Mechanisms Of Purine Alkaloids In Green Tea And Coffee Inhibiting Acrolein In Vitro And In Vivo

Increasing evidence has identified dietary intake and endogenous acrolein(ACR)could accumulate in the body,which is a pathogenic factor in the progression of many pathological conditions.Recently,the inhibition pathway and mechanism of ACR in vivo and in vitro have attracted extensive attention of scientists in related fields all over the world.Eliminating ACR by dietary active substances has been found to be one potential strategy to prevent ACR-associated chronic diseases.Purine alkaloids are one of the most important active ingredients in tea and coffee,and become food additives and health care drugs in people's daily life.In this paper,the mechanism and structure-activity relationship of purine alkaloids scavenging ACR in vitro and in vivo were systematically studied.Firstly,the mechanism of purine alkaloids scavenging ACR in vitro was studied.Four purine alkaloids,theophylline(TP),theobromine(TB),paraxanthine(PXT)and caffeine(CAF),with the same xanthine ring,were compared for their ACR scavenging activities.TP exhibited an outstanding ACR scavenging capacity,while CAF had little activity.Our result suggested that the order of the capacity of the active site to trap ACR on the xanthine core was N-7>N-1>N-3,and N-9 could not trapping ACR directly.Furthermore,in order to study the kinetics of TP and ACR,the effects of different reaction ratios and incubation times on the formation of adducts were investigated by LC-MS/MS.The results showed that mono-ACR-TP appeared after 1 min reaction of TP with ACR at the ratio of 1:1;Di-ACRTP appeared after 60 min reaction at the ratio of 1:3;tri-ACR-TP appeared after 60 min reaction at the ratio of 1:10.And then,to verify the mechanism of TP trapping ACR,the adducts of ACR conjugated with TP were synthesized and purified.And their structures were elucidated by high resolution mass spectrometry(HRMS)and one-and two-dimensional nuclear magnetic resonance(1D-,2D-NMR).Through the determination of the scavenging activity of ACR by the adducts,combined with LC-MS/MS analysis,it was determined that the N-7 position of TP had Michael addition with ACR to form Mono-ACR-TP.And then the ?-C of Mono-ACR-TP reacted with ACR again to produce Di-ACR-TP.Next,the mechanism of purine alkaloids scavenging ACR in vivo was explored.The mice model was established by oral gavage of TP/CAF.After collecting the urinary samples of mice in metabolic cage,the adducts of TP and its main metabolites [1-methyluric acid(1-MU)and 1,3-dimethyluric acid(1,3-DMU)],CAF and its main metabolites [TP,paraxanthine(PXT),1-MU and 1,3-DMU] with ACR were analyzed by UPLC-MS/MS.The results showed that TP could still trap ACR to form mono-and di-adducts in vivo,and the metabolites of TP/CAF could also trap ACR to form corresponding adducts.The changes of adducts in urinary samples of mice treated with different doses of TP/CAF(200/400 mg/kg bw)was quantitatively analyzed.The results showed that there was a positive correlation between the content of adducts and the intragastric dose;when comparing the differences between the groups with or without additional administration of ACR,it was found that with the increase of ACR level in vivo,the content of the adducts of ACR with TP and the metabolites of TP/CAF increased,especially in TP group.Experiments were carried out in vitro: ACR was added to the brewed tea or instant coffee samples for incubation and the results of LC-MS/MS analysis showed that TP could also trap ACR to form adducts in tea/coffee.Then in vivo experiment: six volunteers were recruited and randomly divided into two groups.Each group drank four cups of green tea(2 g tea/cup)or two cups of coffee(4g coffee powder/cup)every day for four consecutive days.24 h urine samples were collected on the fourth day,and the results showed that the mono-and di-ACR-adducts conjugated with TP,mono-ACR-adducts conjugated with PXT,1-MU and 1,3-DMU were detected by UPLC-MS/MS,which proved that purine alkaloids in diet could trap ACR directly or through the formation of metabolites.Finally,the scavenging activities of four purine alkaloids and the ACR adducts of TP at high temperature were investigated under simulated food processing conditions.The results showed that TP and it's adducts had certain scavenging effects,and was verified in cookie model.Finally,the effects of 12 phenolic acids on the trapping effect of ACR by TP were studied.The results showed that myricetin,quercetin,kaempferol,galangin and curcumin could promote the reaction of TP with ACR,while phloretin,epicatechin,EGCG,ferulic acid,chlorogenic acid,caffeic acid and resveratrol could inhibit it.Furthermore,the combined scavenging activity of EGCG and TP on ACR was further analyzed.According to Chou-Talalay method,there was a significant synergistic effect between TP and EGCG when Fa was in the range of 0.05-0.75 at 37 °C and 0.05-0.55 at 100 °C.In addition,both of them had good synergistic effect at low concentrations,but with the increasing of concentration,the synergistic effect gradually weakened and the antagonistic effect gradually enhanced.