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PTIO自由基清除-最简单的自由基清除法 -李熙灿-XICAN LI |
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2?Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3?Oxide(PTIO
?
)
RadicalScavenging:ANewandSimpleAntioxidantAssayInVitro
XicanLi
SchoolofChineseHerbalMedicineandInnovativeResearchandDevelopmentLaboratoryofTCM,GuangzhouUniversityof
ChineseMedicine,232WaihuanEastRoad,GuangzhouHigherEducationMegaCenter,PanyuDistrict,Guangzhou,Guangdong
510006,People’sRepublicofChina
SSupportingInformation
ABSTRACT:Currentinvitroantioxidantassayshaveseverallimitations,whichfrequentlycauseinconsistentresults.Thestudy
developsanewantioxidantassayusingthe2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxideradical(PTIO
?
).Afterthe
investigationofvariousfactors,theexperimentalprotocolwasbrie?yrecommendedasfollows:PTIO
?
andthesamplesolution
wereaddedtophosphatebu?er(pH7.4,50mM),incubatedat37°Cfor2h,andthenspectrophotometricallymeasuredat557
nm.Thevalidationtestbasedon20purecompoundsand30lyophilizedaqueousextractssuggestedthatPTIO
?
scavenginghada
goodlinearrelationship,stability,andreproducibility.Intheultra-performanceliquidchromatographycoupledwithelectrospray
ionizationquadrupoletime-of-?ighttandemmassspectrometryanalysis,PTIO
?
wasobservedtogivem/z234when
encounteringL-ascorbicacid.Asanantioxidantassay,PTIO
?
scavengingpossessesfouradvantages,i.e.,oxygen-centeredradical,
physiologicalaqueoussolution,simpleanddirectmeasurement,andlessinterferencefromthetestedsample.Itcanalso
satisfactorilyanalyzetheantioxidantstructure?activityrelationship.PTIO
?
scavenginghasnostereospeci?cityandisatleast
involvedinH
+
transfer.
KEYWORDS:antioxidantassay,PTIO
?
scavenging,oxygen-centeredradical,analyticalmethod,
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide,H
+
transfer
1.INTRODUCTION
Excessivereactiveoxygenspecies(ROS)levelsareknowntobe
harmfultomajorbiomoleculesincells.ROSscavengingthus
playsacriticroleinthe?eldsoffood,chemistry,medicine,
nutrition,pharmacology,toxicology,andtraditionalChinese
medicine.Nowadays,variousantioxidantassayshavenowbeen
developedtocharacterizeROSscavenginglevels.However,
theseassaysfrequentlyresultininconsistentexperimental
results.Thisinconsistencymaybeattributedto?vefactors:
indirectmeasurements,non-oxygen-centeredradicals,solvent
e?ects,shortwavelengthdeterminations,andlimitationofthe
experimentalprinciple.
1?3
Ideally,anantioxidantassayshouldbebasedondirect
scavengingofROS.However,typicalROSformsaretransient
andhaveaveryshorthalf-life,e.g.,hydroxylradicals(
?
OH,10
?9
s),superoxideradicals(
?
O
2
?
,10
?6
s),andlipidperoxide
radicals(LOO
?
,10
?2
s).Theseradicalscanbetrappedonlyby
speci?celectronspinresonance(ESR)spectroscopyatan
extremelylowtemperature.However,ESRspectroscopycannot
quantitativelyevaluatetheROSscavenginglevelofan
antioxidant.
4
Thus,regularanalyticaltechnologies[e.g.,
spectrophotometry,high-performanceliquidchromatography
(HPLC),and?uorimetry]areusedtoindirectlymeasurethe
ROSscavenginglevel.Forexample,theanalysisofdeoxyribose
degradationcancharacterize
?
OHradicalscavenging;
5
the
measurementofpyrogallolauto-oxidationcanre?ect
?
O
2
?
radicalscavenging;
6
andthedeterminationofmalondialdehyde
(MDA)amountsre?ectsLOO
?
radicalscavenging.
7
These
indirectmeasurementsundoubtedlyintroduceuncertaintyinto
theevaluationofROSscavenging.
Ontheotherhand,somedirectradicalscavengingassaysare
notbasedonoxygen-centeredradicals.Forexample,1,1-
diphenyl-2-picryl-hydrazylradical(DPPH
?
)and2,2′-azino-
bis(3-ethylbenzthiazoline-6-sulfonicacid)radicalion
(ABTS
?+
),whicharewidelyusedforinvitroantioxidant
assays,arenitrogen-centeredradicals.Hence,theDPPH
?
assay
andtheABTS
?+
assayarepreferredfor“reactivenitrogen
species(RNS)scavenging”modelsandnot“ROSscavenging”
models.Inaradicaladductformation(RAF)-basedantioxidant
process,
8
thedi?erencebetweenRNSscavengingandROS
scavengingwillbeenlarged.AnOatominaphenolic
antioxidantcanlinkaNatominRNStoformastableO?N
σbondandhardlylinkanotherOtoyieldaO?Oσbond.A
superoxidebond(O?O)iswell-knowntobeveryunstable.
Thereby,itisscienti?callyunsoundtouseaRNSscavenging
modeltoestimatetheROSscavenginglevelofaphenolic
antioxidant.
Aphenolicantioxidantmayalsoundergoahydrogentransfer
pathwaytoscavengeROS.Theso-calledhydrogentransfer
actuallyincludesseveralsubtypes,i.e.,sequentialproton-loss
electrontransfer(SPLET),
9
hydrogenatomtransfer
(HAT),
10,11
proton-coupledelectrontransfer(PCET),
12
sequentialelectronprotontransfer(SEPT),
13
etc.Mostof
thesetransfersinvolvetheproton(H
+
).Thus,thepHvalueof
theexperimentalsolutioncancausestronginterference.
6,14,15
Received:May15,2017
Revised:July9,2017
Accepted:July9,2017
Published:July10,2017
Article
pubs.acs.org/JAFC
?2017AmericanChemicalSociety6288DOI:10.1021/acs.jafc.7b02247
J.Agric.FoodChem.2017,65,6288?6297
Moreover,becausetheprotonationextentsofthesolventsare
di?erentfromoneanother,thereisagreatsolvente?ect;i.e.,an
antioxidantmaypresentdi?erentROSscavengingactivitiesin
someantioxidantassaysamongdi?erentsolvents.
