Rev. bras. farmacogn. vol.26 número6

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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

Co-extracted

bioactive

compounds

in

Capsicum

fruit

extracts

prevent

the

cytotoxic

effects

of

capsaicin

on

B104

neuroblastoma

cells

Viktorija

Maksimova

a,∗

,

Liljana

K. Gudeva

b

,

Rubin

Gulaboski

a

,

Karen

Nieber

c a_Faculty_of_Medical_Sciences,_Goce_Delcev_University,_Stip,_Macedonia

b_Faculty_of_Agriculture,_Goce_Delcev_University,_Stip,_Macedonia

c_Faculty_of_Pharmacy,_Biology_and_Psychology,_Institute_of_Pharmacy_University_of_Leipzig,_Leipzig,_Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21April2016

Accepted13June2016

Availableonline11August2016

Keywords:

Antioxidants Capsaicin Capsicum Cytotoxicity

Neuroblastomacells

Voltammetry

a

b

s

t

r

a

c

t

TheaimofthisstudywastoinvestigatetheeffectofcapsaicinandethanolicCapsicumextractsonB104 neuroblastomacellsasapotentialanticanceragent.Additionally,thisstudyalsoaimstoexaminethe influenceofco-extractedbioactivecompounds(vitaminE,vitaminCandquercetin)inCapsicumfruit extractsonthecytotoxiceffectsofcapsaicininneuroblastomacells.MTTandLDHassayswereusedto determineviabilityandcelldeathinB104neuroblastomacells.Antioxidativepropertiesofcapsaicin, vitaminE,vitaminCandquercetinwereestimatedbymeansofcyclicandsquarewavevoltammetry. Therewasasignificantcytotoxicityofcapsaicin(100␮mol/l)after24hincubationandforcapsaicin (250␮mol/l),evenwhencellsaretreatedfor1h.Ontheotherhand,ethanolicCapsicumextractswhich containedcapsaicin(0.5–2.1mmol/l)didnotshowanycytotoxiceffect.Wesuggesttherefore,thatother co-extractedcompoundswithintheethanolicextractsinteractantagonisticwiththecytotoxiceffectof capsaicinandtheirinteractionsshouldbefurtherinvestigated.Ourresultsindicatethatcapsaicininhigh concentrationinducescytotoxiceffectsinadosedependentmanner,butotherbioactivecompounds presentinCapsicumfruitspreventthecytotoxiceffectsoftheextractsonneuroblastomacells.

Introduction

Capsaicin(N-vanillyl-8-methyl-6-nonenamide,1)isthemajor componentofcapsaicinoids.Thisalkaloidisasecondary metabo-liteindifferentspeciesofthegenusCapsicum(Buczkowskaetal., 2013).Itgivesthepungencyofhotpeppersandisresponsiblefor manyphysiologicalandpharmacologicalpropertiesofthisplant. Althoughtopicalcreamswithcapsaicinareusedtotreat periph-eralneuropathicpainconflictingepidemiologicdata,manybasic researchstudiesresultssuggestthatcapsaicincanactasacytotoxic orasacytoprotectiveagent(BodeandDong,2011).Themajority ofresearchstudiessuggestthatcapsaicininducescell-cyclearrest orapoptosisorinhibitsproliferationindifferentmalignantcells includinglungcancer,adenocarcinoma,pancreaticcancer,breast cancerDíaz-LaviadaandRodríguez-Henche(2014)hepatocellular carcinomaBaeketal.(2008),osteosarcomaandmanyothersWon etal.(2013).Variousmechanismsforcapsaicin-induced apopto-sishave beenproposedfor differentcellsystems. Physiological

∗ Correspondingauthor.

E-mail:viktorija.maksimova@ugd.edu.mk(V.Maksimova).

processeslinkedtotheintracellularcalciumincrease,reactive oxy-gen species generation, disruptionof mitochondrial membrane transitionpotentialandactivation ofsometranscriptionfactors areinvolved ClarkandHo-Lee(2016)are closelyrelated tothe capsaicinactivity.

