Compounds Derived from the Bhutanese Daisy, Ajania nubigena, Demonstrate Dual Anthelmintic

18 Pages·2016·6.18 MB·English

RESEARCHARTICLE
Compounds Derived from the Bhutanese
Daisy, Ajania nubigena, Demonstrate Dual
Anthelmintic Activity against Schistosoma
mansoni and Trichuris muris
PhurpaWangchuk*,MarkS.Pearson,PaulR.Giacomin,LukeBecker,JavierSotillo,
DarrenPickering,MichaelJ.Smout☯,AlexLoukas☯
a11111 CentreforBiodiscoveryandMolecularDevelopmentofTherapeutics,QueenslandTropicalHealthAlliance,
AustralianInstituteofTropicalHealthandMedicine,JamesCookUniversity,CairnsCampus,Cairns,
Australia
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS Abstract
Citation:WangchukP,PearsonMS,GiacominPR,
BeckerL,SotilloJ,PickeringD,etal.(2016)
CompoundsDerivedfromtheBhutaneseDaisy, Background
Ajanianubigena,DemonstrateDualAnthelmintic
Whipwormsandbloodflukescombinedinfectalmostonebillionpeopleindevelopingcoun-
ActivityagainstSchistosomamansoniandTrichuris
muris.PLoSNeglTropDis10(8):e0004908. tries.Onlyahandfulofanthelminticdrugsarecurrentlyavailabletotreattheseinfections
doi:10.1371/journal.pntd.0004908 effectively;thereisthereforeanurgentneedfornewgenerationsofanthelminticcom-
Editor:PatrickJ.Lammie,CentersforDisease pounds.Medicinalplantshavepresentedasaviablesourceofnewparasiticides.Ajania
ControlandPrevention,UNITEDSTATES nubigena,theBhutanesedaisy,hasbeenusedinBhutanesetraditionalmedicinefortreat-
Received:February2,2016 ingvariousdiseasesandourpreviousstudiesrevealedthatsmallmoleculesfromthisplant
haveantimalarialproperties.Encouragedbythesefindings,wescreenedfourmajorcom-
Accepted:July15,2016
poundsisolatedfromA.nubigenafortheiranthelminticproperties.
Published:August4,2016
Copyright:©2016Wangchuketal.Thisisanopen Methodology/PrincipalFindings
accessarticledistributedunderthetermsofthe
HerewestudiedfourmajorcompoundsderivedfromA.nubigenafortheiranthelminticprop-
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany ertiesagainstthenematodewhipwormTrichurismurisandtheplatyhelminthbloodfluke
medium,providedtheoriginalauthorandsourceare SchistosomamansoniusingthexWORMassaytechnique.Offourcompoundstested,two
credited.
compounds—luteolin(3)and(3R,6R)-linalooloxideacetate(1)—showeddualanthelmintic
DataAvailabilityStatement:Allrelevantdataare activityagainstS.mansoni(IC range=5.8–36.9μg/mL)andT.muris(IC range=9.7–
50 50
withinthepaperanditsSupportingInformationfiles
20.4μg/mL).Usingscanningelectronmicroscopy,wedeterminedluteolinasthemosteffi-
(S1FigandS2Fig).
caciouscompoundagainstbothparasitesandadditionallywasfoundeffectiveagainstthe
Funding:Thisresearchwasfundedbyaprogram
schistosomula,theinfectivestageofS.mansoni(IC =13.3μg/mL).Luteolininducedtegu-
grantfromtheNationalHealthandMedicalResearch 50
Council(NHMRC)awardedtoAL,grantnumber mentaldamagetoS.mansoniandaffectedthecuticle,bacillarybandsandbacillaryglands
APP1037304.URL:https://www.nhmrc.gov.au/grants- ofT.muris.Ourinvivoassessmentofluteolin(3)againstT.murisinfectionatasingleoral
funding.PWwassupportedbyaNHMRCPeter
dosingof100mg/kg,despitebeingsignificantly(27.6%)betterthantheuntreatedcontrol
DohertyEarlyCareerResearcherfellowshipnumber
group,wasmarkedlyweakerthanmebendazole(93.1%)inreducingthewormburdenin
APP1091011.URL:https://www.nhmrc.gov.au/grants-
funding.Thefundershadnoroleinstudydesign, mice.
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AnthelminticActivityofMedicinalPlant-DerivedCompounds
datacollectionandanalysis,decisiontopublish,or Conclusions/Significance
preparationofthemanuscript.
