Rev. bras. farmacogn. vol.27 número4

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

Original

Article

Chemical

composition

and

antibacterial

activity

of

essential

oil

from

leaves

of

Croton

heliotropiifolius

in

different

seasons

of

the

year

José

M.T.

de

Alencar

Filho

a

_,

_Luciana

_da

_C.

_Araújo

a

_,

_Ana

_P.

_Oliveira

a

_,

_Amanda

_L.

_Guimarães

a

_,

Alessandra

G.M.

Pacheco

a

_,

_Fabrício

_S.

_Silva

a

_,

_Leonardo

_S.

_Cavalcanti

b

_,

_Angélica

_M.

_Lucchese

c

_,

Jackson

R.G.

da

S. Almeida

a

_,

_Edigênia

_C.

_da

_C.

_Araújo

a,∗

a_Núcleo_de_Estudos_e_Pesquisas_de_Plantas_Medicinais,_Universidade_Federal_do_Vale_do_São_Francisco,_Petrolina,_PE,_Brazil

b_Instituto_de_Pesquisa_de_Substâncias_Bioativas,_Universidade_Federal_do_Vale_do_São_Francisco,_Juazeiro,_BA,_Brazil

c_Laboratório_de_{Fitoquímica,}_Departamento_de_Saúde,_Universidade_Estadual_de_Feira_de_Santana,_Feira_de_Santana,_BA,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11November2016 Accepted8February2017 Availableonline28March2017

Keywords:

␤-Caryophyllene Antibacterialactivity Essentialoil Seasonalvariation

a

b

s

t

r

a

c

t

Thispaperreportsthefirststudyofthevariationofthechemicalcompositionandabundanceofthe essentialoilofCrotonheliotropiifolius,infourseasons,andtheevaluationofitsantibacterialactivity. EssentialoilobtainedfromleavesofC.heliotropiifoliuswereanalyzedbyGC/MSandevaluatedagainst eightbacteriastrainsbybrothmicrodilutionmethod.Thechemicalconstituentsidentifiedwerethesame inallsamples,butwithdifferentproportions.Thetotalpercentagesidentifiedwere96.58%insummer, 92.08%inautumn,98.44%inwinterand90.78%inspring.Themajorsconstituentsare␤-caryophyllene, bicyclogermacrene,germacrene-D,limoneneand1,8-cineole.␤-Caryophyllenewasthemajorcompound inallsamples.Theresultsoftheantibacterialevaluationsshowedweaktomoderateactivityagainstthe analyzedstrains.Inallanalyzeswasobservedthatessentialoilsamplecollectedinsummerstandsout fromtheothers,displayingstrongeractivityagainstGram-positiveasGram-negativebacteria.

Introduction

CrotonisthesecondlargestgenusoftheEuphorbiaceae fam-ilyandcomprisesaround1200species.PredominantlyinAmerica, Croton has a pantropical distribution and about 350 occur in Brazil (Berry et al., 2005). Several biological assays were car-riedout withessential oilfromspeciesof thisgenusindiverse modelsof activity todemonstrate their popular use, including anti-inflammatory(Ramosetal.,2013),antinociceptive(Bighetti etal.,1999),gastroprotective(Coelho-de-Souzaetal.,2013),wound healing(Cavalcantietal.,2012),anticancer(Sylvestreetal.,2006), synergisticeffectwithantibiotics(Rodriguesetal.,2009)and car-diovascularactivity(Siqueiraetal.,2006).

Popularlyknownas“velame”or“marmeleiro”,Croton heliotropi-ifolius Kunth is traditionally used as remedy in folk medicine and is commonly foundin the Northeast,Midwest and South-eastregionsofBrazil.Theleavesareusedasinfusionparticularly forthetreatmentofgastrointestinaldisturbancesandweightloss (Govaertsetal.,2000).Doriaandco-workers(2010)demonstrated

∗ Correspondingauthor.

E-mail:edigenia.araujo@univasf.edu.br(E.C.Araújo).

thelarvicidalactivityagainstAedesaegyptioftheessentialoilofC. heliotropiifolius.NevesandCamara(2012)demonstratedthatthe majorconstituentofdeessentialoiloftheleavesofthisspeciewas ␤-caryophyllene(20.82±0.48),unlikethemajorfoundinthestem,

␤-elemene (17.28±0.06). The ethanolic extract from leaves of C.heliotropiifoliuspresentedinsecticidalactivityagainstSitophilus zeamais(Silvaetal.,2012).

