[6]-GingerolProtectsagainstDNADamage&AntioxidantMechanismBull.KoreanChem.Soc.2014,Vol.35,No.61633
http://dx.doi.org/10.5012/bkcs.2014.35.6.1633
ProtectiveEffectAgainstHydroxylRadical-inducedDNADamageand
AntioxidantMechanismof[6]-gingerol:AChemicalStudy
JingLin,
a
XicanLi,
a,
LiChen,WeizhaoLu,XianwenChen,
?
LuHan,andDongfengChen
?,
SchoolofChineseHerbalMedicine,GuangzhouUniversityofChineseMedicine.
E-mail:lixican@126.com
?
SchoolofChineseHerbalMedicine,FujianUniversityofChineseMedicine
?
SchoolofBasicMedicalScience,GuangzhouUniversityofChineseMedicine,Guangzhou510006,China
E-mail:CDF27212@21cn.com
ReceivedJanuary24,2014,AcceptedFebruary9,2014
[6]-Gingerolisknownasthemajorbioactiveconstituentofginger.Inthestudy,itwasobservedtoeffectively
protectagainstOH-inducedDNAdamage(IC
50
328.60±24.41μM).Antioxidantassaysindicatedthat[6]-
gingerolcouldefficientlyscavengevariousfreeradicals,includingOHradical(IC
50
70.39±1.23μM),O
2
?
radical(IC
50
228.40±9.20μM),DPPHradical(IC
50
27.35±1.44μM),andABTS
+
radical(IC
50
2.53±0.070
μM),andreduceCu
2+
ion(IC
50
11.97±0.68μM).Inordertoinvestigatethepossiblemechanism,thereaction
productof[6]-gingerolandDPPHradicalwasfurthermeasuredusingHPLCcombinedmassspectrometry.
Theproductshowedamolecularionpeakatm/z316[M+Na]
+
,anddiagnosticfragmentloss(m/z28)for
quinone.Onthisbasis,itcanbeconcludedthat:(i)[6]-gingerolcaneffectivelyprotectagainstOH-induced
DNAdamage;(ii)apossiblemechanismfor[6]-gingeroltoprotectagainstoxidativedamageisOHradical
scavenging;(iii)[6]-gingerolscavengesOHradicalthroughhydrogenatom(H)transfer(HAT)and
sequentialelectron(e)protontransfer(SEPT)mechanisms;and(iv)bothmechanismsmake[6]-gingerolbe
oxidizedtosemi-quinoneorquinoneforms.
KeyWords:[6]-Gingerol,Hydrogenatomtransfer,Sequentialelectronprotontransfer,Antioxidantmecha-
nism,Hydroxylradical
Introduction
Asthemostharmfulreactiveoxygenspecies(ROS),
hydroxylradical(?OH)canoxidativelydamageDNA,lead
todeleteriousbiologicalconsequences,includinggenetic
mutation,
1
carcinogenesis,
2
andcelldeath.
2
Therefore,itis
criticaltosearchforpotentialtherapeuticagentsforoxida-
tiveDNAdamage.Sincegingerhasbeendemonstratedto
possessbeneficialeffectsoncells,
3
itsrelevantbioactive
compoundshadattractedconsiderableattentioninrecent
years.Asamajorpungentprincipleofginger,[6]-gingerol
(Fig.1)hasthereforebeenintensivelyinvestigatedfor
pharmacologicalandphysiologicalactivities.
Severalstudiessuggestedthat[6]-gingerolcanberespon-
siblefortheanti-inflammatory,anti-tumourandantioxidant
activitiesofginger.[6]-Gingerolcanpreventagainstvarious
cancers,
4,5
especiallyprostatecancer,
6
skincancer,
7
colon
cancer.
8
Inaddition,ithasbeenobservedtoattenuateoxida-
tivecelldeath
9
andpreventgenotoxicity.
10
Theseeffectsarethoughttoberelatedtoitsprotective
effectsagainstoxidativeDNAdamageandantioxidantability.
