Rev. bras. farmacogn. vol.25 número2

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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

Cytotoxicity

screening

of

essential

oils

in

cancer

cell

lines

Pollyanna

Francielli

de

Oliveira

a,∗

,

Jacqueline

Morais

Alves

a

,

Jaqueline

Lopes

Damasceno

a

,

Renata

Aparecida

Machado

Oliveira

a

_,

_Herbert

_Júnior

_Dias

b

_,

_Antônio

_Eduardo

_Miller

_Crotti

b

_,

Denise

Crispim

Tavares

a

a_Universidade_de_Franca,_Av._Dr._Armando_Salles_de_Oliveira,₂₀₁_–_Parque_{Universitário,}_14404-600_Franca,_São_Paulo,_Brazil

b_Faculdade_de_Filosofia,_Ciências_e_Letras_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Departamento_de_Química,_Av._{Bandeirantes,}_3.900_Monte_Alegre,_14040-901_Ribeirão_Preto,

SãoPaulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2December2014 Accepted14February2015 Availableonline1April2015

Keywords: Tageteserecta Tetradeniariparia Bidenssulphurea Foeniculumvulgare

Cytotoxicity

a

b

s

t

r

a

c

t

ThisstudyevaluatedthecytotoxicityactivityoftheessentialoilsofTageteserectaL.,Asteraceae (TE-OE),Tetradeniariparia(Hochst.)Codd,Lamiaceae(TR-OE),Bidenssulphurea(Cav.)Sch.Bip.,Asteraceae (BS-OE),andFoeniculumvulgareMill.,Apiaceae(FV-OE),traditionallyusedinfolkmedicine,against thetumorcelllinesmurinemelanoma(B16F10),humancoloncarcinoma(HT29),humanbreast ade-nocarcinoma(MCF-7),humancervicaladenocarcinoma(HeLa),humanhepatocellularlivercarcinoma (HepG2),andhumanglioblastoma(MO59J,U343,andU251).Normalhamsterlungfibroblasts(V79 cells)wereincludedascontrol.Thecellsweretreatedwithessentialoilconcentrationsrangingfrom 3.12to400␮g/mlfor24h.Thecytotoxic activitywasevaluated usingtheXTTassay;resultswere expressedasIC50,andtheselectivityindexwascalculated.Theresultswerecomparedwiththose achievedforclassicchemotherapeuticagents.TE-OEwasthemostpromisingamongtheevaluatedoils: itaffordedthelowestIC50valuesforB16F10cells(7.47±1.08␮g/ml)andHT29cells(6.93±0.77␮g/ml), aswellasselectivityindicesof2.61and2.81,respectively.ThemajorBS-EO,FV-EOandTE-EO chemi-calconstituentswereidentifiedbygaschromatographymassspectrometryasbeing(E)-caryophyllene (10.5%),germacreneD(35.0%)and2,6-di-tert-butyl-4-methylphenol(43.0%)(BS-EO);limonene(21.3%) and(E)-anethole(70.2%)(FV-EO);limonene(10.4%),dihydrotagetone(11.8%),␣-terpinolene(18.1%)and (E)-ocimenone(13.0%)(TE-EO);andfenchone(6.1%),dronabinol(11.0%),aromadendreneoxide(14.7%) and(E,E)–farnesol(15.0%)(TR-EO).2,6-di-tert-butyl-4-methylphenol(43.0%),(E)-anethole(70.2%)and

␣-terpinolene(18.1%),respectively.TheseresultssuggestthatTE-OEmaybeusedtotreatcancerwithout affectingnormalcells.

Introduction

Thesearchfornewdrugsthatdisplayactivityagainstseveral typesofcancerhasbecomeoneofthemostinterestingsubjects inthefieldofnaturalproductsresearch.Inthisarea,plantshave playedadominantroleinthedevelopmentofsophisticated tra-ditionalmedicinesystems,especiallythosewithalonghistoryin thetreatmentofcancer(DeMesquitaetal.,2007).Reportsonthe useofherbsareasoldashumanityandhavedemonstratedthat plant-derivedessentialoilsexertbettertherapeuticactivitythan theirisolatedmajorcompounds.Inaddition,theessentialoilsare theproductsofextractionofaplantspecies,sotheyaremore

con-∗ _{Corresponding}_author.

E-mail:pollyanna.oliveira@unifran.edu.br(P.F.d.Oliveira).

centratedandmayexhibithighertoxicitythantheoriginalplant (Bisset,1994).

Tagetes erecta L., Asteraceae,an ornamentalplant knownas marigold,is commonlyusedtotreatbronchitis,rheumaticpain, cold,andrespiratorydiseases,andwhichcanalsobeemployedas stimulantandmusclerelaxant(Neher,1968).Theessentialoilfrom T.erectaleavesdisplaysschistosomicidalpropertiesandisutilized asantihelminthicintheAmazoniaregion(StasiandHiruma-Lima, 2002;Tonucietal.,2012).Themonoterpenes␣-terpinolene,(E )-ocimenone, limonene, (Z)-␤-ocimene, linalool,dihydrotagetone, piperitone,piperitenoneand(E)-tagetonearethemainchemical constituentsofthisessentialoil(BaslasandSingh,1981;Krishna etal.,2004;OgunwandeandOlawore,2006;Sefidkonetal.,2004; Sharmaetal.,1961;Singhetal.,2003;Tonucietal.,2012).

