Studies of the structure-antioxidant activity relationships and antioxidant activity mechanism of

15 Pages·2017·4.3 MB·English

RESEARCHARTICLE
Studies of the structure-antioxidant activity
relationships and antioxidant activity
mechanism of iridoid valepotriates and their
degradation products
FeifeiWang1,2,YumeiZhang2*,ShouhaiWu3,YiHe2,ZhongDai2,ShuangchengMa2*,
BinLiu1*
1 SchoolofChineseMateriaMedica,BeijingUniversityofChineseMedicine,Beijing,China,2 National
InstitutesforFoodandDrugControl,Beijing,China,3 TheSecondAffiliatedHospital,GuangzhouUniversity
a1111111111 ofChineseMedicine,Guangzhou,China
a1111111111
a1111111111 *[email protected](YZ);[email protected](SM);[email protected](BL)
a1111111111
a1111111111
Abstract
Oxidativestresshasbeenassociatedwithdiversediseases,includingobesity,cancerand
neurodegeneration.Infact,ValerianajatamansiJones(valerian)anditsextractspossess
OPENACCESS
strongantioxidantactivitiesthatextendtheirapplicationinclinicalpracticetothetreatment
Citation:WangF,ZhangY,WuS,HeY,DaiZ,Ma oftheseillnesses,eventhoughtheunderlyingmechanismsarenotwellunderstood.Iridoid
S,etal.(2017)Studiesofthestructure-antioxidant
valepotriate,acharacteristiciridoidesterinvalerianwithpoorchemicalstability,possesses
activityrelationshipsandantioxidantactivity
mechanismofiridoidvalepotriatesandtheir considerableantioxidantcomponents.Theoriginalcompoundsandtheirdegradationprod-
degradationproducts.PLoSONE12(12): uctshavebeenfoundtoexhibitstrongantioxidantactivities.However,therelationship
e0189198.https://doi.org/10.1371/journal.
betweentheirstructureandantioxidanteffectsandthemechanismunderlyingtheiroxida-
pone.0189198
tionresistanceremainunclear.Aforceddegradationstudyusingthreeiridoidvalepotriates
Editor:LuisEduardoMQuintas,Universidade
(valtrate,acevaltrateand1-βacevaltrate)wasperformedinthiswork,andthestructuresof
FederaldoRiodeJaneiro,BRAZIL
theirdegradationproductswereestimatedbyTLC-MSandLC-MS.Comparisonoftheanti-
Received:June27,2017
oxidantactivitiesoftheiridoidvalepotriatesbeforeandafterforceddegradationrevealed
Accepted:November21,2017 thatdegradationreducedtheactivitiesoftheiridoidvalepotriatesinfreeradicalscavenging
Published:December12,2017 andcytotoxicandcellapoptosistests.Theresultssuggestedthattheoxiranenucleusis
importantfordefiningtheantioxidantprofileofiridoidvalepotriate.Weuncoveredpossible
Copyright:©2017Wangetal.Thisisanopen
accessarticledistributedunderthetermsofthe mechanismsthatcouldexplaintheantioxidantactivities,includingthegenerationoftwo
CreativeCommonsAttributionLicense,which hydroxylgroupsthroughintramoleculartransferofanH•fromanoxiraneringandareduc-
permitsunrestricteduse,distribution,and
tioninROSlevelsthroughinteractionswithGABAergicsignallingpathways.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files.
Introduction
Funding:Thisworkwasfinanciallysupportedby
theNationalNaturalScienceFoundationofChina Thehumanbodyconstantlyreactswithoxygentoproducehighlyreactivemoleculesknown
[grantnumber81774101]. asreactiveoxygenspecies(ROS)orfreeradicals.Thestrongcellularoxidizingpotentialof
excessROSmightcauseoxidativedamagetoproteins,membranesandgenes[1,2].Oxidative
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. stresshasbeenimplicatedasamajorcauseofcellularinjuriesinawidevarietyofclinical
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
abnormalities,particularlyinthecentralnervoussystem(CNS).Conversely,intumourcells,
thegenerationofROShascontributedtothedevelopmentofsophisticatedsystemstocounter-
balanceoxidativestressthroughrapidproliferation[3].Basedonthisinformation,drugswith
antioxidantactivitiesarewidelyusedforthetreatmentofcancer,anxietyneurosis,andParkin-
son’sdisease,amongotherdiseases.
