Rev. bras. farmacogn. vol.27 número6

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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

Biological

activities

and

phytochemical

profile

of

Passiflora

mucronata

from

the

Brazilian

restinga

Marlon

H. de

Araujo

a,b

_,

_Isabel

_C.V.

_da

_Silva

c

_,

_Pollyana

_F.

_de

_Oliveira

a

_,

_Arielly

_R.R.

_Barreto

a

_,

Tatiana

U.P.

Konno

d

_,

_Francisco

_de

_A.

_Esteves

d

_,

_Thiago

_Barth

a

_,

_Fernando

_A.

_Aguiar

e

_,

Norberto

P. Lopes

e

_,

_Renee

_K.

_Dermenjian

f

_,

_Denise

_O.

_Guimarães

a

_,

_Ivana

_C.R.

_Leal

a,c

_,

Elena

B. Lasunskaia

b

_,

_Michelle

_Frazão

_Muzitano

a,∗

a_Laboratório_de_Produtos_Bioativos,_Curso_de_Farmácia,_Universidade_Federal_do_Rio_de_Janeiro,_Campus_Macaé,_Macaé,_RJ,_Brazil

b_Laboratório_de_Biologia_do_Reconhecer,_Centro_de_Biociências_e_{Biotecnologia,}_Universidade_Estadual_do_Norte_Fluminense_Darcy_Ribeiro,_Campos_dos_Goytacazes,_RJ,_Brazil c_Laboratório_de_Produtos_Naturais_e_Ensaios_Biológicos,_Departamento_De_Produtos_Naturais_e_Alimentos,_Faculdade_de_Farmácia,_Universidade_Federal_do_Rio_de_Janeiro,_Rio_de

Janeiro,RJ,Brazil

d_Núcleo_de_Estudos_em_Ecologia_e_{Desenvolvimento}_{Sócio-Ambiental}_de_Macaé,_Universidade_Federal_do_Rio_de_Janeiro,_Macaé,_RJ,_Brazil

e_Núcleo_de_Pesquisa_em_Produtos_Naturais_e_Sintéticos,_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil f_Department_of_Process_and_Analytical_Chemistry,_MRL,_Rahway,_NJ,_United_States

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7April2017

Accepted6July2017

Availableonline7September2017

Keywords:

Passifloraceae Terpenes Flavonoid Antimycobacterial Immunomodulatory Antioxidant

a

b

s

t

r

a

c

t

Ingeneral,Passifloraspecies havebeenreportedfortheirfolkmedicinaluseassedativeand anti-inflammatory.However, P.caeruleahasalreadybeenreportedtotreatpulmonarydiseases.Severe pulmonarytuberculosis,generallycausedbyMycobacteriumtuberculosisstrainsresistanttomultiple drugs,canleadtodeleteriousinflammationandhighmortality,encouragingnewapproachesindrug discovery.Thus,theaimofthisworkwastoevaluatethePassifloramucronataLam.,Passifloraceae, poten-tialfortuberculosistreatment.Specifically,relatedtoantimycobacterialactivityandanti-inflammatory relatedeffects(basedoninhibitionofnitricoxide,tumornecrosisfactor-alphaproductionand antioxi-dantpotential),aswellasthechemicalprofileofP.mucronata.Highperformanceliquidchromatography coupledwithdiode-arrayultravioletandmassspectrometeranalysesofcrudehydroalcoholicextractand ethylacetatefractionshowedthepresenceofflavonoids.Ethylacetatefractionshowedtobeas antiox-idantasGinkgobilobastandardextractwithEC50of14.61±1.25␮g/ml.Onemajorflavonoidisolated fromethylacetatefractionwascharacterizedasisoorientin.Thehexanefractionanditsmainisolated compound,thetriterpene␤-amyrin,exhibitedsignificantgrowthinhibitoryactivityagainst Mycobac-teriumbovisBCG(MIC501.61±1.43and3.93±1.05␮g/ml,respectively).Inaddition,Passifloramucronata samples,speciallyhexaneanddichloromethanefractions,aswellaspure␤-amyrin,showeda dose-relatedinhibitionoflipopolysaccharide(LPS)-inducednitricoxideproduction.Inconclusion,Passiflora mucronatapresentedrelevantbiologicalpotentialandshouldbeconsideredforfurtherstudiesusing invivopulmonarytuberculosismodel.

Introduction

The Passiflora genus, Passifloraceae, comprises about 520 species(Wohlmuthetal.,2010)thatarefoundpredominantlyin tropicalandsubtropicalregionsoftheworld(Dhawanetal.,2004). Somespecies,suchasPassifloraalata,P.edulisandP.incarnataare knownfortheirsedativepropertiesandhaverelevantinterestfor thefoodandpharmaceuticalindustry(Dhawanetal.,2004;Zeraik etal.,2010).

∗ _{Corresponding}_author.

E-mails:[email protected],[email protected](M.F.Muzitano).

