Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes

20 Pages·2015·1.46 MB·English

RESEARCHARTICLE
Cissampelos pareira
Linn: Natural Source of
Potent Antiviral Activity against All Four
Dengue Virus Serotypes
RuchiSood1¤a,RajendraRaut2,PoornimaTyagi2,PawanKumarPareek1¤a,Tarani
KantaBarman1¤a,SmitaSinghal1¤a,RajKumarShirumalla3¤a,VijayKanoje3¤a,
RameshSubbarayan3¤a,RavisankarRajerethinam3¤a,NavinSharma1¤b,AnilKanaujia1¤c,
GyaneshShukla1¤a,Y.K.Gupta4,ChandraK.Katiyar1¤d,PradipK.Bhatnagar1¤e,Dilip
J.Upadhyay1¤a,SathyamangalamSwaminathan2¤f*,NavinKhanna2*
1 DepartmentofMicrobiology,NewDrugDiscoveryResearch,RanbaxyResearchLaboratories,Gurgaon,
Haryana,India,2 RecombinantGeneProductsGroup,InternationalCentreforGeneticEngineeringand
Biotechnology,NewDelhi,India,3 DepartmentofPharmacology,NewDrugDiscoveryResearch,Ranbaxy
ResearchLaboratories,Gurgaon,Haryana,India,4 DepartmentofPharmacology,AllIndiaInstituteof
MedicalSciences,AnsariNagar,NewDelhi,India
¤a Currentaddress:DaiichiSankyoIndiaPharmaPrivateLimited,VillageSarhaul,Gurgaon,Haryana,India
¤b Currentaddress:VLCCPersonalCareLimited,Gurgaon,India
OPENACCESS ¤c Currentaddress:R&DAyurvetLimited,VillageKatha,Baddi,HimanchalPradesh,India
¤d Currentaddress:EmamiLimited,Kolkata,India
Citation:SoodR,RautR,TyagiP,PareekPK, ¤e Currentaddress:Exton,Pennsylvania,UnitedStatesofAmerica
BarmanTK,SinghalS,etal.(2015)Cissampelos ¤f Currentaddress:VirologyLaboratory,DepartmentofBiologicalSciences,BirlaInstituteofScienceand
pareiraLinn:NaturalSourceofPotentAntiviral Technology-Pilani,HyderabadCampus,JawaharNagar,Shamirpet,Hyderabad,India
*[email protected](SS);[email protected](NK)
ActivityagainstAllFourDengueVirusSerotypes.
PLoSNeglTropDis9(12):e0004255.doi:10.1371/
journal.pntd.0004255
Abstract
Editor:DavidW.C.Beasley,UniversityofTexas
MedicalBranch,UNITEDSTATES
Received:June1,2015
Background
Accepted:November1,2015
Dengue,amosquito-borneviraldisease,posesasignificantglobalpublichealthrisk.In
Published:December28,2015
tropicalcountriessuchasIndiawhereperiodicdengueoutbreakscanbecorrelatedtothe
Copyright:©2015Soodetal.Thisisanopen
highprevalenceofthemosquitovector,circulationofallfourdengueviruses(DENVs)and
accessarticledistributedunderthetermsofthe
thehighpopulationdensity,adrugfordengueisbeingincreasinglyrecognizedasanunmet
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany publichealthneed.
medium,providedtheoriginalauthorandsourceare
credited.
Methodology/Principalfindings
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. UsingtheknowledgeoftraditionalIndianmedicine,Ayurveda,wedevelopedasystematic
bioassay-guidedscreeningapproachtoexploretheindigenousherbalbio-resourcetoiden-
Funding:ThisworkwasfundedbytheDepartment
ofBiotechnology,GovernmentofIndia,throughGrant tifyplantswithpan-DENVinhibitoryactivity.Ourresultsshowthatthealcoholicextractof
No.BT/PR7356/PBD/17/465/2006toSSwandNK. CissampelosparieraLinn(Cipaextract)wasapotentinhibitorofallfourDENVsincell-
Thefunderhadnoroleinstudydesign,data
basedassays,assessedintermsofviralNS1antigensecretionusingELISA,aswellas
collectionandanalysis,decisiontopublish,or
viralreplication,basedonplaqueassays.VirusyieldreductionassaysshowedthatCipa
preparationofthemanuscript.
extractcoulddecreaseviraltitersbyanorderofmagnitude.Theextractconferredstatisti-
CompetingInterests:RSo,RKS,VK,RSu,RRaj,
callysignificantprotectionagainstDENVinfectionusingtheAG129mousemodel.Aprelim-
NS,AK,GS,CKK,PKBandDJUwereemployedby
RanbaxyResearchLaboratoriesatthetimeofthis inaryevaluationoftheclinicalrelevanceofCipaextractshowedthatithadnoadverse
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004255 December28,2015 1/20
HerbalExtractwithPan-DengueInhibitoryActivity
work.Allotherauthorshavedeclaredthatno effectsonplateletcountsandRBCviability.InadditiontoinherentantipyreticactivityinWis-
competinginterestsexist.Thisdoesnotalterour tarrats,itpossessedtheabilitytodown-regulatetheproductionofTNF-α,acytokineimpli-
adherencetoallPLOSpoliciesonsharingdataand
catedinseveredenguedisease.Importantly,itshowednoevidenceoftoxicityinWistar
materials.
rats,whenadministeredatdosesashighas2g/Kgbodyweightforupto1week.