AccordingtoSanchezetal.(2006),capsaicincanactascytotoxic agentthroughevokingapoptosisinprostatecancercellsthrough mechanismwhichincludesincreasedproductionofreactive oxy-genspecies(ROS),disruptionofinnermitochondrial membrane potentialandactivationofcaspase-3.Pramaniketal.(2011)and Zhangetal.(2008)showedthatapoptosisprovokedbycapsaicin in pancreatic cells is accompanied by 4–6fold increase of the concentrationoffreeradicalsandconsequentlydisruptionofthe mitochondrialmembranepotential.Thereforecapsaicinhas pro-vokedaninhibitionofcellproliferationandinducedapoptosisina dosedependentmanner.

Incontrast,hotpeppersfruitsarewidelyusedineveryday nutri-tionandhaveshownmanybenefitsforhumanhealth.Asknown fromtheliterature,beside capsaicin,theseextractsrepresent a complexmixtureofmanyotherbioactivecompoundsasvitamin C(2),vitamin E(3), carotens,quercetinandluteolin (Asninand Park,2015).Thesemoleculesarereportedascompoundswhich

http://dx.doi.org/10.1016/j.bjp.2016.06.009

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haveshownhighantioxidativepotentialandprotectiverolein car-cinogenesis(MaterskaandPerucka,2005).Actingasantioxidants, thesemoleculesarecapabletoneutralizeorscavengethefree radi-calswhichareresponsibleformanydegenerativediseasesaswell asprogressionofcancer(Uttaraetal.,2009).Therefore,thetotal anitoxidativecapacityofpepperextractscanmodulatethe cyto-toxicityofcapsaicinpresentintheextracts.

1 2 3

Theaimofthepresentworkwastostudythecytotoxic proper-tiesofcapsaicinonB104neuroblastomacells,andalsotoexamine thetoxicityoftheCapsicumfruitextractsobtainedfromaseveral differentvarietiesofhotpeppers.Thecurrentstudyalsoaimsto addressthepossibleinteractionsandthesynergisticantioxidant effectsoftheco-extractedcompoundswithcapsaicin.Tothebest ofourknowledge,theinfluenceoftheotherco-extractedbioactive compoundsinCapsicumfruitextractsonthecytotoxiceffectsof capsaicinonB104neuroblastomacells,havenotbeenevaluatedso far.

Materialsandmethods

Plantmaterials

CapsicumfruitsfromfourdifferentgenotypesofC.annuumL. Solanaceae,(hotpeppers)weretakenforthisexperiment.Different plantseedswerestoredinthegenbankatGoceDelcev Univer-sity,FacultyofAgriculture,atthecampusofStrumica,Macedonia. Plantnamehasbeencheckedontheweb:www.theplantlist.orgon February10,2015.Theseseedshavebeentakenforcultivationand theirfruitswerecollectedfromthefieldinthephaseofbotanical maturity.Thefruitsfromfourgenotypesofhotpepperswithlocal names:Bombona,Feferona,Vezena,andSivrija,weredriedonroom temperatureforabouttwoweeks.Afterward,theyweregrounded andthepowderwasusedforextraction.

Cellline

Cells of the rat neuroblastoma line B104 (ATCC, Manassas, VA,Schubertetal.1974)weremaintainedinDMEM/Ham’swith l_-glutamine _{(Dulbecco’s}_modified_Eagle’s_medium)_(PAA_GmbH)

supplementedwith15%fetalbovineserum(FBS),andantibiotics1% penicillin/streptomycinsolution.Themediumwaschangedevery 2–3days.Cellswereincubatedat37◦_C_in_an_atmosphere_containing 5%CO2andsaturatinghumidity.Cellswereallowedtobeadherent for24hbeforetreatmentwithcapsaicinorCapsicumextracts.

