the inhibition of PhIP increased with the concentration of caffeic acid, and the trend toward greater inhibition gradually slowed as the concentration continued to increase. This finding may be explained by the fact that increased concentration promotes the reaction between the polyphenol (caffeic acid) and the intermediate of PhIP formation (phenylacetaldehyde). However, if the concentration of caffeic acid is increased beyond a certain level, in contrast, its inhibitory effect is weakened, implying that its reaction with phenylacetaldehyde is suppressed. In addition, at the same concentration, the inhibitory effect of caffeic acid against PhIP was always higher under MW than under CV. For example, at 5 mmol/L, the inhibition of PhIP was 93.7% under MW and 73.8% under CV. This difference suggests that at lower concentrations, MW either more strongly promotes the dissolution of caffeic acid or increases its activity, which leads to better inhibitory effects (Bouras et al., 2015; Wang et al., 2008). In addition, caffeic acid (3 mmol/L) was added to the reaction system to investigate its inhibitory effect on PhIP during heating times of 0–2 h. As shown in Fig. 1 (b), PhIP content increased with the heating time, 0.063 and 0.012 ng/mL at 2.0 h under CV (control, 0.128 ng/mL) and MW (control, 0.078 ng/mL), respectively. While in Fig. 1 (d), both under CV and MW, the inhibition of PhIP decreased with prolonged heating time. Moon and co-workers (Moon & Shin, 2013) investigated the inhibitory effect of epigallocatechin gallate (EGCG) on PhIP as a function of heating time at different temperatures. Their results showed that the inhibition of PhIP decreased at all temperatures with the extension of heating time due to the thermal instability of EGCG, the degradation constant of which increased by 180% when heated. Other studies found that decarboxylation or free radical reactions occurred when caffeic acid was heated, accompanied by various degrees of degradation (Khuwijitjaru et al., 2014; Moon & Shibamoto, 2010; Müller, Lang, & Hofmann, 2006). Therefore, we speculate that the decreasing inhibitory effect of caffeic acid on PhIP with time may be caused by thermal degradation during heating. In addition, the inhibition rate under MW always declined less than under CV and remained higher. This indicates that microwave heating not only promotes a high level of PhIP inhibition, but also weakens the decrease of the inhibition rate caused by prolonged heating time. This may be due to the difference in electromagnetic behavior of caffeic acid during MW, which is reflected in the change of microwave absorption ability or dielectric properties.
3.3. Effect of heating time on reflection loss, dielectric loss and caffeic acid degradation products formation In order to show whether the difference of electromagnetic characteristics (RL and ε″) under CV and MW contributes to better inhibitory effects on PhIP formation, firstly, the RL of caffeic acid (3 mmol/L) heated for 0–2 h were evaluated. As shown in Fig. 3 (a), with the extension of heating time, there were no differences of RL under MW and CV, which indicates that the overall microwave absorption abilities of caffeic acid are basically the same. Based on this, the ε″ of caffeic acid was further determined to evaluate whether there are differences in the ability to convert microwave energy into heat. As can be seen from Fig. 3 (b), the ε″ of caffeic acid was higher under MW than under CV, which indicates that the systems under microwave field are able to absorb more microwave energy and further convert it into heat, and finally, indirectly promotes the inhibition on PhIP formation under MW. Additionally, in order to verify that whether the decreasing inhibitory effect of caffeic acid on PhIP with time was caused by thermal degradation during heating, here, the effect of heating time on the formation of main degradation products (4-vinylcatechol) was evaluated. UPLC-MS was carried out to analyze the caffeic acid standards (3 mmol/L) treated with CV or MW at 100℃for 0–2 h. As shown in Fig. 3 (c), under both CV and MW, the amount of 4-vinylcatechol increased with the heating time while the similar trend was not found in the inhibition of PhIP. This phenomenon may attribute to the basically unchanged RL values with heating time, and further indicate that the thermal degradation of caffeic acid may not dominate the inhibitory effect. In addition, at each stage of heat treatment, the concentration of 4-vinylcatechol under MW was always higher than that of CV. When heated for 2 h, the formation of 4-vinylcatechol under MW reached 0.092 ng/mL while that under CV was 0.065 ng/mL. Combined with the results above, the ε″ of caffeic acid under MW was always higher than under CV, it can be concluded that the effect of ε″ on PhIP formation was dominant during the heating times, which may compensate for or even exceed the reduced inhibitory effect caused by the degradation of caffeic acid.
