Rev. bras. farmacogn. vol.27 número4

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

Original

Article

Phytochemical

study

of

Pilosocereus

pachycladus

and

antibiotic-resistance

modifying

activity

of

syringaldehyde

Severino

Gonc¸

alves

de

Brito-Filho

_,

_Jéssica

_Karina

_da

_Silva

_Maciel

_,

_Yanna

_Carolina

_Ferreira

_Teles

_,

Milen

Maria

Magalhães

de

Souza

Fernandes

_,

_Otemberg

_Souza

_Chaves

_,

_Maria

_Denise

_Leite

_Ferreira

_,

Pedro

Dantas

Fernandes

_,

_Leonardo

_P.

_Felix

_,

_Isis

_Caroline

_da

_Silva

_Cirino

_,

_José

_Pinto

_{Siqueira-Júnior}

_,

Raimundo

Braz-Filho

_,

_Maria

_de

_Fátima

_Vanderlei

_de

_Souza

a_{ProgramadePós-graduac¸ãoemProdutosNaturaiseSintéticosBioativos,CentrodeCiênciasdaSaúde,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil}

b_{ProgramadePós-graduac¸ãoemDesenvolvimentoeInovac¸ãoTecnológicaemMedicamentos,CentrodeCiênciasdaSaúde,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil}

c_{DepartamentodeQuímicaeFísica,CentrodeCiênciasAgrárias,UniversidadeFederaldaParaíba,Areia,PB,Brazil}

d_{DepartamentodeAgroecologiaeAgropecuária,CentrodeCiênciasAgráriaseAmbientais,UniversidadeEstadualdaParaíba,CampinaGrande,PB,Brazil}

e_{DepartamentodeCiênciasBiológicas,CentrodeCiênciasAgrárias,UniversidadeFederaldaParaíba,Areia,PB,Brazil}

f_{LaboratóriodeGenéticadeMicroorganismos,DepartamentodeBiologiaMolecular,UniversidadeFederaldaParaíba,JoãoPessoa,PB,Brazil}

g_{LaboratóriodeCiênciasQuímicas,UniversidadeEstadualdoNorteFluminenseDarcyRibeiro,CamposdosGoitacazes,RJ,Brazil}

h_{ProgramadePós-graduac¸ãoemQuímica,UniversidadeFederalRuraldoRiodeJaneiro,Seropédica,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25March2017

Accepted5June2017

Availableonline5July2017

Keywords:

Foragecactus

Caatingaecoregion

Cactaceae

Phytochemicalstudy

Antibacterialactivity

ResistantStaphylococcusaureus

a

b

s

t

r

a

c

t

PilosocereuspachycladusF.Ritter,Cactaceae,popularlyknownas“facheiro”,isusedasfoodand tradi-tionalmedicineinBraziliancaatingaecoregion.Theplantisusedtotreatprostateinflammationand urinaryinfection.ThepresentworkreportsthefirstsecondarymetabolitesisolatedfromP.pachycladus. Therefore,theisolatedcompound4-hydroxy-3,5-dimethoxybenzaldehyde(syringaldehyde)was evalu-atedasmodulatorofStaphylococcusaureuspumpefflux-mediatedantibioticresistance.Theisolationof compoundswasperformedusingchromatographictechniquesandthestructuralelucidationwascarried outbyspectroscopicmethods.InordertoevaluatesyringaldehydeabilitytomodulateS.aureus antibi-oticresistance,itsminimuminhibitoryconcentrations(␮g/ml)wasfirstdeterminate,then,thetested antibioticsminimuminhibitoryconcentrationsweredeterminedinthepresenceofthesyringaldehyde inasub-inhibitoryconcentration.Thechromatographicproceduresledtoisolationoftwelvecompounds fromP.pachycladusincludingfattyacids,steroids,chlorophyllderivatives,phenolicsandalignan.The syringaldehydedidnotshowanyantibacterialactivityat256␮g/mlagainstS.aureus.Ontheotherhand thecompoundwasabletoreducetheantibioticconcentration(tetracycline,norfloxacin,ethidium bro-mide)requiredtoinhibitthegrowthofdrug-resistantbacteria,showingtheabilityofsyringaldehydeof inhibitingtheeffluxpumponthesebacteria.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TheCactaceaefamily consistsof about127generaand1405 species(Huntetal.,2006).Brazilisconsideredthethirdlargest centerofdiversityofthisfamilywherethecaatingaecoregion com-prises58species(Zappietal.,2010).

