Rev. bras. farmacogn. vol.25 número4

(1)

RevistaBrasileiradeFarmacognosia25(2015)419–421

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

Short

communication

Alkaloids

isolated

from

the

leaves

of

atemoya

(

Annona

cherimola

_×

Annona

squamosa

)

Suzana

V. Rabêlo

a

_,

_Emmanoel

_V.

_Costa

b

_,

_Andersson

_Barison

c

_,

_Lívia

_M.

_Dutra

c

_,

_Xirley

_P.

_Nunes

d

_,

José

C. Tomaz

e

_,

_Gibson

_G.

_Oliveira

e

_,

_Norberto

_P.

_Lopes

e

_,

_Maria

_de

_Fátima

_C.

_Santos

f

_,

Jackson

R.G.

da

Silva

Almeida

a,_∗

a_Programa_de_{Pós-graduac¸}_ão_em_{Biotecnologia,}_Universidade_Federal_Rural_de_Pernambuco,_Recife,_PE,_Brazil b_Universidade_Federal_do_Amazonas,_Manaus,_AM,_Brazil

c_Centro_de_RMN,_Universidade_Federal_do_Paraná,_Centro_{Politécnico,}_Curitiba,_PR,_Brazil d_Universidade_Federal_do_Vale_do_São_Francisco,_Petrolina,_PE,_Brazil

e_Departamento_de_Física_e_Química,_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil f_Universidade_Federal_de_Sergipe,_São_Cristóvão,_SE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29May2015 Accepted7July2015 Availableonline26July2015

Keywords: Annonaceae Annona Alkaloids Atemoya Caatinga

a

b

s

t

r

a

c

t

AtemoyaisaninterspecificannonaceoushybridbetweenAnnonacherimolaMill.andAnnonasquamosa L. Itsphytochemical investigation ledto sevenalkaloids, including two aporphine (anonaineand asimilobine),three oxoaporphine(lanuginosine,liriodenine andlysicamine)andtwo proaporphine (pronuciferineandstepharine).Thesealkaloidswereidentifiedbyaseriesofspectrometricmethods, mainlyMSandNMR(1Dand2D),aswellasbycomparisonwithliteraturedata.Ourfindingsshowed thatthisspeciesisanimportantsourceofaporphinealkaloidsandhavehighrelationshipwithother Annonaspecies.

Introduction

Annonaceaeisalargefamilycomprisingabout135generaand

morethan2500species distributedmainlyintropicaland

sub-tropicalregions(Chatrouetal.,2004).Chemically,this familyis

characterizedby thepresence of isoquinoline alkaloids,mainly

aporphines.

Regardingthe species of Annonaceae,those from thegenus

AnnonaL.comprisesapproximately175speciesoftreesandshrubs.

InBrazilthegenusAnnonacontainsaround60species,withthe

largestpartoccurringinforestsandfewrepresentativesinopen

areas(Costaetal.,2011).Moreover,economically,thisgenusisthe

mostimportantofthefamilyAnnonaceaeduetoitsediblefruits

andmedicinalproperties(Dutraetal.,2012).

AtemoyaisafruitfulplantoftheAnnonaceaefamily,beinga

hybridresultingfromacrossbetweenthe“cherimoia”(Annona

che-rimolaMill.)andthe“pinha”or“fruta-do-conde”(Annonasquamosa

L.).ItwasachievedatthebeginningofthecenturyintheFlorida

∗ _{Corresponding}_author.

E-mail:jackson.guedes@univasf.edu.br(J.R.G.daSilvaAlmeida).

(USA),thecrosses beingrepeatedinothercountriesin orderto

obtainhybrids adaptedto tropicalclimate, asA. squamosa,and

subtropicalasA.cherimola(SilvaandMuniz,2011).

TheintroductionofatemoyaintheNortheastregionofBrazilis

recent,withapredominanceofthecultivarGefnerfromIsrael,

orig-inallygrownintheirrigationprojectsoftheValedoSãoFrancisco.A

recentstudycarriedoutbyourresearchgroupquantifiedthelevels

oftotalphenolsandflavonoidsaswellasevaluatedtheantioxidant

andantimicrobialactivitiesofextractsobtainedfromstemsand

leavesofatemoya(Rabêloetal.,2014a).

