Rev. bras. farmacogn. vol.26 número5

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

Original

Article

Potent

inhibition

of

Western

Equine

Encephalitis

virus

by

a

fraction

rich

in

flavonoids

and

phenolic

acids

obtained

from

Achyrocline

satureioides

María

Carola

Sabini

a,∗

,

Laura

Noelia

Cariddi

a

,

Franco

Matías

Escobar

a

,

Fernando

Ma˜nas

b

,

Laura

Comini

c

_,

_Delvis

_Iglesias

b

_,

_Mariana

_Larrauri

e

_,

_Susana

_Nú˜nez

_Montoya

c

_,

_José

_Sereno

b

_,

Marta

Silvia

Contigiani

d

_,

_Juan

_José

_Cantero

b

_,

_Liliana

_Inés

_Sabini

a

a_Departamento_de_{Microbiología}_e_{Inmunología,}_Universidad_Nacional_de_Río_Cuarto,_Río_Cuarto,_Córdoba,_Argentina b_Facultad_de_Agronomía_y_Veterinaria,_Universidad_Nacional_de_Río_Cuarto,_Río_Cuarto,_Córdoba,_Argentina

c_{Farmacognosia,}_Departamento_de_Farmacia,_Universidad_Nacional_de_Córdoba_Ciudad_{Universitaria,}_Córdoba,_Argentina d_Instituto_de_Virología,_Facultad_de_Ciencias_Médicas,_Universidad_Nacional_de_Córdoba,_Ciudad_{Universitaria,}_Córdoba,_Argentina e_Facultad_de_Ciencias_{Agropecuarias,}_Universidad_Nacional_de_Córdoba,_Ciudad_{Universitaria,}_Córdoba,_Argentina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received12October2015 Accepted2May2016 Availableonline14June2016

Keywords:

Achyroclinesatureioides Antiviralactivity Cytotoxicity Genotoxicity

WesternEquineEncephalitisvirus

a

b

s

t

r

a

c

t

Achyroclinesatureioides(Lam.)DC.Asteraceae,‘marceladelcampo’,possessseveralpharmacological

properties.PreviouslywereportedantiviralactivityofanaqueousextractofA.satureioidesagainstan alphavirus,WesternEquineEncephalitisvirus.Alphavirusesarehighlyvirulentpathogenswhichcause encephalitisinhumansandequines.Therearenoeffectiveantiviraltotreatitsinfections.Theaimof thisstudywastoevaluateinvitrocytotoxicandantiviralactivitiesagainstWesternEquineEncephalitis

virusoffivewaterextractchromatographicfractionsfromA.satureioidesandidentifythemain com-poundsofthebioactivefraction.Also,itwastoassessinvivocytogenotoxicabilityoftheactivefraction. CytotoxicitystudiesrevealedlowtoxicityofthemostoffractionsinVeroandinequineperipheralblood mononuclearcells.Antiviralstudiesshowedthatthewatercrudeextract–SephadexLH20–fraction3 MeOH–H2O(Fraction3)wasactiveagainstWesternEquineEncephalitisviruswithEffective Concentra-tion50%=5␮g/ml.SelectivityIndiceswere126.0onVeroand133.6onperipheralbloodmononuclear

cells,fourtimeshigherthanaqueousextractselectivityindex.Regardingthemechanismofactionwe demonstratedthatF3exerteditsactioninintracellularreplicationstages.Further,fraction3showed importantvirucidalaction.Fraction3contains,inorderofhighesttolowest:chlorogenicacid,luteolin, 5,7,8-trimethoxyflavone,3-O-methylquercetinandcaffeicacid.Fraction3didnotinduceinvivotoxic normutageniceffect.Therefore,itissafeitsapplicationasantiviralpotential.Furtherstudiesofantiviral activityinvivowillbedevelopedusingamurinemodel.

Introduction

Medicinalplantsarearelevantsourceofbiologicallyactive sub-stances. Achyroclinesatureioides (Lam.)DC. is a medicinalplant commonlyknownas‘marceladelcampo’whichbelongsto fam-ilyAsteraceae,isnativetoAmericaandalsogrowsupinEurope andAfrica.Thisplantiswidelyusedinfolkmedicine.Itis pop-ularlyconsumedininfusion,decoction,macerated,andassyrup

(García et al., 1990;Alonso Paz et al., 1992)for the treatment

∗ Correspondingauthor.