Essentialoilsarevolatilemixtureandhavecomplex composi-tion,characterizedbyastrongodor.Formedbyaromaticplants assecondarymetabolites,itisknownfortheirantiseptic, bacte-ricidal,virucidal,fungicidalandothermedicinalproperties.These volatileoilsmaybeusedasfoodpreservativesandasanalgesic, sedative,anti-inflammatory,spasmolytic andaslocalanesthetic (Bakkalietal.,2008).

Severalstudiesshowntheinfluenceoftheyear’sseasonsnot onlyonthecontentofchemicalconstituentsofessentialoils,but alsoontheirbiologicalactivities(Angionietal.,2006;Hussainetal., 2008;Al-Hamwietal.,2011;Almeidaetal.,2014).Ina continua-tiontoourinvestigationsonofthemedicinalplantsfromBrazilian CaatingaBiome,theaimofthisstudywastoinvestigatethe pos-siblevariationofthechemicalconstituentsofessentialoilsfrom leavesofC.heliotropiifoliusandtoevaluateitsantibacterialactivity. Coveringaregionofapproximatelyonemillionsquarekilometers,

http://dx.doi.org/10.1016/j.bjp.2017.02.004

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theCaatingaBiomeispresentinmuchoftheBrazilianNortheast, especiallythesemiaridregion(Menezesetal.,2012).

Materialandmethods

Plantmaterial

Wild populations of Croton heliotropiifolius Kunth, Euphor-biaceae,growinginCaboclo locality,Afrânio,PernambucoState (08◦₂₈′_37,50′′ _S, ₀₄₀◦₅₆′_13,30′′ _W, ₅₆₅_m), _Brazilian _northeast werecollected.ThesampleswereidentifiedbythebotanistJosé Alves de Siqueira Filho from Centro de Recuperac¸ão de Áreas DegradadasdaCaatinga–CRAD.Avoucherspecimenwasdeposited intheHVASFFederalUniversityofSanFranciscoValley Herbar-ium(vouchernumber13963).LeaveshavebeencollectedinAugust (winter),November(spring)2010andFebruary(summer)andMay (autumn)2011(SouthernHemisphereseasons),alwayseverythree months.

Essentialoilextraction

Freshleavesweresubmittedtohydro-distillationinaClevenger apparatus.Atotalof250goffreshplantmaterialand2500mlof waterwereused,andthedistillationwascarriedoutfor2h.Traces ofwaterwereremovedbyfreezingthesamplebelow0◦_C_followed bytransferringunfrozenessentialoiltoanewvialtoyield yel-lowishvolatileoils.Samplesweredriedwithanhydroussodium sulfate.

AnalyticalconditionsandGC–MSandGC–FIDanalysis

Toanalyzethechemicalcompositionoftheessentialoils,they werepreviouslydilutedin1mlofethylacetate.Theanalysisby GC/FIDchromatographusedaShimadzuGC-2010®

equippedwith autosampler AOC-20i.Conditions were: Rtx-5 capillary column (30m×0.25mm),positioned00:25filmthicknessmicrons

injec-tortemperatureof220◦_C_and_detector₂₄₀◦_C;_helium_carrier_gas (1.2mlmin−1₎_with_the_oven_temperature_program_60–240◦_C_at 3◦_C_min−1 _maintained_at₂₄₀◦_C _for₂₀_min; _split_1:20,_injection volume 1␮l. Analyses by GC/MS was performed on Shimadzu chromatograph®

CG-2010MassSpectrometercoupledtoGC/MS; ShimadzuQP2010®

withAOC-20iautosampler;capillarycolumn DB-5ms(30m×0.25mm)thickness00:25filmmicronsinjector

temperature220◦_C;_helium_carrier_gas₍₁_ml_min−1_{);temperature} of the interface and the ionization source 240◦_C; _ionization energy 70eV; ionization current 0.7kV, and program tempera-ture and split similar to that described above. Co-injection of the sample with a homologous series of n-alkanes (C8–C24) was held at the GC/MS for the standardization of retention time.

Qualitativeandquantitativeanalysis

ThedatawereacquiredandprocessedwithaPCwithShimadzu GC-MS–Solutionsoftware.Theidentificationoftheconstituents wasassigned onbasisof comparison oftheirrelative retention indicestoan-alkanehomologousseries(C8–C24)obtainedby co-injecting theoil sample witha linear hydrocarbon mixture,as wellas,bycomputerizedmatchingoftheacquiredmassspectra withthosestoredinWiley8andNIST05massspectrallibraries of the GC–MS data system and other published mass spectra (Adams, 1995; Leffingwell and Alford,2005; Özel et al., 2006). The results are expressed in relative percentage of each com-pound,calculatedbynormalizationofthechromatographicpeak areas.