SeveralspeciesofPassifloraexhibitbiologicalactivitythrough productionofdifferenttypesofsecondarymetabolites,especially flavonoids,phenolsandalkaloids(Dhawanetal.,2004).Theaerial partsofPassifloracaeruleaareusedinfolkmedicineasmild antimi-crobialagentindiseaseslikecatarrhandpneumonia(Anesiniand Perez,1993).TheethanolicextractofPassifloraincarnataexhibits anti-inflammatorypropertiesatadoseof125–500mg/kginrats (Borrellietal.,1996).Passifloraedulisleafextractshowedpotential antioxidantactivity(SunithaandDevaki,2009;Silvaetal.,2013).

PassifloramucronataLam.wasfoundasaclimbingplantina tropicalsandycoastalplant community,a habitatlocallycalled restinga,inthesoutheasternstateofRiodeJaneiro,Brazil(Garbin etal.,2012).TheplantcommunitiesaroundtheAtlanticrainforest

https://doi.org/10.1016/j.bjp.2017.07.005

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complex,suchasrestinga,differfromthecoreformationwhere theyexhibit more extremeenvironmental conditions;drought, salinity,hightemperaturesandpoorsoilnutritionarethemain lim-itingfactorsintherestingavegetationopenscrubhabitat(Scarano, 2002).

Toourknowledge,therehavebeennopharmacologicalstudies forP.mucronata,butratherecologicalandbotanicalinvestigations. Thepollinationbiologyoffourpassionflowerspecieswas stud-iedinsoutheastBrazil,specificallytheimportanceoffloralnectar chemicalfeatures,pigmentsandodors.Allspeciesrequired pol-linatorstoproducefruitsanditwasobservedthatP.mucronata waspollinatedbybats(Varassinetal.,2001).Gas chromatography-massspectrometry(GC–MS)analysisofdichloromethaneextracts fromP.mucronataflowerfringefilamentsrevealedeicosene, ben-zylalcoholandlimoneneasthemainconstituents(Varassinetal., 2001).P.mucronatahasbeenusedtotreatinsomnia,hemorrhoid, andassedativeandvermifugeformedicinalpurposesinQuissamã, RiodeJaneiroState,arestingaareawhichbelongstoJurubatiba NationalPark(BoloscoandValle,2008).ThisNationalPark com-prisedthecitiesofCarapebus,Quissamã,Macaéand hasa total areaof148.6km2₍_Imbassahy_et_al.,₂₀₀₉_).

BecauseotherspeciesofthegenusPassiflorashowantimicrobial, anti-inflammatoryandantioxidantactivitiesandinviewofthelack ofinformationonthisspecies,itisimportanttoinvestigatethe sec-ondarymetabolitesfoundinP.mucronataspeciesfromthisregion, andtoquestiontheirpossiblebiologicalactivity.Thisstudyaimed to:(1)evaluatetheactivityof leafcrude hydroalcoholicextract ofP.mucronataagainstMycobacterium,aswellasactivityofits fractionsandisolatedcompound(s);(2)verify thattheextracts, fractionsandisolatedcompoundsinhibitNOandTNF-␣ produc-tionbythemacrophagesandtheirabilitytoactasanantioxidant; (3)evaluatethecytotoxicityinthemacrophages,whicharethe Mycobacteriumhostcells;and(4)investigatethephytochemical profileofP.mucronataleaves.

Materialsandmethods

Generalexperimentalprocedures

1_H_NMR _and 13_C _NMR _spectra_were_obtained_from ₄₀₀_MHz

and100MHz,respectively,onaBrukerDRX-400NMR spectrom-eterand aVarianMERCURY-VXfrom400MHz.Chemicalsshifts (ı)werereferenced tointernal TMSstandards (ı=0, 1H)being expressed in parts per million (p.p.m.) units and the coupling constants(J)inhertz(Hz).Preparativeflashchromatographywas performedonsilica(SiliaflashGCO;70–230mesh).Eluateswere monitoredbythin-layerchromatography(TLC)onsilica60F254 usingbutanol/aceticacid/water(BAW8:1:1),visualizedunderUV light and revealed withNP-PEG or using hexane/ethyl acetate (9.5:0.5)revealedwithanisaldehyde/sulphuricacid.Thefractions wereanalyzedby HPLC-DADonShimadzuSCL-10A withDiode ArrayDetector SPD-M10Awithabsorptionsmeasuredfrom200 to450nm.Additionally,GC–MSanalysiswasperformedon GCMS-QP2010system(Shimadzu).

Plantmaterial

PassifloramucronataLam.,Passifloraceae,leaveswerecollected in February 2011 at the Restinga de Jurubatiba National Park, Quissamã,RiodeJaneiro,Brazil(22.19828◦_S;_41.46338◦_W;₁₀_m altitude).VoucherspecimensweredepositedatUniversityFederal RiodeJaneiroHerbarium,Brazil(RFA38758)afteridentificationby thebotanistTatianaU.P.Konno.Thisresearchhascompliedwith allrelevantfederalguidelinesandinstitutionalpoliciesrelatedto thebotanicalmaterialforresearchpurposes.

