Rev. bras. farmacogn. vol.27 número1

(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

Alkaloids

and

biological

activity

of

beribá

(

Annona

hypoglauca

)

Maria

V.N.

Rinaldi

a

_,

_Ingrit

_E.C.

_Díaz

b

_,

_Ivana

_B.

_Suffredini

b,∗

_,

_Paulo

_R.H.

_Moreno

c

a_Programa_de_{Pós-graduac¸}_ão_em_Fármaco_e_Medicamento,_Faculdade_de_Ciências_{Farmacêuticas,}_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

b_Núcleo_de_Pesquisas_em_{Biodiversidade,}_Laboratório_de_Extrac¸_ão,_Universidade_Paulista,_São_Paulo,_SP,_Brazil

c_Instituto_de_Química,_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18May2016 Accepted4August2016

Availableonline22September2016

Keywords:

Annonahypoglauca

Antibacterialactivity Cytotoxicity Isoquinolinealkaloids Isoboldine

Actinodaphnine

a

b

s

t

r

a

c

t

AnnonahypoglaucaMart.,Annonaceae,popularlyknownas“beribá”,wascollectedinfloodedareasofthe AmazonianRainForest.Thecrudeextractobtainedfromthisspecieswasfoundtobecytotoxicagainst humancancercells.ChemicalinformationonA.hypoglaucaisscarce.So,thepresentworkaimedthe isolationandidentificationofitsalkaloidsandtotesttheircytotoxicactivity.Alkaloidswereobtained fromstembyacid–basepartitioningandtheremainingalkaloid-freeextractwaspartitionedwithorganic solvents.Gaschromatography–massspectrometryGC/MSanalysisoftotalalkaloidsallowedthe iden-tificationoffouraporphinealkaloids:actinodaphnine,anonaine,isoboldineandnornuciferine.Total alkaloidswerefractionatedbycolumnchromatographyandwerepurifiedbypreparative thin-layer-chromatography,whichallowedtheisolationoftwoaporphinealkaloids,actinodaphnineandisoboldine, characterizedbyNMRandCG–MSanalyses.Thisisthefirstreportfortheoccurrenceofactinodaphnine inAnnonaspecies.Allthesamplesweretestedincytotoxicandantibacterialassays.Totalalkaloidextract anditsfractionsshowedantimicrobialactivityagainstStaphylococcusaureusandEnterococcusfaecalis. Inthecytotoxicityassay,thecrudeextractshowedalethaleffectagainstbreastandcoloncancercells. Isoboldine-containingFA5andactinodaphnine-containingFA6showedactivityagainstbreastcancercell line,whilethealkaloid-freefractionsdidnotshowsignificantactivityagainstcancercelllines.

Introduction

Annonaceaeisapantropicalfamilycontainingapproximately 130generaand2300speciesoftreesandshrubs(Heywood,1993; Cordelletal.,2001).ThetropicalAmericangenusAnnonaL.is rep-resentedby 140species, which arerecognized mainlyby their ediblefruits,suchasA.muricata(graviola),A.squamosa (fruta-do-conde),A.coriaceae(araticum)andA.cherimolia(cherimoya)plants belongingtothegenusAnnonaarealsousedinfolkmedicineas antitumor,anti-parasiteand antidiarrhea(Pimenta etal.,2003), antiprotozoal (Siqueira et al., 2011), anti-inflammatory (Siebra etal.,2009),cytotoxicandanti-ulcerogenicagents(Hamidetal., 2012).Some species have also been pharmacologically studied for theirplateletanti-aggregationand antiulcerogenicactivities (Villaretal.,1997;Padmaetal.,1998).ThegenusAnnonaisarich sourceofisoquinoline–particularlyaporphinealkaloids(Leboeuf etal.,1982;Rabêloetal.,2014)–andacetogenins(Bermejoetal., 2005).Thestructuraldiversityoftheisoquinolinealkaloidsisas wideastherangeofitsbiologicalactivities,whichcanbeusedas

∗ Correspondingauthor.

E-mail:extractlab@unip.br(I.B.Suffredini).

antimicrobial(Simeónet al.,1990), cytotoxic(Wu et al.,1993), antitumoral(SonnetandJacobson,1971),antiprotozoal(Tempone etal.,2005),antiviral(Montanhaetal.,1995)besidesmanyother applications, especially in demonstrating antimicrobial activity againstGram-positivebacteria(Villaretal.,1987;Abbasogluetal., 1991;Pauloetal.,1992).

AnnonahypoglaucaMart.occursinthefloodedareas(igapós)of thenorthernAmazonianforests,aswellasinterrafirmeforests. Thespeciescanbefoundasatreeupto10mheightwhen occur-ringinterrafirmeorasalianawhenoccurringintheigapóswhere itispopularlyknownasberibá(Gottsberger,1978),orbiribá,in theBrazilianAmazonregion,orasguanábanahuascaandtortuga blancabyotherSouthAmericancommunities,oraswildsoursop, itsEnglishname.Itsfruitsareeatenbylocalsandtheteamadewith thebarkisusedasmedicineagainstparasites,anemiaandchronic diarrhea(Revilla,2002).A.mucosa,A.sylvatica,Duguetia marcgra-vianaandFusealongifoliaarealsoknownasbiribá,inBrazil(Reflora, 2016).

Previousbiologicalscreeningaimingtheidentificationof cyto-toxicity of 1277 Amazon plant extracts against human breast cancercelllines(Suffredinietal.,2007a),prostatecancercelllines (Suffredinietal.,2006a),colon,lung,centralnervoussystemand leukemiacancercelllines(Suffredinietal.,2007b)havebeenmade,

http://dx.doi.org/10.1016/j.bjp.2016.08.006

(2)

togetherwiththeevaluationoftheantimicrobialactivityofthe extractsagainstsomepathogenicmicroorganismsas Staphylococ-cusaureus(Suffredinietal.,2004),Enterococcusfaecalis(Castilho et al., 2013, 2014), Streptococcus mutans (Barnabéet al., 2014) andEscherichiacoli(CamargoandSuffredini,2014).Fromthe pre-viousscreening,thecrude extractobtainedfromthestemof A. hypoglaucaMart.,Annonaceae,theso-calledEB1109,hasshowna significantactivityagainstbreastcancercelllineMCF-7(Suffredini etal.,2007a)andagainstS.mutans(Barnabéetal.,2014).Despite thesefirstresults,littleisknownaboutthechemicalcomposition andbiologicalactivitiesofA.hypoglauca,despiteawideliterature consideringotherAnnonaspecies.

Thepresentstudyaimstoreporttheisolationofaporphine alka-loids fromthestems of A. hypoglauca,as wellasto reportthe cytotoxicactivityofEB1109 and itsfractions. Asliteraturealso indicatesaputativeantimicrobialactivityrelatedtotheAnnona

alkaloids,allsampleswerealsotestedagainstthepathogenic bacte-ria.