Asanimportantneuroprotectiveandanticancerplant,Valerian jatamansiJones(valerian)
hasbeenextensivelyusedasanherbmedicineordietarysupplement.Thisspecieshasbeen
traditionallyusedastherapyforsleepingdisorders,obesity,epilepsy,insanity,snakepoisoning,
andeyeandskinproblems[4,5].Itsantioxidantactivities,cytotoxiceffects,andanti-leishman-
ial,anti-Parkinsonandanti-HIVactivitieshavebeenofparticularinterestrecently[4,6–13].
Valerianalsocontainsvarioussecondarymetabolites,includingiridoidvalepotriates,valerenic
acids,flavonoidsandtnnins[14].
Iridoidvalepotriates,thecharacteristiciridoidestersinvalerian,haveattractedgreatinter-
estduetotheirvariousbiologicallysignificantproperties[15–18].Previousstudieshaveindi-
catedthatiridoidvalepotriatesdisplaypotentialdose-dependentoxidationresistanceabilities.
However,thepoorstabilityofiridoidvalepotriateiswell-known:itcaneasilydecompose
understrongsunlightorunderhightemperature,acidicoralkalineconditions[19,20].Para-
doxically,boththenaturalanddecompositionproducts(baldrinalandhomobaldrinal)have
multiplebiologicalactivities[21–23],butthedegradationpathwaysandantioxidativemecha-
nismshavenotyetbeensystematicallydocumented.
Weinitiallyidentifiedthestructuralcharacteristicsofiridoidvalepotriates[valtrate(V),
acevaltrate(AV)and1-βacevaltrate(BAV),Fig1]andtheirdegradationproducts.Iridoid
valepotriatesarelabiletothermaldegradationandbasehydrolysis.Theirstructure-activity
relationshipswereexploredthroughanevaluationoftheirantioxidanteffects.Ourresults
demonstratethatiridoidvalepotriatescouldpromotecytotoxicityinthreecancercellswithout
affectingnon-malignantcells.Thestabilityoftheoxiranenucleuswasfoundtobeimportant
duetotheabilityofiridoidvalepotriatestodonatetwohydrogenmoleculesthroughintramo-
leculartransferofH•fromtheoxiranering,whichmightbeonemechanismcontributingto
Fig1.StructuralformulasofV,AVandBAV.
https://doi.org/10.1371/journal.pone.0189198.g001
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
theantioxidantactivityprofileofthesecompounds.Additionally,GABAergicsignallingpath-
wayswerediscussedasanotherpossiblepathwaythroughwhichiridoidvalepotriatesreduce
ROSandinduceHepG2cellapoptosis.
Materialsandmethods
Reagentsandsamples
HPLC-grademethanolandacetonitrilewerepurchasedfromFisherScientific(FairLawn,NJ,
USA).AMilli-Qwaterpurificationsystem(Millipore,SanDiego,CA,USA)wasusedtofur-
therpurifydistilledwater.Othermaterialsincludedcisplatin(CP),N,N-Dimethylformamide
(DMF),2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonicacid)diammoniumsalt(ABTS),
1,1-diphenyl-2-picrylhydrazylradical2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl(DPPH),
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide(MTT),dimethylsulfoxide
(DMSO),butylatedhydroxytoluene(BHT),2,7-dichlorodihydrofluoresceindiacetate(DCFH-
DA)and2,7-dichlorofluorescein(DCF),allofwhichwerepurchasedfromSigmaAldrich
Co.(St.Louis,MO,USA).AnnexinV-fluoresceinisothiocyanate(AnnexinV-FITC)anda
propidiumiodide(PI)kitwereobtainedfromBD(SanJose,CA,USA).Valtrate(lot:111840–
201402),acevaltrate(lot:111841–201401)and1-βacevaltratewereobtainedfromtheNational
InstitutesforFoodandDrugControlofChina.Theanalyticalreagent-gradesodiumhydrox-
ide,hydrochloricacidandhydrogenperoxide(H O ,30%)usedinthisstudywerepurchased
2 2
fromBeijingChemicalWorks(Beijing,China).
