Rev. bras. farmacogn. vol.26 número6

RevistaBrasileiradeFarmacognosia26(2016)759–762

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Short

communication

Spermidine

alkaloid

from

Banara

parviflora

Maria

Izabel

G.

Moritz

_,

_Lara

_A.

_Zimmermann

_,

_Sérgio

_A.L.

_Bordignon

_,

_Miguel

_S.B.

_Caro

_,

Gabriela

M.

Cabrera

_,

_Jorge

_A.

_Palermo

_,

_Eloir

_P.

_Schenkel

a_{DepartamentodeCiênciasFarmacêuticas,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil}

b_{CursodeCiênciasBiológicas,CentroUniversitárioLaSalle,Canoas,RS,Brazil}

c_{DepartamentodeQuímica,UniversidadeFederaldeSantaCatarina,Florianópolis,SC,Brazil}

d_{DepartamentodeQuímicaOrgánicayUMYMFOR,FacultaddeCienciasExactasyNaturales,UniversidaddeBuenosAires,BuenosAires,Argentina}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17November2015 Accepted7June2016 Availableonline20July2016

Keywords:

Banaraparviflora

N1_,N8_{-dibenzoylspermidinyl-N}4_-acetamide

Spermidinealkaloids

a

b

s

t

r

a

c

t

Banaraparviflora(A.Gray)Benth.isaspeciesthatbelongstoSalicaceaefamilyandisnativetoSouthern Brazil.FractionationoftheethanolicextractfromBanaraparvifloraleavesafforded,anewcompound iden-tifiedasN1_,N8_{-dibenzoylspermidinyl-N}4_{-acetamide,aspermidinealkaloid.Thestructurewaselucidated}

byspectroscopicmethods(IR,MS,1_H,13_Cand2-DNMR).

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TheSalicaceaefamilyhasacosmopolitandistribution, includ-ingabout50generaand1000species.MostgeneraofSalicaceae occurringinBrazilcompriseonlyafewspeciesandhavearestricted distribution,exceptforthespeciesofthegenusCaseariaandBanara

(SouzaandLorenzi,2005).ThegenusBanaraAubl.wasoriginally describedbyAubletin1775andwasinitiallyclassifiedas Flacour-tiaceae(Sobraletal.,2006).InChaseetal.(2002)approximately two-thirds of all Flacourtiaceae genera, including Banara Aubl., wereincludedin theSalicaceae family basedonmorphological andmolecularstudies(Chaseetal.,2002;Mosaddiketal.,2007). Thegenuscomprisesabout64species,mainlydistributedinSouth America.ThespeciesBanaraparviflora(A.Gray)Benth.ispopularly knowninRioGrandedoSulas“farinha-seca”,meaning“dryflour” (Sobraletal.,2006).OtherspeciesofFlacourtiaceaenowclassified asSalicaceaefamily,presentsmainlyphenylpropanoids, sesquiter-penesandclerodanediterpenesintheirconstitution(Vieira-Júnior etal.,2011;Xiaetal.,2015).Otherwise,wedidnotdetect diter-penescompoundsintheinvestigationofB.parviflora.Amongthe scarcechemicalstudiestothegenusBanara,thereisonescreening studythatindicatesthepresenceofacyanogenicglycosidefromB. parviflora(SpencerandSeigler,1985).

This paper describes the isolation and identification of the flavoneorientinandalsotheisolationandstructuralelucidation

∗ _{Correspondingauthor.}

E-mail:eloir.schenkel@ufsc.br(E.P.Schenkel).

ofanewderivativeofspermidinefromB.parvifloraleavesand con-tributeswithcompletespectraldatatocharacterizethiskindof alkaloid.

