Rev. bras. farmacogn. vol.25 número4

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Activity

of

Fabaceae

species

extracts

against

fungi

and

Leishmania:

vatacarpan

as

a

novel

potent

anti-Candida

agent

Dandara

Braga

Santana

a

_,

_Raphaella

_Correia

_da

_Costa

a

_,

_Renata

_Mendonc¸

_a

_Araújo

b

_,

José

Elias

de

Paula

a,1

_,

_Edilberto

_Rocha

_Silveira

c

_,

_Raimundo

_Braz-Filho

d,e

_,

_Laila

_Salmen

_Espindola

a,∗

a_Laboratório_de_{Farmacognosia,}_Faculdade_de_Ciências_da_Saúde,_Universidade_de_Brasília,_Campus_{Universitário}_Darcy_Ribeiro,_Brasília,_DF,_Brazil

b_Instituto_de_Química,_Universidade_Federal_do_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

c_Departamento_de_Química_Orgânica,_Universidade_Federal_do_Ceará,_Fortaleza,_CE,_Brazil

d_Laboratório_de_Ciências_Químicas,_Universidade_Estadual_do_Norte_Fluminense,_Campos_dos_Goytacazes,_RJ,_Brazil

e_Departamento_de_Química,_Universidade_Federal_Rural_do_Rio_de_Janeiro,_Seropédica,_RJ,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28May2015 Accepted28July2015 Availableonline15August2015

Keywords:

Enterolobiumellipticum

Sclerolobiumaureum

Vataireamacrocarpa

Vatacarpan

Leishmania

Fungi

a

b

s

t

r

a

c

t

Leishmaniasisandfungalinfectiontreatmentefficacyislimitedbytoxicityandeverincreasing resis-tancetoavailabledrugs,requiringdevelopmentofalternativecompounds.TherichnessofCerradoplant antimicrobialsecondarymetabolitesjustifiesscreeningofFabaceaespeciesextracts:Enterolobium ellip-ticumBenth.,Sclerolobiumaureum(Tul.)Baill.andVataireamacrocarpa(Benth.)Ducke,againstLeishmania (Leishmania)amazonensis,yeastsanddermatophytes.Amongthe26extractstested,morethan50%ofthe totaldemonstratedsignificantantifungalactivityincomparisontothedrugcontrols(minimalinhibitory concentration0.12to≤31.25␮g/ml).Sixextractscapableofcompleteparasiticgrowthinhibitionhadthe

inhibitoryconcentrationindexfor50%valuesfrom9.23to78.65␮g/ml.Theresultsledtotheselection oftheV.macrocarpaethylacetaterootbarkextractforchemicalfractionation.Thisplant, tradition-allyreferredtoasangelim-do-cerradoormaleiteira,isusedtotreatsuperficialmycosesinAmazonia.A previouslyunreportedpterocarpanvatacarpantogetherwiththeknowncompoundmusizinwas iso-lated.Vatacarpandemonstratedaminimalinhibitoryconcentrationvalueof0.98␮g/mlagainstCandida albicansATCC10231,andthuscomparableorsuperiortofluconazoleandamphotericinB.Theresults addtoliterature’sinformationtheabilityofpterocarpanstoactasantimicrobialagents.

Introduction

Natural products and their ethnopharmacological activities havebeenhistoricallyusedasaprimarysourceofcompoundsfor drugdiscovery.However,forthelasttwentyyears,thetechnical requirementsforhighthroughputscreeningledthe pharmaceuti-calindustrytofocusoncombinatorialchemistrylibrariesinstead. Thispathwaywasonlypartlysuccessfulsinceitcameupwith struc-turesthatlackedthecomplexscaffoldofsecondarymetabolites. Currently,naturalproductsareagainasourceofdrugdiscovery, providingleadcompoundsforclinicaltrials,including antimicro-bialagents(Diasetal.,2012;Harveyetal.,2015).

Theincidenceoffungalinfections hassignificantlyincreased sincethelate1960s,primarilywithcasesofimmune-compromised orhospitalizedpatients.Researchdedicatedtothedevelopment

∗ _{Corresponding}_author.

E-mail:darvenne@unb.br(L.S.Espindola).