5,13?16
For
example,resveratrolwasshowntohaveconsiderablydi?erent
DPPH
?
scavengingrateconstantsinethanol(k=95.1M
?1
s
?1
)
andethylacetate(k=1.6M
?1
s
?1
).
17
Finally,thebackgroundabsorbanceoftestedsamplescan
alsodisturbtheassay.Forexample,aH
2
O
2
scavengingassay
basedonaredoxreactionisperformedunderashort
wavelength(λ=230nm).
18
Agalvinoxylradicalscavenging
assay,however,iscarriedoutusing432nm.
1
Thebackground
absorbancefromthetestedsamplesmayeasilyinterferewith
suchshortwavelengthabsorbances.
Therefore,itisvitaltosearchforastable,hydrophilic,
oxygen-centeredradicaltoevaluateROSscavenginglevels.The
2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxideradical
(PTIO
?
),aspeciescurrentlyusedtodetectnitricoxide(NO)
levels,
19,20
ishypothesizedtobesuchacandidateinthepresent
study.AsshowninFigure1A,theunpairedelectronislocated
intheOatomand,thus,isanoxygen-centeredradical;the
amineoxidezwitterionmoietymakesitahydrophilicspecies.A
benzeneringlinkedtoaCNdoublebondisthoughtto
producealargeπ?πconjugativesystemwithlongwavelength
absorbance.Thus,asanavybluepowder,PTIO
?
isstableunder
usualtemperature.Itsscavengingmaybeeasilyanddirectly
detectedbyaspectrophotometer.
ThepresentstudythereforeattemptstodevelopPTIO
?
scavengingasanewinvitroantioxidantassay.Tothebestof
ourknowledge,thisisthe?rstreportregardingtheapplication
ofPTIO
?
inthe?eld.
2.MATERIALSANDMETHODS
2.1.Materials.PTIO
?
(CASRegistryNumber18390-00-6,
>98.0%),catechol,andglutathione(GSH)werepurchasedfrom
TCIChemicalCo.(Shanghai,China).±-6-Hydroxyl-2,5,7,8-tetra-
methlychromane-2-carboxylicacid(Trolox)wasobtainedfromSigma-
AldrichShanghaiTradingCo.(Shanghai,China).D-Ascorbicacidand
melatoninwerefromJ&KScienti?c(Beijing,China).Proanthocyani-
din,chlorogenicacid,sodiumnitroprusside(SNP),andresveratrol
werepurchasedfromAladdinChemistryCo.(Shanghai,China).
Ca?eicandferulicacidswerepurchasedfromtheNationalInstitutefor
theControlofPharmaceuticalandBiologicalProducts(Beijing,
China).EllagicandgallicacidswerepurchasedfromGuangdong
GuanghuaChemicalPlantsCo.,Ltd.(Shantou,China).Quercetin,
sinapine,anddaidzeinwereobtainedfromSichuanWeikeqiBiological
TechnologyCo.,Ltd.(Chengdu,China).3,5-Dica?eoylquinicacid,
baicalein,scutellarein,andbaicalinwerepurchasedfromChengdu
BiopurifyPhytochemicals,Ltd.(Chengdu,China).(+)-Catechinand
L-ascorbicacidwerefromGuangzhouChemicalReagentFactory
(Guangzhou,China).Allotherchemicalreagentswereofanalytical
grade.Atotalof30lyophilizedaqueousextractsfrommedicinalor
edibleplantswerepreparedinourlaboratory(SupplementalMaterial
1oftheSupportingInformation).
2.2.Ultraviolet(UV)SpectraofPTIO
?
.PTIO
?
radicalswere
dissolvedindistilledwateratconcentrationsof1.1and2.2mmol/L.
Theaqueoussolutionswerescannedfrom300to1000nmbyan
ultravioletandvisible(UV/vis)spectrophotometer(Jinhua754PC,
Shanghai,China).Theaboveexperimentalprotocolwasrepeated
usingmethanolanddimethylsulfoxide(DMSO)insteadofdistilled
water.
2.3.SolventE?ect.Intheexperiment,weused(+)-catechin,
chlorogenicacid,andproanthocyanidinasreferencecompounds.The
initialexperimentalprotocolwasbasedonthe2,2-diphenyl-1-
picrylhydrazyl(DPPH)assay.
21
Inthe(+)-catechincomparativeexperimentbetweendi?erent
solvents,a(+)-catechinaqueoussolutionwasaddedtoaPTIO
?
aqueoussolution,andthen,thesolutionwasthoroughlymixed.Afterit
wasincubatedatroomtemperaturefor30min,theabsorbanceofthe
mixturewasdeterminedat557nmusingaUV/visspectrophotometer
(Jinhua754PC,Shanghai,China).ThepercentageofPTIO
?
inhibitionwascalculatedusingthefollowingformula:
=
?
×
AA
A
inhibition(%)100%
0
0
whereA
0
istheabsorbancewithoutthesampleandAisthe
absorbancewiththesample.
Forcomparison,a(+)-catechinmethanolicsolutionwasaddedtoa
PTIO
?
methanolicsolution,andthen,theabsorbancewasdetermined
at586nm.Additionally,a(+)-catechinDMSOsolutionwasmixed
withaPTIO
?
DMSOsolution,andtheabsorbancewasthenmeasured
at584nm.ThepercentagesofPTIO
?
inhibitionof(+)-catechinwere
similarlycalculatedusingtheaboveformula.
Theabovecomparativeexperimentswererepeatedusingchloro-
genicacidandproanthocyanidin.
2.4.StabilityandReactionTimeTest.Tostudythestabilityof
thePTIO
?
radical,a1.0mmol/LPTIO
?
aqueoussolutionwas
kineticallymonitoredat557nmfor20hatroomtemperatureusinga
UV/visspectrophotometer(Jinhua754PC,Shanghai,China).To
determinethebestreactiontime,thereactionmixturesofPTIO
?
with
(+)-catechin(0.25and0.5mg/mL),chlorogenicacid(0.25and0.5
mg/mL),andproanthocyanidin(0.25and0.5mg/mL)were
kineticallymonitoredat557nmfor20hatroomtemperature.
