PTIO自由基清除-最简单的自由基清除法 -李熙灿-XICAN LI

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

benzeneringlinkedtoaCNdoublebondisthoughtto

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.

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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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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:[phone].Fax:[phone].E-mail:

[e-mail].

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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