Thecacti areplants that showspecial morpho-physiological characteristicswithseveralanatomicalvariationsthatensuretheir

∗ _{Correspondingauthor.}

E-mail:[email protected](M.F.Souza).

capacitytoretainwaterandtosurviveinhotandaridenvironments (Hernández-Hernándezetal.,2011).Cactiareusedas supplemen-talfoodforruminantsandindroughttimesthespeciesarealso usedasfoodforhuman,mainlyinpreparationofflourandcakes (CavalcantiandResende,2013;Macieletal.,2016).

PilosocereuspachycladusF.Ritterisaperennialshrubpopularly

knowninBrazilas“facheiro”(Abudetal.,2010).Itplays impor-tantroleinruralcommunitiesduetheuseofthisspeciestomake biscuits,cakesandcandies.Therefore,localpeoplementiontheuse ofitsrootstotreatprostaticinflammation,andthestemdecoction isusedagainsturinaryinfections,fluandfuruncle(Lucenaetal., 2013;Agraetal.,2008).Accordingly,theantimicrobialactivityof Cactaceae species hasbeenpreviously reported(Sanchezetal.,

http://dx.doi.org/10.1016/j.bjp.2017.06.001

0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

2014).Infact,plantextractsand compoundshave beenstudied aimingtodiscovernewbactericides,bacteriostaticandbacterial resistancemodulatoractiveproducts.Thisfacthasgainattention especially in recent years, when the raising of bacterial resis-tancehasbecomeaseriouspublichealthconcerningtoalmostall antibacterialagents.Amongtheresistantstrainsstudied,themain mechanismofbacterialresistanceisthepresenceofeffluxpumps inbacterialcellmembrane,whichallowtheleavingofantibiotics fromthecell(Piddock,2006).

ConsideringthegreatimportanceofCactaceaefamily,thisstudy reportsthe firstphytochemicalstudy ofP.pachycladus and the evaluation of theisolated compound 4-hydroxy-3,5-dimethoxy benzaldehyde (syringaldehyde) as modulator of Staphylococcus

aureuspumpefflux-mediatedantibioticresistance.

Materialsandmethods

Instrumentsandreagents

Isolatedcompoundswereidentifiedby1Dand2DNMRanalysis (1_H500MHz,13_{C125MHz,MercuryVarianand}1_H200MHz,13_C

50MHz,MercuryVarian)usingdeuteratedsolventsandinfrared (IR)Perkin-ElmerFT-IR-1750modelusingKBrdiscs;medium pres-sureliquidchromatography(MPLC)wascarriedoutusingaBUCHI (Switzerland)Pump ManagerC-615. High pressureliquid chro-matography(HPLC)wasperformedusingaShimadzuHPLCwith detectordiodearray.

Plantmaterial

Theplant material of Pilosocereuspachycladus F. Ritter, Cac-taceae,wascollectedinBoaVista(Paraíba,Brazil),inNovember 2010. The botanical identification wascarried out by Prof. Dr. LeonardoPessoaFelix(CCA/UFPB)andavoucherspecimen(19544) wasdeposited in the Herbarium Prof. Jaime Coelho de Morais (CCA/UFPB).

Extraction,fractionationandisolationofcompounds

Freshplantmaterialofthestems,fruitandflowersofP.

pachy-cladusweredriedseparatelyinanovenwithcirculatingairat40◦_C

for72handeachthematerialwasgroundinamechanicalmill, pro-viding4.934g,2.884gand10.022gofpowder,respectively.The powderofthestem,fruitand theflowerswereseparately sub-mitted tomacerationin ethanol (EtOH) (95%)for 72hatroom temperature.Theobtainedethanolicextractswereconcentrated with a rotatory evaporator yielding 205.58g of crude ethanol extractofstem(CES),206.69goffruit(CEFR)and253.53g(CEFL) offlowers.

Thecrudeethanolicextractofstem(CES)wassolubilizedusing EtOH:H2O(7:3)andtheobtainedsolutionwassequentially

parti-tionedinseparationfunnelusinghexane,dichloromethane,ethyl acetate(EtOAc)andn-butanol.Theobtainedsolutionswere con-centratedinrotatoryevaporator,yielding 33.18gof thehexane phase,16.70gofthedichloromethanephase,6.11gofethylacetate phaseand29.34gofn-butanolphaseandthehydroalcoholicphase (90.20g).

