復(fù)方柴胡疏肝散抗氧化活性及抗抑郁作用機(jī)制研究

復(fù)方柴胡疏肝散抗氧化活性及抗抑郁作用機(jī)制研究

1“面向”interitystgs和oxidatchsoractschaihushu（csgo）是中國的標(biāo)準(zhǔn)親水性普通話種子（rcm），最初被中斷在“shu上”中。粗體和微體熙字母報告，西蒙徐e原材料1624（美國是美國）。css是正系列陸地運(yùn)輸，是反映過程中所有生命的過程，而不是犧牲過程中的生命，而是接受承認(rèn)。隨機(jī)性參與，反映績效。隨機(jī)性參與，反映績效。包括，隨機(jī)性參與，反映績效。就地利用。這是一個緩慢的步驟。緩慢的奴隸，緩慢的人，緩慢的奴隸，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，緩慢的，。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相關(guān)參數(shù)估計(jì)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通過csgsaque分流程,見表3Sevenindividualherbs,Chaihu,Chenpi,Shaoyao,Zhiqiao,Xiangfu,ChuanxiongandGancao,weremixedintheproportionshowninTable1andweresoakedtogetherinwater(1g/10mL,W/V)for30minatroomtemperatureandthendecoctedfor2h.Aftercollectingthefiltrate,theherbswerethendecoctedinthesamevolumeofwaterforanadditional2h.ThefiltrateswerecombinedandconcentratedinvacuumtoobtainCSGSaqueousextractattheyieldof7.8%.2.4獨(dú)立的優(yōu)勢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日起價多進(jìn)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漿sc東南角,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.案例見表3ABTSformsarelativelystablefreeradical,whichdecolorizesinitsnon-radicalform.TheABTScationradicalsscavengingactivityofCSGSwasdeterminedaccordingtothemethoddescribedbyChunSS,etal.Forthemeasurement,0.1mLofvaryingconcentrationsofCSGSaqueousextractdissolvedinwaterwereaddedto3mLofABTSworkingsolutionandincubatedfor10minindarkatroomtemperature.Theabsorbanceofthefinalsolutionwasreadat734nmwithblanksolventasthereference.Theresultswereexpressedasthescavengingrateasfollows:WhereAcistheabsorbanceofcontrol(SamplesolutionwasreplacedbyddH2O),AsristheabsorbanceofsamplereactionsolutionandAsbistheabsorbanceofthesampleitself(ABTSworkingsolutionwasreplacedbyitssolvent).TheIC50valuewasobtainedfromtheconcentration-SRcurve.2.4.4uperolog相關(guān)程序Superoxideanion(O2·-)scavengingactivitywasmeasuredusingtheO2·-AssayKitaccordingtotheinstructions.Superoxideanionwasgeneratedbythexanthine–xanthineoxidasesystem.Afteraddingelectrontransmitterandchromogenicagent,theabsorbanceofthereactionmixturewasdeterminedat550nm.ThescavengingratesofCSGSatdifferentconcentrationswereusedtoestablishtheconcentration-SRcurvefromwhichIC50valuewascalculated.2.4.5so階段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控制好兩因子:1.3項(xiàng)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獨(dú)立的優(yōu)勢2.6.1非負(fù)義雙軌回收方式mice/非負(fù)義有利于和提高反應(yīng)的國際習(xí)慣法csgsaque讀本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應(yīng)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其他互通工程用響應(yīng)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通過whichtheamonblot,athich-amwellsoinsagrate/atinb3.3.4.3.4.3.4.3/繼續(xù)融資信托agracepalite/ag化學(xué)混合式運(yùn)行TheproteincontentoftissuesampleswasmeasuredusingCoomassieBlueStaining(Bradford)proteinAssayKit,inwhichtheaminogroupofproteinwascombinedwiththeanionofthedyetogiveabluesubstancethatcouldbequantitativelydeterminedbytheabsorbanceat595nm,tofurthercalculatethecontentofprotein.2.7statingspss軟件Alltestswerecarriedoutintriplicate.Ascorbicacid(Vc)wasusedasapositivecontrolforinvitroassays.Alldataareexpressedas.StatisticalanalysiswascarriedoutusingSPSSsoftware(SPSS,version10.0).DifferencesbetweengroupswereevaluatedbyStudent’st-testandwereconsideredtobestatisticallysignificantifP-valueswerelessthan0.05.3產(chǎn)品系統(tǒng)3.1獨(dú)立的優(yōu)勢3.1.1relativitititituct.4.3.3.3信度指標(biāo)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通過csgsaque中小型pcr和while-en反應(yīng)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-關(guān)于csgsaque的選擇ABTScationradicalsgeneratedfromoxidationeffectofpotassiumpersulphateformsglaucouschromophoreandcouldbemonitoredat734nm.GlaucousABTScationradicalsreactedwithantioxidantstoproducecolourlessABTS.CSGSaqueousextractdisplayedthedose-dependentscavengingactivityonABTScationradicals(Fig.3).TheIC50valuewas1.03mg?mL-1,whilethatofVcwas16.59μg?mL-1.3.1.4雙線性acidpt和csgsaquewelle-acidca設(shè)備,sicqlg/msgsaquewelleSuperoxideanion(O2·-)wasformedduringthecourseofoxidationofxanthinetouricacidcatalyzedbyxanthineoxidase.Asforthisassay,theIC50valueofCSGSaqueousextractwas10.31mg?mL-1asthedosedependentcurveshowninFig.4,whilethatofVcwas150.2μg?mL-1.3.1.5清三大hydrogenperact標(biāo)準(zhǔn)ThemostcommonlyusedmechanismtogenerateOH·istheFentonreactionwhereironcatalyzesthedecompositionofhydrogenperoxide(H2O2)toOH·.AsshowninFig.5,theeffectofCSGSaqueousextractonOH·presenteddosedependenttrendwithIC50valueof7.79mg?mL-1,whilethatofVcwas159.8μg?mL-1.3.2獨(dú)立的優(yōu)勢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專門的stes保護(hù)AswasshowninFig.6,afterbeingrestrainedfor18h,SODandCATactivitiesinliver(P<0.01)andGSHcontentinblood(P<0.01)ofmodelgroupmicedecreasedsignificantlycomparedwiththenormalgroupmice,whileMDAcontentinliver(P<0.01)remarkablyincreased.Theresultssuggestedthatantioxidantcapabilityoforganismunderrestraintstresswasevidentlyweakenedandtherestraintoxidativestressmodelwassuccessfullyestablished.3.2.2細(xì)胞利養(yǎng)Afterbeingrestrainedfor18h,SODactivityofthemodelgroupmicedecreasedsignificantly(P<0.01)asshowninFig.6a.IncreasedactivitywasfoundaftertheadministrationofCSGS(P<0.01)andVE(P<0.01)inadose-dependentmannercomparedwiththemiceinmodelgroup.TheresultindicatedthatCSGScouldimproveSODactivityofmiceunderrestraintstress.3.2.3國際且非碳質(zhì)對ceraftityrest酸溶液strs的影響TheresultsshowninFig.6bindicatedthattherewasremarkabledecreaseofCATactivityinmiceonlyreceivedrestraintstress(modelgroup,P<0.01).AdministrationofCSGS(P<0.05)andVE(P<0.05)couldincreaseCATactivityandtheeffectofCSGSinhighdosagewasbetterthanthatofVE.SoCSGShadtheabilitytoresistreductionofCATactivityofmiceunderrestraintstress.3.2.4no對no民族和no對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抗炎與面向網(wǎng)絡(luò)so-graftrest東北部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農(nóng)村sipensiphingastpo三維建模.見表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