Conclusions/Significance
Ourfindingsabove,takeninthecontextofthehumansafetyofCipa,basedonitsusein
Indiantraditionalmedicine,warrantfurtherworktoexploreCipaasasourceforthedevelop-
mentofaninexpensiveherbalformulationfordenguetherapy.Thismaybeofpracticalrele-
vancetoadengue-endemicresource-poorcountrysuchasIndia.
AuthorSummary
Indiarepresents~50%oftheglobalpopulationestimatedtobeatriskofdengue.Severe
dengue,whichispotentiallyfatal,correlateswithveryhighvirusload,reductioninplatelet
countsandhaemorrhage.Antiviraltherapytoreducehighvirusloadmaybebeneficialin
attenuatingdiseaseseverity.WehaveexploredIndiantraditionalmedicine,Ayurveda,to
identifyplantsthatcouldbeasourceofdengueinhibitoryactivity.Weshowthatanalco-
holicextractpreparedfromCissampelospareiraLinninhibitedthereplicationofdengue
virusesinlivingcellsincultureandprotectedmiceagainstdengueinfection.Italso
showedantipyreticandanti-inflammatoryeffects.Importantly,thisextractdidnotshow
anytoxiceffectsinratsanddidnotaffectplateletsandredbloodcells.Thisobservationis
criticalasdenguefeveriscommonlytreatedwithantipyretics.Inadengue-endemic
resource-poorcountryasIndia,theC.pareiraplantmayserveasasourceforthedevelop-
mentofaninexpensiveherbalformulationagainstdengue.
Introduction
Denguediseaseisamajorpublichealthconcernaroundtheworld.Itisspreadtohumans
throughthebiteofAedesmosquitoeswhichserveascarriersofthedisease-causingviruses.
Therearefourserotypesofdengueviruses(DENV-1,-2,-3and-4),belongingtothefamily
Flaviviridae[1],whichareprevalentinmorethanahundredmosquito-infestedcountriesin
thetropicalandsub-tropicalregionsoftheglobe[2,3].Accordingtorecentestimates,there
areannually~400millioninfectionsaroundtheworldandafourthoftheseareassociatedwith
symptomaticdengueillness[3].Symptomaticdengueillnesscanrangefrommilddenguefever
(DF)tosevereandpotentiallyfataldenguehaemorrhagicfever(DHF)anddengueshocksyn-
drome(DSS)[4].InDF,theappearanceofvirusintheserumcoincideswithfeveronset,with
titersreaching106infectiousunits/ml,atthepeakofthefebrilephase.InDHF/DSSpatients,
theviremiacanescalateby1–2logs[5,6].Thisisaccompaniedbypotentiallyfatalmanifesta-
tionsofthrombocytopenia,haemorrhage,vascularleakageandshock[1,2,7].Unlesshospital-
izedandgivensupportivecareandfluidreplacement,DHF/DSScanbeassociatedwithhigh
casefatalityrates[4].Controllingthespreadofdenguecontinuestobeanintractableproblem
duetotheinabilitytoeradicatethevectormosquitoesandthelackofsafe,potentpreventive
vaccine[8,9].Despitenumeroushurdles,persistenteffortsovertheyearshaveresultedinthe
developmentofseveralliveattenuateddenguevaccinecandidates[9],oneofwhichhas
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004255 December28,2015 2/20
HerbalExtractwithPan-DengueInhibitoryActivity
undergonephaseIIIclinicaltesting[10–12]andisexpectedtobelicensedinthenearfuture.
Thechallengesbeingfacedindenguevaccinedevelopment[13]haveemphasizedtheneedfor
antiviraldrugsandspurredneweffortsinthisdirection[14–16].
Inregardtodrugs,amajorityusedintreatinghumanillness[17]andseveralinclinical
development[18]canbetracedtonaturalorigins.Indiahasarichherbalrepertoirewhichis
usedintraditionalethnomedicine.Thecurrentworkwasundertakenwiththeobjectiveof
exploringthepossibilityofidentifyinganti-DENVactivitythatmaybeassociatedwithindige-
nousplantsinIndia.Tochooseplantslikelytoprovideusefulleads,thestudyutilizedknowl-
edgefromtraditionalIndianmedicine,Ayurveda,aspecificbranchofIndianethnomedicine
[19].Ayurvedaprescribesmedicinesbasedonvariousherbsforavarietyofillnesses.Though
dengueassuchisnotdescribedinAyurvedicliterature,thereareillnessesidentifiedbysymp-
tomsthatcanbecorrelatedwithsomeoftheclinicalmanifestationsofdenguedisease.Plants
shortlistedonthisbasisfromthevastindigenousherbalbio-resourceavailableinIndiawere
screenedforanti-DENVactivity.Thisworkdescribesthescreeningassaysthatwereset-upto
identifyanti-DENVactivityandpresentsdataidentifyingtwoplantsthatmanifestedinhibitory
potencyagainstall4DENVs.Oneofthese,chosenforfurtherstudytoassessitsinvivoefficacy
andclinicalrelevance,wasfoundtobeapromisingcandidateforfurtherdevelopmentasa
pan-DENVinhibitoryformulation.