Tetradeniariparia(Hochst.)Codd.,Lamiaceae,possessesa vari-ety ofmedicinalproperties incases ofcough, dropsy,diarrhea,

http://dx.doi.org/10.1016/j.bjp.2015.02.009

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fever,headaches,malaria,andtoothache(Campbelletal.,1997). TheessentialoilfromT.riparialeavesdisplaysrepellent(Omolo etal.,2004),insecticidal(Dunkeletal.,1990),ascaricidal(Peterand Deogracious,2006),antimalarial(Campbelletal.,1997), antimi-crobial and antinociceptive actions(Gazim et al., 2010).Its oil presents a complex mixture of monoterpenes, sesquiterpenes andditerpenes. Theoxygenatedditerpenescalyculone, 9␤,13␤ -epoxy-7-abietene and 6,7-dehydroroyleanone; the oxygenated sesquiterpenes14-hydroxy-9-epi-caryophyllene, cis -muurolol-5-en-4-␣-ol,␣-cadinolandledolandtheoxygenatedmonoterpene fenchone,perillylalcohol,␣-terpineoland␤-fenchylalcoholhave beenreportedasthemainchemicalconstituentsoftheessentialoil ofT.riparia(Fernandezetal.,2014;Gazimetal.,2010,2014;Omolo etal.,2004).

Bidens sulphurea (Cav.) Sch. Bip., Asteraceae, many times referredtoCosmossulphureusCav.,asynonymousofB.sulphurea intheliterature,hasanti-ictericandhepatoprotectiveeffectsand istraditionallyusedtotreatmalariainBrazil(Botsaris,2007).The essentialoilextractedfromtheflowersof B.sulphureadisplays schistosomicidalpropertiesandexhibitedsignificantantibacterial activitythatsupportfolkloricuseinthetreatmentofsomediseases asbroadspectrumantibacterialagents(Aguiaretal.,2013;Ram etal.,2013).2,6-di-tert-butyl-4-methylphenolandthe sesquiter-penes␤-caryophylleneandgermacreneDarereportedasthemajor constituentsoftheessentialoilfromB.sulphureaflowers(Aguiar etal.,2013).

FoeniculumvulgareMill.,Apiaceae,commonlyknownas “fen-nel”, is a medicinal and aromatic plant used as carminative, digestive,lactogogue,anddiureticagent,andwhichcanalsohelp totreatrespiratoryandgastrointestinaldisorders(Agarwaletal., 2008).Theessentialoiloffennelisusedasadditiveinthefood, pharmaceutical,cosmetic,andperfumeindustries(Tinocoetal., 2007), besides having important medicinal properties, such as diuretic, anti-inflammatory, analgesic,antioxidant (Gross et al., 2002),antiseptic,sedative,carminative,stimulant,andvermifugal activities(HeandHuang,2011;Tinocoetal.,2007).Inthe litera-ture,(E)-anetholeandthemonoterpeneslimoneneandfenchone areoftenreportedasthemainconstituentsofthisessentialoilof fennel(AkgulandBayrak,1988;Anwaretal.,2009;Cosgeetal., 2008;Garcia-Jimenezetal.,2000).

Inthepresentstudy,wescreenedthecytotoxicityofessential oilsextractedfromT.erecta,T.riparia,B.sulphureaandF.vulgare againstdifferentcelllines.Despitethereportsonthebiological activitiesoftheseessentialoils,dataontheircytotoxicityarestill scarcein the literature (Fabio et al., 2007; Gazim et al., 2014; Villarinietal.,2014).

Materialsandmethods

Plantmaterialandessentialoilextraction

Specimens ofTagetes erecta L., Asteraceae, Tetradeniariparia (Hochst.)Codd.,Lamiaceae,Bidenssulphurea(Cav.)Sch.Bip., Aster-aceae,andFoeniculum vulgareMill.,Apiaceae, werecollectedat “Sítio13demaio”(20◦₂₆′_S₄₇◦₂₇′_W₉₇₇_m)_near_Franca,_State_of SãoPaulo,BrazilonMay10,2012andidentifiedbyProf.DrMilton Groppo(DepartamentodeBiologia,FaculdadedeFilosofia,Ciências eLetras deRibeirãoPreto,UniversidadedeSãoPaulo).Voucher specimens(SPFR 10014,12421,12020and 12024,respectively) weredepositedattheHerbariumofthis institution(Herbarium SPFR).

Freshleaves(450g)ofF.vulgare(FV-EO),T.erecta(TE-EO)and T.riparia(TR-EO)andfreshflowers(300g)ofB.sulphurea(BS-EO) weresubmittedtohydrodistillationinaClevenger-typeapparatus for3h.Aftermanualcollectionoftheessentialoils(EO),anhydrous

sodiumsulfatewasusedtoremovetracesofwater,whichwas fol-lowedbyfiltration.TheEOwerestoredinanamberbottleand keptintherefrigeratorat4◦_C _until_further_analysis._The essen-tialoilyieldswerecalculatedfromtheweightoffreshleavesand expressedastheaverageoftriplicateanalysis.

