Rev. bras. farmacogn. vol.26 número1

w w w.jo u r n als . e l s e vie r . c o m / r e v i s t a - b r a s i l e i r a - d e - f a r m a c o g n o s i a

Original

Article

Characterization

and

evaluation

of

the

cytotoxic

potential

of

the

essential

oil

of

Chenopodium

ambrosioides

Ruth

T.

Degenhardt

_,

_Ingrid

_V.

_Farias

_,

_Liliane

_T.

_Grassi

_,

_Gilberto

_C.

_Franchi

_Jr.

_,

Alexandre

E.

Nowill

_,

_Christiane

_M.

_da

_S.

_Bittencourt

_,

_Theodoro

_M.

_Wagner

_,

Marcia

M.

de

Souza

_,

_Alexandre

_Bella

_Cruz

_,

_Angela

_Malheiros

a_{NúcleodeInvestigac¸õesQuímico-Farmacêuticas,ProgramadeMestradoemCiênciasFarmacêuticas,UniversidadedoValedoItajaí,Itajaí,SC,Brazil} b_{CentroIntegradodePesquisasOncohematológicasdaInfância.UniversidadeEstadualdeCampinas,Campinas,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13May2015 Accepted18August2015 Availableonline28November2015

Keywords:

Chenopodiumambrosioides

Ascaridole

p-Cymene Essentialoil Cytotoxic

Gaschromatographic

a

b

s

t

r

a

c

t

TheessentialoilofChenopodiumambrosioidesL.,Amaranthaceae,wasobtainedbysteamdistillationin aClevengerapparatusandcharacterizationwasperformedusingchromatographicandspectroscopic assays(GC-FID,GC/MS,1_{HNMR).Twomajorcompoundswereidentified:p-cymene(42.32%)and}

ascari-dole(49.77%).Theethanolicextractandhydrolatewerefractionatedbyliquid–liquidpartitioningandthe compoundswerecharacterizedbyGC/MS.Theessentialoil,ethanolextractandfractionsbypartitioning withdicloromethane,ethylacetateandbutanolweretestedintumorcelllines(K562,NALM6,B15,and RAJI).Significantcytotoxicactivitywasfoundforessentialoil(IC50=1.0␮g/ml)forRAJIcellsandfraction

dicloromethane(IC50=34.0␮g/ml)andethanolextract(IC50=47.0␮g/ml)forK562cells.Theactivityof

theessentialoilofC.ambrosioidesisprobablyrelatedtothelargeamountofascaridol,sincetheother majorcompound,p-cymene,isrecognizedasapotentanti-inflammatoryandhaslowcytotoxicactivity. ©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

ChenopodiumambrosioidesL.,Amaranthaceae,popularlyknown as “erva-de-santa-maria” or “mastruc¸o” (Kokanova-Nedialkova etal.,2009),hasbeenwidelyusedinfolkmedicineinthemidwest, southandsoutheast ofBraziland isfoundmainlyin temperate andsubtropicalcountries(LorenziandMatos,2002).Theleaves areusedasananthelminticandvermicide(Alitonouetal.,2012) and this species is also used in the treatmentof gastrointesti-nal,respiratory, vascular, and nervous diseases and to combat diabetesandhypercholesterolemia.Furthermore,itpresents seda-tive,antipyretic,andantirheumaticeffects(DeFeoandSenatore, 1993).Duetotheseproperties,in2009,theBrazil’sHealth Min-istryselectedtheC.ambrosioidesasoneoftheplantsofinterestto theHelthSistem(Renisus)thatcanbeusedasanherbalmedicine. ThegeographicalareawhereC.ambrosioidesisobtained,with variablehumidity,temperatureandgeneralenvironmental condi-tionsandthedegreeofevolutionaryandgeneticvariability(the possibleexistenceofchemotypeswithinthespecies)arefactors thatdirectlyinfluencethechemicalcompositionoftheessential

∗ Correspondingauthor.

E-mail:angela@univali.br(A.Malheiros).

oilobtainedfromthisplant(Gobbo-NetoandLopes,2007;Chekem etal.,2010).However,despitethisvariability,theessentialoil con-sistsmainlyofmonoandsesquiterpenes(Kliks,1985;Cruzetal., 2007).