Methods
Cellsandviruses
ThemosquitocelllineC6/36,themonkeykidneycelllinesLLCMK andVero,andhuman
2
hepatomacelllineHepG2werefromAmericanTypeCultureCollection,Virginia,U.S.A.C6/
36cellsweremaintainedinLeibovitzL-15mediumsupplementedwith0.03%tryptosephos-
phatebrothand10%heat-inactivatedfetalcalfserum(ΔFCS)inaCO -freeincubatorat28°C.
2
ThemonkeykidneycellsweremaintainedinDulbecco’sModifiedEaglemedium(DMEM),
supplementedwith10%ΔFCS,ina5%CO humidifiedincubator,at37°C.ForHepG2cells,
2
DMEMwasreplacedbyRoswellParkMemorialInstitute(RPMI)medium.Theremaining
conditionswerethesame.RepresentativesofthefourDENVserotypes(strainandaccession
numbersareindicatedinparentheses)usedinthisstudywere:DENV-1(NauruIsland,
U88535);DENV-2(NewGuineaC,AF038403),DENV-3(H87,M93130)andDENV-4(Dom-
inica,M14931).ThesewerepropagatedinC6/36cellsandtitratedonLLCMK cellsusinga
2
standardplaqueassayasdescribedbelow.
Plantsandpreparationofextracts
PlantsshortlistedforscreeningareindicatedinTable1.Thesewereprocuredthroughlocalsup-
pliersandidentifiedandauthenticatedbytheresidentherbaltaxonomistatRanbaxyResearch
Laboratories,Gurgaon,India.Extractswerepreparedusingpart(s)oftheplantsusedinconven-
tionalAyurvedicpreparations.Ineachcase,threedifferentextracts,methanolic,hydroalcoholic
andaqueous,werepreparedusingmethanol,methanol:water(1:1),andwater,respectively.Plant
partswerewashed,shade-driedandpulverizedpriortoextraction.Toprepareextracts,~100g
plantmaterialwasextractedthreetimeswith500mlextractionsolventatboilingpointfor3
hours,eachtime.Thethreeextractionswerepooled,filteredandconcentratedatlowpressure
andtemperature,anddriedinavacuumovenatroomtemperature(RT)for16–18hours.The
resultantmaterialwasdissolvedindimethylsulfoxide(DMSO),at20mg/mlandstoredat4°C.
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HerbalExtractwithPan-DengueInhibitoryActivity
Table1. Screeningofplantextractsforanti-DENVactivity.
No. NameofPlant Partuseda AntiviralActivityb
1 Aconitumheterophyllum Rhizome -
2 Andrographispaniculata Wholeplant -
3 Azadirachtaindica Bark -
4 Cedrusdeodara Resin -
5 Cissampelospareira Aerialpart +
6 Coptisteeta Root +
7 Cyperusrotundus Rhizome -
8 Desmodiumgangeticum Wholeplant +
9 Enicostemmalittorale Wholeplant -
10 Fumariaindica Wholeplant +
11 Ocimumsanctum Wholeplant -
12 Phyllanthusamarus Wholeplant +
13 Picrorhizakurroa Rhizome ±
14 Premnamucronata Bark +
15 Swertiachirayata Wholeplant -
16 Terminaliachebula Fruit +
17 Tinosporacordifolia Stem +
18 Aloevera Leaf -
19 Citrulluscolocynthis Root -
aMethanolicextractswerepreparedusingpartsoftheplantmentionedinAyurvedicliterature
bAntiviralactivityagainstDENV-3screenedusingType-1assay.“+”indicatesactiveextract(IC <25μg/ml);
50
“±”indicatesborderlineextracts(IC =~25μg/ml);“-”indicatesinactiveextract(IC >25μg/ml)
50 50
doi:10.1371/journal.pntd.0004255.t001
Plaqueassay
ThetiterofinfectiousDENVinvirusstocksandinculturesupernatants(inthetype-3screen-
ingassay,seebelow)weredeterminedusingastandardplaqueassayasdescribedpreviously
[20].Briefly,LLCMK monolayersin6wellplateswereinfectedinduplicatewithserial10-fold
2
dilutions(preparedinDMEM+2%ΔFCS)ofthevirus-containingsamples(250μl/well).Mock-
infectionswereperformedinparallelusinganequivalentvolumeofvirusdiluentalone.Two
hourslater,theinfectedmonolayers(afteraspiratingoffthevirusinoculum)wereoverlaid
withDMEM+6%ΔFCScontaining1%methylcellulose(2ml/well),andincubatedfor6days
(37°C,5%CO ).Onday6post-infection,theoverlaywasremovedandthecellsfixedwith4%
2
formaldehydesolution(1ml/well).Fixedcellswerewashedandstainedwith0.05%(w/v)crys-
talvioletsolutionin20%ethanol.Revealedplaqueswerecountedtodeterminethevirustitre,
expressedasplaque-formingunits(PFUs)/ml.