AportionofthehexanephaseoftheCES(10.5g)wassubjected tochromatographic column(Column 1)usingsilica gelandthe solventshexane,dichloromethaneandmethanol,pureorin mix-turesincreasingpolarity,resultingin124fractions.Theobtained fractionswereconcentratedandanalyzedbythinlayer chromatog-raphy(TLC).Thefraction31/59(0.7883g)waschromatographed atthesameconditions,which werecombinedafteranalysisby TLC.Thesub-fraction1/33(0.151g)wasagainchromatographed

followingthesamemethodtoobtainapurewhiteamorphoussolid (39mg),codedascompound1.

The fraction 81/113 (0.72g) from column 1 was chro-matographedin silicacolumn using hexane,CH2Cl2 and MeOH

andprovided a purewhiteprecipitateencoded ascompound 2 (250mg).

Thefraction114/124(0.90g) waschromatographed in silica flashcolumnusinghexane,EtOAcandMeOH.Fromthiscolumn 73fractionswerecollectedandcombinedbyTLC.Thecombined fractions22/24and41/46(darkgreensolids)werepurewhen ana-lyzedbyTLC,beinglabeledascompound3(20mg)andcompound 4(40mg),respectively.

Thefraction60/80(1.28g) fromcolumn1 wassubmittedto silica column using as eluents hexane, CH2Cl2 and MeOH. The

columnyielded52fractionsanalyzedandcombinedbyTLC.The combinedfractions08/16provided600mgofpurewhitecrystals (compound5).

AnaliquotofdichloromethanephaseoftheCES(10.07g)was submittedtovacuumliquidchromatography(VLCI),usingsilica gelina Büchnerfunnelwithporousplate.Thefollowing gradi-entmixtureofsolventswereused:hexane:CH2Cl2(7:3),CH2Cl2

(100%), CH2Cl2:MeOH (9:1), CH2Cl2:MeOH (8:2), CH2Cl2:MeOH

(7:3),CH2Cl2:MeOH(1:1)and EtOAc:MeOH (1:1). Thefractions

were concentrated under reduced pressure to obtain: 0.019g, hexane:CH2Cl2 (7:3); 0.162g, CH2Cl2; 8g, CH2Cl2:MeOH (9:1);

2.239g,CH2Cl2:MeOH(8:2);0.244g,CH2Cl2:MeOH(7:3),0.400g,

CH2Cl2:MeOH(1:1)and0.220g,EtOAc:MeOH(1:1).

ThefractionCH2Cl2(0.162g)fromVLCIwassubjectedtoMPLC

using silica flash as stationary phase and hexane, CH2Cl2 and

methanolasmobilephase(pureorbinarymixtures)obtainingfifty fractionsanalyzedandcombinedbyTLC.Thefraction14/18showed pureyellowcrystalsbeinglabeledascompound6(25mg).

ThefractionCH2Cl2:MeOH(9:1)(7.9139g)fromVLCIwas

chro-matographedinsilicacolumnusinghexane,CH2Cl2andmethanol

(pureorbinarymixtures).Thisprocedureresultedinapurewhite amorphoussolid,namedascompound7(32mg).

The CEFR was partitioned in separation funnel using the samemethoddescribedforCES.Fromthepartitionthefollowing phases were obtained: 87.13gof thehexane phase, 20.14g of thedichloromethanephase,4.69gofethylacetatephase,41.83g of n-butanolic phase, and 16.76gof hydroalcoholic phase. The dichloromethanephase oftheCEE(10g) wassubmittedtoVLC (VLCII) using hexane, CH2Cl2 and MeOH alone or mixtures in

gradientwise.Theobtainedfractionswereconcentratedinrotary evaporator.

ThefractionCH2Cl2:MeOH(9:1)(3.4g)fromVLCIIwas

sub-jected to silica column chromatography using hexane, CH2Cl2

and MeOH. The combined fractions 6/12 (0.95g) by from this columnchromatographywererechromatographedfollowingthe samemethodresulting in33 fractions.The fraction12showed whitecrystals(12mg),namedascompound8.