Previously,Dugasaniandcolleagueshavecomparedthe
antioxidantlevelsoffourbioactivecomponentsinginger
(including[6]-gingerol).
11
Unfortunately,someexperimental
dataarenotreliable,includingsuperoxideradicalscaveng-
ingandhydroxylradicalscavengingassays.Inhydroxyl
radicalscavengingassay,theyusedDMSOforsamplesolu-
tionpreparation,andthesamplesolutionwasdirectlyused
forhydroxylradical-scavengingassay.Asmentionedinour
previousreport,
12
DMSOitselfcanscavenge?OHradical
andbringaboutconsiderableinterference.Infact,the
reactionofDMSOwith?OHhadbeenrecognizedforover
30years.
13,14
Thereactionratewascalculatedas9×10
9
L
mol
?1
s
?1
.
15
Theproductof?OHreactionwithDMSOhas
beendemonstratedtobe?CH
3
.
16
Inaddition,inxanthine-
luminal-xanthineoxidaseassay,sincealkalineDMSOcan
generatesuperoxideanionradicals,
17,18
DMSOasthesolv-
entundoubtedlycauseinterferencewiththedeterminationof
superoxideradicalscavenging.Inaword,itisnecessaryto
reevaluatetheantioxidantlevelof[6]-gingerolbyareliable
method.
Ontheotherhand,despitethattherehavebeenseveral
computationalstudiesforfreeradical-scavengingmech-
anisms
19
andsomehydrogenatomtransferkineticstudies
forDPPH?scavenging,
20
nomechanisticstudybasedon
oxidizedproductanalysishasbeenreported.Thepresent
study,however,triedtouseHPLCandMStoexplainthe
a
Theseauthorscontributedequallytothiswork.
Figure1.Thechemicalstructureof[6]-gingerol.
1634Bull.KoreanChem.Soc.2014,Vol.35,No.6JingLinetal.
mechanism.Thestructureelucidationofmassspectrumcan
beusedtointerprettheantioxidantmechanismof[6]-gin-
gerol.Obviously,thepresentstudywillprovideimportant
insightsintothemechanismsunderlyingtheantioxidantof
[6]-gingerol.
Experimental
Chemicals.[6]-Gingerol(CASnumber:519-34-6,98%)
wasobtainedfromWeikeqiBiologicalTechnologyCo.,
Ltd(Chengdu,China).1,1-Diphenyl-2-picrylhydrazylradical
(DPPH?),(±)-6-hydroxyl-2,5,7,8-tetramethlychromane-2-
carboxylicacid(Trolox),andBHA(butylatedhydroxy-
anisole)werefromSigma-AldrichShanghaiTradingCo.
(Shanghai,China).Deoxyriboseand2,2′-azino-bis(3-ethyl-
benzothiazoline-6-sulfonicaciddiammoniumsalt)(ABTS
diammoniumsalt)wereobtainedfromAmrescoInc.(Solon,
OH,USA).DNAsodiumsalt(fishsperm,98%)waspur-
chasedfromAladdinChemistryCo.(Shanghai,China).
WaterandmethanolwereofHPLCgrade.Allotherreagents
wereofanalyticalgrade.
ProtectiveEffectAgainst?OH-inducedDNADamage.
Theexperimentwasconductedusingthemethoddeveloped
byourlaboratory.