Cell-basedbioassaysforantiviralscreening
Theinitialantiviralscreeningassay,designatedasthetype-1assay,wasbasedontheplaque
reductionneutralizationtest(PRNT)describedearlierfordeterminingDENV-neutralizing
antibodytitresinsera[20].LLCMK cellswereseededin24-wellplates(5x105cells/well),a
2
dayinadvance.DENV-1,-2,-3and-4(100PFUeach)wereseparatelypre-incubatedwith
serialdilutionsofherbalextracts(correspondingto0–100μg/mlfinalconcentration)in300μl
volume,at4°Covernight.Thepre-incubationmixturewasdilutedwithanequalvolumeof
medium(DMEM+2%ΔFCS)andusedtoinfectLLCMK cells(3wellsforeachconcentration
2
at200μl/well)inthe24-wellplate.After2hoursofadsorptionintheincubator(37°C,5%
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HerbalExtractwithPan-DengueInhibitoryActivity
CO ),infectedcellswereoverlaidwithmethylcellulose-containinggrowthmediumandpro-
2
cessedthereafterasdescribedfortheplaqueassayabove.Toassessanypotentialcytotoxicity,
cellswereexposedtotheherbalextracts(inthesameconcentrationrange)intheabsenceof
DENVinfection.Additionalcontrolexperiments,runinparallel,includedcellswhichwere
eithermock-infected(negativecontrol)orinfectedwithDENVintheabsenceofherbalextract
(positivecontrol).Thehalf-maximalinhibitoryconcentration(IC value)foreachherbal
50
extractagainsteachDENVserotype,withreferencetothepositivecontrolwhichrepresented
100%infection(or0%inhibition),wasdefinedastheconcentrationofherbalextract,inμg/ml,
resultingin50%inhibitionoftheplaquecount.
Inthetype-2screeningassay,LLCMK cellsin24-wellplateswereinfectedwithDENVs
2
(moi=0.002)withoutpre-incubatingwiththeherbalextracts.After2hoursofadsorption,the
virusinoculumwasaspirated,themonolayerrinsed,andthenfedwithcompletemediumcon-
tainingtheherbalextracts(correspondingto0–200μg/mlfinalconcentration).After24hours
ofexposuretotheextract,themonolayerwasaspiratedandoverlaidwithgrowthmediumcon-
tainingmethylcelluloseandplaquesdeveloped6dayslaterasabove.
Thetype-3assaywasdoneusingVerocells,asthesecellssecretetheviralantigenNS1and
theinfectiousvirionsefficientlyintotheculturemediumuponDENVinfection.Theassay
designwassimilartothetype-2assay,exceptthatfollowingsequentialexposureofcellsto
DENVandherbalextract,cellswerefedwithliquidgrowthmedium,insteadofthemethylcel-
luloseoverlay.Aliquotsoftheculturesupernatantwerewithdrawnatperiodicintervalsupto9
daysforestimationofNS1antigenlevels(usingacommercialELISAkit)andvirustitres(by
plaqueassay,asdescribedabove).
DeterminationofcytotoxicityofCipaextract
Cytotoxicitywasevaluatedintwocelllines,LLCMK (inwhichtheantiviralactivityofthe
2
extractswereassayed)andHepG2,acommonlyusedlivercellsurrogateforinvitrocytotoxic-
itytesting.Cellsseededin96-wellplateswereexposedtoawideconcentrationrangeofCipa
extract(1–200μg/ml)for3days.CellviabilitywasassessedusingacommercialMTT(3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide)assaykit(Sigma,cat.#M5655)
withreferencetocontrolcellsthatwerenotexposedtotheextract.Thehalf-maximalcytotoxic
concentration(CC value)fortheherbalextract,withreferencetothepositivecontrol
50
(untreatedcells)whichrepresented100%cellviability(or0%cytotoxicity),wasdefinedasthe
concentrationofherbalextract,inμg/ml,resultingin50%cytotoxicity.Selectivityindex(SI)of
anextractisdefinedastheratioofCC toIC valuesobtainedusingtheLLCMK cellline.
50 50 2
Effectofpre-incubationtimeandvirusdoseontheanti-DENVactivityof
Cipaextract
Toassessifthedurationofpre-incubationoftheCipaextractwithDENVinfluencedantiviral
activityinthetype-1assayformat,pre-incubationtimesrangingfrom0–24hoursweretested
using~50PFUsofDENV-3.
