Rev. bras. farmacogn. vol.26 número2

(1)

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

Original

Article

Antiviral

activity

of

Myracrodruon

urundeuva

against

rotavirus

Alzira

B. Cecílio

a,∗

_,

_Pollyana

_de

_C.

_Oliveira

a

_,

_Sérgio

_Caldas

a

_,

_Priscilla

_R.V.

_Campana

b

_,

Fernanda

L. Francisco

b

_,

_Maria

_Gorette

_R.

_Duarte

c

_,

_Lorena

_de

_A.M.

_Mendonc¸

_a

b

_,

_Vera

_L.

_de

_Almeida

b

a_Divisão_de_Plataformas_{Tecnológicas,}_Diretoria_de_Pesquisa_e_{Desenvolvimento,}_Fundac¸_ão_Ezequiel_Dias,_Belo_Horizonte,_MG,_Brazil

b_Divisão_de_{Desenvolvimento}_Tecnológico_{Farmacêutico,}_Diretoria_de_Pesquisa_e_{Desenvolvimento,}_Fundaç_ão_Ezequiel_Dias,_Belo_Horizonte,_MG,_Brazil c_Divisão_de_{Epidemiologia}_e_Controle_de_Doenç_as,_Diretoria_do_Instituto_Octávio_Magalhães,_Fundaç_ão_Ezequiel_Dias,_Belo_Horizonte,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26December2014 Accepted7October2015 Availableonline2December2015

Keywords: Medicinalplant Myracrodruonurundeuva Crudeextract

Leaves

Antiviralactivityrotavirus

a

b

s

t

r

a

c

t

MyracrodruonurundeuvaAllemão,Anacardiaceae,isamedicinalplantwidelyfoundinBrazil,especially inthenorthernregion.Inourpreviousstudy,theethanolicextractfromleavesofM.urundeuvashowed antiviralactivityagainstsimianrotavirusSA-11.Here,thecrudeextractwassubjectedtofractionationsin ordertosubsequentlyworkwithmoreconcentratedandpurebioactivecompounds,whichwereanalyzed byTLCandHPLCmethodstosupportabetterunderstandingoftheirvirucidaleffect.Theantiviralactivity wasevaluatedusingarotavirusinfectionmodelinMA-104cellstreatedwiththemaximumnon-cytotoxic concentrationofthecrudeextractanditsfractions.Datawereexpressedasthepercentageinhibitionof viralreplicationcalculatedbytheinhibitionofcytopathiceffectinthetreatedcellscomparedtountreated controlsafter48hofincubation.First,weconductedafractionation,generatingfivefractions(F1–F5) whichweresubmittedtoantiviralassay.Then,thefractionthatshowedthehighestvirucidaleffect(F3, PI=75%)wassubjectedtoalargerpartition,yieldingeighteensubfractions,whichweresubmittedto newantiviralassays.Terpenes,flavonoidsandtanninswerethemajorsecondarymetabolitesdetected byTLCanalysisinF3.SF1,aflavonoid-enrichedfraction,showedthestrongestinvitroactivityagainst rotavirus(PI=92%),preventingcytopathiceffect.ChromatographicprofileswereobtainedbyHPLCforthe crudeextractandSF1,themostpotentsubfraction.Overall,ourdatapointtothepotentialanti-rotavirus activityofflavonoid-enrichedfraction(SF1)ofM.urundeuvaleaves,corroboratingthetraditionaluseof thisspeciestotreatdiarrheaandbroadeningourperspectivesoninvivoassaysinmicewithSF1isolated orassociatedwithotherfractions.

