Rev. bras. farmacogn. vol.26 número4

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Cissampelos

sympodialis

has

anti-viral

effect

inhibiting

dengue

non-structural viral protein-1 and pro-inflammatory mediators

Fagner Carvalho Leite

, Cíntia da Silva Mello

, Luciana Gomes Fialho

, Cintia Ferreira Marinho

,

Ana Luisa de Araujo Lima

_{, José Maria Barbosa Filho}

_{, Claire Fernandes Kubelka}

_,

Marcia Regina Piuvezam

a_{Laboratório de Imunofarmacologia, Departmento de Fisiologia e Patologia, Universidade Federal da Paraíba, João Pessoa, PB, Brazil}

b_{Faculdade São Francisco da Paraíba, Cajazeiras, PB, Brazil}

c_{Laboratório de Imunologia Viral, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil}

d_{Laboratório de Fitoquímica, Departmento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, PB, Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2November2015 Accepted6March2016 Availableonline19May2016

Keywords:

Cissampelossympodialis

Dengue Huh-7cells Cytokines NS1 Warifteine

a

b

s

t

r

a

c

t

Dengueisthemostimportantviralinfectiontransmittedamonghumansbyarthropod-borne.Thereare currentlynovaccinesorspecifictherapeuticaltreatment.Therefore,immunomodulatorycompounds fromplantshavebeenwidelyexaminedfortheirantiviraleffects.CissampelossympodialisEichler, Menis-permaceae,hasscientificallyproventopresentimmunomodulatoryactivities.Hereweassessedthe antiviralactivityofleafhydroalcoholicextract,warifteineormethylwarifteinefromC.sympodialisin aninvitrodenguevirusinfectionmodel.Theresultsdemonstratedthatleafhydroalcoholicextractor warifteine/methylwarifteinetreatmentdidnotreducedenguevirus-Ag+hepatocyte(Huh-7cell)rates inpresentexperimentalconditions.However,weassessedthepotentialantiviraleffectofleaf hydroalco-holicextractorwarifteine/methylwarifteineondenguevirus-infectionbytheproductionofinflammatory molecules,TNF-␣,MIF,IL-8andPGE2.DenguevirusinfectionenhancedTNF-␣,MIF,IL-8andPGE2 pro-ductionininfectedHuh-7cellsandleafhydroalcoholicextractbutnotwarifteine/methylwarifteine treatments,significantlyreducedthesemoleculesininfectedcells.Indenguevirus-infectedHuh-7cells, non-structuralprotein-1isproducedandleafhydroalcoholicextractsignificantlyinhibitedit indepen-dentlyofalkaloids.Ourfindingsimplythatleafhydroalcoholicextractmayattenuatedenguevirus infectioninHuh-7cellsbyinhibitingtheenhancedofpro-inflammatorymediatorsandnon-structural protein-1productioninducebydenguevirusindependentlyofwarifteine/methywarifteineitsmajor compound.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Dengue virus (DENV) is an important human infectious

pathogeninthetropics andsubtropics;itremainsanimportant

publichealth burden requiring continuing attention. The

clini-calmanifestations of dengue diseaserange fromasymptomatic

infection,undifferentiatedfeveranddenguefever,todengue

hem-orrhagicfeverwithplasmaleakageandpotentiallylife-threatening

dengueshocksyndrome(Amorimetal.,2014).

∗ Correspondingauthor.

E-mail:[email protected](M.R.Piuvezam).

Ethnobotanicalstudieshavebeentheprimarysourcefor

selec-tionofmoleculesinscientificinvestigationsandtheyrepresenta

richtrialforimmunomodulation products.Cissampelos

sympodi-alisEichler,Menispermaceae,popularlyknownasmilonaoccurs

inseveralBrazilianstatessuchasParaíba.Theaqueousinfusionof

theleaveshasbeenusedinfolkmedicinetotreatinflammatory

dis-eases(Piuvezametal.,2012).Theleafhydroalcoholicextract(AFL)

and its bisbenzylisoquinoline alkaloidspresented

immunomod-ulatory effectin severalexperimental modelsof inflammations

(Piuvezametal.,2012).

