Effects Of Phenolic Compounds On PAHs Formation In Grilled Chicken And It's Mechanism Of Alleviating Cell Damage

The rich nutrients in meat and meat products provide the necessary substance basis for maintaining the nutritional balance and health of the human body.The digestibility of processed meat and meat products is significantly improved,and the microbial damage can be effectively inhibited,while some carcinogenic chemicals such as polycyclic aromatic hydrocarbons(PAHs)in grilled meat and meat products are also produced.It is reported that a large amount of polycyclic aromatic hydrocarbons(PAHs)are produced when meat is processed under high temperature condition(?200?).Although the formation mechanism of PAHs is not clear,it is generally believed that the free radicals generated by the pyrolysis of organic matter,and then formed by thermal aggregation.Epidemiological studies have shown that frequent consumption of meat and meat products containing PAHs may cause a variety of diseases,such as pancreatic cancer,lung cancer and colon cancer.In recent years,more and more attention has been paid to the formation and cytotoxicity of PAHs.It is urgent to find suitable processing methods to reduce the content of PAHs in foods and effective mitigating agents to inhibit the damage of PAHs in vitro.As the most consumed beverages,the effects of tea and beer on the formation of PAHs in charcoal-grilled chicken wings and the key compounds of this action have not been reported.In addition,the effects of these compounds on BaP-induced Caco-2 cell damage and the effect mechanism has not been reported.Therefore,this study mainly discusses the above problems,and provides a theoretical basis for reducing the formation of PAHs in grilled meat and exploring the mechanism of cell damage induced by BaP.The details are as follows:1.Effects of tea infusion and beer on the formation of PAHs in charcoal-grilled chicken wingsThe effects of 6 kinds of tea(green tea-GT,white tea-WT,yellow tea-YT,oolong tea-OT,dark tea-DT and black tea-BT)and 6 kinds of beer(Beer-1,2,3,4,5 and 6)on the formation of PAH8 in charcoal-grilled chicken wings were studied.The chicken wings were marinated in 12 kinds of marinades at 4? for 4 h.The DPPH free radical scavenging activity and total phenolic content of all marinades,stable free radical and PAH8 content in chicken wings were detected.The results showed that GT exhibited the strongest DPPH scavenging activity(50.7%),followed by WT(45.7%),YT(45.5%),OT(45.1%),DT(40.6%)and BT(38.2%);the total phenolic content ranged from 186.90 to 589.49 mg GAE/L and 147.30 to 395.31 mg GAE/L in tea infusions before and after marinating,respectively.The strongest DPPH free radical scavenging activities of the six types of beer was Beer-1(27.0%),followed by Beer-2(23.6%),Beer-3(21.9%),Beer-4(21.4%),Beer-5(9.5%)and Beer-6(8.1%);the total phenolic content ranged from 186.90 to 393.86 mg GAE/L and 177.80 and 370.02 mg GAE/L in beers before and after marinating,respectively.The relative free radical content of charcoal-grilled chicken wings was negatively correlated with DPPH free radical scavenging activity and total phenolic content(p<0.05).The content of PAH8 in charcoal-grilled chicken wings was negatively correlated with DPPH free radical scavenging activity and total phenolic content,whereas positively correlated with the relative content of stable free radicals.Green tea and Beer-1 were the best marinades among tea infusions and beers,respectively.2.Effects of phenolic compounds in tea infusions and beers on the formation of PAHs in charcoal-grilled chicken wingsThe phenolic compounds in tea and beer were identified and quantified by liquid chromatography-mass spectrometry.The phenolic compounds which may inhibit the the PAHs formation were selected from tea infusions and beers according to the content changes before and after the marinating.The DPPH free radical scavenging activity,effect on PAH8 formation,content changes before and after marinating and dose-effect relationship of pure phenolic compounds were studies.The results showed that 24 and 32 phenolic compounds were identified and quantified from tea infusions and beers,respectively.The total content of phenolic compounds was consistent with the trend of total phenolic content measured previously;eight and nine phenolic compounds were screened out from the tea infusions and beers.After marinating with pure phenolic compound solutions,all pure phenolic compounds were effective in inhibiting the formation of PAH8.There was no correlation between PAH8 content and DPPH free radical scavenging activity.However,it was worth noting that there is a negative correlation