Introduction

Rotavirusesarerecognizedasamajorcauseofnon-bacterial

gastroenteritisespeciallyininfantsandyoungchildrenworldwide

(Parasharetal.,2006;Junaidetal.,2011).Itisanunpredictable

diseasehighlycontagiousandmayleadtoseveredehydrationand evendeath(Offit andClark,2000).Itstransmissionispersonto personthroughthefecal-oralroute.Controlandpreventionofthis infectionaredifficultduetothelackofanyeffectivetreatment

otherthanpalliativemeasuresandthepresenceofasymptomatic

children shedding virus(Dennehy, 2000). Despite two liveoral rotavirusvaccinesthathavealreadybeenlicensed,amonovalent

human rotavirus vaccine (Rotarix, GlaxoSmithKline Biologicals)

and a pentavalent bovine-human reassortant rotavirus vaccine

(RotaTeq,Merck),theireffectivenessindevelopingcountrieshas

∗ Correspondingauthor.

E-mail:alzira.cecilio@funed.mg.gov.br(A.B.Cecílio).

shownapooledefficacyofabout51%requiringeffortstooptimize

(Jiangetal.,2010).

Nevertheless,inseveralcases,wherethehostissufferingfrom prolongeddiarrheaandfever,virus-specifictreatmentwillbe nec-essaryifpossible(Takahashietal.,2001).Naturalproductshave

beenthe sourceofmostof theactive ingredientsof medicines

andmorethan80%ofdrugsubstanceswerenaturalproductsor

inspiredbyanaturalcompound(Harvey,2008).Inthiscontext, invitroassays have beenestablishedand usedby ourresearch grouptoscreenantiviralactivitiesofextracts,fractionsandnatural substanceswithpotentialtherapeuticalaction.

Inpreviouswork,usingsimilarmethodologyemployedhere,

weevaluatedethanoliccrudeextractsofdifferentBrazilian medic-inalplantsandweobservedaninvitroanti-rotavirusactivityof Myracrodruon urundeuva Allemão, Anacardiaceae (Cecílio et al., 2012),amedicinalplantwidelyusedinBrazil,mainlyduetoits

anti-inflammatory, antimicrobialand wound healing properties

(Monteiroetal.,2006;Souzaetal.,2007;Sáetal.,2009a).Thisplant

isarepresentativespeciesoftheAnacardiaceaefamilyoccurringin

http://dx.doi.org/10.1016/j.bjp.2015.10.005

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BrazilandotherSouthAmericancountries,notablyintheCerrado region(Leite,2002).

Analgesic, anti-inflammatory, antioxidant, antifungal, and

antibacterial, among other activities, have been reported from

preparationsmadewithM.urundeuvaextracts.Vianaetal.(1997,

2003)haveobserved analgesicandanti-inflammatoryeffects of

thetannin and chalcones fractions isolated from M.urundeuva

barksinstudiesconductedinmice.Souzaetal.(2007)havealso shownthattannin-enrichedfractionfromstembarkofthisplant presentsanti-inflammatoryandantiulcer effectsin mice,partly duetoitsantioxidantaction,knowntobepresentinpolyphenols, includingtannins.Interestingly,DeMendonc¸aAlbuquerqueetal.

(2011)demonstrated inhibitionofmyeloperoxidaseactivityand

antioxidanteffectsofchalconesfromM.urundeuvastembarkson anallergicconjunctivitismodelinguineapig,indicatingthemas candidatesforthetreatmentofallergicconjunctivitisandother

inflammatoryconditions.

AccordingtoSáetal.(2009a),lectinsisolatedfromM. urun-deuvaheartwoodshowedantimicrobialactivityagainstbacteria andfungithatattackplants,includingwoods.Inaddition,stembark hydroethanolicextractwasactiveagainstStaphylococcusaureus, Klebsiellapneumonia,EnterococcusfaecalisandCandidaspp.(Alves

etal.,2009;Gomesetal.,2013),andtheseactivitieswereattributed

tothepresenceofbioactivecompoundssuchastannins,flavonoids andalkaloids.