Amongthefourcompoundstested,luteolindemonstratedthebestbroad-spectrumactivity
CompetingInterests:Theauthorshavedeclared againsttwodifferenthelminths—T.murisandS.mansoni—andwaseffectiveagainstjuve-
thatnocompetinginterestsexist.
nileschistosomes,thestagethatisrefractorytothecurrentgoldstandarddrug,praziquan-
tel.Medicinalchemistryoptimisationincludingcytotoxicityanalysis,analoguedevelopment
andstructure-activityrelationshipstudiesarewarrantedandcouldleadtotheidentification
ofmorepotentchemicalentitiesforthecontrolofparasitichelminthsofhumansand
animals.
AuthorSummary
Schistosomiasisandtrichuriasisaffectsmillionsofpeopleworldwideandarecausedby
bloodflukesandwhipworms,respectively.Onlyahandfulofanthelminticdrugsexistto
treattheseinfectionsandthepipelineforthenextgenerationofanthelminticdrugsis
sparse,precipitatingtheneedfornewdrugdevelopment.Inthiscontext,medicinalplants
presentaviablesourceofnovelanthelminticcompounds.Thisinspiredustostudythe
selectednaturallyoccurringcompoundsderivedfromaBhutanesedaisymedicinalplant,
Ajanianubigenafortheiranthelminticactivities.Here,usingthexWORMmotilityassay,
wedemonstratethattwocompounds,luteolin(3)and(3R,6R)-linalooloxideacetate(1),
displaysignificantbroad-spectrumanthelminticactivityagainsttwoofthemostimportant
generaofhumanhelminthparasites,thenematodewhipwormandtheplatyhelminth
bloodfluke.LuteolinexhibitedthebestactivitieswithIC valuesof5.8μg/mLagainst
50
schistosomesand9.7μg/mLagainstwhipworms.Usingscanningelectronmicroscopywe
showedthatluteolindamagesthetegumentofbloodflukesandinducesabnormalitiesin
thebacillarybands/glandsandcuticlesofwhipworms.Intriguingly,ourpreviousstudy
showedthatluteolin(3)waseffectiveagainstmulti-drugresistantPlasmodiumfalciparum
malaria.Duetoitsbroad-spectrumanti-parasiticactivities,luteolin(3)isadesirabledrug
leadscaffold,whichcouldbeusedfordevelopingeffectivecompoundstocontrolandtreat
numeroustropicaldiseases.
Introduction
TheWorldHealthOrganization(WHO)recognises17different‘neglectedtropicaldiseases’
(NTDs)thataffectmorethan1.4billionpeoplein149countries[1].Helminthinfections
causedbyroundworms(nematodes)andflatworms(platyhelminths)comprisethelargest
groupofNTDs[2].Whipworms(Nematoda)causetrichuriasisandinfectabout800million
peopleworldwide,secondamongthenematodesonlytoAscarisinfection[3].Theschistosome
bloodflukes(Platyhelminthes)causeschistosomiasis,adiseasethatafflictsmorethan240mil-
lionindividualsandkillshundredsofthousandseachyear[4].
Avarietyofapproacheshavebeenemployedtocombattheseinfectionsincludingeduca-
tion,vectorcontrol,sanitationandhygiene,behaviouralchangeandmassdrugadministration
(MDA)programs.Variousinvitroandanimalmodelstudieshavehighlightedtherepurposing
ofexistingdrugsanddiscoveryanddevelopmenteffortsfornewdrugs[2,5]butallthingscon-
sidered,thepipelineforthenextgenerationofanthelminticdrugsissparse.Indeed,asystem-
aticassessmentofdatabasesofdrugregulatoryauthoritiesandtheWHO,aswellasclinical
trialregistries,revealedthatnonewantiparasiticdrugshavebeenapprovedduringthelast
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decade[6].Thereareonlyahandfulofanthelminticdrugsonthemarket,someofwhichhave
unwantedsideeffectsorachievepoorcureratesduetoprimarydrugresistancedevelopingin
theparasites[7–9].