Generalexperimentalprocedures
NMRspectrawereobtainedusingaBrukerAvance-III600MHzspectrometer(BrukerCorp,
Karlsruhe,Germany)inCD ODwithtetramethylsilane(TMS)astheinternalstandard.The
3
liquidchromatography-massspectrometry(LC-MS)system(Agilent6410TripleQuad,CA,
USA;Q-TOF,Waters,USA)wasusedtoanalysetheforceddegradationproducts.LCwasper-
formedonanAgilentSB-C column(1μL/mL,4.6mm×250mm,5μm)at30˚C,andthe
18
mobilephaseconsistedofamixtureofmethanolandwater(70:30,v/v).Massspectrometry
analyseswereperformedinthepositiveionizationmodewithascanningrangeofm/z80~1,
000.Thefollowingelectrosprayionization(ESI)parameterswereset:thecapillaryvoltagewas
4,000Vwithatargetmass-to-chargeratioof150,thenebulizer(N )pressurewassetto30psi,
2
thedryinggas(N )temperaturewas325˚C,andthedryinggasflowratewas7L/min.Forthe
2
MS/MSinvestigations,theionsofinterestwereisolatedthroughquadrupoletandemmass
spectrometry.Thin-layerchromatography(TLC)wasperformedonsilicagel60F TLC
254
plates(Merck,Darmstadt,Germany)anddevelopedusingacyclohexane:acetonemixture(5:
1,v/v).Afterdrying,theTLCplateswereexaminedunderUVlightat254nminaTLCvisual-
izer(Camag,Muttenz,Switzerland).
Stresseddegradationconditions
ThestressdegradationstudiesofiridoidvalepotriateswereperformedbasedontheICH
guidelines[24,25].Thefollowingoptimizedstressconditionswereused:acidichydrolysis
(0.01MHClat25˚Cfor5h),alkalinehydrolysis(0.01MNaOHat25˚Cfor10min),oxidative
hydrolysis(3%~6%H O at25˚Cfor24h),andthermaldegradation(mentholsolutionat
2 2
60˚Cfor0,1,2,4,or6h;solidsat4˚Cfor7days;solidsat25˚Cfor7days).Thedrugswere
exposedtolight(intensityof5000lxfor3h)[26]andthendissolvedanddilutedinmentholto
afinalconcentrationof1mg/mL(forcurrentuse).
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
TLC-DPPH•bioautography/MSanalysis
FortheDPPH•analysis,2mLofthedrugsatvariousconcentrationswasaddedto2mLof
DPPH•solution(0.1mM,2mL).After20minofincubationat37˚Cinthedark,theabsor-
bancewasrecordedat517nmusingaUV-Visiblespectrophotometer(Evolution260Bio,
ThermoScientific,USA).
TheTLC-DPPH•assaywasconductedasascreeningtesttoestimatetheantioxidantprop-
ertiesofthetestedfractions[27,28].TwoTLCplatesweredevelopedinparallel.TheTLC
operationwascompletedasdescribedinthe‘Generalexperimentalprocedures’section.After
developmentanddrying,oneTLCplatewasexaminedunderUVlightat254nm;thefluores-
cence-quenchingspotsweremarkedandthensubjectedtoTLC-MSanalysis.TheotherTLC
platewassprayedwith0.04%DPPH•-methanolsolution(20mgofDPPH•dissolvedin50mL
ofmethanol).Thefreeradicalscavengingcompoundsappearedasyellowishspotsonthepur-
plebackground.Theyellowishwhitebandsindicatedthatthosecompoundspossessedantioxi-
dantactivities.Theintensityillustratedtheantioxidantcapacitiesofthesamples.
FortheMSanalysis,theTLC-MSinterface(Camag,Muttenz,Switzerland)inletcapillary
wasconnectedtoanHPLCpump.Theextractionwasperformedwithmethanolataflowrate
of0.1mL/minanddirectlyconnectedwiththeESI-MSinstrument.Thezoneswerethen
extractedduringa1.8-mintimeperiodwithanovalextractionplungerandindividuallytrans-
ferredintotheESI-MSinstrument.TheplungerheadwascleanedwithN gasfor4sbetween
2
theextractionsteps.
ABTSradicalscavengingactivity
Thereactionmixturecomprised950μLofABTSsolutionand50μLofthesamplesatvarious
concentrationsinafinalvolumeof1mL.Thereactionmixturewashomogenizedandincu-
batedfor20min.Theabsorbanceofthesolutionsat734nmwasmeasuredwithUV-Visible
spectrophotometer.