Extractionandisolation

Freshleaves(50.4g)weretrituratedandextractedwith500ml ofethanol/water(85:15)atroomtemperaturebymacerationfor 24h.Thesolventwasrenewedfivetimes,completingthe extrac-tionafter120h.Theextractwaslyophilizedandthenanaliquotof 6.96gofthetotaldriedcrudehydroalcoholicextract(codePMCE) (7.42g) was re-suspended withmethanol and partitioned with hexanetoobtainPMH(1.14g).Theresidualmethanolphasewas driedandre-suspendedwithpurewaterandpartitioned sequen-tiallywithdichloromethane,ethylacetateandbutanol,affording PMDM(532.9mg),PMEA(134.6mg),PMB(1.2465g),respectively. TheresidualaqueousphasewasnamedPMA(2.5915g).Analiquot of 501.6mg of PMH was chromatographed on a silica column (62.0×2.0cm; hexane/ethyl acetate/methanol gradient), yield-ing 271 fractions. Fraction PMH-113 crystallized via the slow evaporationofhexane/ethylacetate(8:2),affordingcompound1 (108.6mg)aswhitecrystallineneedles.Compound1was identi-fiedastriterpene␤-amyrinbasedonMSdata(GC–MSanalysis), m/z498[M+SiMe3]+(trimethylsilylated␤-amyrin),andNMR spec-troscopydata,inaccordancetoliteraturereports(Carvalhoetal., 1998).Analiquotof54.2mgofPMEAwaschromatographed on a reverse-phase C18-bonded silica column (61.0×1.5cm; dis-tilledwater/methanolgradient),yielding48fractions.Thefractions 18–19(85:15)and20–22(80:20)werecombinedaffording com-pound2(5.3mg).Compound2wasidentifiedasflavoneisoorientin by NMR spectroscopy data, in accordance to literature reports (Costaetal.,2011;Wenetal.,2007).

Gaschromatography–massspectrometry(GC–MS)analyses

Thehexanefractionandisolatedcompound1weredissolved in N-methyl-N-trimethylsilyltrifluoroacetamide, and resultant trimethylsilylatedproductsweredirectlysubjectedtoGC–MS anal-ysisonDBcolumn(␸0.25mm×15m,0.25␮mfilmthickness,J andWScientific)withheliumcarrier(flowrate1ml/min). Injec-torandinterfaceweremaintainedat270and230◦_C,_{respectively.} Columntemperatureincreasedfrom60to280◦_C,_with_a₁₅◦_C/min temperatureramp.

ChromatographicanalysisbyHPLC-DAD

Crudeextractfromleavesandethylacetatewereanalyzedby HPLC-DAD(Shimadzu)withanRP-18reverse-phasecolumn(5␮m particle, 250mm×4.60mm, Supelcosil, Supelco) maintained at 30◦_C. _The _eluents_were _H2O _adjusted _to_pH ₃ _with_H3PO4 _(A) and acetonitrile (ACN, B). Thefollowing mobile phase gradient (v/v)wasused:0–10min,A–B(100:0→82:18);10–35min,A–B (82:18→80:20);35–40min,A–B(80:20→79:21);40–45min,A–B (79:21→78:22);45–50min,A–B (78:22→0:100).Theinjection volumewas10␮landtheflowratewas1.00ml/min.HPLC analy-seswereperformedoneachsample,afterdilutionof5mgin1ml ofultrapurewater.

ChromatographicanalysisbyHPLC-MS

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(65:35→55:45);50–59min,A–B(55:45→40:60);60–69min,A–B (40:60→20:80);69–74min,A–B(20:80–0:100);75–76min,A–B (0:100→95:5).Theinjectionvolumewas10␮landtheflowrate was1ml/min.HPLCanalyseswereperformedoneachsample,after dilutionof5mgin1mlofultrapurewater.

Flavonoidquantificationinthecrudeextractanditsethylacetate fraction

TheflavonoidquantificationbyHPLCwascarriedoutusinga ten-pointcalibrationcurveobtainedusingrutin (Sigma–Aldrich ≥94%purity)amountsrangingfrom0.20to10.0␮g.The detec-torresponselinearityrangewasverifiedusingaseriesoftwo-fold dilutedrutinsolutions.Therelationshipbetweenpeakareas (detec-torresponses)andamountofrutinwaslinearover1000–20␮g/ml (r2₌_0.9999)._To_evaluate_the_injection_integration_{repeatability,}_the rutinstandardsolutionandsampleswereinjectedthreetimesand therelativestandarddeviationvaluescalculated.