1_In_memoriam.

of new therapeutic strategies has culminated in four antifun-gal classes currently used in clinicalpractice: fluoropyrimidine analogs, polyenes(suchasamphotericinB, themostcommonly usedmedicationfor systemicinfections)azolesand equinocan-dines(Vandeputteetal.,2012).

Cutaneous leishmaniasis is considered a neglected tropical diseases group,with 1.5million peopleworldwide manifesting skinlesionswhichpersistforseveralmonths,orevenyears(Silva etal.,2013).Pentavalentantimonials(Sb+5₎_remain_the_primary

treatmentoption,despitetheirtoxicityandlowpatienttolerance, witheitherpentamidineoramphotericin Bused assecondline treatments.

The failure of the aforementioned drug strategies has been attributed to microbial resistance. The use of plants by tradi-tional local communities and the richness of Cerrado species antimicrobialsecondarymetaboliteshavebeenreported(DeAssis etal., 2014;Albernazetal., 2012;MeloeSilvaetal., 2009;De Mesquitaet al.,2005).We screenedFabaceaeextractsfromthe Cerrado:EnterolobiumellipticumBenth.,Sclerolobiumaureum(Tul.) Baill.andVataireamacrocarpa(Benth.)Ducke,againstLeishmania

http://dx.doi.org/10.1016/j.bjp.2015.07.012

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(Leishmania) amazonensis, yeasts and dermatophytes. These antimicrobialresultsledtotheselectionofV.macrocarparootbark ethylacetateextractforfurtherchemicalstudies.Here,wepresent theisolation,structureelucidationandantimicrobialactivityofone pterocarpan.Thisnewnaturalproductwasmoreactivethanthe antifungalreferencefluconazoleandamphotericinB.

Materialsandmethods

Plantextracts

Plant species Enterolobium ellipticum Benth., Sclerolobium aureum (Tul.) Baill. and Vatairea macrocarpa (Benth.) Ducke, Fabaceae,werecollectedin2010fromtheCerradobiomeinthe LagoaFormosaarea,Planaltina,FederalDistrictofBrazil,south lat-itude15◦₂₇′_34.2′′_; _south _longitude₄₇◦₉₂′_3.3′′_; _at_an _altitude_of 1071m, and subsequently identifiedbybotanist Prof.JoséElias dePaula.VouchernumberswerekeptintheUniversityofBrasilia (UB/UnB)Herbarium(Table1).Plantorganswereseparated,dried, stabilized,pulverizedandextractedbymacerationwithhexane, ethylacetateandethanol.Theextractivesolutionswere concen-tratedin a rotary evaporator, yielding different crude extracts, whichwerestoredat−20◦C.

Antifungalactivitymethod

Theminimalinhibitoryconcentration(MIC)ofeachextractwas determinedforyeasts:CandidaalbicansATCC10231,C.parapsilosis

ATCC 22019 and C. glabrataLMGO 44, using CLSIM27-A3 and M27-S4 protocols(CLSI, 2008a, 2012); and for dermatophytes:

TrichophytonmentagrophytesLMGO 09and Trichophytonrubrum

LMGO 06, following the CLSI M38-A2 protocol (CLSI, 2008b); withminormodificationsaspreviouslydescribedbytheresearch group(DaCosta etal., 2014)(see Supportinginformation).The LMGO (Laboratório de Micologia de Goiás) strains are clinical isolatesfrompatientsattheFederalUniversityofGoiásHospital, Brazil.Itraconazole(Sigma),flucytosine(Sigma)andamphotericin B (Sigma) were used as reference compounds for yeasts and dermatophytes.Afourthpositivecontrolfluconazole(Sigma)was usedforyeastsonly.

Invitroantileishmanialactivity

The extracts were tested against Leishmania (Leishmania)

amazonensis(L(L)a)(MHOM/BR/PH8)promastigotesmaintainedin Schneiderculturemedium(Sigma)with20%heat-inactivatedfetal calfserum,at22–25◦_C._Extracts_were_dissolved_in_DMSO_(Sigma) anddilutedinSchneider’smedium(Sigma).Theinhibitory concen-trationindexfor50%(IC50)ofthepromastigoteswasdetermined

bytheMTTmethod.Experimentswereperformedaspreviously described(DaCostaetal.,2014)(seeSupportinginformation)with amphotericinB(Sigma)usedasthereferencedrug.