Materialsandmethods

Plantmaterial

Thestem ofAnnona hypoglaucaMart.,Annonaceae,was col-lected in water-flooded forests (named igapó forest), Amazon rain forest, Manaus, (Lat. 2◦₅₈′ _and _Long. ₆₀◦₂₇′_), _Anavilhanas Ecological Station). A voucher specimen was deposited under identificationnumber [AAOliveira,3577 (UNIP Herbarium] and wasidentifiedbyDr.MateusL.B.Paciencia.Plantcollectionwas doneunder Brazilian Governmentplant collectlicense number MMA/ICMBio/SISBIO#14895andlicensetoaccessgeneticmaterial Ibama/MMA/CGen#012A-2008.

Extractionprocedures

Crudeextract:Thestemsoftheplant(1.074kg)wereair-dried at40◦_C_in_an_{air-circulating}_oven._Plant_material_was_ground_in_a hammermillbeforebeingsubmittedto24hmacerationwith10l ofamixturecontainingdichloromethane:methanol(1:1,v/v).The resultingorganicextractwasconcentratedtodryness(46.14g)and yielded4.3%ofEB1109(Younesetal.,2007).

Totalalkaloidfraction(TA):EB1109(5g)wereextractedwith 30ml0.1Mphosphoricacidunderagitationfor30min.Extraction wasrepeatedfourtimes.Acidsolutionswerefilteredandreunited tothesamefunnel.Acidsolutionwasthreetimesextractedwith 50ml hexane in order tohave non-polar compoundsremoved. TheacidsolutionwasbroughttopH∼9withammonia

hydrox-idesolution(25%,w/w)andwaspartitionedwithportionsof50ml chloroform,untilnegativetoDragendorff’sreagent.Theorganic phasewasdriedwithanhydroussodiumsulphateandconcentrated togivethetotalalkaloid(TA)fraction(3.1g;0.28%).TAyieldwas obtainedconsideringthecrudeplantmaterial.

Alkaloid-freeextracts: Thecake, consideredas theremaining insolublematerialresultedfromthealkaloidextraction,wasdried and the remainingsolids were dissolved in a series of solvent systems. Hexane (30ml) was added to the cake and the sys-temremainedunderagitationfor30min.Afterthat,thesystem was decanted and filtered. This procedure was repeated three timesunderthesameconditions.Thecombinedhexanesolutions were evaporated and originated fraction hexane (FHex,2.64g; 0.25%).Aseconddissolutiondoneinasimilarwaywasperformed withamixtureofdichloromethane(DCM)andmethanol(MeOH) (1:1, v/v), resulting fraction DCM/MeOH (DCM/MeOH; 1.71g; 0.16%).Thethirddissolutionwasmadewithethylacetate, result-ingin fractionFEAC (0.21g;0.02%).Finally, theremainingcake

material waspartitioned with a 20% ethanol:H2O solution and extractedwiththreeportionsof250mleachofbutanol,originating thefractionbutanol(FBuOH;0.65g;0.06%).Fractionsyieldswere obtainedconsideringthecrudeplantmaterial.

Isolationofalkaloids

TA(2g)wasfractionatedbycolumnchromatography (70cm lengthx40mmdiameter)on50gnormalphasesilicagel60.Elution wasmadewiththefollowingsolventmixtures:DCMandMeOH in order ofincreasing polarity,as follows: 100% DCM(200ml), DCM:MeOH9:1(400ml),DCM:MeOH8:2(400ml),DCM:MeOH 7:3(400ml),DCM:MeOH1:1(400ml)and100%MeOH(200ml). Theelutionresultedinthefollowingfractions:100%DCMyielded fraction1(FA1, 7.6mg;0.0011%);DCM:MeOH9:1(v/v)yielded fractions2(FA2,0.4mg;0.00005%)and3(FA3,738.4mg;0.1055%); DCM:MeOH8:2(v/v)yieldedfractions4(FA4,161.4mg;0.0231%) and5(FA5,176.1mg;0.0252%);DCM:MeOH7:3(v/v)yielded frac-tions6(FA6,395.8mg;0.0565%)and7(FA7,43.4mg;0.0062%); andfinallyfractions8(FA8, 26.9mg;0.0038%),9(FA9,22.1mg; 0.0031%) and 10 (FA10, 16.0mg; 0.0023%) were eluted with DCM:MeOH1:1(v/v).Fractionsyieldswereobtainedconsidering thecrudeplantmaterial.

Fractions FA4, FA5 and FA6 were again fractionated using preparative thin layer chromatography (PTLC) precoated with 1mmofsilicagel60F254,withoutbeingactivatedandasolvent mix-tureofCHCl3:MeOH(92:8)asmobilephase.Spotswereobserved underU.V.light(=366nm)andrevealedafterreactionwith Dra-gendorff’sreagent.Bandsrelatedtoeachisolatedcompoundwere elutedwithDCMandwerethensubmittedtoRMNanalysis. Frac-tionFA4originatedfourfractions,FA5originatedthreefractions, andFA6originatedsixfractions.Compound1wasthenisolated fromFA5.2usingPTLCasabrownamorphoussolid(3.7mg). Com-pounds2and3wereanalyzedby1_H_NMR_as_a_mixture._Lastly, compound4wasisolatedfromFA6.2,obtainedfrompreparative TLC,asalightbrownamorphoussolid(7.8mg).Fouralkaloidswere identified.

Compound1,isoboldine:(1_H_NMR,₃₀₀_MHz,_CDCl

3,TMS inter-nalstandard):ı7.94(1H,s,H-11),6.74(1H,s,H-8),6.47(1H,s,H-3), 3.84–3.85(6H,s,br,OMe-10andOMe-2),2.49(3H,s,N-CH3)(Soares etal.,2015;JacksonandMartin,1966);13_C_NMR₍₇₅_MHz,_CDCl

3): ı145.74(C-2),144.90(C-9),144.43(C-10),140.47(C-1),129.7 (C-7a),124.4(C-11a),119.7(C-1a),113.87(C-11),111.55(C-8),108.64 (C-3),62.51(C-6a),56.16(C-2OMe),56.09(C-10OMe),53.36(C-5), 43.80(C-6N-CH3),33.9(C-7),28.79(C-4))(Jackmanetal.,1979). MS/EI (M+)RT=27.99:327(M+_), ₃₂₆_(M+_-1),₃₁₀_(M+_-17), ₂₈₄ (M+_-43),₂₆₉_(M+_-58),₂₅₃_(M+_-74).

Compound 2, nornuciferine (present in fraction FA5.3): (1_H NMR, 200MHz, CDCl3, TMS internal standard): ı 8.32 (1H, d,

J=7.8Hz,H-11),7.28(1H,m,H-8,H-9,H-10),6.58(1H,s,H-3),3.59 (3H,s,2-OCH3),3.32(3H,s,1-OCH3)(Dutraetal.,2012;Hasratetal., 1997).MS/EI(M+)RT=20.12:281(M+_),₂₈₀_(M+_-1),₂₆₆_(M+_-15), 250(M+_-31),₂₃₇_(M+_-43),_221,_178,_165,_152.

