Rev. bras. farmacogn. vol.25 número4

(1)

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Antiproliferative

activity,

antioxidant

capacity

and

chemical

composition

of

extracts

from

the

leaves

and

stem

of

Chresta

sphaerocephala

Larissa

Saito

da

Costa

a

_,

_Nathalia

_Luiza

_Andreazza

a

_,

_Wallace

_Ribeiro

_Correa

a,b

_,

Ildenize

Barbosa

da

Silva

Cunha

a

_,

_Ana

_Lúcia

_Tasca

_Gois

_Ruiz

c

_,

_João

_Ernesto

_de

_Carvalho

c

_,

Elisandra

Cristina

Schinor

d

_,

_Diones

_Aparecida

_Dias

d

_,

_Marcos

_José

_Salvador

a,∗

a_Departamento_de_Biologia_Vegetal,_Programa_de_{Pós-graduac¸}_ão_em_Biociências_e_Tecnologia_de_Produtos_Bioativos_e_Programa_de_{Pós-graduac¸}_ão_em_Biologia_Vegetal,_Instituto_de

Biologia,UniversidadeEstadualdeCampinas,Campinas,SP,Brazil

b_Instituto_Federal_de_Educac¸_ão,_Ciência_e_Tecnologia_do_Sul_de_Minas_Gerais,_{Inconfidentes,}_MG,_Brazil

c_Divisão_de_Farmacologia_e_Toxicologia,_Centro_{Pluridisciplinar}_de_Pesquisas_Químicas,_Biológicas_e_Agrícolas,_Universidade_de_Campinas,_Campinas,_SP,_Brazil

d_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4December2014

Accepted7April2015

Availableonline18June2015

Keywords:

Chrestasphaerocephala

Steroids Terpenoids Flavonoids

Antiproliferativeandantioxidantactivities

a

b

s

t

r

a

c

t

Inthisstudy,antiproliferativeandantioxidantactivitiesofcrudeextracts(hexane,ethylacetateand methanol)fromleavesandstemofChrestasphaerocephalaDC.,Asteraceae,wereinvestigated. Antipro-liferativeactivitywastestedinvitroagainsttenhumancancercellsandagainstVERO(nocancercell). AntioxidantactivitiesweredeterminedusingDPPHandORAC-FLassaysandthetotalphenoliccontent wasestimatedbyFolin–Ciocalteumethod.Hexaneandethylacetateextracts(leavesandstem)exhibited antiproliferativeactivityagainstcancercelllineswithtotalgrowthinhibition(TGI)between50.40and 250␮g/ml.ForVEROcell,TGIvalueswere>250␮g/mlforallextracts,excepttohexaneextractofthe stem(TGI80.92␮g/ml).Inaninitialevaluation,ethylacetateandmethanolextracts(leavesandstem) haveshownlevelsofphenoliccompoundsbetween6.94and30.96mgGAE/kginFolin–Ciocalteuassay, DPPHfree-radicalscavengingactivitywithSC50intherangeof75.22and400␮g/mlandantioxidant

capacitybetween290.08and1088␮molTE/gofextractinORAC-FLassay.HPLC-DADandESI-MS analy-sisallowedtheidentificationofflavonoidsinthemethanolextractfromtheleavesofC.sphaerocephala. ThreesteroidsandninetriterpenoidswereidentifiedinthebioactivehexaneextractsusingHRGC.

Introduction

Naturalproductsaresmallmoleculesthatcanpresentbioactive

structurebyitselforbeemployedasprecursorsforthesynthesis

ofdifferentdrugs(Kumaretal.,2014).Therefore,theyaremajor

sourcefor drugdiscoveryor canhelp inthecomprehension of

biochemicalpathwayrelatedtopathologicalprocesses(Rishton,

2008).A great diversity of organisms canbe a sourceof

natu-ral productswithemphasisonterrestrial plants. Plantsarethe

basis oftraditional medicine aroundtheworld,in which many

drugsusedtodaywerediscovered.Ethnobotanyand

ethnophar-macognosystudiesassistintheidentificationofplantspecieswith

pharmacologicalpotential,aswellastoidentifysourcesofdifferent

compoundclasses(Gurib-Fakim,2006).

∗ _{Corresponding}_author.

E-mail:[email protected](M.J.Salvador).

Nonetheless, studies for the development of validated, safe

andeffectivenaturalproducts,coupledwithsustainableuse,are

increasinglyneeded(Farias,2004).Indevelopingcountries,about

twothirdsofthepopulationusesplantsasasourceofdrugs

with-outanyscientificbasis,apracticethatcanleadtoacuteorchronic

poisoning.InBrazil,itisalsoareality,furtheraggravateddueto

anabundanceofplantspeciesunknown,bothtaxonomicallyand

chemically.

Moreover,Brazildisplaysagreatbiodiversityandanintrinsic

chemicaldiversity,whichensuresapotentialforbioprospecting

fromitsnaturalresources.Therefore,studiestotracechemicaland

pharmacologicalprofileofthisbiologicalwealth,seenasa

promis-ingalternativefortheprimaryhealthcare,arerequired(Maciel

etal.,2002).