Due to expansion of atemoya cultivation and its increasing

consumptioninthemainBrazilianmarkets,itisnecessaryto

inves-tigatethechemicalcompositionofthisplant.Inthepresentpaper,

wereportresultsofthefirstphytochemicalstudyoftheatemoya

collectedintheValedoSãoFrancisco,andtheisolationand

chem-icalcharacterizationofsevenalkaloidsbyspectrometricmethods.

Materialsandmethods

NMRexperimentswereacquiredinCDCl3,at303KonaBruker

AVANCEIII600NMRspectrometeroperatingat14.1Tesla,

observ-ing1_H_and13_C_at₆₀₀_and₁₅₀_MHz,_{respectively.}_The_spectrometer

http://dx.doi.org/10.1016/j.bjp.2015.07.006

(2)

420 S.V.Rabêloetal./RevistaBrasileiradeFarmacognosia25(2015)419–421

wasequippedwitha5-mmmultinuclearinversedetectionprobe

withz-gradient.One-bondandlongrange1_H–13_C_correlation_from

HSQCandHMBCNMRexperimentswereoptimizedforaverage

couplingconstants1_J

(H,C)andLRJ(H,C)of140and8Hz,respectively.

All1_H_and13_C_NMR_chemical_shifts_are_given_in_ppm_related_to

theTMSsignalat0.00ppmasinternalreference,andthecoupling

constants(J)inHz.High-resolutionESI-MSdataweretakeninthe

positiveionmode,onaBrukermicrOTOFII–ESI-TOFMass

Spec-trometer.Silica gel 60 (F254) wasusedfor analytical thin layer

chromatography (TLC),while silica gel60 (230–240mesh)was

usedforcolumnchromatography(CC).Spotsonchromatograms

weredetectedunderexposuretoUVlight(254and365nm).When

necessary,Dragendorff’sreagentwasusedtovisualizethespotson

theTLCplates.

Leavesofatemoya(AnnonacherimolaMill.×Annonasquamosa

L.) were collected in Petrolina (Coordinates: 9◦20′30′′S and

40◦₄₀′₄₂′′_W), _state_of _Pernambuco, _Brazil,_in _July _of _2013._The

specieswasidentifiedbyProf.JoséAlvesdeSiqueiraFilho,anda

voucherspecimen(16310)wasdepositedintheHerbárioValedo

SãoFranciscooftheUniversidadeFederaldoValedoSãoFrancisco.

Driedandpowderedleavesofatemoya(1163g)wereextracted

withhexane(3l,threetimes),followedbyMeOH(3l,threetimes),

yieldingofhexane (63g) andMeOH (120g) extracts,aftereach

solventremovalunderreducedpressure.

TLCanalysisalsoindicatedahighconcentrationofalkaloidsin

theMeOHextract,whichwasinitiallysubjectedtoanacid–base

extraction(Costaetal.,2006)togivealkaloid(1.5g)andneutral (7.5g)fractions.Thealkaloidalfractionwassubjectedtosilicagel

CCpreviouslytreatedwitha 10%NaHCO3 solution,elutedwith

increasingconcentrations of hexane, CH2Cl2,EtOAc and MeOH,

giving297fractions(40mleach).Thesefractionswereevaluated

and pooledaccording toTLCanalysis yieldingeighteen groups.

Group 5 (81.8mg) was subjected to a preparative TLC eluted

withCH2Cl2:MeOH(95:05,

v

/

v

, three times), givingasimilobine

(1)(2.3mg), amixture ofalkaloids asimilobineand

pronucifer-ine(2)(6.5mg),and a mixtureofthree oxoaporphinealkaloids

lanuginosine(3),liriodenine(4)andlysicamine(5)(5.9mg).Group

7 (66.9mg) was subjected to a preparative TLC eluted with

CH2Cl2:MeOH(95:05,

v

/

v

,threetimes),affordingamixtureof

alka-loidsasimilobineandanonaine(6)(3.3mg)andtheproaporphine

alkaloidstepharine(7)(2.5mg).