Generalexperimentalprocedures

Column chromatography was performed using silica gel 60 (230–400mesh, Merck).ThinLayer Chromatography(TLC) was

Table1

Fabaceaespeciesextractactivity–determinationoftheminimuminhibitoryconcentration(MIC–␮g/ml)againstyeastsanddermatophytes,andIC50(␮g/ml)against

Leishmania(Leishmania)amazonensis.

Fabaceaefamily species Vouchernumber

Extract Plantpart (solvent)/%yield

MIC(␮g/ml) IC50(␮g/ml)

L.(L.)amazonensis

C.albicans ATCC10223

C.parapsilosis ATCC22019

C.glabrata LMGO44

T.mentagrophytes LMGO09

T.rubrum LMGO06

Enterolobiumellipticum Benth

(UB)3739

RW(h)/0.37 250 250 125 500 250 >100 RW(ea)/0.56 1.95 3.91 >1000 500 62.5 >100 RW(e)/9.05 >1000 >1000 >1000 >1000 1000 70.05 RB(ea)/1.44 500 125 >1000 500 500 >100 SW(h)/0.32 >1000 1000 500 1000 500 78.65 SW(ea)/0.61 0.98 31.25 >1000 250 250 >100 SB(h)/0.55 1000 500 250 500 1000 25.87 SB(ea)/2.18 >1000 15.62 >1000 125 500 9.23 L(ea)/5.64 125 15.62 >1000 250 >1000 >100

Sclerolobiumaureum (Tul.)Baill. (UB)3818

RW(h)/0.42 >1000 >1000 >1000 1000 250 >100 RW(ea)/0.62 0.48 0.12 31.25 31.25 31.25 >100 RW(e)/6.06 1.95 3.91 15.62 31.25 31.25 >100 RB(ea)/1.98 7.81 7.81 15.62 15.62 31.25 >100 SW(h)/0.25 1000 500 500 500 31.25 >100 SW(ea)/0.66 0.48 15.62 15.62 15.62 31.25 >100 SW(e)/3.47 0.12 0.12 0.12 15.62 31.25 >100 SB(ea)/2.05 7.81 15.62 7.81 15.62 31.25 >100 L(ea)/4.74 250 7.81 1000 >1000 >1000 >100

Vataireamacrocarpa (Benth.)Ducke (UB)3815

RW(h)/0.48 0.98 1.95 500 62.5 500 72.39 RW(ea)/0.90 3.91 3.91 250 125 500 >100 RB(ea)/9.78 0.98 0.98 31.25 62.5 125 71.47 SW(h)/0.43 250 250 >1000 1000 125 >100 SW(ea)/0.78 1.95 1.95 500 62.5 125 >100 SW(e)/4.25 0.24 1.95 500 31.25 62.5 >100 SB(ea)/4.84 1.95 1.95 250 31.25 31.25 >100 L(ea)/8.58 1.95 1.95 250 62.5 62.5 >100 Itraconazole 0.125 0.0625 0.125 0.25 0.125 –

Flucytosine 0.25 0.0635 0.125 – –

Fluconazole 1 0.5 4 4 2 –

AmphotericinB 4 4 16 4 – 0.067

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performedonprecoatedsilicagelaluminumplates(TLCSilicagel 60 F254, Merck).The compounds werevisualizedby UV

detec-tionand/orsprayedwithasolutionofvanillin/sulphuricacid/EtOH. OpticalrotationwasmeasuredonaPerkinElmer341polarimeter. TheIRspectra(KBRpellets)wererecordedusingaPerkin-Elmer FT-IR1000spectrometer.Thehighresolutionelectrospray ioniza-tionmassspectra(HRESIMS)wereacquiredusingaLCMS-IT-TOF (225-07100-34)Shimadzuspectrometer.Themassspectrawere recordedonaShimadzuQP5000/DI-50spectrometer,operating withelectron impactat 70eV. The1D and 2D NMR datawere acquiredonaBrukerAvanceDPX-300spectrometer,equippedwith a5mmmultinuclearinverseprobe,usinggradientfieldintheZ directionandamagnitudeof10A.Chemicalshifts,givenonthe ıscale,werereferencedtotheresidualundeuteratedportionof theCDCl3,deuteratedsolvent.Allstandardpulsesequenceswere

providedbytheBrukerTOP-SPINsoftware.Allexperimentswere conductedatroomtemperature.