Forexample,praziquantel,whichisthesolefrontlinedrugusedinthemasstreatmentof
schistosomiasis,isefficaciousbuthasmanydisadvantages:a)itisineffectiveagainstjuvenile
stagesoftheparasite,b)reducedefficacyhasbeenreportedinfieldstudies[10],c)thereisa
strongpossibilitythatpraziquantelresistancecouldappearifsufficientselectionpressureis
appliedandmassdrugadministrationiscontinued[11],andd)itsactive(S)-enantiomerand
inactive(R)-enantiomercomponentsremaininseparableintheproductionprocess,rendering
bulkytabletsthatdiscouragespatientsfromtakingtherightdosesorthecompletedosingregi-
men,whichcouldtriggerthedevelopmentofdrugresistance[12].Untilnewarsenalsofsafe
andeffectivedrugsand/orvaccinesaremadeavailable,helminthinfectionswillcontinueto
affecttheworld’smostimpoverishedpopulations,causingsignificantmorbidityandmortality
worldwide.
Whilenewdrugscanbedevelopedsynthetically,naturalproducts—especiallythemedicinal
plants—havebeenanimportantpoolofantiparasiticdrugs.Quinineandartemisinindiscov-
eredfrommedicinalplantscontinuetosavethelivesofmillionsofpeopleworldwide.Assuch,
thenotionoftherapeuticsderivedfrommedicinalplantshasre-surfaced[13].Crudeextracts
andcompoundsofplantoriginhavebeendemonstratedtopossessbroadbiologicalactivities
ininvitroandexvivoassaysandanimalmodelsofparasiticinfections[14–19].Edwardsetal.
[20]showedthat7-keto-sempervirolisolatedfromtheboxthornfromwhichgojiberriesare
harvested,Lyciumchinense,waseffectiveagainstSchistosomamansoniandFasciolahepatica.
Acompoundthatdisplayssuchbroadanti-parasiticactivityagainstvariouslifestagesofmulti-
pleparasitesishighlydesirable.ExtractsoftheBhutanesemedicinalplantfromtheflowering
daisyfamily,Ajanianubigena(Syn.TanacetumnubigenumDC.)havebeenpreviouslyshown
topossessbroadbiologicalactivitiesincludingantiparasiticeffectsagainstPlasmodiumfalcipa-
rumandantimicrobialproperties[21].Itislocallyknownasm.khan-d.karandhasbeenused
inBhutanesetraditionalmedicine(derivedfromTibetanscholarlymedicine)forthousandsof
yearsasincenseandfortreatinganarrayofconditionsandinfectionsincludingwounds,bleed-
ingandswelling[21].Althoughthisplantisnotspecificallyindicatedfortreatingintestinal
worms,thedecoctionofitscloselyrelatedspecies,TanacetumpartheniumL.(feverfew)and
TanacetumdolichophyllumKitamhasbeentraditionallyusedbytheLadakhisAmchis(medical
systemderivedfromTibetanmedicineandsimilartoBhutanesetraditionalmedicine)[22]and
CostaRicanshealers[23]againstintestinalworms.Theseplantshavereservesofhighlyaro-
maticessentialsoilsthathaveevolvedtoaidinplantprotectionandcompetitionagainstplant
parasitesandherbivorousinsects.Chemically,theseplantscontainsimilarchemotypesinclud-
ingsesquiterpenesandflavonoids[23].Encouragedbytheseleadinformation,wehaveinvesti-
gatedtheanthelminticpropertiesoffourcompoundsisolatedfromtheBhutaneseA.nubigena
againsttwoofthemostimportantgeneraofhumanhelminthparasites,thenematodewhip-
worm(Trichuris)andtheplatyhelminthbloodfluke(Schistosoma).Tomonitorwormviability
weusedxWORM,atechniquethatmonitorshelminthmotilityinrealtimeusingxCELLigence
[24–25].TheadvantageofusingxWORMoverothermethodsisthatitenableshigh-through-
putscreeningofalargenumberofcompoundsinafullyautomated,label-freemanner.