GC-FIDandGC–MSanalyses

BS-EO, FV-EO, TE-EO and TR-EO were analyzed by gas chromatography(GC)onaHewlett-PackardG1530A6890gas chro-matographfittedwithFIDanddata-handlingprocessor.AnHP-5 (Hewlett-Packard,PaloAlto,CA,USA)fused-silicacapillarycolumn (30m×0.25mmi.d.;0.33␮mfilmthickness)wasemployed.The operationconditionswereasfollows:thecolumntemperaturewas programmedtorisefrom60to240◦_C_at₃◦_C/min_and_then_held at240◦_C_for₅_min;_carrier_gas₌_H

2,ataflowrateof1.0ml/min;

injectionmode;injectionvolume=0.1␮l(splitratioof1:10); injec-toranddetectortemperatures=240and280◦_C,_{respectively.}_The componentsrelativeconcentrationswereobtainedbypeakarea normalization(%).Therelativeareasweretheaverageoftriplicate GC-FIDanalyses.

GC–MSanalyseswerecarriedoutonaShimadzuQP2010Plus (Shimadzu Corporation,Kyoto,Japan) systemequippedwithan AOC-20iautosampler.ThecolumnconsistedofRtx-5MS(Restek Co.,Bellefonte,PA,USA)fusedsilicacapillary(30-mlength× 0.25-mmi.d.×0.25-␮mfilmthickness).Theelectronionizationmode wasusedat70eV.Helium(99.999%)wasemployedasthecarrier gasataconstantflowof1.0ml/min.Theinjectionvolumewas0.1␮l (splitratioof1:10).Theinjectorandtheion-sourcetemperatures weresetat240and 280◦_C,_{respectively.}_The_oven_temperature programwasthesameastheoneusedforGC.Massspectrawere takenwithascanintervalof0.5s,inthemassrangefrom40to 600Da. BS-EO,FV-EO,TE-EO andTR-EOcomponents identifica-tionwasbasedontheirretentionindicesonanRtx-5MScapillary columnunderthesameoperatingconditionsasinthecaseofGC rel-ativetoahomologousseriesofn-alkanes(C8–C24);structureswere

computer-matchedwiththeWiley7,NIST08,andFFNSC1.2 spec-tralibraries,andtheirfragmentationpatternswerecomparedwith literaturedata(Adams,2005).Standardcompoundsavailableinour laboratorywerealsoco-elutedwiththeessentialoilstoconfirmthe identityofsomeoftheircomponents.

Celllines

Eightdifferenttumorcelllineswereusedduring the exper-iments:murinemelanoma(B16F10),courtesybyDepartamento deBioquímicadaFaculdadedeMedicinadaUniversidadedeSão Paulo,Campus de Ribeirão Preto, São Paulo; colon adenocarci-noma(HT29),humanglioblastoma(MO59J,U343,andU251)and humancervical adenocarcinoma(HeLa),obtainedfromthe Cell Bank of Universidade Federaldo Rio de Janeiro;human breast adenocarcinoma (MCF-7) and human hepatocellular carcinoma (HepG2),courtesyofLaboratóriodeMutagênesedoDepartamento deCiênciasBiológicasdaUniversidadeEstadualPaulista,Campus deAraraquara,SãoPaulo.Inordertocomparethecytotoxiceffects andtheselectivityobtainedontumorcellsafterthetreatmentwith theessentialoils,wealsoincludedtreatmentsinanormalcellline (Chinesehamsterlung fibroblasts;V79),courtesyofLaboratório de Mutagênese da Universidade Estadual de Londrina, Paraná. Thedifferentcelllinesweremaintainedasmonolayers in plas-ticcultureflasks(25cm2₎_in_culture_medium_(HAM-F10₊_DMEM,

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(Sigma–Aldrich),at36.5◦_C,_with_5%_CO

2orinaBOD-typechamber.

Thecellswereusedfromthe4thpassage.

Cytotoxicactivityoftheessentialoils

The cytotoxic activity of the essential oils against differ-ent cell lines was screened using the Colorimetric Assay In Vitro Toxicology– XTT Kit (Roche Diagnostics). For the experi-ments,1×104_cells_were_seeded_into_microplates_with₁₀₀_␮_l_of

culturemedium(1:1 HAMF10/DMEMor DMEMalone) supple-mentedwith10% fetal bovineserum containingconcentrations of the essential oils that ranged from 3.12 to400␮g/ml. Neg-ative (no treatment), solvent (0.02% DMSO, dimethylsulfoxide, Sigma-Aldrich),andpositive(25%DMSO)controlswereincluded. Theclassicchemotherapeuticagentsdoxorubicin(DXR,Pharmacia BrasilLtda.,98%purity),(S)-(+)-camptothecin(CPT,Sigma–Aldrich,

≥90%purity),andetoposide(VP16,Sigma–Aldrich,≥98%purity) were also tested. After incubation at 36.5◦_C _for ₂₄_h, _the cul-turemediumwasremoved.Thecellswerewashed with100␮l ofPBS(phosphatebufferedsaline)toremovethetreatmentsand exposedto100␮lofculturemediumHAM-F10withoutphenol red.Then,25␮lofXTTwasadded,andthecellswereincubated at 36.5◦_C _for ₁₇_h. _The _absorbance _of _the _samples _was deter-minedusingamulti-platereader(ELISA–Tecan–SWMagellan vs5.03STD2P)atawavelengthof450nmandreferencelengthof 620nm.