Allisolatedcompoundswereidentifiedbyaseriesof

spectro-metricmethods,mainlyMSandNMR(1Dand2D)data,aswell

ascomparisonwiththosereportedintheliterature.Alkaloidsin

mixtureswereestablishedthroughtotallyindependentsdatasets

fromthe1Dand2DNMRexperimentsincludingsignalareas.Each

datasetwasconsistentwithonlyonestructure.Moreover,itwas

observedthesameresultsinseveralsamplesinvestigated.

Asimilobine(1): yellowamorphous solid,molecular formula

C17H17NO2. Positive ESI-MS m/z: 268.1354 [M+H]+. 1H NMR

(600MHz,CDCl3):ı8.27(1H,d,J=7.8Hz,H-11),7.22–7.33(3H,

m,H-8,9,and10),6.72(1H,s,H-3),3.59(3H,s,C1–OCH3).TheMS

and1_H_NMR_data_are_in_agreement_with_the_literature₍_Guo_et_al.,

2011;Costaetal.,2015).

Pronuciferine(2):brownamorphoussolid,molecularformula:

(600MHz,CDCl3):ı 6.41(1H,dd,J=8.1 and 2.8Hz, H-12),6.89

(1H,dd,J=7.1and2.8Hz,H-8),7.00(1H,dd,J=8.1and2.8Hz, H-9),6.29(1H,dd,J=8.10and2.00Hz,H-11),6.64(1H,s,H-3),3.60 (3H,s,C1–OCH3),3.80(3H,s,C2–OCH3),3.05(3H,s,N–CH3).13C

NMR(150MHz,CDCl3):ı184.42(C-10),153.34(C-8),128.49(C-9),

127.10(C-11),149.80(C-12),61.15(1-OMe),56.48(2-OMe),44.83

(N-Me).TheMS,1_H_NMR_and13_C_NMR_data_are_in_agreement_with

theliterature(Thuyetal.,2005).

Lanuginosine (3): dark brown solid, molecular formula:

C18H11NO4.PositiveESI-MSm/z:306.0762[M+H]+.TheMSdata

areinagreementwiththeliterature(Wijeratneetal.,1996).

Liriodenine(4):yellowamorphous solid,molecularformula:

areinagreementwiththeliterature(Guoetal.,2011;Costaetal., 2011).

Lysicamine(5): yellowamorphoussolid, molecularformula:

areinagreementwiththeliterature(Harriganetal.,1994).

Anonaine(6):yellowpowder,molecularformula:C17H15NO2.

PositiveESI-MSm/z:266[M+H]+_.1_H_NMR₍₆₀₀_MHz,_CDCl₃_):_ı_8.15

(1H,d,J=7.74Hz,H-11),7.22–7.32(3H,m,H-8,9,10),6.58(1H,s,

H-3),6.09and5.94(each1H,s,–OCH2O–).TheMSand1HNMR

dataareinagreementwiththeliterature(Guoetal.,2011;Costa

etal.,2015).

Stepharine (7): amorphous brownsolid, molecularformula:

(600MHz,CDCl3):ı6.39(1H,dd,J=8.4and2.0Hz,H-12),6.87(1H, dd,J=7.4and2.0Hz,H-8),6.98(1H,dd,J=7.4and2.00Hz,H-9), 6.28(1H,dd,J=8.4and2.0Hz,H-11),6.64(1H,s,H-3),3.60(3H,

s,C1–OCH3),3.80(3H,s,C2–OCH3).TheMSand1HNMRdataare

inagreementwiththeliterature(Thuyetal.,2005;Costaetal.,

2015).

Resultsanddiscussion

Thephytochemicalinvestigationfromtheleavesofthehybrid

atemoya,anediblefruitoftheAnnonaceaefamily,resultedinseven

alkaloids, includingtwo aporphine(anonaine and asimilobine),

threeoxoaporphine(lanuginosine,liriodenineandlysicamine)and

twoproaporphine(pronuciferineandstepharine).Allofthemare

beingdescribedforthefirsttimeinthehybridatemoya.