Antimycobacterialactivity

Samples were evaluated for their antimycobacterial activ-ityusinga[3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide]MTTassaytomeasuremycobacterialgrowthinaliquid medium.Initially,Mycobacteriumbovis BCGstrainMoreau (pro-videdbyButantanInstitute–Brazil)orM.tuberculosisH37Rv(ATCC 27294)suspensionwasgrowninMiddlebrook7H9medium sup-plementedwith0.05%Tween80andalbumin,dextrose,catalase (ADC).Duringthemiddlelogarithmicgrowthphase,thebacterial suspensionwasplatedina96-wellmicroplate(1×106_CFU/well) inthepresenceofeachsampleatfourconcentrations.Thesealed platewasincubatedat37◦_C_and_5%_CO2 _for₇_days_for_M._bovis BCGand5daysforM.tuberculosisH37Rv.Afterthisperiod,the mycobacteriawereincubatedfor3hwithMTTsolutionandlysed viatreatmentwithlysisbuffer(20%w/vsodiumdodecylsulfate –SDS/50%dimethylformamide–DMFindistilledwater–pH4.7) (Gomez-Floresetal.,1995).Theplatewasincubatedovernightat 37◦_C _and_the_reading_was_made_using_a_UV _{spectrophotometer} at570nm.Aspositivecontrol,abacterialsuspensiontreatedwith isoniazid(Sigma–Aldrich≥99%purity),atconcentrationsof0.032, 0.16,0.8and4␮g/ml.Asanegativecontrol,anuntreated bacte-rialsuspensionwasused.Concentrationsarereportedintermsof ␮g/mlsothatisolatedcompoundcouldbeplottedtogetherwith fractionsandextract.

DeterminationofNOandTNF-˛productionfromLPS-activated RAW264.7cells

MacrophagesRAW264.7(ATCC,TIB-71)wereculturedin Dul-becco’sModifiedEagle’smedium(DMEM-F12)supplementedwith 10%fetalbovineserum(FBS)andgentamicin(0.2%)inthepresence 5%CO2at37◦_C._These_cells _were_seeded_in_96-well_microplates (2×104_cells/well)_in_the_presence_of_each_sample_at_three concen-trationsandstimulatedwith1␮g/mlLPS(Escherichiacoli0111:B4; Sigma-Aldrich).After24hincubationperiod,culturesupernatants werecollectedforNOandTNF-␣assays.IntheNOexperiments,a nitricoxideinhibitor,NG_-methyl-_l_-arginine_acetate_salt₍_l_-_N_MMA – Sigma–Aldrich 98% purity), was used as positive control at 20␮g/ml.Asanegativecontrol,untreatedmacrophageswereused. Nitrite,astableNOmetabolite,wasquantifiedbyusingtheGriess method(Griess,1879).Thenitriteconcentrationwascalculated fromaNaNO2standardcurve.Theopticaldensitywasmeasuredby spectroscopyat570nm.TNF-␣wasmeasuredbyanL929 fibrob-last(ATCC,CCL-1)bioassay,basedonsensitivityofL929cellsto cytotoxiceffectofTNF-␣.Forthis,theL929cellswereseededin 96-wellmicroplates(2.5×104_cells/well)._After₂₄_h_incubation,_the

resultingcellmonolayersweretreatedwiththemacrophage cul-turesupernatantsinthepresenceofactinomycinD(2␮g/ml).After anadditional24hincubation,theL929cellviabilitywasassayed bytheMTTmethod(Mosmann,1983).Thecytokineconcentrations weredeterminedbyusingarecombinantmousecytokinetoobtain astandardcurvecorrelatingcellularviabilityandTNF-␣ concentra-tion.Eachwell’sopticaldensitywasmeasuredat570nmemploying amicroplatereader(DynatechMR5000).

Cytotoxicity

CytotoxiceffectsofP.mucronatasamplesonmacrophageswere examinedusingacommercialLDHkit(Doles®).Releaseoflactate dehydrogenase(LDH,cytoplasmicenzyme)fromRAW264.7cells treated with the samples was determined colorimetrically, as described previously (Muzitano et al., 2006). Briefly, the cells (2×104 _cells/well),_seeded_in_96-well_microplates,_were_treated withsamplesat0.8,4,20and100␮g/mlfor24h.ExtracellularLDH concentrationswerequantifiedinsupernatantculture.Celllysates obtainedthroughthetreatmentwith1%TritonX-100wereused asapositivecontrol.DMSOwasusedassolventforthesample dilutions,andwastestedinparallelascontrol.