E-mail:[email protected](M.C.Sabini).

ofdigestiveandrespiratoryproblems,aswellasforviral infec-tions (Filot Da Silva and Langeloh,1994; Instituto Nacional de

Investigacion Agropecuaria, 2004; Taylor, 2005). Several

phar-macological properties, suchas anti-inflammatory, antioxidant, hypocholestorolemic, immunomodulatory,antimicrobial, antitu-moralandantiviralhavebeenscientificallyconfirmed(Ruffaetal.,

2002;Bettegaetal.,2004;DeSouzaetal.,2007;Ferraroetal.,2008;

GonzálezandMarioli,2010;Espi˜naetal.,2012;Barionietal.,2013;

Caseroetal.,2015).Inpreviousstudieswedemonstratedabsence

ofcytogenotoxicityinvitroandinvivoofcoldaqueousextractof

A.satureoidesathighconcentrations(Sabinietal.,2013).Inother studies,wereportedstrongantiviralactivityofthis crude aque-ousextractagainstWesternEquineEncephalitisvirus(Sabinietal.,

2012).

http://dx.doi.org/10.1016/j.bjp.2016.05.004

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Western Equine Encephalitis virus (WEEV) is a member of alphaviruses(Togaviridaefamily),whichareagroupofenveloped viruses with a positive sense, single-stranded RNA genome

(Contigiani,1996)which generatesencephalitis in humans and

equines.Theencephalitic alphaviruses alsoinclude Eastern and

VenezuelanEquineEncephalitisviruses(EEEVandVEEV).This com-plexof virus is arboviruses, arthropod-borne viruses,and they causefebrileillnessandencephalitis.Theyarealsothemost impor-tantemergingpathogensandthathavecausedmany epidemics inrecentdecades(WeaverandReisen,2010).Alphavirusesinfect neuronsresultinginCNSinflammationandneuronaldestruction

(SteeleandTwenhafel,2010).Thesehighlyvirulentpathogenscan

causesevere disease in humans withhighlethality, as wellas long-termneurologicalsequelaeinmostsurvivors(Ravehetal., 2013).Ininfantsandchildren thisdiseaseiseven moreserious and isoften associated withseizures(Griffin,2001).Moreover, this illness has economic importance becauseit causes signifi-cantmorbidity and mortality in humans and animals. Besides, therearecurrentlynoeffectiveantiviraldrugsforalphavirus infec-tions(Steineretal.,2010).Theseviruseshavebeenclassifiedas potentialagentsofbiologicalweaponsthatcouldendangerpublic safety(ZacksandPaessler,2010).Therefore,itisapublichealth prioritytodevelopantiviralagentsfortreatmentofalphaviruses infection.

Ontheotherhand,ithasbeenshownthatseveralflavonoids andphenolicacidshaveantiviralactivityagainstviruses’spectrum

(Orhanetal.,2009;Andresetal.,2009;Ozc¸eliketal.,2011;Ikeda

etal., 2011).Therefore, vegetal fractionsrich in flavonoids and phenolicacidsobtainedofA.satureioidescouldbeactiveagainst

WEEV.

Theaimofthepresentstudyistoevaluateinvitrocytotoxic abilityandantiviralactionoffivewaterextractchromatographic fractions obtained from A. satureioides against Western Equine Encephalitisvirusandidentifiesthemaincompoundsofthe bioac-tivefractionbyHPLC–ESI-MS/MS.Otheraimistoevaluateinvivo

cytogenotoxicability oftheactivefraction, inordertoconsider itsfutureapplicationasantiviralinthetreatmentofviraldiseases causedbyalphaviruses.

Materialandmethods

Plantmaterial

Achyroclinesatureioides(Lam.)D.C.,Asteraceae,plantswere col-lectedmanuallyfromVillaJorcoricó,locatedinsouthernCórdoba hills (32◦₄₁′_S; ₆₄◦₄₃′_W; ₈₀₀_m.a.s.l.) _in _March_2010. _The _plant materialwasidentifiedbyDr.LuisDelVitto,FacultyofPharmacy andBiochemistry,Universityof SanLuis,SanLuis,Argentina. A voucherspecimenwasdepositedintheherbariumoftheUniversity ofSanLuis(no.6362).

Obtentionofwaterextractchromatographicfractionsof

Achyroclinesatureioides

Aerialvegetalparts(leaves,stemsandblooms)were mechan-ically grinded using a knife mill (Retsch K.G. 5657Haan West-Germany) with a mesh no. 5 (sieve opening 4mm) and were submittedtoextractionwithcoldwater(4◦_C)₍₁₅_g_of_dried_and pulverizedmaterialper700mlofwater)for2days.Thissuspension wasidentifiedascoldaqueousextract(CAE).Thesuspensionwas filteredandlyophilized.Thedriedextract(1g)wasresuspendedin waterandwassubmittedtofractionationonSephadexLH20 col-umn(2.5cm×20cm)elutedsuccessivelywithCH3OH(100%)to CH3OH:H2O(9:1).FivefractionswerecollectedfromCAE(fractions 1–5),whichweresubjectedtodryinginrotaryevaporator.

Cellcultureandvirus

BioassayswereperformedonVerocells(ATCCCCL-81)grown in Eagle’s minimal essential medium (MEM; Gibco, USA) sup-plemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS)(Natocor,Argentina),l-glutamine(30_␮g/ml)and gentami-cin(50␮g/ml)(bothfromSigma–Aldrich,Italy).Cellcultureswere maintained at 37◦_C _with _5% _CO₂ _in _a _humidified _atmosphere. WEEVstrainAg80-646,anenzooticstrain,wasisolatedinChaco (Argentina)fromCulex(Melanoconion)ocossamosquitoes(Mitchell etal.,1985).Theviruswaspropagatedbyintracerebralinoculation ininfantRockefellermice.Viralstockswerestoredat−70◦_C.