Furthermore,experimentsinratsthatpointtoanti-diarrheal

(Chavesetal.,1998)andneuroprotectiveactivities(Nobre-Júnior

etal.,2009),colonicanastomotichealing(Goesetal.,2005),and evenlarvicidaleffectagainstAedesaegypti(Napoleãoetal.,2012) andtermiterepellentaction(Sáetal.,2009b)showthe biologi-calpotentialofthisplant,whichhidesarichsourceofcompounds thatcouldbeemployedtotherapeuticandbiotechnological appli-cations,amongotherpurposes.

ConsideringthepharmacologicalpotentialofM.urundeuvaand ourpreviousdataofanti-rotavirus activity(Cecílioetal.,2012), thecrudeextractfromtheleavesofthisplantwasfractionatedand subjectedtoantiviralassay,beingtheactivefractionscharacterized

byTLCmethodinanattempttoidentifythebioactivecompounds

involvedinthevirucidaleffect.

Materialandmethods

Plantmaterial

MyracrodruonurundeuvaAllemão,Anacardiaceae,leaveswere collectedfromadultplantsinthe“cerrado”areaofSantanado Pira-pama,intheStateofMinasGerais,Brazil,betweenSeptember2006 andFebruary2007.Theplantwasidentifiedasavoucherspecimen

andwasdepositedattheHerbárioPAMGdaEmpresadePesquisa

AgropecuáriadeMinasGeraisunderthenumberPAMG53312.

Preparationofextractandfractionation

Thecrudeextract (MUL)waspreparedby percolationofthe

driedandpowderedmaterialwithethanol95GL(VetecQuímica

Fina)untilexhaustionatroomtemperatureandevaporatedunder

reducedpressureat40◦_C._The_ethanolic_extract₍₂₅₀_g)_was

frac-tioned by filtrationchromatography in silica gel (silica gel 60, 0.040–0.063mm,Merck),givingfivefractionsafterelutionwith

hexane(F1,30g),dichloromethane (F2,21g),

dichloromethane-ethyl acetate (1:1) (F3, 14.7g), ethyl acetate (F4, 45.5g) and

methanol(F5, 120g). A portionof themostpotentfraction, F3

(10g), was further fractioned in a silica gel column (silica gel

60,0.040–0.063mm,Merck),withsolventsofincreasing

polari-ties(dichloromethane,ethylacetate,ethylacetate,methanol,water

andformicacid1%andmixturesofthese),yieldingeighteen sub-fractions.SF1(47mg),obtainedwithDCM:EtOAc(1:1),showedthe bestantiviralactivityanditschemicalcompositionwasevaluated byTLCandHPLCprofiles.Thelowamountobtainedprevented fur-therfractionation.

Phytochemicalscreening

MUL,fractions(F1–F5)andthesubfractionSF1weresubjected tophytochemicalscreeningtodeterminethepresenceofdifferent classesofnaturalproductsusingmethodsdescribedbyWagnerand

Bladt(2001).Theanalysiswasperformedbythin-layer

chromatog-raphy(TLC)onMercksilicagel60F254aluminumplates,which

weredevelopedaccordingtoTable1.Thepresenceoftanninswas determinedusingaproteinprecipitationtest(Matos,1997).

HPLCanalysis

MULand SF1were preparedat10mg/mland at5mg/mlin

MeOHHPLCgrade,respectively(VetecQuímicaFina).After

cen-trifugation at 9300×g (Eppendorf, model, 5415D) thesolution

wasinjected in an Agilent Technologies 1200 seriesHPLC

sys-temequippedwithDADdetector.Chromatographicanalysiswas

obtainedinaZorbaxXDBC18column(50×4.6mm,1.8␮m),at

40◦_C,_flow_rate_of_0.3_ml/min_and_detection_at₂₁₀_nm._The_profiles

wereobtainedinlineargradientelutionofwater(A)and acetoni-trile(B),from15to90%ofBfrom0to50min,andfrom90to95%

ofBbetween50and60min.TheUVspectrawererecordedonline

inthe190–400nmrangeforallretentiontimes.