ToassesstheeffectofthesizeoftheDENVdoseontheanti-DENVefficacyofCipaextract
inthepre-incubationstep(at4°C,overnight),type-1assayswereperformedusingDENV-3
rangingfrom50to5000PFUs.EachdoseofDENV-3wasassayedagainstCipaextractranging
inconcentrationfrom0–200μg/ml.
EvaluationofinvivoefficacyofCipaextractintheAG129mousemodel
ToprepareastockofchallengeviruscapableoflethalinfectioninAG129,weusedapreviously
publishedstrategy[21,22]withsomemodifications.EssentiallyDENV-2(NGC)was
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004255 December28,2015 5/20
HerbalExtractwithPan-DengueInhibitoryActivity
alternatelypassagedbetweenAG129(intracranialinoculationof106PFU)andC6/36cellsin
tissueculture.After4–5suchcyclesofpassaging,theviruswastestedinAG129micetodeter-
minetheminimumlethaldose(MLD)byi.p.injections.MLDisdefinedasthedosethatcan
causeclinicalsymptomsand90–100%death3–4weekspostchallenge.Thechallengevirus
stockthusobtainedwastitrated,aliquottedandstoredinliquidN untiluse.Totestprotective
2
efficacyofCipaextract,AG129mice(9–12weeksold,20–24gbodyweight)werechallenged
with106PFU(permouse,0.4ml,i.p)ofthechallengeDENV-2stockdescribedabove.Chal-
lengedmiceweredividedintogroups(n=6)andtreatedorallywithvehiclealone(0.25%
methylcellulose)orwithtwodifferentdosesofCipaextract(at125mgand250mg/kgbody
weight).ThemethanolintheCipaextractsadministeredtomicewasremovedcompletelyby
evaporation.Theresultantmethanol-freeCipapastewasthoroughlyre-suspendedin0.25%
methylcellulosewaterandadministeredorallytoinfectedmice.Thevolumeoftheoraldose
wasadjustedinaccordancewiththebodyweightofeachanimal(10ml/Kg/dose)andadminis-
teredbyatrainedveterinarianusingaspeciallydesignedmousefeederneedlefittedwitha
graduated1mldisposablesyringe.Thetreatmentwasinitiated2hourspost-infectionandcon-
tinuedtwicedailyfor5consecutivedays.Animalsweremonitoredtwicedailyforaperiodof
35daysforclinicalsymptomsandmortality.Acontrol(sham)groupthatwasnotvirus-chal-
lenged,butwhichreceivedCipaextract(250mg/kg),wasalsotestedinparallel.Attheendof
theexperiment,thesurvivaldatawasusedtoplotKaplanMeiersurvivalcurvesandanalysed
bythelogranktest(Mantel-Cox)testforstatisticalsignificanceusingGraphPadPrism5
software.
DeterminationofeffectofparacetamolonCipaextractinvitroandinvivo
InteractionbetweenparacetamolandCipaextractwasassessedinvitrousingtype-1assay
formatasfollows.DENV-3(~50PFUs)andCipaextract(ranginginconcentrationfrom
0–50μg/ml)werepre-incubatedovernightat4°Cinavolumeof100μl,andusedtoinfect
LLCMK cellsin24-wellplates.Parallelinfectionsweresetupusingpre-incubationmixtures
2
containingparacetamol(1–100μg/ml),inadditiontoDENVandCipaextract.Asbefore,
mock-infectionsandDENVonlyinfections(intheabsenceofCipaandparacetamol)werealso
setupandanalysedinparallel.
TheinvivoeffectofCipaextractinthepresenceandabsenceofparacetamolwasassessed
usingtheWistarratpyrexiamodel.Wistarrats(weighing180–220g)ofeithersexwereused.
Basaltemperatureoftheratswasmeasuredusingadigitalrectalthermometer(Experimetria
Ltd.,Hungary)andtheninjectedsubcutaneously(intheintra-scapularregion)with20%brew-
er’syeast(10ml/kgbodyweight)andallowedtofastovernightwithfreeaccesstowater.At18
hourspost-injection,rectaltemperatureswererecordedagaintoidentifyanimalsthatregis-
tered(cid:1)0.7°Criseinbodytemperatureforinclusioninthestudy.Groups(n=7–9)offebrile
ratswereorallyadministeredparacetamol(200mg/kg),orCipaextract(200mg/kg)orboth.
Ratsinthecontrolgroupreceivedjustthevehicle(0.5%methylcellulose).Thiswasfollowed
byrecordingofrectaltemperaturefor3hoursat30minuteintervals.
EffectofCipaextractonerythrocytesandplateletsexvivoandinvivo
Forexvivostudies,humanbloodwascollectedfromhealthyadultdonorsafterinformedcon-
sent.Erythrocyteswerepelleteddowninacentrifuge(1500xg,5minutes)fromfreshlycol-
lectedheparinizedblood,rinsedthoroughlywithPBS(pH7.4),andusedtomakea1%cell
suspensioninPBS.Cipaextractranginginconcentrationfrom12.5to400mg/Lwasaddedto
theerythrocytesuspensionandincubatedat37°Cfor1hour.Afterthis,thesampleswerespun
down,andtheabsorbanceofthesupernatantmeasuredat576nmtodeterminetheextentof
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HerbalExtractwithPan-DengueInhibitoryActivity
erythrocytelysis.Controlswhereinerythrocyteswereincubatedwithbufferalone(0%lysis),
DMSOalone(Cipasolvent)and0.1%TritonX-100(100%lysis)wereprocessedinparallel.