Compound3,anonaine(presentinfractionFA5.3):(1_H_NMR, 200MHz,CDCl3,TMSinternalstandard):ı8.00(1H,d,J=7.8Hz; H-11),7.28–7.14(1H,m,H-8,H-9,H-10),6.68(1H,s,H-3),5.89 (1H,d,J=0.8Hz,1-OCH2O-2)and6.28(1H,d,J=0.8Hz,1-OCH2 O-2,)(Costaetal.,2012;Hasratetal.,1997).MS/EI(M+)RT=21.32: 265(M+_),₂₅₄_(M+_-1),₂₃₆_(M+_-29).

(3)

(300MHz,CDCl3):CH:ı114.4;110.1;106.9;CH3:56.1;53.3;CH2: 100.6;42.55;35.4;28.3.13_C _NMR₍₇₅_MHz,CDCl

3):ı146.99 (C-2),145.65(C-10),145.35(C-9),141.70(C-1),128.09(C-7a),125.98 (C-1b),125.65(C-3a),122.74(C-11a),116.57(C-1a),114.41(C-8), 110.11(C-11),106.96(C-3),100.62(OCH2O), 56.08(C-10OMe), 53.27(C-6a),42.56(C-5),35.37(C-7),28.32(C-4))(Stévignyetal., 2002).MS/EI(M+)RT=27.48:311(M+_),₃₁₀_(M+_-1),_279,_251,_181.

NCH

₃

H

₃

CO

HO

OH

H

₃

CO

1 NH

O

NH

H

3

CO

H

₃

CO

2 NH

OH

H

₃

CO

O

4

3

Spectroscopicanalysis

NMRspectraof1_H_and13_C_were_obtained_on_a_Bruker spectrom-eter,operatingat200MHzorat300MHz(1_H)_and_at₇₅_MHz₍13_C). GC–MSwasperformedinanAgilentSeries6890chromatograph, equippedwithaHP-5column(30m×0.25mm,filmof0.25␮m), theinjectorwassetto290◦_C,_the_carrier_gas_(He)_flow_was_adjusted to1ml/min,theinitialtemperaturewas95◦_C_for₂_min,_followed_by anincrementof8◦_C/min_until₃₁₀◦_C,_the_temperature_was main-tainedfor2min.TheMSdetectoroperatedintheelectronimpact (EI;70eV),wasusedtoqualitativeevaluatethealkaloidcontents.

Biologicalassays

Samplespreparation

Samplepreparationforcytotoxicassay:allthesampleswere diluted to 2mg/ml in 50% dimethylsulfoxide (DMSO) in water beforebeingevaluatedinasingle-concentrationassay(finaltest concentration 100␮g/ml). Sample preparation for antibacterial assay:samplesweredilutedinDMSO50%tothefollowing concen-trationsof200,180,160,140,120,100,80,60,40and20mg/ml, inordertoproportionatefinaltestconcentrationsof100,90,80, 70, 60, 50, 40, 30, 20 and 10␮g/ml. Vehicle wastested for its antimicrobialactivity,aswellasdoxorubicin(Adriamicin®₎_at_a concentrationof25mMwasusedasstandarddruginthe cytotox-icityassayandgentamycin andtetracyclinepreparedatvarious concentrationswereusedasstandarddrugsintheantibacterial assay.AntibioticfinaltestconcentrationsforMIC/MBCassayranged from120␮g/mldownto0.16␮g/ml.

Antibacterialmicrodilutionbrothassay

Testswereperformedin asepticconditions(Suffredinietal., 2015). Bacteria inoculum was prepared at the concentra-tion of 1.5×102CFU/ml, starting from a 0.5 McFarland (or

1.5×108CFU/ml), as follows (Younes et al., 2007). S. aureus

(ATCC29213),Escherichiacoli(ATCC25922)andE.faecalis(ATCC 29212) were the bacterial strains tested. Bacteria inoculum of eachstrainwasobtainedfromfresh24-hculturecoloniesgrown onMüeller-Hinton agarplates. Eachstrain wasinoculated into 5ml of Müeller–Hinton broth in order to obtain a concentra-tionof1.5×108CFU/ml(0.5McFarland),whichwasdetermined

by a turbidimeter (Oxoid) adjusted to the 0.5 McFarland con-centration.Eachinoculumwasthendilutedinbrothmediumto 1.5×102CFU/ml.Onehundredandninetymicrolitersofthis

sus-pensionwastransferredtoeachmicroplatewell.Tenmicrolitersof eachtreatmentwereaddedtothemicroplatewellsandincubated at35◦_C_for_18–20_h._Treatments_that_have_visually_inhibited bacte-riafromgrowweresubculturedinMüeller-Hintonagarplatesthat wereincubatedat35◦_C_for₁₈_to₂₀_h._Observations_of_bacteria_grow orlackofbacteriagrowdeterminedwhichtreatmentwaseffective. Allsamplesthatshowedeffectivenessinthesingle-concentration assayweresubmittedtothedeterminationofMICandMBC.

Determinationofminimalinhibitoryconcentrationandminimal bactericidalconcentration

Minimalinhibitoryconcentration(MIC)andminimal bacteri-cidalconcentration(MBC)weredeterminedforthetreatmentsthat showedtotalgrowthinhibitioninthesingle-concentrationassay usingtheprotocoldescribedabove.Thesameprotocolwasused todetermineMICandMBC.Sampleswerepreparedin concentra-tionsof0.2upto2mg/ml,ina0.200mg/mlfold(testconcentrations rangingfrom10to100␮g/ml,in10␮g/mlfold)wereevaluated. Turbidity wasobserved and those identified ashaving a visual lackofturbidityweresubculturedinMüeller–HintonagarPetri dishes,whichwerethenincubatedat35◦_C_for_18–20_h._After_the incubation, bacteriagrowthwasdeterminedand supportedthe establishmentofMIC’sandMBC’sasfollows:thelower concentra-tionobservedashavingalackofturbiditybutpresentingbacteria growthwasdeterminedastheMICandthelowerconcentration havingalackofturbidityanddidnotpresentanybacteriagrowth aftersubculturewasconsideredastheMBC.

Cytotoxicityassay

(4)

treatment(extract,fractions,isolatesordoxorubicin)wasadded to thewells in sextuplicates. Also, in order to obtain a better experiment control,two wellshaving only medium plus treat-mentwere added totheplates, as wellas16 wells containing mediumpluscellswithouttreatment,inordertoestablisha reg-ularcellgrow(positivecontrol).Plateswerekeptincubatingfor 48hbeforeevaluation.Afterthat,plateswerealsofixedwithTCAat thepre-stipulatedconcentration.TCAwasremovedwithwaterand sulforodamineB(SRB)colorimetricassaywasperformed. Accord-ingtoVoigt(2005),theproteindyeSRBassayiscurrentlyused formeasuringdrug-inducedcytotoxicityandcellproliferationfor bindingelectrostaticallyandpHdependenttoproteinbasicamino acidresiduesofTCA-fixedcellsinalinearcorrespondencetothe amountofcells.So,timezero(T0),cellcontrol(C)andtreatment(T) opticaldensitiesat515nmwereusedtocalculatethepercentage ofcellgrowinhibitionor,asfollows:[(T−T0)/(C−T0)×100=%of

growth].Resultswereinterpretedbythecomparisonofcellgrowth aftertreatment(T)topositivecontrolcells(C),bothconsideringcell growthatTimeZero(T0).If100>T>T0wasobservedthepresence ofcellgrowthinhibition;ifT=T0=0wasobservedthatcellsdonot growinrelationtothecelldensityatT0;ifT<T0,itmeansthat treatmentiseffectiveenoughtocausecelllethality.Treatments wereevaluatedataspecifictestconcentrationof100␮g/ml.