Biologicalassays

Samplepreparation

For thebioassays, each samplewas solubilizedin

dimethyl-sulfoxide(DMSO,Sigma–Aldrich)at50mg/mlandcentrifugedat

9300×g(Eppendorf,model,5415D).Thesamplesweredilutedto

workconcentrationusingculturemedia.

Cellsandviruses

MA-104cells(arhesusmonkeykidneycellline)werecultivated

inDulbecco’smodifiedEaglemedia(DMEM)supplementedwith

10%fetalbovineserum(FBS),100␮g/mlofstreptomycin(Gibco)

and100U/mlpenicillinG(Invitrogen).Thecellcultureswere main-tainedat37◦_C_in_a_humidified_5%_CO₂_atmosphere._Simian_rotavirus

SA11wereactivatedwith10␮g/mltrypsinfor60minat37◦_C_and

propagatedinMA-104cellsmonolayersinthepresenceof10␮g/ml

trypsin.Thevirustiterswere estimatedfromcytopathogenicity

bythelimit-dilutionmethodandexpressedas50%tissueculture infectiousdoseperml(TCID50/ml)(ReedandMuench,1938).

Cytotoxicity

Thecytotoxicityofthesamples(MUL,fractions and

subfrac-tions)wasdeterminedusingthemethoddescribedbyMirandaetal.

(1999)basedoncellularmorphologicalterations.Several

concen-trations(5000,500,50,5and0.5␮g/ml)ofsampleswereplacedin

contactwithconfluentMA-104cellsmonolayerspreparedin

12-wellmicroplatesandwereincubatedat37◦_C_in_a_humidified_5%

CO2atmospherefor48h.Aftertheincubationperiod,thecellswere examinedusinganinvertedopticalmicroscope(Nikon)andtreated

anduntreatedcultures(control)werecompared.Thehigher

con-centrationofeachextractshowingnocellularmorphologicchanges

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Table1

TLCconditionstotestthepresenceofdifferentclassesofnaturalproductsintheethanolicextract.

Class Eluent Spray

Tannins Toluene:aceticacid:formicacid(70:167:14) K3Fe(CN)61%:FeCl32%(1:1)

Flavonoids Ethylacetate:methanol:water(176:22:22) AlCl3reagent(2%)

Anthraquinones Toluene:acetone:chloroform(40:25:35) KOHreagent(5%)

Terpenes Hexane:ethylacetate(1:1) Anisaldehyde-sulfuricacidreagent

Cardiotonicglycosides Ethylacetate:methanol:water(20:3:2) Keddereagent Alkaloids Ethylacetate:formicacid:aceticacid:water:ethylmethylcetone(86:16:23:47:78) Dragendorffreagent

andwasconfirmedusingPromega’sCytoTox96®_{Non-Radioactive} CytotoxicityAssay,accordingtothemanufacturer’sinstructions.

Antiviralassay

Antiviralactivitywasevaluatedbasedontheabilityofsamples todiminishthemultiplication of thevirus, includinginhibiting thecytopathiceffect(CPE)of rotavirusontreatedMA-104 cells monolayers(4×104cells/well)cultivatedin96-wellmicroplates.

Thus,MULandfractions(atMNTC)wereaddedtothecells, fol-lowedbytheadditionof10TCID50ofactivatedrotaviruses,and theplateswereincubatedina5%CO2atmosphereat37◦Cfor48h. Theantiviralactivitywasexpressedasapercentageofinhibition

(PI)(Mirandaet al.,1999)usingantilogarithmvalues ofTCID50

for rotavirus,as follows:PI=[1−(antilogarithm T/antilogarithm

C)]×100being“T”viraltiteroftreatedcellsand“C”viraltiterof

untreatedcells(positivecontrol).Experimentswerecarriedoutin octoplicate.ThepresenceofrotaviruswasalsoverifiedbyRT-PCR.