Virustitration

Viruswastitratedbyquantificationplaques-formingunit(PFU) methodforarbovirus(Earlyetal.,1967).Cellmonolayersgrownin 24-wellcultureplates(Cellstar,GreinerBio-One,Germany)were infectedwith100␮loften-foldserialdilutionsofvirusperwell, induplicateandwereincubatedfor1hat37◦_C._After_that, resid-ualviruswasremoved.CellswerewashedwithPBS,andoverlaid withMEM-0.5%UltraPureAgarose(Invitrogen,USA)andfurther incubatedfor96hat37◦_C._After_incubation,_cell_monolayers_were fixedwith10%formalin(Cicarelli,Argentina)andfurtherstained with1%crystalvioletsolution.

Cytotoxicityassays

CytotoxicityonVerocells

For cytotoxicity assays, the cells (2×105cells/ml)were cul-turedin96-wellcultureplates.Afterincubationfor24hat37◦_C, cellswereexposedtoincreasingconcentrationsofF1,F2,F4,F5 (0–200␮g/ml)andF3(0–1000␮g/ml).Assayswerecarriedoutin triplicate.Monolayersincubatedonlywithmaintenancemedium (MM:MEMwith2%ofFBS)wereusedascontrolsofcellular via-bility.Thecytotoxicconcentrationoffractionwhichreducedthe viablecellnumberby50%(CC50)wasdeterminedbyneutralred uptake(NRU)assay.

Aftertreatmentof cellswithwater extract chromatographic fractions(Fs)for96h,themicroplateswereincubatedwithNR solu-tionfor3hat37◦_C_and_finally,_with_an_extraction_solution_(49% distilledwater:50%ethanol:1%aceticacid)for15mininashaker. Theabsorbancewasreadat540nmonamultiwell spectropho-tometer (Bio-Tek, ELx800, USA) (Rajbhandari et al., 2001; Seth etal.,2004).Percentagesurvivalfractionwascalculated consid-eringopticaldensity(OD)oftreatedculturesversuscontrols.

Isolationofequineperipheralbloodmononuclearcells

Peripheralblood wasdrawnfromhealthy individuals. PBMC (PeripheralBlood MononuclearCells)were isolatedfrom blood samplesusingHystopaque-1077centrifugation(Sigma–Aldrich,St. Louis,USA).Fromanoptimalsuspension1×106cells/ml,cell via-bilitywasdeterminedbyTrypanbluedyeexclusionassay(Mongini

andWaldner,1996).ThestudywasapprovedbytheComitéde

ÉticadelaInvestigaciónCientífica(COEDI)withnumber73/2012, UniversidadNacionaldeRíoCuarto.

CytotoxicityonequinePBMC

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for24h.Afterthattime,cellviabilitywasevaluatedbyTrypanblue dyeexclusionusingNeubauerchamberforcountingofviablecells, asdescribedbyMilitaoetal.(2006).Eachexperimentwasdonein triplicate.

Antiviralactivity

Monolayersgrownin24-wellcultureplates(Cellstar,Greiner Bio-One,Germany)wereexposedto100PFUofWEEV perwell for1hat37◦_C_and_treated_with_one_{concentration}_of_each_F:_F1 (50␮g/ml), F2 (20␮g/ml), F3(50␮g/ml), F4(50␮g/ml)and F5 (50␮g/ml). Cultureswerewashedand overlaidwithMEM-0.5% UltraPureAgarose(Invitrogen,USA)containingeachFatthesame concentrationwhichintheadsorptionandpenetrationand incu-batedfor96hat37◦_C._After_incubation,_cell_monolayers_were_fixed with10%formalin(Cicarelli,Argentina)andthenstainedwith1% crystalvioletsolution.Controlsofvirus,cellsandFwereincluded inallassays.Apositiveantiviralcontrolwasnotincludedbecause therearenoeffectiveantiviraldrugsagainstWEEV.Thenumberof plaquesoftreatedcellswascomparedtountreatedviralcontrols tocalculatetheplaquereductionpercentage.

DeterminationofthemechanismofactionofF3

InordertostudytheantiviralactivityoftheF3,three exper-imentswereperformedbyaddingthefractionatdifferenttimes andevaluatingtheinhibitoryactionbyaplaquereductionassay.

Adsorptionandpenetration

Monolayersgrownin24-wellcultureplates(Cellstar,Greiner Bio-One,Germany)wereexposedto100PFUofWEEV perwell for1hat37◦_C_in_the_presence_of₅₀_␮_g/ml_of_F3._After_adsorption, residualinoculumwasremoved,andMEM-0.5%agarosewasadded andincubatedfor96hat37◦_C._After_incubation,_cell_monolayers werefixedwith10%formalinandstainedwith1%crystalviolet solution.Controlsofvirus,cellsandF3wereincludedinallassays. Thenumberofplaquesoftreatedcellswascomparedtountreated viralcontrolstocalculatetheplaquereductionpercentage.