Cytotoxicityassay
Celllinesandculture. Humanlivercarcinoma(HepG2)cells,humancervixcarcinoma
(HeLa)cells,breastcancer(MDA-MB-231)cellsandhumanumbilicalveinendothelialcells
(HUVECs)wereobtainedfromZhongshanUniversity,Guangzhou,China.Allofthecells
wereculturedinDulbecco’smodifiedEaglemedia(DMEM)supplementedwith10%foetal
calfserum,100units/mLpenicillinGand100μg/mLstreptomycin.Allmediaandserumwere
obtainedfromGibco(USA).Thecellsweregrownandmaintainedat37˚Cina5%CO
2
humidifiedatmosphere.
CytotoxicityanalysisviaMTTassay. Thecytotoxicitiesofthedrugsandtheirdegrada-
tioncomponents(storedat25˚Cfor7day)wereevaluatedthroughamodifiedMTTassay
[29].Thecellswereplatedataconcentrationof104cells/wellin96-wellplates(Corning,
Sigma,USA)containing100μLofgrowthmediumperwellfor24h.Thestocksolutionwas
preparedbydissolving10mgofthesamplein1mLofDMSO(AR).Thestocksolutionwas
thendilutedwithDMSOtoproducesolutionswithdifferentconcentrations.Finally,1μLof
eachofthesolutionswasaddedto100μLofgrowthmediumin96-wellplatestoproducesam-
plesolutionswithconcentrationsrangingfrom10to100μM.Noprecipitationwasobserved
ineachofthesolutionsbyvisualinspection,whichsuggestedthatthechemicalcompounds
werecompletelydissolvedinthesolvent.After24hofincubation,thecellsweretreatedwith
20μLofMTT(5mg/mL)andincubatedat37˚Cfor4h.Themediumwasremoved,and
100μLofDMSOwasaddedtoeachwell.Aftershakingfor10min,theabsorbanceat490nm
wasmeasured.Themitochondrialactivityrelatedtoviabilityisexpressedastheconcentration
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
ofdrugthatinhibitedcellgrowthby50%(IC )comparedwiththenegativecontrol(cells
50
treatedwith1.0%DMSO)andthepositivecontrol(seriallydilutedcisplatinsolution).The
testsandanalyseswererunintriplicate(n=3),andaveragesoftheresultsarereported.
FlowcytometryanalysisforROSmeasurements. DCFH-DAwasusedforROScapture
inthecells.TheaveragefluorescentintensityofDCFisproportionaltotheintracellularROS
levels.HepG2cellswereseededin6-cmdishesandtreatedwiththedrugs.Twenty-fourhours
aftertreatment,theHepG2cells(approximately105cells)werewashed,resuspendedinPBS
andtreatedwithDCFH-DAatafinalconcentrationof10μM.Thecellswereincubatedat
37˚Cfor30mininthedark,andtheROSlevelswerethenmeasuredbyflowcytometry(BD,
SanJose,CA,USA).Atleast20,000eventswereanalysed.
Flowcytometryanalysisforapoptosisassessment. Thecellsweretreatedwiththedrugs
for24h,washedtwicewithpre-chilledPBSandthenre-suspendedin100μLofbindingbuffer.
ThefractionofapoptoticcellswasassessedviaflowcytometryusinganAnnexinV-FITC/PI
kit.Thedrugswerepreparedaccordingtothemanufacturer’sinstructionsandmeasuredby
flowcytometryanalysis.Atleast20,000eventswereanalysed.
Statisticalanalyses
SPSS17.0softwarewasusedforthestatisticalanalyses.Thedataarepresentedasthe
means±SDs.Analysisofvariancewasappliedforthecomparisonsofthemeansamongtwo
ormultiplegroups.ANOVAwasfurtherusedforcomparisonsbetweentwogroups.Thedif-
ferencesbetweensampleswerestatisticallysignificantat(cid:3)p<0.05.
Resultsanddiscussion
StructuralidentificationofAVandBAV
AVandBAVareisomers,eventhoughtheirstructureshavebeenconfusedintheliterature[7,
30].Thus,LC-MS,UVandNMRanalyseswereperformedtoconfirmtheirchemicalstruc-
tures.Heteronuclearmultiplebondcoherence(HMBC)andheteronuclearmultiplequantum
coherence(HMQC)studieswereconductedtoconfirmtheacylgroups’chemicalshiftdata
(S1Fig).Theacylgroups,whichconnecttodifferentpositionsontheiridoidrings,showed
chemicalshiftdifferencesof0.2–0.3ppmintheir13CNMRspectra(theγ-effect)[6,31,32].