Resultsanddiscussion

Thealkaloid yieldfromA. hypoglaucastems was0.28%. Pre-viousstudies(Debourgesetal.,1987)relateyieldsrangingfrom 0.3%and 0.2%for Duguetia species, while (Fischeret al., 2004) describesyieldsof0.5% for Annona.The yieldof total alkaloids hereobtainedfromA.hypoglaucaissomewhatamongtheexpected values.

TAfractionwasanalyzedbygasGC–MS,andthepresenceoffour aporphinealkaloidscouldbedetermined:isoboldine(1)isolated fromFA5.2,nornuciferine(2)andanonaine(3),identifiedas mix-tureinfractionFA5.3andactinodaphnine(4),isolatedfromFA6.2. Compoundswerecharacterizedbythecomparisonoftheir frag-mentationpatterninthemassspectra(MS)tothelibraryfromthe equipment(Wiley25)andtotheliteraturedata.Majorcompound (Rt20.12min)showedthefollowing fragmentationpattern:m/z

281(M+._),₂₈₀_(M+_-1),₂₆₆_(M+_-15),₂₅₀_(M+_-30),₂₅₂_(M+_-29),_237, 221,178,165,152whichiscompatibletonornuciferine(2).The peakatm/z280with100%intensityischaracteristicofaporphines, whicheasilylosethehydrogennexttotheNHgroupatC-6a(Ohashi etal.,1963).Nornuciferine(2)wasisolatedfromA.hayesiistemas

thesecondmajorcompound(Rasamizafyetal.,1987).This alka-loidwasalsoreportedtooccurinAnnonaandinotherAnnonaceae genus,suchasEnantia,Guatteria,Isolona,PseudovariaandXylopia

(Lebouefetal.,1982;Lúcioetal.,2015).ThecompoundhavingRt

21.32minwascharacterizedasanonaine(3),bycomparisonofits fragmentationpattern(m/z265(M+._), ₂₆₄_(M+_-1),₂₃₆_(M+_-29), withtheliterature(JacksonandMartin,1966;Bhakunietal.,1972). CompoundshowingRt27.48min,m/z 311(M+._), ₃₁₀_(M+_-1), 282(M+_-29),₂₇₉_(M+_-31),₂₆₆_(M+_-15),_251,₁₈₁_was_identified_as actinodaphnine(4),whichfragmentationpatternisinaccordance topreviousreport(McLaffertyandStauffer,1989).Isoboldine(1) wascharacterizedatRt27.9minandbyitsfragmentationpattern,

m/z327(M+._),₃₂₆_(M+_-1),₃₁₀_(M+_-17),₂₈₄_(M+_-43),₂₆₉_(M+_-58), 253(M+_-74)₍_Jackson_and_Martin,₁₉₆₆_)._Isoboldine₍₁₎_previously isolatedfromfiveAnnonaspecies:A.cherimola,A.glabra,A. mon-tana,A.salzmanniandA.senegalensis(Lebouefetal.,1982;Simeón etal.,1990;Philipovetal.,1994).Inthepresentwork,both isobol-dineandactinodaphninewerealsoisolatedfromTA,asdescribed below.

Thealkaloidfractions(FA5andFA6),originatedfromcolumn chromatography(CC),werepurifiedbyPTLCandyielded isobol-dine (1)as a brownamorphous solid.Isoboldine structure was proposed based on its molecular weight (M 327Da), that was determinedbyGC–MS, andit iscompatiblewiththemolecular formulaC19H21O4N.Moreover,1Hand13CNMRchemicalshift val-ueswereinaccordancetothosereportedintheliterature(Jackson andMartin,1966),thesingletsat6.47and6.74ppmweredueto thehydrogensincarbons3and8;respectively;abroadsingletat 3.83–3.84ppm(6H)relativetothemethoxygroupsfoundcarbons 2and10andasingletat2.49ppm,relativetothehydrogensinthe NCH3group.Jackmanetal.,1979;Soaresetal.,2015haveassigned thecarbonssignalsintheNMRspectraforthiscompound,andit ispossibletoverifyagainthelevelofaccordancebetweenthedata hereobtainedandtheliteratureresults.Isoboldine(1)was pre-viouslyisolatedfromA.cherimola(Simeónetal.,1990),A.glabra,

A.montanaandfromseveralotherAnnonaceaespecies.However, actinodaphnine(4)hasbeenexclusivelyisolatedfromGuatteria scandens(Hocquemilleretal.,1983),anditisbeingherereported as novelty for the Annona genus. This alkaloid was reported to occur ubiquitously in Hernandiaceae and Lauraceae species (Guinaudeauetal.,1983;Hocquemilleretal.,1983;Sulaimanetal., 2011).

Nornuciferine(2)andanonaine(3)(Hasratetal.,1997)hadtheir structuresproposedbasedonchemical shiftvalues recordedin a200MHzspectrometer,once theywerefoundasamixturein

Table1

Minimalinhibitoryconcentration(MIC)andminimalbactericidalconcentration(MBC)fromthecrudeextractobtainedfromthestemofAnnonahypoglaucaMart.andits alkaloidfractionsagainstthreebacteriastrains.

Extractsandfractions Staphylococcusaureus ATCC29213

EnterococcusfaecalisATCC29212 Escherichiacoli ATCC25922

MIC(␮g/ml) MBC(␮g/ml) MIC(␮g/ml) MBC(␮g/ml) MIC(␮g/ml)

Crudeextract >100 >100 >100 >100 >100

FHex >100 >100 >100 >100 >100

DCM/MeOH >100 >100 >100 >100 >100

FEAC >100 >100 >100 >100 >100

FBuOH >100 >100 >100 >100 >100

Totalalkaloids 60 70 50 50 >100

FA4.4 NT NT NT NT NT

FA5a ₇₀ ₇₀ ₄₀ ₄₀ _>100

FA6a ₇₀ ₈₀ ₄₀ ₄₀ ₉₀

Gentamycin 0.20 0.20 8 8 0.40

Tetracyclin 0.50 0.50 32 32 2

MIC,minimalinhibitoryconcentration;MBC,minimalbactericidalconcentration,NT,nottested.FHex,fractionhexane,DCM/MeOH,fractiondichloromethaneandmethanol, FEAC,fractionethylacetate,FBuOH,fractionbutanol.