Basalplateletcountinfreshlycollectedheparinizedbloodandinbloodpre-incubatedwith
DMSO(vehicle)orCipaextract(2–10μg/ml)fordifferentdurations(1–4hours)wasdeter-
minedusingaBeckmanCoulterhematologyanalyser.
Forinvivostudies,fourgroups(n=5)ofWistarratswerefastedovernightandadminis-
teredorallywithvehicle(0.25%methylcellulose)orCipaextract,atthreedifferentdosages
(100,300and1000mg/kgbodyweight).BloodwascollectedjustbeforeCipaextractadminis-
tration(0hour)andat1and4hourspost-administration.Hematologyparametersweremea-
suredusingADVIA-120hematologyanalyser.
Cytokinereleaseassay
Humanperipheralbloodmononuclearcells(PBMCs)wereobtainedasfollows.Freshlycol-
lectedheparinizedbloodwasdilutedwithanequalamountofRPMI1640mediumandlay-
eredonFicollHypaque1077andcentrifugedat2,500rpmfor25minutesatRT.Theupper
layerwasdiscardedandthefluffylayerattheinterphasewasharvested,rinsedandre-sus-
pendedinRPMI1640at5x105cells/ml.FreshlycollectedPBMCswereseededin96-well
plates(105cells/well)andtreatedwithCipaextractatdifferentdilutions(inRPMI1640),fol-
lowedby30minutesincubationatRTonarotaryshaker(200rpm).Next,wellsweretreated
with50μl(4μg/ml)lipopolysaccharide(SigmaCat.#L2654)andallowedtoincubatefora
further30minutesatRT.Thevolumeperwellwasmadeupto200μlusingRPMI+10%FCS
andtheplatesincubatedovernightat37°CinaCO incubator.Negativecontrols(nolipo-
2
polysaccharidetreatment)wereruninparallel.Theplateswerecentrifuged(3000rpm,
10minutes)toobtainclarifiedsupernatantsforTNF-α(e-Bioscience,cat#BMS223/4TEN,)
andIL-1β(BDBioscience,cat#557953)determinationsusingcommercialELISAkitsasper
thevendors’instructions.
Toxicology
Groupsof5adultWistarratswereorallyadministered4ml0.25%methylcellulose(vehicle)/kg
or4mlvehiclecontaining400mgto2000mgCipaextract/kg,oncedailyfor7days(inaccor-
dancewithOECDguidelines-407).Duringthisperiod,foodintake,bodyweight,andclinical
signsweremonitoreddaily.Attheendoftheexperiment,animalswereeuthanized,followedby
determinationofhematological(Hb,WBCcount,RBCcount,plateletcountandhematocrit)
andbiochemical(SGOT,SGPT,totalprotein,serumalbumin,totalcholesterol,urea,creatinine
andrandomsugar)parameters.Necropsywasperformed.Organweightswererecordedandhis-
topathologywasdone.
Ethicalclearance
Humanbloodfromhealthyvolunteerswascollectedafterwritteninformedconsentinstrict
accordancewithapprovedguidelinesoftheHumanEthicsCommittee,RanbaxyLaboratories,
whichapprovedthe‘Clinicalprotocolforcollectionofbloodsamplesfromhealthyadult
humansubjectsforscreeningofpotentialnewchemicalentitiestoinhibitcytokinereleaseand
otherinvitroparametersinanassaysystem’throughEthicalApprovalnumber:5001_cok_11.
Allanimalexperimentswereperformedinstrictaccordancewithguidelinesspecifiedbythe
CommitteeforthePurposeofControlandSupervisionofExperimentsonAnimals(CPCSEA)
oftheGovernmentofIndia.AnimalexperimentalprotocolswereapprovedbytheInstitutional
AnimalEthicsCommittee(IAEC)ofRanbaxyResearchLaboratories,IndiathroughApproval
number151/07.