Thisfamilyisconsideredthecentreofdistributionof

isoquino-linealkaloids. Recent chapterpublished byour researchgroup

presentedanoverviewofthechemistryandpharmacologyofthe

alkaloidsfoundinspeciesoftheAnnonaceaefamily.Withinthe

Annonaceaefamily, thegenera Annona, Duguetia,and Guatteria

haveledtomanyimportantpublications.Thealkaloidsofthe

apor-phinetyperepresentthepredominantgroupinthisfamily.Many

of theisolated alkaloids exhibit uniquestructures. The chapter

couldbeconsideredasacontributionforthescientificcommunity,

mainlytoenablethesearchforalkaloidsinspeciesbelongingto

theAnnonaceaefamily(Lúcioetal.,2015).Anotherworkreviewed

articlespublishedintheliteratureregardingalkaloidsisolatedfrom

plantsofthegenusAnnona.Thisreviewcoversaperiodfrom1930

to2013andshowstheidentificationof147alkaloidsinAnnona

species(Rabêloetal.,2014b).

The aporphine asimilobine and anonaine, and the

oxoapor-phineliriodeninehavebeendescribedinseveralspeciesofAnnona.

Recentstudiesdescribetheisolation ofasimilobineand

liriode-nine in some species of Annona, suchas Annona foetida Mart.,

Annona pickelii(Diels)H. Rainer, AnnonasalzmanniiA. DC., and

AnnonasericeaDunal,allnativetoBrazil(Camposetal.,2008;Costa etal.,2011;Cruzetal.,2011;Dutraetal.,2012).Thesealkaloids

werefoundinleavesandbarksofA.crassiflorafromtheGuianas

(Hocquemilleretal.,1982).Anonaine,asimilobineandliriodenine

(3)

S.V.Rabêloetal./RevistaBrasileiradeFarmacognosia25(2015)419–421 421

consideredaschemotaxonomicmarkersofthisgenus(Cruzetal.,

2011).

Otherstudies described the isolation of thealkaloid

pronu-ciferineinAnnonacherimola(Chenetal.,1999);lanuginosinein

A.cherimola,A.rugulosaandA.squamosa;lysicamineinA. acumi-nata,A.cherimola,A.glabra,A.hayesii,A.pickelii,A.purpureaandA. sericea;andstepharineinAnnonacacans,A.glabra,A.hayesiiand

A.spinescens(Rabêloetal.,2014b).Allthesecompoundsarebeing

describedforthefirsttimeinthehybridatemoyacollectedinthe

ValedoSãoFrancisco.

Authors’contributions

SVR,XPNandMFCSwereresponsibleforthecollection,

prepa-rationoftheextractsandphytochemicalstudies.EVC,ABandLMD

conductedtheexperimentsof nuclearmagneticresonance. JCT,

GGOandNPLconductedtheexperimentsofmassspectrometry.

EVCandJRGSAanalyzedandinterpretedthedata,anddraftedthe

manuscript.Alltheauthorshave read thefinal manuscript and

approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultoCNPq(Processes:470594/2013-6,

442209/2014-2,and303587/2014-8),CAPES,FINEPandUFPRfor

financialsupportand fellowshipsaswellastoCentrode

Refer-ênciaparaRecuperac¸ãodeÁreasDegradadas(CRAD/UNIVASF)for

botanicalidentificationoftheplantmaterial.

References

Campos,F.R.,Batista,R.L.,Batista,C.L.,Costa,E.V.,Barison,A.,Santos,A.G.,Pinheiro, M.L.B.,2008.IsoquinolinealkaloidsfromleavesofAnnonasericea(Annonaceae). Biochem.Syst.Ecol.36,804–806.

Chatrou,L.W.,Rainer,H.,Maas,P.J.M.,2004.Annonaceae(Soursopfamily).In:Smith, N.,etal.(Eds.),FloweringPlantsofNeotropics.BotanicalGarden,NewYork,pp. 18–20.