21
Briefly,thesamplewasfirstlydissolved
in95%ethanolat2mg/mL.Thesamplesolutionwasthen
aliquotedintotubes.Afterevaporatingthesamplesolutions
inthetubestodryness,300μLofphosphatebuffer(0.2M,
pH7.4)wasaddedtothesampleresidue.Subsequently,50
μLofDNAsodium(10mg/mL),75μLofH
2
O
2
(33.6mM),
50μLofFeCl
3
(3.2mM)
and100μLofNa
2
EDTA(0.5mM)
solutionwereadded.Thereactionwasinitiatedbyadding75
μLofascorbicacid(1.2mM).Afterincubationinawater
bathat55°Cfor20min,thereactionwasterminatedby
adding250μLoftrichloroaceticacid(10%,w/w).Thecolor
wasthendevelopedbyadditionof150μLofTBA(5%,in
1.25%NaOHaqueoussolution)andheatinginanovenat
105°Cfor15min.Themixturewascooledandthe
absorbancewasmeasuredat530nmagainstthebuffer(as
theblank).Theinhibitionpercentagefor?OHisexpressedas
follows:
Protectiveeffect%=
WhereA
0
istheabsorbanceat530nmofthecontrol
withoutsample,andAistheabsorbanceat530nmofthe
reactionmixturewithsample.
HydroxylRadical(OH)ScavengingAssay.Theexperi-
mentof?OHradical-scavengingwasconductedaccordingto
ourmethod.
12
Inbrief,thesampleethanolicsolution(1mg/
mL)wasseparatelyaddedintotubes.Afterevaporatingthe
samplesolutionsinthetubestodryness,550μLofphos-
phatebuffer(0.2M,pH7.4)wasaddedtothesample
residue.Then,50μLglucose(2.8mM),50μLNa
2
EDTA(1
mM),50μLFeCl
3
(3.2mM)and50μLH
2
O
2
(2mM)were
added.Thereactionwasinitiatedbymixing50μLascorbic
acid(1.8mM)andthetotalvolumeofthereactionmixture
wasadjustedto800μLwithbuffer.Afterincubationat50
°Cfor20min,thereactionwasterminatedby500μLtri-
chloroaceticacid(5%,w/w).Thecolorwasthendeveloped
byadditionof500μLTBA(5%,in1.25%NaOHaqueous
solution)andheatedinanovenat105°Cfor15min.The
mixturewascooledandabsorbancewasmeasuredat532nm
(Unico2100,spectrophotometer,Shanghai,China)against
thebuffer(asblank).Thehydroxylradicalscavenging
activitywasexpressedas:
Inhibition%=
WhereA
0
istheabsorbanceofthecontrolwithoutsample;
andAistheabsorbanceofthereactionmixturewithsample.
SuperoxideAnion(O
2
?
)Radical-scavengingAssay.
Measurementofsuperoxideanion(?O
2
?
)scavengingactivity
wasbasedonourmethod.
22
Briefly,thesamplewasdis-
solvedinmethanolat2mg/mL.Thesamplesolution(xμL,
wherex=0,30,60,90,120and150μL)wasmixedwith
2960-xμLTris-HClbuffer(0.05mol/L,pH7.4)containing
Na
2
EDTA(1mmol/L).When40μLpyrogallol(60mmol/L
in1mmol/LHCl)wasadded,themixturewasshakenat
roomtemperatureimmediately.Theabsorbanceat325nm
ofthemixturewasmeasured(Unico2100,Shanghai,China)
againsttheTris-HClbufferasblankevery30sfor5min.
The?O
2
?
scavengingabilitywascalculatedas:
Inhibition%=
Here,ΔA
325nm,control
/TistheincreaseinA
325nm
ofthemix-
turewithoutthesampleandΔA
325nm,sample
/Tisthatwiththe
sample;T=5min.Theexperimenttemperaturewas37°C.
DPPHRadical-scavengingAssay.TheDPPH?radical-
scavengingactivitywasdeterminedasdescribed.
23
Briefly,
500μLofDPPH?solution(0.1mM)wasmixedwith250μL
sample95%ethanolsolutionwithvariousconcentrations.
Themixturewaskeptatroomtemperaturefor30min,and
thentheabsorbancewasmeasuredat519nmagainst95%
ethanol(asblank).TheDPPH?inhibitionpercentagesofthe
sampleswerecalculated:
Inhibition%=
WhereA
istheabsorbancewithsamples;whileA
0
isthe
absorbancewithoutsamples.TroloxandBHAwereusedas
thepositivecontrols.
