Rev. bras. farmacogn. vol.25 número1

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

Polycarpol

in

Unonopsis

,

Bocageopsis

and

Onychopetalum

Amazonian

species:

chemosystematical

implications

and

antimicrobial

evaluation

Felipe

M.A.

da

Silva

,

Bruna

R.

de

Lima

,

Elzalina

R.

Soares

,

Richardson

A.

de

Almeida

,

Francinaldo

A.

da

Silva

Filho

_,

_Wallace

_R.

_Corrêa

_,

_Marcos

_J.

_Salvador

_,

_Antonia

_Q.L.

_de

_Souza

_,

Hector

H.F.

Koolen

_,

_Afonso

_D.L.

_de

_Souza

_,

_Maria

_L.B.

_Pinheiro

a_{DepartamentodeQuímica,UniversidadeFederaldoAmazonas,69077-000Manaus,AM,Brazil} b_{InstitutodeBiologia,UniversidadeEstadualdeCampinas,13083-970Campinas,SP,Brazil} c_{FaculdadedeCiênciasAgrárias,UniversidadeFederaldoAmazonas,69077-000Manaus,AM,Brazil} d_{InstitutodeQuímica,UniversidadeEstadualdeCampinas,13083-970Campinas,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7December2014 Accepted8January2015 Availableonline11February2015

Keywords:

Antimicrobialactivity Bocageopsis

Chemotaxonomicmarker Onychopetalum Polycarpol Unonopsis

a

b

s

t

r

a

c

t

Polycarpol,arecurrentlanostane-typetriterpeneinAnnonaceaefamily,wasconfirmedbythinlayer chromatographyandmassspectrometryanalysisintheaerialparts(twigsandtrunkbarks)ofUnonopsis duckeiR.E.Fr.,U.floribundaDiels,U.rufescens(Baill.)R.E.Fr.,U.stipitataDiels,Onychopetalum amazon-icumR.E.Fr.andBocageopsispleiospermaMaas.Itschemotaxonomicsignificancewasdiscussedforthese threegenera,aswellfortheAnnonaceaefamily.Inaddition,theantimicrobialactivityagainstseveral strainsofmicroorganismswasevaluatedforthefirsttimeforthiscompound,beingobservedsignificant antibacterialactivityagainstStaphylococcusaureus(ATCC6538),Staphylococcusepidermidis(ATCC1228) andEscherichiacoli(ATCC10538andATCC10799)withminimalinhibitoryconcentrationvaluesbetween 25and50␮gml−1_.

©2014SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

The Annonaceae family comprises about 2500 species dis-tributedin130genera.Thisfamilyconsistsoftrees,shrubsand climbers,witha predominantdistributioninlowlands of tropi-calandsubtropicalregions,beingconsideredapantropicalfamily (Richardsonetal.,2004).Thisfamilyischemicallycharacterized bythepresenceofalkaloids,particularlyisoquinoline-derivedand terpenoids,wheremonoterpenesandsesquiterpenesare predom-inantin thecomposition ofessential oils (Leboeuf et al.,1982; Fournieretal.,1999).Anotherremarkablefeatureisthepresenceof annonaceousacetogenins,aclassexclusivefromthisfamily,that hasattractedinterestduetothegrowinglistofnewlydescribed structuresandbiologicalactivities(Changetal.,1999;Cunhaetal., 2009).Acetogeninsalongwiththelanostane-typetriterpene poly-carpolhavebeensuggestedaspotentialchemotaxonomicmarkers forthisfamily(Leboeufetal.,1982;Goulartetal.,1986;Jungetal., 1990).

∗ Correspondingauthor.

E-mail:felipemas@ufam.edu.br(F.M.A.d.Silva).

Polycarpol displays a growing list of biological activities describedintheliterature,suchasantifilarial(Nyasseetal.,2006), antineoplastic(Matosetal.,2006),antitrypanosomal(Ngantchou etal.,2009)andmorerecentlyanti-inflammatory(Saadawietal., 2012).Althoughtriterpenespossessprovenantimicrobial activi-ties(Haraguchietal.,1999;Katerereetal.,2003;Djoukengetal., 2005;Angehetal.,2007)alackofstudiesdescribingthe antibacte-rialactivitiesofpolycarpolisobserved.Thecontinuoussearchfor newantimicrobialdrugsisstimulatedbytheincreasing appear-anceofantibiotic-resistantorganisms,suchasindividualsofthe Staphylococcus,Pseudomonas,Enterococcus,andPneumococcus gen-era(Pachecoetal.,2012).

