Study Of Dietary Flavonoids Trapping Acrolein

Acrolein(ACR),as a kind of highly-reactive unsaturated aldehyde,mainly produced by excessive heating of oil and fat in food,endogenous metabolism and incomplete combustible of organic materials.Acrolein can accumulate in human body by cross-linking with protein,DNA,RNA,and further induces a series of chronic dieases:cardiovascular dieases,diabete metillus,lung cancer,Alzhehimer etc.Numbers of studies demonstrate that dietary flavonoids can trap ACR through Michael addition.This paper aimed to investigate,in in vitro and in vivo environments,the ACR-trapping activity and mechanism of four different flavonoids,including phloretin,EGCG,quercetin and genistein,and also studied on their structural related activity.The in vitro study focused on the flavonoids' structural effect on ACR-trapping percentage,and deeply exploit the key-effect of opening C-ring,this study also synthesized the ACR adduct of genistein for the first time.In addition,through mouse study,we also studied with LC-MSn the metabolic pathway and mechanism of flavonoids scanvaging ACR in vivo,and discussed the bioavailbility and biotransformation effect on ACR-trapping activity of flavonoids.In vitro study1.Comparing the ACR-trapping efficacy of four different flavonoids,including phloretin(dihydrochalcone),EGCG(flavan-3-ol with galloyl group),quercetin(flavone),genistein(isoflavone),the result showed that the trapping ACR percentage in decreasing order was:phloretin,EGCG,quercetin and genistein.Particularly,the ACR-trapping efficacy of phloretin was evidently better than that of other flavonoids,which can trap 99.4%ACR at 1.5 h.2.Choosing four polyphenols with typical structure(hesperitin,dihydrochalcone-hesperitin,maclurin and phloroglucinol)to exploit the effect of opening C-ring on ACR-trapping activity.The results showed that the hesperitin could only trap 32.4%ACR at 6 h,however,after its C-ring being opened,the new flavonoids Di-HES could reach 99.2%ACR-trapping rate at 1.5 h,which indicated that opening C-ring facilitated ACR-trapping activity of flavonoids.Besides,maclurin and phloroglucinol shared the same A-ring structure with dihydrochalcone,and also possesed high ACR-trapping rate,which verified that was the integrity of A-ring contributing to the advancement effect of opening C-ring structure on trapping ACR.3.Establishing the reaction system between genistein and acrolein,the reaction mixture would be seperated and purified by Sephadex LH-20?TLC and Silica colomn,and the final purity of ACR adducts with genistein(GEN-ACR)could reach 94%approximately.The structure of GEN-ACR would be elucidated by LC-MS and NMR,which found a hemiacetal structure was formed on A-ring of genistein via Michael addition.In vivo studyIn vivo study built rich ACR mouse model in which mice received both flavonoids and ACR via oral gavage,and normal mouse model in which mice only received flavonoids.The linear ion trap mass spectrometry was used to detect and analyse the flavonoids and their metabolites conjugated with ACR in mouse urine and fecal samples,so as to illustrate the metabolic pathway and mechanism of flavonoids scanvaging ACR in vivo.1.Phloretin and its metabolites can efficiently quench ACR in vivo.In phloretin+ACR-treated mouse group,both Mono-and Di-Ph-ACR could be detected in urine samples collected at 4,8,24 h using LC-MSn,while in mouse urine samples gained from 200,100,50 mg/kg phloretin-treated mouse groups,only mono-Ph-ACR could be found and its yield(based on NL value)was positively related to the phloretin dose.Afterwards,further researches were performed on mouse urine samples collected from 50 mg/kg phloretin-treated mouse groups,in which the hydroxylated,glucuronidated and sulfated metabolites of phloretin conjugated with ACR(3'-OH-Ph-ACR,GUL-Ph-ACR,SUL-Ph-ACR)were detected using LC-MS2.In 400,200 mg/kg EGCG-treated-groups,mono-EGCG-ACR could only be detected in mouse fecal but not urine samples after 24 hours.Wheras the methylated and further glucuronidated EGCG adducts with ACR(MeEGCG-ACR,GUL-MeEGCG-ACR)were detected in mouse urine samples collected from 200 mg/kg EGCG-treated-group at 4,8,24 h.However,the NL value of all these ACR adducts was low,which indicate that the ACR-trapping ability of EGCG in vivo was not effective.3.ACR adduct of quercetin and its metabolites(QUE-ACR,3'-MeQUE-ACR,8-MeQUE-ACR,GUL-QUE-ACR)can be detected in mouse urine samples collected from quercetin and ACR-treated but not quercetin-treated group,however,QUE-ACR,3'-MeQUE-ACR,8-MeQUE-ACR could be found in fecal samples obtained from quercetin-treated group.Therefore,we supposed that quercetin may tend to trap ACR in gut because of its relatively low bioavailbility.4.In both established genistein+ACR-and genistein-treated mouse groups(model and normal groups,respectively),ACR adduct of genistein(GEN-ACR)could be detected in mouse fecal samples,and the NL value of GEN-ACR in model group was 10 times higher than that in normal group,but no metabolites of genistein conjugated ACR were found in feces.However,in mouse urine samples gained from genistein-treated mouse groups,orobol(phase ? metabolite of genistein)and 6'-DMA-OH(gut microbiota metabolite of genistein)conjugated with ACR could be dtected as well as GEN-ACR.5.Bioavailability and biotransformation can affect the trapping ACR efficacy of flavonoids in vivo.For bioavailability,genistein is highest followed by phloretin,quercetin and EGCG is lowest,under this influence,ACR-trapping ability of EGCG and genistein in vivo contradicted that in vitro,generally,in in vivo study,phloretin also showed the strongest trapping ability,followed by genistein and quercetin,EGCG is weakest.Besides,the way biotransformation affect the in vivo ACR-trapping ability of flavonoids was adhere to the structural-related-activity law concluded from in vitro study,for example,because the glucumide or sulfate substitutes the hydroxyl group on A-ring,the NL value of GUL-Ph-ACR,SUL-Ph-ACR was weaker than that of 3-OH-Ph-ACR,even though phase ? metabolites is the main biotransformation of phloretin.Moreover,6'-DMA-OH,as a microbiota metabolite of genistein,possess the opening C-ring,thus its NL value was 2 and 10 times more than that of GEN-ACR,Orobol-ACR,respectively.