The Asteraceae family has many species with

ethnophar-macological and medicinal uses, being popularly used for its

anti-inflammatory,antiseptic and anti-tumor properties.

Phyto-chemical studiesofAsteraceaespecies reporttheoccurrenceof

http://dx.doi.org/10.1016/j.bjp.2015.04.005

(2)

Table1

AntiproliferativeactivityofChrestasphaerocephalaextracts.

Samples TGI(␮g/ml)a

U251 UACC-62 MCF-7 NCI-ADR/RES 786-0 NCI-H460 PC-03 OVCAR-3 HT-29 K562 VERO

Chrestasphaerocephala

Extractsofleaves

HEL 98.6 125.2 >250 210.2 72.3 210.8 135.9 58.3 198.9 152.1 >250

EEL >250 >250 >250 250.0 >250 >250 >250 172.3 >250 >250 >250

MEL >250 >250 >250 >250 >250 >250 >250 >250 >250 250.0 >250

Extractsofstem

HES 77.6 143.9 250.0 87.0 94.6 90.1 250.0 50.4 72.6 250.0 80.9

EES 90.8 >250 >250 >250 >250 >250 >250 199.3 >250 118.6 >250

MES >250 >250 >250 >250 >250 >250 >250 >250 >250 >250 >250

Doxorubicinb _6.3 _1.2 _>25 _>25 ₂₅ _>25 _3.5 _3.8 _>25 _1.0 _6.9

a_TGI_–_Total_growth_inhibition_–_{concentration}_that_inhibited_cell_growth_by_100%._The_coefficients_of_variation_obtained_in_these_analyses_were_below_to_5%.

b _Positive_control.

Celllines:U251(glioma);UACC-62(melanoma);MCF-7(breast);NCI-ADR/RES(ovarian-resistant);786-0(kidney);NCI-H460(lung);PC-03(prostate);OVCAR-3(ovarian);

HT-29(colon);K562(leukemia);VERO(normalepithelialrenalcellline,greenmonkey).

triterpenes, sesquiterpene lactones, lignans,flavonoids and

caf-feoylquinicacidderivatives(Schinoretal.,2006;Gobbo-Netoand

Lopes,2008;Salvadoretal.,2009),relatedtoantioxidant,analgesic

andantiprotozoalproperties.

ThegenusChresta,comprisestwelveendemicspeciesincentral

Brazil,andsomespeciesshowedantibacterialandantiprotozoal

activites(Schinor et al.,2006, 2007a,b; Robinson, 1999). These

speciesarerecognizedby thepopulationof northeasternBrazil

astraditional herbsusedtotreatgastricdiseasesand other

dis-orders,hencepresenting ethnopharmacological relevance(Silva

etal.,2012).

ChrestasphaerocephalaDC.,isanherbaceousspeciesfromthe

tribe Vernonieae, Asteraceae, known as “joão-bobo”. It strictly

occursintheregionofBraziliancerrado.FewspeciesoftheChresta

genushavebeenstudiedregardingitschemicalcompositionand

pharmacologicalpotential.C.exsuccaand C.scapigerapresented

significantpotentialregardingantimicrobialactivityagainst

bacte-ria,fungiandprotozoaandsteroids,terpenoidsandflavonoidswere

identifiedontheirbioactiveextracts(Schinoretal.,2006,2007a,b).

Theseresultsindicatethatspecies ofthis genuscanbesources

ofactivecompounds. Tothebestof ourknowledge, noreports

aboutcytotoxicandantioxidantpropertiesC.sphaerocephalahas

beendocumentedintheliterature.Thisfactpromptedusto

inves-tigatetheseactivitiesofcrudeextracts(hexane,ethylacetateand

methanol)fromtheleavesandstemofC.sphaerocephala,aswell

as,exploreinmoredetail itschemicalcompositionbydifferent

chromatographictechniques.

Materialsandmethods

Plantmaterial

ChrestasphaerocephalaDC.,Asteraceae,wascollectedinits

nat-uralhabitat,Furnas-MG,Brazil.Thebotanicalidentificationwas

carriedoutbyProf.Dr.JoãoSemir(DepartamentodeBiologia

vege-tal,InstitutodeBiologia,UniversidadedeCampinas)andavoucher

specimenhasbeendepositedin theherbariumofFaculdadede

Filosofia,CiênciaseLetras deRibeirãoPretodaUniversidadede

SãoPaulo(SPFR06875).

Plantextracts

Afterdrying theplantmaterial(leavesand stem)ina

circu-latingair ovenat 40◦_C,_it _was_pulverized_in _grinder_knives_and

subjectedtotheprocessofmacerationwithorganicsolvents,

hex-ane,ethylacetate(EtOAc)andmethanol,atapowder/solventratio

of1:20(w/v).Afterfiltration,solventswereremovedunderreduced

pressuretoobtainthecrudeextractsdenominatedasfollows:

hex-aneextractsfromtheleaves(HEL)andstem(HES),ethylacetate

extractsfromleaves(EEL)andstem(EES)andmethanolextracts

fromleaves(MEL)andstem(MES).Theseextractswereproperly

packedandstored.