Antioxidantactivityassay

Passiflora mucronataextract and fractionsradical scavenging activitywasestimated usingstable freeradical 2,2-diphenyl-1-picryl-hydrazylhydrate(DPPH,Sigma).Theassaywasdetermined in96-wellmicroplatesusinga modifiedmethod(Mensoretal., 2001).Theextractsand fractionsweredilutedtothefinal con-centrationsof1, 5,10, 25,50, 100and 200␮g/ml inmethanol. Ginkgo bilobaextract (GBE) is an importantantioxidant extract and is composedmainlyby terpenoids, flavonoids (Jiang et al., 2017),aswellas,P.mucronatacrudeextractandethylacetate frac-tion.Foraninterestingcomparison,theGBEwasusedaspositive control and prepared using the same samples dilution proce-dures. In each well wereadded 125␮lof sample solution and 50␮lofDPPHsolution(300␮M).Themicroplatewasallowedto reactatroomtemperatureandabsenceoflight.After30minthe absorbancevaluesweremeasuredat518nmin spectrophotome-terSpectraMaxandconvertedintopercentageantioxidantactivity (AA%=100−{[(Abssample−Absblank)×100]/Abscontrol} (Gonc¸alves etal.,2015).Methanol(50␮l)plussamplessolutionorGBE(125␮l) wasusedasblank.DPPHsolution(50␮l)plusmethanol(125␮l) wasusedasnegativecontrol.TheEC50valueofeachsamplewas calculatedbynon-linearregressionfromtheplottedgraphof per-centageDPPHneutralizationvs.concentrationofsample.

Statisticalanalysis

The tests were performed in triplicate and values were expressedasmean±standarddeviation(SD).Statisticalanalyses wereperformedbyone-wayANOVA,followedbyTukey’s post-test.Theresultswereconsideredstatisticallysignificantforp<0.05. EC50,IC50andMIC50valueswerecalculatedbynon-linear regres-sionbasedontheconcentration-responsecurveofeachsampleby GraphPadPrism5.

Resultsanddiscussion

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37.35%.FromthePMEA,theflavonoidisoorientin(luteolin6-C -␤-d-glucopyranoside),compound2,wasisolatedandcharacterized byNMR.

Inviewofnewperspectivesforresearchoftuberculosis(TB) treatment, the antimycobacterial activity of the samples from P. mucronata was investigated. The search for new promising compoundsthatcouldbeusedasadjuvanttreatmentforsevere pulmonarytuberculosishasbeenfocusedbyourgroup(Machado etal.,2014;Venturaetal.,2015a,b,c).P.mucronatawaschosen becausePassifloraspecieshavebeenreportedfortheirpopularuse totreatpulmonarydiseases(AnesiniandPerez,1993)andfortheir anti-inflammatorypotential(Borrellietal.,1996).

Currently,tuberculosisstillrepresentsamajorthreattopublic healthinseveralregionsoftheworld.Mycobacteriumtuberculosis isthemaincausativeagentforthisinfectiousdiseaseinhumans. In 2015therewerean estimated10.4 millionnewcasesof TB, including580.000casesofmultidrug-resistantTB(MDR-TB)and 1.4milliondeathsfromthisdisease(WHO,2016).Thisdatashows theimportance ofa constant search fornew antimycobacterial agents.

Naturalproductsplayanimportantroleinthissearch,asplant specieshaveshownpromisinginvitroactivityagainstM. tubercu-losis,withalkaloids,terpenoidsandpolyphenolsrepresentingthe mostpromisingclassesofsecondarymetabolitesrelatedto antimy-cobacterialbioactivity (Okunade et al.,2004; Copp and Pearce, 2007;SalomonandSchmidt,2012).Thisstudyrepresentsthefirst timethatantimycobacterialactivitywasdescribedforP.mucronata and,toourknowledge,forPassifloragenus.

Intheinitialscreening,samplesfromP.mucronatawereable toinhibitM.bovisBCGgrowth.ThisattenuatedstrainofM.bovis is non-virulent,but closely related toM.tuberculosis (Mahairas et al., 1996). As shown in Fig. 1a, at 100␮g/ml, P. mucronata crudeextract(PMCE)inhibited79.22±3.48%oftheMycobacterium growth.PMH,PMDM,PMEA,PMBand aqueous(PMA)fractions inhibited97.92±0.56,86.21±0.0,38.63±2.40,47.50±2.40 and 47.42±7.98%,respectively,at100␮g/ml(Fig.1b–f).PMH,themost active,showedMIC50of1.61±1.43␮g/ml anddichloromethane fraction showed MIC50 of 8.83±1.17␮g/ml. Compound 1, at

100␮g/mlshowed86.76±0.21%ofMycobacteriumgrowth

inhi-bition(MIC50 3.93±1.05␮g/ml)(Fig.1g).Compound 2wasnot evaluatedduetothelowactivityfoundtoPMEA.

Inthenextstep,thethreemostpromisingsamples:PMH,PMDM andcompound 1wereevaluatedagainst avirulent strainofM. tuberculosisH37Rv.Althoughinhibitionpercentagesprovedlow, thefractionsandcompound1werestillabletoinhibitthegrowthof thisvirulentstrain(Fig.1i–k).Theseresultsareinagreementwith literaturedata,which reporteda lowactivityfor M.tuberculosis growthinhibitionby␤-amyrin(Martinsetal.,2011).Astudy eval-uatedamixtureoflupeoland␣-and␤-amyrinshowingpromising antimicrobialactivity,butconfirmedlowactivityof␤-amyrinonce isolated(Higuchietal.,2011).