Post-penetration

Cellswereinfectedwith100PFUofvirusperwellandincubated for1hat37◦_C._Then_any_unadsorbed_virus_was_removed._Cells_were washedwithPBS,andthenMEM-0.5%agarosecontaining50␮g/ml ofF3wasadded.Afterincubation,cellmonolayerswerefixedwith 10%formalinandstainedwith1%crystalvioletsolution.Controls ofvirus,cellsandF3wereincludedinallassays.Thenumberof plaquesoftreatedcellswascomparedtountreatedviralcontrols tocalculatetheplaquereductionpercentage.

Virucidalactivity

TodeterminetheabilityofF3toinactivatedirectlythevirus par-ticles,equalvolumesofWEEV(200PFU/100␮l)andF3(50␮g/ml) weremixedandincubatedfor1hat37◦_C._Afterwards,_each_mixture wasaddedtocultures(100␮lperwell).Itwasincubatedfor1hat 37◦_C._Then,_monolayers_were_washed_and_covered_with_MEM-0.5% agarose.Controlsofvirus,cellsandF3wereincluded.After incuba-tionforfourdaysat37◦_C,_the_cells_were_fixed_with_10%_formalin andstainedwith1%crystalvioletsolution.Theplaquereduction percentagewascalculated.

Determinationof50%effectiveconcentration(EC50)

Cellmonolayersculturedin24-wellmicroplateswereinfected with100PFU perwell,and incubatedfor1hat37◦_C. _Residual inoculumwasremoved;cellswerewashedwithPBSand MEM-0.5%agarosecontainingincreasingconcentrationsofF3wasadded.

Afterfourdaysat37◦_C,_the_cultures_were_fixed;_stained_and_viral plaqueswerecounted.TheEC50wascalculatedastheF concentra-tionthatreducedthenumberofPFUto50%withrespecttoviral control.EC50andCC50valueswereestimatedbynon-linear regres-sionofconcentration–responsecurves generatedfromthedata. Cytotoxicityand antiviralactivityresultswereusedtocalculate theSelectivityIndex(SI)ofF3(SI=CC50/EC50).

Genotoxicityassays

Animalsandtreatment

MaleandfemaleBalb/cmiceaged8–12-weeksold,(weighing 20–25g)wereobtainedfromtheCentral Bioterioofthe Univer-sidad Nacional de Río Cuarto. Animals had access tofood and water adlibitumand were housedin a temperature controlled environmentona12hlight/12hdarkcyclethroughoutthe exper-imentalperiod. Allexperimentalprocedureswereconductedin accordancewithrecentlegislation.Thisstudywasapprovedwith number 73/2012byComité deÉticade laInvestigación Cientí-fica (COEDI), UniversidadNacional deRío Cuarto. Threegroups of mice were inoculated by intraperitoneal injection with F3 at concentrationsof 3, 6 and 12mg/kg body weight(b.w.) dis-solved in saline solution and 2 control groups were included. Thenegativecontrolgroupreceivedsalinesolutionandthe pos-itivecontrolgroupreceived30mg/kgb.w.ofcyclophosphamide (Sigma–Aldrich,St.Louis,US).Eachtreatmentgroupconsistedof fouranimals.

Micronuclei test in mouse bone marrow. The assay was carried out following standard protocols as recommended by Schmid

(1975). The animals were sacrificed by cervical dislocation

at 24h post-injection. The bone marrow samples of femoral bone were obtained withFBS, were homogenized, centrifuged, and plated on slides which were fixed by soft flutter. Then, theslides werestained withMay-Grunwald–Giemsa.To estab-lish genotoxic capacity of F3, the frequency of micronuclei in 1000 polychromatic erythrocytes per slide was determined. To detect possible cytotoxiceffects, theratio of polychromatic erythrocyte/normochromaticerythrocyte(PCE/NCE)in1000 poly-chromatic erythrocyte was calculated. The slides were scored usingalightmicroscopeata1000×magnification.Average num-ber of micronucleated polychromatic erythrocytes (MNPCE) in individual mice was used as the experimentalunit, with vari-ability based on differences among animals within the same group.

Single-cellgelelectrophoresis(cometassay). After24hoftreatment withF3,peripheralblood wasdrawnfromthetailveinofmice inheparinizedtubestoperformthecometassay.Cometassay(CA) wascarriedoutfollowingamethoddescribedbySinghetal.(1988)

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electrophoresistoallowtheDNAtounwind.Electrophoresiswas carried out in ice bath (4◦_C) _for ₂₀_min _at ₂₅₀_mA _and ₃₀_V (0.722V/cm).Theslidesweresubmergedinneutralizationbuffer (0.4MTris–HCl,pH7.5)for 15min,driedat roomtemperature andfixedin absoluteethanol for10min. Theslides weredried andstored overnightor longerbeforestaining.For stainingthe slideswere brieflyrinsedin distilledwater, coveredwith25␮l 1×ethidiumbromidestainingsolutionpreparedfroma200␮g/ml 10×stocksolution,andcoverslipped.Thematerialwasevaluated immediatelyat400×magnificationusingfluorescencemicroscope (Axiophot,CarlZeiss,Germany)attachedtotheimage-analysis sys-tem(PowershotG6,7.1megapixels,CanonINC,Japanwithsoftware AxioVisionRelease4.6.3,CarlZeiss,Germany),with515–560nm excitationfilteranda590nmbarrierfilter.Fromeachtreatment, imagesfrom100“nucleoids”werecapturedwithacameraattached tothefluorescentmicroscopeandlinkedtotheCometScore®_1.5 software.Highlydamagedcellswerenotincludedinthescoring (cloudswerenotanalyzed).Tailmoment(TM)wasusedtoestimate DNAdamage(arbitraryunits).