RNAextraction

SupernatantsfromtheantiviralassaysweresubmittedtoRNA

extractionbyamodifiedsilicamethod(Boometal.,1990).Briefly,

60␮lofeach sample wastreated with200␮llysis buffer(60g

guanidineisothiocyanate,Invitrogen,50mlof0.1MTris–HClpH 6.4,Invitrogen, 11mlof 0.2MEDTApH8.0, Invitrogen, 1.3gof TritonX-100,PackardInstrumentCo.)and50␮lofsterilized sil-icasolution(preparedaccordingtoBoometal.,1990).Afterbeing

centrifuged,thesilica waswashed witha washing buffer(60g

guanidine isothiocyanate) (Invitrogen), 50ml of 0.1M Tris–HCl

pH 6.4 (Promega), followed by two washes with 70% ethanol

(Merck)and acetone(Merck).Thematerial wasresuspendedin

watertreatedwithdiethylpyrocarbonate0.1%,andafterbeing cen-trifuged,theRNAwascollectedintheupperphaseandmaintained at−80◦_C_until_required.

cDNAsynthesis

ThecDNAsynthesiswasconductedina20␮lreaction contain-ing7%dimethylsulfoxide,7␮lofviralRNAand1␮Moftheprimers

RotaA–Fwd1:5′_{GGATGTCCTGTACTCCTTGTCAAAA3}′_and_Rota_A

–Rev1:5′_{TCCAGTTTGGAACTCATTTCCA3}′_,_which_amplify_a_144-bp

product(Loganetal.,2006)fromrotavirusVP6region.The reac-tionwasincubatedat95◦_C_for₅_min_and_then_chilled_on_ice_for

5min.Next,1×reactionbufferwasadded,3mMMgCl2,0.5mMof eachdNTPand1␮lImpromIITM_Reverse_{Transcriptase}_(Promega). Finally,thereactionwasincubatedfor5minat25◦_C,₆₀_min_at

42◦_C, _and _the_enzyme _was_inactivated_for ₁₅_min_at ₇₀◦_C. _The

cDNAsweremaintainedat−80◦Cuntilrequired.

PCRassay

ThePCRassaywasconductedinareactionof50␮lcontaining 5␮lofcDNA,22mMTris–HClpH8.4,55mMKCl,1.65mMMgCl2,

220␮Meachdeoxynucleosidetriphosphate(dNTP),800nMofthe

sameprimersusedinthecDNAsynthesisand0.5Urecombinant

TaqDNAPolymerase(Invitrogen).Thecyclingprogramconsistedof aninitialdenaturationat95◦_C_for₁₀_min,_followed_by₃₀_cycles_of

95◦_C_for₁₅_s,₆₀◦_C_for₁_min_and₇₀◦_C_for₁_min._Each_reaction_set

containedthereferencesampleSA11asapositivecontrol. Nega-tivecontrolconsistedofareactionwithallreagentsincludedand waterinsteadofcDNA.Then,10␮lofthePCRamplifiedproductwas analyzedonasilver-stained8%polyacrylamidegelelectrophoresis (PAGE).

Results

Determiningthemaximumnon-cytotoxicconcentration

Tocarryouttheinvitroantiviralassays,wefirstdeterminedthe concentrationofMULandfractionatedsamplesthatcouldbeused withoutcausingdamagetothecell,basedoncellularmorphologic

alterationsandPromega’sCytoToxAssaydata.Themaximum

non-toxicconcentrations(MNTC)observedwere500␮g/mlforMUL,

and50␮g/mlformostofthegeneratedfractions,exceptforF2and SF3,4,6and15,whichshowedMNTCat5␮g/ml(Table2).

Fig.1Ashowsacytotoxiceffectcharacterizedbytypical

mor-phological alterations including detachmentof cells from their

substratum and disruption of cell monolayer, compared with

healthycontrolcells(Fig.1C).

Table2

Maximumnon-toxicconcentration(MNTC)andpercentinhibitionofrotavirus(PI) inMA-104cellstreatedwithcrudeextract(MUL)fromMyracrodruonurundeuvaleaf anditsfractions(F)andsubfractions(SF).

