复方柴胡疏肝散抗氧化活性及抗抑郁作用机制研究

复方柴胡疏肝散抗氧化活性及抗抑郁作用机制研究

1“面向”interitystgs和oxidatchsoractschaihushu（csgo）是中国的标准亲水性普通话种子（rcm），最初被中断在“shu上”中。粗体和微体熙字母报告，西蒙徐e原材料1624（美国是美国）。css是正系列陆地运输，是反映过程中所有生命的过程，而不是牺牲过程中的生命，而是接受承认。随机性参与，反映绩效。随机性参与，反映绩效。包括，随机性参与，反映绩效。就地利用。这是一个缓慢的步骤。缓慢的奴隶，缓慢的人，缓慢的奴隶，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，缓慢的，。Exposuretostressisafactorofriskformentaldiseasesincludingdepression.Oxidativestress,brieflydescribedasincreasedproductionofreactiveoxygenspecies(ROS),lipidperoxidationandoxidantrelatedreactions,playsaroleinthepathogenesisofmajordepression.Ithasbeenreportedthatoxidativestresscausesthedestructionofcellsbydecreasingthevolumeofhippocampusinpatientswithmajordepression.Responseforstressisaphysiologicallyadaptablecourseoforganismsunderburdenandleadstochangesinthephysiologicalfunctionandmetabolism.Whenthepatientswithmajordepressionwereexposedtooxidativestress,thepotencyoftheoxidativestresswassignificantlyrelatedtotheseverityofthedepression.Antidepressantscouldsignificantlyimproveantioxidantenzymeactivities,increaseGSHlevelanddecreaselipidperoxidationlevelindepressionmodelanimalsontheassumptionthatantioxidantstatusinvivoisaputativetargetofantidepressantaction.Tothebestofourknowledge,theantioxidanteffectofCSGS,nomatterinvitroandinvivo,hasnotbeenexplored.InordertounderstandthepossiblerelationshipbetweenantioxidantpropertiesandtheantidepressiveeffectofCSGS,weexaminedtheinvitrotheantioxidativepotentialofCSGSandalsoinvestigatedtheantioxidativeactivityinvivousingmicewithoxidativestress.2杏仁杏仁2.1相关参数估计1,1-Diphenyl-2-picryl-hydrazyl(DPPH),2,4,6-tri(2-pyridyl)-S-triazine(TPTZ)and2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonicacid)diammoniumsalt(ABTS)werepurchasedfromSigma-Aldrich(Shanghai,China).TheassaykitswerepurchasedfromtheInstituteofBiologicalEngineeringofNanjingJianchen(Nanjing,China).VitaminE(VE)camefromtheCentralPharmaceticalCo.,Ltd.(Tianjin,China).AlltheotherreagentswereofanalyticalgradeandwerwpurchasedfromSinopharmReagentChemicalCo.,Ltd.(Beijing,China).2.2ijrafta-medicinalpoAllrawherbswereprovidedbyTongrentangTraditionalHerbalMedicineCompany(Beijing,China)andidentifiedbyProf.YULin-LinofInstituteoftheMedicinalPlantDevelopment,ChineseAcademyofMedicalSciences,China.ThevoucherspecimensaredepositedintheNaturalMedicinalChemistryResearchCenterofthisInstitute.2.3通过csgsaque分流程,见表3Sevenindividualherbs,Chaihu,Chenpi,Shaoyao,Zhiqiao,Xiangfu,ChuanxiongandGancao,weremixedintheproportionshowninTable1andweresoakedtogetherinwater(1g/10mL,W/V)for30minatroomtemperatureandthendecoctedfor2h.Aftercollectingthefiltrate,theherbswerethendecoctedinthesamevolumeofwaterforanadditional2h.ThefiltrateswerecombinedandconcentratedinvacuumtoobtainCSGSaqueousextractattheyieldof7.8%.2.4独立的优势2.4.1indexa&mertrace4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3日起价多进路的sox-axisreaclizacth3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3Ferric-reducingantioxidantpower(FRAP)ofCSGSaqueousextractwasdeterminedaccordingtothemethoddescribedbyBenzieandStrainwithmodifications.Inbrief,theFRAPreagentcontaining300mmol⋅L-1sodiumacetatebuffer(pH3.6),10mmol⋅L-1TPTZsolutionand20mmol⋅L-1FeCl3solutionwasfreshlyprepared.3mLofFRAPreagentwasmixedwith0.1mLofvariousconcentrationsofCSGSextractinddH2O.Afterincubationfor20minat37°C,theabsorbanceofthefinalsolutionwasreadat593nmwithddH2Oastheblankreference.ODvalueofthereactionwasexpressedasfollows:WhereAcistheabsorbanceofcontrol(ThesamplesolutionwasreplacedbyddH2O),AsristheabsorbanceofsamplereactionsolutionandAsbistheabsorbanceofthesampleitself(ThereactionreagentwasreplacedbyddH2O).TheFeSO4calibrationcurvewasconstructedasy=1.7451x+0.0013(r=0.9993)usingconcentrationsofFeSO4solutionsintherangeof0.025mmol⋅L-1,whereyrepresentsODvalues,andxistheconcentrationofFeSO4(mmol⋅L-1).FRAPvaluewasdefinedasthecorrespondingFeSO4concentrationsofdifferentsamples’ODinthecalibration.Differentconcentrationsofthesampleswereusedasthex-axisandthecorrespondingFRAPvalueswerethey-axistoplottheconcentration–FRAPcurve.TheresultwasexpressedasmmolFeSO4pergramsubstancefromthecurve.2.4.2sc浆sc东南角,whichDPPHfreeradicalsscavengingactivitywasdeterminedusingthemethodpreviouslydescribedbyLiuJK,etal.withslightmodifications.Briefly,3mLofaDPPHsolutionin70%methanol(0.06mmol⋅L-1)wasincubatedwith0.3mLofvaryingconcentrationsofCSGSextractdissolvedin70%methanol.Thereactionmixtureswerethenshakenwellandincubatedfor30minindarkatroomtemperature.Theabsorbanceoftheresultingsolutionwasreadat515nmwith70%methanolasthereference.Theradicalscavengingactivitywasexpressedasscavengingrate(SR),whichwascalculatedasfollows:WhereAcistheabsorbanceofcontrol(Thesamplesolutionwasreplacedby70%methanol),AsristheabsorbanceofsamplereactionsolutionandAsbistheabsorbanceofthesampleitself(DPPHsolutionwasreplacedby70%methanol).Theconcentrationofthesampleneededtoscavenge50%oftheDPPH(IC50)wasobtainedbyplottingtheconcentration–SRcurve.2.4.3as必要interpersonals.s.asieieges.s.s.s.s.n.sgisis.n.案例见表3ABTSformsarelativelystablefreeradical,whichdecolorizesinitsnon-radicalform.TheABTScationradicalsscavengingactivityofCSGSwasdeterminedaccordingtothemethoddescribedbyChunSS,etal.Forthemeasurement,0.1mLofvaryingconcentrationsofCSGSaqueousextractdissolvedinwaterwereaddedto3mLofABTSworkingsolutionandincubatedfor10minindarkatroomtemperature.Theabsorbanceofthefinalsolutionwasreadat734nmwithblanksolventasthereference.Theresultswereexpressedasthescavengingrateasfollows:WhereAcistheabsorbanceofcontrol(SamplesolutionwasreplacedbyddH2O),AsristheabsorbanceofsamplereactionsolutionandAsbistheabsorbanceofthesampleitself(ABTSworkingsolutionwasreplacedbyitssolvent).TheIC50valuewasobtainedfromtheconcentration-SRcurve.2.4.4uperolog相关程序Superoxideanion(O2·-)scavengingactivitywasmeasuredusingtheO2·-AssayKitaccordingtotheinstructions.Superoxideanionwasgeneratedbythexanthine–xanthineoxidasesystem.Afteraddingelectrontransmitterandchromogenicagent,theabsorbanceofthereactionmixturewasdeterminedat550nm.ThescavengingratesofCSGSatdifferentconcentrationswereusedtoestablishtheconcentration-SRcurvefromwhichIC50valuewascalculated.2.4.5so阶段asociectlitutionAccordingtoFentonreactionsystem,FeSO4reactswithH2O2togenerateOH·whichcanbecapturedbysalicylicacid.ThescavengingactivityofCSGSaqueousextractonOH·wasmeasuredbythemodifiedmethod.Thereactionsystemincluded1mLofFeSO4solution(9mmol⋅L-1),1mLofH2O2solution(8.8mmol⋅L-1),1mLofsalicylicacidethanolsolution(9mmol⋅L-1)and1mLofvaryingconcentrationsofCSGSaqueousextractdissolvedinwater.AfteraddingH2O2,themixturewasincubatedfor30minat37°C.Theabsorbanceofthereactionmixturewasmeasuredat510nmwithblanksolventasthereference.ScavengingactivitywasexpressedasSRthatcanbecalculatedasfollows:WhereAcistheabsorbanceofcontrol(SamplesolutionwasreplacedbyddH2O);AsristheabsorbanceofsamplereactionsolutionandAsbistheabsorbanceofthesamplewithoutreaction(H2O2solutionwasreplacedbyddH2O).Theaveragevalueofthreeparalleltestswasobtainedtoplotconcentration-SRcurvefromwhichIC50valuewascalculated.2.5控制好两因子:1.3项聚合物tempratorFifty(30±5)gICRmicewerepurchasedfromtheLaboratoryAnimalCenteroftheAcademyofMilitaryMedicalSciencesofChina,Beijing,China.Theanimalswerekeptundercontrolledconditions[temperature:(23±2°C);humidity:55%±5%;14hlight–10hdarkcycle]andwereallowedfreeaccesstostandardlaboratorydietandwaterthroughouttheexperimentalperiod.ThebreedingandexperimentalprotocolwasinaccordancewiththeethicalprinciplesofanimaluseandcareoutlinedbyEthicsCommitteeoftheInstituteofMedicinalPlantDevelopment,CAMS&PUMC.2.6独立的优势2.6.1非负义双轨回收方式mice/非负义有利于和提高反应的国际习惯法csgsaque读本Theanimalswererandomizedintofivegroups,lasttimeeachbeingadministered0.4mLliquorfor11daysconsecutive.Aftertheadministrationofmedicineforthelasttime,allgroupsexceptnormalcontrolwererestrainedinwell-ventilated50mLofpolypropylenetubesforonce18hwithoutaccesstofoodandwateraspreviouslydescribed.Thetreatmentgroupsofmicewereorallyadministeredby2.5g⋅kg-1bodyweight(lowdosagegroup)and10g⋅kg-1bodyweight(highdosagegroup)ofCSGSaqueousextract.MicetreatedbyVEatthedosageof50g⋅kg-1bodyweightservedaspositivecontrol.Themedicinesweredissolvedin50%PEG400.Inparallel,themiceinthenormalcontrolgroupandthemodelgroupwereadministeredwater/PEG400(1∶1)eachdayinthesamevolumeasthetreatmentgroups.2.6.2determinationof应用程度Allmiceweresacrificedrightafterrestraintstresstreatment.Bloodsampleswerecollectedintoheparinizedtubesandnine-foldvolumeofddH2Owasaddedtomake1∶9hemolysateforthedeterminationofGSHcontent.Liverswereweighedandhomogenizedincoldnormalsalinetoobtain10%homogenate.Thehomogenatewascentrifugedat3500r⋅min-1for15min,andthesupernatantwasusedforthedeterminationofMDAcontent.The10%liverhomo-genatewasthendilutedto1%homogenateusingcoldnormalsalineandalsocentrifugedat3500r⋅min-1for15min.ThesupernatantwasusedtodetermineSODandCATactivity.2.6.3sodactitySuperoxideanion(O2·-)canpromotetheoxidationofhydroxylamineintoitsnitriteformswhichtakeonamaranthundertheeffectofchromogenicagent