Unonopsis,BocageopsisandOnychopetalumarebotanicallyclose generadistributedthroughneotropicalregions.The morphologi-calsimilaritiesamongthesegenerawereexpressedbyFries,when he placed them in the informal “Unonopsis-Gruppe”. Recently thiscloserelationshipwassupportedbyphylogeneticresearches (Maasetal.,2007).Unonopsisisthelargestgenusamong these three,comprising approximately50 species.Ontheotherhand,

Bocageopsisand Onychopetalumcomprise few species, fourand

two, respectively (B. mattogrossensis, B. canescens, B. multiflora andB.pleiosperma,O.amazonicumandO.periquino)(Maasetal., 2007).Unonopsisisalsothemostexploredfromthechemicaland

http://dx.doi.org/10.1016/j.bjp.2015.01.003

biologicalpointsof view(Siqueiraet al.,1998; Waechteretal., 1999; Silva et al., 2012b,c, 2014). The chemical information regardingtheBocageopsisandOnychopetalumgeneraisstilllimited, beingfocuseddirectedtothestudyoftheessentialoilsand alka-loidcompositions(Almeidaetal.,1976;Oliveiraetal.,2014;Soares etal.,2015).Inthiswork,thepresenceofthetriterpenepolycarpol (1)intheaerialparts(trunkbarksandtwigs)ofAmazonian

Boca-geopsis, Onychopetalum and Unonopsis species was investigated

bythinlayerchromatography(TLC)andmassspectrometry(MS) approaches.Thechemotaxonomicsignificanceofpolycarpolwas discussedforthesethreegeneraandfortheAnnonaceaefamily. Inaddition,polycarpolwassubmittedtoinvitroassaysto evalu-ateitsantimicrobialactivityagainstStaphylococcusaureus(ATCC 6538),Staphylococcusepidermidis(ATCC1228),Pseudomonas aeru-ginosa(ATCC27853),Enterobacterfaecalis(Ef),Bacillussubtilis(Bs),

Escherichiacoli(ATCC10538and10799),Candidaalbicans(ATCC

10231and1023),Candidaparapsilosis(ATCC22019),Candida tropi-calis(ATCC157andct),Candidaglabrata(ATCC30070)andCandida dubliniensis(ATCC778157).

OH

(1) HO

Materialsandmethods

Plantmaterial

Theaerialparts (twigsand trunkbarks)of Unonopsisduckei R.E.Fr.,(voucherno.3289), Onychopetalumamazonicum R.E.Fr. (voucherno.218341)andBocageopsispleiospermaMaas(voucher no.183125)werecollectedintheReservaFlorestalAdolphoDucke, atthemunicipalityofManaus,Brazil,fromindividualspreviously identifiedbyspecialists.Thevoucherspecimensaredepositedin theherbariumoftheInstitutoNacionaldePesquisasda Amazô-nia(INPA).The samepartsfor U.floribunda (voucherno.6701)

and U. rufescens (voucherno. 3767) were collectedin the

Dis-tritoAgropecuáriodaSUFRAMAinthesamecity,fromindividuals previously identified byspecialists. The voucher specimens are depositedinthebotanycollectionofPDBFF/INPA(ProjetoDinâmica BiológicadeFragmentosFlorestais).Theplantmaterial(twigsand trunkbarks)of U.stipitata(voucher no.8250) wascollectedin thecampusoftheUniversidadeFederaldoAmazonas(UFAM).A voucherspecimenwasdepositedintheHerbariumofthe institu-tion.ThespecimencollectedonthecampusofUFAMwasidentified byProf.AntonioCarlosWebberfromtheDepartamentode Biolo-giaoftheUFAM.TheSISBIOlicensenumberforthecollectionis 34677-1.

Extractionandconfirmationofthepolycarpol

TheextractionofpolycarpolfromU. duckei,U. floribunda, U. rufescens,U.stipitata,O.amazonicumandB.pleiospermawasmade accordingto theadapted methoddescribed for U. guatterioides (Silvaetal.,2012a),briefly:10gofpowderedmaterial(twigsand trunkbarks)weremaceratedforthreedayswithhexane(80ml). Theextractwasconcentrated atreduced pressure. The precipi-tateformedwaswashedwithhexaneandre-crystallizedinethyl

Table1

AntimicrobialactivityinvitroofpolycarpolwiththeirMICvalues(␮gml−1_).