Various studies have been undertaken to characterize the composition oftheessential oilof C.ambrosioides bygas chro-matography coupled tomass spectrometry (GC/MS). The main compoundsfoundare:(Z)-ascaridole,(E)-ascaridole,carvacrol,p -cymene,˛-terpinene andlimonene (Cavalliet al., 2004;Jardim et al., 2008; Chekem et al., 2010; Vieira et al., 2011). Studies ontheessentialoilshowedantifungalactivityagainstAspergillus fumigatus,Aspergillus niger,Botryodiplodia theobromae, Fusarium oxysporum,Sclerotiumrolfsii,Macrophominaphaseolina, Cladospo-rium cladosporioides, Helminthosporim oryzae, and Pythiumdeba ryanumataconcentrationof100g/ml(Matos,2011).Monzonte etal.(2007)notedthattheessentialoilofC.ambrosioidespresents

invitroactivityagainsttheprotozoanLeishmaniadonovani,causing irreversibleinhibitionoftheirgrowth.

Kinupp (2007) describes this species as also being rich in flavonoidsandterpenoidsandhavingverydiverse pharmacologi-calactivities,includingantioxidantandchemopreventiveeffects againstcancer, aswellas antimicrobial,anti-inflammatory,and analgesicproperties (Cruz et al., 2007; Dembitskyet al., 2008; Grassi,2011).AccordingtoHmamouchietal.(2000),theextract

http://dx.doi.org/10.1016/j.bjp.2015.08.012

obtainedfromthisspeciespresentspotentialmolluscicidal activ-ityagainst snailstransmittingschistosomiasis, Bulinustruncatus

(LC90=2.23mg/l).Patrícioetal.(2008),ontheotherhand,studied theeffectoftheaqueouscrudeextractoftheleavesofC. ambro-sioidesonskinulcersinducedbyLeishmaniaamazoniensisinmice. Intralesionaltreatmentwasabletoinhibittheprogressionofthe ulcer.

Someauthors,citedbyMatos(2011),reportedthatthe anti-inflammatoryactivityofChenopodiumisduemainlytoascaridole, whichisoneofthemajorcomponentsoftheessentialoilofthis plant.Thissamecompoundcanalsoexhibitantipyreticeffectsand hasbeenindicatedasbeingresponsibleforgrowthinhibitionin differenttumorcelllines.Itsactionissosignificantinvitrothatisa strongcandidateforthetreatmentofcancer(Efferthetal.,2002).

Given the above, the objective of this study was to obtain andcharacterizetheessentialoilandfractions fromthe hydro-late and ethanol extract of leaves of C. ambrosioides through chromatographic and spectrometricassays (GC-FID, GC/MS, 1_H NMR)and evaluateitscytotoxicityinvitroby theMTTmethod (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)in differenttumorcelllines:myeloidleukemia(K562),acuteB lym-phoblasticleukemia(NALM6and B15),and Burkitt’slymphoma (RAJI),sincecancerisa majorpublichealth problemworldwide (INCA,2011).

Materialsandmethods

Plantmaterial

ChenopodiumambrosioidesL.,Amaranthaceae,wasobtainedin March2010inCáceres,acityinthestateofMatoGrosso,Brazil. Tax-onomicidentificationofthecollectedmaterialwasdonebybotanist M.Sc.OscarBenignoIzabycomparisonwithauthenticsamples.The exsiccatewasdepositedintheHerbariumBarbosaRodrigues(HBR), Itajaí,SC,withtheregistrynumber52802.

Extractionoftheessentialoil

TheessentialoiloftheleavesofC.ambrosioides,whichhadbeen driedandweighed(148g),wasobtainedbysteamdistillationina Clevengerapparatus.Theleaveswereextractedwith3lofdistilled waterintheflask,andtheextractionwasperformedoveraperiod of4h.Theoilwasremovedwithamicropipetteand,toprevent oxidationreactionsduetotheremainingwater,anhydroussodium sulfatewasadded.Theoilwasstoredfrozen,protectedfromlight.

Hydrolateandethanolicextract

Aftertheextractionoftheessentialoil,thewaterwasfiltered toyieldabout2.75lofhydrolate.Theleavesusedforoil extrac-tion(approximately185g)weresubjectedtomacerationinethanol for7days.Approximately1leachofthehydrolateandethanolic extractweresubjectedtoliquid–liquidpartitioningwith immisci-blesolventsinincreasingorderofpolarity(dichloromethane,ethyl acetateandbutanol).Foreachsolvent,twoextractionswere per-formedusingfirst400mlandthen200mlofthesolvent.Afterthe separationofthephases,anhydroussodiumsulfatewasaddedto theorganicphase toremovetheremainingwater.Theobtained fractionswerefilteredandconcentratedinarotaryevaporatorat amaximumtemperatureof50◦_{Ctoobtainadryresidue.Aliquots}

ofthesefractionsweresentforanalysisbyGC/MS.