HPLCAnalysisandMassSpectrometryofReaction
Productof[6]-gingerolandDPPHRadical.Theproduct
mixtureof[6]-gingerolandDPPH?radical(10:3,mol/mol)
wasfilteredusing0.45μmfiltersthenanalyzedbyapre-
parativeHPLCsystem(ProminenceLC-20A,Shimadzu,Japan),
equippedwithaDiamonsilC
18
(250mm×4.6mm,5μm)
column(DikmaCo.,Beijing,China).Themobilephase
consistedofmethanol-0.5%aceticacid(90:10,v:v)andthe
flowratewas0.5mL/min,injectionvolumewas27μL,
A
0
A–
A
0
---------------100%×
A
0
A–
A
0
---------------100%×
ΔA
325nm,control
T
-------------------------------
??
??
ΔA
325nm,sample
T
------------------------------
??
??
–
A
325nm,sample
T
--------------------------
??
??
----------------------------------------------------------------------------100%×
A
0
A–
A
0
---------------100%×
[6]-GingerolProtectsagainstDNADamage&AntioxidantMechanismBull.KoreanChem.Soc.2014,Vol.35,No.61635
detectionwavelengthwas225nm.[6]-GingerolandDPPH?
radicalwerealsocomparativelymeasuredunderthesame
chromatographicconditions.
ThereactionproductisolatedbyHPLCwasfurtherana-
lyzedbyamicroflexLTMALDITOF-QII(BrukerDaltonics,
USA)massspectrometerwhichwasequippedwithan
electrosprayionisation(ESI)sourceandruninpositive
mode.Thescanrangewas50-3000m/z.ESIparameters
wereoptimisedwithdirectinfusionofdansylatedamine
mixturebyanexternalsyringeandsetasfollows:capillary,
+4.5kV;nebulizerpressure,0.3bar;drygasflow,4.0L/
min;drygastemperature,180
o
C.Argonwasappliedasthe
collisiongas,andthecollisionenergywassetto10eVto
providesomestructuralinformationandtofocusionflux.
Highpuritynitrogenwasappliedbothasanebulizergasand
adryinggas.TheQ-TOF/MSparameterswereoptimisedto
thefollowing:funnel1was300.0Vppand2was400.0Vpp;
hexapoleRfwas400.0Vpp;quadrupoleionenergywas5.0
eV;collisionRfwas650.0Vpp.Theiontransfertimeand
prepulsestoragetimeweresetto120and10us,respectively.
ABTS
+
Radical-scavengingAssay.TheABTS
+
scaveng-
ingactivitywasevaluatedbythemethod.
23
TheABTS
+
?was
producedbymixing350μLABTSdiammoniumsalt(7.4
mM)with350μLK
2
S
2
O
8
aqueouspersulfate(2.6mM).The
mixturewaskeptinthedarkatroomtemperaturefor12hto
allowcompletionofradicalgeneration,thendilutedwith
95%ethanol(about1:50)sothatitsabsorbanceat734nm
was0.70±0.02.Then,0.6mLdilutedABTS
+
?reagents
werebroughtto150μLsampleethanolicsolutions(0.01
mg/mL).Afterincubationfor6min,theabsorbanceat734
nmwasreadonaspectrophotometer(Unico2100,Shanghai,
China).Thepercentageinhibitionwascalculatedas:
Inhibition%=
WhereA
0
istheabsorbanceofthecontrolwithoutany
samples,Aistheabsorbanceofthemixturewith[6]-
gingerol,Trolox,orBHA.