Microorganism Polycarpol Positivecontrolsb

MICa _MIC

Staphylococcusaureus(ATCC6538)c _{25 25} Staphylococcusepidermidis(ATCC1228)c _{50 50} Bacillussubtilis(Bs)d _–e ₅₀ Escherichiacoli(ATCC10538)c _{50 50} Escherichiacoli(ATCC10799)c _{50 50} Pseudomonasaeruginosa(ATCC27853)c _{– >500} Enterobacterfaecalis(Ef)d _{– 50} Candidaalbicans(ATCC10231)c _{250 12.5} Candidaalbicans(ATCC1023)c _{250 12.5} Candidaparapsilosis(ATCC22019)c _{– 12.5} Candidatropicalis(ATCC157)c _{– 12.5} Candidatropicalis(ct)d _{– 12.5} Candidaglabrata(ATCC30070)c _{– 12.5} Candidadubliniensis(ATCC778157)c _{250 12.5}

a_{MICminimuminhibitoryconcentrationin␮gml}−1_.

b_{Positivecontrol:chloramphenicolforbacteriastrainsandketoconazoleforyeast} strains.

c _{Standardstrain.} d _Fieldstrain.

e_{(–)withoutinhibitionofdevelopment.Compoundsevaluatedintherangeof10} and500␮gml−1_.

acetate.Approximately1mgoftheeachsolidwasseparatedfor TLCandMSanalysis,beingpolycarpol(1)identifiedbycomparison withanauthenticstandard(Silvaetal.,2012a).

Biologicalassay

Thepurestandardofpolycarpolwasevaluatedforits antimi-crobial activity using the brothmicrodilution method(96-well microtiterplates),aspreviouslydescribed(Salvadoretal.,2002; Barrosetal.,2009; Costaet al.,2010;Bataglionet al.,2014)to giveconcentrations between 10 and 500␮gml−1. The minimal inhibitoryconcentration(MIC)wascalculatedasthelowest con-centrationshowingcompleteinhibitionofatestedstrain.Inthese tests, chloramphenicol and ketoconazole were used as experi-mentalpositivecontrol,whilethesolutionpropyleneglycol-sterile distilledwater(5:95,v/v)servedasthenegativecontrol.Each sensi-tivitytestwasperformedinduplicateforeachmicroorganismand repeatedthreetimes.Thestrainsutilizedintheassaysareshown inTable1.

Instrumentsandmaterials

200 300 400 500 600 700 800 900 1000

m/z 0

10 20 30 40 50 60 70 80 90

100 423.33

441.31 405.34

455.27

207.24 338.42

121.15 269.37

535.26 577.45 637.71 674.49 727.48 775.74 810.48 882.82 934.80 HO

HO HO

[M–2H₂O+H]+ _[M+H]+

[M–H2O+H]+

H

H

H H O

+

+ + +

Fig.1. MassspectrumandinsourcethermaldissociationproposalforthesequentiallossesobservedforAPCIionizationofpolycarpol.

Resultsanddiscussion

ConfirmationofpolycarpolbyTLCandMSanalyses

Fromtheaerialparts ofU. duckei,U. floribunda,U. rufescens, U.stipitata,O.amazonicumand B.pleiospermawhitesolidswere obtainedafterextractionandre-crystallizationprocedures.Allthe samplesweredissolvedinethylacetateandanalyzedbyTLCin comparison withan authentic polycarpolstandard (Silvaet al., 2012a).Thesamplesandthestandardpresentedthesame reten-tion factor (Rf 0.6) when eluted with a hexane–ethyl acetate (7:3)mixtureandrevealedwithvanillin–sulphuricacidreagent (bluespot) andunderUV light(254nm).Theobservationofan intense blue spot in all samples is in agreement with results expected for triterpene compounds (Oleszek et al., 2008). The sampleswhensubjected toMSanalysisinfull scanmode, pre-sented three major ions at m/z441 [M+H]+_{, 423 (}

−18Da) and

405 (−18Da) (Fig. 1). The ion at m/z 441 corresponds to the

protonatedmoleculeexpectedforpolycarpol,whilethem/z423 and 405are consistent with sequential neutral losses (−H2O). Hydroxilatedtriterpenespresentthisdissociativebehaviordueto theapplicationofhighdesolvationtemperaturesinAPCI exper-iments (Koolen et al., 2013). All samples presented the same threemajorionswhensubjectedtoMSanalysis.Theseevidence along withTLC observations assisted theconfirmation of poly-carpol (1) in aerial parts (twigs and trunk barks) of U. duckei, U. floribunda, U. rufescens, U. stipitata, O. amazonicum and B. pleiosperma,being this substance reportedfor the first time in

thisUnonopsisspeciesaswellinOnychopetalumandBocageopsis

genus.