Microorganismsanddeterminationofminimuminhibitory concentration(MIC)andminimumbactericidalconcentration (MBC)

The referencebacterial strainsusedin this study were pur-chased from National Institute of Quality Control in Health (INCQS/FIOCRUZ–Brazil).MicroorganismsusedwereBacilluscereus (ATCC11778),Enterococcusfaecalis(ATCC19433),Escherichiacoli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Salmonella enterica(ATCC10708),Serratiamarcescens(ATCC13880),Shigella flexneri(ATCC12022)andStaphylococcusaureus(ATCC25923).

Antibacterialactivity wasevaluatedby themethodof broth microdilution(Santosetal.,2012)asrecommendedbyTheNational CommitteeforClinicalLaboratoryStandards(CLSI,2003).Initially astocksolutionof1mg/mlofessentialoilswaspreparedusingan aqueoussolutionof20%DMSO(v/v).Ahundred␮lofthis dilu-tionhavebeentransferredtothemicroplatecontaining100␮lof Müller-Hintonbroth.Then,serialdilutionswereperformed result-ing in concentrationsof 500, 250, 125, 62.5, 31.25, 15.625and 7.8125␮g/ml.Inoculumcontaining5×105CFUml−1(0.5in

McFar-landscale)wasaddedtoeachwell.Wellsinmicroplatehavebeen dedicatedtosterility controlof thebroth, bacterialgrowthand actionofantimicrobialreference(Gentamicin).Asolutionof gen-tamicinwasusedpreparedataninitialconcentrationof1.6mg/ml, whichwasdilutedtoconcentrationsof0.8,0.4,0.2,0.1,0.05,0.025, 0.0125mg/ml.Microplateswereincubatedunderconditionsof aer-obicallyfor24hat37◦_C.₁₀_␮_l_of_{2,3,5-triphenyl-tetrazolium}_(CTT) 2%wereaddedtoeachwelltodetectthecolorchangeoftheCTT (colorless)tored,reflectingthebacterialmetabolismasactive.The MICwasdefinedasthelowestconcentrationofthesamplesthat visiblyinhibitedbacterialgrowth.TodeterminetheMBC,before addingtheCTTtodeterminedeMIC,aliquotsof10␮lwere with-drawnfromeachwellcontainingextractsandtransferredtoPetri dishescontainingMüller-Hintonagar.Theplateswereincubated for24hat37◦_C._The_appearance_of_bacterial_colony_for_a_given concentrationindicatesthatthesampleswasnotabletokill99.9% ofbacterialinoculum used,sothat,theMBCwasdefinedasthe lowestconcentrationofthesamplesthatvisiblyinhibitedbacterial growth.Assayswereperformedintriplicate.Thedensityoftheoils wasemployedtoconvert␮l/mltomg/ml.

Resultsanddiscussion

Harvestofleaves(250g)wascarriedoutat7am,rangingfrom threemonthsaccordingtotheseasonsofyearsummer,autumn, winterandspring.Chemicalconstituentsidentifiedwerethesame inallsamples(Table1).␤-Caryophylleneisthemajorconstituent inallsamplesandtheirpercentagewere46.99%inwinter,43.85% in spring, 41.04% in summer and 28.61% in autumn, followed bybicyclogermacrene,germacrene-D,limonene and1,8-cineole. Sesquiterpeneswereinhighestproportionsinallsamples.

Co-occurrenceof␣/␤-pinenemightbeacharacteristicofthe genusCroton(BrachoandCrowley,1966);indeed,alongwith␤ -caryophyllene,theseterpenoidsoccurinessential oilof several species as C. cordiifolius (Nogueira et al., 2015), C. conduplica-tus(Almeidaetal.,2014),C.campestris(Almeidaetal.,2013),C. antanosiensis,C.decaryiandC.sakamaliensis(Radulovicetal.,2006).

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Table1

EssentialoilchemicalcompositionfromleavesofCrotonheliotropiifoliusindifferentseasonsoftheyear.