Cu
2+
-reducingPowerAssay.Thecupricions(Cu
2+
)re-
ducingpowercapacitywasdeterminedbasedonthemethod,
23
withaslightmodification.Inbrief,100μLCuSO
4
aqueous
solution(10mM),100μLneocuproineethanolicsolution
(7.5mM)and400μLCH
3
COONH
4
buffersolution(100
mM,pH7.0)werebroughttotesttubeswithdifferent
volumesofsamples(0.1mg/mL,15-75μL).Then,thetotal
volumewasadjustedto800μLwiththebufferandmixed
vigorously.Absorbanceagainstabufferblankwasmeasured
at450nmafter15min.Therelativereducingpowerofthe
sampleascomparedwiththemaximumabsorbance,was
calculatedusingtheformula:
Relativereducingpower%=
Here,A
max
isthemaximumabsorbanceinthetestandA
min
istheminimumabsorbanceinthetest.Aistheabsorbance
ofsample.
StatisticalAnalysis.Eachexperimentwasperformedin
triplicateandthedatawererecordedasmean±SD(standard
deviation).TheIC
50
valuewasdefinedasthefinalconcen-
trationof50%radicalinhibition(relativereducingpower,or
chelatingeffect).Statisticalcomparisonsweremadebyone-
wayANOVAtodetectsignificantdifferenceusingSPSS
13.0(SPSSInc.,Chicago,IL)forWindows.P<0.05was
consideredtobestatisticallysignificant.
ResultsandDiscussion
Inthepresentstudy,weusedFentonreaction(Equation1)
toproduce?OHradicalsfortheinvestigationonthepro-
tectiveeffectof[6]-gingerolagainstoxidativeDNAdamage.
Fe
2+
+H
2
O
2
→Fe
3+
+?OH
+OH
?
(1)
Thedataindicatedthat[6]-gingerolandthepositivecontrols
dose-dependentlyincreasedtheprotectiveeffectagainst
oxidativeDNAdamageat0-200μg/mL(Fig.2).Basedon
theIC
50
values(328.60±24.41and690.76±12.31μM,
respectivelyfor[6]-gingerolandTrolox,Table1),itcanbe
inferredthat[6]-gingerolpresented2.10timeshigherpro-
tectiveeffectthanthestandardantioxidantTroloxinour
model.Thisisconsistentwiththepreviousstudy,inwhich
[6]-gingerolwasfoundtopreventUVB-inducedROSpro-
ductionandoxidativeDNAdamage.
24
Itsprotectiveeffect
againstDNAoxidativedamagemaybeprimarilyresponsible
forthepharmacologicaleffects,includinganti-inflammatory
andanti-cancereffects.
4-8
Infact,oxidativeDNAdamage
hasbeenobservedtoplayakeyroleininflammation-related
carcinogenesis.
25
Previousworkhasshownthattherearetwoapproaches
fornaturalphenolicantioxidantstoprotectagainstoxidative
DNAdamage:oneistoscavengethe?OHradicalpriorto
DNAdamage;andtheotheristopreventtheDNAradicals
resultingfrom?OHradicalattack.
26
Tofurtherconfirmwhethertheprotectiveeffectof[6]-
gingerolagainstoxidativeDNAdamageisrelevanttoits
A
0
A–
A
0
---------------100%×
AA
min
–
A
max
A
min
–
-------------------------100%×
Figure2.TheprotectiveeffectagainstOHradical-inducedDNA
damageof[6]-gingerolandpositivecontrolTrolox(Eachvalueis
expressedasmean±SD,n=3).
1636Bull.KoreanChem.Soc.2014,Vol.35,No.6JingLinetal.
radical-scavengingability,wethendeterminedtheradical-
scavengingabilitiesof[6]-gingerolon?OHand?O
2
?
.Inthe
?OHradical-scavenging
assay(Suppl.Fig.S1),[6]-gingerol
couldeffectivelyscavenge?OHradicalsat0-22.5μg/mL(0-
77.7μm)anditsIC
50
valuewascalculatedas70.39±1.23;
Inthe?O
2
?
radical-scavenging
assay(Suppl.Fig.S2),[6]-
gingerolalongwiththepositivecontrolsalmostlinearly
increasedthe?O
2
?
radical-scavengingpercentages,andthe
IC
50
valueof[6]-gingerolwas228.40±9.20μM(Table1).