BasedontheHMBCdata,thechemicalshiftsofthefourestercarbonylswereassignedasδ
C
172.6(C-13),171.2(C-16),171.8(C-22)and172.2(C-2’)forAVandδ 172.6(C-13),172.9
C
(C-16),168.9(C-22)and172.3(C-2’)forBAV;theseresultscorrespondtothedataintheliter-
ature[32,33](S1Fig).
CharacterizationoftheV,AV,BAVandtheirdegradationproducts
Iridoidvalepotriatesarethermolabileanddecomposeeasilyunderacidoralkalineconditions
orinalcoholicsolutions[34].Inthiswork,forceddegradations(e.g.,acid,alkaline,oxidation,
photolysisandthermaldegradation)wereperformedtoobtainthedesiredcompounds.The
degradationpathwayswereestimatedbyLC-MSanalysis(Fig2).
IntheLC-MSchromatogramobtainedduringalkalinehydrolysis,atotaloffourAVdegra-
dationcomponentsweredetectedandidentified(Fig2B).AVshowedapseudo-molecularion
atm/z503[M+Na]+,andtheMS/MSspectrumofthem/z503ionshowedproductionsatm/z
219,177,149and131(S2Fig).Thedegradationproductsfromalkalinehydrolysisshowedthe
sameUVmaximumabsorptionatapproximately256nm,suggestingthattheiridoidringwas
stableduringdegradation[35].
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
Fig2.Schematicrepresentationofacevaltratedegradation(AVD).
https://doi.org/10.1371/journal.pone.0189198.g002
AB ,themainalkalinedegradationproductofAV,showedapseudo-molecularionatm/z
1
461(C H O Na)intheMSspectra,whichsuggestedthatthecompoundwasproducedby
22 30 9
cleavageoftheacetylgroupatAVO-1’orO-12.TheMS/MSspectraofAB andAB had
1 2
almostidenticalpredominantproductionsatm/z255[M-C H O -C H O].Asaconse-
5 9 2 5 9
quence,AB wasidentifiedasabasicdegradationproductofAB withthepseudo-molecular
2 1
ionatm/z361(C H O Na)duetolossofaC H O groupfromAB .AB hadapseudo-
17 22 7 5 9 2 1 3
molecularionatm/z319(C H O Na),whichis42DalessthanthatofAB duetocleavage
15 20 6 2
ofanacetylgroup.ThepredominantproductionintheAB MS/MSspectrumwaslocatedat
3
m/z277[M-H O].TheMSspectrumofAB showedapseudo-molecularionatm/z277
2 4
(C H O Na),indicatingthatAB wasthehydrolysisproductofAB generatedbylossofa
15 20 6 4 2
C H Ogroup.
5 9
AtotaloffivedegradationcomponentswereidentifiedintheAVthermalsolutionobtained
underthedegradationcondition.ThethermaldegradationproductsAH -AH showeda
1 4
maximumUVabsorptionatapproximately240nm,whichmightbetheresultofaring-open-
ingreaction[10].TheMSspectrumofAH showedapseudo-molecularionatm/z535
1
(C H O Na),andthedaughterionsofthepseudo-molecularionwerelocatedatm/z251
25 36 11
and191.TheAH ,AH andAH degradationproductswereallformedbyopeningoftheoxi-
1 2 4
raneringandpossessedsimilarMS/MSspectrums,includingdaughterionsatm/z305,233
and191.AH m/z423(C H O H)andAH m/z423(C H O H)wereobtainedbylossofa
3 22 30 8 4 22 30 8
C H O groupfromAH .AH (hydroxymethylformationofV)andAH m/z393
3 6 3 1 4 5
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
(C H O H)displayedionsatm/z293[M-H O-C H O].ThemaximumUVabsorption
20 24 8 2 5 9
wavelengthofAH was263nm,whichsuggestedthatformationofanaldehydegroupiniri-
5
doidvalepotriatecouldreinforcetheconjugatedsystem.AH wasobtainedbythelossoftwo
5
oxoethylgroupsfromatO-12andO-2’positionsofAV.
