Formation Of Rutin-methylglyoxal Adducts In Foods And In Vivo,and Their Cytotoxicity Evaluation

Methylglyoxal(MGO)is a highly reactive α-dicarbonyl compound(α-DCs)widely present in food and in vivo,which can pose a threat to human health.Polyphenols with meta-phenol structure have been reported to easily react with MGO,and scavenge it.Rutin(3’,4’,5,7-tetrahydroxy-flavone-3-rutinoside)is a dietary flavonoid widely distributed in plants,which possess the scavenging capacity for MGO with the typical meta-phenol structure.However,the safety of the products formed between rutin and MGO after the elimination of MGO by rutin is unknown,which brings potential threats to food safety.In this study,the elimination capacity of rutin for MGO was evaluated under the simulated physiological conditions,and its elimination mechanism was investigated.Five rutin-MGO adducts were detected and prepared for structure elucidation.The determination methods of adducts in food and physiological samples were established to evaluate the formation levels of adducts in food and in vivo.Moreover,the cytotoxicity of adducts against gastrointestinal tract,blood vascular and neural cell lines was evaluated.The main results are as follows:1.The elimination capacity and scavenging mechanism of rutin for MGOUnder simulated physiological conditions(37 ℃,pH=7.4),the elimination of MGO by rutin increased significantly with the extension of the incubation time.At 15 min,the elimination percentage of MGO was 7.3%,which reached 50.0% finally at24 h.Rutin can scavenge MGO through adducts formation via the nucleophilic addition reaction.In the rutin-MGO reaction solution,five main adducts were discovered,and their concentrations were positively correlated with the incubation time.The total yield of adducts was 12.3 pHM at 15 min and increased to 175.6 pHM as the time prolonged to 24 h.According to the HPLC chromatogram,three rutin-MGO adducts with higher amounts were named adducts a,b,and c,while the other less abundant compounds were named adducts d and e.Adduct c was always the most abundant product,accounting for 40%-63% of the total amount of adducts formed throughout the incubation period,followed by adducts b(16%-25%),a(7%-16%),e(3%-16%)and d(3%-12%).2.The preparation,purification and structural characterization of rutin-MGO adductsThe preparation conditions of rutin-MGO adducts were optimized.Finally,the adducts a,b and c were prepared by reacting 10 m M rutin with 100 m M MGO at 70 ℃under pH 7.4 for 13 h.The mixed reaction solution was separated and purified by Sephadex LH-20 and octadecylsilyl-A-HG(ODS)to obtain the standards(purity >97%).The adducts d and e were prepared with solution containing 10 m M rutin and50 m M MGO at pH 7.4,50 ℃ for 4 h,and then separated by a preparative HPLC to obtain high purity(> 95%)adducts.HPLC-MS/MS and HRMS analysis showed the molecular weights of adducts a and b were identical to be 752.17,that of c was 750.15,and those of d and e were both 754.18.The molecular formulas of adducts a,b,c,d,e were C33H36O20,C33H36O20,C33H34O20,C33H38O20,C33H38O20,respectively.Their structures were further identified by NMR.Adducts d and e were generated by the electrophilic addition of two molecules of MGO on the C6 and C8 in the A ring of rutin.Adducts a,b,c was formed through further oxidations of adducts d and e at different extents,which resulted in dione structures in MGO substitute groups.3.The formation levels in food and in vivo and cytotoxicity of rutin-MGO adductsThe formation of adducts in food and in vivo was analyzed with HPLC-MS/MS.It was found that the contents of oxidized adducts a,b and c in commercial food were higher,with the highest values being 1.43,2.85 and 2.79 mg/kg,respectively.However,in the freshly prepared biscuits fortified with rutin,the contents of unoxidized adducts d and e were found to be higher,with the values up to 76.63 pHg/kg and 160.33 μg/kg.The adducts were also detected in serum and tissues when rutin(100 mg/kg BW)was administrated to rats.The concentration of adducts a,b,and c in serum reached maximum at 0.25 h(2.37,1.61 and 1.02 pHg/L,respectively),and then decreased rapidly,the turns to stable after 4 h;The concentration of d and e only reached the detection limit at 0.25 h.In tissue samples collected after 24 h of administration,adducts a,b,c were only detected at quantitative levels in heart,brain,and kidney,but hardly accumulated in stomach,intestine,and liver.These results indicated that the adducts could be formed in vivo,absorbed into blood circulation,and transported to organs after rutin was ingested.Therefore,GES-1,Caco-2,HUVEC and PC-12 cells were used to evaluate the cytotoxicity of the five adducts.The results showed that the viability of GES-1 and PC-12 cells was reduced to 17%and 21%,and that of HUVEC decreased to 49% after treatment of 1 m M MGO for 24 h.In comparison,the treatments of 1 m M adducts a,b,c,d and e elevated the cell viability of GES-1 to 83%,73%,77%,73% and 75%,that of PC-12 to 83%,76%,87%,82% and 83%,respectively,and that of HUVEC cells to all over 80%.This indicated that the formation of adducts between rutin and MGO remarkably reduced the cytotoxicity of MGO.Caco-2 cells showed resistance toward the treatment of MGO.But the formation of adducts still improved the cell viability at the highest treatment concentration(1 m M).In this study,it was demonstrated that rutin possesses good scavenging capacity on MGO,and the formation of the adducts between rutin and MGO lowers the cytotoxicity of MGO.The results suggested that rutin can be used as a dietary supplement to reduce the deleterious health effects of MGO and inhibit the formation of harmful substances such as AGEs.It is expected to become a new and safe food additive,which can be used in food,medicine and other fields.