ThesedataclearlysuggestthatROS-scavenging(especially
?OH-scavenging)isonepossiblemechanismfor[6]-gin-
geroltoprotectagainstoxidativeDNAdamage.
Toexplorethepossiblemechanismfor[6]-gingerolto
scavengeROS,wefurtherexploredtheradical-scavenging
effectonDPPH?andABTS
+
?radicals.
TheDPPHassayrevealedthat[6]-gingerolpossesseda
concentration-dependenteffectat0-10μg/mL(0-34.01μM,
Suppl.Fig.S3),andtheIC
50
wascalculatedas27.35±1.44
μM(Table1).Itmeansthat[6]-gingerolcaneffectively
eliminateDPPH?radical.
Inordertofurtherexplorethemechanismfor[6]-gingerol
toscavengeDPPH?radical,thereactionproductof[6]-
gingerolwithDPPH?wasmeasuredusingHPLC(Fig.3).
TheHPLCprofileclearlyindicatedapeakastheproductat
11.98min(retentiontime).
Whentheproductwasfurtheranalyzedbymassspectro-
metry,itgaveamolecularionpeakatm/z316[M+Na
+
]
whichwasobviouslyonelessthanreactant[6]-gingerolat
m/z317[M+Na
+
].Itindicatedahydrogenatomtransferof
[6]-gingeroltoproduct.Inaddition,fragmentunitsofm/z
302.30,274.27,and246.24intheproductwerealsoobserved.
Obviously,thesefragmentunitsshowedalossofC=O(m/z
28)whichisregardedasthecharacteristicdiagnosticfrag-
mentlossforquinone(Fig.4(a)).Ontheotherhand,the
reactant[6]-gingerol,however,didnotexhibitthecharac-
teristiclossesatm/z28(Fig.4(b)).Inaword,thehydrogen
atomhasbeenshowntobetransferredfrom[6]-gingerolto
DPPH?,and[6]-gingerolhasbeenoxidizedtosemi-quinone
orquinonebyDPPH?.
Basedontheabovedata,andpreviousreportwhichDPPH?
maybescavengedthroughhydrogenatom(H?)transfer
(HAT)toformDPPH-Hmolecule,
27
theproposedreaction
of[6]-gingerolwithDPPH?canbeillustratedinFigure5.As
showninFigure5,thereactionof[6]-gingerolwithDPPH?
wasthoughttoyieldaphenoxylradical(I),whichcanbe
convertedtosemi-quinone(II).Semi-quinone(II),however,
possessedvariousresonancesformula,e.g.semi-quinone
(III),semi-quinone(IV),andphenoxylradical(I)whichis
someextentstableastablespecies.Thus,step1for[6]-
gingeroltotransferahydrogenatomiseasilytobeinitiated.
UndertheconditionsofexcessiveDPPH?,however,semi-
quinone(IV)maybefurtherbeextractedhydrogenatomto
Figure3.TypicalHPLCchromatogramofthereactionproductof
[6]-gingerolwithDPPH(1,1-Diphenyl-2-picrylhydrazylradical).
Figure4Massspectraofthereactionproduct[6]-gingerolwith
DPPH(a),and[6]-gingerol(b).
Figure5.Theproposedreactionof[6]-gingerolwithDPPHvia
HAT(hydrogenatomtransfer)mechanismbasedonmassspectro-
metryanalysis.
[6]-GingerolProtectsagainstDNADamage&AntioxidantMechanismBull.KoreanChem.Soc.2014,Vol.35,No.61637
producebenzoquinone.However,sincetheortho-positionof
–OHis–OCH
3
not–OH,thehydrogenextractionisn’tso
easy,therefore,theyieldofbenzoquinoneisveryless,and
thepeakatm/z246.24isverylowinMSspectrometry(Fig.
4(a)).