MaterialsandMethods
Plantmaterialsandpreparationofcompounds
TheaerialpartofwildAjanianubigenawascollectedfromalpinemountains(altituderangeof
3600–4800metersabovesealevel)ofLingzhi,BhutaninAugust2009.Thecollectedplant
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materialwasair-driedandaherbariumspecimenwithvouchernumber73wasdepositedat
theherbariumcollectionsectionofMenjongSorigPharmaceuticals,MinistryofHealth,Bhu-
tan.Theair-driedplantmaterial(2kg)waschoppedintosmallpiecesandwasrepeatedly
extractedwithmethanol(AR/HPLCgrade,3Lover48h).Theextractwasfilteredandthen
concentratedusingaBuchirotaryevaporatortogenerateacrudemethanol(MeOH)extract
(58.2g).Theisolationtechniquewasdescribedpreviously[21].MeOHextractwasdissolvedin
MeOH:H O(200mL,1:9)andthenfractionatedwithhexanefollowedbyethylacetateto
2
obtainthehexaneextract(28.0g)andtheethylacetateextract(12.5g),respectively.Subse-
quently,essentialoil(EO)extractionwasperformedusinghydro-distillation(60°C).Onekgof
driedplantmaterialyielded7mLofpalegreenEO.ThecrudeMeOHextractandEOweresub-
jectedtoextensivenaturalproductsisolationprocesses.Flashcolumnchromatographypacked
withMerckKieselgel60PF254andpre-coatedsilicaplates(0.2mmsilicathickness,Merck)
wereusedforrepeatedseparationandpurificationofcompounds.UVlight(shortwavelength
of254nm,longwavelengthof366nm)andcericammoniummolybdate(CAM)wereusedfor
visualizationanddetectionofcompoundsonThinLayerChromatography(TLC)plates.Eight
compoundswereisolatedandcharacterisedintotalfromtheMeOHandEOextractsusing
Infrared(IR)Spectroscopy,MassSpectrometry(ESI-MS,HR-EI-MS),GasChromatography
MassSpectrometry(GCMS),andNuclearMagneticResonance(NMR-1H,13C,gCOSY,
gNOESY,TOCSY,gHSQCandgHMBC)[21].Inthisstudy,wehaveselectedfourmajorcom-
poundswhosestructuresareproducedinFig1:(3R,6R)-linalooloxideacetate(1),(E)-spir-
oether(2),luteolin(3)andluteolin-7-O-β-D-glucopyranoside(4).
Thestocksolutionsofthefourtestcompoundswerepreparedattheconcentrationof100
mg/mLinDMSOandthensubsequentlydilutedthemwithrespectivetissueculturemediato
make10xsolutions.20μlof10×drugconcentrationwasaddedto180μlofmediacontaining
helminthsintheE-platewells.ControlwormswereculturedinthepresenceofDMSOequiva-
lenttothatusedforthehighestdrugconcentration;thisgroupwasusedtodetermine100%
motility.Forschistosomuladrugassays,stocksolutionsweredilutedinculturemediawith
two-folddilutionswithin-wellconcentrationsof(2–1000μg/mL).
Fig1.Structureofcompounds[21].(3R,6R)-linalooloxideacetate(1),(E)-spiroether(2),luteolin(3),luteolin-7-O-β-D-glucopyranoside(4)werederived
fromA.nubigenaandtestedfortheiranthelminticactivitiesusingxWORM.Plantphotocourtesy–PW.
doi:10.1371/journal.pntd.0004908.g001
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AnthelminticActivityofMedicinalPlant-DerivedCompounds
PreparationofSchistosomamansoni
S.mansonicercariaewereshedfrominfectedBiomphalariaglabratasnails(Biomedical
ResearchInstitute,MD,USA)byexposuretolightat26°Cfor2hoursandusedtoinfect12–14
weekoldmaleBALB/cmice(120cercariae/mouse)byabdominalpenetration[26].Adult
flukeswereperfusedfromthemesenteries7weekspost-infectionandthentransferredto
Baschmediumforculturingaspreviouslyreported[27].TomonitortheeffectsofdrugsonS.
mansonischistosomula,sheddingofcercariaefromsnailsandsubsequentinvitrotransforma-
tiontoschistosomulawasperformedasdescribedbyPeaketal[28].
PreparationofTrichurismuris
Geneticallysusceptiblemice(STAT6-1-)wereorallyinfectedwithT.muriseggs(200μLvolume
containing~200eggs)andsacrificedafter4weeks.Adultwormswereharvestedfromthecae-
cum,washedwithPBS/2xantibiotic/antimycotic(AA)andresuspendedin100μlofRPMI
containing10%foetalcalfserumandAA(culturemedium)thentransferredtoE-platesfor
motilityassessmentusingthexWORMassay.
xWORMassay
ThetrematocidaleffectsoftestcompoundsagainstS.mansoniwereevaluatedusinganxCEL-
LigenceSPsystem(ACEABiosciences)asdescribedbyus[25].Adultflukes(1flukeperwell)
wereplacedintriplicateforeachcompoundinto96wellE-plates(ACEABiosciences)contain-
ing180μlofculturemediumandculturedovernightat37°Cwith5%CO toobtainabaseline
2
motilityreading.TestcompoundswereaddedtoE-platesandmotilitywasmonitoredfor12–
40hr.Theinter-wellspacesofE-plateswerefilledwith100μLculturemedia.Allexperiments
werecarriedoutaspermanufacturer’sinstructionswith15secreadintervalsusingthereal
timecellassay(RTCA)software(ACEABiosciences)asdescribedpreviously[24–25].