Compound Leaves(%)a _LRIcalcb _LRIlitc

Summer (February)

Autumn (May)

Winter (August)

Spring (November)

␣-Pinene 0.31 3.95 0.61 0.19 933 932

Sabinene 0.29 2.36 0.82 0.17 973 969

␤-Pinene 0.30 0.73 Trace Trace 977 974

␤-Myrcene 0.47 1.51 0.58 0.18 990 988

p-Cymene 2.83 2.61 0.58 0.31 1024 1020

Limonene 5.24 9.56 4.54 1.76 1028 1024

1,8-Cineole 8.66 16.31 4.60 1.12 1031 1026

␥-Terpinene 1.32 2.21 1.98 1.05 1058 1054

Terpinolene Trace Trace 0.17 Trace 1089 1086

Linalool 1.40 1.47 0.39 0.25 1095 1100

Borneol 4.58 2.09 0.38 0.39 1166 1165

Terpinen-4-ol 2.31 1.18 Trace 0.21 1178 1174

␣-Terpineol 1.31 0.51 0.26 Trace 1191 1186

Bornylacetate 2.70 0.58 1.14 0.41 1287 1287

␤-Caryophyllene 41.04 28.61 46.99 43.85 1421 1417

␣-Humulene 4.57 3.62 4.73 4.81 1456 1452

Germacrene-D 4.03 3.06 7.80 6.11 1484 1484

Bicyclogermacrene 9.24 8.47 21.83 22.60 1502 1500

Spathulenol 2.92 3.03 0.40 2.78 1581 1577

Caryophylleneoxide 2.49 3.38 0.64 3.56 1586 1582

␣-Cadinol 0.57 0.79 Trace 1.03 1658 1652

Monoterpenes 10.76 18.98 9.28 3.66

Monoterpenoids 20.96 22.14 6.77 2.38 Sesquiterpenes 58.88 43.76 81.35 77.37 Sesquiterpenoids 5.98 7.20 1.04 7.37 Totalidentified 96.58 92.08 98.44 90.78

a_Compound_percentage_by_CG/FID_analysis.

b _LRIcalc₌_linear_retention_index_calculated_on_a_DB5_column_(comparison_with_n_-alkanes_C8–C24). c _LRIlit₌_linear_retention_index_from_literature.

Table2

Minimumandmaximumtemperature,relativehumidityofharvestday,monthlyrainfallandtheyieldofextractions.

Minimum(◦_C) _Maximum₍◦_C) _Relative_humidity_(%) _Rainfall_(mm) _Yield_(%)

Winter 17.7 32.4 47.5 0.0 0.60

Spring 20.2 32.4 52.2 1.1 0.24

Summer 23.4 33.6 58.5 66.4 0.36

Autumn 21.7 27.9 70.0 9.4 0.16

yield(0.6%),aswellasthehighestproportionof␤-caryophyllene (46.99%),happenedinwinter,thesamemonthoflowerrainfall

(0.0mm),lowerrelativehumidity(47.5%)andmaximum

temper-ature(32.4◦_C) _very_close_to_the_highest _observed_(33.6◦_C)._The worstoilyield(0.16%)andproportionof␤-caryophyllene(28.61%)

happenedintheautumn,thesamemonthwiththehighest

rela-tivehumidity(70.0%)andlowermaximumtemperatures(27.9◦_C) amongthemonthsobserved.Themostprobableexplanationtofind ␤-caryophyllene inlargerproportionsintheessentialoil, when temperaturesarehigher,isthatthiscompoundisasesquiterpene, andtheirlosstotheenvironmentinanaturalwayoccursmore

slowlythanthemonoterpenes,whicharecompoundswithlower

molecularweightthatevaporatefaster. Followingasimilar rea-soning,themonoterpene1,8-cineolwasfoundinhigherproportion duringfall(16.31%),exactlytheseasonwhere␤-caryophyllenewas insmallerproportion.Thus,wecanobserveforsamplesobtained inrainyseasonthat,asthesesquiterpeneslike␤-caryophyllene, germacrene-Dandbicyclogermacreneoccurredinreduced propor-tionintheessentialoilofC.heliotropiifolius,somemonoterpenes increase, such as p-cymene, 1,8-cineol, limonene, borneol and linalool.

Geneticfactorsdeterminechemical composition of essential oils,butotherfactorsmayresultinsignificantchangesinthe pro-ductionofsecondarymetabolites.Thesemetabolitesrepresenta chemicalinterfacebetweenplantsandenvironment,andstimuli arisingfromenvironmentinwhichtheplantislocatedmay

redi-rectthemetabolicpathway,leadingtothebiosynthesisofdifferent

compounds,orthesamecompoundsbutindifferentproportions

(Morais,2009).

