The Changes Of Representative Flavonoids During Thermal Processing And Their Impacts On Formation And Toxicity Of AGEs

Polyphenols are plant secondary metabolites and the most abundant source of antioxidants in human diets.They have various bioactivies such as anti-inflammatory,neuroprotective,cardioprotective,cancer-preventing and anti-obesity effect.In fact,the health benefits of polyphenols on the human body depend on the processing.Thermal treatment is the most effective and widely used method for both quality improvement and shelf life extention during food processing and preservation.For foods rich in dietary polyphenols,thermal processing can result in changes in the nutritional value.The addition of polyphenolic compounds that are beneficial to human health into foods may trigger a series of complex chemical reactions and changes in biological activity,which might in turn affect the final quality of food and human health.However,the current research in this area has not received the attention it deserves.Few studies have focused on the changes in the amount of polyphenols and biological activities during food processing process as well as the resulting changes in the health value of food.Moreover,experiments are limited to the evaluation of single kind of polyphenols,lacking of comparison among polyphenols with different structural characteristics in food systems.Flavonoids are a large class of natural polyphenolic compounds,especially in the plant kingdom,accounting for almost two-thirds of human dietary polyphenol intake.Flavonoids,widely found in natural foods,might affect food qualities during thermal processing.Naringenin is a typical flavonoid.Besides,the structural derivatives of naringenin,including naringin,hesperetin and dihydromyricetin are also representatives of typical flavonoids,all of which are the main natural components in human diet.Baked foods,as a common diet,have a large consumer base.In addition,some Maillard Reaction products such as advanced glycation end products produced during the processing of baked goods are potentially harmful to human health.This work aims to study the thermal response characteristics of these representative flavonoids,their effects on the Maillard reaction model system as well as baking model systems,especially the formation of advanced glycation endproducts,and effects and possible mechanisms of typical flavonoids on oxidative stress and inflammation induced by advanced glycation end products.In this study,the methods mainly used and important results are listed as follows:1.Glycine,phenylalanine and cysteine were selected as representatives of neutral,aromatic and sulfur-containing amino acids,respectively.The Maillard chemical model reaction systems were constructed by reactions between the above three typical kind of amino aicds with glucose,resepectively,under different pH values of four,seven and ten.Their final concentration of model systems was controlled at 50 mM and heated at 140°C for 60 min.The effects of naringenin(10 mM)on the characteristic Maillard reaction products in the model reaction systems were studied.Through MMSE-GC-MS combined with electronic nose technology,significant effects by naringenin on the characteristic Maillard volatile components,such as pyrazines,furans and thiophenes,in three types of model systems were found.Under neutral conditions,the overall odor substances of the three model systems were significantly reduced by the addition of naringenin,and the types and contents of volatiles varied according to the type of the specific model.In addition,the results of spectra as well as TEAC assay showed that naringenin could increase the browning intensity of the glycine model system,inhibit the formation of fluorescent AGEs and enhance the antioxidant capacity as well.HPLC-DAD results showed that naringenin itself did not undergo thermal degradation after heat treatment at 140°C for 60 min.No Loss of antioxidant capacity neither.However,the content of naringenin decreased exponentially over time in the model system.2.To further investigate the role of naringenin in Maillard reaction,a bread model was constructed.The enzyme-linked immunosorbent assay,LC-MS/MS and fluorescence analysis were used.In fact,in addition to volatile components,certain types of harmful Maillard reaction products can not be ignored in bread,a typical baking staple.With the increase content of naringenin fortified in bread(0.25-1%w/w),the formation of carboxymethyllysine CML and total fluorescent AGEs in the crust were significantly inhibited,with the inhibition rates of 19.67-54.27%and 11.79-35.19%,respectively.In addition,the intensification of 0.25%w/w naringenin significantly inhibited the formation of acrylamide in the crust(35.85%).3.In view of the positive effect of naringenin on the biological activity of the Maillard model system,further verification was carried out.To be more specific,the antioxidant and antibacterial activities were studied in bread model.Resulsts showed that the baking process could lead to the reduction of antioxidant capacity of naringenin,but its fortification could still significantly enhance the antioxidant activity of the bread crumb,with the evidence that ABTS~+radical scavenging ability increased with higher fortification levels.In addition,studies on total number of colonies showed that antibacterial activity of test bread was increased,following the similar trend with antioxidant capacity of the bread model.The above phenomena further demonstrated that the addition of naringenin can have positive effects on bioactivities of the bread model.Moreover,the analysis of electronic eye,texture and low field nuclear magnetic resonance showed that proper fortification with naringenin