Extractionandisolation

A 115.70g portion of V. macrocarpa root bark was dried, powderedandsubsequentlymaceratedinethylacetateatroom temperature,yielding11.83gofextractaftersolventevaporation underreducedpressure.An8.28gportionoftheEtOAcextractwas fractionatedwithhexane,EtOAcandMeOHasthebinarymixture ofincreasingpolarityandyielded65fractionswhichwereanalyzed byTLC.

Flashchromatographyoffractions 19–22(749.9mg),by elu-tionwithhexane,ethylacetateandMeOHasabinarymixtureof increasingpolarityyielded316fractionsanalyzedbyTLC.

Fraction3(50–61)(5.10mg)correspondedtomusizin(2). Fraction 9 (176–179) (27.5mg) and fraction 10 (180–183) (40.3mg)wereregroupedfollowingTLCanalysis.Anewflash chro-matographywasconductedwhichyielded141fractionsanalyzed byTLC.Fraction3(16–21)(4.60mg)wasfoundtobeapreviously unreportedpterocarpan–namedhereinasvatacarpan(1).

Vatacarpan(1).Whitesolid;m.p.135.2–136.4◦C;[_␣]

D=+63◦ (c.0.1,MeOH);1_H_and13_C_NMR_(CDCl

3,300MHz)seeTable2;

HR-ESI-TOFMS(1)m/z423.2166[M+H]+_(calcd_for_C

26H30O5).

Musizin(2).Browncrystals,1H(CDCl3,300MHz):6.99(s,H-4),

7.08(d,7.8,H-5),7.48(t,7.8,H-6),6.84(d,7.8,H-7),2.76(s,H-12), 2.66(s,H-13),17.35(s,OH-1),10.24(s,OH-8)and13_C_NMR:_205.2

(C-11),168.8(C-1),158.8(C-8),138.4(C-10),133.0(C-6),132.4 (C-3),122.1(C-4),117.7(C-5),113.8(C-2),113.2(C-9),111.4(C-7), 32.4(C-12),25.5(C-13).

2'

2'' 3'

3''

4'' 4'

5'

5''

1'

4 4a

1a 11a

10a 8 7

7 6

5 4

10 3

9

8 1

11 2 12

13

6

7a 6a 3

2

1

1''

HO

H

₃

CO

OH

O

2

10 9

OH

1

Resultsanddiscussion

Atotalof26extractswerepreparedfromtheFabaceae fam-ily:ninefromE.ellipticum,ninefromS.aureum,andeightfrom

V.macrocarpa.Ofalltheextracts,85%presentedactivityagainst aminimumofonefungalstrainusingtheactiveextractcriterion (MIC≤125␮g/ml)specifiedbyAlbernazetal.(2010).Inaddition,

Table2

1_H₍₃₀₀_MHz)_and13_C₍₇₅_MHz)_NMR_data_assignments_for_vatacarpan_(CDCl 3).

Carbon number

HSQC HMBC

ıC ıH 2JCH 3JCH

1 114.3 6.95(s) H-11a 1a 123.2 –

2 143.7 –

3 146.6 – H-1/MeO-3

4 116.0 –

4a 147.3 – H-1/H-11a

6 70.6 4.02(d,10.0) 3.64(d,10.0)

H-11a

6a 46.7 – 7 124.4 7.01(d,7.9)

7a 123.9 – H-8/H-10

8 107.7 6.36(dd,7.9,2.2) H-10/H-7a

9 156.8 – H-7

10 98.6 6.34(d,2.2)

10a 160.9 – H-7

11a 83.1 5.10(s) H-1 1′ _26.7 _3.34_(d,_6.5)