2.5.TemperatureandIrradiationE?ect.Similarly,(+)-cat-
echin,chlorogenicacid,andproanthocyanidinwereusedasreference
compoundsintheexperiments.Eachreferencecompound(inan
aqueoussolution)wasaddedtoaPTIO
?
aqueoussolutionandmixed
thoroughly.Afterincubationina37°Cwaterbathfor2h,the
absorbanceofthemixturewasdeterminedat557nmusingaUV/vis
spectrophotometer(Jinhua754PC,Shanghai,China).Thepercentage
ofPTIO
?
inhibitionwascalculatedusingtheaboveformula.The
aboveexperimentwasrepeatedat15,25,45,and55°C.
Toexploretheirradiatione?ectbetweenirradiationandnon-
irradiation,eachofthethreereferencecompoundswascomparatively
determinedat37°Cinaqueoussolutionfor2h.TheUVirradiation
wascarriedoutusingaZXF-LCAUV-lightcatalyticreactor(Zhengxin
InstrumentFactory,Binhai,China.)
2.6.pHValueE?ect.ConsideringthecomplexityofthepHe?ect,
20commonantioxidants(C1?C20;Figure2andTable1)were
selectedfortheinvestigation,including(+)-catechin,chlorogenicacid,
proanthocyanidin,ca?eicacid,ferulicacid,GSH,(+)-ascorbicacid,
(?)-ascorbicacid,ellagicacid,gallicacid,Trolox,resveratrol,and
quercetin.TheexperimentwasconductedwithoutUVirradiation,and
thedistilledwaterpreviouslyusedwasreplacedbya50mMphosphate
bu?eratpH5.0,6.0,7.4,8.0,and9.0.Then,thepercentagesofPTIO
?
inhibitionwerecalculatedusingtheaboveformula.
2.7.ValidationofthePTIO
?
ScavengingAssay.Inthe
validationexperiment,20purecompounds(C1?C20;Figure2and
Table1)and30plantextracts(lyophilizedpowders,E1?E30;Table
2)wereinvestigatedusingthefollowingprotocol:PTIO
?
radicalswere
dissolvedinphosphatebu?er(pH7.4,50mM)atapproximately0.05
mg/mLandthenincreasedbysamplesolution.Thetotalvolumeof
thereactionmixturewasadjustedbythebu?er.Afterthereaction
Figure1.(A)Structureand(B)ball-and-stickmodelbasedonthe
preferentialconformationofPTIO
?
.
JournalofAgriculturalandFoodChemistryArticle
DOI:10.1021/acs.jafc.7b02247
J.Agric.FoodChem.2017,65,6288?6297
6289
solutionwasthoroughlymixed,itwasincubatedat37°Cinawater
bathwithoutUVirradiationfor2h,andthen,theabsorbancewas
measuredat557nm.Thetestedsamplecouldbepreparedusing
distilledwater,bu?er,ororganicsolvents.AccordingtotheA
557nm
value,thePTIO
?
inhibitionpercentageswerecalculatedanddose?
responsecurveswerecreated.
Onthebasisofthedose?responsecurves,thelinearityofthe
methodwasevaluatedat?veconcentrations.Acalibrationcurvewas
preparedbyplottingthemeaninhibitionpercentagesoftriplicate
analysesagainstthe?nalconcentrations.Theprecisionwasevaluated
usingtherelativestandarddeviation(RSD,%)inhibitionpercentages
ofthetriplicateanalyses.Inaddition,reproducibilitywasalsoassessed
usingU.S.FoodandDrugAdministration(FDA)guidance.
22
The
threeparametersbasedonFDAguidancewereusedtovalidatethe
PTIO
?
scavengingassayinthestudy.
2.8.Ultra-PerformanceLiquidChromatographyCoupled
withElectrosprayIonizationQuadrupoleTime-of-Flight
TandemMassSpectrometry(UPLC?ESI?Q?TOF?MS/MS)
AnalysisfortheReactionProductofPTIO
?
withSNPand
AscorbicAcid.Atotalof4mgofSNP(orL-ascorbicacid)wasadded
to1mLofaPTIO
?
aqueoussolution(4mg/mL).Afterthemixture
wasultrasonicallyincubatedfor20min,itwasleftstandingfor24h.
Theproductmixturewasdiluted40times,then?lteredusinga0.22
μm?lter,andfurtheranalyzedusinganUPLC?ESI?Q?TOF?MS/
MSsystemequippedwithaC
18
column(2.1mminnerdiameter×
100mm,1.6μm,Phenomenex,Torrance,CA,U.S.A.).Themobile
phasewas100%methanolwitha?owrateof0.2mL/min.The
injectionvolumewas3μLandwasappliedforseparation.Q?TOF?
MS/MSwasperformedonaTripleTOF5600
plus
massspectrometer
(ABSCIEX,Framingham,MA,U.S.A.)equippedwithanESIsource.
Thescanrangewas100?2000Da.Thefollowingparametersettings
wereused:ionsprayvoltage,+4500V;ionsourceheater,550°C;
curtaingas(CUR,N
2
),30psi;nebulizinggas(GS1,air),50psi;and
TISgas(GS2,air),50psi.Thedeclusteringpotential(DP)was+100
V,andthecollisionenergy(CE)was+40Vwithacollisionenergy
spread(CES)of20V.
Forcomparison,1μLofaPTIO
?
aqueoussolution(50μg/mL)
wasalsoinjectedfortheanalysisusingtheaboveconditions.
2.9.StatisticalAnalysis.Eachexperimentwasperformedin
triplicate,andthedatawererecordedasthemean±standard
deviation(SD).TheIC
50
valueforeachantioxidantwasde?nedasthe
?nalconcentrationneededfor50%PTIO
?
inhibition.Statistical
comparisonsweremadebyone-wayanalysisofvariance(ANOVA)to
detectthesigni?cantdi?erenceusingSPSS13.0(SPSS,Inc.,Chicago,
IL,U.S.A.)forWindows.p<0.05wasconsideredtobestatistically
signi?cant.
Figure2.Structuresof20purecompounds.