The experiments were performed in triplicate; 50% inhibi-tion of cell growth (IC50) was used as the analysis parameter

calculated byPrism Graphpad(version5.0) software.The One-Way ANOVA analysis was used to compare the mean values (p<0.05).

Resultsanddiscussion

Table1shows thechemicalcompositionof theessentialoils investigatedin this study.Mostofthecompoundsidentified in theseEOaremonoterpenes,sesquiterpenesandphenylpropanoids. DiterpenesweredetectedonlyintheessentialoilofT.riparia (TE-EO).

(E)-caryophyllene (10.5%), germacrene D(35.0%) and 2,6-di-tert-butyl-4-methylphenol(43.0%) were identified as themajor constituentsintheessentialoilofB.sulphurea(BS-EO).These com-poundswerealsothemainchemicalconstituentsofasampleofthe essentialoilfromaspecimenofB.sulphureafromSoutheastBrazil (Aguiaretal.,2013).

Limonene(21.3%)and(E)-anethole(70.2%)werethemajor com-poundsintheessentialoilofF.vulgare(FV-EO).Thesecompounds havealsobeenfoundtobethemajorconstituentsinessentialoils ofspecimensofF.vulgarefromdifferentcountries(Cosgeetal., 2008;Garcia-Jimenezetal.,2000;Wakabayashietal.,2015).Onthe otherhand,methyl-chavicoland␣-phellandrene,whichareoften detectedintheessentialoilofF.vulgareandwerereportedasits majorcompoundsinsomestudies(Chungetal.,2011;Ozcanand Chalchat,2006),werenotdetectedinFV-EO.

Limonene (10.4%), dihydrotagetone (11.8%), ␣-terpinolene (18.1%) and(E)-ocimenone(13.0%) werefoundtobethemajor compoundsintheessentialoilofT.erecta(TE-EO).These monoter-peneshave beenreported among themain constituents ofthe essentialoilofotherT.erectaspecimens(BaslasandSingh,1981; Krishnaetal.,2004;OgunwandeandOlawore,2006;Sefidkonetal., 2004;Sharmaetal.,1961;Singhetal.,2003).Ontheotherhand, theoxygenatedmonoterpenes(Z)-ocimenone,(Z)-tagetone, linal-lylacetateandlinalool,whichwerefoundintheessentialoilof otherT.erectaspecimens(BaslasandSingh,1981;Sharmaetal., 1961;Singhetal.,2003),werenotdetectedinTE-EO.

Finally,themajorconstituentsintheessentialoilofT.riparia (TR-EO) were identified as being fenchone (6.1%), dronabinol (11.0%),aromadendreneoxide(14.7%)and(E,E)–farnesol(15.0%). ThechemicalcompositionofTR-EOdiffersfrommostofthe pre-viously investigated T. riparia essential oils in the low content ofditerpenes.13-Epimanoyloxide(7.2%)wastheonlyditerpene identified in TR-EO.The oxygenatedditerpenes calyculone, 14-hydroxy-9-epi-caryophyllene,cis-muurolol-5-en-4-ol,whichwere identifiedasmajorconstituentsintheessentialoilofotherT.riparia specimens(Fernandezetal.,2014;Gazimetal.,2010,2014)were notdetectedinTR-EO.

Table2showstheIC50values forthenormal andtumorcell

linestreated withTE-OE,TR-OE,BS-OE,and FV-OE.Our results showedthattheIC50valuesfortumorcelllinestreatedrangedfrom

6.93±0.77 to 161.60±1.41␮g/ml for TE-OE; from 77.46±1.75 to 272.37±18.45␮g/ml for TR-OE; from 229.23±10.40 to 334.17±15.50␮g/ml for BS-OE, and from 112.78±13.74 to 406.00±1.57␮g/mlforFV-OE.RegardingthenormalcelllineV79, theIC50 valueswere19.50±5.96,76.33±3.44, 96.50±1.19and

448.00±19.52␮g/mlforTE-OE,TR-OE,BS-OE,andFV-OE, respec-tively.

TE-OE exerted the most pronounced antiproliferative effect againstthetumorcelllines.ThelowestIC50valueswere7.47±1.08

and6.93±0.77␮g/mlfortheB16F10andHT29celllines, respec-tively,whichweresignificantlylowerthantheIC50valueobtained

forthenormalcellline(19.50±5.96␮g/ml).Ontheotherhand, recent studiesusingtheMTTassayreportedthat Tageteserecta extractsinEtOHandEtOAcdidnotsignificantlyaffectthecell via-bilityofH460(pleuralcarcinoma)andCaco-2(coloncarcinoma) cellsascomparedwiththecontrolgroups(Vallisutaetal.,2014).

FV-EO was more cytotoxic to B16F10 tumor cells (IC50=

112.78±13.74␮g/ml) than to normal cells (IC50=448.00±

19.52␮g/ml); the selectivity index was 3.97 (Tables 2 and 3). However,accordingtoAmerican NationalCenter Institute,only extractswithIC50valueslowerthan30␮g/mlagainst

experimen-taltumorcelllinesconstitutepromisinganticanceragentsfordrug development(Suffness andPezzuto, 1990).Inthis sense,TR-EO andBS-EOshowedIC50valuesgreaterthan30␮g/mlforallcell

linestested,andweremorecytotoxictonormallinetowhichthe tumorcelllines.