.Superoxidedismutase(SOD)isaspecialinhibitortoO2·-.SODactivitywasdeterminedusingSODAssayKit.Asaresult,theactivityinliverwasexpressedasunitspermilligramprotein.2.6.4其他互通工程用响应面模型Catalasecandirectlydecomposehydrogenperoxide(H2O2)underacertainconditiontodecreasetheconcentrationofH2O2andtofurtherreducetheopticaldensityat240nm.TheactivitywasdeterminedusingCATAssayKitandtheresultswereexpressedasunitspermilligramproteininliver.2.6.5u2004index,methodmethodusingdrasingmartingmartyfig因地制宜,u2004.Malondialdehyde(MDA)isafinalproductionoflipidperoxidationandthecontentwasdeterminedbythiobarbituricacid(TBA)methodusingMDAAssayKit.MixtureofsampleandTBAwereheatedinanacidicandhightemperatureenvironmentforaperiodoftime,andthentheabsorbancewasdeterminedat532nm.Thecontentinliverwasexpressedasnmolpermilligramprotein.2.6.6dtizactizactizactizactdtnbwhichrectebdtnbGSHcontentwasdeterminedusingGSHAssayKit.Thecontentwasmeasuredusingdithiobisnitrobenzoicacid(DTNB)whichreactedwiththefreethiolinGSHtoproduceyellowsubstance,whichcanbequantifiedbytheabsorbanceat420nm.TheresultswereexpressedasgramGSHperliterblood.2.6.7通过whichtheamonblot,athich-amwellsoinsagrate/atinb3.3.4.3.4.3.4.3/继续融资信托agracepalite/ag化学混合式运行TheproteincontentoftissuesampleswasmeasuredusingCoomassieBlueStaining(Bradford)proteinAssayKit,inwhichtheaminogroupofproteinwascombinedwiththeanionofthedyetogiveabluesubstancethatcouldbequantitativelydeterminedbytheabsorbanceat595nm,tofurthercalculatethecontentofprotein.2.7statingspss软件Alltestswerecarriedoutintriplicate.Ascorbicacid(Vc)wasusedasapositivecontrolforinvitroassays.Alldataareexpressedas.StatisticalanalysiswascarriedoutusingSPSSsoftware(SPSS,version10.0).DifferencesbetweengroupswereevaluatedbyStudent’st-testandwereconsideredtobestatisticallysignificantifP-valueswerelessthan0.05.3产品系统3.1独立的优势3.1.1relativitititituct.4.3.3.3信度指标的合成Underacidicconditions,antioxidantsreduceFe3+–TPTZcomplexesintotheFe2+–TPTZforms,thecolorofwhichchangestodarkandcouldbemonitoredat593nm.Theresultsareexpressedasferrousionequivalentantioxidantcapacity.Fig.1showedexcellentlinearrelativitybetweenconcentrationsandFRAPvaluesofCSGSaqueousextractwithequation:y=0.2379x+0.0133(r=0.9984),whilethatofthepositivecontrolVcwas:y=0.5794x+0.313(r=0.9993).Thetotalantioxidantcapacitieswere0.24mmolFeSO4pergramCSGSaqueousextractand5.79mmolFeSO4pergramVc,respectively.3.1.2通过csgsaque中小型pcr和while-en反应表1.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4和4.4.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3DPPHisastablenitrogen-centeredfreeradicalthecolorofwhichchangesfrompurpletoyellowuponreductionbyeithertheprocessofhydrogenorelectrondonating.CSGSaqueousextractshowedadose-dependentscavengingactivityonDPPHradicalswithIC50valueof0.83mg⋅mL-1(Fig.2),whiletheIC50ofthepositivecontrolVcwas14.95μg⋅mL-1.3.1.3ssgsaquewelle-关于csgsaque的选择ABTScationradicalsgeneratedfromoxidationeffectofpotassiumpersulphateformsglaucouschromophoreandcouldbemonitoredat734nm.GlaucousABTScationradicalsreactedwithantioxidantstoproducecolourlessABTS.CSGSaqueousextractdisplayedthedose-dependentscavengingactivityonABTScationradicals(Fig.3).TheIC50valuewas1.03mg⋅mL-1,whilethatofVcwas16.59μg⋅mL-1.3.1.4双线性acidpt和csgsaquewelle-acidca设备,sicqlg/msgsaquewelleSuperoxideanion(O2·-)wasformedduringthecourseofoxidationofxanthinetouricacidcatalyzedbyxanthineoxidase.Asforthisassay,theIC50valueofCSGSaqueousextractwas10.31mg⋅mL-1asthedosedependentcurveshowninFig.4,whilethatofVcwas150.2μg⋅mL-1.3.1.5清三大hydrogenperact标准ThemostcommonlyusedmechanismtogenerateOH·istheFentonreactionwhereironcatalyzesthedecompositionofhydrogenperoxide(H2O2)toOH·.AsshowninFig.5,theeffectofCSGSaqueousextractonOH·presenteddosedependenttrendwithIC50valueof7.79mg⋅mL-1,whilethatofVcwas159.8μg⋅mL-1.3.2独立的优势3.2.1rest酸4.4.4inblotstsiptraftrest酸capa化合物rest酸sigraftingrest5.4.4.4.4.4.3.4.3.4.3.4.3.4.3sraftingraftingraftingpo专门的stes保护AswasshowninFig.6,afterbeingrestrainedfor18h,SODandCATactivitiesinliver(P<0.01)andGSHcontentinblood(P<0.01)ofmodelgroupmicedecreasedsignificantlycomparedwiththenormalgroupmice,whileMDAcontentinliver(P<0.01)remarkablyincreased.Theresultssuggestedthatantioxidantcapabilityoforganismunderrestraintstresswasevidentlyweakenedandtherestraintoxidativestressmodelwassuccessfullyestablished.3.2.2细胞利养Afterbeingrestrainedfor18h,SODactivityofthemodelgroupmicedecreasedsignificantly(P<0.01)asshowninFig.6a.IncreasedactivitywasfoundaftertheadministrationofCSGS(P<0.01)andVE(P<0.01)inadose-dependentmannercomparedwiththemiceinmodelgroup.TheresultindicatedthatCSGScouldimproveSODactivityofmiceunderrestraintstress.3.2.3国际且非碳质对ceraftityrest酸溶液strs的影响TheresultsshowninFig.6bindicatedthattherewasremarkabledecreaseofCATactivityinmiceonlyreceivedrestraintstress(modelgroup,P<0.01).AdministrationofCSGS(P<0.05)andVE(P<0.05)couldincreaseCATactivityandtheeffectofCSGSinhighdosagewasbetterthanthatofVE.SoCSGShadtheabilitytoresistreductionofCATactivityofmiceunderrestraintstress.3.2.4no对no民族和no对grestcition.SeenfromFig.6c,MDAcontentinliverofthemodelgroupmicesignificantlyincreased(P<0.01)comparedwiththenormalgroupafter18hrestriction.AdministrationofCSGS(lowdosagegroup:P<0.05;highdosagegroup:P<0.01)andVE(P<0.01)couldinhibittheformationofMDA.Besides,highdosageofCSGSshowedbettereffectthanVE.TheresultindicatedthatCSGScouldreducelipidperoxidationcausedbyoxidativestressandrelievedamageinliver.3.2.5抗炎与面向网络so-graftrest东北部asrest鞣TherewasaremarkabledecreaseofGSHcontentinbloodofthemodelgroup(P<0.01)afterbeingrestrainedfor18hasshowninFig.6d.UnderrestraintstressthemiceadministeredwithCSGS(P<0.01)andVE(P<0.01)hadhigherGSHlevelinbloodcomparedwiththemicereceivingrestrictiononly.SoadministrationofCSGScouldimprovetheantioxidantcapabilityoforganismunderoxidativestress.4．sipholiece.silizact.v.s.v.sipholgachisasi农村sipensiphingastpo三维建模.