Selectedmaterials MNTC(␮g/ml) PI(%)

Crudeextract

MUL 500 100

Fractions

F1 50 0

F2 5 21

F3 50 75

F4 50 21

F5 50 68

Subfractions

SF1 50 92.06

SF2 50 0

SF3 5 80.05

SF4 5 0

SF5 50 68.38

SF6 5 36.90

SF7 50 20.57

SF8 50 20.57

SF9 50 0

SF10 50 0

SF11 50 0

SF12 50 20.57

SF13 50 0

SF14 50 0

SF15 5 0

SF16 50 0

SF17 50 60.19

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Fig.1.TherhesusmonkeykidneycelllineMA-104.(A)Typicalcytotoxiceffectinducedbytoxicconcentrationofextract.(B)Typicalcytopathiceffectobservedincells infectedwiththesimianrotavirusSA11.(C)Uninfectedanduntreatedcontrolcells(magnification,20×).

Antiviralassays

TheMNTCofthesampleswereusedtoconducttheantiviral assaystoassesstheirpotentialactivityagainstrotavirus.Basedon cytopathiceffect,wedeterminedthepercentinhibition(PI)ofthe samplesonthereplicationofrotavirus.Therewerenocytopathic effects(CPE)incellstreatedwiththeMUL.However,weobserved CPE(datanotshown)anddifferentPI(rangingfrom0to75%)inthe cellstreatedwiththefractions(Table2).Thus,thefractionwiththe highestPI(F3,PI=75%)wasselectedforfurtherfractionation.The testswiththesubfractionsrevealedthatnoneofthemwereable toinhibit100%ofviralreplication,andmostofthemshowedno antiviraleffect.Interestingly,SF1(asubfractionfromF3)showed thehighestPI(92%),evenhigherthanF3(Table2).Fig.1Bshowsthe typicalCPEinducedbythevirusinMA-104cells.The morpholog-icaldataobservedinopticalmicroscopywereconsistentwiththe moleculardataassayedbyRT-PCR.Theresultsoftheamplifications werevisualizedinasilver-stained8%polyacrylamidegelshowing thebandcorrespondingtothe144-bpfromrotavirusVP6region.

PhytochemicalandHPLCanalysis

MUL,F1–F5andSF1weresubjectedtophytochemical

analy-sistodeterminethemainclassesofnaturalproductswhichcould beinvolvedintheantiviralactivity.Tannins,flavonoidsand

ter-penes were detected in MUL. F1 and F2 showed terpenes, F4

andF5showedflavonoidsandtannins,andF3showedterpenes,

flavonoidsandtannins(Table3).BecauseF3showedthehighest PI,it wasselectedforanadditionalfractionation,yielding eigh-teensubfractions.Then,SF1,thesubfractionwiththehighestPI, wasalsoanalyzed,revealingflavonoidsandterpenes.Finally,

chro-matographicprofilesofMULandSF1wereobtainedbyHPLC.MUL

showedapredominanceofcompoundsoflowpolarityasshownin

Fig.2A.SF1,whichhadthehighestantiviralactivity,showedpeaks distributedthroughoutthechromatogram,indicatingthepresence

ofcompoundswithdifferentpolarities.Mostofthecompounds

wereelutedwithretentiontimelowerthan20min.Inthistime

range,weobservedtwomajorpeaks(r.t.of17.16and18.84min) withUVprofilecharacteristicofflavonoids(Fig.2B).Thisfraction

alsoshowedpeakswithretentiontimehigherthan40min,which

hadlowUVabsorption,withmaximumabsorptionatca.210nm.

Discussion

Our tests were conductedusing a simple but very effective

modelfordetectionofbioactivesamples.Ourstrategywasto deter-minetheworkingconcentrationofcrudeandfractionatedextract, andthendeterminethepercentageinhibitionofrotavirus replica-tioninMA104cells.TheSimianrotavirusstrainSA11waschosen becausethis strainis wellcharacterizedasa groupArotavirus andisone ofthemostwidelyusedreferencestrains(Gutiérrez etal.,2010).Rotavirusesaredifferentiatedintosevengroups(A–G).