BesidesDPPH?scavenging,ABTS
+
?scavenginghasalso
beenusedforinvestigationontheantioxidantmechanismof
[6]-gingerolinthestudy.AsseeninSuppl.Fig.S4,[6]-
gingerollinearly(R=0.99925)increaseditsABTS
+
?scaven-
gingpercentagesat0-1.33μg/mL(0-4.55μM)anditsIC
50
was2.53±0.070μM(Table1).Asweknow,ABTS
+
?
scavengingisanelectron(e)transferprocess.
28
Inthepro-
cess,etransferisalwaysaccompaniedbydeprotonation,so
itistermedasequentialelectronprotontransfer(SEPT)
mechanism,
29
orprotoncoupledelectron-transfer(PCET)
mechanism,
30
sequentialprotonlosssingleelectrontransfer
(SPLET).
31
TheSEPTmechanismfor[6]-gingeroltoscavenge
ABTS
+
?wasproposedasdescribedinFigure6.Through
SEPTmechanism,[6]-gingerolmightchangetophenoxyl
radical(I).TheSEPTmechanismisalsosupportedbyCu
2+
assay,inwhich[6]-gingerolincreasedthereducingpercent-
ageinadose-dependentmanner(Suppl.Fig.S5)withIC
50
valuebeing11.97±0.68μM(Table1).Asweknow,reduc-
ingreactionsareactuallyanelectron(e)transferprocess.
Similarly,phenoxylradical(I)wasthoughttobefurther
convertedintosemi-quinones,evenquinoneunderexcess
ABTS
+
?radicalsorCu
2+
ions.
Thefactthat[6]-gingerolcouldeffectivelyscavengeboth
ABTS?
+
andDPPH?radicals,suggeststheprocessof[6]-
gingerolscavengingofROS(especially?OHradicals)in
cellswouldbemediatedviaHATandSEPTmechanisms.
AsshowninEq.(1),thegenerationof?OHradicalsmay
alsobringaboutequalOH
?
ionsincells.Therefore,athigh
levelsof?OHradicals,massiveOH
?
ionscouldalkalizethe
cellularenvironment.Underalkalineconditions,however,
theaciditymaypredominateoverthechemicalactionof[6]-
gingerol.Asaphenol,[6]-gingerolpresentsaweakacidity
(pK
a
~10).
32
Thus,[6]-gingerolmighteasilyionizetoyield
H
+
ions,and[6]-gingerol
?
anionwhichcouldfurtherdonate
anelectron(e)to?OHradicalstoformphenoxylradical(I)
(Fig.7),evensemi-quinonesandbenzoquinone.Thisisa
possiblemechanismfor[6]-gingeroltodirectlyscavenge
?OHviatheSEPTmechanism.Themechanismissimilarto
thatoftrans-resveratroltoward?OHradicals.
33
However,atlowlevelsof?OHradicals,thecellularenviron-
mentwasalmostneutralandtheaciditycouldnotpredo-
minateoverthechemicalactionof[6]-gingerol.Therapid
anddirectattackof?OHradicalsmaycausehomolysisof
[6]-gingeroltogeneratephenoxylradical(I)andhydrogen
atom(?H).Hydrogenatom,however,wouldimmediatelybe
donatedto?OHtoformthestableH
2
Omolecule(Fig.8).
Figure6.Theproposedreactionof[6]-gingerolwithABTS
+
via
SEPT(sequentialelectronprotontransfer)mechanism.
Figure7.Theproposedreactionfor[6]-gingeroltoscavengeOH
viaSEPT(sequentialelectronprotontransfer)mechanism.
Figure8.Theproposedreactionfor[6]-gingeroltoscavengeOH
viaHAT(hydrogenatomtransfer)mechanism.