(5)

fractionFA5.3notallhydrogenscouldbeassigned.So, nornucifer-ineshowedadoubletat8.32ppm(J=7.8Hz)correspondingtotheH atcarbon11,asingletat6.58ppmfortheHatcarbon3,andsinglets at3.59and3.32ppmrelativetothetwomethoxygroups1-and 2-OCH3.Theotheralkaloid,anonaine,showedadoubletat8.00ppm (7.8Hz)relativetotheHatcarbon11,asingletat6.68relativeto theHatcarbon3andtwodoubletsat5.89and6.28belongingto themethylenedioxyatcarbons1and2(1-O-CH2-O-2).

Actinodaphnine(4)wasisolatedfromFA6.2asa lightbrown amorphous solid. The molecularweight(M 311Da)was deter-minedbyGC–MSanditwascompatiblewiththemolecularformula C18H17O4N. The structure of actinodaphine (4) was attributed basedonthecomparisonofNMRspectraldatawithvaluesreported intheliterature(Stévignyetal.,2002).Itispossibletoobservethe presenceoftwoapparentsingletsat5.86and6.01ppmthatwere attributedtothehydrogensinthemethylenedioxyinposition1,2. Thepresenceofasingletreferentto3Hin3.85ppmmaycorrespond toamethoxygroupasasubstituentatcarbon10.Theabsenceof asingletwithintegrationto3Hnextto2.82ppmeliminatesthe possibilityofaNCH3group,confirmingthatcompound4isa nora-porphine.Withtheseobservations,itwaspossibletoattributethat compound4isactinodaphnine.TheattributionsoftheJvalueswere comparedtoresultsfoundintheliterature(Stévignyetal.,2002). Thepuritygradeofthesamplepermitteditsanalysiswithdifferent carbonNMRexperimentstoconfirmthestructure.Actinodaphnine wasfirstisolated fromAnnonaceaespecies suchasG. scandens

asabrowncrystal(Guinaudeauetal.,1983;Hocquemilleretal., 1983).

EB1109, hexane fraction, DCM/MeOH fraction, ethyl acetate fraction butanol fraction (Table 1) did not show a significant antibacterialactivity,consideringthatcrudeextractsandfractions shouldhaveantibacterialactivity<100␮g/ml(Padmaetal.,1998) tobeconsideredasapotentialantimicrobialnaturalproductagent. Ontheotherhand,TA,FA5(majorcompoundisoboldine(1),other compoundsnornuciferine(2)andanonaine(3))andFA6 (actin-odaphnine(4))showedamoderateactivityagainstGram-positive bacteria(S.aureusandE.faecalis),MICaslowas70and40␮g/ml, respectively,andgivesusaprospectionofgoodantibacterial activ-itytoactinodaphnine. Almostallsampleswereinactiveagainst theGram-negativeorganism(E.coli), onlyFA6(MIC>90␮g/ml) showedsomeactivity.Previousreportsshowedthatisoboldine(1) wasnot consideredashavingantibacterialactivity(Villaretal., 1987; Bermejo etal., 2005)whereas anti-S. aureusactivity has beenconfirmedtoactinodaphnine(4)(Hoetetal.,2004), suppor-tingthepresentfindings.Accordingtothepresentachievements, thefractionationofEB1109uptoFA5andFA6ledtoan improve-mentoftheantibacterialactivityandconfirmedactinodaphnineas apotentialantibacterialagent.Theantimicrobialactivityagainst Gram-positivebacteriaofdifferentisoquinolinecompoundshave alreadybeenstudied,regardingtheirstructure–activity relation-ships. In these studies, aporphines demonstrated lower or no activity.Onlynor-aporphinesbearinga1,2-methylendioxygroup couldbeconsideredactive.A.hypoglaucaaccumulatedatleasttwo aporphinealkaloids,anonaine(3)andactinodaphnine(4),that con-tainthestructuralrequirementslikelytobetheresponsibleforthe resultsobserved.

Resultsshown inFig.1 andTable2 describethepercentage ofgrowthand lethalityobservedforalltreatmentsagainst can-cercelllines.EB1109showedcytotoxicityagainstbreast(MCF-7) andcolon (KM-12)cancercelllines.Thepercentageoflethality foundforbreastcelllineswas−32.80%.Thisnegativevalueshall

beinterpretedbasedonthetimezero(T0)opticaldensityrelated tocellgrowthbeforetreatmentaddition,soEB1109notonlykilled 100%ofthesecells(whencomparedtoacellgrowthcontrol with-outtreatment), but alsokilleda number ofthe cellsthat grew beforetreatment.EB1109hasalsoshownlethalityagainstKM-12

Percentage of growth inhibition

DOXO

FA6*

FA5*

FA4.4

TA

FBuOH

T

reatments [100

µ

g/ml]

FEAC

DCM/MeOH

FHex

Crude extract

–100 –50 50

% LE-% GI

100 0

MCF-7 PC-3 NCI-H460 KM-12 SF-268 RPMI-8226

Fig.1.Cytotoxicactivityobservedaftertreatmentswithcrudeextract,total alka-loids(TA),alkaloid-freefractionsdescribedashexanefraction(FHex),butanol fraction(FBuOH),dichloromethane/methanolfractionandethylacetatefraction (FEAC),from stemofAnnona hypoglauca againstsixhuman tumorcelllines. MCF-7:breastcarcinoma;PC-3:prostatecarcinoma;NCI-H460:non-smallcells lungcarcinoma;KM-12:colonadenocarcinoma;SF-268:glioblastomaand RPMI-8226:multiplemyeloma.*FA4.4,FA5andFA6:columnfractionsobtainedfrom TAcontainingasmajorcomponentsisoboldineandactinodaphnine,respectively. NI:noinhibition;NT:nottested.Graphicis interpretedasthepercentageof growthobservedforthesixcellsafterbeingtreated.Calculationsusetheformula [{(T−T0)/(C−T0)×100}−100=%ofgrowthinhibition].Resultsareinterpretedby

thecomparisonofcellgrowthaftertreatment(T)tothecellgrowthatTimeZero (T0).If100>T>T0itmeansthattreatmentpreventedapercentageofcellsfrom grow;ifT=T0=0itmeansthattreatmentiseffectivetoavoidcellsfromgrow,in relationtoT0;ifT<T0itmeansthattreatmentiseffectiveenoughtocauselethality.

(−2.5%)andcytotoxicityagainstcentralnervoussystemcellline

SF-268(41.5%growthinhibition),prostate cancercelllinePC-3 (88.6%ofgrowthinhibition),leukemiacelllineRPMI-8226(82.7% ofgrowthinhibition)andlungcancercelllineNCI-H461(98.7%of growthinhibition).Totalalkaloidfractionshowedexpressive cyto-toxicityagainstMCF-7(−8.90%oflethality)andhasalsoshown

(6)

Table2

PercentageofgrowthofcancercelllinesMCF-7(breast),PC-3(prostate),NCI-H460(lung),KM-12(colon),SF-268(centralnervoussystem)andRPMI-8226(leukemia),after treatmentwithorganicextractofstemofAnnonahypoglaucaanditsfractions.NegativenumbersmeanlethalitytocelllinesinrelationtocellgrowthcontrolandTimezero growthcontrol(cellgrowthbeforetreatmentaddition).