Similarly,thenematocidaleffectsoftestcompoundsagainstT.muriswereassessedusing
thesamexCELLigenceSPsystemasdescribedabove.Wedeterminedtheoptimalculturedura-
tionandwormconcentrationtomaximizethesignaltonoiseratiousingthexWORMtech-
niqueforthefirsttimewithT.muris.DifferentnumbersofadultT.muris(2,4and8)were
addedtoindividualE-platewellsandmotilitywasmonitoredovernight.Fourwormsofmixed
genderperwellinafinalvolumeof200μlofculturemediumwasdeterminedtobeoptimalfor
thisstudy.TheE-platescontainingwormsweretreatedwithpreparedconcentrationsofthe
testcompoundsandweremonitoredusingthexCELLigenceSPsystemfor12–40hr.Inter-well
spaceswerefilledwith100μLofculturemediumorPBStopreventevaporation.Eachsetof
conditionswasmonitoredintriplicate.
Determiningtheeffectsofcompoundsagainstschistosomula
The96wellplatescontainingculturemediawereloadedwithschistosomula(100μLvolume
containing~100schistosomula)intriplicateandtreatedwiththetestcompoundsatvarious
in-wellconcentrationsof2–1000μg/mL.Plateswereculturedat37°Cwith5%CO for12–40
2
hrandwerefinallystainedwithtrypanbluesolutiontoassessfinalviabilityaftertreatment.
Thestainedschistosomulawereobservedbylightmicroscopy,andliveanddeadflukesineach
wellwerecountedmanuallyand50%inhibitoryconcentration(IC )valueswereobtained.
50
DeterminationofIC valuesfortestcompounds
50
TheIC valuesoftestcompoundsweredeterminedbasedonthemotilityindexforadult
50
wormsasdescribedbyus[25].Briefly,motilityindexwascalculatedasthestandarddeviation
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(SD)over800datapoints(i.e.4readingsperminfor200min)ofthecellindex(CI)difference
fromtherollingaverageover20datapoints(10proceedingandprecedingCIvalues—5min
total).Onehundredpercentmotilitywasdeterminedfromtheaveragemotilityindexofthe
untreatedwells,while0%motilitywasdeterminedfromamediaonlywell(nowormspresent).
Themotilityindexaveragedover100datapoints(25min)wasconvertedtopercentagemotility
andthisfigurewasusedinGraphPadPrism6.0tocalculatedoseresponsecurves.Weusedalog
(testcompoundconcentration)vsnormalisedresponse(100%–0%)formula,withvariableslope
whendataweresufficientorset-1hillslopewhendatawaslimited,andautomaticremovalof
outliers(withdefaultROUTcoefficientused:Q=1.0%).IC valuesforeachdoseconcentration
50
werecalculatedat1hr,6hr,and12hrpost-treatmentofthewormswiththetestcompounds.
CompoundswithIC valuesofhigherthan100μg/mLwereconsideredineffectiveinthisstudy.
50
Statisticalanalysis
StatisticalanalyseswereundertakenusingGraphPadPrism6.0.Whendataweresufficienttousethe
variableslopeanalysis,theHillSlopeandtheLogIC valueweretogethercomparedforsignificant
50
differencesusinganextrasum-ofsquaresF-test.Fortheinvivomouseexperiments,1-wayANOVA
withHolm-Sidak’smultiplecomparisonstestwasusedfordeterminingsignificancep-values.
Scanningelectronmicroscopy
Wormstreatedwiththetestcompoundswerepreparedforscanningelectronmicrocopy
(SEM)asfollows:a)fixedin3%gluteraldehydeinSorensen’sbufferovernight,b)dehydrated
for15mininagradedethanolseries(50%,60%,70%,80%,90%,100%),mixtureofethanol
andhexamethyldisilizane(HMDS)(1:1ratio)andthenfinallywithpureHMDS(100%),c)the
dehydratedwormswerecoveredandleftovernightinafumehoodtoallowtheHMDStoevap-
orate.Completelydriedwormswereplacedonanaluminumstub(atleastthreewormsfrom
eachtreatmentregimen),sputteredwithgoldandvisualizedusingaJEOLJSMscanningelec-
tronmicroscopeoperatingat10kV.Eachwormonastubwasscannedfromheadtotailto
determineifthecompoundshadaltereditsgrossmorphology.Digitalimageacquisitionwas
performedontheaffectedregionofthewormsusingSemaphoresoftware.