Iridoidvalepotriateswerestableunderlight(3h)andthermaldegradation(solidat60˚C
for24h)conditions.ThedegradationproductsintheH O solution(24h)hadnotypicalLC
2 2
profiles.Thedegradationproductsobtainedbyacidichydrolysisweresimilartothoseofthe
basereaction.VandBAVdisplayedcomparabledegradationpathwaystoAV(Fig2Aand2C),
althoughthestabilityofBAVwasnotablylowerthanthoseofVandAV.
Analysisofantioxidantactivities
Toevaluatetheantioxidantactivities,weassessedtheirperformancesinABTSand
TLC-DPPH•bioautography/MSassays.Alkalinehydrolysisisstronglyexothermic,andthe
solutionobtainedfromthisreactionturnsblackwithin10min.Thedegradationproducts
obtainedthroughthermaldegradation(methanolsolutionat60˚Cfor1,2,4,or6h;methanol
solutionat25˚Cfor7days)weretestedthroughTLC-DPPH•bioautographyassay.
TLC-DPPH•bioautography/MSanalysis
TheoriginalcompoundsshowedgoodseparationfromtheirdegradationproductsbyTLC(S3
Fig).Inthechromatograms,theyellowspotsforV,AVandBAVappearedimmediatelyafter
DPPH•stainingonthesilicagel,whereaslightyellowspotsforthedegradationproducts
appearedslowly.
Thestructuresoftheoriginalcompoundsandtheirdegradationproductsweresubse-
quentlyconfirmedby“dot-blot”MSanalysis.Thestructuresofthedegradationcompounds
estimatedfromtheTLC-DPPH•bioautography/MSanalysiscomparedwellwiththose
obtainedwiththeLC-MSanalysis.TheMSdataandidentifiedcompoundsareshownin
Table1.
ABTSandDPPH•assays
Thesolutionconditionsproducedthesamedegradationproductsat60˚C(for1,2,4,and6h)
and25˚C(7days),andthelatterconditionwasselectedforfurtherstudies.Antioxidantactivi-
tieswereevaluatedthroughABTSandDPPH•assaysusingBHTasareferenceorpositivecon-
trol.Theconcentrationsthatinhibited50%oftheactivityineachtest(IC values)areshown
50
inTable2.ThemaximumactivitywasrecordedwithV(IC rangingfrom1.6±0.3μMfrom
50
theABTSassayto3.6±0.2μMfromtheDPPH•assay),andtheminimumactivitywas
obtainedwith1-βacevaltrate,aBAVdegradationproduct(IC rangingfrom63.4±1.2μM
50
fromtheABTSassayto89.4±2.3μMfromtheDPPHassay).Theactivitiescouldbeordered
asfollows:V>AV>BAV>valtratedegradationproducts(VD)>AVD>BAVD.
Cytotoxictests
Effectoncancercellproliferation. Theanti-proliferativeeffectsofV,AV,BAVandtheir
degradationproductswereinvestigatedonthreehumancancercellsthroughanMTTassay
(Table3).ThedatashowedthatBAVDexertedrelativelyweakanti-proliferativeeffectsonthe
threecancercelllines.Vdisplayedbetteranti-proliferativeactivities,withIC valuesranging
50
from25.4±0.6μM(HepG2cells)to37.1±1.1μM(HeLacells).Allofthesamplesdemon-
stratedrelativelyhighanti-proliferativeactivitiesinHepG2cells.
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Table1. MSandMS/MSspectroscopicdatafortheiridoidvalepotriatesandtheirdegradationproducts.