Chen,C.Y.,Chang,F.R.,Teng,C.M.,Wu,Y.C.,1999.Cheritamine,anewn-fattyacyl tryptamineandotherconstituentsfromthestemsofAnnonacherimola.J.Chin. Chem.Soc.46,77–86.

Costa,E.V.,Pinheiro,M.L.,Xavier,C.M.,Silva,J.R.,Amaral,A.C.,Souza,A.D.,Barison, A.,Campos,F.R.,Ferreira,A.G.,Machado,G.M.,Leon,L.L.,2006.A pyrimidine-␤-carbolineandotheralkaloidsfromAnnonafoetidawithantileishmanialactivity. J.Nat.Prod.69,292–294.

Costa,E.V.,Pinheiro,M.L.B.,Souza,A.D.L.,Barison,A.,Campos,F.R.,Valdez,R.H., Nakamura,T.U.,Dias-Filho,B.P.,Nakamura,C.V.,2011.Trypanocidalactivity ofoxoaporphineandpyrimidine-␤-carbolinealkaloidsfromthebranchesof

AnnonafoetidaMart.(Annonaceae).Molecules16,9714–9720.

Costa,E.V.,Sampaio,M.F.C.,Salvador,M.J.,Nepel,A.,Barison,A.,2015.Chemical constituentsfromthestembarkofAnnonapickelii(Annonaceae).Quim.Nova,

http://dx.doi.org/10.5935/0100-4042.20150069(inpress).

Cruz,P.E.O.,Costa,E.V.,Moraes,V.R.S.,Nogueira,P.C.L.,Vendramin,M.E.,Barison,A., Ferreira,A.G.,Prata,A.P.N.,2011.ChemicalconstituentsfromthebarkofAnnona

salzmannii(Annonaceae).Biochem.Syst.Ecol.39,872–875.

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

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

Guo,Z.F.,Wang,X.B.,Luo,J.G.,Luo,J.,Wang,J.S.,Kong,L.Y.,2011.Anovelaporphine alkaloidfromMagnoliaofficinalis.Fitoterapia82,637–641.

Harrigan,G.G.,Gunatilaka,A.A.,Kingston,D.G.,Chan,G.W.,Johnson,R.K.,1994. Iso-lationofbioactiveandotheroxoaporphinealkaloidsfromtwoannonaceous plants,XylopiaaethiopicaandMiliusacf.banacea.J.Nat.Prod.57,68–73.

Hocquemiller,R.,Cavé,A.,Jacquemin,H.,Touche,A.,Forgacs,P.,1982.Alkaloids oftheAnnonaceae.XXXVI.AlkaloidsofAnnonacrassifloraMart.Plant.Med. Phytother.16,4–6.

Lúcio,A.S.S.C.,Almeida,J.R.G.S.,Cunha,E.V.L.,Tavares,J.F.,Barbosa-Filho,J.M.,2015.

AlkaloidsoftheAnnonaceae:occurrenceandacompilationoftheirbiological activities.TheAlkaloids:ChemistryandBiology,vol.74.,1sted.Elsevier,pp. 233–409.

Rabêlo,S.V.,Costa,M.M.,Libório,R.C.,Almeida,J.R.G.S.,2014a.Atividade antiox-idanteeantimicrobianadeextratosdeatemoia(AnnonacherimolaMill.×A.

squamosaL.).Rev.Bras.Frutic.36,265–271.

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.In:Brar,S.K.,Kaur,S.,Dhillon,G.S.(Org.),Nutraceuticals andFunctionalFoods:NaturalRemedy,1sted.,vol.1.NovaSciencePublishers, Inc.,NewYork,USA,pp.41–60.

Silva,A.V.C.,Muniz,E.N.,2011.Qualityofatemoyafruitsharvestedattwomaturity stages.Rev.Caatinga24,9–13.

Thuy,T.T.,Sung,T.V.,Franke,K.,Wessjohann,L.,2005.Aporphineandproaporphine alkaloidsfromStephaniarotunda.J.Chem.43,619–623.