Statisticalanalysis

StatisticalanalysiswasperformedusingGraphPadPrism pro-gram, version 5.00.288 (San Diego, USA, 2007). The CC50 and EC50werecalculatedfromconcentration–effectplotsbynon-linear regressionanalysis(Boltzmannsigmoidal).Theresultsaccountfor the mean±standard error of the mean values of three differ-entexperiments.Results obtainedbymicronucleusassayswere submitted to a oneway analysis of variance (ANOVA) and the

Tukey’smultiplecomparisontest. Comet assayresultswere sub-mitted toKruskal Wallis test and Dunns multiplecomparison as

a posteriori test were used in all the experiments. The Spear-manstatisticaltestwasusedtoexaminepossibledose–response effects. In allcases,the level ofsignificance wasestablished at

p<0.001.

Identificationandquantificationofpolyphenolandflavonoid derivativesinF3byHPLC–ESI-MS/MS

Samplepreparation

AsolutionofF3(2.25mg/ml)waspreparedtocarryoutits qual-itativeandquantitativeanalysisbyHPLC–ESI-MS/MS.MeOH–HPLC grade(Merck)wasusedinallsamples,whichwerefilteredthrough aMilliporemembrane(0.45␮m)beforeHPLCanalysis.

HPLC–ESI-MS/MSinstrumentsandchromatographicconditions

AnAgilentSeries1200LCSystem(Agilent,USA)coupledtoa MicrOTOF QII(Bruker Daltonics, USA) wasusedfor HPLC–ESI-MS/MSanalysis.TheHPLCsystemconsistedina microvacuum degasser,binarypumps, an autosampler(40␮lsample loop), a thermostatedcolumncompartment, andadiodearraydetector. Themassspectrometerequippedwithelectrosprayionsourceand qTOFanalyzer,wasusedinMSandMS/MSmodeforthestructural analysisofphenolicsandflavonoids.

HPLCanalysiswereperformedonathermostated(40◦_C) Hyper-sil5columnC18(30×4.6mm,Phenomenex)ata0.4ml/minflow rateusingMeOH–formicacid0.16M(53:47)asmobilephase(De

Souzaetal.,2002).Theinjectionvolumewas40␮l.

ESI-MSdetectionwasperformed innegative ionmodewith massacquisitionbetween100and1500Da.Nitrogenwasusedas dryingand nebulizergas(7l/minand 3.5bar,respectively),and 180◦_C_for_drying_temperature._For_MS/MS_experiments fragmen-tationwasachievedbyusingAutoMS2_option._DAD_analyses_were carriedoutintherangebetween200and700nm.

Calibration standard samples were prepared by appropriate dilutions with MeOH from the stock solutions and filtered on

Milliporemembranebeforeuse.MSanalysiswasusedfor quan-tificationofthecompoundswithspecificcalibrationcurve.When referencecompoundswerenotavailable,thecalibrationof struc-turally related substance was used.Compounds concentrations were calculated in triplicate and reported as means±standard deviationineachcase.

Results

Obtentionofwaterextractchromatographicfractionsof

Achyroclinesatureioides

Five water extract chromatographic fractions were obtained fromCAE of A. satureioides. Afterdrying them, 202.4mg of F1, 68.6mgofF2,39.9mgofF3,48.6mgofF4and277.3mgofF5were obtained.It indicateda yieldof20.24%,6.86%,3.99%, 4.86%and 27.73%forF1,F2,F3,F4andF5,respectively.

Cytotoxicityassays

First, fivewater extractchromatographic fractions of A. sat-ureioideswereevaluated in theircytotoxiccapacity for thento evaluateof theirantiviralaction.Cytotoxicity onVerocells was determinedbyneutralreduptake(NRU)assayand,equinePBMC viabilitywasdetermined bytrypan bluedye exclusionmethod (TB).Cytotoxicconcentrations50%(CC50)ofwater extract chro-matographic fractions are presented in Table 1. F1, F4 and F5 indicatedCC50valuesabove200␮g/mlinbothtypeofcells.The viabilitypercentat200␮g/mlwasgreaterthan80%forthese frac-tions. F2 wasthe most toxic on Vero cells with CC50 value of 35␮g/ml.However,itwasnottoxicinequinePBMCswhoseCC50 washigherthan200␮g/ml.Ontheotherhand,F3indicatedCC50 valuesof630␮g/mlonVerocellsand668␮g/mlonequinePBMC

(Figs.1and2).TheeffectofF3onviabilityofVerocellsandequine

PBMCsshowedadose-dependentdecreaseinthenumberofviable cells.