MCF-7 PC-3 NCI-H460 KM-12 SF-268 RPMI-8226

Crudeextract −32.80 11.4 1.3 −2.5 58.5 17.3

FHex NI 77.4 48.1 NI NI 53.5

DCM/MeOH 2.90 59.6 44.9 NI 93.7 53.4

FEAC Nottested 67.1 38.6 NI 77.4 NI

FBuOH 11.90 46.6 65.6 NI NI 83.8

TA −8.90 17.04 71.7 32.4 73.3 NI

FA4.4 Nottested −3.8 −26.0 11.7 45.4 29.7

FA5a

−11.60 Nottested Nottested −14.4 Nottested Nottested

FA6a

−3.10 Nottested Nottested −8.4 Nottested Nottested

DOXO −16.31 −50.0 −46.2 −5.1 −34.0 −14.3

FHex,fractionhexane;DCM/MeOH,fractiondichloromethaneandmethanol;FEAC,fractionethylacetate;FBuOH,fractionbutanol;TA,totalalkaloidfraction;DOXO, doxorubicin.

a_FA5_and_FA6_are_column_fractions_obtained_from_the_total_alkaloids_containing_as_major_components_isoboldine_and_{actinodaphnine,}_{respectively.}

inhibitedbreastcancercelllines,andthatmayindicatethatthe alkaloidsareresponsible tothecytotoxicityagainstbreast can-cercellline.Noneofthefractionsobtainedfromthecakeshowed lethality,althoughhaveshowncytotoxicity,asDCM/MeOH frac-tion, which showed growth inhibition of 97.1% against breast cancercelllineandBuOHfraction, whichinhibitedin88.1%the growthofthesamecell.FA5andFA6weretestedonlyagainst MCF-7andKM-12.Itwasobservedalethalityof11.6%againstMCF-7 breastcancercelllineand14.4%againstcoloncancercellline KM-12forfractionFA5.Isoboldineisthemajorcompoundoffraction FA5,whichcontainsyetnornuciferineandanonaine. Nornucifer-ineshowedleishmanicidalactivityagainst Leishmania mexicana

andL.panamensis(Montenegroetal.,2003)Anonaine,asisolated fromdiverseAnnonaceaeandMagnoliaceaespecies isfoundto beactiveasantiplasmoidal,antimicrobial,antioxidant,anticancer, antidepressantandvasorelaxant(Lietal.,2013).Insilico analy-sisofanonaineledtotheidentificationofitspotentialtoinhibit topoisomeraseII,oneofthecrucialtargetsagainstcancers(Singh etal.,2016).FA6hasalsoshownalethaleffect,althoughweaker, againstthecelllinesofbreastcancerMCF-7(−3.1%)andcolon

can-cerKM-12(−8.4%).Actinodaphnine(4)wasfoundinthisfraction.

Aporphinoidalkaloidsbearinga methylenedioxygroup,suchas actinodaphnine(4),exhibitedahighaffinitytotheDNAmolecule demonstratingacytotoxicactivityinvitroandanon-specific inhi-bitionoftheTopoisomeraseIactivitythroughDNAintercalation (Hoetetal.,2004).Inourfindings,fractionFA5,which contains isoboldine(1),nornuciferine(2)andanonaine(3),wasmore effi-cient thanFA6,the fractioncontainingactinodaphnine. Further studiesrelatedtostructure–activityrelationshipareneededto elu-cidatedifferencesinbiologicalactivityrelatedtoAnnonaalkaloids. Moreover,fractionFA4.4,obtainedfromfractionFA4byCCD,was testedandresultsshoweditssignificantlethalityagainstlung can-cercelllineNCI-H460(−26.0%)andprostatecancerPC-3(−3.8%),

andshowingagrowthinhibitionagainstthecelllinesofCNS can-cerSF-268(54.6%),leukemiaRPMI-8226(70.3%)andcoloncancer KM-12(88.3%).Itisclearthatthetotalalkaloidfractionandtheir fractionsFA5andFA6haveshownanexcellenttumorcelllethality incomparisontoanyofthefractionsobtainedfromthecake,and clearly,thealkaloidsareresponsibleforthesignificantcytotoxic activity,butmaybethepresenceofothercompoundsmayinduce somesynergismobservedintheextremegoodactivityofthecrude extract.Also,fractionFA4.4hasshownadifferentcytotoxicity pro-file,becauseitwastheonlyfractionamongalltestedthatrevealed anextremegoodactivityagainstlungcancercellline,althoughno alkaloidhasbeenidentifiedfromthatfractionsofar.Thestronger effectobservedforthecrudeextractmightbeexplainedbya syn-ergismamongthealkaloidsandtheothermetabolitespresentin theextract.

Conclusions

ThepresentresultsdemonstratedthatA. hypoglaucashowed animprovementofitsantibacterialactivity,asfractionationwas performed,maybeduetothepresenceofactinodaphnine. Actin-odaphnine,thealkaloidpresentinFA6,mayalsobetheresponsible forthefractioncytotoxicity,butthepresenceofisoboldine, nornu-ciferineandanonainemayhavesynergisticallycontributedtothe expressivelethalitytobreastcancercells,ifcomparedtoFA6.This isthefirstreportfortheoccurrenceofactinodaphnineinAnnona

species.Also,EB1109,TA,FA5andFA6showedsignificant cyto-toxicactivityagainstbreastandcoloncancercelllines.Finally,the identificationofsuchenthusiasticresultsledustopursuenew evi-dencesofbiologicalactivitiesrelatedtoA.hypoglauca,aswellasto achieveresultsonitstoxicactivitiesinthefuture.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authorscontributions

MVNRexecutedalltheexperiments;IECDmadeNMRanalyses; IBSexecuted/designedcytotoxicityassays andmanuscript writ-ingandtranslation;PRHMwriting/translationofthemanuscript, designedthechemicalexperiments,conductedasasupervisor.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsareindebtedtoFAPESP(#99/05904-6; #08/58706-8)forthefinancialsupportandtheCNPqforresearchgrant.

References

(7)

Barnabé,M.,Saraceni,C.H.C.,Dutra-Correa,M.,Suffredini,I.B.,2014.Theinfluence ofBrazilianplantextractsonStreptococcusmutansbiofilm.J.Appl.OralSci.22, 366–372.

Bermejo, A., Figadere, B., Zafra-Polo, M.C., Barrachina, E.E., Cortes, D., 2005. AcetogeninsfromAnnonaceae:recentprogressinisolation,synthesisand mech-anismsofaction.Nat.Prod.Rep.22,269–303.

Bhakuni,D.S.,Tewari,S.,Dhar,M.M.,1972.AporphinealkaloidsofAnnonasquamosa. Phytochemistry11,1819–1822.

Camargo,L.R.P.,Suffredini,I.B.,2014.Atividadeanti-Escherichiacolideextratosde plantasbrasileiras.Novastendênciasempesquisaveterinária.Arq.Bras.Med. Vet.Zootec.66,617–620.