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HerbalExtractwithPan-DengueInhibitoryActivity
Results
Designofscreeningassays
ThreebioassaysweredevelopedtoidentifyherbalextractswithpotentialDENV-inhibitory
activity,asshownschematicallyinFig1.Thetype-1assaywasdesignedtoidentifyherbal
extractsthathadtheabilitytoblockDENVfromenteringsusceptiblecells.Asastandardized
referenceisnotavailabletoassesstheefficacyofDENVinhibitorypotency,PRNTdataof
monoclonalantibodies(mAbs)wereusedtodecideonthecut-offvalueinthetype-1assay.For
example,twopan-DENVmAbs,4G2andDB13-19,showhalfmaximalinhibitionofDENV-2
plaquecountsat11and33μg/ml,respectively[23].Basedonthisobservation,anextract
whichshowedanIC value(cid:3)25μg/ml,avaluewhichfallsbetweenthesetwo,wasdesignated
50
tobeactiveasaDENVinhibitor.Consideringthatthisvalueisforacrudemulticomponent
extract,itrepresentsastringentcut-off.Inthecontextofantiviralscreening,thisassayhadthe
followingpossibleoutcomes:theextractcouldbecytotoxic(compromisedmonolayer),inactive
(noreductioninplaquesincomparisontocontrolinfection),oractive(reducednumberofpla-
ques).However,thetype-1assayisnotlikelytonecessarilyrevealiftheactiveextractsalsopos-
sessedtheabilitytoinhibitpost-entrystepsintheDENVlifecycle.
Thetype-2assaywasdesignedtoassessthecapacityoftheherbalextractstoinhibitDENVs
withinthecell.Therefore,DENVinfectionprecededexposuretotheherbalextract.Asthe
herbalextractmustbeinternalizedbeforeitexertsanyinhibitoryeffect,wedecidedtouse
higheramountsoftheextract.Accordingly,thecut-offinthetype-2assaywasincreasedto
150μg/ml.Possibleoutcomesinthetype-2assayareasfollows:onceagain,theextractcouldbe
Fig1.Schematicrepresentationoftheantiviralscreeningassays.Anoutlineofthethreetypesofscreeningassays(indicatedbyArabicnumbers1,2
and3)isshown.Themulti-colouredsphererepresentsDENVandtheEppendorftubewithgreenliquidrepresentstheherbalextract.Thesetwowerepre-
incubated(1)beforeadditiontothemonolayeroraddedsequentially(2,3)tothemonolayer.Inassays1and2,thetreated-monolayerswereoverlaidwith
methylcellulose-containinggrowthmedium.Shownatthebottomarethepossibleoutcomesofthetype1and2assays.The‘x’markdenotesfailureofentry
intocells.Inassay3,liquidgrowthmediumwasaddedinsteadofthemethylcelluloseoverlay,andfollowedbyanalysisofNS1andvirusreleasedintothe
culturesupernatant.
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HerbalExtractwithPan-DengueInhibitoryActivity
cytotoxic.Ifnot,itmaybeineffectiveduetooneoftworeasons:theextractfailedtoenterthe
cells,oritwasineffectivedespitesuccessfulentry.Thefinalpossibilityisthattheextractwould
reducetheplaquecount,indicatingantiviralactivity.Thetype-3assaywasdesignedtomonitor
inhibitionbasedonreductioninviralantigenproductionandvirusyieldsfromDENV-infected
cellsfollowingtreatmentwithherbalextracts.TheamountsofviralNS1antigenandinfectious
virusreleasedintotheculturesupernatantweremeasuredovertimetogaugethemagnitudeof
inhibition.
Inordertoidentifyaneffectiveherbalextractagainstdengue,itwasreasonedthatitmust
targetallfourDENVs.Further,itwouldbedesirabletobeabletoinhibitthevirusesboth
beforeaswellasafterentryintosusceptiblecells.Towardsthisend,allextractswerescreened
againstasingleDENVserotypeinthetype-1assay,followedbyscreeningoftheactiveextracts
againsttheremainingthreeserotypes.Next,extractsmanifestingpan-DENVinhibitorypoten-
tialinthetype-1assaywereprogressedtoscreeninginthetype-2assay.Extractsidentifiedas
activeagainstall4DENVsinthetype-1and-2assaysweretestedintype-3assaytoassesstheir
abilitytoreducevirustitresby(cid:1)ten-fold.
Identificationofplantextractswithpan-DENVinhibitoryactivity
NineteenplantsdescribedinAyurvedicliteratureasbeingbeneficialinthetreatmentofill-
nesseswithdengue-likesymptomswereshortlistedforbioassayguidedscreening(Table1).
Initialscreeningshowedthatonlythemethanolicextractsmanifestedantiviralactivitywhen
assayedagainstDENV-2orDENV-3.Thehydroalcoholicandaqueousextractsofall19plants
selectedforthestudydidnotmanifestanyantiviralactivitywhentestedagainstthesetwo
DENVserotypes(IC >>100μg/ml).Asaresult,allsubsequentstudieswerecarriedoutusing
50
themethanolicextracts.
MethanolicextractswerepreparedfromeachoftheseandscreenedagainstDENV-3ina
type-1assay.ExtractsthatmanifestedIC valuesbelowthepre-designatedcut-offwerescored
50
aspositiveandtheremainingasnegativeforantiviralpotency.Theresultsaresummarizedin
Table1.Thisscreeningassayrevealed8of19extractstopossessdefiniteanti-DENV-3inhibi-
toryactivity,whileonemanifestedborderlineinhibition.These9extractswerethenscreened
againsttheremainingthreeDENVserotypesaswellinthetype-1assay.Theseresults,shown
inTable2,revealedthat4ofthe9anti-DENV-3extractsmanifestedpotentinhibitoryactivity
Table2. ExtractswithPan-DENVinhibitoryactivity.