Invivoanimalmodelstudiesofluteolin(3)
Fourtofiveweek-oldSTAT6-1-miceweregrouped(eachgroupwith9mice)as:solventcon-
trol,positivecontrolandluteolin(3).Eachmousewasorallyinfectedwith200μlofPBScon-
tainingapproximately200liveembyronatedeggsofT.muris.Thesemicewerehousedfor4
weekswithconstantaccesstowaterandpelletedfood.After4weeks,luteolin(3)andtheposi-
tivecontroldrug(mebendazole)preparedin1%DMSO/PBSwereorallyadministeredatasin-
gledoseof100mg/kg(9miceintotalforeachgroup)aspertheprotocol[29–30].Fivedays
afteronedoseoforaltreatmentthemiceweresacrificed,wormswereharvestedfromthecae-
cum,andcountedmanuallyusinglightmicroscopy.Therecordednumbersofwormswere
averagedtofindthepercentagereductioninwormburdenforeachgroupofmice.
Ethicsandapprovals
ThepermittocollectmedicinalplantsfromtheparkmanagementareasaroundLingzhi,Bhu-
tanwasobtainedfromtheDepartmentofForest,MinistryofAgricultureandForestryinBhu-
tan.ThematerialtransferagreementandapprovalwassoughtfromtheNationalBiodiversity
CentreofBhutan.MeOHextractsoftheplantsweretransportedtoAustraliawithpriorapproval
fromtheBhutanAgricultureandFoodRegulatoryAuthority,UniversityofWollongongand
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AnthelminticActivityofMedicinalPlant-DerivedCompounds
sampleinspectionsbyAustralianQuarantine&InspectionServicein2010.TheJamesCookUni-
versity(JCU)animalethicscommitteeapprovedallexperimentalworkinvolvinganimals(Ethic
approvalnumberA2213).MiceinfectedwithS.mansoniandT.muriswereraisedincagesinthe
JCUanimalfacilityfor4–7weeksincompliancewiththeAustralianCodeofPracticeforthe
CareandUseofAnimalsforScientificPurposes,7thedition,2007andtheQueenslandAnimal
CareandProtectionAct2001.Micewerekeptundernormalconditionsatregulatedtemperature
(22°C)andlighting(12hrlight/darkcycle)withfreeaccesstopelletedfoodandwater.Allrea-
sonableeffortsweremadetominimisethesufferingofthemice.
Results
TheisolationandcharacterizationofcompoundsfromtheA.nubigenaplantwereperformed
asreportedpreviouslybyus[21].Outofeightcompoundsisolatedfromthisplant,several
werereportedtohaveantimalarial,antibacterial,antifungalandcytotoxicityactivities[21].
Encouragedbytheantimalarialactivities,fourmajorcompounds(Fig1):(3R,6R)-linalool
oxideacetate(1),(E)-spiroether(2),luteolin(3)andluteolin-7-O-β-D-glucopyranoside(4),
weretestedforbroad-spectrumanthelminticactivitiesagainsttwodistinctphylaofhelminths,
theplatyhelminthtrematodebloodflukeS.mansoniandthenematodewhipwormT.muris.
TrematocidalactivityofcompoundsagainstadultS.mansoni
Ofthefourcompoundstested,(3R,6R)-linalooloxideacetate(1),luteolin(3)andluteolin-7-O-
β-D-glucopyranoside(4)showedanti-schistosomedose-dependentanti-schistosomeeffects
(Fig2A).Atthehighestconcentrationtested(1mg/mL)allthreecompoundskilledflukes
within1–12hr.Lowerdrugconcentrations,however,tooklongertokillflukesreflectedby
highermotilityindexvalues(Fig2A).
WhentheIC valuesofeachcompoundwereaveragedorcombined(calculatedforthe
50
doseconcentrationsof0.1–1000μg/mL)foreachtimepoint(1hr,6hrand12hr),luteolin(3)
andluteolin-7-O-β-D-glucopyranoside(4)appearedtobefastactingonwormsastheirIC
50
valuesdidnotchangesignificantlybetweentheinitialandfinal12hrdosingtimepoints(Fig
2B).Ontheotherhand,(3R,6R)-linalooloxideacetate(1)wasslow-acting,showingtwo-fold
decreasesinIC ateach6hrtimepoint.Ofthecompoundsassessed,luteolin(3)exhibitedsig-
50
nificantlybettertrematocidalactivityatboth6hrand12hrtimepointswithIC valuesof
50
4.6μg/mLand5.8μg/mL,respectively.