Compound HPLC-MS TLC-MS UV MSSpectrum Formula
λmnmax
Knowncompounds V + + 256 445[M+Na]+ C H O
22 30 8
AV + + 256 503[M+Na]+ C H O
24 32 10
BAV + + 256 503[M+Na]+ C H O
24 32 10
Alkalinehydrolysis B + - 256 403[M+Na]+ C H O
1 20 28 7
B + - 256 361[M+Na]+ C H O
2 17 22 7
B + - 256 319[M+Na]+ C H O
3 15 20 6
B + - 256 277[M+Na]+ C H O
4 15 14 6
AB + - 256 461[M+Na]+ C H O
1 22 30 9
AB + - 256 361[M+Na]+ C H O
2 17 22 7
AB + - 256 319[M+Na]+ C H O
3 15 20 6
AB + - 256 277[M+Na]+ C H O
4 12 14 6
1-βB + - 256 461[M+Na]+ C H O
1 22 30 9
1-βB + - 256 419[M+Na]+ C H O
2 20 28 8
1-βB + - 256 283[M+Na]+ C H O
3 14 18 4
Thermaldegradations H + - 254 535[M+Na]+ C H O
1 25 36 11
H + + 240 423[M+H]+ C H O
2 22 30 8
H + + 243 423[M+H]+ C H O
3 22 30 8
H + + 236 407[M+H]+ C H O
4 22 30 7
AH + + 254 535[M+Na]+ C H O
1 25 36 11
AH + + 240 423[M+H]+ C H O
2 22 30 8
AH + + 243 423[M+H]+ C H O
3 22 30 8
AH + + 236 407[M+H]+ C H O
4 22 30 7
AH + + 264 391[M+H]+ C H O
5 21 28 7
1-βH + - 254 535[M+Na]+ C H O
1 25 36 11
1-βH + + 245 313[M+H]+ C H O
2 15 20 7
1-βH + + 263 337[M+H]+ C H O
3 17 18 7
1-βH + + 241 232[M+H]+ C H O
4 14 14 3
+identified-undetected
https://doi.org/10.1371/journal.pone.0189198.t001
HepG2cellswereselectedforfurthermechanisticstudiesbecausetheyweremoresensitive
tothetestedcompounds.TheMTTresultssuggestedthattheiridoidvalepotriatesweremore
activeagainstcancercellsthannon-malignantcells(HUVECs;IC =25.4vs.IC =97.5μM
50 50
forV).Thecellswerethentreatedwiththedrugsataconcentrationofapproximately40μM
forfurthertests.
Table2. AntioxidanteffectoftheiridoidvalepotriatesassessedthroughABTSandDPPT•assays.
Compounds IC (μM,n=3)
50
ABTS DPPH
V 1.6±0.3 3.6±0.2
AV 2.5±0.2 5.4±0.1
BAV 14.9±0.4 38.4±0.8
VD 20.5±2.1 35.5±2.7
AVD 29.5±1.5 56.2±1.2
BAVD 63.4±1.2 89.4±2.3
BHT 28.6±0.8 110.4±1.1
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
Table3. CytotoxicityofiridoidvalepotriatesagainstHepG2,HeLaandMDA-MB-231cellsandHUVECs.
Compound CancerCellLines(IC ,μM,n=3)
50
HepG2 HeLa MDA-MB-231 HUVEC
V 25.4±0.6 37.1±1.1 30.5±0.5 97.5
AV 30.4±0.7 42.3±1.3 37.5±0.5 92.2
BAV 48.5±2.1 50.3±0.8 60.3±1.8 >100
VD 50.5±1.9 60.1±2.1 75.1±1.9 89.1±1.9
AVD 62.4±0.9 >100 80.2±1.4 >100
BAVD >100 >100 >100 >100
CP 15.2±1.2 12.7±0.6 13.1±1.1 95.6±1.3
https://doi.org/10.1371/journal.pone.0189198.t003
ROSmeasurements
TheROSconcentrationsinHepG2cellsweremeasuredtodeterminewhetheriridoidvale-
potriatescanaltertheintracellularROSlevels.Theindependentexperimentswereperformed
intriplicate(n=3).TheROSlevelsweredeterminedusingthecell-permeableoxidation-sensi-
tivedyeDCFH-DA,andthefluorescenceinthecellswasquantifiedbyflowcytometry.As
showninFig3A–3G,theiridoidvalepotriatesinducedasignificantdecreaseinROSconcen-
trationscomparedwiththeuntreatedcontrolgroupandthedegradationproducts.Themaxi-
mumactivitywasrecordedforV(4.0±0.3%),andtheminimumactivitywasobtainedwith
AVD(19.2±0.9%)(Fig3H).
Treatmentwithiridoidvalepotriatesandtheirdegradationproducts
inducedapoptosisinHepG2cells
ApoptosistestswereperformedinHepG2cellstoconfirmtheanti-apoptoticeffectsofthese
compounds.ApoptoticcellsweredefinedasAnnexinV-FITC-positivecellsandspecifically
referstothesumofearly(quadrant4,Q4)andlate(Q2)apoptoticcells[36,37].