Analysisbychromatographicandspectroscopicassays

The essential oil obtained from the dried leaves of C. ambrosioides was analyzed by gas chromatography with a

flame ionization detector (GC-FID) and by gas chromatogra-phy coupled to mass spectrometry (GC/MS Shimadzu QP2010 S). The analysis by GC-FID, with an Rtx-1 capillary column (30m×0.25mm×0.10␮m), used helium as the carrier gas (0.8ml/min);theinjectortemperaturewas180◦_{Candthedetector}

temperaturewas250◦_{C,1:50split,usingthefollowing}

temper-atureprogram:80–200◦_Cat20◦_{C/min,200–300}◦_Cat15◦_C/min;

300–310◦_Cat12◦_{C/min;FID(310}◦_{C);H2:40ml/min.}

Thefractionsobtainedfromthehydrolateandethanolextract and theessential oilwereanalyzed byGC/MS withaninjector temperatureof250◦_{C;thetemperatureprogramwas80–200}◦_C

at20◦_{C/min;200–300}◦_Cat15◦_{C/min,withotherconditionsequal}

tothoseofGC-FID.Theidentificationofthechemicalcomposition oftheessentialoilandfractionswasperformedbycomparingthe massspectraobtainedwiththedataavailableinthelibrary(NIST version8.0).

The1_{HNMRspectrawereobtainedonaBrukerAC-300MHz} 300F. Spectra were obtained in deuterated chloroform (purity 99.8%+0.05%TMS)obtainedfromCambridgeIsotopeLaboratories Inc.,withtetramethylsilaneastheinternalreference(TMS). Chemi-calshiftswererecordedindimensionlessvalues␦(ppm)indicating thesignassinglet(s),doublet(d),triplet(t),etc.

1_{H NMR (CDCl}

3,300MHz)essential oilconsisting mainlyof ascaridoleandp-cymene.Ascaridole␦:1.03(d,J=6.9,H9,H10), 1.39(s,H7),1.52(d,J=9.0,H5),1.91(sept,J=6.9,H8),2.07(d,J=9.0, H6),6.43(d,J=8.7,H3),6.51(d,J=8.7,H2).p-Cymene␦:1.26(d,

J=6.9,H9,H10),2.34(s,H7),2.90(n,J=6.9,H8),7.16(s,H3,H4,H5, H6).

Theessentialoil, ethanolextract anddichloromethane,ethyl acetateandbutanolfractionsfromtheextractwereevaluatedfor cytotoxicityusingK562(myeloidleukemia),Nalm6andB15(acute Blymphoblasticleukemia),and RAJI(Burkitt’slymphoma)cells. CellviabilitywasassessedbytheMTT(3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)assay,whichisbasedona methodusingadye,i.e.atetrazoliumsaltsolublethatisinwater whichisconverted topurpleformazanafterreduction by mito-chondrialdehydrogenasesinviablecells(Mosmann,1983).

Cells were grown in appropriate plastic dishes withDMEM (Dulbecco’sModified Eagle’sMinimum EssentialMedium), sup-plemented with fetal calf serum (FBS) 10% inactivated for 1h at 56◦_{C, 10mM of ethanesulfonicacidhydroxyethyl piperazine}

(HEPES),1.5g/lofsodiumbicarbonate,1%ofpenicillinG(100U/ml), 100mg/ml of streptomycin, 50␮g/ml of amphotericin B in an incubatorhumidifiedat37◦_{Cwith5%CO2emissions.Beforethe}

experiments,thenumberofviablecellswasdeterminedbythe try-panblueexclusionmethod,withcountsperformedinaNeubauer chamber.