EffectsofcompoundsagainstschistosomulaofS.mansoni
ThecompoundsthatwereactiveagainstadultS.mansoniwerealsotestedagainsttheintra-
mammalianlarvalstage,theschistosomulum.Monitoringofschistosomulasurvivalinthe
presenceofdifferentdrugconcentrationusingTrypanblueexclusionshowedthatluteolin(3)
startedtoshowlethaleffectsatthelowestdilutionof3.91μg/mLandachieved98–100%killing
atthedilutionof31.3μg/mL.(3R,6R)-linalooloxideacetate(1)howeverstartedtoshowlethal
effectsataconcentrationof125μg/mLandonlyachievedabout43%killingatthemaximum
dosetestedof250μg/mL.Schistosomulatreatedwith1%DMSOalone(solventcontrol)had
100%survivalasmeasuredbyTrypanblueexclusion.Adoseresponsecurveofschistosomula
survivalaftertreatmentwithluteolin(3)revealedanIC of13.3μg/ml(Fig3B).
50
Effectsofluteolin(3)onthemorphologyofadultS.mansoni
Luteolin(3)demonstratedthebestanthelminticactivitywhenmotilitywasassessedusing
xWORM.Theeffectofthiscompoundandpraziquantelonadultflukemorphologyat
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Fig2.Invitroanti-schistosomeactivitiesoffourcompoundsagainstadultS.mansoni.A)Motilityindexdose
responsecurveofwormsatthe12hrtimepointwhentreatedwith(3R,6R)-linalooloxideacetate(1),(E)-spiroether(2),
luteolin(3),luteolin-7-O-β-D-glucopyranoside(4)(abbreviatedasCom-1,Com-2,Com-3andCom-4)atdifferent
concentrations(0.1–1000μg/mL).Motilityindexwascalculatedasthestandarddeviation(SD)over800datapoints
(i.e.4readingsperminfor200min)ofthecellindex(CI)differencefromtherollingaverageover20datapoints(10
proceedingandprecedingCIvalues—5mintotal).B)50%inhibitoryconcentration(IC )curvesovertime.Errorbars
50
represent95%confidenceintervalsofnonlinearcurvefit.Thecurvesweremarginallyshiftedonthex-axistoaid
viewing.Thesefiguresrepresentthedatafromthreeindependentstudies.
doi:10.1371/journal.pntd.0004908.g002
4–20μg/mLconcentration,withparticularemphasisonthetegument,wasassessedbySEM.
Adultflukestreatedwithpraziquanteladoptedatightlycoiledappearanceduetocontraction.
Bothmaleandfemaleflukestreatedwithluteolin(3)werecontractedandcoiledcomparedto
controlflukesculturedin1%DMSOinmedia,butnotastightlycoiledaspraziquantel-treated
parasites(Fig4A–4F).
ObservationoffluketegumentsbySEMunderhighmagnificationrevealedthatthetegu-
mentofDMSO-treatedflukesdisplayednumeroustubercles,withwell-formedspinesinthe
males(Fig5A)andclearlydefinedsurfacegrooveswithsensorypapillaeinfemales(Fig5B),
andoralsuckerswithclearlydefinedpitscontainingsharpspines(Fig5C).Flukestreatedwith
praziquantel(Fig5D–5F)andluteolin(3)(Fig5G–5I)exhibitedseveremorphologicalalter-
ationsofthetegument.
Atthelowestconcentrationtestedof4μg/mLweobservedcleartegumentaldamage
inducedbyluteolin(3)(Fig5G–5I),similartothatinducedbypraziquantel(Fig5D–5F).
Whilemaleflukes(Fig5G)sufferedpartiallossofpitsandtheirencasedspines,femaleflukes
(Fig5E)showedsurfaceerosion,constrictionofgrooves,burstingofsmallsensorypapillaeand
formationofholesonthetegument.Athigherconcentrationsof20–1000μg/mL,luteolin(3)
andpraziquantelcompletelydestroyedthebodysurfacesandexhibitederosionoftubercles,
oralandventralsuckers(S1Fig).
