Inhibitory effects of dietary antioxidants on the formation of carcinogenic polycyclic aromatic hydrocarbons in grilled pork

Objective The inhibitory effects of dietary antioxidants, diallyl disulfide (DADS) and quercetin, in marinade were investigated on the formation of carcinogenic polycyclic aromatic hydrocarbons (EPA priority 16 PAHs) in grilled pork. Methods The formation of PAHs in grilled sirloin pork with different marinades after charcoal-grilling for 2 min/side were evaluated using high performance liquid chromatography with a photodiode array detector (HPLC-DAD). Results Compared with the control marinade treatment (without antioxidant), the addition of DADS (500 mg/kg meat sample) in marinade significantly decreased benzo[a]pyrene (BaP) (100%) and heavy PAHs (84%) in charcoal-grilled pork, while the addition of quercetin at the same concentration could reduce 23% and 55% of BaP and heavy PAHs, respectively. Conclusion The results of this study suggested that the addition of DADS in the marinade could be important in decreasing the levels of PAHs in grilled meat.


INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) that form mainly as a result of pyrolysis process, especially the incomplete combustion of organic compounds are a diversified classes of car cinogenic chemicals [1]. These compounds are hydrophobic molecules where the solubility in water decreases as the molecular weight increases and easily dissolve in oil phase [2]. PAHs composed of two to four fused aromatic rings are called light PAHs and those containing more than four aromatic rings are known as heavy PAHs. The heavy PAHs are more stable and more toxic than the light ones [3]. As PAHs represent an important class of carcinogens, PAHs are not toxic but their metabolites that are converted from hydrophobic compounds into relatively hydrophilic compounds during detoxification by the organism are the cause of DNA damage [4]. The International Agency for Research on Cancer [5] has determined that benzo[a]pyrene (BaP) is carcinogenic to humans (group 1), whereas benz[a]anthracene (BaA), chrysene (Chry), benzo[k]fluoranthene (BkF) and benzo [b]fluoranthene (BbF) are possibly carcinogenic to humans (group 2B). BaP is the most widely known and studied of the PAHs due to its importance as one of the most potent animal carcinogenic PAHs, however, the European Food Safety Authority [6] suggested that the sum content of the four PAHs (PAH4: BaP, BaA, BbF and Chry) is a more suitable marker than only BaP. European Union (EU) Regulation No 835/2011 stipulated that the maximum level of BaP was 2.0 μg/kg and the sum of PAH4 was set at 12.0 μg/kg in smoked meats and smoked meat products [7]. Moreover, PAH16 (Table 1) are classified as priority pollutants by Environmental Pro tection Agency (EPA) based on carcinogenicity and their occurrence in contaminated foods and the environment is of concern [8]. The definite mecha nism of the formation of PAHs is not exactly proven; however, previous researches have proposed that they might be formed through a complicated mechanism (free radical reactions, intramolecular addition, or the polymerization) of small mole cules [9] and intramolecular cyclization of lipid peroxides [10]. Various antioxidant compounds have shown effective inhibitory effects on PAH formation in a meat model system [10] and in different types of meat [11,12].
The occurrence of PAHs in food is mainly due to processing at high temperature especially in the charcoalgrilling process because charcoal contains many hydrocarbon compounds which are activated via incomplete combustion to form PAHs [13]. The PAH contamination levels in grilled meats depends on many factors such as types of heating source, grilling time, distance from the heating source, amount of fat, and marinade ingredients [14]. Marinades often contain a lot of additives, oil, herbs and spices to improve the sensory properties (tex ture, color, etc.) of meat products [15]. Various spices especially garlic and onion are added in the marinade to contribute the unique flavor and taste. Some studies have shown that the ad dition of ingredients with an antioxidant activity (garlic, onion, lemon juice, etc.) could reduce PAH levels and other carcin ogens such as heterocyclic aromatic amines (HCAs) in meat products [16]. The addition of garlic (5 g/kg meat) and onion (65 mL/kg meat) into a marinade could reduce the sum of PAHs in grilled beef from 74.0 μg/kg to 45.2 μg/kg, compared to marinade without the addition of antioxidant [17]. Garlic contains an abundance of chemical compounds that have been shown to possess high antioxidative compounds [18]. Diallyl disulfide (DADS) is the predominant oilsoluble organo sulfides in essential garlic oil which has demonstrated an inhibition effect on carcinogenic compounds in meat model system [19]. Quercetin is a flavonoid compound, which is widely contained in onion, tomato, etc. and has been shown to be a strong antioxidant. Quercetin is one of the most power ful scavengers of reactive oxygen species which cause many diseases such as cancers [20].
The reduction of PAH levels in grilled meat by adding in gredients which have antioxidant activities were confirmed on the basis of previous research, however, studying the inhi bition effects of pure dietary antioxidants would give a better understanding. Thus, the main objective of this study was to illustrate the effect of the addition two dietary antioxidants, DADS and quercetin, that represent the antioxidant com pounds in garlic and onion, respectively in marinade treatments on PAHs formation in charcoalgrilled pork. Our results could provide a theoretical basis for the use of these compounds as potential inhibitors of the PAH formation.