3.2. Identification of main degradation products of caffeic acid Considering that caffeic acid degrades easily when heated, here, the main degradation products was first identified for further analysis. The caffeic acid standard was treated under CV or MW at 100 °C for 2 h. The mass spectrum of the main degradation products with low collision energy is shown in Fig. 2 (a), wherein the m/z of the parent ion was 135.0436 and the relative molecular weight was 136.0514. Further analysis of the mass spectrum with high collision energy, as shown in Fig. 2 (b), shows that there were ion fragments with m/z of 135.0456, 117.0345 and 107.0526, which were consistent with previous studies (Terpinc et al., 2011) and further used to qualitatively identify the main 4
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Fig. 2. The mass spectra with low collision energy (a) and high collision energy (b), and the FTIR spectra (c) of the main degradation products of caffeic acid and 4vinylcatechol standard.
3.4. Identification of caffeic acid degradation product – phenylacetaldehyde adduct
inhibitory effect of caffeic acid on PhIP formation may be due to the capture of phenylacetaldehyde by its degradation product. To further identify the adducts, the mass spectra of the two target products were analyzed, as shown in Fig. 5. It was found that for the primary mass spectrometry fragments with m/z of 255, the main products had m/z of 135 and 119 (Fig. 5 (a)), which coincide with the fragments of 4-vinylcatechol and phenylacetaldehyde. When increasing the collision energy, the content of the ion with m/z of 255 decreased while that of the above-mentioned fragments increased (Fig. 5 (b)). Therefore, it can be concluded that the parent ion of the compound has m/z of 255, and its molecular weight is 256, making it an adduct of one molecule of 4-vinylcatechol with one molecule of phenylacetaldehyde. Similarly, the compound with m/z of 391 has main products with m/z of 135 and 271 (Fig. 5 (c)), which coincides with the fragments of one molecule and two molecules of 4-vinylcatechol, respectively. When increasing the collision energy, the content of the ion with m/z of 391 decreased, while that of the fragment with m/z of 135 increased (Fig. 5 (d)). This indicated that the parent ion of the compound has m/z of 391, and the molecular weight is 392. This compound is formed by fragmentation of phenylacetaldehyde to form a fragment of 271, together with a dimer structure of 4-vinylcatechol, so the compound is an adduct of two molecules of 4-vinylcatechol with one molecule of phenylacetaldehyde.
Studies have shown that several polyphenols, such as quercetin, naringenin, dihydromyricetin and EGCG, can capture phenylacetaldehyde, an intermediate during PhIP formation, and form new adducts to inhibit PhIP formation (Cheng, Chen et al., 2008; Cheng, Wong et al., 2008; Cheng et al., 2009). Given that caffeic acid differs from other polyphenols both structurally and in terms of thermal stability, its inhibition pathway against PhIP may be unique, which means that the trapping of phenylacetaldehyde may be achieved by caffeic acid or its degradation products. Therefore, to further clarify the role of caffeic acid in trapping phenylacetaldehyde and further inhibiting PhIP formation, a caffeic acid–phenylacetaldehyde mixture was heated under MW, in which the concentrations of the two reactants were 25 and 125 mmol/L, respectively. The mixture was then extracted with ethyl acetate and n-hexane at a ratio of 1: 2: 1, concentrated, and separated by an LH-20 Sephadex column. Each tube of the eluate was 20 mL, and the sequence of elution of the target was determined by HPLC. A total of 24 tubes were collected from the 300 mL reaction system. The parent ion of m/z = 255 was found in the first eluate tube, which was confirmed by selecting ions for which the retention time of the target was 14.21 min, as shown in Fig. 4 (a) and 4 (b). The parent ion of m/ z = 391 was found in the second tube, which was further verified by the fact that the retention time of the target was 15.72 min, as shown in Fig. 4 (c) and 4 (d). Table S2 showed the proposed molecular structure of new adducts, both m/z = 255 and m/z = 391 were formed from 4vinylcatechol and phenylacetaldehyde. The results indicate that the
3.5. Structural characterization of caffeic acid degradation product – phenylacetaldehyde adduct Mass spectral analysis showed that the two targets clearly contained 5
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Fig. 3. Reflection loss (a), dielectric loss (b) and the formation of 4-vinylcatechol (c) as a function of the heating time. Asterisks in Fig. 3 (b) indicate the significant differences between CV and MW treatments. Values are mean ± standard deviation, n = 3, *p < 0.05, **p < 0.01.