Materialsandmethods

Theinfrared spectra(IR)wereobtainedin anIRPrestige-21 FTIR-8400S(Shimadzu)usingKBr.TheNMRspectrawereobtained onanapparatusBrukerAvance2(1_H:500MHz,13_C:125MHz)in CDCl3.Chemicalshiftsaregivenin ı(ppm)usingTMSas inter-nalstandard.The 2Dexperiments(HSQC,HMBC,COSY,NOESY) were performed using standard Brukerpulse sequences. High-resolutionAPPI(MSandMS2)massspectrawereobtainedona BrukerspectrometerMicrOTOF-QII,inpositiveionmode.During theisolationprocedures,chromatographicseparationswere per-formedonsilicagel60columns(0.04–0.063mm,0.063–0.2mm), activatedcharcoal (Nuclear®_{)and gelpermeationonmolecular} SephadexLH-20(GEHealthcare®_{).ForTLCanalysis,silicagel60} F256(Merck®_{)plateswereused.Thesubstances weredetected} byultravioletabsorptionat254and366nm,andalsoby spray-ingwithDragendorffreagentanddiphenylboryloxy-ethylamine1% inmethanol(NaturalReagent).ForHPLCanalysis,aSCL-10A Shi-madzuchromatographwasused,consistingofa LC-10ADpump andNT-10AVultravioletdetector.HPLCgrademethanol, acetoni-trile and acetic acid were provided by MTedia®_{. The purified} waterMilli-Q(Millipore®_{)wasalsousedasmobilephase.A} Shim-packpreparativeHPLCcolumn(ODS10mm,250mm×21.2mm)

was used for the separations and 0.45␮m Nylon membranes http://dx.doi.org/10.1016/j.bjp.2016.06.003

760 M.I.Moritzetal./RevistaBrasileiradeFarmacognosia26(2016)759–762

(Millipore®_{)forthefiltrationof themobilephase andsamples.} Theflavonoidorientin(3′_,4′_{,5,7-tetrahydroxyflavone-8-glucoside,}

≥98.0%)purchasedfromExtrasynthèsewasusedasstandard.

Theplantmaterial(B.parviflora(A.Gray)Benth.,Salicaceae)was identifiedbyoneofus(SALB)andwascollectedinJanuaryof2009 inTaquarí,RioGrandedoSul.Voucherspecimens(ICN189972) weredepositedattheHerbariumofDepartmentofBotany,UFRGS. Thefreshleaves(400g)weregroundandextractedwithethanol 96◦_{C for seven daysat roomtemperature. Theextract was}

fil-teredand concentrated ona rotaryevaporatorattemperatures below40◦C resultinginthecrude ethanolextractof theleaves

ofB.parviflora(15g).Thedriedethanol extract(15g)was frac-tionatedby Sigel vacuumliquid chromatography,using a step gradientfromhexanetoethylacetateandthenethylacetateto methanol as mobilephase, 200ml of each fraction. Eight frac-tionswereobtained.ThelastfractionH,whichhadthegreatest yield(9g),wasmostly composedofchlorophyllsand wasused to perform furtherfractionation on activated charcoal and sil-ica (1:1) using 200ml of methanol and methanol:acetonitrile (50:50, v/v) as mobile phase. The methanol fraction yielded 234mg whilethemethanol:acetronitrile fractionyielded45mg. Themethanolfraction(234mg)waspermeatedthroughSephadex LH-20 in methanol resulting mainly in two fractions (PS20-25: 8.2mg and PS60: 4.6mg) that were subsequently purified bypreparativeHPLC(mobilephase:acetonitrile:methanol:Milli-Q water(30:30:40,v/v/v),flowrate3ml/min,detectionat230nm) resultinginsubstances1and2.Theyieldofthesubstance1inthe

leaveswas0.004%(w/w)ofthedriedleaves.

Resultsanddiscussion

Asaninitialapproachtothechemicalcompositionoftheleaves, theethanolcrudeextractwasfirstanalyzedbyTLC,using differ-entsprayreagentsasDragendorff,EhrlichandNaturalReagentand twomajorcompoundsweredetected.Thefractionsandsubstances wereanalyzedbythinlayerchromatographytomonitorthe isola-tion.Theorientinstandardwasusedinbothtests,TLCandHPLC (injectedinthesameconditionswiththesubstance2)toconfirm theidentityofthecompound2.Thesubstance1waspositiveto Dragendorffreagent.Thesubstance2wasobtainedasyellowish

powderandshowedanUVspectrumsimilartoflavonoid deriva-tives.Afterwards,itwasidentifiedasorientinbyUV,TLCandHPLC comparisonwithanauthenticsample.