Sample preparations
Sirloin pork and all marinade ingredients were obtained from a grocery store in Bangkok, Thailand. Two pieces of 0.5cm thickness sirloin pork were randomly chosen and immersed in each marinade treatment at 4°±2°C for 1 h. Control mari nade (C) (without added pure antioxidant) was composed of 50 g water, 50 g sugar, 20 g oyster sauce, 10 g salt and 7.5 g spice powder per 1 kg meat. DADS and quercetin were added into different marinade treatments: 100 mg/kg diallyl disul fide marinade (D100), 500 mg/kg diallyl disulfide marinade (D500), 100 mg/kg quercetin marinade (Q100), and 500 mg/kg quercetin marinade (Q500). The marinated samples were charcoalgrilled 2 min/side. The heating source including 500 g of charcoal and 60 g of wood was placed at the bottom of the grill and changed for each sample. The grilled samples were cooled to reach room temperature and packed in alu minum foil bags until used for PAH extraction.

Extraction and clean up
Sample extraction and cleanup procedures followed our pre vious study [9]. Meat samples (10 g) were ground and saponified with 100 mL of 2 mol/L of potassium hydroxide in methanol/ water (80:20, v/v) and then extracted with 50 mL of nhexane. This extraction procedure was repeated four times, the collect ed hexane layers were evaporated using a parallel evaporator at 60°C under reduced pressure. The residue was dissolved in 3 mL of acetonitrile, transferred to an activated SepPak Florisil cartridge (6 mL/1,000 mg, MachereyNagel, Langer wehe, Germany) and purged to dryness. Acetonitrile was added to the residue to a final volume of 400 μL and the mixture was subjected to highperformance liquid chromatography photodiode array detector (HPLCDAD) analysis. Each sample were conducted in triplicate.

HPLC-DAD analysis of PAHs
PAH analysis was performed by HPLCDAD (Waters, Milford, MA, USA) and followed our previous study [9]. Chromato graphic resolution was achieved using a reverse phase C18 column (ZORBAX Hypersil ODS column of 250 mm×4.6 mm, 5 μm particle size). PAHs were detected at 254 nm and con firmation by comparing the retention time and DAD spectra (scanned wavelength from 200 to 600 nm) with reference standards. PAHs were quantified using an external standard method. The concentration of each PAH was calculated from its respective calibration curve which obtained by plotting the peak area against the standards at concentration ranged be tween 0.1 to 20.0 μg/mL (the correlation coefficient ranged from 0.994 to 0.999). The limit of detection (LOD) and quan tification (LOQ) of PAHs were determined using signalto noise of S/N = 3 and S/N = 10 of the lowest concentration of reference standards, respectively [17].

Statistical analysis
Experiments was performed by a completely randomized design. All experiments were carried out at least in triplicate. Data analysis was processed using analysis of variance, Dun can' s multiple range test and ttest using the SPSS 10.0 software (SPSS, Chicago, IL, USA) to determine whether differences between mean values were significant (p<0.05).

Limit of detection, limit of quantification, and recovery study
The LOD and LOQ of PAH16 standards were observed using HPLCDAD with detection performed at 254 nm and the results ranged from 0.57 to 7.29 μg/L and 1.91 to 22.10 μg/L, respectively. These results showed enough sensitivity for the detection of the compounds in the samples. According to ex traction and cleanup method in this study, the recoveries (in percent) of PAHs were in the range of 67.9% to 112.1%, with a mean recovery of 98.5% which satisfactory for determina tions at the applied detection level (mg/kg). Some PAHs (Nap, DahA, and Anl) produced a low recovery level because they may have undergone partial loss during cleanup procedure using SPE cartridge [21]. However, based on the AOAC man ual guidelines, these recovery values were acceptable [22].