2′ _122.8 _5.14_(t,_6.5) _3H-4′_/3H-5′ 3′ _131.0 _– _3H-4′_/3H-5′

4′ _18.1 _1.76_(s) _3H-5′ 5′ _25.9 _1.66_(s) _3H-4′ 1′′ _31.2 _2.45_(d,_7.5) _H-6_␤ 2′′ 118.9 5.23(t,7.8) 3H-4′′/3H-5′′ 3′′ _135.6 _– _3H-4′′_/3H-5′′

4′′ _18.2 _1.54_(s) _3H-5′′ 5′′ _26.2 _1.70_(s) _3H-4′′ MeO-5 61.7 3.79(s)

morethan50%ofthetotalsamplesdemonstratedsignificant activ-ityincomparisontothedrugcontrols(MIC0.12to≤31.25␮g/ml) (Table1).Theinhibitoryconcentrationindexfor50%ofthe pro-mastigotesfromL.(L.)amazonensis(IC50determinedbytheMTT

method)wascalculatedforthesixextractscapableofcomplete parasiticgrowthinhibitionataconcentrationof100␮g/ml:four extractsfromE.ellipticumandtwofromV.macrocarpa(IC509.23to

78.65␮g/ml);withnoactivityobservedforS.aureum(Table1). InspiredbytheclinicaluseofamphotericinBinthetreatmentof fungalinfectionsandleishmaniasis,weselectedtheV.macrocarpa

rootbarkEtOAcextractforchemicalstudiesbasedonitsactivity, yield,quantityandpolarity.Silica-gelopencolumn chromatogra-phyofthis8.28gextract(fungalMIC0.98–125␮g/mlandparasite IC5071.47␮g/ml)yielded65fractions.

Fractionationrevealedanunreportedcompoundnamedherein asvatacarpan(1),togetherwithmusizin(2)(Covelletal.,1961).All compoundstructureswereestablishedusingspectroscopic tech-niques,including1Dand2DNMRanalysis,andcomparisonwith previouslyreporteddata.

Compound 1, was a white solid (m.p. 135.2–136.4◦C). The infrared (IR) spectrum showedabsorption bands at 3384cm−1 characteristicofhydroxylgroupsandat1620and1570cm−1_of_the skeletalvibrationsofthebenzenering.Asymmetricandsymmetric C O Cstretchingofaryl-alkyletherat1261–1199and1085cm−1, respectively,werealsoobservedinadditiontophenolabsorptions at1380and1165cm−1_._The_HRESIMS_of₁_displayed_a_molecular ionpeakatm/z423.2166correspondingtothemolecularformula C26H30O5.

Inthe1_H_NMR_spectrum₍₃₀₀_MHz,_CDCl

3),amethoxygroupat

ıH3.79(s)andanaromaticprotonatıH7.26(H1,s)wereobserved,

consistentwithapentasubstitutedbenzenemoietyandthree aro-maticprotonscompatiblewiththepresenceofanABXspinsystem atıH6.34(H10,d,2.2),6.36(H8,dd,7.9,2.2)and7.01(H7,d,7.9).

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5.14(H2′,t,J=6.5Hz)and5.23(H₂′′,t,J=7.5Hz),inadditiontothe twomethyleneprotonsatıH3.34(H1′,d,J=6.5)and2.45(H₁′′,d, J=7.5).ThecorrelationofthesesignalsintheCOSYspectrum con-firmedthetwoprenylmoieties.Inaddition,the1_H_NMR_spectrum

revealedsignalsforadiastereotopicmethyleneatıH4.02(H6␣,d, 10.0)and3.64(H6␤,d,J=10.0Hz)andanoxymethineprotonatıH

5.10(H11a,s)typicaloftheoxymethyleneandoxymethineprotons

oftheheterocyclicringB(2H-6)andC(H-11a)ofa6a-substituted pterocarpanskeleton,respectively.Thepterocarpanskeletonwas supportedbythecorrelationdisplayedbetweenH-6andH-11ain theNOESYspectrum.