JournalofAgriculturalandFoodChemistryArticle
DOI:10.1021/acs.jafc.7b02247
J.Agric.FoodChem.2017,65,6288?6297
6290
Table
1.
IC
50
Values
(
μ
mol/L)
and
Validation
Parameters
of
20
Pure
Compounds
in
the
PTIO
Scavenging
Assay
a
IC
50
values
number
antioxidant
resource
and
characterization
pH
5.0
pH
6.0
pH
7.0
pH
7.4
pH
8.0
pH
9.0
IC
50
(RSD,
%)
R
value
C1
(+)-catechin
exogenous,
natural,
tea
polyphenol
115.9
±
2.8
e
50.3
±
5.5
d
26.6
±
2.1
c
25.2
±
1.9
c
14.8
±
0.7
b
12.4
±
0.9
a
7.5
0.9440
C2
chlorogenic
acid
exogenous,
natural,
phenolic
acid
ester
33.9
±
1.7
e
26.3
±
2.3
d
13.0
±
2.0
b
19.5
±
2.0
c
9.9
±
0.6
a
13.8
±
1.4
b
10.2
0.9213
C3
procyanidine
exogenous,
natural,
anthocyanidin
28.1
±
1.7
e
17.2
±
0.3
d
13.1
±
1.0
c
16.3
±
1.9
d
10.1
±
0.8
b
8.8
±
0.8
a
11.6
0.9849
C4
ca
?
eic
acid
exogenous,
natural,
phenolic
acid
294.4
±
20.0
f
118.3
±
12.8
c
105.6
±
6.7
b
70.0
±
0.7
a
177.8
±
30.0
d
244.4
±
5.0
e
1.0
0.8811
C5
ferulic
acid
exogenous,
natural,
phenolic
acid
289.9
±
11.8
f
343.5
±
22.1
c
284.8
±
17.0
e
259.0
±
12.9
d
216.8
±
13.9
b
92.7
±
5.1
a
5.0
0.9380
C6
GSH
b
endogenous,
oligopeptide,
mercapto
molecule
245.3
±
13
e
211.0
±
5.2
d
144.3
±
12.3
a
151.1
±
17.1
b
143.3
±
27.7
a
198.0
±
38.1
c
11.3
0.8874
C7
D
-ascorbic
acid
exogenous,
synthetic,
vitamin
38.6
±
1.7
c
36.9
±
0.6
c
29.5
±
1.1
b
29.5
±
0.6
b
22.2
±
0.6
a
23.3
±
0.6
a
2.1
0.9839
C8
L
-ascorbic
acid
exogenous,
natural,
vitamin
44.3
±
2.3
d
38.0
±
0.6
c
33.0
±
0.6
b
31.2
±
0.6
b
26.1
±
0.6
a
25.0
±
0.6
a
1.9
0.9904
C9
ellagic
acid
exogenous,
natural,
phenolic
lactone
2498.3
±
220.8
f
2058.3
±
526.0
e
621.2
±
220.5
b
511.3
±
53.0
a
663.2
±
81.1
c
983.4
±
147.4
d
10.4
0.8915
C10
gallic
acid
exogenous,
natural,
tannin
79.0
±
7.2
d
49.9
±
2.9
c
10.7
±
0.2
b
5.9
±
0.4
a
6.5
±
0.18
a
9.4
±
0.9
b
6.8
0.9209
C11
Trolox
exogenous,
synthetic,
vitamin
53.7
±
2.8
a
56.5
±
2.3
a
130.0
±
7.4
b
128.6
±
5.3
b
232.0
±
3.7
c
272.0
±
28.4
d
5.7
0.8844
C12
resveratrol
exogenous,
natural,
stilbene
4421.0
±
466.7
e
4719.0
±
7.0
f
1079.0
±
44.7
d
785.0
±
30.7
c
443.0
±
14.5
b
350.0
±
9.2
a
3.9
0.9390
C13
catechol
exogenous,
natural,
polyphenol
612.6
±
12.6
f
414.4
±
14.4
c
387.4
±
11.7
b
353.2
±
36.0
a
445.0
±
29.7
d
525.2
±
18.9
e
10.1
0.9239
C14
sinapine
exogenous,
natural,
phenolic
alkaloid
830.0
±
33.7
b
757.6
±
11.7
a
723.8
±
44.5
a
879.9
±
58.6
c
835.5
±
62.3
b
1034.5
±
88.9
d
6.6
0.9880
C15
quercetin
exogenous,
natural,
?
avonol
66.6
±
2.0
c
67.5
±
1.0
c
55.3
±
2.0
a
61.6
±
3.3
b
57.3
±
3.3
a
113.2
±
10.6
d
5.4
0.9844
C16
2
′
-hydroxydaidzein
exogenous,
natural,
iso
?
avone
ND
ND
ND
512.6
±
13.8
ND
ND
2.7
0.9989
C17
melatonin
endogenous,
endocrine
hormone
ND
ND
ND
341.8
±
10.8
ND
ND
3.1
0.9885
C18
baicalein
exogenous,
natural,
?
avone
ND
ND
ND
110.1
±
2.7
ND
ND
2.4
0.9978
C19
scutellarein
exogenous,
natural,
?
avone
ND
ND
ND
97.7
±
2.3
ND
ND
2.3
0.9745
C20
baicalin
exogenous,
natural,
?
avone
glucoside
ND
ND
ND
212.2
±
10.5
ND
ND
4.9
0.9987
a
The
IC
50
value
was
de
?
ned
as
the
concentration
of
50%
radical
inhibition
and
expressed
as
the
mean
±
SD
(
n
=
3).
The
IC
50
value
was
obtained
from
the
dose
?
response
curves
in
Supplemental
Material
4
of
the
Supporting
Information
and
further
converted
into
molar
units
(
μ
mol/L).
The
R
value
was
calculated
from
linear
regression,
and
the
linear
regression
was
analyzed
by
Origin
6.0
Professional
software.
Mean
values
with
di
?
erent
letters
in
the
same
row
are
signi
?
cantly
di
?
erent
(
p
<
0.05).
RSD
=
S
D
÷
mean
×
100%.
b
GSH
=
glutathione.
JournalofAgriculturalandFoodChemistryArticle
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Table
2.