Castilho,A.L.,daSilva,J.P.C.,Saraceni,C.H.C.,Díaz,I.E.C.,Paciencia,M.L.B.,Varella, A.D.,Suffredini,I.B.,2014.InvitroactivityofAmazonplantextractsagainst

Enterococcusfaecalis.Braz.J.Microbiol.45,769–779.

Castilho,A.L.,Saraceni,C.H.C.,Diaz,I.E.C.,Paciencia,M.L.B.,Suffredini,I.B.,2013. Newtrendsindentistry:plantextractsagainstEnterococcusfaecalis.Theefficacy comparedtochlorhexidine.Braz.OralRes.27,109–115.

Cordell,G.A.,Quinn-Beattie,M.L.,Farnsworth,N.R.,2001.Thepotentialofalkaloids indrugsdiscovery.Phytother.Res.15,183–205.

Costa,E.V.,Sampaio,M.F.C.,Salvador,M.J.,Nepel,A.,Barison,A.,2012.Chemical constituentsofthestembarkofAnnonapickelii(Annonaceae).Quim.Nova38, 769–776.

Debourges,D.,Roblot,F.,Hocquemiller,R.,Cavé,A.,1987.AlkaloidesdesAnnonacées 77.AlcaloidesdesDuguetiaspixiana.J.Nat.Prod.50,664–673.

Dutra,L.M.,Costa,E.V.,Moraes,V.R.S.,nogueira,P.C.L.,Vendramin,M.E.,Barison, A.,Prata,A.P.N.,2012.ChemicalconstituentsfromtheleavesofAnnonapickeii

(Annonaceae).Biochem.Syst.Ecol.41,115–118.

Fischer,D.C.H.,Gualda,N.C.A.,Bachiega,D.,Carvalho,C.S.,Lupo,F.N.,Bonotto,S.V., Alves,M.O.,Yogi,A.,DiSanti,S.M.,Avila,P.E.,Kirchgatter,K.,Moreno,P.R.H., 2004.Invitroscreeningforantiplasmodialactivityofisoquinolinealkaloidsfrom Brazilianplantspecies.ActaTrop.92,261–266.

Gottsberger,G.,1978.SeeddispersalbyfishininundatedregionsofHumaita, Ama-zonia.Biotropica10,170–183.

Guinaudeau,H.,Leboeuf,M.,Cavé,A.,1983.Aporphinealkaloids,III.J.Nat.Prod.46, 761–835.

Hamid,R.A.,Foong,C.P.,Ahmad,Z.,Hussain,M.K.,2012.Antinociceptiveand anti-ulcerogenicactivitiesoftheethanolicextractofAnnonamuricataleaf.Rev.Bras. Farmacogn.22,630–641.

Hasrat,J.A.,Pieters,L.,deBacker,J.P.,Vauquelin,G.,Vlietnick,A.J.,1997.Screening ofmedicinalplantsfromSurinamefor5-HT1Aligands:bioactiveisoquinoline alkaloidsfromthefruitofAnnonamuricata.Phytomedicine4,133–140. Heywood,V.H.,1993.FloweringPlantsoftheWorld.BTBatsfordLtd,London. Hocquemiller,R.,Rasamizafy,S.,Cavé,A.,1983.AlcaloïdesdesAnnonaceesXXXVII:

AlcaloïdesduGuatteriascandens.J.Nat.Prod.46,335–341.

Hoet,S.,Stévigny,C.,Block,S.,Opperdoes,F.,Colson,P.,Baldeyrou,B.,Lansiaux,A., Bailly,C.,Quetin-Leclercq,J.,2004.AlkaloidsfromCassythafiliformisandrelated aporphines:antitrypanosomalactivity,cytotoxicity,andinteractionwithDNA andtopoisomerases.PlantaMed.70,407–413.

Jackman,L.M.,Thewella,J.C.,Moniot,J.L.,Shamma,M.,Stephens,R.L.,Wenkert, E.,1979.Thecarbon-13NMRspectraofaporphinealkaloids.J.Nat.Prod.42, 437–449.

Jackson,A.H.,Martin,J.A.,1966.Stericeffectsinthemassspectraofaporphine alkaloids.J.Chem.Soc.(C)23,2181–2183.

Leboeuf,M.,Cavé,A.,Bhaumilk,P.K.,Mukherjee,R.,1982.Thephytochemistryofthe Annonaceae.Phytochemistry21,2783–2813.

Li,H.T.,Wu,H.M.,Chen,H.L.,Liu,C.M.,Chen,C.Y.,2013.Thepharmacological activ-itiesof(−)-anonaine.Molecules18,8257–8263.

Lúcio,A.S.S.C.,Almeida,J.R.G.S.,Cunha,E.V.L.,Tavares,J.F.,Barbosa-Filho,J.M.,2015. AlkaloidsoftheAnnonaceae:occurrenceandacompilationoftheir biologi-calactivities.TheAlkaloids:ChemistryandBiology,74.,1sted.Elsevier,pp. 233–409.

McLafferty,F.W.,Stauffer,D.B.,1989.TheWiley/NBSRegistryofMassSpectralData. Wiley-Interscience,NewYork.

Monks,A.,Scudiero,D.,Skehan,P.,Shoemaker,R.,Paull,K.,Vistica,D.,Hose,C., Langley,J.,Cronise,P.,Vaigrowolff,A.,Graygoodrich,M.,Campbell,H.,Mayo,J., Boyd,M.,1991.Feasibilityofahigh-fluxanticancerdrugscreenusingadiverse panelofculturedhumantumorcelllines.J.Natl.CancerInst.83,757–766. Montanha,J.A.,Amoros,M.,Boustie,J.,Girre,L.,1995.Anti-herpesvírusactivityof

aporphinealkaloids.PlantaMed.61,416–424.

Montenegro,H.,Gutiérrez,M.,Romero,L.I.,Ortega-Barría,E.,Capson,T.L.,Rios, L.C.,2003.AporphinealkaloidsfromGuatteriaspp.withleishmanicidalactivity. PlantaMed.69,677–679.

Ohashi,M.,Wilson,J.M.,Budzikiewicz,H.,Shamma,M.,Slusarchyk,W.A.,Djerassi, C.,1963.MassspectrometryinstructuralandstereochemicalproblemsXXXI. Aporphinesandrelatedalkaloids.J.Am.Chem.Soc.85,2807–2810.

Padma,P.,Chansouria,J.P.N.,Khosa,R.L.,1998.Effectofsomeindigenousdrugson coldimmobilizationstressinducedgastriculcer.Phytother.Res.12,127–128. Paulo,M.Q.,Barbosa-Filho,J.M.,Lima,E.O.,Maia,R.F.,Barbosa,R.C.B.B.C.,Kaplan,

M.A.C., 1992. Antimicrobial activity of benzilisoquinoline alkaloids from

AnnonasalzmaniiD.C.J.Ethnopharmacol.36,36–41.

Pimenta,L.P.S.,Pinto,G.B.,Takahashi,J.A.,Silva,L.G.F.,Boaventura,M.A.D.,2003. Bio-logicalscreeningofAnnonaceusBrazilianmedicinalplantsusingArtemiasalina

(BrineShrimpTest).Phytomedicine10,209–212.