TheethylacetatephaseofCEFR(4.69g)waschromatographed incolumnofSephadexLH-20elutedwithmethanolyielding37 fractions.Thecombinedfractions4/14wererechromatographed using the same method. An aliquot (0.100g) of the obtained sub-fractions 4/10, was injected at a concentration of 5mg/ml in an HPLC ( 269nm) using a C18 column and gradient method H2O:MeOH (5:95) by 1h. The chromatogram showed

threepeaks.Thesampleswerecollectedandanalyzed.The sec-ond peak collected was named as compound 9 (6mg) (white solid).

Analiquot ofhexane phase ofCEFL (10g)wassubmittedto columnchromatographyusinghexane,EtOAcandMeOHpureor ingradientmixtures.Fromthiscolumn,82fractionscollectedof 20mlwerecollected.Thesub-fraction36/42showedaprecipitate andasupernatant.Thesupernatant(0.20g)waschromatographed following thesamemethodologypurifyingtransparent crystals, labeledascompound10(46mg).Theprecipitatewaswashedwith hexaneandnamedascompound11(18mg).Thecombined frac-tion56/60(0.065g)fromCEFLhexanephaseshowedaprecipitate (whitecrystals).Itwaswashedwithhexanetoyieldthecompound 12(25mg).

Minimuminhibitoryconcentrationandmodulationofbacterial

resistance

The antibiotics norfloxacin, erythromycin and tetracycline were obtained from Sigma Chemical Co. (USA). The antibiotic solutions were prepared according to the guidelines of CLSI (2005).Stocksolutionsof4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)werepreparedusingDMSOandatthehighest concentrationafterdilutioninthebroth(4%)causednobacterial growthinhibition.

Theminimuminhibitoryconcentrations(MIC)oftheantibiotics andthetestedcompoundsyringaldehydeweredeterminedinBHI bythemicrodilutionassayusing asuspension ofca.105_cfu/ml

andadrugconcentrationrangeof256-0.5␮g/ml(two-foldserial dilutions). TheMIC wasdefinedas thelowest concentrationat which nobacterialgrowthwasobserved. For theevaluation of syringaldehydeasmodulatorofantibioticresistance,theMICof theantibioticsweredeterminedinthepresenceofthecompound inasub-inhibitoryconcentration(Falcão-Silvaetal.,2009).

TheS.aureusstrainsusedwere:SA1199B,whichoverexpresses

thenorAgeneencodingtheNorAfluoroquinolones(suchas nor-floxacin) efflux protein; RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide (such as erythromycin)effluxprotein;andIS-58,whichpossessestheTetK tetracyclineeffluxprotein.Thestrains,kindlyprovidedbyprofessor SimonGibbons(UniversityCollegeLondon,UK),weremaintained inbloodagarbase(LaboratóriosDifcoLtda.,Brazil)slantsand,prior touse,thecellsweregrownovernightat37◦_{Cinbrainheart}

infu-sionbroth(BHI–LaboratóriosDifcoLtda.,Brazil).

Results

Byusingtypicalchromatographicproceduresandspectroscopic methods (IR,NMR 1_{H and} 13_{C) 13 compounds wereidentified,}

includingfattyacids,steroids, chlorophyllderivatives,phenolics andalignan.

Compound1: IR(KBr,cm−1_{):3401,2948,2868.NMR} 1_H(ı,

CD3OD,200MHz):3.63(t,J=6.56Hz,H-1),1.24–1.69(m),0.87(t,

J=6.26Hz,H-10).NMR13_C(ı,CD

3OD,50MHz):63.09(C-1);32.77

(C-2),31.92(C-4),29.70,29.61,29.42,29.36,25.72(C-3),22.69, 14.13(C-10).

Compound2:MS(m/z):M−_{255.1;IR(KBr,cm}−1_):2918,2849,

1771,1448,1305.NMR1_H(ı,CDCl

3,200MHz):2.34(t,J=7.46Hz,

H-2),1.60(q,J=7.35Hz,H-15),1.25(bs),0.87(t,J=6.44Hz,H-16). NMR13_C(ı,CDCl

3,50MHz):180.29(C-1),34.07(C-2),31.92(C-4),

29.69(C-5,C-12,C-13,C-14),29.59(C-11),29.43(C-6),29.36 (C-7),29.24(C-8),29.05(C-9,C-10),24.65(C-3),22.69(C-15),14.13 (C-16).