Groups A,B, and C areassociated withacute gastroenteritisin

humansandanimalswhilegroupsD,E,F,andGonlyinfectanimals

(SaifandJiang,1994;Westermanetal.,2006;EstesandGreenberg,

2013).Besides,groupA rotavirusesare themost commonviral

agentscausingdiarrhealinfectionsinchildrenyoungerthanthree years(Parasharetal.,2006;Junaidetal.,2011).Thesevirusesare internalizedintoMA104 cellsshowingtypicalcytopathiceffects

(MalherbeandStrickland-Cholmley,1967;LópezandArias,1992;

Gonc¸alvesetal.,2005;Loganetal.,2006;Gutiérrezetal.,2010;

Wolfetal.,2011)whichareessentialfeaturesforthesuccessofour test.

Rotaviruses are nonenvelopedviruses composedof a

triple-layeredproteincapsidthatsurroundstheviralgenome. Nonstruc-turalglycoprotein4(NSP4)encodedbyrotavirusistheonlyviral proteincurrentlybelievedtofunctionasanenterotoxin. Identifi-cationofNSP4asthefirstviralenterotoxinisofinterestbecause itshowsthisproteinhaspleiotropicpropertiesbesidesits intra-cellularroleinviralreplication.Theoutermostlayeriscomposed oftheproteinVP7 andprotrudingspikesoftrimericVP4(López

andArias,2006;EstesandGreenberg,2013).TheVP4cleavageby

trypsinpromotesrearrangementsintheviralparticlesthatrigidify thespikesandisrequiredforreceptorbindingandcellpenetration

(Lópezetal.,1985;Ariasetal.,1996).

In ourwork,thechemical fractionationof thecrude extract

resultedinfivefractions,andoneofthem(F3)showedpotential antiviralactivitywithCPEinhibitionat50␮g/ml(datanotshown) andasatisfactoryPI(75%),althoughlowerthantheactivityfound tothecrudeextract(PI=100%).Asexpected,thefractionation

pro-ducedsampleswithhighercytotoxicityduetotheconcentration

ofsomecompoundclasses,forcingtheevaluationoftheantiviral activityatlowerconcentrationsthanthatusedforthecrudeextract.

Table3

Phytochemicalscreeningandyieldofcrudeethanolicextract(MUL),fractions(F)andsubfractions(SF)fromMyracrodruonurundeuvaleaf.

Selectedmaterials Extractyield(%,w/w) Tan Fla Ant Ter CarGly Alk

MUL 19.4 + + − + − −

F1 12.0 − − ND + ND ND

F2 8.4 − − ND + ND ND

F3 5.9 + + ND + ND ND

F4 18.2 + + ND − ND ND

F5 48.0 + + ND − ND ND

SF1 0.5 − + ND + ND ND

(5)

min

10 20 30 40 50 60

mAU

0 200 400 600 800 1000

1373

1833

1977

2060

2452

9431

12 001 _{13 173}

47 179

50 296

56 080

57 215

57 626

60 129

60 840

61 437

61 811

62 385 62 926 63 752 _{64 244}

min

10 20 30 40 50 60

mAU

0 200 400 600 800

1371

1482

1646

1821

1983

2057

2.225 2.455 2692

11

122

11

817

15

892

17

164

17

836

40

433

54

317

55

703

60

167

64

248

A

B

1

2

Fig.2.ChromatographicprofileofcrudeextractMUL(A)andfractionSF1(B)withUVspectraobtainedonlineforpeaks1and2.