CellswereplatedusingepMotion®_{5070equipment(Eppendorf,}

Vaudaux, Schonenbuch, Switzerland) which distributed 2×104 cellsperwellin96-wellplates,which wereincubatedwiththe testsubstancesatdifferentconcentrations(0.01,0.1,1,10,100and 1000mg/ml)for48hat37◦_Cin5%CO

2.DMSOwasusedfor solubi-lizationofthetestsubstancesinamaximumconcentrationof0.1% well/treatment;thisconcentrationisnotcytotoxictothecells. Dox-orubicinatthesameconcentrationstestedwasusedasapositive standard.

theequation below. Each experiment wasperformed in tripli-cateand repeatedatleastthreetimes.Resultsareexpressedas mean±standarddeviationvalues. ForIC50 calculationwasused nonlinearregressionPrism5.0softwareforwindows

Relativeviability=Teste_Controlsample_absorbanceabsorbance×100

Resultsanddiscussion

TheprocessofextractingtheessentialoilfromC.ambrosioides

was conducted by steam distillation with a Clevenger appara-tus. The essential oil obtained was called CAEO, with a yield of 1.15%. After extraction, an aliquot of the oil was analyzed by GC-FID (Fig. 1), displaying two major peaks with retention times of 3.614 and 5.064min, which were identified by com-parison of the mass spectra in the standards of the NIST 8.0 libraryas1-isopropyl-4-methyl-benzene(p-cymeneorp-cymol1) (42.32%)and1-isopropyl-4-methyl-2,3dioxabicyclo[2.2.2] oct-5-ene(ascaridole2)(49.77%),respectively.Thesetwomonoterpenes amountedto92.09%oftheessentialoil.Oftheremaining7.91%of theessentialoil,onlytwocompoundsshowedpercentagesabove 1%,butthesewerenotidentifiedinNISTlibraryversion8.0.

O

1 2

O

Accordingtotheliterature,itisknownthatthechemical com-positionoftheoilvariesgreatlyaccordingtoitsregionoforigin anditsmethodofextraction.Asaresult,variationsinthe percent-ageofanalyzedcompoundscanbeobserved.p-Cymene,shownto makeup42.32%oftheessentialoil,differedlittlefromastudyby Tapondjouetal.(2002),whofoundthattheoilcontained50%p -cymene.ResearchbyJardimetal.(2008)ontheessentialoilofC. ambrosioidesobtainedinBrazil,Cavallietal.(2004)inMadagascar, Chekemetal.(2010)inCameroonandSinghetal.(2008)inIndia,

showedlowerconcentrationsofp-cymene,withpercentagesof2.0, 16.2,23.4and25.77%,respectively.

Ascaridoleappearedataconcentrationof49.77%intheoil.This percentagewasclosetothatfoundinastudybyCavallietal.(2004), whofoundthattheoilcontained55.3%ascaridole.Thispercentage differedgreatlycomparedtothatfoundinastudybyVieiraetal. (2011),whichfoundascaridoleasthemajorcompound(87%).On theotherhand,Borgesetal.(2012)foundlessascaridoleintheoil obtainedfromtheplantcollectedinnortheasternBrazil(17.1%).In astudyontheessentialoilfromIndia,ascaridolewaspresentas only7%oftheoilcomposition(Guptaetal.,2002).

AccordingtoJohnsonandCroteau(1984),ascaridoleisa sensi-tiveheatcomponent,whichhasabiosyntheticpathwayrelatedto theformationof˛-terpinene.Ascaridolemayundergochangesin itsconformationandbecomeisoascaridole.JohnsonandCroteau (1984)assessedthethermalisomerizationinascaridoleto isoas-caridolebyGCanalysisofleavesandfruitsofC.ambrosioideswhen theinjectortemperaturewasabove200◦_{C.Thethermalstructural}

rearrangementofascaridoleisknown,but therehavebeenfew studiesonthisreaction(BocheandRunquist,1968).Cavallietal. (2004)reportedthatduringthecharacterizationoftheessential oilofC.ambrosioidesfromMadagascar,usingGC-FIDandGC/MS assays,thepartialisomerizationof ascaridoleintoisoascaridole occurred.

Inthepresentstudy,whentheoilwasanalyzedbyGC/MSwith aninjectiontemperature of250◦_{C it wasobservedthat}

ascari-dole(retentiontimeof5.550min)wasconvertedintoitsisomer, isoascaridole (retentiontime of6.001min). So,theoilwasalso evaluatedbynuclearmagneticresonanceofhydrogen(1_HNMR) (Fig.2).Thespectrumwasobtainedatroomtemperatureandno signwasdetectedthatcouldbeattributedtoisoascaridole.This techniquehasbecomeanexcellenttoolfortheanalysisof essen-tialoilsbecauseitallowsforevaluatingallhydrogenspresentin amatrix, whichcanbecomplex.Allsignalsobserved inthe1_H NMRspectrumwereassignedtothehydrogensofthetwomajor compounds.Thesignalsrelativetotheotherhydrogensofthe com-poundswereobservedasnoiseinthebaseline.Fromtheanalysis of theintegral of the signals, theratio betweenp-cymene and ascaridolecouldbeestablished,whichwas1:1.2.Thesedataare consistentwiththeinformationobtainedbyGC-FID.