The13_C_and_DEPT₁₃₅◦NMRspectroscopicdataof₁(Table2) revealed26carbonatoms,correspondingto:fourmethyls(␦C18.1,

18.2,25.9and26.2);amethoxyl(␦C61.7);threemethylenes(␦C

70.6,31.2and 26.7);one oxymethine(␦C 83.1); one

nonhydro-genated(␦C46.7);fourolefiniccarbons(ıC118.9,122.8,131.0and

135.6);fourhydrogenatedaromaticcarbons(␦C98.6,107.7,114.3

and124.4);andeightquaternaryaromaticcarbons(␦C116.0,123.2,

123.9,143.7,146.6,147.3,156.8and160.9).The1_H,13_C-HSQC

spec-trumexhibitedcorrelationbetweenthehydrogensat␦H4.02(H6␣,

d,10.0),3.64(H6␤,d,10.0Hz)and5.10(H11a,s)withthe

oxycar-bonsat␦C70.6(C-6)and83.1(C-11a),respectively.TheseNMR

signalstogetherwiththemethinecarbonat␦C46.7(C-6a),ratified

thepresenceofa6a-substitutedpterocarpanskeleton. Thedeshieldingofthemethylenehydrogens2H-1′ (_␦

H 3.34)

when compared with the 2H-1′′ ₍_␦

H 2.45), was associated to

anisotropiceffectofthearomaticringa-locatedattheC-1′_carbon. Inaddition,theHMBCcorrelationofthe2H-1′(_␦

H3.34)withthe

carbonsat␦C116.0(C-4),and147.3(C-4a)establishedtheposition

theoneprenylfragmentatC-4inthearomaticringA.OtherHMBC correlationofthemethoxylprotons(s,3.79)withthesignalat␦C

146.6(C-3),andconcomitantcorrelationoftheH-1(s,6.95)with thesignalsat␦C123.2(C-1a),146.6(C-3),and83.1(C-11a),

indi-catedthepositionofthemethoxylandhydroxylgroups,atC-3and C-2,respectively,confirmedthesubstitutionpatterninaromatic ringA.ThelocationofthehydroxylgroupattheC-9carbonofthe trisubstitutedaromaticringDwasbasedonHMBCcorrelationof thehydrogenat␦H7.01(H-7)tothecarbonsat␦C156.8(C-10a)

and160.9(C-9)andconcomitantcorrelationofthehydrogensat ␦H6.36(H-8)and6.34(H-10)withthecarbonat␦C123.9(C-7a),

inaccordancewithbiogeneticbackgrounds(Dewick,2002). TherelativestereochemistryoftheBandDpterocarpanrings wasdeterminedbytheNOESYexperiment,whichrevealeddipolar interactionsofthemethylene2H-1′′withthehydrogensH-6and H-11a.Inaddition,thepositiveopticalrotationvalue[␣]D=+63◦

(c.0.1,MeOH)wasdecisivefortheassignmentoftheabsolute ste-reochemistryofthestereogeniccentersC-6aandC-11aasS(Araújo etal.,2008).Thus,compound1wasidentifiedas(+)-6aS,11aS -2,9-dihydroxy-3-methoxy-4,6a-diprenyl-pterocarpanonthebasis of theNMR(1Dand2D,Table2)spectraldataandHRESIMS(positive mode).

Pterocarpans,thesecondlargestgroupofnaturalisoflavonoids, receivedthis name fromPterocarpusspecies,thefirst sourceof thisclassofcompounds.Sincethen,manystructurallydiverse pte-rocarpanshavebeenisolatedand somedemonstrateinteresting biological activities(Goel et al.,2013). Pterocarpanshave been mainlyfoundinalargenumberofFabaceaespecies,andhavebeen reportedtoexhibitantifungal(Jiménez-Gonzálezetal.,2008)and antiprotozoal(Vieiraetal.,2008)activities.

Compound2wasobtainedasbrowncrystals.TheIRspectrum showedabsorptionbandsofO Hstretchingat3297cm−1and car-bonylconjugatedC Ostretchingat1628cm−1_._Phenol_absorptions werealso observedat 1378–1317cm−1 and 1280–1160 associ-atedtoC Ostretching,andat1580and1440cm−1oftheskeletal vibrationsof the aromaticring. The NMR spectra (see Suppor-tinginformation)showedthepresenceoftwomethylgroups,an

chelatogenichydroxylgroup,acarbonylgroup,andanaphthalene ring.Followinganalysisbyspectroscopicmeansandcomparison withthedatafromtheliterature,compound2wasidentifiedas musizin(Covellet al.,1961)otherwise named dianellidin (Dias etal.,2009)andnepodin(LiandMcLaughlin,1989).