Antiproliferativeassay

HumantumorcelllinesU251(glioma),UACC-62(melanoma),

MCF7 (breast),NCI-ADR/RES (ovarian expressing multipledrug

resistancephenotype),786-0(kidney),NCI-H460(lung,non-small

cells),PC-3(prostate),OVCAR-3(ovary),HT29(colon)andK-562

(leukemia)werekindlyprovidedbyNationalCancerInstituteat

FrederickMA-USA(NCI).Also,anormalcellline(VERO,renal,green

monkey)wasused.Stockculturesweregrowninmedium

contain-ing5mlRPMI1640(GIBCOBRL)supplementedwith5%fetalbovine

serum.Peniciline:streptomicinemixture(1000UI/ml:1000␮g/ml,

1ml/LRPMI 1640) wasadded toexperimentalcultures. Cells in

96-wellplates(100␮lcells/well)wereexposedtodifferent

sam-pleconcentrationsinDMSO/RPMI(0.25,2.5,25,and250␮g/ml)

and incubated at 37◦_C, _5% _of _CO

2 in air for 48h. Final DMSO

concentrationdidnotaffectcellviability.Afterwards,cellswere

fixedwith50%trichloroaceticacidandcellproliferationwas

deter-minedbyspectrophotometricquantification(540nm)ofcellular

proteincontentusingsulforhodamineBassay.Doxorubicin

chlo-ridrate(0.1mg/mg;Europharma)wasusedasapositivecontrol.

Three measurements were obtained: at time zero (T0, at the

beginningofincubation)and48hpost-incubationfor

compound-free (C) and tested (T)cells. Cell proliferation wasdetermined

accordingtotheequation100×[(T−T0)/C−T0],forT0<T≤C,and

100×[(T−T₀)/T₀],forT≤T₀andaconcentration–responsecurve

foreachcelllinewasplottedusingsoftwareOrigin8.0(OriginLab

Corporation).Fromtheconcentration–responsecurveforeachcell

line,TGI(concentrationthatproduces100%ofcellgrowth

inhibi-tionorcytostaticeffect)valuewasdeterminedthroughnon-linear

regressionanalysisusingthesoftwareOrigin8.0®_(OriginLab

Cor-poration)(Monksetal.,1991;Shoemaker,2006).Theresultsare

depictedinTable1andFig.1.

DPPHradicalreduction

TheradicalDPPH(2,2-diphenyl-1-picrylhydrazyl)isstable,pale

purple,andwhenreduced,turnsyellow.Inthisassay,itwas

evalu-atedstandardsandsamplesabilitytoreduceDPPHradical.Briefly,

1mgofeachextractwasdissolvedinmethanol(10ml),yielding

astocksolution.Severaldilutionswereprepared(20–200␮g/ml)

(3)

Hexane extract leaves (HEL) EtOAC extract leaves (EEL) Methanol extract leaves (MEL)

Methanol extract leaves (MEL) EtOAC extract stem (EEL)

Hexane extract stem (HEL)

Concentration (μg/ml) Concentration (μg/ml)

Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml)

Gro

wth cell (%)

Gro

wth cell (%)

Gro

wth cell (%)

Gro

wth cell (%)

Gro

wth cell (%)

100

75

50

25

–25

–50

–75

–100

10–2 ₁₀0 ₁₀1 ₁₀2 10–3 ₁₀–1

0.25 2.5 25 250 10–3 10–2 10–10.251002.5 10125 102250 ₁₀–3 10–2 ₁₀–1 100 10125 102

0.25 2.5 250

0

100

75

50

25

–25

–50

–75

–100

100

75

50

25

–25 0

–50

–75

–100 0

100

75

50

25

–25

–50

–75

–100

100

75

50

25

–25

–50

–75

–100 0

10–2 10–3 10–3 ₁₀–2 ₁₀–1 ₁₀0 ₁₀1 ₁₀2

0.25 2.5 25 250 101 ₁₀2

100

10–3 10–2 ₁₀–1 ₁₀0 ₁₀1 ₁₀2

0.25 0.5 25 250 10–1

0.25 2.5 25 250

0

Gro

wth cell (%)

100

75

50

25

–25

–50

–75

–100 0

Fig.1.AntiproliferativeactivityofChrestasphaerocephalaextracts.

(10mg/ml).After30min,theabsorbancewasmeasuredbya

spec-trophotometer (=517nm) and the percentage of anti-radical

activitywas calculated (Huang et al., 2005).Asa positive

con-trolit wasused quercetinand trolox and the dilutionsolution

(methanol)asnegativecontrol.Antioxidantactivityofeach

sam-plewasexpressedastheSC50value,whichistheconcentration

in␮g/mlofeachextractthatscavenged50%ofDPPHradical.All

experimentswereperformedintriplicate(Table2).