Validation
of
30
Lyophilized
Aqueous
Extracts
from
Medicinal
or
Edible
Plants
in
the
PTIO
Scavenging
Assay
(
μ
g/mL)
a
number
plant
Chinese
name
solution
color
IC
50
IC
50
(RSD,
%)
R
value
number
plant
Chinese
name
solution
color
IC
50
IC
50
(RSD,
%)
R
value
E1
Typhae
pollen
puhuang
yellow
2455.1
±
53.2
2.2
0.9887
E16
epimedii
folium
yinyanghuo
green
174.2
±
6.7
3.8
0.9590
E2
artemisiae
argyi
folium
aiye
brown
387.0
±
2.9
0.7
0.9569
E17
Tibetan
tea
zangcha
red
144.6
±
7.6
5.2
0.9234
E3
rubiae
radix
et
rhizoma
qiancao
red
1863.8
±
142.0
7.6
0.9911
E18
Camellia
sinensis
L.
jinjunmei
tea
red
142.0
±
3.99
2.8
0.9766
E4
callicarpae
formosanae
folium
zizhu
purple
619.1
±
14.2
2.3
0.9632
E19
Camellia
sinensis
L.
green
tea
green
46.2
±
4.1
8.
9
0.9835
E5
lonicerae
japonica
?
os
jinyinhua
yellow
2833.3
±
115.7
4.1
0.9218
E20
Camellia
sinensis
var.
assamica
pu-er
tea
red
123.4
±
1.2
0.9
0.9538
E6
rosae
rugosae
?
os
meiguihua
rufous
369.2
±
39.5
10.7
0.9962
E21
Camellia
sinensis
L.
tieguanyin
tea
green
54.6
±
3.8
6.9
0.9791
E7
Lavandula
pedunculata
xunyicao
brown
2179.8
±
124.7
5.7
0.8059
E22
paeoniae
radix
alba
baishao
colorless
656.9
±
30.3
4.6
0.9618
E8
cimicifugae
rhizoma
shengma
brown
3188.3
±
80.4
2.5
0.9935
E23
Angelicae
sinensis
radix
danggui
fawn
9203.9
±
253.3
2.8
0.9366
E9
polygonati
rhizoma
huangjing
fawn
3784.0
±
413.0
10.9
0.9771
E24
spatholobi
caulis
jixueteng
orange
163.8
±
1.1
0.7
0.9903
E10
atractylodis
macrocephalae
rhizoma
baizhu
colorless
658.8
±
57.5
8.7
0.9927
E25
notoginseng
radix
et
rhizoma
sanqi
fawn
4929.7
±
175.6
3.6
0.9746
E11
Gynostemma
pentaphyllum
(Thunb.)
Makino
jiaogulan
fawn
4041.0
±
194.0
4.8
0.9793
E26
chuanxiong
rhizoma
chuanxiong
fawn
3590.7
±
254.1
7.2
0.9762
E12
eucommiae
cortex
duzhong
fawn
4181.5
±
253.3
6.1
0.9162
E27
drynariae
rhizoma
gusuibu
orange
6095.9
±
192.3
3.2
0.9725
E13
psoraleae
fructus
buguzhi
brown
691.5
±
54.6
7.9
0.9155
E28
carthami
?
os
honghua
yellow
1820.4
±
175.2
9.6
0.9597
E14
astragali
complanati
semen
shayuanzi
colorless
717.9
±
38.1
5.3
0.9466
E29
vaccariae
semen
wangbuliuxing
fawn
6024.1
±
77.6
1.3
0.9712
E15
cynomorii
herba
suoyang
yellow
233.6
±
26.1
11.2
0.9902
E30
lycopi
herba
zelan
orange
1793.8
±
121.1
6.7
0.9736
a
The
IC
50
value
is
de
?
ned
as
the
concentration
of
50%
radical
inhibition
and
expressed
as
the
mean
±
SD
(
n
=
3).
The
R
value
was
calculated
from
linear
regression,
and
the
linear
regression
was
analyzed
by
Origin
6.0
Professional
software.
RSD
=
S
D
÷
mean
×
100%.
Their
dose
?
response
curves
were
shown
in
Supplemental
Material
5
of
the
Supporting
Information.
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3.RESULTS
3.1.UVSpectra.TheUVspectraofPTIO
?
(Figure3)
suggestedthatPTIO
?
showedalongwavelengthabsorbanceat
557,586,and584nminwater,methanol,andDMSO,
respectively.
Accordingtothemaximumabsorbancevaluesandthe
PTIO
?
concentrations,themolarabsorptioncoe?cients(ε)of
PTIO
?
werecalculatedas1022,677,and477Lmol
?1
cm
?1
in
water,methanol,andDMSO,respectively.
3.2.SolventE?ects.Thedose?responsecurvesareplotted
inSupplementalMaterial2oftheSupportingInformation.It
wasobservedthateachreferencecompoundexhibitedadose-
dependente?ect;however,theIC
50
valuesvariedwiththetype
ofsolvent.Ingeneral,thelowestIC
50
valueswereobservedin
theaqueoussolution,whileintermediateIC
50
valueswere
foundinthemethanolsolution,andthehighestIC
50
values
werefoundintheDMSOsolution(Figure4).Thisresult
stronglyindicatedasolvente?ect.
3.3.StabilityandReactionTimeTest.TheA
557nm
decay
ofthePTIO
?
aqueoussolution(1mg/mL)within20his
showninFigure5.ThefactthattheA
557nm
valueofthePTIO
?
aqueoussolutionremainedstablefor20handthattheA
557nm
valueofthePTIO
?
reactantreachedastableminimumwithin2
hindicatesthatthePTIO
?
aqueoussolutioncanbestoredat
roomtemperatureforatleast20handthereactionofPTIO
?
withanantioxidantcanreachabalancewithin2h.
3.4.TemperatureandIrradiationE?ect.Inthe
temperaturee?ectexperiment,eachofthethreereference
compoundshadadose-dependente?ect(Supplemental
Material3oftheSupportingInformation).Nevertheless,the
IC
50
valuesofeachreferencecompounddecayedwiththe
reactiontemperature.This?ndingsuggeststhatahigher
temperatureacceleratedthePTIO
?
scavengingreaction.