Table3showstheselectivityindicesoftheessentialoilstested againstthetumorcelllinesandthenon-tumorcellline.Forall natu-ralproductstested,theselectivityindexesweregreaterthanthose observedforthechemotherapeuticagents,especiallyVP16. Liter-aturepapershaveconsideredthatavaluegreaterthanorequalto 2.0isaninterestingselectivityindex(SuffnessandPezzuto,1990). Thisvaluemeansthatthecompoundismorethantwicemore cyto-toxictothetumorcelllineascomparedwiththenormalcellline. AccordingtoBézivinetal.(2003),theselectivityindexisinteresting inthecaseofvaluesgreaterthanthree.

TreatmentswithTE-EOandFV-EOaffordedthehighest selec-tivityindices,whichrangedfrom0.23to2.81forTE-EOandfrom 1.10to3.97forFV-EO(Table3).Thesefindingsdemonstratedthat TE-EOconstitutesapromising essentialoilforthedevelopment ofanticancerdrugs,becauseitprovidedindicesgreaterthan2.For FV-EO,althoughtheselectivityindicessuggestedpromising antitu-moractivity,theIC50valuesweregreaterthanthatrecommended

bySuffnessandPezzuto(1990).Inaddition,itwasnotpossibleto calculatetheselectivityindexforthemajorityofthecancercell linestreatedwithFV-EO,sinceitwasmorecytotoxictothenormal cellline.

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Table1

Chemicalcompositionandyields(w/w)oftheessentialoilsofB.sulphurea(BS-EO),F.vulgare(FV-EO),T.erecta(TE-EO)andT.riparia(TR-EO).

Compound RIexp RIlit %RA Identification

BS-EO FV-EO TE-EO TR-EO

␣-pinene 938 939 1.5 1.3 RL,MS,Co

camphene 954 953 0.2 0.6 RL,MS

sabinene 978 976 0.8 0.8 1.0 RL,MS

␤-pinene 983 980 tr 0.5 RL,MS,Co

myrcene 988 991 1.7 RL,MS

␣-phellandrene 1007 1005 0.5 tr RL,MS

limonene 1033 1031 21.3 10.4 1.2 RL,MS,Co

(Z)-␤-ocimene 1040 1043 0.5 4.2 0.5 RL,MS

(E)-␤-ocimene 1043 1050 0.9 0.7 RL,MS

dihydrotagetone 1049 1054 11.8 RL,MS

␣-terpinolene 1084 1088 18.1 RL,MS

fenchone 1095 1094 1.2 6.1 RL,MS,Co

(E)-tagetone 1151 1146 6.9 RL,MS

camphor 1151 1143 2.0 RL,MS,Co

borneol 1173 1165 0.8 RL,MS,Co

terpinen-4-ol 1184 1177 0.7 RL,MS

␣-terpineol 1197 1189 1.0 RL,MS

estragol 1198 1195 1.8 RL,MS

verbenone 1218 1230 9.7 RL,MS

(E)-ocimenone 1238 1239 13.0 RL,MS

piperitone 1252 1252 8.8 RL,MS

(E)-anethole 1287 1293 70.2 RL,MS,Co

piperitenone 1335 1342 9.7 RL,MS

␣-copaene 1399 1376 0.8 RL,MS

␤-elemene 1416 1391 2.1 1.5 RL,MS

␣-gurjunene 1426 1409 4.5 RL,MS

(E)-caryophyllene 1441 1428 10.5 1.2 1.6 RL,MS,Co

␣-trans-bergamotene 1460 1436 0.4 RL,MS

precoceneI 1458 1467 1.4 RL,MS

␣-humulene 1467 1467 0.5 0.7 RL,MS,Co

germacreneD 1488 1480 35.0 0.9 tr RL,MS

aromadendrene 1490 1491 0.5 RL,MS

viridiflorene 1500 1493 1.3 RL,MS

bicyclogermacrene 1507 1494 5.8 0.6 RL,MS

E,E-␣-farnesene 1503 1508 0.4 3.1 RL,MS

2,6-di-tert-butyl-4-methylphenol 1523 1519 43.0 0.3 RL,MS,Co

␦-cadinene 1527 1524 0.4 2.7 RL,MS

cis-nerolidol 1531 1539 1.5 RL,MS

germacrene-D-4-ol 1572 1574 tr 5.4 RL,MS

spathulenol 1575 1576 0.5 0.3 RL,MS

caryophylleneoxide 1577 1581 1.0 tr tr RL,MS

viridiflorol 1590 1590 2.4 RL,MS

␣-cadinol 1650 1653 3.0 RL,MS

␣-muurolol 1655 1657 0.5 RL,MS

t-cadinol 1664 1660 5.6 RL,MS

aromadendreneoxide 1672 1668 14.7 RL,MS

E,E-farnesol 1702 1706 15.0 RL,MS

13-epimanoyloxide 1996 2002 7.2 RL,MS

dronabinol 2190 2202 11.0 RL,MS

Total 99.7 99.4 99.8 99.2

Monoterpenehydrocarbons 0.5 26.2 18.1 4.1 Oxygenatedmonoterpenes 1.2 78.0 10.6 Sesquiterpenehydrocarbons 54.7 2.1 17.9 Oxygenatedsesquiterpenes 1.5 0.2 55.6