EvaluationofantioxidantcapacitybyORACassay

Antioxidant activity of extracts was measured applying

ORAC-FLassay,withfluoresceinas fluorescentprobeandAAPH

[2,2′_-azobis _{(2-amidiopropane)} _{dihydrochloride]} _as _a _source _of

radical peroxyl. The experiments were performed in 96 wells

microtiterplatesasdescribedbyOuetal.(2001)andPrioretal.

(2003)withmodifications(Salvadoretal.,2006).Forthisreason,

extracts(50mg/ml)andfractions(5mg/ml)stocksolutionswere

prepared in phosphate buffer/DMSO (99:1) and diluted 100,

500, 1000,5000and 10,000times withphosphate buffer. Both

compounds and trolox (referencesubstance,

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid–vitamin Eanalog)were

analyzedatconcentrationsof6.25,12.5,25,50,100and200␮M,

usingthesamesolventsystem.Thereadingwasperformedusing

fluorescentfilter(excitation=485nmandemission=528nm)in

amicroplatereaderbymonitoringthereationkineticsevery2min

foraperiodof70min,at37◦_C._Results_were_expressed_as_␮mol_of

troloxequivalent(TE)pergramofextractindrybasis(␮molTE/g)

andastroloxequivalentrelativetoisolatedcompounds(positive

controls).Quercetin,isoquercitrin,caffeicacidandchlorogenicacid

wereusedaspositivecontrolsandthedilutionsolutionasnegative

control.Allexperimentswereperformedintriplicate(Table2).

Table2

TotalphenolcontentandantioxidantcapacitybytheDPPHandORACassaysofChrestasphaerocephalaextracts.

Samples Phenolcontenta_(mg_of_GAE/kg)b _DPPH_assay,_SC

50a(␮g/ml)c ORACa(␮molTE/g)d

Hexaneextractleaves(HEL) <5.0 – –

EtOAcextractleaves(EEL) 30.9(0.07) >400 290.1(0.23)d

Methanolextractleaves(MEL) 14.6(0.05) 75.22(3.00) 1088(0.12)d

Hexaneextractstem(HES) <5.00 – –

EtOAcextractstem(EES) 6.94(0.06) >400 <200d

Methanolextractstem(MES) 17.24(0.04) 400(6.80) 471.2(0.17)d

Quercetin* _– _12.40_(2.00) _5.60_(0.75)e

Isoquercitrin* _– _– _5.15_(1.20)e

Caffeicacid* _– _– _2.98_(1.90)e

Chlorogenicacid* _– _– _2.40_(1.70)e

Trolox* _– _9.60_(1.80) _–

*_Experimental_positive_controls.

–Notevaluated.

a_Mean_(%RSD,_relative_standard_deviation)_of_triplicate_assays.

b_Total_phenolics_data_expressed_as_milligrams_of_gallic_acid_equivalents_per_gram_(mg_of_GAE/kg)_of_extract.

c _DPPH_assay_data_expressed_as_SC

50(concentrationthatinhibited50%oftheDPPHradical)inmicrogramspermilliliters(␮g/ml).

d _ORAC_data_expressed_as_micromol_of_Trolox_equivalents_per_gram₍_␮_mol_of_TE/g)_of_extract.

(4)

TLCautographicassayforDPPHradical-scavengingandEASI-MS analysisofantioxidantextracts

TLCautographicchromatography assay wasperformedwith

antioxidantmethanolextractfromleavesofC.sphaerocephalaand

TLCchromatographyyellowspotwithantioxidantactivitywere

analyzedby EASI-MS (easy ambientsonic-spray ionization) via

ambientdesorption/ionizationand (tandem)massspectrometry

detection (Haddad et al., 2008).Thus, 10␮lof extract solution

(1mg/ml) in methanol wasapplied into two TLC plates (silica

gel60GF254,Fluka,AG,Switzerland,n-butanol/aceticacid/water,

65:15:25,v/v).OneoftheTLCplatewassprayedwitha0.2%

2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma–Aldrich,St Louis, MO)

solutioninMeOHandleftatroomtemperatureandtheotherplate

(control,withoutDPPHspraying)wasanalyzedonlybyEASI-MS.

Plateswereobserved30minafterspraying.Activecompoundsare

observedasyellowspotsagainstapurplebackground.AsbothTLC

weredevelopedtogether,theyellowspotsinfirstTLC(sprayed

withDPPH)had servedasa guidetochooseareasintheother

TLCplatethatwouldbeanalyzedbyEASI-MSaccordingtoHaddad

etal.(2008)andMSorMS/MScharacterizationwereperformed.

Allexperimentswereperformedintriplicate.

Quantitativedeterminationoftotalsolublephenols

Extracts,dissolvedinmethanol,wereanalyzedfortheirtotal

solublephenoliccontentaccordingtoFolin–Ciocalteucolorimetric

method(Piccinellietal.,2004;Wuetal.,2004),usinggallicacid

asreference(FCRassay).Resultswereexpressedasmilligramsof

gallicacidequivalents(GAE)pergramofextract(mgofGAE/g).

Analyseswereperformedintriplicate(Table2).

Cleanupprocedure

TheexperimentswereperformedasdescribedbySchinoretal.

