RAF Mechanisms Of Fifty-six Phenolic Antioxidants:Experimental Evidence And Analysis

ObjectivePhenolic antioxidants(especially phenolic antioxidants in traditional Chinese medicine)scavenge free radicals and release oxidative stress in cells through a variety of chemical mechanisms.Radical addition(RAF)is one of the chemical mechanisms.At present,the antioxidant mechanism of RAF are studyed in the theoretical study of quantum chemistry,which lacking experimental evidence.Therefore,the antioxidant mechanism of RAF is needed rather more realistic conditions.The products of the reaction of 56 phenolic antioxidants with DPPH·free radicals were analyzed by UPLC-ESI-Q-TOF-MS/MS.Discuss their mass spectra information furtherly and study the possible mechanism of reaction.MethodsFifty-six phenolic antioxidants,including monophenylcyclophenol(e.g.6-gingerol)and biphenylcyclophenol(e.g.magnolol),were selected to scavenge DPPH·free radicals.Free radicals were reacted with antioxidants in different proportions for 12 hours.The reaction solution was diluted 20 times and put into the liquid bottle.The reaction solution was separated and analyzed by UPLC-ESI-Q-TOF-MS/MS.According to the fragment ion decomposition rule of mass spectrometry,the products and the possible RAF mechanism of the reaction was inferred.ResultsSolvent effectIn different solvents(in methanol and acetone),the mass spectra of gallic acid dimer products are different.In this case,3-methoxybenzene-1,2-diol reacts with DPPH·(the mass spectra of adducts are different).Compared with methanol solution,the adduct and dimerization peaks of reaction products in acetone can be detected more easily on UPLC-MS chromatogram.Monobenzene ring systemIn addition to catechol and hydroquinone,monophenols can detect adduct and dimerization peaks on UPLC-MS chromatogram.Generally speaking,the number of adduct and dimerization peaks of phenol is more than that of catechol and pyrogallol.One of the phenol dimer's retention time were similar to those of 2,2-biphenylcatechol,and so was mass spectrometric decomposition.The other one was similar to magnolol in the retention time and mass spectrometric decomposition of piperol dimer.Biphenyl ring systemWhen biphenylcyclophenols react with DPPH·free radicals,a large number of adduct and dimerization peaks are generated.For example,the number of adduct and dimerization peaks of 2,2'-biphenylcatechol is more than 4,4'-biphenylcatechol and the number of dimerization peaks.Adduct and dimerization peaks of oxyresveratrol,oxyresveratrol-3 '-O-?-D-glucose and mulberroside A can be extracted.However,their peak areas are quite different.The peak areas are oxyresveratrol>oxyresveratrol-3 '-O-?-D-glucose>mulberroside A.Both piceatannol and piceatannol-glucose can be extracted into adduct and dimeric peaks.The peak area of piceatannol-glucose is less than that of piceatannol.Similarly,there are hapontigenin and rhaponticin.However,resveratrol,?-viniferin and a-viniferin can extract adduct and dimerization peaks,however,The smaller of molecular size,the fewer dimers peak area was generated.Both the dihydroresveratrol and resveratrol can extract adduct and dimerization peaks,but the number of chromatographic peaks of dihydroresveratrol is lower than that of resveratrol.The adduct and dimerization peaks of tetrahydrocurcumin,Demethoxycurcumin and dioxycurcumin can be extracted,but the amount of adduct products produced by tetrahydrocurcumin is smaller than others,and there is no secondary mass spectrometry sign of tetrahydrocurcumin.Conclusions1.Solvent effect:When phenolic antioxidants scavenge DPPH·free radicals,the solvents will affect the stability and pyrolysis of the products.2.RAF product types:the combination of phenolic antioxidants and DPPH·free radicals;the dimerization of phenolic antioxidants with phenolic antioxidants.3.Phenolic hydroxyl groups:in the phenolic substances of the monophenyl ring system,the reaction rate of monophenol with DPPH·radical is slower than that of dihydroxyphenol and trihydroxyphenol.On the contrary,the number of monophenol adduct peaks is much larger than that of dihydroxyphenol and trihydroxyphenol.The RAF of phenolic antioxidants with DPPH·free radicals can be explained by the following:the first step was the reaction sequence of dehydrogenation;then the formation of quinones.Quinones are more stable than semiquinones in this process.4.The number of benzene rings:(1)monophenyl rings:when monocyclic phenols scavenge free radicals,dimerization products can be detected;when monocyclic phenols are excessive,trimerization and tetramerization products may also be detected.The number of adduct peaks of monocyclic phenols is mainly related to the ratio of reaction products,the ortho and para substituents of phenolic hydroxyl groups.(2)the complex structure of phenolic compounds in the biphenyl ring system,many aspects are taken into account about the mechanism of RAF,such as the conjugated system of phenolic compounds.5.Conjunction sites:Take DPPH·free radicals as an example,the linking mode of adduct reaction is mainly N-C and N-O;the linking mode of dimerization reaction is mainly C-C and C-O.The dimerization sites of Monocyclic phenol,at least in-OH ortho-junction.