Inthepresentstudy,wehypothesizedthatAFL,warifteineor

methylwarifteinedisplayantiviraleffectviaits

immunomodula-toryproperties.ToproveitweusedtheinvitromodelofDENV

infectioninHuh-7cells(humanhepatocytecelllineage).

http://dx.doi.org/10.1016/j.bjp.2016.03.013

Materialsandmethods

Plantmaterial,obtainingandpreparationofleafhydroalcoholic extract(AFL)ofCissampelossympodialis

TheleavesofCissampelossympodialisEichler,Menispermaceae,

wereobtainedfromtheBotanicalGardenoftheFederalUniversity

ofParaiba(vouchespecimenAGRA1456)andtheextractofthe

leaves(AFL)aswellasitalkaloidswarifteine(WAR)and

methyl-warifteine(MWAR)weregentlyprovidedbyDrJoseMariaBarbosa

Filho(DeFreitasetal.,1996;Vieiraetal.,2013).Inbrief,3kgoffresh

leaveswascollected,driedandpulverized.After,threesuccessive

alcoholextractionswereperformedinapercolatoratroom

tem-perature(25–30◦_{C).TheAFLwasobtainedwithamixtureofwater}

andethanol(30/70,v/v).Thensolventwasremovedandthedry

weightoftheextractwas79.9%basedonthepresentsolidwaste.

TheAFLwaspreparedinsterilesaline(Piuvezametal.,1999)to

posteriorstandardizationusingthealkaloidwarifteineasapattern

(Cerqueira-Limaetal.,2010).AFLwasdissolvedindimethyl

sulf-oxide(DMSO)andstocksolutions(1mg/ml)storedat−20◦_C.AFL

wasdilutedtotheindicatedconcentrations(0.1–100␮g/ml)with

culturemediumbeforeuseinexperiments.DMSOconcentration

didnotexceed0.01%.

Bisbenzylisoquinolinealkaloidsextractionandpurification

Toobtainwarifteineandmethylwarifteine,theC.sympodialis

extractwasdissolvedin3%HClandextractedwithCHCl3.

Aque-ousfractionwasbasifiedwithNH4OHatpH9andagainextracted

withCHCl3.TheCHCl3 extractwaswashedwithH2Oanddried

withMgSO4togetthetertiaryalkaloidfraction.After,thetertiary

alkaloidfractionwassubjectedtochromatography columnover

alumina,elutingwithhexanecontainingCHCl3/MeOH.Fractions

elutedwithCHCl3–MeOH(49:1)werefurtherpurifiedbythinlayer

column(1mmlayer)forisolationofWARandMWAR(

Cerqueira-Limaetal.,2010;Meloetal.,2003;Vieiraetal.,2013).WARor

MWARpowder(purity90%)wasdissolvedin0.1NHCl.Foreach

experiment,thestocksolutionwasfurtherdilutedwith0.1NHCl

todesiredconcentrations(1,2.5,5,10␮M).

CulturemediumandpreparationofHuh-7cells

Huh-7cells(hepatocarcinomacellline),obtainedfrom

Ameri-canTypeCultureCollection(ATCC,cellline-615),weremaintained

inDulbecco’sModifiedEagleMedium(DMEM)(Sigma, St.Louis,

MO)supplementedwith10%fetalbovineserum(FBS)(Biological

Industries,KibbutzBeitHaemek,Israel),100U/mlpenicillin,and

100␮g/mlstreptomycin(InvitrogenLifeTechonologies).