(2004).Analiquot(10mg)ofHEL and HESwasresuspended in

analyticalgradechloroform(3ml)andpercolatedthrougha

sep-kap column (Alltech, silica-gel 200mg, 3ml). The column was

elutedwithhexane(10ml)andchloroform(10ml).Fractionswere

collectedseparatelyandevaporatedtodrynessatroom

temper-ature.ChloroformphasewasanalyzedbyHRGCbelow.MELand

MESextracts(10mg)wereresuspendedinmethanol–water(7:3,

v/v,3ml)andpercolatedthroughasep-kap column(Alltech,

C-18,200mg,3ml).Thecolumnwaseluted withanalytical grade

methanol(10ml)andthisphasewasanalyzedbyHPLC–ESI-MS.

Gaschromatographicanalysis

Chloroform phase was analyzed by HRGC on a

Hewlett-Packard model 5890 Series II Gas Chromatograph with a split

injector (split ratio 1:60, v/v) at 260◦_C _and _a _flame

ioniza-tiondetectorat330◦_C._The_injected_volume_was₂_␮l._Hydrogen

was employed as carrier gas at an average linear velocity of

44cm/s(HP-50)and42cm/s(HP-1).AnHP-50(cross-linked50%

phenyl-methylsilicone, 30m×0.25mm×0.25mm) and anHP-1

(cross-linked methyl-silicone, 30m×0.25mm×0.25mm)

capil-larycolumnswereemployed.ForHP-50thecolumntemperature

was280◦_C_(isotherm)_and_for_HP-1,_the_column_temperature

pro-gramwas250◦_C_held_for₁₂_min,_increased_at₆◦_C/min_to₂₈₀◦_C,

andheldthistemperaturefor30min.Eachsamplewasanalyzedin

duplicate.DatawereprocessedonaHewlett-Packardmodel3395

injector.Thestandardsusedinthegaschromatographyarelisted

inTable3.

Table3

Chemicalcompositionofhexaneextracts(HELandHES)ofChrestasphaerocephala

(steroids,triterpenoidsandtriterpenoidacetates)analyzedbygaschromatography

(HP-50column).

Compounds Leaves Stem RRa

Campesterol − + 1.236

Stigmasterol + + 1.306

␤-Sitosterol − + 1.450

Stigmast-7en-3␤-ol + + 1.509

Spinasterol − − 1.676

Taraxerone − − 1.628

Epitaraxerol − − 1.656

Taraxerol − − 1.698

␤-Amyrin + + 1.760

␣-Amyrin + + 1.983

Lupeol + + 2.041

␤-Friedelanol − + 2.476

Friedelin − + 2.709

Pseudotaraxasterol − − 2.467

Taraxasterol − + 2.561

11-Oxours-12-ene − − 3.047

11-Oxolean-12-ene − − 3.386

Taraxerolacetate − − 1.866

␤-Amyrinacetate + + 1.923

␣-Amyrinacetate + + 2.145

Lupeolacetate + + 2.224

Bauerenylacetate − − 2.497

11␣-12␣-Oxidetaraxeryl − − 2.795

␤-Friedelanolacetate − − 2.857

␣-Amyrinononilacetate − − 3.637

␤-Amyrinononilacetate − − 4.107

−Compoundnotdetected.

+Compounddetected.

a_Relative_retention_time_related_to_the_internal_standard_{(cholesterol)}_in_minutes.

HPLC-DAD

Methanolextracts,MELandMES,wereinvestigatedbyHPLC

andtheanalysiswascarriedoutwithaWatersAlianceequipment,

usingaC-18Polaris-Variananalyticalcolumn(4.6mm×250mm).

Themobilephaseconsistedofalineargradientcombiningsolvent

A(methanol)andsolventB(water/formicacid,99:1,v/v)as

fol-lows:15%A(0min),15–90%A(30min),90%A(5min),90–15%A

(5min).Theanalyseswereperformedintriplicateataflowrate

of1.0ml/minwiththeUVdetectorsetat=280nmandan

injec-tionvolumeof10␮l.Subsequentsampleswereinjectedwithan

intervalof10minaftertheprevioussamplerun.

HPLC–ESI-MSanalysis

TheESI-MSspectrawereacquiredonaQ-trap(Applied

Biosys-tems) Mass Spectrometer with an ESI source operated in the

negativeionmode.Thesourceanddesolvationtemperatureswere

70◦_C_and₁₀₀◦_C,_{respectively.}_Cone_voltage_was₃₀_V._The_parent

ionandtheretentiontimewerecomparedwithpreviouslyisolated

flavonoids(Table4)ofourresearchgroup,asdescribedbySchinor

etal.(2006,2007a,b).

Statisticalanalysis

Dataarereportedasmean(%RSD,relativestandarddeviation)

andanalyzedbyANOVAtheTukeytest,p-value<0.05.

Resultsanddiscussion

TheantiproliferativeactivityoftheextractsofC.sphaerocephala

(leavesandstem)wasinvestigatedonaseriesofcancercelllinesby

sulforhodamineBassay.Theresultsshowedantiproliferativeeffect

againstallcelllinesonlyforhexaneextractswithtotalgrowth

(5)

Table4

FlavonoidsidentifiedbyHPLCandESI-MSonmethanolextracts(MELandMES)ofChrestasphaerocepahala.