Inhibitory effect antioxidants to marinade treatments on the formation of PAHs in grilled pork
The contents of PAH compounds in grilled sirloin pork with five different marinade treatments (C, D100, D500, Q100, and Q500) are shown in Table 2. The results illustrated that the highest concentrations of PAH4 (318.5 μg/kg) and PAH16 (1,173.4 μg/kg) were observed in grilled sirloin pork without the addition of antioxidant in marinade treatment (C) while the samples that treated with all antioxidant marinade treat ments (D100, D500, Q100, and Q500) had lower PAH16 concentrations (878.5, 249.4, 504.4, and 508.5 μg/kg, respec tively). Our result was agreed with the complete reduction (100% reduction) of BaA and BghiP that was found in grilled sirloin pork treated with D500, Q100, and Q500 marinade treatments compared to control. The definite mechanism of PAHs formation in food is not well understood; however, our results assented with the study reported by Min et al [10] who proposed that the major mechanism of PAHs formation might be formed through free radical reactions. Therefore, our re sult confirmed that the antioxidant compounds that have free radical scavenging activity could inhibit PAHs formation dur ing charcoal grilling process. Considering the level of addition antioxidants, Q100 mari nade treatment exhibited significantly lower concentration of light PAHs (438.0 μg/kg) than D100 marinade treatment (823.6 μg/kg). However, a greater effect of reduction for heavy PAHs was presented by D100 marinade treatment. Fur thermore, grilled sirloin pork treated with D500 marinade treatment contained the lowest concentrations in both light PAHs and heavy PAHs (223.1 μg/kg and 26.3 μg/kg, respec tively) compared to Q500 marinade treatment (433.4 μg/kg and 75.1 μg/kg, respectively). The results demonstrated that two levels of addition DADS (100 and 500 mg of antioxidant per 1 kg meat sample) in this experiment resulted in a greater reduction of heavy PAHs than that of quercetin at the same concentration. This may have been due to the lipophilic nature of PAHs, DADS that are oil soluble antioxidants (easily dis solved in oil phase similar to PAHs) have a stronger effect on reduction of BaP and heavy PAHs in grilled sirloin pork than does quercetin which contains hydroxyl groups and is a more polar antioxidant. Moreover, DADS were reported to be a powerful terminator of lipid peroxidation [18]. DADS was also found to inhibit oxidation reaction by means of radical scavenging but were not involved in chainbraking antioxidant mechanisms [23]. In addition, a comparative computational modeling and analysis of transition state mechanism has suggested that organosulfur compounds derived from garlic with the existence of a thioallyl group, i.e. DADS, play an important role of scavenging • OH and ROO • [24]. Based on its protective effects against free radicals, DADS can be pro posed to be involved with the inhibition of PAH formation.
Antioxidant marinade treatments (D100, D500, Q100, and Q500) illustrated inhibitory effects on PAH formation during charcoal grilling process of marinated sirloin pork in comparison to control (C). Figure 1 presents the percentage of reduction of BaP as a marker for PAHs contamination, PAH4, PAH16, light PAHs, and heavy PAHs. D500 marinade treatment showed the highest effect on reducing the genera  Nap  ND  ND  ND  ND  ND  Anl  ND  ND  ND  ND  ND  Ane  ND  ND  ND  ND  ND  Flu ND ND ND ND ND Phen tion of BaP (100%), PAH4 (85%), PAH16 (79%), light PAHs (78%), and heavy PAHs (84%) in grilled sirloin pork. Reduc tion of BaP level was the highest when the sirloin pork treated with D500 marinade treatment (100%), followed by D100 marinade treatment (33%), Q500 marinade treatment (21%), and Q100 marinade treatment (16%). According to this experi ment, D500 marinade treatment effectively reduced individual light and heavy PAHs as well as sum of PAH contamination levels (PAH4 and PAH16) in grilled sirloin pork.
In this study, the addition of DADS and quercetin in mar inade could reduce the PAH levels in charcoalgrilled pork. Due to the lipophilic property and the presence of thioallyl group, the addition of DADS significantly decreased the PAH concentrations lower than did quercetin in the grilled sirloin pork, especially BaP which is considered as carcinogenic to humans (group 1) and heavy PAHs that are more stable and more toxic. As previously mentioned, DADS is antioxidant which naturally present in garlic and onion and has been re cognized as part of healthful diet throughout history. DADS is noted in many scientific publications as being associated with medicinal properties and health benefits such as anti cardiovascular disease, antineurological disease, antiliver disease effects as well as effects for prevention of cold, flu, and arthritis [25]. Importantly, DADS could be a prospec tive agent for multitargeted prevention and/or treatment against human cancers because DADS has no toxic effects in healthy cells [26]. Although the adverse effects of very high dose of DADS has been documented, which includes a burning sensation in the mouth and gastrointestinal tract, nausea, diarrhea, vomiting and body odor; in fact, due to the limiting effect of its strong flavor and malodor, it is nearly impossible to add DADS to food at the level that causes ad verse effects [26]. In summary, we successfully demonstrated the effectiveness of DADS and quercetin in the inhibition of PAH formation. Our results provide a theoretical basis for the use of these compounds as potential inhibitors of the PAH formation in grilled meat which could be applied to household cooking and the food industry.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.