MTTassayforcellviabilitycytotoxicityofAFL,warifteineand methylwarifteine

Huh-7cells wereincubatedin96-wellplates at1×105 _cells

perwellcontaining100␮lofDMEMmediumanddifferent

con-centrationsofAFL(0at200␮g/ml),warifteine(WAR,0–200␮M)

or methylwarifteine (MWAR, 0–200␮M) for 72h. Cells were

washedoncebeforeadding50␮lFBS-freemediumcontainingMTT

(5mg/ml).After4hofincubationat37◦_{C,themediumwas}

dis-cardedandtheformazanbluethatformedinthecellswasdissolved

inDMSO(SIGMA).Theopticaldensitywasmeasuredat540nm

(Mosmann,1983).

Cellcultures,virusstockpreparationandvirustitration

TheDENVserotype2strain16681hasprovidedbyDr.SB

Hal-stead(Naval Medical Research Center, USA) and propagated in

Aedes albopictusC6/36cellclonetoobtainedthevirusstock,as

describedbefore(Reisetal.,2008).Inbrief,A.albopictusC6/36cell

clonewasgrownasmonolayersat28◦_{ConLeibovitzmedium}

(L-15)supplementedwith200mMglutamine,1%non-essentialamino

acid solution, 0.5% tryptose phosphate broth, 100 U/penicillin,

10␮g/streptomycinand5%fetalbovineserum(FBS)andinfected

withDENV-2for8days.After,thesupernatantcontainingthevirus

particleswasultracentrifuged(100,000×g)for1hat4◦_C.The

pel-letwasstoredat−70◦_{CandVirustiterwascalculatedas50percent}

tissuecultureinfectiousdose(TCID50).Thevirusstockusedwas

ataconcentrationof1.6×109_{TCID50/ml(Reisetal.,2008;}

Lima-Junioretal.,2013).

Huh-7cellsinfectionandtreatmentwithAFL,warifteineor methylwarifteine

Huh-7 cells were resuspended in supplemented RPMI 1640

medium,plus10%FCSandseededat2×106_{cells/mlon96-or}

24-wellplates.Afteranovernightincubation,infectionwaseffected

witha diluted inoculum (30 or300␮l) in cell culturemedium

containing1.6×109_{TCID50/ml.Aftera2h-incubationperiodfor}

adsorption,thecellculturesupernatantwasreplacedwitha2%FBS

mediumandincubatedwithleafhydroalcoholicextract(AFL)ofC.

sympodialis(0.1,1or10␮g/ml),WAR(0.1,1or10␮M)orMWAR

(0.1,1 or 10␮M)and subsequently incubatedat 37◦C with5%

CO2.After24,48or72h,supernatantswerecollectedandstocked

at−20◦_{Cforcytokinemeasurementandcellsrecoveredforviral}

antigendetermination,cellviabilitydeterminedinculturebyMTT

assay.Wellcontentwithcellcontrol,inactivatedandinfectious

DENVwasassayed.

ViralantigendeterminationinHuh-7cellsbyflowcytometry

Huh-7cellswererecoveredinacoldcellculturemedium,set

at1×106_{cells/microtube,thencentrifuged(350}

×g,10min)and

washedoncewithphosphatebufferedsalinepH7.4containing1%

bovineserumalbuminand0.1%NaN3(PBS/BSA).Afterwards,cells

werefixedwithparaformaldehyde2%inPBS/BSAat4◦_Cfor20min

andpermeabilizedwithsaponin0.15%inPBS/BSA.Permeabilized

cellswerethenblockedwith5%inactivatedplasmainPBS/BSAat

4◦_{Cfor30minandincubatedwithmouseanti-DengueComplex}

monoclonalantibody(MAB8705,Millipore)at4◦_{Cfor60min.Cells}

werethenwashedandincubatedfor30minat4◦_{Cwithanti-mouse}

IgGAlexaFluor488(A20181,LifeTechnologies).Afterincubation,

cells werewashed withPBS/BSA,resuspendedin

paraformalde-hyde2%,andkeptat4◦_{C untilcellacquisition(5000 eventsfor}

gated monocytes) by FACS® _{Calibur flow cytometer (Beckon &}

Dickinson)andanalyzedwithFlowJoSoftware(TreeStarInc.).An

isotype-matchedantibodywasadoptedasastainingnegative

con-trol(Lima-Junioretal.,2013).