Accordingtothetemperature?IC
50
curves,weconducteda
?rst-orderexponentialdecay?t,inwhichanin?ectionpoint
wasfoundatapproximately37°C(Figure6A).
Intheirradiatione?ectexperiment,however,noneofthe
referencecompoundsshowedasigni?cant(p<0.05)di?erence
inIC
50
valuesbetweenirradiationandnon-irradiation(Figure
6B).
3.5.pHValueE?ect.Thedose?responsecurvesofthe20
referencecompounds(C1?C20)arelistedinSupplemental
Material4oftheSupportingInformation.TheIC
50
valueswere
calculatedaccordingtothedose?responsecurvesandare
detailedinTable1.
3.6.ValidationExperiments.Thedose?responsecurves
areshowninSupplementalMaterials4and5oftheSupporting
Information.TheIC
50
valuesandvalidationparameterswere
calculatedaccordingtothedose?responsecurvesandare
detailedinTables1and2.
Figure3.UVspectraofPTIO
?
in(A)water,(B)methanol,and(C)DMSO.
Figure4.IC
50
valuesof(+)-catechin,chlorogenicacid,and
proanthocyanidinindi?erentsolventsinthePTIO
?
assay(μg/mL).
EachIC
50
valueisexpressedasthemean±SD(n=3).TheIC
50
value
isde?nedastheconcentrationthatgivesa50%e?ectandwas
calculatedbylinearregressionanalysis.Thelinearregressionwas
analyzedbyOrigin6.0Professionalsoftware.Meanvalueswith
di?erentlettersinthesamereferencecompoundaresigni?cantly
di?erent(p<0.05).Theirdose?responsecurvesareshownin
SupplementalMaterial2oftheSupportingInformation.
Figure5.A
557nm
decayofaPTIO
?
aqueoussolution(0.25mg/mL,
1.1mmol/L)andreactionmixtureofPTIO
?
with(+)-catechin,
chlorogenicacid,andproanthocyanidin.
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3.7.UPLC?ESI?Q?TOF?MS/MSAnalysisforthe
ReactionProductofPTIO
?
withSNPandAscorbic
Acid.TherelevantspectraarelistedinSupplementalMaterial
6oftheSupportingInformation.ThedataimplythatPTIO
?
,
PTIO
?
withL-ascorbicacid,andPTIO
?
withSNPshowed
molecularionpeaksm/z234(235),235(236),and218(219),
respectively.However,SNPwasusedasadonatorofNOinthe
experiment.Intermsofthesedataandpreviousliterature,
19
we
proposedthereactionofPTIO
?
withNOandwithL-ascorbic
acidasthefollowing(eqs1and2):
4.DISCUSSION
AsseeninFigure3,PTIO
?
presentedcharacteristicabsorption
maximumsat557,586,and584nminwater,methanol,and
DMSO,respectively.However,themolarabsorptioncoe?cient
wasthelargest(ε=1022Lmol
?1
cm
?1
)intheaqueous
solution.Eachreferencecompoundalsoshowedthehighest
PTIO
?
scavengingcapacitiesintheaqueoussolution(Figure
4).Therefore,weselectedwater(notorganicsolvents)asthe
reactionsolventfortheassay,andaccordingly,557nmwas
usedasthemonitoringwavelength.Therearethreeadvantages
tousingwaterasareactionsolvent:(1)thecellular
environmentishydrophilic,andaqueousextractsfromChinese
herbalmedicineshavebeenusedintraditionalChinese
medicineforthousandsofyears;thus,theassay-basedaqueous
solutionisofrelevancetophysiology,biology,andtraditional
Chinesemedicine;(2)someinterferencefromorganicsolvents
previouslyreported
5,13,15?17
canbee?ectivelyavoided;and(3)
suchalongwavelength(λ=557nm)caneliminatethe
interferencefromthebackgroundabsorbanceofthetested
samples.Infact,ourdataindicatedthatevensomeextractswith
deepercolor(suchasredandpurple)couldnotprovide
absorptionat557nm(SupplementalMaterial1ofthe
SupportingInformation).
Consequently,thestabilityofthePTIO
?
aqueoussolution
wasstudiedonvthebasisofmonitoringtheabsorbanceat557
nm.Asillustratedinthedecaycurve(Figure5),PTIO
?
remainedstableforatleast20h,whichisconvenientforthe
experimentalprotocol.Somecommonlyusedfreeradicalscan
bestoredforonlyseveralhours.Forexample,aDPPH
?
solutioncanbestoredfor5h.Similarly,thereactionmixtures
ofPTIO
?
andthereferencecompoundswerealsokinetically
monitoredfor20hatroomtemperatureinanaqueous
solution.TheresultspresentedinFigure5suggestedthatthe
reactiontimeshouldbedesignatedas2h.
Intheexplorationofthetemperaturee?ect,wefoundthat,
althoughhighertemperatures(40?55°C)acceleratethe
scavengingreaction,anin?ectionpointwasobservedat
approximately37°C(Figure6A).Thus,thephysiological
temperatureof37°Cwasassignedastheexperimental
temperature.
Thefreeradicalreactioniswell-knowntobeassociatedwith
irradiation;therefore,weinvestigatedthepossibilityofan
irradiatione?ect.Ourdatasuggestedaslightradiatione?ect
(Figure6B),andwedecidedtoadoptnon-irradiationforthe
experimentalconditiontoreducethepossibilityofthe
irradiatione?ect.
Subsequently,weused20referencecompoundstowidely
investigatethee?ectofpH.AsseeninSupplementalMaterial4
oftheSupportingInformationandTable1,therewasalarge
pHe?ectinthePTIO
?
scavengingassay.Nevertheless,
physiologicalpH7.4wasusedforthisassaytoobtainreliable
results.ThispHisparticularlyimportantforsomeacidic
antioxidants(e.g.,ca?eicacid).
6,15
Takentogether,ourdatasuggestthattheexperimental
protocolcanbeoptimizedasfollows:PTIO
?
radicalsdissolved
inaphosphatebu?er(pH7.4,50mM)atapproximately0.05
mg/mLandthenincreasedbythesamplesolution.Thetotal
volumeofthereactionmixturewasadjustedusingthebu?er.