Substance1wasobtainedintheformofawhiteviscousoilthat aftersuccessivepurificationprocedureswaselucidatedby spectro-scopicanalysis(UV,IR,MSandNMR).Theultravioletmaximum absorptionwasobserved at232nm and 276nm (methanol).IR spectrumshowedacharacteristicabsorptionin3410vcm−1_(>NH)

andat1643and1635vcm−1(>C O)suggestingthepresenceofan

aromaticamidederivative.Theatmosphericpressure photoioni-zation(APPI)highresolutionmassspectrumexhibiteda[M+H]+_ion peakatm/z396.22623,thereforeamolecularformulaC23H29N3O3 wasdeduced,inaccordancewiththeNMRdata.

The13_{CNMRspectrumandtheDEPTexperimentsrevealedthe} presenceof23carbonatoms,threeofwhicharecarbonyls(ıC171.8, 168.0and167.6),12carbonscorrespondtoaromaticrings,while theremainingcarbonscorrespondtosevenCH2andonlyoneCH3 group.Thismethylgroupwasattributedtoasingletmethyl reso-nanceatıH2.13.Somesignalsofthe1HNMRspectrum,namelyıH 6.85andıH7.92(broaddd),showednocorrelationsinthe HSQC-DEPT135spectrumandwereassignedtoNHprotons,takinginto accounttheinformationobtainedbyIRandMS.TheCOSY spec-trumclearlyshowedcorrelationsbetweentheabovementioned amideprotonswiththealiphaticmethylenehydrogensatıH3.47 and3.37.ThesemethylenehydrogensandtheotherCH2hydrogens

Table1

13_Cand1_{HNMRdataofcompound1(}1_H,500MHz;13_{C,125MHz;CDCl} 3,ppm).

Position 1

ıCa ıHb(multiplicity,JinHz)

1 36.2(CH2) 3.37(m,6.0;6.8)

2 27.1(CH2) 1.78(m,6.2;6.8)

3 42.5(CH2) 3.46(dd,6.2)

5 48.4(CH2) 3.32(dd,7.1)

6 26.0(CH2) 1.68(m)

7 27.0(CH2) 1.65(m)

8 39.1(CH2) 3.47(m)

9 171.8(C) –

10 21.3(CH3) 2.13(s)

8_{C-NH – 6.85(dd,6.0;7.2)}

1_{C-NH – 7.93(dd,6.1;7.1)}

1′ _{167.6(C) –}

2′ _{134.2(C) –}

3′_;7′ _{127.0(CH) 7.87(dd,7.1;1.7)}

4′_;6′ _{128.5(CH) 7.43(dd,7.1;1.7)}

5′ _{131.3(CH) 7.48(m,7.1)}

1′′ 168.0(C) –

2′′ _{134.3(C) –}

3′′_;7′′ _{126.8(CH) 7.77(dd,7.2;1.5)}

4′′_;6′′ _{128.5(CH) 7.43(dd,7.2;1.5)}

5′′ _{131.5(CH) 7.50(m,7.2)}

a_125MHz,CDCl 3. b_500MHz,CDCl

3.

couldbesequentiallyassignedbyCOSYcorrelations.Twochains couldbedistinguished,onewith3carbonsandtheotherwith4 carbonsseparatedbya NH group,characterizingaspermidine derivative(Table1).

DetailedexaminationoftheHMBCcorrelationsallowed estab-lishingtheconnectivityasshowninFig.1,mainlythehydrogens of both monosubstituted aromatic rings and their correlations. Thus, the high-resolution mass spectra and its fragmentation pattern (Scheme 1), together with the NMR (1_H, 13_{C and 2-D} NMR) dataallowed us topropose thestructure of 1 asN1,N8 -dibenzoylspermidinyl-N4-acetamide.