Others 43.0 72.0 1.4 11.0

Yield(w/w) 0.24% 1.60% 0.53% 1.26%

RIexp:Retentionindexdeterminedrelativeton-alkanes(C8–C20)ontheRtx-5MScolumn.RIlit:Retentionindexfromtheliterature(Adams,2005).Compoundidentification:

RL,comparisonoftheRIwiththoseoftheliterature(Adams,2005);RA:relativearea(peakarearelativetothetotalpeakareaintheGC-FIDchromatogram),averageof threereplicates;MS,comparisonofthemassspectrawiththoseoftheWiley7,NIST08,andFFNSC1.2spectrallibrariesaswellaswiththoseofliterature(Adams,2005); Co:co-elutionwithstandardcompoundsavailableinourlaboratory.tr:relativearealowerthan0.1%.Yieldswereexpressedastheaverageofthreereplicates(3×150gfor FV-EO,TE-EOandTR-EOand3×100gforBS-EO).

onethemajorcompoundsfoundontheBS-EOandTE-EO,combined withdocetaxelsignificantlyenhancedthecytotoxicitytonormal prostateepithelialcells(DU-145)(RabiandBishayee,2009).

Ourresultsdemonstrated thatTE-EOexerteda selectiveand cytotoxicactivityagainsttumorcelllines.Therefore,thisessential oilshouldbeconsidereda promisingsourcetodevelopspecific antitumordrugs.

Author’scontributions

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Table2

IC50valuesfoundforthedifferentcelllinesaftertreatmentwiththeessentialoilsofT.erecta(TE-EO),T.riparia(TR-EO),B.sulphurea(BS-EO),F.vulgare(FV-EO)andthe

positivecontrol:etoposide(VP-16),(S)-(−)camptothecin(CPT)anddoxorrubicin(DXR).

Treatments IC50(␮g/ml)a

V79 B16F10 HT29 MCF-7 HeLa HepG2 MO59J U343 U251

TE 19.50±5.96 7.47±1.08 6.93±0.77 63.42±2.42 26.02±5.52 161.60±1.41 38.69±5.51 83.56±10.81 75.56±7.11 TR 76.33±3.44 272.37±18.45 77.46±1.75 129.57±10.68 155.70±19.09 140.97±11.29 217.97±13.55 221.30±9.80 109.90±2.83 BS 96.50±1.19 230.00±19.50 268.70±8.23 253.75±15.91 334.17±15.50 241.07±9.49 229.23±10.40 243.53±0.87 236.30±2.33

FV 448.00±19.52 112.78±13.74 NE NE NE NE 406.00±1.57 NE NE

VP-16 1.28±0.05 48.91±8.53 325.40±6.79 82.67±15.62 225.50±31.82 235.37±6.47 58.94±3.10 2.18±0.99 42.97±0.40 CPT 3.27±0.14 20.17±1.98 7.34±1.47 36.09±12.46 19.38±0.76 11.87±1.96 15.55±0.65 5.71±1.05 11.14±2.79 DXR 0.57±0.27 3.81±0.18 101.62±24.15 5.39±1.35 21.90±9.09 62.13±2.04 6.98±2.13 0.70±0.35 16.28±2.51

a_The_values_are_mean_±_standard_deviation._IC

50(concentrationinhibiting50%growth).V79(normalhamsterlungfibroblasts);B16F10(murinemelanoma);HT29(human

coloncarcinoma);MCF-7(humanbreastadenocarcinoma);HeLa(humancervicaladenocarcinoma);HepG2(humanhepatocellularcarcinoma);andMO59J,U343andU251 (humanglioblastoma).NE–noteffective.

Table3

SelectivityofthecytotoxicityofT.erecta(TE-EO),T.riparia(TR-EO),B.sulphurea(BS-EO)andF.vulgare(FV-EO)essentialoilstotumorcellsascomparedwithV79cells.

Treatments Selectivityindexa

B16F10 HT29 MCF-7 HeLa HepG2 MO59J U343 U251

TE 2.61 2.81 0.30 0.74 0.12 0.50 0.23 0.25

TR 0.28 0.98 0.58 0.49 0.54 0.35 0.34 0.34

BS 0.41 0.35 0.38 0.28 0.39 0.42 0.39 0.40

FV 3.97 NE NE NE NE 1.10 NE NE

VP16 0.00 0.00 0.00 0.00 0.00 0.00 0.60 0.00

CPT 0.10 0.40 0.40 0.00 0.10 0.20 0.60 0.30

DXR 0.10 0.00 0.00 0.00 0.00 0.00 0.70 0.00

a_The_selectivity_index_is_the_ratio_of_the_IC

50valuesofthetreatmentsonV79cellstothoseinthecancercelllines.B16F10(murinemelanoma);HT29(humancolon

carcinoma);MCF-7(humanbreastadenocarcinoma);HeLa(humancervicaladenocarcinoma);HepG2(humanhepatocellularcarcinoma);andMO59J,U343,andU251 (humanglioblastoma).NE–noteffective

thedraftofthemanuscript.DCTsupervisedthebiologicaltests.All theauthorshavereadthefinalmanuscriptandapprovedthelate submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authorsthank the FAPESP Brazil, for a PhD scholarship (P.F.O;grant#2009/21310-2)andthefinancialsupport(grant# 2007/54241-8).TheauthorsarealsogratefultoCNPqfor fellow-ships.