Afterthereactionsolutionwasthoroughlymixed,itwas
incubatedat37°Cinawaterbathfor2h,andthen,the
absorbancewasmeasuredat557nm.Thetestedsamplecould
bepreparedusingdistilledwater,bu?er,ororganicsolvents.
Figure6.IC
50
valuesfrom(A)temperaturee?ectand(B)irradiatione?ectexperimentswith(+)-catechin,chlorogenicacid,andproanthocyanidin
inthePTIO
?
assay.TheIC
50
value,de?nedastheconcentrationprovidinga50%e?ect,wascalculatedbylinearregressionanalysisandisexpressed
asthemean±SD(n=3).Thelinearregressionanalysisand?rst-orderexponentialdecay?twereconductedusingOrigin6.0Professionalsoftware.
Signi?cantdi?erenceanalyses(p<0.05)wereconductedbySPSS.Theirdose?responsecurvesareshowninSupplementalMaterial3ofthe
SupportingInformation.
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6294
Usingtheoptimizedexperimentalprotocol,wedetermined
thePTIO
?
scavengingabilitiesof20purecompounds(C1?
C20)and30lyophilizedplantextracts(E1?E30).Asshownin
Table1,all50samplesexhibitedgoodlinearity(R=0.8811?
0.9989;theaveragecorrelationcoe?cientwas0.956966)and
lowRSDvalues(0.5?11.6%;theaveragevaluewas5.4%).To
testtheprecisionofthemethodunderthesameoperating
conditionsoverdays,areproducibilityexperimentwas
conductedfor3days.TheRSD(%)fortheIC
50
valuesof
(+)-catechinwas8.5%(SupplementalMaterial7ofthe
SupportingInformation),suggestingthatthemethodwas
reproducible.Inshort,themethodhashigherlinearity,
precision,andreproducibility.
AsseeninFigure2,the20purecompounds(C1?C20)
coveredalmosteverytypeofantioxidant,suchasexogenous
andendogenous,naturalandsynthetic,vitamin,polyphenol,
phenolicacid,phenolicacidester,?avone,iso?avone,?avonol,
?avoneglucoside,tannin,oligopeptide,anthocyanidin,stilbene,
phenoliclactone,alkaloid,endocrinehormone,andmercapto
compounds.The30plantmaterialslistedinTable2appearas
variouscolors,suchasyellow,red,green,andpurple,and
includevariousplantparts,suchaswholeherb,leaf,root,bark,
rhizome,?ower,fruit,andseed.Thesuccessfuldetermination
ofthesesamplesimpliesthatPTIO
?
scavengingissuitablefor
alltypesofaqueousantioxidants(includingpurecompounds
andextracts).Itisworthmentioningthat,intheassayforthe
30extracts,thecorrelationbetweentotalphenolicsandPTIO
?
scavenginglevelwashigherthanthatwithDPPHscavengingor
withABTSscavenging(thecorrelationcoe?cientRvalues
betweentotalphenolicswiththePTIO
?
scavenginglevel,
DPPH
?
scavenginglevel,andABTS
?+
scavenginglevelwere
0.63551,0.60993,and0.54887,respectively;Supplemental
Material8oftheSupportingInformation);suchanadvantage
mayresultfromthelongwavelengthabsorptionandaqueous
solubilityofPTIO
?
.
AsseeninTable1,ca?eicacidwithacatecholmoiety
showedastrongerPTIO
?
scavengingabilitythanthatofferulic
acidwithaguaiacolmoiety,ca?eicacidasaphenolicacid
exhibitedahighere?ectivePTIO
?
scavenginglevelthanits
esterchlorogenicacid,andthree?avonoidsinScutellaria
baicalensisGeorgidecreasedthePTIO
?
scavengingabilities
withthefollowingorder:scutellarein(witha4′-OHgroup)>
baicalin(withouta4′-OHgroup)>baicalein(withouta4′-OH
groupandglucoside).Alloftheseresultsagreewithprevious
studiesabouttheantioxidantstructure?activityrelation-
ship,
23?26
suggestingthatthePTIO
?
scavengingassaycan
alsobeusedfortheantioxidantstructure?activityrelationship
analysis.
Asmentionedabove,PTIO
?
radicalsarecurrentlyusedto
measurethelevelsofNO.
19,20
InthereactionwithNO,PTIO
?
isreportedtodonateOtoNO.
19
Infact,inourUPLC?ESI?
Q?TOF?MS/MSanalysisoftheproductmixtureofPTIO
?
andSNP,alossoftheOatomwasobserved(m/z234?218).
However,initsreactionwithL-ascorbicacid,onlyalossofH/
H
+
(m/z235?234)wasfoundwithoutOatomtransfer.These
?ndingssuggestthatPTIO
?
scavengingbythephenolic
antioxidantisinvolvedinahydrogentransfer,especiallyH
+
transfer.ThepossibilityofH
+
transferisfurthersupportedby
theabove-mentionedpHe?ectandsolvente?ect.Asillustrated
inTable1,20referencecompounds(C1?C20)similarly
presentedtheweakestPTIO
?
scavengingatpH5.0or6.0,
regardlessofwhethertheyhadanacidic?COOHgrouporan
alkalineNatom.This?ndingmeansthat,underalowerpH
value,ahigherproton(H
+
)concentrationinthesolutioncan
considerablyinhibitH
+
-transfer-basedPTIO
?
scavenging.In
thecontextofthesolvente?ect,threereferencecompounds
(C1?C3)decreasedtheirPTIO
?
scavenginglevelsintheorder
ofwater>methanol>DMSO(Figure4).Thissequenceis
parallelwiththeirextentsofprotonation(water>methanol>
DMSO)butnotwiththeirpolarityindexes:water(10.2)>
DMSO(7.2)>methanol(5.1).Inshort,ournewobservations
aboutthepHe?ect,solvente?ect,andUPLC?ESI?Q?TOF?
MS/MSanalysisfurthersupporttheprevioushypothesis
involvingH
+
transfer.
19
Electrontransfer(redoxreaction),
accompaniedbyH
+
transfer,shouldhappenearlyorlate.Thus,
therearethreepossibilitiesinPTIO
?
scavenging,i.e.,PCET,
SPLET,orSEPTsubtypes.However,furtherworkisrequired
topreciselyidentifywhichsubtypesareinvolved.