between PAH8 content and DPPH free radical scavenging activity by the same parent structure of phenolic compounds.The effect of phenolic compound on inhibiting the formation of PAH8 is complicated,that was,the inhibitory effect of different phenolic compound at the same concentration was significantly different;the inhibitory effect of phenolic compound with higher concentration was not necessarily the best;the inhibitory effect of phenolic acid was generally better than flavonoids.After marinating,the content of all phenolic compound marinades decreased significantly,indicating that the active ingredients in the marinade could adhere to the surface of the chicken wings or interact with the oxides produced in the chicken wings during grilling.With the same phenolic compound marinades,the PAH8 content gradually decreases as the concentration of the marinade increases.Finally,we selected six kinds of phenolic compounds which showed the best inhibitory effect of PAH8 formation,namely Eriodictyol,Naringenin,Quinic acid,Homovanillic acid,ferulic acid and Trolox,providing a material basis for further investigation of cytotoxic effects induced by BaP.3.The study of phenolic compounds on alleviating BaP-induced Caco-2 cells damageFirstly,different concentrations of BaP(1,2,5,10,20,50 and 100 ?M)and different treatment times(0.5,1,2,3,4,5,6,12,24 and 48 h)on the effect of Caco-2 cell viability was determined,to be the most significant inhibitory effect on cell viability at 50 ?M for 3 h,with an inhibition rate of 44.4%.According to the determined treatment time,the effects of different concentrations(1,2,5,10 and 20 mg/mL)of phenolic compounds on Caco-2 cell viability at 3 h were studied.The results showed that there was no significant inhibition of cell viability when treated with 5,10 and 20 mg/mL Eriodictyol and 20 mg/mL Naringenin.Subsequently,the effects of phenolic compound pretreatment or simultaneous BaP treatment on the decrease of cell viability caused by BaP were studied.The results showed that no matter what concentration of phenolic compound was pretreated,it could not alleviate the viability of BaP,only 20 mg/mL Eriodictyol and 20 mg/mL Naringenin can significantly alleviate the cell viability when treated with BaP.Finally,Control,50 ?M BaP,20 mg/mL Eriodictyol,20 mg/mL Naringenin,20 mg/mL Eriodictyol+50 ?M BaP and 20 mg/mL Naringenin+50 ?M BaP were used to investigate the apoptosis,cell cycle,reactive oxygen species(ROS),lipid oxidation(MDA)and total superoxide dismutase(T-SOD)and alleviation mechanism of phenolic compounds on BaP-induced cytotoxicity.The results showed that BaP treatment increased apoptosis,shortened the G1 and G2 phases of the cell cycle and prolonged the S phase,increased the production and accumulation of ROS and MDA,and inhibited the activity of T-SOD.When the two phenolic compounds were treated simultaneously with BaP,the negative effects of BaP on Caco-2 were effectively alleviated,and the alleviating effect of Eriodictyol was significantly better than that of Naringenin.Based this result,only Eriodictyol treatment was studied in subsequent experiments.4.Proteomic study of the alleviation mechanism of phenolic compounds on BaP-induced Caco-2 damageTo further investigate the alleviation mechanism of phenolic compounds on BaP-induced Caco-2 damage,we used proteomics techniques investigating the protein expression for Control,50?M BaP,20 mg/mL Eriodictyol,20 mg/mL Eriodictyol+50 ?M BaP.The results of the two comparison groups of BaP vs Control and Eriodictyol+BaP vs Control were analyzed,and Venn,GO,KEGG and protein interactions were performed.The results showed that 1355 proteins were identified in the four treatment groups,and the protein abundance showed a normal distribution.The Pearson correlation coefficients of different replicates in each group were greater than 0.9,indicating that the proteome experiments were reliable.A total of 234 proteins were identified in BaP vs Control and Eriodictyol+BaP vs Control.Based on the molecular function and interaction of protein,we screened 91 key proteins that may be alleviating,and participated in 28 KEGG pathways.After further analysis,BaP is involved in the inhibition of some key proteins in the transcription and translation,cell cycle and apoptosis processes of Caco-2 cell,and Eriodictyol show significantly alleviation effect on BaP-induced Caco-2 cell damage by regulating these proteins in these three pathways.The above results indicate that phenolic-rich additives can be used as effective candidates for inhibiting the formation of PAHs during meat processing,and phenolic compounds are also important mitigators of cell damage.