Compound6:IR(KBr,cm−1_{):1670,1514,1423,1172,1038.NMR} 1_H(ı,CDCl

3,200MHz):9,92 (s,H-7),7.26(s, H-2/H-6),4.07(s,

OCH3C-3/C-5).NMR13C(ı,CDCl3,50MHz):190.78(C-7),147.29

(C-3/C-5),140.75(C-4),129.30(C-1),106.63(C-2/C-6),56.43(OCH3

C-3/OCH3C-5).

Compound8: IR(KBr,cm−1_{):3354,1655,1597,1516,1126,}

1026.NMR1_H(ı,C

3D6O,500MHz):7.13(2H,d,J=8.6Hz,H-2/H-6),

6.78(2H,d,J=8.6Hz,H-3/H-5),3.50(s,2H,H-7).NMR13_C(ı,C 3D6O,

100MHz):173.29(C-8),157.14(C-4),131.15(C-2/C-6),126.50 (C-1),115.91(C-3/C-5),40.50(C-7).

Compound9:IR(KBr,cm−1_{):3450,1450,1160.NMR}1_Hand 13_C:Table1.

Minimuminhibitoryconcentrationandmodulationofbacterial

resistance

ThesyringaldehydeisolatedfromP.pachycladusstemsdidnot show any antibacterial activity at 256␮g/ml against all tested strainsofS.aureus(MICused≥512␮g/ml).Whenthecompound

wasaddedinthegrowthmedium at128␮g/ml(MIC≤¼),a32

timesreductioninMICwasobservedfortetracycline,adramatic reductionintheMICoftheantibiotic.TheMICofnorfloxacinwas reducedbytwotimesandtheMICoferythromycindidnotdecrease (Table2).

Discussion

Structuralelucidation

TheIRspectrum(KBr)ofcompound1showedabsorptionsat 2948and2868cm−1 _{indicatingthepresenceofsp}3 _carbon;and

abroadbandat3401cm−1 _{characteristicofO H.}1_HNMR

spec-trum of 1 showed one terminal methylas a tripletat ıH 0.87

(3H, J=6.26Hz), multiplets for eight methylene hydrogens (ıH

1.24–1.69)andonemethylenegrouplinkedwithhydroxyl(ıH3.63

(t),J=6.56Hz).The13_{CNMRspectrumof1presentedeight}

methy-lenecarbons(ıC32.77,25.72,31.92,29.70,29.61,29.42,29.36,and

22.69),onesignalinupfieldforonemethylcarbon(ıC14.13)and

onecarbonatıC63.09bondedtooxygen.Thespectraldataand

comparisonwithliteraturedata(Paviaetal.,2010)allowedto iden-tifythecompound1asthealiphaticalcoholdecanol,producedby plantswithecologicalrelevancebyactingasgrowthregulatorand commerciallyusedaspesticide(ArnandAcree,1998;EFSA,2010). TheIRspectrum(KBr)ofcompound2showedabsorptionsat 2918and2849cm−1_(C-sp3_{);anintensebandat1771cm}−1_{(C O),}

andbandsat1448and1305cm−1_{(C C).The}1_{HNMRspectrumof}

2showedoneterminalmethylatıH0.87(3H,t,J=6.44Hz),

sig-nalsforthirteenmethylenehydrogens(ıH1.25–1.60)aswellasa

signalforanothermethyleneproton(H-2)nexttoacarbonyl(ıH

2.34,t,J=7.46Hz).The13_{CNMRspectrumof2exhibitedone}

car-boxylgroup(ıC 180.29),signals tofourteenmethylene carbons

(ıH 34.07,31.92,29.69,29.59,29.69,29.43,29.24,29.36,29.05,

24.65,22.69),andonemethylcarbon(ıH14.13).Themassspectrum

(EI/MS)showedthemolecularionpeak(M−_{m/z)255.1,confirming}

theidentityofcompound2 aspalmiticacid(Bangetal.,2002), afattyacidwidelyproducedandpreviouslyreportedfrommany speciesofCactaceae(López-Cervantesetal.,2011;Maywormetal., 1998).