Antiviralactivityoffivewaterextractchromatographicfractions

AntiviralstudiesindicatedthatF1,F2andF4didnotinhibitto WEEV.Thepercentofinhibitionwerelowerthan30%.While,F5 andF3indicated45%and100%ofinhibition,respectively(Fig.3). F3wasthemosteffectiveagainstWEEV.Thus,itwassubmittedat antiviralmechanismstudies.

DeterminationofthemechanismofactionofF3

To elucidate the mechanism of action of F3, WEEV was treatedduringadsorptionandpenetrationandpost-adsorptionand penetrationinseparatetrials.WEEVwasnotinhibitedin adsorp-tionandpenetrationstages,indicating15%ofinhibition.Whileit wasinhibitedin 100%whenitwastreatedpost-adsorptionand

Table1

CytotoxicityofF1,F2,F3,F4andF5ofAchyroclinesatureioidesonVerocellsand equinePBMCsdeterminedbyneutralreduptake(NRU)andtrypanblueexclusion (TB)assays,respectively.

Fraction Cytotoxicity

VerocellsbyNRU (CC50␮g/ml)

EquinePBMCsbyTB (CC50␮g/ml)

F1 >200 >200

F2 35 >200

F3 630 668

F4 >200 >200

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120

100

80

60

40

20

0

Viability

, %

0 200 400

CC50=630 µg/ml

600

Concentration (µg/ml)

800 1000 1200

Fig.1. ViabilityofVerocellsexposedtodifferentconcentrationsoffraction3 (F3)ofAchyroclinesatureioidesfor96h.Theresultsarepresentedaspercentage (mean±SD).Cellviabilitywasevaluatedbyneutralreduptake(NRU)assay.

120

100

80

60

40

20

0

0 200 400 600 800 1000 1200

Viability

, %

CC₅₀=668 µg/ml

Fig.2.ViabilityofequinePBMCsfromhealthyindividualsexposedtodifferent con-centrationsoffraction3(F3)ofAchyroclinesatureioidesfor24h.Theresultsare presentedaspercentage(mean±SD).Cellviabilitywasevaluatedbytrypanblue dyeexclusion(TB)method.

120

100

80

60

40

Vir

al inhibition, %

20

0

F1 F2 F3

Vegetal fraction

F4 F5

Fig.3.PercentofinhibitionofWesternEquineEncephalitisvirus(Ag80-646)treated withfivewaterextractchromatographicfractionsobtainedfromAchyrocline sat-ureioides.

penetrationwith50␮g/mlofF3.Therefore,thefractionexertsits

bioactivityinstagesintracellularofthereplication.Moreover,the virucidalactionindicatedasignificantinactivation(60%)ofWEEV whenitwastreatedwith50␮g/mlofF3.

100

80

60

40

20

Vir

al inhibition, %

0

0 20 40 60

80 100 120

EC50=5 µg/ml

Fig.4. Determinationofeffectiveconcentration50%(EC50)ofF3ofAchyrocline sat-ureioidesagainstWesternEquineEncephalitisVirus.Note:Verocellmonolayerswere infectedwithabout100PFUperwell,andincubatedfor1hat37◦_C._MEM-0.5% agarosewithincreasingconcentrations(5–100␮g/ml)ofF3wasadded.After

incu-bationforfourdaysat37◦_C,_viral_plaques_were_counted._Thereafter_the_percentage inhibitionwascalculated,andtheEC50wasdetermined.Dataaccountforthemeans

ofthreeseparateexperiments.

Determinationof50%effectiveconcentration(EC50)

Cellculturestreatedwithconcentrationsfrom5to100␮g/mlof

F3wasusedtoconstructthedose–responsecurvewhichallowed todeterminetheEC50value,seeFig.4.

Table2summarizestheresultsofEC50, CC50 and Selectivity

Indices(SI) determined onVero cells and equine lymphocytes. Thesevalues(SI)were126.0and133.6onVeroandPBMC, respec-tively.TheSIofF3isfourtimeshigherthantheSIofcoldaqueous extract(CAE)ofA.satureioideswhichwas32byNRU.

Genotoxicityassays

Micronucleitestinmousebonemarrow

TheresultsofcytogenotoxicabilityofF3areshowninTable3. Inthisstudy,thenegativecontrolgroupdemonstratedlowMNPCE values, as expected, and the positive control group’s MNPCE frequencywassignificantlyhigher(p<0.001),confirmingthe sen-sitivityofthetest.TheF3genotoxicityanalysis,foralldosestested (3,6,and12mg/kgb.w.),indicatednosignificantincreaseinthe MNPCEfrequencyat24hwhencomparedwiththenegativecontrol group(p<0.001).Thepositivecontrolwhichindicateda genotox-icityindexof31.75(±2.21)showedsignificantdifferencewithall treatments(p<0.001).

RegardingF3toxicity,statisticalanalysisofthePCE/NCEratio revealednosignificantdifferencesbetweenanyofthetreatments

versusthenegativecontrol.Therefore,F3ofA.satureioidesdoesnot havecytogenotoxiceffects.