AsclearlyshowninFig4H,theearlyandlateapoptoticratesweresignificantlyincreased
((cid:3)p<0.05)inHepG2cellscomparedwiththecontrolgroup.Themaximumactivitywas
observedwithtreatmentwithV(rangingfrom4.8±0.9%to23.1±0.7%forearlyapoptosisand
Fig3.EffectofV,AV,BAVandtheirdegradationproductsonROSinHepG2cells.ROSinthecellswas
labelledwithDCFH-DAandmeasuredbyflowcytometry.(A—G)Resultsforthe(A)control,(B)V(-20˚C),(C)V
(25˚Cfor7days),(D)AV(-20˚C),(E)AV(25˚Cfor7days),(F)BAV(-20˚C)and(G)BAV(25˚Cfor7days)groups.
(H)Thedataarepresentedasthemeans±SDsfromthreeseparateexperiments(*p<0.05).
https://doi.org/10.1371/journal.pone.0189198.g003
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Studiesofthestructure-antioxidantrelationshipsandmechanismofiridoidvalepotriatesanddegradations
Fig4.ApoptosiswasmeasuredbyflowcytometrythroughAnnexinV-FITCandPIstaining.Representative
plotsfromonesetofAnnexinV-FITCflowcytometryandPIstainingexperimentsperformedintriplicateareshown.
Earlyapoptoticcells(Annexin-V+andPI-)areshowninthelowerrightquadrant,andlateapoptoticcells(Annexin-V
+andPI+)areshownintheupperrightquadrant.ThepercentagesofapoptoticcellsareindicatedasAnnexin-V
+cellsandareshownasthemeans±theSDfromthreeindependentexperiments.(A—G)Resultsfromthe(A)
control,(B)V(-20˚C),(C)V(25˚Cfor7days),(D)AV(-20˚C),(E)AV(25˚Cfor7days),(F)BAV(-20˚C)and(G)
BAV(25˚Cfor7days)groups.(H)Thedataarepresentedasthemeans±theSDfromthreeseparateexperiments
(*p<0.05).
https://doi.org/10.1371/journal.pone.0189198.g004
from5.1±1.0%to11.7±0.6%forlateapoptosis)(Fig4B).Theothertestedcompoundsalso
yieldedincreasedapoptosisratescomparedwiththecontrolgroup(p<0.05)(Fig4A–4G).
Discussion
Inthisstudy,forceddegradationswereperformedtoassessthebehavioursofiridoidvalepotri-
atesandtheirdegradationproductsundervariousstressconditions.V,AVandBAVwere
labiletobasehydrolysislargelyduetotheiresterfunctionality[33].AccordingtotheFDAand
ICHguidelinesandpublishedpapers,thedesireddegradationlevelsweresettobetween15
and20%degradationinthiswork[24,25].Thus,LC-MSandTLC-DPPH•bioautography/MS
analyseswereperformedtoelucidatethestructuresofthedegradationproducts.
ToreducetheinterruptionofpHduringthecellulartests,theacidicandalkalinehydrolysis
sampleswereneutralizedpriortotheinitialdilutionofthesample.However,theLCprofiles
werealteredbyneutralization,whichsuggestedthatsomeoftheby-productswereproduced
byneutralization.
Inpharmacokineticsanddecompositionstudies,hydrolysisoftheesterlinkagesoftheiri-
doidvalepotriatesreadilyproducedbaldrinals[33].Inthestresstests,baldrinalwasdetectedas
adegradationproductandshowedreducedantioxidantactivities.Themainreactingcom-
poundsmightplaycriticalrolesasdonatingelectroncentres.AstudybyMarcetal.demon-
stratedthattheoxiranenucleuswasimportantforexertinganti-proliferativeactivitiesin
neuroblastomacells,andourworkagreeswiththeliterature[38–40].
Basedontheforceddegradationdata,thedonationoftwohydroxylgroupsfromtheoxi-
ranenucleuswasdeducedasapossibleantioxidantmechanism.ThereactionsshowninFig5
areexampleDPPH•reactions.Theoxiraneringcouldgeneratehydroxylgroupsthroughthe
intermoleculartransferofanH•,whichcouldinteractwiththeoxidationreagent[41].The
acetoxygroupfromR (Fig5)possessesaconjugatedgroupintheorthoposition,whichmight
3
enterintoresonancewiththeoxiranering.TheacetoxygroupfromR wouldthereforebe
5
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