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 carcinogenic 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 (B
aP) is carcinogenic to humans (group 1), whereas benz[
a]anthracene (B
aA), chrysene (Chry), benzo[
k]fluoranthene (B
kF) and benzo[
b]fluoranthene (B
bF) are possibly carcinogenic to humans (group 2B). B
aP 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: B
aP, B
aA, B
bF and Chry) is a more suitable marker than only B
aP. European Union (EU) Regulation No 835/2011 stipulated that the maximum level of B
aP 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 Protection Agency (EPA) based on carcinogenicity and their occurrence in contaminated foods and the environment is of concern [
8]. The definite mechanism 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 molecules [
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 charcoal-grilling 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 (texture, 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 addition of ingredients with an antioxidant activity (garlic, onion, lemon juice, etc.) could reduce PAH levels and other carcinogens 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 oil-soluble organosulfides 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 powerful scavengers of reactive oxygen species which cause many diseases such as cancers [
20].
The reduction of PAH levels in grilled meat by adding ingredients which have antioxidant activities were confirmed on the basis of previous research, however, studying the inhibition 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 compounds in garlic and onion, respectively in marinade treatments on PAHs formation in charcoal-grilled pork. Our results could provide a theoretical basis for the use of these compounds as potential inhibitors of the PAH formation.
MATERIALS AND METHODS
Standards and reagents
PAH16 standards as listed in
Table 1 were purchased from Supelco (Bellefonte, PA, USA). HPLC-grade solvents (acetonitrile and dichloromethane) and analytical-grade solvents/chemical (methanol, 2-propanol, 1-buthanol, n-hexane and potassium hydroxide) were purchased from RCI Labscan (Bangkok, Thailand) and Ajax Finechem (Silverwater, NSW, Australia). The DADS and quercetin hydrate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Sample preparations
Sirloin pork and all marinade ingredients were obtained from a grocery store in Bangkok, Thailand. Two pieces of 0.5-cm thickness sirloin pork were randomly chosen and immersed in each marinade treatment at 4°±2°C for 1 h. Control marinade (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 disulfide marinade (D-100), 500 mg/kg diallyl disulfide marinade (D-500), 100 mg/kg quercetin marinade (Q-100), and 500 mg/kg quercetin marinade (Q-500). The marinated samples were charcoal-grilled 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 aluminum foil bags until used for PAH extraction.
Extraction and clean up
Sample extraction and clean-up procedures followed our previous 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
n-hexane. This extraction procedure was repeated four times, the collected 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 Sep-Pak Florisil cartridge (6 mL/1,000 mg, Macherey-Nagel, Langerwehe, Germany) and purged to dryness. Acetonitrile was added to the residue to a final volume of 400 μL and the mixture was subjected to high-performance liquid chromatography-photodiode array detector (HPLC-DAD) analysis. Each sample were conducted in triplicate.
HPLC-DAD analysis of PAHs
PAH analysis was performed by HPLC-DAD (Waters, Milford, MA, USA) and followed our previous study [
9]. Chromatographic 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 confirmation 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 between 0.1 to 20.0 μg/mL (the correlation coefficient ranged from 0.994 to 0.999). The limit of detection (LOD) and quantification (LOQ) of PAHs were determined using signal-to-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, Duncan’s multiple range test and t-test using the SPSS 10.0 software (SPSS, Chicago, IL, USA) to determine whether differences between mean values were significant (p<0.05).