Single-cellgelelectrophoresis(cometassay)

Theresultsofcometassayanalyzedinmice’sbloodareshown inFig.5.Basedonthetailmomentresults,positivecontrolrevealed significantdifferencewithnegativecontrol(p<0.001).Allthe treat-mentswithF3ofA.satureioidesdidnotshowsignificantdifference withnegativecontrolgroupwhileitdidshowstatistically signifi-cantdifferencewithmitomycinC(positivecontrolgroup).

HPLC–ESI-MS/MSanalysisoffraction3(F3)ofA.satureioides

The chemical evaluationof F3obtainedfrom A. satureioides, wasorientedtowardthesearchfortheactiveprinciplesreported previouslyinthevegetalspecies(DeSouzaetal.,2002;Polydoro

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Table2

SelectivityindicesofF3ofAchyroclinesatureioidesagainstWEEV.

Fraction CC50(␮g/ml) EC50(␮g/ml) SI(CC50/EC50)

Verocells EquinePBMCs againstWEEV Verocells EquinePBMCs

F3 630 668 5 126.0 133.6

Table3

Frequencyofmicronucleatedpolychromaticerythrocytes(MNPCE)andpolychromaticerythrocytes/normochromaticerythrocytes(PCE/NCE)ratio(TI)inmicebonemarrow cellstreatedwithdifferentdosesofF3ofAchyroclinesatureioidesfor24h,andrespectivecontrols.

Treatments Dose(mg/kg) MNPCE(‰₎

(mean±SD)

PCE/NCE(TI) (mean±SD)

Total Total

Negativecontrol(salinesolution) 0 7.00(±0.70) 1.83(±0.41)

A.satureioidesF3 3 7.25(±0.95) 1.79(±0.09)

A.satureioidesF3 6 7.60(±1.34) 1.71(±0.19)

A.satureioidesF3 12 6.50(±1.76) 1.64(±0.34)

Positivecontrol(cyclophosphamide) 30 31.75(±2.21)a 1.61(±0.12)

SD,standarddeviation;MNPCE,micronucleatedpolychromaticerythrocytes;PCE,polychromaticerythrocytes;NCE,normochromaticerythrocytes;TI,ToxicityIndex.Inall cases2000polychromaticerythrocytes(PCE)wereanalyzed.

a_{Significantly}_different_from_negative_control₍_p_<_0.001)_(ANOVA_Tukey_test).

Table4

ChemicalcompositionofF3ofAchyroclinesatureioides.

Compound Parention[M−1]−_m/z _t_R_(min) _%_P/P_(mg_compound/100_mg_F3)

Chlorogenicacid 353 6.8 1.360±0.008

Caffeicacida ₁₇₉ _6.3 _0.004

±0.001

3-O-Methylquercetinb ₃₁₅ _22.3 _0.009_±_0.002

Quercetin-3-metoxi 331 5.1 nc

5,7,8-Trimethoxyflavoneb ₃₁₁ _6.6 _0.018

±0.002

Luteolin 285 21.9 0.537±0.010

a_3-_O_{-methylquercetin}_and_{5,7,8-trimethoxyflavone}_expressed_in_quercetin. b _Caffeic_acid_expressed_in_chlorogenic_acid.

nc=Isbelowthedetectionlimit.

1.5×1007

1.0×1007

5.0×1006

0.0

CN

3 mg/kg 6 mg/kg _{12 mg/kg}

CP

Treatment with F3

Tail moment

Arbitr

ar

y units

Fig.5. CometassayinbloodofBalb/cmiceinoculatedwith3,6and12mg/kgb.w. ofF3ofAchyroclinesatureioides.Resultsareexpressedastailmoment.Datashown aremeansoffourreplicatecellssamples(p<0.001;Dunnstest).

HPLC-ESI-MSanalysis,wewereabletoidentifythepresenceof differentflavonoidsand organicacids(Table4).The amountof eachcompoundinthefractionunderstudyisshowedinTable4

(quantificationanalysis).Dataareexpressedasmeans±standard deviation(SD)ofthreeseparateexperiments.

Thecomponentsdetectedinthesamplewere,inorderofhighest tolowest:chlorogenicacid,luteolin,5,7,8-trimethoxyflavone,3-O -methylquercetinandcaffeicacid.

Discussion

Medicinalplantsarearichsourceofmoleculesthatmayexert differentbioactivities.Consideringthatthereisnoeffective antivi-ral to treat diseases caused by alphavirus and, moreover, the disadvantages of usesynthetic drugs, is very relevant tostudy medicinalplantsasapossiblesolutiontosolvethisproblem.

CytotoxicitystudiesrevealedthatthemajorityoffractionsofA. satureioideshadlowtoxicityinbothtypeofcells.F1,F4andF5 indi-catedviabilitypercentsgreaterthan80%at200␮g/ml.F2showed highercytotoxiccapacityagainstVerocells(CC50=35␮g/ml)than againstequinelymphocytes(CC50>200␮g/ml).Probably,the com-poundspresentinF2donotaffecttheintegrityofthemembranes oflymphocytesatconcentrationsthatgeneratelysosomaldamage inVerocells.