Amongthesefactors,onecanstresstheplantinteractionswith microorganisms, insector otherplants,age andstage of devel-opment,and abioticfactors suchaslight, temperature,rainfall, nutrition,season,timeofcollectionandharvestingtechniques.Itis worthnotingthatthesefactorsmayhavecorrelationsamongthem, notactingalone,andexertjointinfluenceinsecondarymetabolism. Temperatureandluminosityhavearelevantroleinphotosynthesis, becausetheinteractionofthesefactorsensuresoptimum environ-mentforphysiologicalprocess.Althoughthespeciescantoadapt totheirnaturalhabitat,plantsareabletowithstandtemperature variations,andthesevariationsareresponsibleforchangesinthe productionofsecondarymetabolites(Souzaetal.,2008;Morais, 2009).

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Table3

AntibacterialactivityofessentialoilfromleavesofCrotonheliotropiifoliusindifferentseasonsoftheyear.

Microorganisms EOCh-Summer EOCh-Autumn EOCh-Winter EOCh-Spring GEN

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Bacilluscereus (ATCC11778)

500 a a a a a a a _0.4 _0.4

Enterococcusfaecalis

(ATCC19433)

62.5 a ₁₂₅ a ₅₀₀ a ₅₀₀ a _0.4 _0.4

Staphylococcusaureus

(ATCC25923)

a a a a a a a a _0.025 _0.025

Escherichiacoli

(ATCC25922)

500 a a a ₅₀₀ a ₅₀₀ a b _0.4

Klebsiellapneumonia

(ATCC13882)

a a a a a a a a _0.05 _0.05

Salmonellaenterica

(ATCC10708)

500 a a a a a ₅₀₀ a _0.05 _0.05

Serratiamarcescens

(ATCC13880)

500 a ₅₀₀ a ₅₀₀ ₅₀₀ ₅₀₀ a b _0.025

Shigellaflexneri

(ATCC12022)

500 500 500 a a a a a b _0.025

MIC,minimalinhibitoryconcentration(␮g/ml).MBC,minimumbactericidalconcentration(␮g/ml).

a_Bacterial_growth_at_all_{concentrations.}

b_Absence_of_bacterial_increase_at_all_{concentrations}_tested₍_n₌_3).

EOCh,essentialoilofCrotonheliotropiifolius;GEN,gentamicin.

thepopularuseofthespeciestotreatgastrointestinaltract dis-orders.Thus,awaytouseasample,whichcouldprobablyhave aneffectagainstthesetwomicroorganisms,wouldperformingthe harvestplantinsummer.

Theincreasingincidenceofmultipleresistanceofpathogenic microorganismstodrugsthatarecurrentlyinclinicalusemakesthe discoveryofnewantibioticsoneintenseresearchfield( Oliveira-Júnioretal.,2012).Cytotoxiccharacteristicsofessentialoilshave beenstudiedtounderstandeffectsonbacteria.Thisbiological activ-ityappearstoberelatedtoitsabilitytocausedamagetocellwall. Thelipophilicconstituentsofessentialoilscanpassthroughcell walland cytoplasmicmembrane andaffectthestructure of dif-ferentlayersofpolysaccharides,fattyacidsandphospholipids.In bacteria,permeabilizationisassociatedwithlossofions, reduc-tionofmembranepotential,collapseprotonpumpanddepletion ofATP(DiPasquaetal.,2006).Thedamagetocellwallandcell membrane causeescape ofmacromolecules, progressingtocell lysis(Gustafson,1998).

Thisstudyshowedthat theperiod of harvestinfluencesthe percentageof chemicalconstituentsof C.heliotropiifolius essen-tialoil,andtheantibacterialactivityobservedmayberelatedto thechemicalcompositionvariation.Tothebestofourknowledge, thisisthefirststudythatshowschemicalcompositionand evalu-ationofantibacterialactivityforessentialoilofC.heliotropiifolius indifferentseasonsoftheyear.

Authors’contributions

JMTAF and LCA contributed in collectingplant samples and obtainingtheessentialoils.ECCAandJMTAFdraftedthepaperand contributedtocriticalreadingofthemanuscript.LSCandJRGSA contributedtocriticalreadingofthemanuscript.AMLandECCA contributedtochromatographicanalysis.FSSdesignedthestudy and supervisedthe laboratorywork.APO, AGMPand ALG con-tributedtomicrobiologicalstudy.Alltheauthorshadreadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors gratefully acknowledge the financial support obtainedfromCAPES,CNPq,andFundac¸ãodeAmparoàPesquisa doEstadodePernambuco.

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