References

Adams,R.,2005.IdentificationofEssentialOilsComponentsbyGas Chromatogra-phy/MassSpectrometry,4thed.AlluredPublishing,CarolStream,IL. Agarwal,R.,Gupta,S.K.,Agarwal,S.S.,Srivastava,S.,Saxena,R.,2008.

Oculohypoten-siveeffectsofFoeniculumvulgareinexperimentalmodelsofglaucoma.IndianJ. Physiol.Pharmacol.52,77–83.

Aguiar,G.P.,Melo,N.I.,Wakabayashi,K.A.L.,Lopes,M.H.S.,Mantovani,A.L.L.,Dias, H.J.,Fukui,M.J.,Keles,L.C.,Rodrigues,V.,Groppo,M.,Silva-Filho,A.A.,Cunha, W.R.,Magalhães,L.G.,Crotti,A.E.M.,2013.Chemicalcompositionandinvitro schistosomicidalactivityoftheessentialoilfromtheflowersofBidenssulphurea (Asteraceae).Nat.Prod.Res.27,920–924.

Akgul,A.,Bayrak,A.,1988.Comparativevolatileoilcompositionofvariouspartsfrom Turkishbitterfennel(Foeniculumvulgarevar.vulgare).FoodChem.30,319–323. Anwar,F.,Ali,M.,Hussain,A.I.,Shahid,M.,2009.Antioxidantandantimicrobial activ-itiesofessentialoilandextractsoffennel(FoeniculumvulgareMill.)seedsfrom Pakistan.FlavourFrag.J.24,170–176.

Baslas,R.K.,Singh,A.K.,1981.Chemicalexaminationofessentialoilfromtheleaves ofTageteserectaLinn.J.IndianChem.Soc.58,104.

Bézivin,C.,Tomasi,F.,Lohézie-Le,D.,Boustie,J.,2003.Cytotoxicactivityofsome lichenextractsonmurineandhuman cancercelllines.Phytomedicine10, 499–503.

Bisset,N.G.,1994.HerbalDrugsandPhytopharmaceuticals–AHandbookforPractice onaScientificBases.London/Stuttgart,Medpharm/CRCPress.

Botsaris,A.S.,2007.PlantsusedtradicionallytotreatmalariainBrazil:thearchives offloramedicinal.J.Ethnobiol.Ethnomed.3,2–8.

Campbell,W.E.,Gammon,D.W.,Smith,P.,Abrahams,M.,Purves,T.,1997. Compo-sitionandantimalarialactivityinvitrooftheessentialoilofTetradeniariparia. PlantaMed.63,270–272.

Chung,I.-M.,Ro,H.-M.,Moon,H.-I.,2011.Majoressentialoilscompositionand immunotoxicityactivityfromleavesofFoeniculumvulgareagainstAedesaegypti L.Immunopharmacol.Immunotoxicol.33,450–453.

Cosge,B.,Gurbuz,B.,Kendir,H.,Ipek,A.,2008.Compositionofessentialoilinsweet fennel(Foeniculumvulgaremill.var.dulce)linesoriginatedfromTurkey.Asian J.Chem.20,1137–1142.

DeMesquita,M.L.,dePaula,J.E.,Pessoa,C.,DeMoraes,M.O.,Costa-Lotufo,L.V., Grougnet,R.,Michel,S.,Tillequin,F.,Espindola,L.S.,2007.Cytotoxicactivityof BrazilianCerradoplantsusedintraditionalmedicineagainstcancercelllines.J. Ethnopharmacol.123,439–445.

Dunkel,F.,Weaver,D.,VanPuyvelde,L.,Cusker,J.L.,Serugend,A.,1990.Population suppressioneffectsofRwandanmedicinalplant,Tetradeniariparia(Hochst.) Codd(Lamiaceae)onstoredgrainandbeaninsects.In:Proc.5thInt.Wkg.Conf. StoredProd.Prot,pp.1609–1617.

Fabio,A.,Carmelli,C.,Fabio,G.,Nicoletti,P.I.,Quaglio,P.,2007.Screeningofthe antibacterialeffectsofavarietyofessentialoilsonmicroorganismsresponsible forrespiratoryinfections.Phytother.Res.21,374–377.

Fernandez,C.M.M.,Barba,E.L.,Fernandez,A.C.M.,Cardoso,B.K.,Borges,I.B., Take-mura,O.S.,Martins,L.D.,Cortez,L.E.R.,Cortez,D.A.G.,Gazim,Z.C.,2014.Larvicidal activityofessentialoilfromTetradeniaripariatocontrolofAedesaegyptilarvae infunctionofseasonvariation.J.Essent.OilBear.Pl.17,813–823.

Garcia-Jimenez,N.,Perez-Alonso,M.J.,Velasco-Negueruela,A.,2000.Chemical com-positionoffenneloil,FoeniculumvulgareMiller,fromSpain.J.Essent.OilRes. 12,159–162.

Gazim,Z.C.,Amorim,A.C.L.,Hovell,A.M.C.,Rezende,C.M.,Nascimento,I.A.,Ferreira, G.A.,Cortez,D.A.G.,2010.Seasonalvariation,chemicalcomposition,and anal-gesicandantimicrobialactivitiesoftheessentialoilfromleavesofTetradenia riparia(Hochst.)CoddinSouthernBrazil.Molecules15,5509–5524.

