Study On Antioxidant Mechanisms And Products Of Phenolic Components Of Eucommiae Cortex And Eucommiae Folium

Objective1.The components and contents of Eucommiae Cortex(EC)and Eucommiae Folium(EF)were detected by ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry(UHPLC-Q-Exactive-Orbitrap MS/MS)and HPLC,and the differences between the two were analyzed.2.The antioxidant activity and mechanisms of the components in EC and EF were explored through quantum chemical calculations and chemical experiments in vitro.3.Using coniferyl alcohol(CA)as an example,the antioxidant products of the components in EC and EF were further elucidated by studying its transformation process under laccase in vitro.MethodsIn the first part of this study,the components of EC and EF were systematically identified based on UHPLC-Q-Exactive-Orbitrap MS/MS method.The contents of four flavonoids(quercetin(QU),kaempferol(KA),isoquercitrin(IS),and astragalin(AS))and four phenylpropanoids(CA,coniferin(CON),chlorogenic acid(CH),and caffeic acid(CAF))in EC and EF were subsequently determined by HPLC.In the second part of this study,QU,KA,IS,and AS were studied to analyze their antioxidant activity and mechanisms.Initially,thermodynamic parameters,including bond dissociation enthalpy(BDE),ionization potential(IP),proton dissociation enthalpy(PDE),proton affinity(PA),and electron transfer enthalpy(ETE),were acquired by quantum chemistry calculation with Gaussian to characterize three antioxidant mechanisms(hydrogen atom transfer(HAT),single electron transfer followed by proton transfer(SET-PT),and sequential proton loss electron transfer(SPLET)),which are based on hydrogen-abstraction reaction.Furthermore,this study calculated theΔG of 2,2-diphenyl-1-picrylhydrazyl(DPPH~·)radical adduct reaction(RAF)to assess the difficulty of this reaction.Subsequently,transient state calculation for the reaction of~·OH-scavenging by these flavonoids was studied.Finally,several radical-scavenging assays and ferric ion reducing antioxidant power assay were performed to detect the antioxidant activity in vitro of these four flavonoids and confirm the calculation results.In the third part of this study,their antioxidant activity and mechanisms of CAF,CH,CA,and CON were investigated using the methods described in the second part.Moreover,this part studied the possible RAF process of phenylpropanoids using CA as an instance.To achieve this,NBO and Gaussian were firstly used to calculate the CA radical and obtain its main resonance structures and their weights based on natural resonance theory.Subsequently,single point energies of structures involved in the theoretical analysis were calculated using Gaussian.Finally,the RAF products of CA catalyzed by laccase were determined by HPLC to confirm the calculation results.ResultsAccording to the results of UHPLC-Q-Exactive-Orbitrap MS/MS identification,this study identified 19 components from EC,including eight phenylpropanoids,five lignans,four organic acids,one coumarin,and one flavonoid,plus 27 components from EF,including18 flavonoids,five phenylpropanoids,two alkaloids,one ligan,and one steroid.The contents of the four flavonoids and the four phenylpropanoids of EC and EF were examined by HPLC,and the results showed that flavonoid glycosides,QU and CH were more abundant in EF.In contrast,KA and other phenylpropanoids were more abundant in EC.In terms of the HAT activity,the four flavonoids exhibit different patterns in gas phase and in solution.In the gas phase,the order of activity is QU>IS>KA>AS,whereas in solution,the order is QU>KA>IS>AS.The HAT activity order of each hydroxyl group varies among the flavonoids.For QU and IS,the order is 4’-OH>(3-OH)>3’-OH>7-OH>5-OH;for KA and AS,the order is(3-OH)>4’-OH>7-OH>5-OH.The SET-PT mechanism was analyzed by combining IP and PDE,and the activity laws of the four flavonoids under the SET-PT mechanism were consistent with those of HAT.The results of frontier molecular orbital(FMO)theory and molecular surface electrostatic potential(ESP)analysis indicated that the hydroxyl and carbonyl groups were the possible sites of electron transfer(ET).The SPLET mechanism was characterized by PA and ETE parameters,and the results also confirmed the above activity laws.The results of RAF calculations for the four flavonoids showed that the5-OH and 7-OH were the relatively active sites for RAF.The calculation results showed that the~·OH-scavenging reaction by the four flavonoids was a spontaneous exothermic reaction,and the activity orders in thermodynamics and kinetics were:QU>KA>IS>AS and KA>QU>IS>AS,respectively.The experiments of the~·OH reaction satisfy the calculation results of thermodynamics,and the results of other antioxidant assays are basically consistent with the calculation results of the hydrogen-abstraction reaction.In terms of the HAT activity of four phenylpropanoids,the HAT activity of CAF and CH were found to be similar,while the HAT activity of CA was stronger than that of CON.The results of FMO theory and ESP analysis indicated that the sites of ET were the benzene ring,as well as the O and C of the double bond conjugated to it.The activity laws of the four phenylpropanoids under the SET-PT and SPLET mechanisms were consistent with those of HAT.The results of the antioxidant assays in vitro were consistent with the above results for the activity of the mechanism based on the hydrogen-abstraction reaction.The calculations of RAF with DPPH~·of the four phenylpropanoids revealed that the active sites were the hydroxyl or carboxyl groups of the side chains of the benzene ring.Furthermore,the study of RAF products catalyzed by laccase showed that CA through hydrogen-abstraction,β-β’oxidation coupling and nucleophilic addition,the products was a pair of enantiomers,namely(+)-pinoresinol and(-)-pinoresinol.Conclusions1.The components of EF mainly consist of flavonoids,and EC mainly consist of lignans;furthermore,flavonoid glycosides,QU and CH are more abundant in EF,while KA and other phenylpropanoids are more abundant in EC.2.The HAT activity of phenolics is reduced by the formation of intramolecular hydrogen bonds by phenolic hydroxyl groups.The stability of radicals after hydrogen-abstraction could characterize the HAT ability,which can be quantified by the standard deviation(SD)of spin population.The lower the SD value,the more stable the radicals are,and therefore the more potent HAT activity for the corresponding hydroxyl groups is.3.The information on the phenolics in the HAT mechanism,such as the activity and active sites,is basically consistent with those in the SET-PT and SPLET mechanisms.However,the thermodynamic priority of the HAT mechanism is higher.4.The activity and active sites of RAF with DPPH~·occurring in phenolics are inconsistent with the hydrogen-abstraction reaction.The active sites of the latter reaction of flavonoids are 3-OH or 4’-OH,and the flavonoid glycosides activity is weaker than the corresponding aglycones.The active sites of the former reaction are 5-OH and 7-OH,and the activity of flavonoid glycosides is more potent than the corresponding aglycones.The active sites of the phenylpropanoids of the latter reaction are the hydroxyl groups,and the active sites of the former reaction are the hydroxyl or carboxyl group of the side chain of the benzene ring.5.CA undergoes an enzymatic process by laccase to produce(+)-pinoresinol and(-)-pinoresinol through hydrogen-abstraction,and oxidative coupling and nucleophilic addition.