This is the first report of the occurrence of this sub-stance in the literature, however, a similar structure, N1,N8 -dibenzoylspermidine,wasreportedbyAlemayehuetal.(1988)as a constituentof Cassiafloribunda Cav., Fabaceae.Theadditional acetamide couldbeincludedin thestructure byan acetylation usingacetyl-CoA(Dewick,2002).The1_Hand13_{CNMRdataofthe} compoundN1,N8-dibenzoylspermidine,providedintheliterature, werecomparedtothesubstance1data,herecharacterized,andthe

similarityofthesignalsallowedustoconfirmthestructure eluci-dation.Otherderivativesofspermidinehavealsobeenreported fromother plants,suchas N1,N10-di-dihydrocaffeoylspermidine fromextractsofIochromacyaneum,Solanaceae,anda feruloylsper-midineofCorylusavellanaL.,Betulaceae(Meureretal.,1986;Sattar

N H N O O H NH O H H H H H H H H H H H H H 3" 4" 5" 6" 7"

2" 1" 8 7 6 5 9 10 3 2 1 3' 7' 6' 5' 4' 1' 2'

M.I.Moritzetal./RevistaBrasileiradeFarmacognosia26(2016)759–762 761

N H

N

O O

NH

O

H

m/z396.2262 = [M+H]+_{= C} 23H30N3O3

N H H2N

O

H

N O

N H HN

OH

H

N O

m/z336.2 = C21H26N3O

-C11H16N2O

N H O

m/z162.0923 = C10H12NO

H N

N O

O N

H OH

H N

N O

O N

H OH

-C7H7NO

H N

O

O

m/z275.1776 = C16H23N2O2

Scheme1.ProposedfragmentationpatternandthemainfragmentionsinhighresolutionMS/MSspectrumofthecompound1.

et al., 1990) as wella siderephore (iron transport compound), parabactinAanditsderivatives,fromMicrococcusdenitrificansand alsoexcretedbythesoilbacteriumParacoccusdenitrificans, fea-turingironchelatorproperties,thissubstanceactslikeatransport systemtoassimilateironinthemicroorganismwithahigh affin-ity totheferric l-parabactin receptor in membranes(Bergeron

etal.,1980,1988).Additionally, asiderophorealsoverysimilar tothesubstance1,agrobactinwasidentifiedasaconstituentof

Agrobacteriumtumefaciens(Ongetal.,1979).Consideringthefact thatthesiderophoresareirontransportcompoundsandcommonly theyarefoundonlyinmicroorganismsandinsomegraminaceous plants,thereisapossibilityofthesubstance1hadbeenisolated fromsomeendophyticmicroorganisms(AhmedandHolmström, 2014).

According to the methodologies described by Bianco et al. (2013), the antimicrobial (against Staphylococcus aureus and

Enterococcus faecalis), antiprotozoal (Leishmania braziliensis and

Trypanosoma cruzi)and antiviral (HerpesSimplex Virus type1) activitieswereevaluatedforthesubstance1,whichdidnotshow activityinthismodels,attheconcentrationtested.

N1_,N8_{-dibenzoylspermidinyl-N}4_{-acetamide(1):whiteoil,R} f:

0.45(ethylacetate,waterandformicacid90:5:5).IR(KBr):3410, 1643,1635,1456,1309,615cm−1_.UV/vis

max(MeOH):232nm; 273.3nm; 526.3nm. 1_{H and} 13_{C NMR: Table 1. APPI MS: m/z} 396.22623 ([M+H]+ _{(100), (calc. for C23H30N3O3}+ _396.22817); MS/MSexperiment(CE15eV,Argon)on[M+H]+_{m/z396.23:m/z} 354.21752[M+H-CH2CO]+(calc.forC21H28N3O2354.21760);m/z 336.20720[M+H-CH2CO-H2O]+,(calc.forC21H26N3O336.20704);

m/z275.17766[M+H-C7H7NO]+(calc.forC16H23N2O2275.17540);

m/z233.16654[m/z275-CH2CO]+(calc.forC14H21N2O233.16484) andm/z162.09237(calc.forC10H12NO162.09134).

Authors’contributions

MIGMcontributedwithlaboratorywork,structuralelucidation, analysisofthedataandwritingthemanuscript.LAZcontributed withtheextractionandisolationofthecompoundsfromB. parv-iflora.SALBsuggestedtheinvestigation,contributedincollecting theplant, identificationand herbarium deposit. JAP,MSBC and GMCparticipatedinthestructuralelucidationandspectroscopic analysisandtocriticalreadingthemanuscript.EPSsupervisedthe work,collectedtheplant,andcontributedwritingthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

Theauthorsgratefullythankthefinancialsupportofthisstudy andalsotheresearchstipendsfromtheCNPqtoMIGM,LAZand EPS.

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