Compounds Rt.(min)a _[M₋_H]−₍_m_/_z₎b _Present_flavonoids_in_methanol_extract

Leaves Stem

Apigenin 24.56 269 + −

Luteolin 22.72 285 + −

Genkwanin 29.20 283 + −

Crysoeriol 24.78 299 + −

Velutin 29.01 313 + +

Luteolin3′_-O-_␤_-_d_{-glucopyranoside} _19.38 ₄₄₇ ₊ ₋

Luteolin7′_-O-_␤_-_d_{-glucopyranoside} _11.08 ₄₄₇ ₊ ₋

Vicenin-2 12.63 593 + −

+Compounddetected.

−Compoundnotdetected.

a_Values_of_retention_time.

b_ESI-MS_analysis.

For both HESand HEL extracts,thecell lineOVCAR-3 (ovarian

cancercellline) wasmore susceptiblywithTGIvalues equalto

50.4 and 58.3␮g/ml,respectively. HESalsoshowed weak

cyto-staticeffectagainstHT29(colon,TGI=72.6␮g/ml),U251(glioma,

TGI=77.6␮g/ml),NCI-ADR/RES(ovarianexpressingmultipledrug

resistance phenotype, TGI=87.0␮g/ml), NCI-H460 (lung,

non-smallcells,TGI=90.1␮g/ml)and786-0(renal,TGI=94.6␮g/ml).

This extract also inhibited cell growth of VERO cells (renal,

greenmonkey)at thesamelevel thataffectedcancercells (TGI

80.9␮g/ml).Ingeneral,bothmethanolandethylacetateextracts

wereinactiveagainstallcelllines.Theseresultsindicatethatthe

antiproliferativeactivityofC.sphaerocephalaareprobablydueto

nonpolarcompounds.

In FCRassay,both methanoland ethylacetateextracts,

pre-sented significant levels of phenolic compounds (Table 2).The

amountofgallicacidinMEL(14.6mg/kg)andMESweresimilar

(17.2mg/kg),whilethiscompoundcontentatEEL(30.9mgGAL/g)

werealmostfivetimeshigherthantheonefoundinEES(6.9mg

GAE/kg).OnlyMELshowedexpressiveDPPHscavengeabilitywith

SC50 valuesequal to75.2␮g/ml whereasfor EEL,EESandMES,

SC50werehigherthan400␮g/ml.Ontheotherhand,atORAC-FL

assay,theseextractsdemonstratedantioxidantcapacitybetween

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A)

Intensity (U

.A) Genkwanin, Rt = 29.20 min, [M-H] = 283 m/z

Velutin, Rt = 29.01 min, [M-H] = 313 m/z

Luteolin, Rt = 22.72 min, [M-H] = 285 m/z

Luteolin-7’-O-β-D-glucopyranoside, Rt = 11.08 min, [M-H] = 447 m/z Luteolin-3’-O-β-D-glucopyranoside,, Rt = 19.38 min, [M-H] = 447 m/z

Vicenin-2, Rt = 12.63 min, [M-H] = 593 m/z

Apigenin, Rt = 24.56 min, [M-H] = 269 m/z

Crysoeriol, Rt = 24.78 min, [M-H] = 299 m/z

(6)

290.1and1088.1␮molTE/gofextract,exceptforEES,whichdid

notreachmeasurableconcentrations accordingtoexperimental

procedure.Hexaneextracts(HELandHES)showednoantioxidant

activityinbothtests.Therefore,itispossibletoassertthatDPPH

radicalreductionandantioxidantcapacityofmethanolandethyl

acetateextractsareingoodcorrelationwiththetotalphenolic

con-tentmeasuredbyFolin–Ciocalteu,oncetheseclassesofcompounds

areknownaspotentantioxidants.

In chemical profile characterization of HES and HEL by GC

analysis, allowed the identification of three steroids

(stigmas-terol,␤-sitosterolandstigmast-7-en-3␤-ol)andninetriterpenoids

(␣-amyrin, ␤-amyrin, lupeol, taraxasterol, friedelin, friedelanol,

␣-amyrin acetate, ␤-amyrin acetate and lupeol acetate) as

shown in Table 3. HPLC-DAD and ESI-MS analyses revealed

that both methanol extracts present flavonoids as major

con-stituents, such as luteolin-3-O-␤-d-glucopyranoside,

luteolin-7-O-␤-d_{-glucopyranoside,} _vicenin-2, _luteolin, _crysoeriol, _velutin,

apigeninand genkwanin(Table4; Fig.2).TLC chromatography

analysisofMELextract(theonewithhigherantioxidantactivity),

followedbyEASI-MSanalysisoftheyellowspotreveledintheTLC

plateforantioxidantactivityshowedthesubstanceasbeing

api-genin.Inthiscase,notonly,theseextract(MEL)antioxidantactivity

isprobablyattributedtotheseflavonoidbutalso,thecombination

ofbothanalyticaltechniquesprovidedasimpleandadvantageous

techniquefortheseparationanddetectionofanactivecompound.

Moreover, the compounds stigmasterol and lupeol found

in hexane extracts have already been described as possessing

promisingantiproliferativeactivitiesandcouldberelatedtothe

resultsobserved inthis study(Yasukawaetal., 1991;Kasahara

et al., 1994; Saleem, 2009). On the other hand, luteolin and

apigenin, observed in methanol extracts, are reported as

out-standing antioxidant/free-radical scavenging substances (Burda

andOleszek,2001;Romanovaetal.,2001;Salaetal.,2003;Shohreh

etal.,2008).

Despite the promising results foundhere, further biological

studies should be performed, including in vivo investigations,

lookingtowardaclinicalemploymentofthesebioactivenatural

products.

Authorcontributions

LSCand NLAcarriedout allexperimentalproceduresof this

study,dataanalysisandinterpretationanddraftedthemanuscript.

WRCassistedinantioxidantassays.ALTGRandJECassistedinthe

antiproliferativebiologicalassays.IBSCcarriedoutESI-MSanalysis

andassistedinspectradatainterpretation.ECS,DADandMJS

con-ceivedthestudyandparticipatedinitsdesignandcoordination

andhelpedtodraftthemanuscript.Allauthorsreadandapproved

thefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsaregratefultoDr.JoãoSemirfromDepartamento

deBiologiaVegetal/UNICAMPforplantidentification;toDr.Marcos

NogueiraEberlinfromLaboratórioThomsondeEspectrometriade

Massas,InstitutodeQuímica/UNICAMPformassspectra;andto

FAPESP,CAPES,CNPqandFAEPEX-UNICAMPforfinancialsupport.

References

Burda,S.,Oleszek,W.,2001.Antioxidantandantiradicalactivitiesofflavonoids.J.

Agric.FoodChem.49,2774–2779.

Farias,M.R.,2004.Avaliac¸ãodaqualidadedematerias-primasvegetais.In:Simões,

C.M.O.,Schenkel,E.P.,Gosmann,G.,Mello,J.C.P.,Mentz,L.A.,Petrovick,P.R.(Eds.), Farmacognosia:daplantaaomedicamento.,5thed.EditoradaUniversidade UFRGS/EditoradaUFSC,PortoAlegre/Florianópolis,pp.263–288.

Gobbo-Neto, L., Lopes, N.P., 2008. Online identification of chlorogenic acids,

sesquiterpenelactones,andflavonoidsintheBrazilianarnicaLychnophora eri-coidesMart(Asteraceae)leavesbyHPLC-DAD–MSandHPLC-DAD–MS/MSand avalidatedHPLC-DADmethodfortheirsimultaneousanalysis.J.Agric.Food Chem.56,1193–1204.

Gurib-Fakim,A.,2006.Medicinalplants:traditionsofyesterdayanddrugsof

tomor-row.Mol.Asp.Med.27,1–93.

Haddad,R.,Milagre,H.M.S.,Catharino,R.R.,Eberlin,M.N.,2008.Easyambient

sonic-sprayionizationmassspectrometrycombinedwiththin-layerchromatography. Anal.Chem.80,2744–2750.

Huang,D.,Ou,B.,Prior,R.L.,2005.Thechemistrybehindantioxidantcapacityassays.

J.Agric.FoodChem.53,1841–1856.

Kasahara,Y.,Kumaki,K.,Katagiri,S.,Yasukawa,K.,Yamanouchi,S.,Takido,M.,

Aki-hisa,T.,Tamuta,T.,1994.Carthamiflosextractanditscomponent,stigmasterol,

inhibittumourpromotioninmouseskintwo-stagecarcinogenesis.Phytother. Res.8,327–331.

Kumar,S.V.,Saravanan,D.,Kumar,B.,Jayakumar,A.,2014.Anupdateonprodrugs

fromnaturalproducts.AsianPac.J.Trop.Med.7,S54–S59.

Maciel,M.A.M.,Pinto,A.A.,Veiga,V.F.J.,2002.Plantasmedicinais:anecessidadede

estudosmultidisciplinares.Quim.Nova25,429–438.

Monks,A.,Scudiero,D.,Skehan,P.,Shoemaker,R.,Paull,K.,Vistica,D.,Hose,C.,

Langley,J.,Cronise,P.,Vaigro-Wolff,A.,Gray-Goodrich,M.,Campbell,H.,Mayo,

J.,Boyd,M.,1991.Feasibilityofahigh-fluxanticancerdrugscreenusingadiverse

panelofculturedhumantumorcelllines.J.Natl.CancerInst.83,757–766.

Ou,B.,Hampsch-Woodill,M.,Prior,R.L.,2001.Developmentandvalidationofan

improvedoxygenradicalabsorbancecapacityassayusingfluoresceinasthe fluorescentprobe.J.Agric.FoodChem.49,4619–4626.

Piccinelli,A.L.,DeSimone,F.,Passi,S.,Rastrelli,L.,2004.Phenolicconstituentsand

antioxidantactivityofWenditacalysinaleaves(burrito),afolkParaguayantea. J.Agric.FoodChem.52,5863–5868.