Gazim,Z.C.,Rodrigues,F.F.G.,Amorin,A.C.L.,deRezende,C.M.,Sokovic,M.,Tesevic, V.,Vuckovic,I.,Krstic,G.,Cortez,L.E.R.,Colauto,N.B.,Linde,G.A.,Cortez,D.A.G., 2014.Newnaturalditerpene-typeabietanefromTetradeniaripariaessentialoil withcytotoxicandantioxidantactivities.Molecules19,514–524.

(6)

He,W.P.,Huang,B.K.,2011.Areviewofchemistryandbioactivitiesofamedicinal spice:Foeniculumvulgare.J.Med.PlantsRes.5,3595–3600.

Krishna,A.,Kumar,S.,Mallavarapu,G.R.,Ramesh,S.,2004.Compositionofthe essen-tialoilsoftheleavesandflowersofTageteserectaL.J.Essent.OilRes.16,520–522. Nakagawa,Y.,Suzuki,T.,2003.Cytotoxicandxenoestrogeniceffectsvia biotransfor-mationoftrans-anetholeonisolatedrathepatocytesandculturedMCF-7human breastcancercells.Biochem.Pharmacol.66,63–73.

Neher,R.T.,1968.TheethnobotanyofTagetes.Econ.Bot.22,317–325.

Ogunwande,I.A.,Olawore,N.O.,2006.Theessentialoilfromtheleavesandflowers ofAfricanMarigold,TageteserectaL.J.Essent.OilRes.18,366–368.

Omolo,M.O.,Okinyo,D.,Ndiege,I.O.,Lwande,W.,Hassanali,A.,2004.Repellencyof essentialoilsofsomeKenyanplantsagainstAnophelesgambiae.Phytochemistry 65,2797–2802.

Ozcan,M.M.,Chalchat,J.C.,2006.Effectofcollectiontimeonchemicalcomposition oftheessentialoilofFoeniculumvulgaresubsppiperitumgrowingwildinTurkey. Eur.FoodRes.Technol.224,279–281.

Peter,W.,Deogracious,O.,2006.Theinvitroascaricidalactivityofselected indige-nousmedicinalplantsusedinethnoveterinarypracticesinUganda.Afr.J.Tradit. Complem.3,94–103.

Rabi,T.,Bishayee,A.,2009.d-Limonenesensitizesdocetaxel-induced cytotoxic-ityinhumanprostatecancercells:Generationofreactiveoxygenspeciesand inductionofapoptosis.J.Carcinog.8,9.

Ram,B.,Mandlik,M.,Kumar,K.,2013.AntimicrobialactivityofCosmossulphureus flowersaroundPune.Int.J.Pharm.Res.Dev.5,27–31.

Sefidkon,F.,Salehyar,S.,Mirza,M.,Dabiri,M.,2004.TheessentialoilofTageteserecta L.occurringinIran.FlavourFrag.J.19,579–581.

Sharma,M.L.,Nigam,M.C.,Handa,K.L.,Chopra,I.C.,1961.EssentialoilofTagetes erecta.Perfum.EssentialOilRec.4,561–562.

Singh,G.,Singh,O.P.,DeLampasona,M.P.,Catalan,C.,2003.Studiesonessentialoils. Part35:ChemicalandbiocidalinvestigationsonTageteserectaleafvolatileoil. FlavourFragr.J.18,62–65.

Stasi,L.C.,Hiruma-Lima,C.A.,2002.PlantasmedicinaisnaAmazôniaenaMata Atlântica,2nded.UNESP,SãoPaulo.

Suffness,M., Pezzuto,J.M., 1990.Assays related tocancer drugdiscovery. In: Hostettmann,K.(Ed.),MethodsinPlantBiochemistry:AssaysforBioactivity. AcademicPress,London,pp.71–133.

Tinoco,M.T.,Martins,M.R.,Cruz-Morais,J.,2007.Antimicrobialactivityof Foenicu-lumvulgareMilleressentialoil.Rev.CiênciasAgrárias30,448–454.

Tonuci,L.R.S.,Melo,N.I.,Dias,H.J.,Wakabayashi,K.A.L.,Aguiar,G.P.,Aguiar,D.P., Mantovani,A.L.L.,Ramos,R.C., Groppo,M.,Rodrigues, V.,Veneziani,R.C.S., Cunha,W.R.,Silva-Filho,A.A.,Magalhães,L.G.,Crotti,A.E.M.,2012.Invitro schis-tosomicidaleffectsoftheessentialoilofTageteserecta.Rev.Bras.Farmacogn22, 88–93.

Vallisuta,O.,Nukoolkarn,V.,Mitrevi,A.,Sarisuta,N.,Leelapornpisid,P., Phrutivo-rapongkul,A.,Sinchaipanid,N.,2014.Invitrostudiesonthecytotoxicityand elastaseandtyrosinaseinhibitoryactivitiesofmarigold(TageteserectaL.)flower extracts.Exp.Ther.Med.7,246–250.

Villarini,M.,Pagiotti,R.,Dominici,L.,Fatigoni,C.,Vanini,S.,Levorato,S.,Moretti,M., 2014.Investigationofthecytotoxic,genotoxic,andapoptosis-inducingeffects ofestragoleisolatedfromthefennel(Foeniculumvulgare).J.Nat.Prod.77, 773–778.