inferred. The compound has an aldehyde-based hydrogen at the chemical shift of 9.76, and three hydrogens are derived from a methyl group at the chemical shift of 1.34. This methyl group may be due to 4vinylcatechol undergoing an addition reaction with phenylacetaldehyde, causing the carbon-carbon double bond to become saturated. In Fig. 6, the presence of hydrogen No. 1, 2 and 3 indicates that caffeic acid undergoes a decarboxylation reaction triggered by the absorption of electromagnetic energy under a microwave field and its subsequent conversion into heat. An addition reaction occurs between the unsaturated bond of 4-vinylcatechol and phenylacetaldehyde, which
fragment ions having m/z of 135 and 119. However, the UV absorption of the two targets was not strong, and that of the ion with a molecular weight of 392 was even weaker. Therefore, the target with a molecular weight of 256 was selected for further separation and purification by preparative chromatography. The 1H NMR spectrum is shown in Fig. 6, wherein δ = 9.76 (d, 1H, J = 2.4 Hz), δ = 7.21–7.19 (m, 5H), δ = 7.01–7.00 (m, 1H), δ = 6.62–6.41 (m, 3H), δ = 5.3–4.9 (m, 2H), δ = 3.65 (dd, 1H, J = 2.8, 10.0 Hz), δ = 3.47–3.43 (m, 1H), δ = 1.34 (d, 3H, J = 6.8 Hz). From the 1H NMR spectrum, the following structural details of the compound with a molecular weight of 256 can be
Fig. 4. The TIC spectrum (a) and selective ion spectrum (b) of the parent ion of m/z = 255, and the TIC spectrum (c) and selective ion spectrum (d) of the parent ion of m/z = 392. 6
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Fig. 5. The mass spectra of the parent ion of m/z = 255 with both low (a) and high (b) collision energies, and the mass spectra of the parent ion of m/z = 392 with both low (c) and high (d) collision energies.
Fig. 6. The 1H NMR spectrum of the compound with a molecular weight of 256.
finally produces 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. In summary, the inhibition pathway of caffeic acid against phenylacetaldehyde formation under a microwave field is shown in Fig. S3. The inhibition mechanism of caffeic acid against PhIP under a microwave field is different from that of meta-dihydroxy polyphenols. The decarboxylation product of caffeic acid under MW forms an adduct with the intermediate of PhIP formation, phenylacetaldehyde, thus
inhibiting PhIP formation by blocking the interaction of phenylacetaldehyde with creatinine. 4. Conclusion The inhibition of PhIP formation by caffeic acid increased with the caffeic acid concentration but decreased with the prolongation of heating time. The inhibitory effect of caffeic acid on PhIP formation was 7
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stronger under MW than under CV, which was dominated by ε″. The effect of ε″ may compensate for or even exceed the reduced inhibitory effect caused by the degradation of caffeic acid. The inhibitory effect of caffeic acid on PhIP under a microwave field involves capturing phenylacetaldehyde, a key PhIP intermediate, which is also the case for inhibition by meta-dihydroxy polyphenols. The difference is that caffeic acid is first degraded to form 4-vinylcatechol, which then forms an adduct with phenylacetaldehyde, finally leading to significant inhibition of PhIP formation.