Cytokinequantification

ELISAcytokinekitswasusedtomeasureTNF-␣,IL-8,andMIF

in the cells supernatant, and the assaywas performed

accord-ing to themanufacturer’s instructions (R&D Systems, CA,USA)

as described previously (Assuncao-Miranda et al., 2010). Data

analyses of all assays were performed with Bio-Plex Manager

software(Bio-Rad). Prostaglandin(PG)E2 concentrationsin the

cellculturesupernatants fromHuh-7cells weredeterminedby

anenzymeimmunoassay(ELISA)kitaccordingtotheprocedures

suppliedbythemanufacturer(CaymanChemical,AnnArbor,MI,

50 60

a

b

c

**

_*

800

600

400

200

0 40

20

0

Inf

ection,

%

TNF-α

pg/ml

MIF pg/ml

40

30

20

10

0

Control DENV-2 AFL 0.1 µg/ml AFL 1 µg/ml AFL 10 µg/ml

Fig.1. EffectofAFLonDenguevirustype2(DENV-2strain16681)Dinfection,TNF-␣andMIFproductionininfectedHuh-7cells.(A)DENVantigenpositivecellsdetected byflowcytometryanalysis.(B)TNF-␣and(C)MIF-␣levelsdetectedbyELISA.Resultswerepresentedasmean±SEMwhere*p<0.05wasconsideredsignificantbyone-way ANOVAandaDunnetttest,asapost-testwhenthetestsubstancewerecomparedwithDENV-2.Dataarerepresentativeofthreeindependentexperimentsperformedin triplicates.

NS1quantification

NS1wasmeasuredinculturemediumfromHuh-7cellsinfected

withdenguevirusandpre-treatedusingacommercialsandwich

ELISAkit(PlateliaNS1;BioRadLaboratories).Thetestswere

con-ductedwithaccordingtothemanufacturer’sprotocols.

Resultsanddiscussion

Immunomodulatory compounds of plants have been widely

examinedfortheirantiviraleffects(AbdKadiretal.,2013;Reis

etal.,2008).Inthisstudy,anti-DENVactivityofleafhydroalcoholic

extract(AFL)ofC.sympodialis,warifteine(WAR)anditsmethylated

formmethylwarifteine(MWAR)themajoralkaloidsisolatedfrom

theplantwereevaluated.TheconcentrationofAFLorWAR/MWAR

usedwastestedbyMTT assay(seesection Culturemediumand

preparationofHuh-7cells)andtheydonotshowtoxiceffectson

Huh-7cells(datanotshown).

To determine whether AFL has anti-DENV effect, we firstly

measuredDENV-antigenexpression,TNF-␣andMIFproductionin

Huh-7cellsinfectedbyDENV-2.Wepre-infectedHuh-7cellsfor

2handtreatedwithAFL(0.1,1and10␮g/ml)andcellswere

col-lectedat72hpost-infection.AsshownonFig.1A,theplantextract

(AFL)didnot interfere withtheexpressionof DENV. However,

AFL(10␮g/ml) reduced the production of TNF-␣ at 72h

post-infection(Fig.1B)andreducedMIFproductionbyHuh-7infected

cells(Fig.1C).

TNF-␣isthemaincytokinethatinducesvascularleakageand

itsserumlevelshavebeenimpliedinDENV-infectedpatientswith

seriousformsofdisease(Bragaetal.,2001;Sellahewa,2013).In

mice,vascularhemorrhagecanbeinducedbyinjectionofDENV

andislesssevereinTNF-␣deficientmiceorblockingby

anti-TNF-␣antibodies(Chenetal.,2007;Shrestaetal.,2006).Furthermore

(Perez et al., 2010)demonstrated an association between high

levelsofexpressionofTNF-␣alleleandsusceptibilityofdengue

hemorrhagicfever(DHF).