Prior,R.L.,Hoang,H.,Gu,L.,Wu,X.,Bacchiocca,M.,Howard,L.,Hampsch-Woodill,M.,

Huang,D.,Ou,B.,Jacob,R.,2003.Assaysforhydrophilicandlipophilic

antiox-idantcapacity(oxygenradicalabsorbancecapacity(ORAC-FL))ofplasmaand otherbiologicalandfoodsamples.J.Agric.FoodChem.51,3273–3279.

Rishton,G.M.,2008.Naturalproductsasarobustsourceofnewdrugsanddrug

leads:pastsuccessesandpresentdayissues.Am.J.Cardiol.101,S43–S49.

Robinson,H.,1999.In:SmithsonianInstitutionPress(Ed.),GenericandSubtribal

ClassificationofAmericanVernonieae.SmithsonianContributionstoBotany, Washington,DC,pp.9–12.

Romanova,D.,Vacakova,A.,Cipak,L.,Ovesna,Z.,Rauko,P.,2001.Studyofantioxidant

effectofapigenin,luteolinandquercetinbyDNAprotectivemethod.Neoplasma 48,104–107.

Sala,A.,Recio,M.C.,Schinella,G.R.,Ma ˜nez,S.,Giner,R.M.,Cerdá-Nicolas,M.,Rios,J.L.,

2003.Assessmentoftheanti-inflammatoryactivityandfreeradicalscavenger

activityoftiliroside.Eur.J.Pharmacol.461,153–161.

Saleem,M.,2009.Lupeol,anovelanti-inflammatoryandanti-cancerdietary

triter-pene.CancerLett.285,109–115.

Salvador,M.J.,Ferreira,E.O.,Mertens-Talcott,S.U.,Castro,W.V.,Butterweck,V.,

Derendorf,H.,Dias,D.A.,2006.IsolationandHPLCquantitativeanalysisof

antioxidantflavonoidsfromAlternantheratenellaColla.Z.Naturforsch.C61, 19–25.

Salvador,M.J.,Sartori,F.T.,Sacilotto,A.C.B.C.,Pral,E.M.,Alfieri,S.C.,Vichnewski,

W.,2009.BioactivityofflavonoidsisolatedfromLychnophoramarkgraviiagainst

Leishmaniaamazonensisamastigotes.Z.Naturforsch.C64,509.

Schinor,E.C.,Salvador,M.J.,Turatti,I.C.,Zucchi,O.L.A.D.,Dias,D.A.,2004.Comparison

ofclassicalandultrasound-assistedextractionsofsteroidsandtriterpenoids fromthreeChrestaspp.Ultrason.Sonochem.11,415–421.

Schinor,E.C.,Salvador,M.J.,Tomaz,J.C.,Pral,E.M.F.,Alfieri,S.C.,Albuquerque,S.,Dias,

D.A.,2006.Biologicalactivitiesandchemicalcompositionofcrudeextractsfrom

Chrestaexsucca.Braz.J.Pharm.Sci.42,83–90.

Schinor,E.C.,Salvador,M.J.,Pral,E.M.F.,Alfieri,S.C.,Albuquerque,S.,Dias,D.A.,2007a.

EffectofextractsandisolatedcompoundsfromChrestascapigeraonviabilityof LeishmaniaamazonensisandTrypanossomacruzi.Braz.J.Pharm.Sci.43,295–300.

Schinor,E.C.,Salvador,M.J.,Ito,I.Y.,Dias,D.A.,2007b.Evaluationoftheantimicrobial

activityofcrudeextractsandisolatedconstituentsfromChrestascapigera.Braz. J.Microbiol.38,145–149.

Shoemaker,R.H.,2006.TheNCI60humantumourcelllineanticancerdrugscreen.

Nat.Rev.Cancer6,813–823.

Shohreh,N.,Mehrdad,H.,MehdI,R.,Heidar,A.T.,2008.DNAadductswithantioxidant

flavonoids:morin,apigenin,andnaringin.DNACellBiol.27,433–442.

Silva,A.A.R.,Bezerra,M.M.,Chaves,H.V.,Pinto,V.P.T.,Franco,E.S.,Vieira,A.M.,

Araújo,E.B.,Cunha,L.R.,Leite,A.C.R.,Maia,M.B.S.,2012.Protectiveeffectof

Chrestamartiiextractonethanol-inducedgastropathydependsonalpha-2 adrenoceptorspathwaysbutnotonnitricoxide,prostaglandinsoropioids.J. Ethnopharmacol.142,206–212.

Wu,X.,Beecher,G.R.,Holden,J.M.,Haytowitz,D.B.,Gebhardt,S.E.,Prior,R.L.,2004.

LipophilicandhydrophilicantioxidantcapacitiesofcommonfoodsintheUnited States.J.Agric.FoodChem.52,4026–4037.

Yasukawa,K.,Takido,M.,Matsumoto,T.,Takeuchi,M.,Nakagawa,S.,1991.Sterol