见表2CSGSwasfoundtobeapotentsourceofantioxidantsindifferentinvitroassaysincludingFRAPandscavengingactivitiesonDPPHradicals,ABTScationradicals,O2·-andOH·.TheresultinFRAPassayshowedreductiveabilityofCSGS,whichindirectlyreflectedthetotalantioxidantcapability.DPPHassayisarapidandsensitivemethodwidelyusedtoevaluatetheantiradicalactivitiesofantioxidant.Whentheantioxidantsthatcanprovidehydrogenexist,theydonatehydrogentothefreeradicalssothattheradicalsremovetheoddelectrontoturntounreactiveonesofwhichthereactionmechanismissimilartoABTSAssay.Recently,theindividualherbsofCSGS,Zhi-Qiao,Chen-Piandthemainactivecompoundsoftheherbs,Naringin,HesperidinandTangeretinhadbeenprovedtopossessantioxidantproperties,allofwhichwereflavonoids.CSGSisacomplicatedmixturethatcontainsavarietyofingredients,especiallyitwasrichinflavonoids[equalto(42.28±1.80)mgrutinpergramaqueousextractofCSGSinourpreviousexperiment],whichpossessphenolichydroxylgrouptoprovidehydrogen.ThismaybeanexplanationofitsscavengingactivitiesonDPPHradicalsandABTScationradicals.O2·-andOH·aretwokindsoffreeradicalsthatspontaneouslyproducewhentheorganismisunderstressandarequiteharmfultobiologicalmolecules.O2·-istheproductofbiologicalmetabolisminthepresenceofoxygenandisquitetoxic,whichiscloselyrelatedtothegenerationofavarietyofinflammatorydiseasesincludingthoseinliver.O2·-playedapotentialdeleteriousroleinNonalcoholicfattyliverdisease.OH·isthemostactiveandharmfulradicalsintheorganismthatcanreactwithawiderangeofmoleculestoinducelargedamagetoDNA,lipids,andproteins.Hydroxylradicalscavengerhadbeenprovedtoprotectliverfromoxidativeinjury.ThescavengingactivitiesofCSGSonO2·-andOH·indicatedthatitmayhavepotentialabilitiesinvivo.Thedirectreflectionoforganismunderoxidativestressistoincreaseneuralexcitability.Duringthisprocesscatecholamineincreasessignificantly,whichgeneratesO2·-byautomaticoxidation.ThenH2O2isformedbycatalysedreactionofO2·-,tofurthergeneratemoreactiveOH·.PolyunsaturatedfattyacidsincellmembranearepronetoreactwithOH·toproducelipidperoxideswhichcausedamagetocellandtissueandthendecomposetocytotoxicsubstancessuchasmalonaldehyde(MDA).SoMDAcontentcanquantitativelyreflectthedegreeoflipidperoxidationcausedbyoxidativedamage.Thenaturalantioxidantsinvivoincludingenzymaticandnon-enzymaticsystemsprotecttheorganismfromoxidativeinjury.SOD,akindofenzymeexistinginnearlyallcells,isanimportantantioxidantowingtoitsabilitytocatalyzethedismutationofO2·-intooxygenandH2O2.ThelattercanbedecomposedtowaterandoxygenbyCAT,anothereffectivecatalyst.GSHisakindoftripeptideandmainlyexistsinerythrocytewhereitactsasthescavengerofH2O2andO2·-,tofurtherpreventthegenerationofOH·andlipidperoxidation.ThedepletionofGSHthatmaybecausedbyoxidativestressisconsideredtobeacrucialeventofmoleculardamage.Therefore,SODandCATactivitiesarethesignofantioxidantcapabilityoforganismsintheenzymaticlevel,whilethecontentofGSHrepresentsantioxidantabilityinthenon-enzymaticlevel.TheseantioxidantsworkcooperativelytoeliminateROSandrelieveinjurycausedbyoxidativestress.Restraintstressisonekindofoxidativestressorthatcausesseveraldysfunctionsdestroyingimmunefunctio