No. Extract Antiviralactivity(IC ,μg/ml)
50
DENV-1a DENV-2a DENV-3a DENV-4a DENV-3b
1 Cissampelospareira 11.11 1.97 3.17 1.23 78
2 Coptisteeta 11.11 5.04 7.87 33.33 >200
3 Desmodiumgangeticum 12.5 6.7 14.62 12.5 >200
4 Fumariaindica 12.5 7.02 11.32 12.5 >200
5 Phyllanthusamarus 3.7 8.33 20.76 11.11 145
6 Picrorhizakurroa 12.5 16.02 27.14 25 >200
7 Premnamucronata 12.5 27.9 21.37 12.5 >200
8 Terminaliachebula 25 70 11.2 50 >100
9 Tinosporacordifolia 50 58.75 16.02 25 >100
aType-1assay(carriedoutusingallDENVserotypes)
bType-2assay(carriedoutusingDENV-3alone)
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Table3. Extractsdisplayingpan-DENVinhibitoryactivityinType-2assay.
No. Extract Antiviralactivity(IC ,μg/ml)
50
DENV-1 DENV-2 DENV-3 DENV-4
1 Cissampelospareira 100 125 78 100
2 Phyllanthusamarus 100 133 145 100
doi:10.1371/journal.pntd.0004255.t003
againsttheremainingthreeDENVserotypesaswell.Therestwereeffectiveatleastagainsttwo
otherDENVserotypes.Whenallthese9weretestedinatype-2assayagainstDENV-3toiden-
tifyextractsthatmayinhibitthevirusafteritsentryintocells,twoextracts,oneobtainedfrom
CissampelospareiraLinn,andtheotherfromPhyllanthusamarus,wereeffectiveincurbing
DENV-3(IC values<150μg/ml).Extendingthetype-2assaytoDENV-1,-2and-4revealed
50
thatboththeseextractspossessedtheabilitytoinhibitallfourDENVsevenaftertheirentry
intocells(Table3).WhilePhyllanthusamarushasbeendocumentedtopossessantiviralprop-
ertiesagainstanotherflavivirus,HepatitisCvirus[24]aswellasmanyotherviruses[25],this
isthefirstreportofantiviralactivityassociatedwithCissampelospareiraLinn.Furtherstudies
inthisworkfocusedonCissampelospareiraLinn,henceforthdesignatedasCipafor
convenience.
Cipaextractsfromdifferentgeographicallocationsaresimilarin
compositionandantiviralpotency
Fromtheperspectiveofevaluatingtheavailabilityofstartingmaterial,itwasconsideredneces-
sarytoascertaintheextentofseasonalandgeographicalvariationsinthecapacityoftheCipa
extracttomanifestDENVinhibitoryactivity.Tothisend,extractswerepreparedfromCipa
plantscollectedduringdifferentseasonsfrommultiplegeographicallocationswithinIndia.
Analysisoftheseextractsrevealedthattheanti-DENVinhibitoryactivityisevidentinplants
collectedduringthefloweringseasonofApriltoSeptember[26].Intheexperiment,theresults
ofwhicharesummarizedinTable4,Cipaextractswerepreparedfromplantsobtainedduring
thefloweringseasonfromfourdifferentlocations,andevaluatedusingthetype-1assay
describedabove.ThedatashowthatCipaextractsmanifestedpan-DENVinhibitoryactivityin
bothassayformats.Further,theobservedmeanpan-DENVIC valueswerenotsignificantly
50
differentforallfourgeographiclocationstested.Fractionationofthemethanolicextractswith
differentsolventsresultedinnominalincreaseinantiviralefficacyandasignificantlossin
Table4. Profileofanti-DENVactivityofextractsfromCipacollectedfromgeographicallydiverselocations.
Extract/Fraction S.India N.India Rajasthan M.P.
IC a(μg/ml) Yieldb(%) IC a(μg/ml) Yieldb(%) IC a(μg/ml) Yieldb(%) IC a(μg/ml) Yieldb(%)
50 50 50 50
Methanolic 7.12 6 8.01 5.96 7.46 8.62 21.38 13.2
Hexane 2.33 1.37 2.10 1.25 2.89 1.5 2.79 2.15
Chloroform 1.02 0.53 1.05 0.66 1.08 0.25 3.62 0.26
Dichloromethane 2.94 0.61 1.61 0.69 1.72 0.22 6.42 0.3
Ethylacetate 2.89 2.57 4.07 2.74 2.7 2.93 4.65 3.44
Acetone 1.68 2.11 3.05 2.02 1.88 2.3 2.93 2.78
aDenotespan-DENVinhibitoryactivity(meanoftheIC valuestargetingeachofthefourDENV-serotypes)
50
b%Yield=(wt.ofextractorfractionobtained/wt.ofstartingmaterial)x100
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