1.0 –0.25

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

O

O 2.50 _Chromatogram

uV(x1 000 000)

2.0 3.0 4.0 5.0 6.0 7.0 8.0 Min

Fig.2.1_{HNMRspectrum(300MHz)oftheessentialoilofChenopodiumAmbrosioides.}

Afterextractionoftheessentialoil,thehydrolatewassubmitted topartitioning with solvents of increasingpolarity. These frac-tionswerecalled:partitionhydrolatedichloromethane(PHDCM), partitionhydrolateethylacetate(PHEA),andpartitionhydrolate butanol(PHB).ThefirsttwoweresubjectedtoanalysisbyGC/MS

forcharacterization,whilethelatter,duetoitshighpolarity,was notanalyzedbythistechnique.

Theleavesusedforextractionoftheoil,inturn,werefurther subjectedtoextractionbymacerationwithethanol.Theextractwas alsosubjectedtotheliquid–liquidpartitionprocess.Thefractions

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 –0.25

–0.50 0.00 0.25 0.50 0.75 1.00 1.25

1

5

6 10

2 4

3 7_{8 9} 1.50

a

(x10 000 000)

PHDCM

b

c

d

e

Table1

ComparisonofpercentageofthecompoundsanalyzedbyGC/MSinthedichloromethane(PHDCM)andethylacetate(PHEA)hydrolatefractionsandinthefractionof dichloromethaneofethanolextract(PEDCM).

Peaks Fraction

PHDCM PHEA PEDCM

RT Area% RT Area% RT Area%

1 4.458 13.36 4.461 19.64 4.454 18.11

2 – – 5.642 1.26 – –

3 5.738 3.43 5.739 7.22 5.735 2.78

4 5.792 1.06 – – – –

5 5.847 3.46 5.848 6.65 5.842 3.24

6 5.986 41.84 5.982 17.65 5.976 23.60

7 6.090 11.11 6.090 11.21 6.084 8.51

8 6.249 3.66 6.249 1.37 6.250 1.33

9 6.333 1.58 – – – –

10 6.467 0.80 6.478 1.94 – –

11 6.569 19.68 6.573 31.27 6.560 17.93

12 – – 6.920 1.79 – –

Table2

Cytotoxiceffect(IC50␮g/ml)ofessentialoilandfractionsoftheextractofChenopodiumambrosioidesandpositivecontrol(doxorubicin)againstK562,NALM6,B15,andRAJI cells.

Sample IC50(␮g/ml)

K562 NALM6 B15 RAJI

Essentialoil(CAEO) 86.1±10.6 25.3±3.6 >100 1.0±0.2 Ethanolicextractoftheleaves(CAEE) 47.0±6.1 61.9±6.4 >100 29.6±6.9 Dichloromethanefraction(PEDCM) 34.0±2.7 45.9±5.6 >100 40.9±6.5 Ethylacetatefraction(PEEA) >100 >100 >100 >100 Butanolfraction(PEB) >100 >100 >100 >100

Doxorrubicin 62.5±18.1 5.8±1.8 >100 13.2±0.5

Dataexpressedasmean±standarddeviationvalues.

werecalled:partitionextractdichloromethane(PEDCM);partition extractethylacetate(PEEA);partitionextractbutanol(PEB).These fractions,exceptforPEB,weresubsequentlyanalyzedbyGC/MS. ThechromatogramsobtainedfromfractionsoftheDCMandAE hydrolatesandtheethanolextractofC.ambrosioideswere com-paredandareshowninFig.3.

ThechromatogramsofthefractionsPHDCM,PHEA,andPEDCM hadpeakswithsimilarretentiontimes,indicatingasimilar chem-icalcomposition,differingonlyintheirconcentrations(Table1). Hydrolatepartitioningwasnotefficientbecausethesame com-poundsweredetectedindifferentfractions.