TheliteraturedescribestheactivityofmusizinagainstC.albicans

ATCC14053andTrichophytonmentagrophytesATCC28185(Dias et al., 2009), and as a potent antiprotozoal compound against malariaparasites(LeeandRhee,2013).

Vatacarpan(1)wasactiveagainstC.albicansATCC10231with a MIC value (MIC 0.98␮g/ml) comparable to fluconazole (MIC 1.00␮g/ml)andsuperiortoamphotericinB(MIC4.00␮g/ml).

The literature reports the production of pterocarpans as a responseto bioticand abioticfactors,suchasfungal andother pathogenchallenges(Jiménez-Gonzálezetal.,2008; Goeletal., 2013).Pterocarpansaccumulateininfectedtissue,thesiteof anti-fungalactivity.Forsomeofthemhighconcentrationsarereached rapidly,then slowlydecrease as the infectionceases ( Jiménez-Gonzálezet al., 2008).Several studies have tried torelate the structureofthepterocarpansystemwiththeirfungicidalactivity.

Investigations have shown that the roots of some plants whichcontainpterocarpanshaveastrongactivitytowardhuman pathogens(Jiménez-Gonzálezetal.,2008).It iswellestablished thatlipophilicpterocarpanspossessincreasedantifungalactivity, sincetheycrossfungalmembranesmoreeasily(Harborne,1978). TherootbarkisthesourceoftheV.macrocarpaextractstudied.This organiscontinuouslychallengedtosoilmicroorganisms,humidity andoxidativestress,conditionsthatmayenhancetheproduction ofpterocarpans.

BiologicalactivityscreeningofV.macrocarpaextracts demon-strated themostpositive resultsin terms of activityspectrum. Thetwoextractsactiveagainsttheprotozoawerefromtheroot (Table1).AlloftheV.macrocarpaextractswereactiveagainstat leastonefungalstrain(MIC≤125␮g/ml)(Table1),withthe

major-ityofMICvaluesforC.parapsilosisATCC22019comparableto,or betterthan,thedrugcontrols.C.parapsilosisisregardedas refer-enceyeast(CLSI,2008a,b,2012).Adecade-spanningmulti-centric studyinBrazilianhospitalsrevealedanincreaseintheincidenceof

Candidanon-albicanspathogens,primarilyC.parapsilosis(Colombo etal.,2006).Anotherstudyin29Spanishhospitalsreported can-didemiain ICU patientscaused by non-albicans species in 48% ofcases,withC.parapsilosisasthemostcommonspecies( Puig-Asensio etal., 2014).In dermatophytes, theMICvalues ranged from31.25to125␮g/ml(Table1).Literaturereportsthetraditional useofV. macrocarparootand stembarkinsuperficialmycoses treatmentinAmazonia (Piedade andFilho, 1988).In this work, theethylacetateextractsfromthesameplantorganswereactive againstclinicalisolatesofTrichophytonmentagrophytesLMGO09 andT.rubrumLMGO06(MIC31.25to≤125␮g/ml)–the

dermato-phytesresponsibleforthemajorityofsuperficialfungalinfections intheAmericas(Grannoumetal.,2013;Havlickovaetal.,2008).

V.macrocarparemainsawidelyusedtreatmentofdiabetes melli-tussymptoms(Bavilonietal.,2010;Oliveiraetal.,2008),orasan antiulcerandanti-inflammatory(Jesusetal.,2009)–suggesting thatitisanon-toxicspecies,asshowninexperimentalanimalsfor heartwoodmethanolextract(Jesusetal.,2012).

V.macrocarpaandV.guianensisareoftendescribedinthe lit-erature as sources of lectins, a very diverse class of proteins. Lectinsfoundintheseedsofthesespecies,amongotherFabaceae, showvariousbiologicalactivities,suchasalternativeantimicrobial agents(Vasconcelosetal.,2014),ashistologicalmarkers,orastools forcancerresearch(Sousaetal.,2015).