deriva-Table1

1_Hand13_{CNMR(C2D6OS,500and125MHz)spectraldataof9.}

HMQC HMBC

ıC ıH 2J 3J

1/1′ _{132.49 –}

3/3′ _{145.26 –}

4/4′ _{144.76 –}

CH

2/2′ _{113.75 6.72(s) C-4/C-6/C-7/C-4}′_/C-6′_/C-7′

5/5′ _{115.46 6.67(d,J=7.82Hz) C-1/C-3/C-1}′_/C-3′

6/6′ _{117.24 6.59(d,J=7.82Hz) C-4/C-2/C-7/C-4}′_/C-2′_/C-7′

7/7′ _{85.19 4.52(d,J=2.88Hz) C-8/C-8}′ _C-8′_/C-9/C-9′_/C-2/C-6/C-2′_/C-6′

8/8′ _{53.70 2.93(sl) – –}

CH2

9/9′ _{70.58 4.07(dd,J=8.34and6.32Hz) C-8 C-7/C8}′_/C-7′

3.67(dd,J=8.34and2.20Hz)

tivesphaeophytinaandthe132_-hydroxy-(132_{-S)-phaeophytina} (Schwikkardetal.,1998;Jerzetal.,2007;Telesetal.,2014; Brito-Filhoetal.,2014).

Theanalysisofthespectraldataandcomparisonwithliterature (Kojimaetal.,1990;Liuetal.,2011)permittedtoidentifythe sub-stances5,7,10,11and12asknownsteroidsandtheirglucosyl derivatives.Thecompound5wasidentifiedas␤-sitosterol; com-pound 7 as sitosterol-3-O-␤-d-glucopyranoside; the compound 10 was defines as a mixture of ␤-sitosterol and stigmasterol; compound 11 was found to be a mixture of ␤-sitosterol and ergosterol; and the compound 12 was identified as a mixture ofsitosterol-3-O-␤-d-glucopyranosideandstigmasterol-3-O-␤-d -glucopyranosoide.Theyhavebeenpreviouslyreportedfromother Cactaceaes such as Pilosocereus gounellei (Maciel et al., 2016). Steroidsare widelyspread in plants. Their biological relevance isrelatedtotheirrolecomposingthevegetablemembrane and cellwall,andasprecursorsofvitaminD.Theyhavebeenshown topossess anti-inflammatoryproperties,and manyofthem are

beingreportedaseffectiveagentstopreventcardiovascular dis-eases(Silvaetal.,2012).

Compound6wasobtainedasyellowcrystals.TheIR absorp-tionsindicatedthepeaksofaldehydecarbonyl(1670cm−1_),C–O

(1038–1172cm−1_{)andaromaticbonds(1514cm}−1_and1423cm1_).

The1_{HNMRspectrumpresentedthree singlets:onedeshielded}

protonat ıH 9.92(1H) (s, H-7);the secondone indicating the

presenceoftwoequivalentmethoxygroupsatı4.07(6H)(s,OCH3 C-3/C-5)andthethirdoneinthearomaticzoneatı7.26(2H)(s, H-2/H-6).The13_{CNMRspectrumshowedsignalscompatiblewitha}

1,3,4,5-tetrasubstitutedaromaticringwithchemicalandmagnetic equivalence.Thesubstituentsandpositionsare:hydroxylatC-4 (ıC140.75),methoxylatpositions3and5(ıC147.29),and

alde-hydegroupatC-1(ıC129.30).Thealdehydecarbonylwasfoundat

ıC190.78(C-7),twomethinecarbonsatıC106.63(C-2/C-6)anda

doubledsignalfortwomethoxygroupsatıC56.43(OCH3C-3/OCH3

C-5).

Thespectralandliteraturedata(Kimetal.,2003)ledto iden-tify the compound 6 as 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde),aphenolicconstituentofthelignincellwallof plants.Theligninisresponsibleformechanicalsupportand vas-cular elements of plants, furthermore, the lignin is the source fromwhich syringaldehyde can beobtainedthrough enzimatic oxidation reactions (Aguiar and Ferraz, 2011; Ibrahim et al., 2012).

TheIRspectrumofcompound8showedabsorptions indicat-ingthepresenceofthefollowinggroups:carbonyl(1655cm−1_),

C–O(1026–1126cm−1_{),aromaticbonds(1597and1516cm}−1_)and

hydroxyl(3354cm−1_).The1_{HNMRspectrumpresentedone}

sin-gletatıH3.50(s,2H)andtwodoubletsatı7.13(2H)andıH6.78

(2H)andJ=8.6Hz,suggestingandAA′_BB′_{substitutedring.The}13_C

NMRspectrum of8arevealed6signals,fromwhich 3are non-hydrogenatedandacarbonylatıC173.29(C-8)characteristicof

carboxylicacid.Additionally,onedeshieldedaromaticcarbonwas foundatıC157.14(C-4).AcoupleofintensesignalsatıC131.15

(C-2/C-6)andıC115.91(C-3/C-5)confirmedtheAA′BB′systemand

thepeakatıC40.50wasattributedtothemethylenecarbon(C-7).