Rabêlo,S.V.,Araújo,C.S.,Costa,V.C.O.,Tavares,J.F.,Silva,M.S.,Barbosa-Filho,J.M., Almeida,J.R.G.S.,2014.OccurrenceofalkaloidsinspeciesofthegenusAnnonaL. (Annonaceae):areview.NutraceuticalsandFunctionalFoods:NaturalRemedy, vol.1.,1ed.NovaSciencePublishers,NewYork,UnitedStates,pp.41–60. Rasamizafy,S.,Hocquemiller,R.,Cassels,B.K.,Cavé,A.,1987.AlkaloidsfromAnnona

hayesii.J.Nat.Prod.50,759–761.

Reflora. 2016. http://reflora.jbrj.gov.br/reflora/listaBrasil/ConsultaPublicaUC/ BemVindoConsultaPublicaConsultar.do?invalidatePageControlCounter=7& idsFilhosAlgas=%5B2%5D&idsFilhosFungos=%5B1%2C11%2C10%5D&lingua=& grupo=5&familia=null&genero=&especie=&autor=&nomeVernaculo=birib%C3% A1&nomeCompleto=&formaVida=null&substrato=null&ocorreBrasil= QUALQUER&ocorrencia=OCORRE&endemismo=TODOS&origem=TODOS& regiao=QUALQUER&estado=QUALQUER&ilhaOceanica=32767& domFitogeograficos=QUALQUER&bacia=QUALQUER&vegetacao=TODOS& mostrarAte=SUBESP VAR&opcoesBusca=TODOS OS NOMES&loginUsuario= Visitante&senhaUsuario=&contexto=consulta-publica(Accessedin26.07.16). Revilla,J.,2002.PlantasúteisdaBaciaAmazônica,II.INPA,Manaus,pp.444. Siebra,C.A.,Nardin,J.M.,Florão,A.,Rocha,F.H.,Bastos,D.Z.,Oliveira,B.H.,

Weffort-Santos,A.M.,2009.PotencialantiinflamatóriodeAnnonaglabraAnnonaceae. Rev.Bras.Farmacogn.19,82–88.

Simeón,S.,Ríos,J.L.,Villar,A.,1990.AntimicrobialactivityofAnnonacherimoliastem barkalkaloids.Pharmazie45,442–443.

Singh, S.,Das, T., Awasthi,M., Pandey,V.P., Pandey,B., Dwivedi,U.N., 2016. DNAtopoisomerase-directedanticancerousalkaloids:ADMET-basedscreening, moleculardocking,anddynamicssimulation.Biotechnol.Appl.Biochem.63, 125–137.

Siqueira,C.A.T.,Oliani,J.,Sartoratto,A.,Queiroga,C.L.,Moreno,P.R.H.,Reimão,J.Q., Tempone,A.G.,Fischer,D.C.H.,2011.Chemicalconstituentsofthevolatileoil fromleavesofAnnonacoriaceaandinvitroantiprotozoalactivity.Rev.Bras. Farmacogn.21,33–40.

Soares,E.R.,Silva,F.M.A.,Almeida,R.A.,Lima,B.R.,SilvaFilho,F.A.,Barison,A.,Koolen, H.H.F.,Pinheiro,M.L.B.,Souza,A.D.L.,2015.DirectinfusionESI-IT-MSn_alkaloid

profileandisolationoftetrahydroharmanandotheralkaloidsfromBocageopsis pleiospermaMaas(Annonaceae).Phytochem.Anal.26,339–345.

Sonnet,P.E.,Jacobson,M.,1971.TumorinhibitorsII:cytotoxicalkaloidsfromAnnona purpurea.J.Pharm.Sci.60,1254–1256.

Stévigny,C.,Block,S.,Pauw-Gillet,M.C.,Hoffmam,E.,Llabres,G.,Adjakidjé,V., Quetin-Leclercq,J.,2002.CytotoxicaporphinealkaloidsfromCassythafiliformis. PlantaMed.68,1042–1044.

Suffredini, I.B.,Paciencia,M.L.B.,Frana,S.A.,Varella, A.D.,Younes,R.N., 2007a.

InvitrobreastcancercelllethalityofBrazilianplantextracts.Pharmazie10, 798–800.

Suffredini,I.B.,Paciencia,M.L.B.,Varella,A.D.,Younes,R.N.,2007b.Invitrocytotoxic activityofBrazilianplantextractsagainstahumanlung,colonandCNSsolid cancersandleukemia.Fitoterapia78,223–226.

Suffredini,I.B.,Paciencia,M.L.B.,Varella,A.D.,Younes,R.N.,2006a.Invitroprostate cancercellgrowthinhibitionbyBrazilianplantextracts.Pharmazie61,722–724. Suffredini,I.B.,Varella,A.D.,Younes,R.N.,2006b.Cytotoxicmoleculesfromnatural sources.TappingtheBrazilianbiodiversity.AnticancerAgentsMed.Chem.6, 67–75.

Suffredini,I.B.,Sader,H.S.,Gonc¸alves,A.G.,Reis,A.O.,Gales,A.C.,Varella,A.D.,Younes, R.N.,2004.Screeningofantibacterialactiveextractsobtainedfromplantsnative toBrazilianAmazonrainforestandAtlanticforest.Braz.J.Med.Biol.Res.37, 379–384.

Suffredini,I.B.,Saraceni,C.H.C.,Díaz,I.E.C.,2015.Canmouthwashescontaining chlorhexidine0.12%beusedassynonymofawatersolutionofchlorhexidine 0.12%?Braz.J.Pharm.Sci.51,367–372.

Sulaiman,S.N.,Mukhtar,M.R.,Hadi,A.H.,Awang,K.,Hazni,H.,Zahari,A.,Litaudon, M.,Zaima,K.,Morita,H.,2011.Lancifoliaineanewbisbenzylisoquinolinefrom thebarkofLitsealancifolia.Molecules16,3119–3127.

Tempone,A.G.,Borborema,S.E.T.,Andrade,H.F.J.,AmorimGualda,N.C.,Yogi,A., Carvalho,C.S.,Bachiega, D.,Lupo, F.N.,Bonotto,S.V.,Fischer,D.C.H.,2005. AntiprotozoalactivityofBrazilianplantextractsfromisoquinoline alkaloid-producingfamilies.Phytomedicine12,382–390.

Villar,R.,Calleja,J.M.,Morales,C.,Cáceres,A.,1997.Screeningof17Guatemalan medicinalplantsforplateletantiaggregantactivity.Phytother.Res.11,441–445. Villar,A.,Mares,M.,Rios,J.L.,Canton,E.,Gobernado,M.,1987.Antimicrobialactivity

ofbenzylisoquinolinealkaloids.Pharmazie42,248–250.

Voigt,W.,2005.SulforhodamineBassayandchemosensitivity.MethodsMol.Med. 110,39–48.

Wu,Y.C.,Chang,G.Y.,Duh,C.Y.,Wang,S.K.,1993.CytotoxicalkaloidsofAnnona montana.Phytochemistry33,497–500.