ThemassspectraobtainedfromthehydrolatefractionDCMwas comparedwiththemassspectrainNISTlibraryversion8.0,butno compoundwasidentified.Itissuggestedthatmanyofthese com-poundsarederivedfromthedegradationofthemaincompoundsin theessentialoilduetoheatingduringextraction.Peaks5,6,and7, withretentiontimesof5.99,6.09,and6.25min,respectively, corre-spondtoisomersofeachother,sincetheyhavethesamemolecular weight(170),oneofwhichispossiblydihydroascaridol,and oth-ersitsisomers.Compound10,correspondingtoaretentiontime of6.57min,isanisomerofascaridol,withanidenticalmolecular mass(168).

Afterthecharacterizationofessentialoilandfractionsobtained from the ethanol extract and hydrolate, samples CAEO, CAEE, PEDCM,PEEAandPEBweretestedondifferenttumorcelllines: myeloidleukemia(K562),acuteBlymphocyticleukemia(NALM6 andB15),andBurkitt’slymphoma(RAJI).Theresults(Table2)show thepotentialcytotoxicityofthetestedsamples,especiallyCAEO, whichshowedanextremelylowIC50of1mg/mlonRAJIcells, com-paredtotheIC50of13.2mg/mlofdoxorubicin(positivecontrol). ThefractionsPEDCMandCAEEalsoshowedlowerIC50valuesin K562cellscomparedtodoxorubicin(62.5mg/ml),withvaluesof 34and47mg/ml,respectively.

Efferth et al. (2002) isolated ascaridole from a commercial preparationoftheessentialoilofC.ambrosioidesandanalyzedin itusingdifferentstrainsoftumorcellsinvitro(CCRF-CEM–human lymphoblasticleukemiaTcells,HL60–promyelocyticleukemia, andMDAMB-231–breastcancer).Accordingtotheirresults,it wasconcludedthatascaridoleshowedcytotoxicityintheanalyzed cells.Thisstudywasthefirsttodemonstratetheimportanceof ascaridoleasapossiblecandidateforthetreatmentofcancer,and revealstheneedforfurtherstudyofitscytotoxicaction.Thus,it canbesuggestedthattheantitumoreffectoftheessentialoilin Burkitt’slymphomacells(RAJI)inthepresentstudymayberelated tothehighconcentrationofascaridole.

Nascimentoetal.(2006),ontheotherhand,investigatedthe treatmenteffectofahydroalcoholicextractofC.ambrosioidesin Ehrlichascitestumorcellsandobservedthatthisspecieswasable toinhibittumorgrowthwhenadministeredpriortotumor implan-tation,i.e.twodaysafterimplantation.Theauthorsalsosuggested thatthiseffectmayberelatedtotheantioxidantpropertiesofC. ambrosioides.

TheactivityoftheessentialoilofC.ambrosioidesisprobably related tothelargeamountof ascaridol, since theothermajor compound,p-cymene,isrecognizedasapotentanti-inflammatory (Chenetal.,2013)andhaslowcytotoxicactivity(Kobaetal.,2009). ThroughtheanalysisoftheessentialoilofC.ambrosioidesby GC/MSand1_{HNMR,itwaspossibletoidentifytwomajor} com-pounds, i.e. ascaridole and p-cymene, which were identified in previousresearchandhavebeenfoundindifferentconcentrations. Thisindicatesthedependenceoftheregionofcollectionand extrac-tionconditionsontheisolationofthesecompounds.

in inhibitingthe proliferationof K562and Raji tumorcells are required.

Ethicaldisclosures

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Authorcontributions

RTD,LTG,andAMcontributedtocollectingplantsampleand identification,ofherbarium,runningthelaboratorywork,analysis ofthedataanddraftingthepaper.RTG,IVF,GCF,AEN,CMSB,MMS, ABC,andAMcontributedtorunningthelaboratorywork, extrac-tionofessentialoil,fractionationandbiologicalstudies.RTG,CMSB, TMW,andAMcontributedtochromatographicanalysis.RTG,MMS, ABC,GCF,andAMcontributedtobiologicalstudies,analysisofthe dataanddraftingthepaper.Alltheauthorshaveread thefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthors thank CNPq,Fundac¸ãode AmparoàPesquisa e Inovac¸ãodoEstadodeSantaCatarinaandtheUniversidadedoVale deItajaíforprovidingfinancialsupport(ProBICProPPEC/UNIVALI).

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