the formation of heterocyclic aromatic amines and sensory quality in fried bacon. Food Chemistry, 168, 383–389. Ito, N., Hasegawa, R., Imaida, K., Tamano, S., Hagiwara, A., Hirose, M., & Shirai, T. (1997). Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) in the rat. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 376(1–2), 107–114. Iwasaki, M., Kataoka, H., Ishihara, J., Takachi, R., Hamada, G. S., Sharma, S., & Tsugane, S. (2010). Heterocyclic amines content of meat and fish cooked by Brazilian methods. Journal of Food Composition and analysis, 23(1), 61–69. Janoszka, B. (2010). Heterocyclic amines and azaarenes in pan-fried meat and its gravy fried without additives and in the presence of onion and garlic. Food Chemistry, 120(2), 463–473. Jia, C., Wang, X., Qi, J., Hong, S., & Lee, K. (2016). Antioxidant properties of caffeic acid phenethyl ester and 4-vinylcatechol in stripped soybean oil. Journal of Food Science, 81(1), C35–C41. Johansson, M., Fredholm, L., Bjerne, I., & Jägerstad, M. (1995). Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues. Food and Chemical Toxicology, 33(12), 993–1004. Khuwijitjaru, P., Plernjit, J., Suaylam, B., Samuhaseneetoo, S., Pongsawatmanit, R., & Adachi, S. (2014). Degradation kinetics of some phenolic compounds in subcritical water and radical scavenging activity of their degradation products. Canadian Journal of Chemical Engineering, 92(5), 810–815. Knize, M., Dolbeare, F., Carroll, K., Moore, D., II, & Felton, J. (1994). Effect of cooking time and temperature on the heterocyclic amine content of fried beef patties. Food and Chemical Toxicology, 32(7), 595–603. Layton, D. W., Bogen, K. T., Knize, M. G., Hatch, F. T., Johnson, V. M., & Felton, J. (1995). Cancer risk of heterocyclic amines in cooked foods: An analysis and implications for research. Carcinogenesis, 16(1), 39–52. Moon, J.-K., & Shibamoto, T. (2010). Formation of volatile chemicals from thermal degradation of less volatile coffee components: Quinic acid, caffeic acid, and chlorogenic acid. Journal of Agricultural and Food Chemistry, 58(9), 5465–5470. Moon, S.-E., & Shin, H.-S. (2013). Inhibition of mutagenic 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) formation using various food ingredients in a model systems. Food Science and Biotechnology, 22(2), 323–329. Müller, C., Lang, R., & Hofmann, T. (2006). Quantitative precursor studies on di- and trihydroxybenzene formation during coffee roasting using “in bean” model experiments and stable isotope dilution analysis. Journal of Agricultural and Food Chemistry, 54(26), 10086–10091. Murkovic, M., Steinberger, D., & Pfannhauser, W. (1998). Antioxidant spices reduce the formation of heterocyclic amines in fried meat. Z Lebensm Unters Forsch, 207(6), 477–480. Oguri, A., Suda, M., Totsuka, Y., Sugimura, T., & Wakabayashi, K. (1998). Inhibitory effects of antioxidants on formation of heterocyclic amines. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis, 402(1), 237–245. Quelhas, I., Petisca, C., Viegas, O., Melo, A., Pinho, O., & Ferreira, I. (2010). Effect of green tea marinades on the formation of heterocyclic aromatic amines and sensory quality of pan-fried beef. Food Chemistry, 122(1), 98–104. Salazar, R., Arámbula-Villa, G., Hidalgo, F. J., & Zamora, R. (2014). Structural characteristics that determine the inhibitory role of phenolic compounds on 2-amino-1methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) formation. Food Chemistry, 151, 480–486. Shirai, T., Sano, M., Tamano, S., Takahashi, S., Hirose, M., Futakuchi, M., ... Ito, N. (1997). The prostate: A target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) derived from cooked foods. Cancer Research, 57(2), 195–198. Sinha, R., Gustafson, D. R., Kulldorff, M., Wen, W. Q., Cerhan, J. R., & Zheng, W. (2000). 2-Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine, a carcinogen in high-temperature-cooked meat, and breast cancer risk. Journal of the National Cancer Institute, 92(16), 1352–1354. Sinha, R., Peters, U., Cross, A. J., Kulldorff, M., Weissfeld, J. L., Pinsky, P. F., ... Hayes, R. B. (2005). Meat, meat cooking methods and preservation, and risk for colorectal adenoma. Cancer Research, 65(17), 8034–8041. Terpinc, P., Polak, T., Šegatin, N., Hanzlowsky, A., Ulrih, N. P., & Abramovič, H. (2011). Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food Chemistry, 128(1), 62–69. Wakabayashi, K., Nagao, M., Esumi, H., & Sugimura, T. (1992). Food-derived mutagens and carcinogens. Cancer Research, 52(7 Suppl), 2092s–2098s. Wang, Y., You, J., Yu, Y., Qu, C., Zhang, H., Ding, L., & Li, X. (2008). Analysis of ginsenosides in Panax ginseng in high pressure microwave-assisted extraction. Food Chemistry, 110(1), 161–167. Warzecha, L., Janoszka, B., Błaszczyk, U., Stróżyk, M., Bodzek, D., & Dobosz, C. (2004). Determination of heterocyclic aromatic amines (HAs) content in samples of household-prepared meat dishes. Journal of Chromatography B, 802(1), 95–106. Zhang, N., Fan, D., Chen, M., Chen, Y., Huang, J., Zhou, W., & Chen, W. (2017). Concentration-related microwave heating processes: Electromagnetic interference of Maillard reaction substrates (glucose and lysine). RSC Advances, 7(39), 24382–24386. Zhang, N., Fan, D., Zhao, Y., Wu, Y., Yan, B., Zhao, J., ... Zhang, H. (2019). Dielectric loss mediated promotion of microwave heating in the Maillard reaction. LWT, 101, 559–566.