AnotherimportantcytokineinDENVinfectionismacrophage

migrationinhibitoryfactor(MIF).Assuncao-Mirandaetal.(2010)

showedenhancedofMIFonplasmaofpatientswithsevereforms

ofdengue.Therefore,endogenousMIFcontributestothe

pathogen-esisofdengueinfection.Inthepresentstudy,invitroinfectionof

Huh-7cellswithDENV-2increasedMIFlevelsthatwere

success-fullysuppressedbyAFLtreatment(Fig.1C)

ToconfirmtheimmunomodulatoryactivityofAFLwemeasured

onsupernatant,IL-8andPGE2levels.AFL(10␮g/ml)inhibitedboth

IL-8andPGE2(Fig.2AandB).Interleukin-8andPGE2induce

alter-ationsinendothelialfunctions(Purwatietal.,2011;Lima-Junior

etal.,2013).Elevatedlevelsofthesemoleculesweredetectedin

serumandpleuralfluidofpatientswithDHF(Raghupathyetal.,

1998;Purwatietal.,2011)suggestingthatthefluideffusionintothe

pleuraandotherserouscavitiesinDHFpatientsmaybeatleastin

partattributedtoIL-8and/orPGE2.TheproductionofIL-8appears

tobedependentonIL-1andTNF-␣(Raghupathyetal.,1998)andas

itwasrelatedaboveAFLdecreasedtheamountofTNF-␣on

DENV-2infectedHuh-7cellssuggestingacytokinedownregulatoryeffect

ofAFL.

In DENV infection, hepatocytes produce and activate

cyclooxygenase-2(COX-2)withproduction ofPGE2(Liou etal.,

2008).Theso-calledbreak-bonepainindenguediseaseis

prob-ably related to the overproduction of PGE2 and this molecule

800

**

*

15

10

5

0

IL-8 pg/ml _{PGE2 pg/ml}

600

400

200

0

Control DENV–2 AFL 10 µg/ml

a

b

Fig.2.EffectofAFLonDENV-2inducedIL-8,PGE2orNS1productionininfectedHuh-7cellstreatedwithAFL(10␮g/ml)for2hpre-infection.Thesupernatantofcellswere

collectedat48hpost-infectionwithDenguevirustype2(DENV-2strain16681).(A)IL-8levelinDENVinfectedHuh-7cellstreatedwithAFL(B)PGE2levelinDENVinfected

hasimportant effect on replicationand infectivity of the virus (SteerandCorbett,2003;Tsatsanisetal.,2006).ThereforeCOX-2

modulationbytheplant extractmaybe a promisingtarget for

controllingnotonlyinflammationandpainbutalsoviralinfection

indicatinganimportantroleoftheplantinthisdisease(Roccaand

FitzGerald,2002;SteerandCorbett,2003).

DENVis anenvelopedvirus,and itsgenomeiscomposedof

apositive-sense,single-strandedRNAcodingforthreestructural

proteinspresentinthevirionand infectedcellsandseven

non-structural(NS)proteins(NS1,NS2a,NS2b,NS3,NS4a,NS4b,and

NS5) not present in the virion (Amorim et al., 2014).NS1 is a

glycoprotein expressed in infected mammaliancells as soluble

monomers that is subsequently transported tothe cell surface

where theproteinremains and/orit is releasedintothe

extra-cellularmilieubeenessentialforviralreplicationandviability.In

addition,thisproteinappearsintothebloodstreamandseveral

lab-oratorialtestsuseNS1todiagnoseDENVinfections(Amorimetal.,

2014).Inthisstudy,weshowedthatAFLsignificantlyreducedNS1

productioninDENV-2infectedHuh-7cells(Fig.3).