Table1.TheIC
50
valuesof[6]-gingerol,Trolox,andBHAinvariousassays(μM)
Assays[6]-Gingerol
Positivecontrols
Ratio
TroloxBHA
DNAprotection328.60±24.41
a
690.76±12.31
b
N.D.2.10
OHscavenging70.39±1.23
a
93.00±1.35
b
124.62±3.68
c
1.32
O
2
?
scavenging228.40±9.20
a
226.54±6.35
a
358.97±11.41
b
0.99
DPPHscavenging27.35±1.44
a
29.26±0.59
a
34.71±0.81
b
1.07
ABTS
+
scavenging2.53±0.070
a
7.36±0.43
c
3.41±0.10
b
2.91
Cu
2+
reducing11.97±0.68
a
36.83±1.43
c
23.16±0.21
b
3.08
Average1.91
IC50valueisdefinedastheconcentrationof50%effectpercentageandcalculatedbylinearregressionanalysisandexpressedasmean±SD(n=3).The
linearregressionwasanalyzedbyOrigin6.0professionalsoftware.Meansvalueswithdifferentsuperscriptsinthesamerowaresignificantlydifferent
(p<0.05),whilewithsamesuperscriptsarenotsignificantlydifferent(p<0.05).ThepositivecontrolisSodiumcitrate.Ratio=IC50,Trolox:IC50,[6]-
gingerol.Thedoseresponsecurvesof[6]-gingerolinantioxidantassaywereshowninSupplemental1.N.D.,notdetermined.BHA,butylated
hydroxyanisole.
1638Bull.KoreanChem.Soc.2014,Vol.35,No.6JingLinetal.
Meanwhile,phenoxylradical(I)changedintosemi-quinones.
ThismaybetheHATmechanismfor[6]-gingeroltodirectly
scavenge?OH.Thismechanismagreeswiththeprevious
findingsthatthedopaminereactiontowards?OHismainly
viaHATatphysiologicalpH7.4.
28
Toquantitativelyevaluatetherelativeantioxidantlevelof
[6]-gingerol,theratiovaluewasdefinedasIC
50,Trolox
/IC
50,[6]-
gingerol
.AsshowninTable1,theratiovaluesofoxidative
DNAdamage,?OH-scavenging,·O
2
?
scavenging,DPPH?
scavenging,ABTS
+
?scavenging,andCu
2+
-reducingwere
2.10,1.32,0.99,1.07,2.91,and3.08,respectively.Theaver-
ageratiovaluewascalculatedas1.91(Table1).Itimplies
that[6]-gingerolhad1.91timeshigherthetotalantioxidant
capacitythanthestandardantioxidantTrolox.
ItmustbeemphasizedthattheIC
50
valuesof[6]-gingerol
inour·O
2
?
and?OHscavengingassayswererespectively
228.40±9.20and70.39±1.23μM,whiletheywerecalcu-
latedas4.05and4.62μMrespectivelyintheprevious
literature.
11
Undoubtedly,thereisaconsiderablydifference.
However,theIC
50
valuesinDPPHscavengingassayare
generallyidentical:AsshowninTable1,itwaslistedas
27.35±1.44μMinourstudy,whilethepreviousliterature
shownas26.3μM.
11
ItclearlyindicatedthatDMSOusedas
asolventinthepreviousworkindeedbroughtabout
considerableinterferencewiththe·O
2
?
and?OHscavenging
assays.
11
Conclusion
Basedontheabovediscussion,itcanbeconcludedthat:
(i)asthemajorbioactiveconstituentofginger,[6]-gingerol
caneffectivelyprotectagainst?OH-inducedDNAdamage;
(ii)apossiblemechanismfor[6]-gingeroltoprotectagainst
oxidativedamageis?OHradicalscavenging;(iii)[6]-gin-
gerolscavenges?OHradicalspossiblythroughhydrogen
atom(H?)transfer(HAT)andsequentialelectron(e)proton
transfer(SEPT)mechanisms;and(iv)radicalscavenging
makes[6]-gingerolbeoxidizedtosemi-quinoneorquinone
forms.
SupplementaryMaterials:Doseresponsecurvesof
antioxidantassaysof[6]-gingerolandpositivecontrols.
ConflictofInterestStatement.Theauthorsconfirmthat
therearenoconflictsofinterest.
Acknowledgments.Thisworkwassupportedbythe
NationalNatureScienceFoundationofChina(81273896).
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