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ethylacetateextractdemonstratedthemostleishmanicidal activ-ity(IC50 9.23␮g/ml) (Table 1).Thisspecies islocally knownas

“brincos-de-saguim”becauseitsfruitresembles“monkey’sears” anditisusedbytraditionalcommunitiestotreatpulmonary infec-tions(Corrêa,1984).Otherspeciesofthesamegenusareusedin Braziltotreatparasitismandgonorrhea(Mimakietal.,2004).

S.aureumextractswereinactiveagainstL.(L.)amazonensis,but exhibitedhighactivityin71%ofthetestsconductedagainstATCC strainsandclinicalisolatesofpathogenicfungi.C.glabrataLMGO 44 results(MIC0.12to 31.25␮g/ml)(Table1)are thereforean importantconsiderationinthesearchforleadercompoundsgiven theincreasedclinicalincidenceofthisspecies(Puig-Asensioetal., 2014).ItisimportanttonotethatS.aureumisusedbytheCerrado communitiesforthetreatmentoffungalinfectionsandasa hep-atoprotector–suggestingthatitisanon-toxicspecies.Quilombola andsomeindigenousBrazilianpeopleuseadecoctionofthestem barkasacontraceptive(Rodrigues,2007).TheMumbuca commu-nityuseaS.aureuminfusionasahepatoprotectorinTocantinsState, Brazil(Coelhoetal.,2005).AnotherSclerolobiumspeciesisusedin thetreatmentofskindiseases(Mu˜nozetal.,2000).

PreviousresultsfromtheCerradoplantextractlibrary,builtby ourresearchgroup,havedemonstratedthatrootandstemextracts, mainlyfrombarkareoftenactiveagainstfungiandprotozoa(Da Costaetal.,2014).Thesepathogenexposedplantorgansshouldbe prioritizedfortargetcompoundsisolation.

Antifungal (85%) and leishmanicidal (23%) activity were observedfor26extractsfromthreespeciesoftheFabaceae fam-ily. E.ellipticumwas distinguishedby its activityagainst L.(L.)

amazonensis,especiallythestembarkethylacetateextract(IC50

9.23␮g/ml).S.aureumpresentednoactivityagainsttheparasite, butcuriouslysixroot/stemwoodandroot/stemethylacetateor ethanolextractsexhibiteddifferentialantifungalactivity(MIC0.12 to31.25␮g/ml).V. macrocarpa extractswerealso activeagainst bothmicro-organisms.Itsrootandstembarkareusedin super-ficialmycosestreatmentinAmazonia–traditionallyreferredtoas

angelim-do-cerrado or maleiteira(Piedade and Filho, 1988).The resultsfoundinTrichophytonmodelsareconsistentwiththis tra-ditionaluse.

FractionationofV.macrocarparootbarkEtOAcextractresulted in the isolation of a previously unreported vatacarpan (1) and knowncompoundmusizin(2).Vatacarpandemonstratednotable activityagainstC.albicansATCC10231(MIC0.98␮g/ml),an oppor-tunisticpathogenicfungiwhichisthemain etiologicalagentof candidiasis(PierceandLopez-Ribot,2013).

This work documentsthe activity of Cerrado plantextracts againstresistantclinicalfungalisolatesandLeishmania. Further-more,itconfirmstheneedtoconservetheCerradobiomehotspot withtheviewtodevelopingleadmolecules(DaCostaetal.,2014).

Authors’contributions

LSE,RBF and ERS conceived and designed the experiments; JEDPcollectedandidentifiedtheplantswiththeresearchgroup; DBS,RCDCandRMAconductedtheexperiments;RBFelucidated thestructuresof thecompounds;LSE,RBF, ERS, RMA,DBS and RCDC,contributedtodataanalysis;LSEandERScontributedwith reagents/materials/analysis tools and LSE, RBF, DBS,RCDC, ERS and RMA for paper writing. Allthe authorsapproved the final manuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswishtothankthefollowingBrazilianagenciesfor theirfinancialsupport:CNPq,CAPES,FAPDFandFINATEC.Weare grateful tothe Ministryof theEnvironment (CGEN/IBAMA) for authorizingbiologicalaccess.WealsoextendspecialthankstoDr. MariadoRosárioRodriguesSilvaforprovidingthefungalstrains.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2015.07.012.

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