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004908 August4,2016 8/18
AnthelminticActivityofMedicinalPlant-DerivedCompounds
Fig3.Effectofcompoundsonthesurvivalofschistosomula.A)Images(20x)ofwellscontaining
schistosomulathatweretreatedwithlinalooloxideacetate(1)andluteolin(3)(abbreviatedasCom-1andCom-3)
atthemaximumconcentrationtestedof250μg/mLandstainedwithtrypanblue.Deepbluestainingsignifiesa
deadparasite.B)Theeffectof(3R,6R)-linalooloxideacetate(1)andluteolin(3)(abbreviatedasCom-1andCom-
3)onschistosomulamortalityobservedattwo-folddilutions(250–1.9μg/mL).Thedatawasgeneratedfrom
triplicatesamplesobtainedfromtwoindependentstudies.
doi:10.1371/journal.pntd.0004908.g003
NematocidalactivitiesofcompoundsagainstadultT.muris
Priortotestingthenematocidaleffectsof(3R,6R)-linalooloxideacetate(1),(E)-spiroether(2),
luteolin(3)andluteolin-7-O-β-D-glucopyranoside(4),westandardizedtheculturingcondi-
tionsofadultT.murisforthexWORMassay.E-platewellscontainingfouradultworms(both
malesandfemales)yieldedoptimalmotilitysignals(S2Fig),andwastheconditionselectedfor
subsequentanthelminticscreeningofthetestcompounds.(3R,6R)-linalooloxideacetate(1)
andluteolin(3)showedthebestanti-TrichurisactivitywithIC valuesof20.4μg/mLand
50
9.7μg/mL,respectively,calculatedoncellmotilityindexatthe12hrtimepoint(Fig6A).
TheIC valuesofthefourcompoundstestedherewereobtainedusingxWORMandcalcu-
50
latedat1hr,6hrand12hrtimepoints(Fig6B).Luteolin(3)wasthemostefficaciousdrugin
termsofreducedmotilityofT.muris,exhibitingthelowestorequallylowIC valuesatall
50
timepointsandafinal12hrvalueof9.7μg/mL.
Effectsofluteolin(3)onthemorphologyofadultT.muris
Basedontheefficacyofluteolin(3)atreducingTrichurismotility,weexaminedthemorpho-
logicalchangesinthecuticleinducedbythiscompoundusingtheSEMprotocolsdescribedby
Stepeketal[30]andTrittenetal[31]specifictoT.muris.LiveadultT.muris(mixedsexes)
weretreatedfor48hrwithluteolin(3)ormebendazoleatdoseconcentrationsof200–1000μg/
mL.Morphologicalchangeswereobservedtowardstheanteriorendofthewormsintheform
ofpartiallydamagedbacillaryband/glandsandadjacentcuticle.Thebacillarybandisa
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004908 August4,2016 9/18
AnthelminticActivityofMedicinalPlant-DerivedCompounds
Fig4.Effectsofluteolin(3)andpraziquantelonthemorphologyofadultS.mansoniat4–20μg/mL
concentrationvisualizedusingscanningelectronmicroscopy.A,CandEshowmaleflukes.B,DandF
depictfemaleflukes.(A,B)FlukesinDMSO/mediacontrol.(C,D)Flukestreatedwithpraziquantel(tightly
coiled).(E,F)Flukestreatedwithluteolin(3)(moderatelycoiled).Threeseparatewells(with3–5adultflukes
perwell)weretreatedwithdifferentdosesoftestsamplesasaboveandeachofthemwasexaminedunder
SEM.
doi:10.1371/journal.pntd.0004908.g004
specializedrowoflongitudinalcellsofsomenematodesconsistingofglandularandnon-glan-
dularcells.Thesebandshosttheglands.WormstreatedwithDMSO/media(solventcontrol)
alonehadamoderatelycoiledappearancewithasmoothcuticledisplayingknittedparallelseg-
mentaljoinsandminimalshrinkageofbands(Fig7A–7C)incomparisontothegroupstreated
withmebendazole(Fig7D–7F)andluteolin(3)(Fig7G–7I).Athighermagnificationwe
observedthattheluteolin-treatedwormsexhibitedblister-likeformationsonthesurfaceofthe
cuticle,swellingandlooseningofcuticleseams/groovesnearthebacillaryglands(Fig7I).
Thesemorphologicalchangesweresimilartothatofthemebendazole-treatedworms(Fig7F).
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004908 August4,2016 10/18