Antibacterialactivity

Thepolycarpolwasevaluatedinvitroforantimicrobialactivity againstelevenstrainsofmicroorganism(Table1).Thissubstance presentedsignificantantimicrobialactivityagainstS.aureus(ATCC

6538),S.epidermidis(ATCC1228)andE.coli(ATCC10538andATCC 10799)withMICvaluesbetween25and50␮gml−1.Forthestrains ofB.subtilis,E.faecalisandP.aeruginosawerenotobserved inhi-bitionofthedevelopment.Theseobservationsareinagreement withliteraturedata,sincelannostanetriterpenesisolatedfromthe vegetalspeciesandfungiwhenassayedagainstgram-negativeand gram-positivebacteria,presentseveralactivestructures(Liuetal., 2010a,b;Mosaetal.,2014).Oleanane,ursane,lupane,friedelane, fernane and others miscellaneous-typetriterpenes alsopresent severalactivecompoundsagainstgram-negativebacteria,mainly P.aeruginosa,E.coli,Klebsiellapneumoniae,andSalmonellatyphi, andgram-positive,comprisingS.aureus,B.subtilis,Bacilluscereus, and S.faecalis.Thetentative understandingof therelationships betweenthechemicalstructuresoftriterpenesandthe antibac-terialactivitiesindicatesthat theactivitymayberelatedtothe presenceofanoxygenatedgroupatC-3(Pachecoetal.,2012).In polycarpolthispositionisoccupiedbyahydroxylgroup,however anothersubstituents,likecarbonyls,O-glycosides,esters(mainly acetylmoieties),orhydroxylaminescanbepresentinthisposition (Pachecoetal.,2012).

Thesignificantantibacterialactivitiesobservedforpolycarpol anddescribedinthisworkforthefirsttimeneedsfurther inves-tigations,which couldhelpin thesearchfor newantimicrobial drugs.

Chemotaxonomicsignificanceofpolycarpol

The chemotaxonomic importance of the polycarpol for the AnnonaceaefamilywasfirstlyrecognizedbyLeboeufetal.(1982)

duringa systematic researchwithseveral species belongingto neighboringfamilies,suchasLauraceae,Monimiaceaeand Menis-permaceae.Atthistime,polycarpolwasnotfound,beingsuggested for the first time in literature as chemotaxonomic marker for Annonaceae.Thisideawassupportedovertheyears(Goulartetal., 1986; Junget al., 1990).Polycarpol was recentlyisolated from Duguetiaglabriuscula (Pereira et al., 2003), Duguetiafurfuraceae (Silva et al., 2007), Artabotrys madagascariensis (Murphy et al., 2008),Piptostigmapreussi(Ngantchouetal.,2009)andArtabotrys spinosus(Sichaemetal.,2011),allspeciesfromAnnonaceae fam-ily,which enhancestheinitialpropositionof thiscompound as achemotaxonomicmarkerforthisfamily.Consideringitsbroad occurrence at genera level, is appropriate to restrict the term “chemotaxonomicmarker”atafamilylevel,beingsuggestedthe useoftheterm“chemicalmarker”togeneralevel.

InUnonopsisgenus,polycarpolhasbeenreportedin

Unonop-sisglaucopetala(Jayasuriyaet al.,2005), Unonopsisguatterioides (Touché et al., 1981; Silva et al., 2012a), Unonopsis spectabilis (Laprévoteetal.,1987)andUnonopsispacifica(Arangoetal.,1988). There-currentobservationofpolycarpolinallthesespecies sub-jectedtophytochemicalapproaches,aswellasinO.amazonicum andB.pleiospermaisanimportantchemicalevidencenotonlyto confirmthebotanicalproximityofthesegenera(Maasetal.,2007), butalsotoreinforcepolycarpolasa chemical markerforthese genera.Theinsufficientphytochemicalknowledgeregarding Ony-chopetalumandBocageopsisgenerastillnotdoesallowsaprofound evaluationofthechemicalsimilaritiesbetweenthemand Unonop-sis.Inthissense,phytochemicalinvestigationsdirectedatbetter understandingofthechemical relationshipsamongthese three close-relatedgeneraareindispensableinthefuture.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Authorscontributions

FMAS,HHFK(PhDstudents),BRL,ERS(MScstudents),RAM(PhD student)andFASF (CIstudent)contributedequallyincollecting plantsamples,confirmationofthecompoundstructureandwritten ofthemanuscript.WRCandMJScontributedtoinvitrobiological assays.ADLS,MLBPandAQLSsupervisedthelaboratoryworkand contributedtocriticalreadingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.

Acknowledgment

TheauthorsaregratefultoCAPES,CNPq,FINEP,FAPEAMand FAPESPforfinancialsupport.WethankPDBFFforproviding logis-ticalandfieldsupport.Thisispublicationnumber663inthePDBFF TechnicalSeries.

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