GiventhatF3wasactiveagainstWEEVitscytotoxicpotential wasstudiedathigherconcentrations.F3showedCC50 valuesof 630and 668␮g/ml onVerocells and onequinePBMC, respec-tively.Moreover,theevaluationofF3againstequinelymphocytes allowsustoexpressthatthisfractionathighconcentrationsdidnot affectthistypeofcellsofimmunesystem.Thisisveryimportant becauseleucopeniaoccursduringviremiaperiodofWEEV(Peters

andDalrymple,1990).

Thereare fewresearcheswhichevaluatetheantiviralaction againstalphavirusesand,inparticular,againstWEEV(Madsenetal.,

2014;Ravehetal.,2013;Sindacetal.,2013).Therearenoantiviral

studiesemployingmedicinalplants.However,inapreviouspaper, wewereabletodemonstratetheabilityofanaqueousextractof

A.satureioidestoinhibitatWEEviruswithagreatselectivityindex

(SI=32)(Sabinietal.,2012).Inthepresentstudy,wedemonstrated

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againstthisalphavirus.ItsSIwasfourtimeshigherthantheSIof aqueousextract.Thisindicateswhichfractioncouldbebetterfor treatingalphavirusinfections.

Moreover, the SI of F3 was higher (126.0 and 133.6) than theSIofsyntheticcompoundssuchasthirdgeneration indole-2-carboxamidederivativeswhoseSIwereatorbelow14(Sindacetal., 2013).Theseresultssupporttheuseofnaturalproductsfortreating diseasesofviraletiology.

Respecttothe mechanismof action,thefractionexertedits bioactivityin stages intracellularof the replication.Further, F3 showedanimportantvirucidalaction.

ThechemicalstudiesindicatedthatF3contains,inorderof high-esttolowest:chlorogenicacid,luteolin,5,7,8-trimethoxyflavone, 3-O-methylquercetinandcaffeicacid.Thepresentstudyshowed that the fractionrich in chlorogenic acid and luteolin (F3) has strong inhibitory activity against Western Equine Encephalitis

virus.

Someinvestigationsreportedantiviralactionofthecompounds present inF3. However,there is nobackgroundaboutantiviral capacityagainstWEEVoralphavirus.Thus,Xuetal.,2014reported theantiviralabilityofluteolinagainstotherviruses,Enterovirus71

andCoxsackievirusA16.Inotherresearchitwasdemonstratedthe inhibitionofreplicationofEnterovirus71bychlorogenicacid(Li

etal.,2013).

Other researchers reported that herpes simplex virus was inhibited by caffeic acid (Ikeda et al., 2011). Also, caffeic acid inhibitedtheinfluenzaAvirusmultiplicationinvitro(Utsunomiya etal.,2014).AlthoughcaffeicacidisinlowproportioninF3itcould betheresponsiblecompoundofitsbioactivityagainstWEEV.

Also, it has been demonstrated antiviral capacity 3-O -methylquercetin, which wasshown tobea potentinhibitor of poliovirusRNAsynthesis(Castrilloetal.,1986).Maybe,the com-pounds presentin F3act aloneor in asynergistic combination againstWEEV.

Ontheotherhand,theresultsofgenotoxicassessmentsofF3 ofA.satureioidesbyMN(mononucleous)testshowedno signifi-cantincreasetheMNPCEfrequencyatalldosestested(3,6,and, 12mg/kg)at24hnorexhibitedtoxic activitybyanalysisofthe PCE/NCEratioinmousebonemarrowcells.Additionally,thecomet assayanalyzedinmice’sbloodindicatedabsenceofgenotoxicity inalltreatmentsofF3.Therefore,inthepresentwork,theinvivo

studiesindicatedthatF3ofA.satureioidesbybothtestsof cytogeno-toxicitywasneithertoxicnorgenotoxic,andtheobtainedresults supporttheapplicationofthisfractionsuchasantiviraldrugwith security.Furtherrelevantstudiesofantiviralactivityinvivowillbe developedusingamurinemodel.

Inconclusion,F3ofAchyroclinesatureioidesshowedlow cyto-toxicityinvitroinVeroandinequinelymphocytescells.Itshowed greatantiviralactioninvitroagainstWesternEquineEncephalitis

virusanditcouldbeusedasneurotropicalphavirusesinhibitor. Furthermore,thefractionatconcentrationstesteddidnotinduce

invivotoxicnormutageniceffects.Therefore,itssafeitsapplication asantiviralpotential.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

Conceived and designed the experiments: MCSand LS. Per-formedtheexperiments:MCS(cytotoxicassays,antiviralactivity andmicronucleitest),FMEandLNC(micronucleitest),FMandDI (cometassay),LC(chemicalstudies).Analyzedthedata:MCS,FM and,SNM.Contributedreagents/materials/analysistools:JJC,ML, JS,MC.Wrotethepaper:MCS.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbyGrantsfromCONICETandSeCyT, Resolution#763,years2009/11,fromUniversidadNacionaldeRío Cuarto.

WethanktheDiagrammaS.A.CompanyandMicrobiologist Car-losCrettonforhelpinguswithlyophilizationoftheaqueousextract ofA.satureioides.

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