Inflammationisstronglycorrelated withthepathogenesisof mostinfectiousdiseases,includingTB.Ingeneral,forprotection againstmycobacteria,theproductionofpro-inflammatory medi-ators, such as NO and TNF-␣, by the infected macrophages is essential.However,inthecasesofsevereformsofTB,suchas mil-itaryTBortuberculousmeningitis,additionalanti-inflammatory therapyisrequiredtopreventexcessiveinflammation(Garlanda etal.,2007).Inaddition,anti-inflammatorytherapyreduces mor-tality in patientsexhibiting hyperinflammatory phenotypethat couldbedeterminedbyhostgeneticpolymorphisms,increased bacterial virulence or specific comorbid states (Critchleyet al., 2013).

Such aspects could also be pre-clinical investigated dur-ing new TB drug development. In vivo studies done by our groupusingTBmodelin C57BL/6miceinfectedwiththehighly

virulentM.tuberculosisstrainM299reproducethe hyperinflamma-toryresponseofaTB-resistantimmunocompetenthosttohighly virulentmycobacteria.Thisresponsewasstronglyassociatedwith excessive recruitment of polymorphonuclear and mononuclear phagocytesandproinflammatorycytokineproductioninthelungs thatcancontributetopulmonarynecrosis(Almeidaetal.,2017).

The needfor suchan alternativetreatmentfor TB therefore exists,especiallyfortheseveredestructiveanddisseminatedforms ofTBfrequentlyassociatedwithexacerbatedinflammation. Per-hapsanidealsolutionwouldbedually-activecompoundswhich exhibitboth antimycobacterialand anti-inflammatoryactivities. Indeed,ourgrouphasdescribedseveralsuchcompounds possess-ingdualactivities(Machadoetal.,2014;Venturaetal.,2015a,b,c). The importance of development of newdrugs withdual, anti-inflammatory andantimycobacterial,activitiesis highlightedby emergencyofincreasingprevalenceofmultidrugresistant(MDR) TBandextensivelydrug-resistant(XDR)TB,whereapproximately oneinfivetuberculosisisolatesworldwideareresistanttoatleast onemajorfirst-line(Dhedaetal.,2017).

IntheevaluationofP.mucronataimmunomodulatory proper-ties, withfocus onpro-inflammatorymediators, itwas verified whethercrude extract,fractions and compound 1couldinhibit nitric oxide (NO) and tumor necrosis factor-alpha (TNF-␣) production, induced in RAW 264.7 macrophages stimulated with lipopolysaccharide(LPS). The PMCE showed IC50 value of

9.43±1.88␮g/ml. PMH, PMDM, PMEA, PMB and PMA showed

IC50 value of 4.24±1.90, 1.95±1.93, 14.69±3.04, 87.27±1.58 and>100␮g/ml,respectively.Compound1wasactiveatalltested concentrations(Fig.2),withIC50<0.8␮g/ml.Thissuggeststhat ␤-amyrincontributestotheactivityobservedforthehexanefraction fromwhichitwasoriginated.Isoorientin,isolatedfromPMEA,was reportedintheliteraturetosignificantlyinhibittheLPS-stimulated productionofNOinRAW264.7cells(Luyenetal.,2014).

With respect to TNF-␣ production, P. mucronata showed poor inhibitory activity (data not shown). Only hexane and dichloromethanefractionsshowedIC50valueof44.43±1.52and 56.52±1.34␮g/ml.However,TNF-␣productioninhibitoryeffects exhibitedbyPMHcanbeassociatedwiththeircytotoxicityinthe highestconcentration(100␮g/ml)(Fig.3).Previously,␤-amyrin obtainedfromEuphorbiahirtawasevaluatedforanti-inflammatory activityandshowedinhibitoryeffectonNOproduction by LPS-activated RAW 264.7 macrophages by inhibiting iNOS protein expression.However,whenotherinflammatoryfactorsasPGE2, TNF-␣andIL-6wereevaluated,onlyaslightinfluenceof␤-amyrin ontheirslevelswasobserved(Shihetal.,2010).

When cytotoxicity was analyzed, only the hexane fraction showedalevelgreaterthan35%intheconcentrationof100␮g/ml, butthisvaluewasnotmaintainedatlowconcentrations,which alsoshowedhighinhibitoryeffectsofNOproductionand Mycobac-teriumgrowthinhibition.Theremainingsamplesinthesmaller concentrationsshowednosignificantcytotoxicitywhencompared tocontrol0%lysis(p<0.05)(Fig.3).