ThestructurewasconfirmedbythecorrelationsobservedatHMBC spectrum,identifyingthecompound8as4-hydroxyphenylacetic

Table2

MICvalues(␮g/ml)ofteststrainstoantibioticsintheabsenceandpresenceof4-hydroxy-3,5-dimethoxybenzaldehyde(syringaldehyde)–128␮g/ml.

Lineage/Antibiotic Antibioticisolated Antibiotic+syringaldehyde(1/4MIC)

IS-58/Tetracycline 64 4(32×)

RN-4220/Erythromycin ≥256 >256

acid(p-hydroxyphenylaceticacid).

The IR spectrum of compound 9 showed absorptions sug-gesting the presence of aromatic bonds(1450–1160cm−1_{) and} hydroxylgroup(3450cm−1_).The1_{HNMRspectrumshowed}

aro-maticabsorptionsindicatingthepresenceofanABXsubstituted ring.Thefollowingsignalswereobserved:ıH6.67(d,J=7.82Hz, H-5/H-5′_{)couplingorthowithı}

H6.59(d,J=7.82Hz,H-6/H-6′),a singletatıH6.72(H-2/H-2′),anoxymethinicprotonatıH4.52(d, J=2.88Hz,H-7/H-7′_{),oxymethylenicprotonsatı}

H4.07(dd,J=8.34 and6,32Hz,H-9/H-9′_)andı

H3.67(dd,J=8.34and2.20Hz, H-9/H-9′_{),andamethinehidrogenatı}

H2.93(bs,H-8/H-8′).The13CNMR spectrumshowed9peaks,characteristicoflignans:sixaromatic carbonsıC145.26(C-3/C-3′),ıC144.76(C-4/C-4′),ıC132.49 (C-1/C-1′_),ı

C117.24(C-6/C-6′),ıC115.46(C-5/C-5′),ıC113.75(C-2/C-2′); oneoxymethiniccarbonoatıC85,19(C-7/C-7′),oneoxymethylenic carbonıC70,58(C-9/C-9′),andonemethinicatıC53,70(C-8/C-8′). TheHSQCandHMBCdataareshowedinTable1.Thespectraldata combinedwithliteraturedata(Kamiyaetal.,2004)allowedto iden-tifythecompound9as3,3′_{-bisdemethylpinoresinol,thefirstlignan}

isolatedfromspeciesofCactaceaefamily(Table1).

Modulationofbacterialresistance

The syringaldehyde modulated theactivity of antibiotics by reducing theconcentration ofantibiotic requiredto inhibitthe growthofdrug-resistantbacteria.Thefoundactivitymayberelated toitslipophilicity,acommonfeatureofseveralinhibitorsofefflux. Thisquality,aspointedoutbyGibbons(2004),isimportantforthe solubilityinthebacterialmembraneandfortheinteractionwith effluxtransporters.Thus,theresultspresentedhereindicatethat syringaldehydeisapromisingmoleculetobeusedasantibiotic adjuvant.

Conclusions

Thepresentstudycontributedtothechemotaxonomic knowl-edgeofCacteaceaefamilythroughtheisolationandidentification oftwelve substances fromP.pachycladus: an alcohol(decanol),

two fatty acids, two chlorophyll derivatives (pheophytin a and hydroxy-pheophytin a), five steroids, one phenolic alde-hyde (syringaldehyde), a phenolic acid (p-hydroxyphenylacetic acid) and a lignan (3,3′_{-bisdemethylpinoresinol). The}

com-poundssyringaldehydeand3,3′_{-bisdemethylpinoresinolarebeing}

reported for the first time from species of Cacteaceae family. Additionally, thesubstancesyringaldehydewasshowedtobea promising moleculetoreversebacterialresistancemediatedby effluxpump.

Authors’contributions

SGBF,JKSM,MMMSF,MDLFworkedontheextraction,partition andchromatographyprocedures.YCFT,OSC,RBF,MFVSworkedon spectroscopyandstructuralelucidation.PDFandLPFcontributedin plantcollection,identificationandherbariumconfection.JPSJand ICSCworkedonbiologicalassays.Alltheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktoCAPES,CNPqandFAPESQ-PB.

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