TodeterminethemechanismofactionofAFLoninvitrodengue

infectionwetestedtwomajoralkaloidsfromtheplant:warifteine

(WAR)anditsmethylateformmethylwarifteine(MWAR).Asitis

shownonFig.4A,WAR/MWARtreatmentdidnotreduceDENV-2

antigenininfectedHuh-7cellsanddidnotinhibitTNF-␣orMIF

production(Fig.4BandC).Inaddition,neitherWARnorMWAR

reducedIL-8,PGE2norNS1productionbyDENV-2infected

Huh-7cells(Fig.5A–C)indicatingthatAFLeffectisindependentlyof

2.0

*

NS1 O

.D

1.5

1.0

0.5

0.0

Control DENV–2 AFL 10 µg/ml

Fig.3.EffectofAFLonDENV-2inducedNS1productionininfectedHuh-7cells treatedwithAFL(0.1,1or10␮g/ml)for2hpre-infection.Thesupernatantofcells

werecollectedat24,48or72hpost-infectionwithdenguevirustype2 (DENV-2strain16681).NS1levelinDENV-infectedHuh-7cellstreatedwithAFLwere determinedbyELISAinthesupernatantsofHuh-7cells72hpostinfection,where *p<0.05,**p<0.01wasconsideredsignificantbyone-wayANOVAandaDunnett test,asapost-testwhenthetestsubstancewerecomparedwithDENV.Dataare representativeofthreeindependentexperimentsperformedintriplicates.

50

a

b

c

Control DENV–2

WAR 0.1 µM

WAR 1 µM MWAR 0.1 µM

MWAR1 µM

MWAR10 µM

WAR 10µM

800

600

400

200

0 40

20

0 40

30

% inf

ection

MIF pg/ml

TNF-α

pg/ml

20

10

0

Fig.4.EffectofWARorMWARonDenguevirustype2(DENV-2strain16681)Dinfection,TNF-␣andMIFproductionininfectedHuh-7cells.(A)DENVantigenpositivecells

detectedbyflowcytometryanalysis.(B)TNF-␣and(C)MIF-␣levelsdetectedbyELISA.Resultswerepresentedasmean±SEMwhere*p<0.05wasconsideredsignificant byone-wayANOVAandaDunnetttest,asapost-testwhenthetestsubstancewerecomparedwithDENV-2.Dataarerepresentativeofthreeindependentexperiments performedintriplicates.

800

a

b

c

1.0 1.5

NS1 O

.D

PGF2 pg/ml

IL-8 pg/ml _0.5

0.0 15

20

10

5

0 600

400

200

0

Control DENV-2

WAR 10µM MWAR 10 µM

WAR/MWARandthattheplantextractpresentsothercompounds

responsiblefortheantiviraleffect.

Conclusion

Inconclusion,DENV2infectedHuh-7cells produceTNF,MIF,

IL-8andPGE2thatarecorrelatedwithNS1productionandAFL

suppressedtheviral-inducedinflammatoryresponsebydecreasing

the cytokine production as well as NS1 release, but failed to

affectvirusproduction.Conversely, warifteine/methylwarifteine

did not affect the inflammatory mediators nor virus

produc-tion in DENV-2 infected Huh-7 cells. These data shed light on

dengue virus infection and C. sympodialis extract capable to

reduceNS1and pro-inflammatorymediatorsdemonstratingthe

potentialanti-inflammatoryandantiviral therapyof thisherbal

medicine.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor

thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

FCL,CFMandLGF(PhDstudents)contributedtothe

develop-ment of thebiological protocols. CSMand ALAL(MS students)

contributedtotheELISAassay.JMBFcontributedinplant

identi-ficationandalkaloidisolation.CFKandMRPdesignedthestudy,

supervisedthelaboratoryworkandcontributedtocriticalreading

ofthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This study was financially supported by CNPq-Universal14/

2012-472853/2012-0;CAPES/Brazil;PROEP, IOCand Plataforma

Tecnológica(RPT11D)/FIOCRUZCSM, LGF and CM are graduated

studentsatPosgbpIOC,Fiocruz.

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