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P. mucronata crude extract

Concentration (µg/ml)

Compound 1 (β-amyrin)

Compound 1 (β-amyrin) Isoniazid

Isoniazid Concentration (µg/ml)

Gro

wth inhibition (% Control)

Gro

Concentration (µg/ml) Concentration (µg/ml) Concentration (µg/ml)

Concentration (µg/ml) Concentration (µg/ml) Concentration (µg/ml) Concentration (µg/ml) Concentration (µg/ml)

P. mucronata hexane fraction

P. mucronata crude extract

P. mucronata ethyl acetate fraction

P. mucronata dichloromethane fraction

P. mucronata butanol fraction P. mucronata aqueous

P. mucronata hexane fraction

P. mucronata dichloromethane fraction

0 0.8 4 20 100 0 0.8 4 20 100 0 0.032 0.16 0.8 4

0 0.8 4 20 100

0 0.032 0.16 0.8 4

0 0.8 4 20 100

0 0.8 4 20 100 0 0.8 4 20 100 0 0.8 4 20 100

0 0.8 4 20 100 0 0.8 4 20 100

100

75

50

25

0 100

75

50

25

0

100

75

50

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Fig.1.Anti-mycobacterialactivityofPassifloramucronataextract,fractionsandcompound1at0.8,4,20and100␮g/ml.InhibitionofthegrowthofMycobacteriumbovisBCG

(A–H).PMCE(A),PMH(B),PMDM(C),PMEA(D),PMB(E),PMA(F),compound1(G).PositiveControl–MycobacteriumbovisBCGtreatedwithantibioticisoniazidat0.032,

0.16,0.8and4␮g/ml(H).NegativeControl–MycobacteriumbovisBCGuntreated.InhibitionofMycobacteriumtuberculosisH37Rv(I–L).PMH(I),PMDM(J),compound1(K).

PositiveControl–MycobacteriumtuberculosisH37Rvtreatedwithantibioticisoniazidat0.032,0.16,0.8and4␮g/ml(L).NegativeControl–MycobacteriumtuberculosisH37Rv

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* * *

*

*** *

* **

*

* *

125

100

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- + 4 20100 4 20 100 4 20 100 4 20 100 4 20 100 4 20100 4 20 100 µg/ml

PMCE PMH PMDM PMEA PMB PMA β-amyrin

Nitr

ic o

xide inhibition

(% control)

Fig.2.Evaluationofnitricoxide(NO)productionfromLPS-activatedRAW264.7macrophagestreatedwithPassifloramucronataextracts,fractionsandcompound1at4,

20and100␮g/ml.Crudeextract(PMCE),hexane(PMH),dichloromethane(PMDM),ethylacetate(PMEA),butanol(PMB)andaqueous(PMA)fractions.Negativecontrol:

macrophagesstimulatedwith1␮g/mlLPS(0.01%inhibitionand45.77±0.56␮MofNOproduction).Treatmentwithl-NMMAwasusedaspositivecontrolforNOinhibition,

reducing43.89±4.60%oftheNOproductionat20␮g/ml.Resultsrepresentthemean±standarddeviationoftwoindependentexperimentswithintriplicate.*p<0.05

comparedtonegativecontrol.

PMCE

PMDM PMEA PMB PMA PMH

β-amyrin 100

90

80

70

60

50

40

30

20

10

0 0

Cytoto

xicity(%)

* * * * *

0.8 4 20 100

Fig.3.CytotoxicactivityofPassifloramucronataextracts,fractionsandcompound

1at0.8,4,20and100␮g/mlonRAW264.7macrophagecells.Cytotoxicitywas

measuredbylactatedehydrogenase(LDH)specificreleasepercentage.Thespecific

releasewascalculatedaspercentageofmacrophagestreatedwithdetergentTriton

X-100(1%)aspositivecontrol(Lise100%)andmacrophagesnon-treatedasnegative

control(Lise0%).Arithmeticmean±standarddeviation(n=3).*p<0.05compared

tocellsnon-treated.

isoorientinwaspreviouslyreportedwithveryhighactivity(EC50 8.0±0.2␮g/ml)andcanbecomparabletothatofascorbicacidin DPPHradicalscavengingantioxidantactivity(Sientzoffetal.,2015). Hexanefractionwasnottestedbecauseitsinsolubilityandlackof probableantioxidantcharacteristiccompounds.

InordertobetterunderstandP.mucronataantioxidant activ-ity,andalsotocontributetophytochemicalknowledgeaboutthis species,PMCEandPMEAwereanalyzedbyHPLC-DAD-MSto ver-ifytheirchemicalprofile(Fig.5a–d).Majorpeaks,14and15,at tR 18.07 and 18.51min, displayed thetypical UV absorptionof flavonoids withmax at 271and 337nm, and 269and 349nm, respectively(Fig.5b).Besidesmajorflavonoids, HPLC-DADdata allowedtheidentificationofadditionalflavonoidpeaksbetweentR 15.77–23.32min.Theethylacetatefractionchromatogramshowed onlyonemajorpeak,number19,attR18.44min,alsocharacteristic offlavonoid(Fig.5c).