AsshowninTable1andSupplementalMaterial4ofthe
SupportingInformation,twostereoisomers,D-ascorbicacidand
L-ascorbicacid,didnotpresentasigni?cant(p<0.05)
di?erenceinthePTIO
?
scavengingassay,indicatingthatthe
PTIO
?
scavengingreactionhasnostereospeci?city.Itmaybe
attributedtotheplanarcon?gurationPTIO
?
,whichgivesan
identicalopportunityforstereoisomerstoattackthePTIO
?
radical(Figure1B).
Nevertheless,thePTIO
?
scavengingassayhastwo
disadvantages:(1)itcannotbeusedforlipid-soluble
antioxidants,suchasbutylatedhydroxyanisole(BHA),and
(2)itrequiresagreateramountoftestedsamplethanother
assays(e.g.,DPPHassay).Todecreasetheconsumptionof
someexpensivetestedsamples,theA
0
valueofthePTIO
?
radicalcanbeassignedaslowaspossible,ifallowedbythe
analyticalapparatus.Inourexperiment,thePTIO
?
concen-
trationwas0.05mg/mLandtheA
0
valuewas0.2±0.01.
Itshouldalsobenotedthat(1)thenewantioxidantassay
focusesontheconcentrationandstoichiometry(notkinetic
analysis),therebyitsresultcannotalwayscorrelatelinearlywith
thatfromsomekineticanalysis-orcell-basedassays,
27
and(2)
theSafetyDataSheetstatesthatthePTIO
?
radicalisnontoxic
tothehumanbody;nevertheless,usersareadvisedtoavoid
swallowingordirectlycontactingit.
Inconclusion,thePTIO
?
scavengingassayisanewinvitro
antioxidantassay.Itsexperimentalprotocolcanberecom-
mendedasfollows:PTIO
?
radicalsaredissolvedinaphosphate
bu?er(pH7.4,50mM)andthenmixedwiththesample
aqueous(oralcoholicorbu?er)solution.Thereactionmixture
isadjustedtoacertaintotalvolumeusingbu?er.Themixtureis
furtherincubatedat37°Cinawaterbathfor2h,andthen,the
absorbancewasmeasuredat557nm.ThePTIO
?
scavenging
assaypossessesfouradvantages(i.e.,oxygen-centered,physio-
logicalaqueoussolution,simpleanddirectmeasurement,and
longwavelengthdeterminationforavoidinginterference)andis
proventobeareliableassay.Itcanbeusedfornotonlythe
evaluationofalltypesofaqueous-solubleantioxidants
(especiallyextracts)butalsotheanalysisoftheirantioxidant
structure?activityrelationship.PTIO
?
scavenginghasno
stereospeci?cityandhasbeendemonstratedtobeinvolvedin
atleastH
+
transfer.AccompaniedbyH
+
transfer,theredox
reactionisthoughttohappenearlyorlateintheprocess.
■
ASSOCIATEDCONTENT
SSupportingInformation
TheSupportingInformationisavailablefreeofchargeonthe
ACSPublicationswebsiteatDOI:10.1021/acs.jafc.7b02247.
JournalofAgriculturalandFoodChemistryArticle
DOI:10.1021/acs.jafc.7b02247
J.Agric.FoodChem.2017,65,6288?6297
6295
Preparationandevaluationof30extracts(Supplemental
Material1),dose?responsecurvesofthesolvente?ect
experiment(SupplementalMaterial2),dose?response
curvesofthetemperaturee?ectexperiment(Supple-
mentalMaterial3),dose?responsecurvesofthepH
e?ectexperimentof20purecompounds(originaldata
forTable1)(SupplementalMaterial4),dose?response
curvesof30extracts(includingoriginaldataforTable2)
(SupplementalMaterial5),spectraofUPLC?ESI?Q?
TOF?MS/MS(SupplementalMaterial6),reproduci-
bilitytest(SupplementalMaterial7),andcorrelation
analysisof30extracts(SupplementalMaterial8)(ZIP)
■
AUTHORINFORMATION
CorrespondingAuthor
Telephone:+86-20-39358076.Fax:+86-20-38892690.E-mail:
lixican@126.com.
ORCID
XicanLi:0000-0002-4358-3993
Funding
Thisworkis?nanciallysupportedbytheNationalNatural
ScienceFoundationofChina(81573558).
Notes
Theauthordeclaresnocompeting?nancialinterest.
■
ACKNOWLEDGMENTS
Theauthorthanksthestudentsfortechnicalworks,including
XiafukaitiXiakeer,WeijuMa,YitongHuang,YuxinMao,Shuqi
Zhang,YingyingPan,TingweiSu,WenyingHuang,Zhiyuan
Zhang,JingzhaoYan,DacaiYang,RuiwangHe,YangliGuo,
DongchanLiang,JieyingChen,HaihuaLuo,HuajinLin,and
WeilinHuang.
■
ABBREVIATIONSUSED
ABTS
?+
,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid)
radicalion;BHA,butylatedhydroxyanisole;DMSO,dimethyl
sulfoxide;DPPH,2,2-diphenyl-1-picrylhydrazyl;ESR,electron
spinresonance;FDA,U.S.FoodandDrugAdministration;
GSH,glutathione;HAT,hydrogenatomtransfer;HPLC,high-
performanceliquidchromatography;MDA,malondialdehyde;
PCET,proton-coupledelectrontransfer;PTIO
?
,2-phenyl-
4,4,5,5-tetramethylimidazoline-1-oxyl3-oxideradical;RAF,
radicaladductformation;RNS,reactivenitrogenspecies;
ROS,reactiveoxygenspecies;RSD,relativestandarddeviation;
SD,standarddeviation;SEPT,sequentialelectronproton
transfer;SNP,sodiumnitroprusside;SPLET,sequential
proton-losselectrontransfer;Trolox,(±)-6-hydroxyl-2,5,7,8-
tetramethlychromane-2-carboxylicacid;UPLC?ESI?Q?
TOF?MS/MS,ultra-performanceliquidchromatography
coupledwithelectrosprayionizationquadrupoletime-of-?ight
tandemmassspectrometry;UV/vis,ultravioletandvisible
■
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