CRediT authorship contribution statement Nana Zhang: Conceptualization, Methodology, Formal analysis, Writing - review & editing. Yanfang Chen: Data curation, Software, Writing - original draft. Yueliang Zhao: Formal analysis, Investigation. Daming Fan: Conceptualization, Supervision, Funding acquisition, Resources. Lijie Li: Software, Validation. Bowen Yan: Visualization. Guanjun Tao: Data curation. Jianxin Zhao: Visualization. Hao Zhang: Project administration. Mingfu Wang: Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province-Outstanding Youth Foundation [BK20170052]; the National Natural Science Foundation of China [31671821]; the National First-class Discipline Program of Food Science and Technology [JUFSTR20180102]. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.126447. References Aguilar, S. M., Al-Joumayly, M. A., Hagness, S. C., & Behdad, N. (2010). Design of a miniaturized dual-band patch antenna as an array element for microwave breast imaging. Antennas and Propagation Society International Symposium (APSURSI) (pp. 1– 4). IEEE. Bouras, M., Chadni, M., Barba, F. J., Grimi, N., Bals, O., & Vorobiev, E. (2015). Optimization of microwave-assisted extraction of polyphenols from Quercus bark. Industrial Crops & Products, 77, 590–601. Cheng, K.-W., Chen, F., & Wang, M. (2008). Preventive potential and mechanism of dietary phenolics on the formation of mutagenic heterocyclic amines. The 14th world conference of food science and technology (pp. 394–395). . Cheng, K. W., Wong, C. C., Chao, J., Lo, C., Chen, F., Chu, I., & Wang, M. (2009). Inhibition of mutagenic PhIP formation by epigallocatechin gallate via scavenging of phenylacetaldehyde. Molecular Nutrition & Food Research, 53(6), 716–725. Cheng, K.-W., Wong, C. C., Cho, C. K., Chu, I. K., Sze, K. H., Lo, C., & Wang, M. (2008). Trapping of phenylacetaldehyde as a key mechanism responsible for naringenin’s inhibitory activity in mutagenic 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine formation. Chemical Research in Toxicology, 21(10), 2026–2034. Dundar, A., Sarıçoban, C., & Yılmaz, M. T. (2012). Response surface optimization of effects of some processing variables on carcinogenic/mutagenic heterocyclic aromatic amine (HAA) content in cooked patties. Meat Science, 91(3), 325–333. Fan, D., Li, L., Zhang, N., Zhao, Y., Cheng, K.-W., & Yan, B. (2018). A comparison of mutagenic PhIP and beneficial 8-C-(E-phenylethenyl) quercetin and 6-C-(E-phenylethenyl) quercetin formation under microwave and conventional heating. Food & Function, 9(7), 3853–3859. Felton, J., Fultz, E., Dolbeare, F., & Knize, M. (1994). Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties. Food and Chemical Toxicology, 32(10), 897–903. Gibis, M., Kruwinnus, M., & Weiss, J. (2015). Impact of different pan-frying conditions on
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