Interestingly,afurtherF3fractionationyieldedtwoactive subfrac-tions(SF1andSF3)withPIvaluesgreaterthanF3(92%and80%, respectively).InSF1,whichshowedthehighestactivity,flavonoids werethemajor class ofcompounds foundin additiontosmall amountsofterpenes,asevidencedbyTLC(datanotshown)and HPLCprofiles.TheTLCanalysisofthisfractionshowed predomi-nanceofspotswithyellowfluorescenceunderUV365nmlight,which wasintensifiedwiththeuseofNP/PEGsprayreagent.Thisfinding wascorroboratedbytheHPLCprofile,whichshowedpeaks asso-ciatedwithUVspectracharacteristicofflavonoidsandmaximum absorptionatca.275and350nm(Fig.2).Terpeneswereevidenced

bythepresenceofspotsofpurplecolorwhentheTLCplatewas

sprayed with anysaldehyde/sulfuric acid and these compounds

maybetheresponsibleforthepeaksobservedinthechromatogram ofSF1withretentiontimesinthe40–65minrange.Thefinal purifi-cationofthechemicalconstituentsofSF1wasnotperformeddue tothelowamountobtainedforthissample(47mg).

The antiviral activities of flavonoids have been extensively

reported.Massspectrometricdatahavedemonstrated the

pres-ence of gallotannins in M. urundeuva (Da Silva et al., 2011).

Interestingly,gallotanninsarepotentcalcium-activatedCl−

chan-nel inhibitors whose biological activity provides antisecretory

benefits(Namkunget al., 2010).On the otherhands, there are

evidencesthatNSP4,anonstructuralglycoproteinreleasedfrom

rotavirus-infected cells, induces the release of Ca2+ _from _the

endoplasmicreticulum,resultinginincreasedparacellular perme-abilityinenterocytes,aswellasincreasedsecretioninthecrypt cells mediated by activation of Cl− _transporter ₍_Ramig, ₂₀₀₄_).

Thus,thesefindingspoint toapossiblecourseofactionforthe anti-rotavirusactivityofM.urundeuva.

Anotherinteresting findingin literature, which corroborates withthepotentantiviraleffectofflavonoid-enrichedfractionSF1, istheinhibitoryactivityrelatedtoseveralflavonoidsonreverse transcriptasesandproteasesevenatlowconcentrations(Koetal., 2009).Trypsininhibitoryactivitieshavealsorecentlybeen

iden-tified in flavonoids extracted from orange peel and green tea

leaves(Shahwaretal.,2013).Sincetheinfectivityofrotavirusesis increasedbytrypsinandthevirusesreplicateprimarilyin intesti-nalenterocytesduringanaturalinfection,itispossiblethatthe

flavonoids present in SF1, may be disrupting viral entry by a

proteaseinhibitormechanism.Therefore,ourdataprovideinsights forfurtherstudieswiththisflavonoid-enrichedsubfraction, focus-ingonthesuppressiveeffectofviralamplificationintheearlyphase ofinfection,andonaninhibitoryactivityonapossiblemechanism ofactioninvolvingtheNSP4enterotoxin.

Finally,itisimportanttoconsiderthatasinglecompoundcould notberesponsibleforthefullantiviralactivityandthecombination ofcompoundswithbalancedproportionsincrudeextractcouldbe actinginanadditiveorsynergisticmoderesultinginmoreeffective virucidalactivity.Theseconsiderationsencourageustoperform

additionalstudieswithSF1andMULusingothercomplementary

testsinvitroand invivoassaywithmurinerotavirusstrainsin mice.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor

(6)

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authorscontributions

ABC,coordinationoftheresearchandwritingofthemanuscript; PCO,biologicalassays;SC,PCRassayandanalysis;PRVC, phyto-chemicalscreeningandfractionation;FLF,preparationofextract;

MGRDandVLA,HPLCanalysis;LAMM,preparationofextract.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsthanktheFundac¸ãodeAmparoàPesquisadoEstado deMinasGerais(FAPEMIG)forfinancialsupport(N◦_EDT-3253/06)

ofthisresearchandtheFundac¸ãoEzequielDias(Funed).

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