HPLC-MSwasusedtoidentifythemajorflavonoidspresentin theethylacetatefraction(Fig.5d,Table1),comparingMSspectrum andelutionorderforisomerspairs.Anumberofflavonoid glyco-sideswhich havebeenreportedinotherPassifloraspecieswere identified.Isochaftoside,schaftoside,isoorientin,orientin,vitexin andisovitexinareflavonesconsideredstandardsusedtoidentify differentPassifloraspecies (Sakalem etal., 2012), suchas Passi-floraedulisfo.flavicarpa,P.incanata,P.tripartite(Sakalemetal., 2012;Zucolottoetal.,2012;Pereiraetal.,2005;Abourashedetal.,

150

135

120

105

90

75

15 30

0 60

45

EC

50

µ

g/ml

CTL+ PMCE PMDM PMEA PMB PMA

*

Fig.4.AntioxidantactivityofPassifloramucronataextractandfractionsevaluated

byDPPH•

assay:crudeextract(PMCE),hexane(PMH),dichloromethane(PMDM),

ethylacetate(PMEA),butanol(PMB)fractionsandaqueous(PMA).Ginkgobiloba

extractwasusedaspositivecontrol(CTL+).Thevaluesshownrepresentthemedian

effectiveconcentration(EC50)andareexpressedas␮g/ml.Resultsrepresentthe

mean±standarddeviationofthreeindependentexperimentswithintriplicate.*No

significantdifferencewhencomparedtoCTL+.

2002).Flavonoidglycosidesareamongthecompoundsresponsible forPassifloraactivityinthecentralnervoussystem,as sedative-hypnotics,anxyoliticand analgesic(Sakalemetal.,2012).These identified and isolated flavonoids seem not to be involved in antimycobacterialactivity,sinceethylacetatefractionpresented lowactivity.However,theycouldparticipateinanti-TBactivityof P.mucronataextract,usinginvivomurinemodel,sincetheypresent anti-inflammatorypotential,describedhereandinliterature(Melo etal.,2005;Zucolottoetal.,2009).

Flavonoidswerequantifiedbasedonanarea×␮gcalibration curve obtainedusing a rutin external standard. The sum of all flavonoidpeaks in chromatogram wasassumed torepresent P. mucronataextracttotalflavonoidcontent.Resultsareexpressedas apercentage(w/w)−g/100goflyophilizedextract.Itwasfound that flavonoids represent13.32% w/wof thecrude extract and 14.78%w/wofthePMEA.ItisimportanttomentionP.mucronata extractflavonoidcontentwashigherthanothersobservedinplant specieswhereflavonoidsareresponsibleforpharmacological activ-ity(Muzitanoetal.,2011).

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mAU

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a

b

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Fig.5. Passifloramucronatacrudeextractchromatogramat254nm(A).Ultravioletspectraofcrudeextractchromatogrammajorpeaks,attR18.07min(peak14)and18.51min

(peak15)(B).ChromatogramofP.mucronataethylacetatefractionat254nm(tR18.44)(C).HPLC-DAD-MSchromatogramofP.mucronataethylacetatefraction,where

1-isoschaftoside;2-isoorientin;3-apigenindi-C-␤-hexoside;4-schaftoside;5-apigenindi-C-␤-hexoside;6-isovitexinorvitexin;8-methoxyluteolin-Chexoside;9-orientin

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Table1

TheflavonoidspresentinethylacetatefractionfromPassifloramucronata.

Flavonoids Retentiontime(min) Molecularweight [M+H]+·_m/z _Referencesa

ApigeninC-pentosideC-hexoside(isoschaftoside) 19.2 564 565 [I,II]

LuteolinC-hexoside(isoorientin) 19.4 448 449 [I–IV]

Apigenindi-C-␤-hexoside 20.2 594 595 [I,II,III]

ApigeninC-pentosideC-hexoside(schaftoside) 20.8 564 565 [I,III]

Apigenindi-C-hexoside 21.6 594 595 [II,III]

ApigeninC-hexoside(isovitexinorvitexin) 21.9 432 433 [I–IV]

Methoxy-luteolin-C-hexoside 22.6 462 463 [II]

LuteolinC-hexoside(orientin) 22.9 448 449 [I–IV]

a_I,_Sakalem_et_al.₍₂₀₁₂₎_;_II,_Zucolotto_et_al.₍₂₀₁₂₎_;_III,_Pereira_et_al.₍₂₀₀₅₎_;_IV,_Abourashed_et_al.₍₂₀₀₂₎_.

steps,itwillbeinvestigatedtheinvivopotentialofP.mucronata activesamplesusingaTBmodel,previouslydescribed(Almeida etal.,2017),usefulfortestingofnewapproachesforthetreatment ofsevereTB,aimedatreducingthehyperinflammatoryresponse andthepreventionorreductionofpulmonarynecrosis.

Authors’contributions

TUPKcontributedincollectingplantsample,identificationand herbariumconfection.Conceivedanddesignertheexperiments: MFM,EBL,ICRL.Performedtheexperiments:MHA,ICVS,PFO,ARRB, FAA.Analyzedthedata:MHA,ICVS,MFM,EBL,ICRL,DOG,TB,NPL, FAE.Wrotethepaper:MHA,MFM,ICVS,RKD.Alltheauthorshave readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankCNPq,FAPERJandInstitutoMacaédeCiência eTecnologia(IMCT)forfinancialsupportandfellowship.

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