would not adversely affect bread qualities.Quantitative analysis with high performance liquid chromatography showed a loss of 31%for naringenin in the bread crust,indicating its participation in the Maillard reaction.Nevertheless,a high retention rate of 91%for naringnenin was observed in the bread crumb.4.In order to systemtically study the role of naringenin as well as the other typical food-borne natural flavonoids in the baking model,a new representative type of highly unsaturated fatty acid baking system was further constructed with the fortification level of naringenin at 0.25%w/w.Though no inhibitory effect on carboxymethyl lysine was observed,the inhibition of fluorescent AGEs and the enhancement of antioxidant activity of the system were confirmed similar to that in bread system.Therefore,naringenin indeed had significant effects on the characteristic Maillard products of the baking system.Moreover,the structural derivatives of naringenin were selected for further comparative study.Four flavonoids were found to be effective to inhibit the formation of Malondialdehyde.Protein carbonylation,fluorescent AGEs were significantly reduced,and antioxidant activity of the system were significantly enhanced with the intensification of the selected flavonoids except for naringin.Among them,dihydromyricetin showed the best effect in both reduction of toxicants as lipid and protein oxidation products,carboxymethyl lysine and enhancement of antioxidant activity in the model system.In order to study the above chemical and bioactivity changes caused by flavonoids intensification,specifically wheather due to the thermal degradation of them or their participation in the chemical reactions occurred in foods,further quantitative analysis by HPLC,total phenol determination,TEAC method and microbial experiment were carried out.The results showed that among the selected flavonoids separately heated at 180°C for 20minutes,naringenin and hesperetin were not degraded,which also confirmed by the constant inhibitory capability of Gram-negative/positive bacteria and the scanveging ability of ABTS~+radicals.A lost of 28.66%and 10.18%was observed in both naringin and dihydromyricetin,respectively,with a corresponding change in antioxidation and antibacterial activities.As for the high-unsaturated fatty acid model baking at 180°C for 20 minutes,naringenin and hesperetin have relatively high retention rates of 73.5,84.8%,yet only 36.1%of dihydromyricetin was remained after baking,accompanied by the great reduction of brightness in system.5.With the accumulation of foodborne AGEs,the body can induce oxidative stress and chronic inflammation,which can lead to chronic diseases.Since the selected typical flavonoid-type polyphenols can reduce the formation of AGEs to some extent,further investigation were carried out to see whether they can also reduce the AGEs induced toxicity by using the RAW264.7 cell line.The cell culture,cytotoxicity experiments,DCFH-DA fluorescence test and qPCR experiments were used to further investigated the effect of naringenin and its structural derivatives to AGEs induced oxidative stress and inflammation.The results of in vitro experiment in RAW264.7cell showed that naringenin,served as a positive control,could significantly reduce AGEs-induced reactive oxygen species(ROS)as well as gene expressions of typical inflammatory factors including interleukin-1β(IL-1β),IL-6,cyclooxygenase-2(COX-2)in RAW264.7 cells.Furthermore,the effects of other three flavonoids on AGEs-induced oxidative stress and inflammation were studied.It was found that dihydromyricetin and naringin(25μM)showed good inhibitory effect on ROS production,yet almost no significant effect on gene expressions of inflammatory factors even at the concentration of 40μM.However,hesperetin could significanlty inhibit AGEs-induced ROS production and gene expressions of TNF-α,IL-1β,IL-6,MCP-1,COX-2 and iNOS,which is similar to naringenin.6.Finally,high-throughput transcriptome analysis of AGEs-stimulated macrophages was performed and the possible protective mechanism of representative flavonoid hesperetin was investigated.By the analysis of differentially expressed genes and corresponding enrichment of GO and KEGG,it was found that AGEs could affect a variety of basic biological processes,cell composition and molecular function,while hesperetin played a regulatory role in inflammation and immunity.Combined with the results of pathway enrichment,Western blot and immunofluorescence,anti-AGEs-induced inflammation of hesperetin might be attributed to the inhibition of MAPK,JAK,NF-κB-related pathway activation.Furthermore,for high-throughput sequencing of all expression profiling data,gene set enrichment analysis(GSEA)was performed using the MSigDB database.The comparison between the AGEs vs BSA,and hesperitin vs AGEs led to the screening of interference related gene sets,which appeared with high frequency with statistical and biological significance.14 typical gene sets were further selected to conduct in-depth investigation in the gene level.Finally,twenty-three key genes,related to the harmful effect of AGEs and the protective effect of hesperetin,were screened and listed as follows:ZBP1,ISG20,ISG15,USP18,DDX60,SAMHD1,SP100,XAF1,MYD88,SAMD9L,IF135,IF144,IRF7,DHX58,SP110,EPST11,WARS,STAT1,GBP4,CXCL10,PARP12,TAP1,OAS2.In summary,this study evaluated the thermal response characteristics of four representative flavonoids,their effects on characteristic Maillard reaction products in both chemical model system and baking model system,especially against the AGEs’formation and induced oxidative stress and inflammation.This study might lay a theoretical foundation for basic research and new product development of polyphenol-rich foods.