Reagents

Stock solutions of capsaicin (1) (>95%, natural capsaicin), vitamin E (2), quercetin and ascorbic acid were freshly pre-paredbyusingstandardsubstancesobtainedfromSigma-Aldrich and 96% ethanol (reagent grade) (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide)(MTT),sodiumdodecylsulfate (SDS),dimethylformamide(DMF),lactate,NAD,diaphorase,HCl, SDSlysisbuffer,DMEM,phosphatebuffersolution)PBS,phosphate buffer(pH7.4),fetalbovineserum,penicillin,streptomycin(PAA GmbH),tritonX,ethanol96%(Sigma–Aldrich).Allsolutionswere storedat4◦_C._In_all_solutions_used_for_{electrochemical} measure-ments,KClwasaddedasanadditionalelectrolyteatconcentration of0.010M.

Methods

Extractionmethod

Extraction wasperformed by maceration using ethanol 96% (v/v) as a solvent (Rafajlovska et al., 2007). Maceration was

performedfor5h,on60◦_C,_and_afterward_the_extracts_were fil-teredbygouchfilter,usingavacuumpump.0.2gofthepulverized plantmaterialwasmaceratedwith25mlsolvent.Stocksolution waspreparedbyusingcapsaicin,with96%ethanolandthendiluted toappropriateconcentrations.

Spectrophotometricmethod

UV/VISspectrophotometrywasusedforquantificationof cap-saicininethanolicextractsandstandardssolutions(Peruckaand Oleszek,2000).Theconcentrationofcapsaicinwasmeasuredby usingaCary100spectrometer,instrumentversionno.9.00,ona specificmaximumwavelengthof280nm.Aserialofstandard dilu-tionsofcapsaicin(0.25,0.125,0.0625,0.0312,0.0156mg/ml)were preparedforobtainingtheregressioncurve.Theethanolicextracts weremeasuredandaccordingtotheabsorbanceobtainedforthem, regressionanalysiswasperformedforcalculationofconcentration ofcapsaicinintheextracts.

Cytotoxicitymethods

TheviabilityofB104cellsaftertreatmentwithcapsaicinor Cap-sicumextractswasassessedusingMTTmethod.Fordetermination ofthecelldeath,theLDHmethodwasused.

MTTassay

TheMTT assayinvolvesthe conversionofthewater soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide)toaninsolubleformazan.Theformazanisthensolubilized anditsconcentrationwasdeterminedbyopticaldensityat570nm (Mosmann, 1983). MTT cell proliferation assay was performed accordingtotheprotocolgivenbythemanufacturerRoche Diag-nosticsGmbHforCellProliferationKitI.Inbrief,B104cellswere seededin96-wellcellcultureplates(2×104_cells/well)_and sub-sequentlytreatedwithcapsaicin(0.5,1,10,100and250␮mol/l) ordifferentethanolicCapsicumextractsat37◦_C_for_1,₆_and₂₄_h, respectively.

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LDHassay

TheLDHassayoffersasimplewaytomeasureplasma mem-branedamage,basedonthereleaseoflactatedehydrogenase(LDH), astablecytoplasmicenzymepresentinmanycells (Chouetal., 2009).Assaywasperformedaccordingtothemanufacturers pro-tocol (RocheDiagnostics GmbH)given for theLDH cytotoxicity detectionkit. Inbrief,afteradherenceof thecells tothewells, testsubstances(capsaicinindifferentconcentrationsandethanolic extracts)dilutedinanappropriateassaymedium(DMEM),were titratedinaseparatemicroplatebyseveraldilutions(final vol-umeupto200␮l/well). Then, theassaymedium wasremoved and100␮lfreshassaymediumwasaddedtoeachwell.100␮l ofthetestsubstancedilutionsweretransferredinto correspond-ingwellscontainingtheadherentcells.Thereafter,100␮lofthe supernatantwereremovedfromthewellscarefullyandtransferred intocorrespondingwellsofanopticallyclear96-wellflatbottom microplate.To determinethe LDH activityin thesupernatants, 100␮lofthereaction mixture(freshlyprepared)wasaddedto eachwellandincubatedfor30minat+15to+25◦_C_in_a_dark atmo-sphere.ThereactionmixturewaspreparedbymixingtheCatalyst (Diaphorase/NAD+_mixture)_and_Dye_solution_(INT_and_sodium lac-tate).

Absorbanceofthesampleswasmeasuredat492nmbyanELISA platereader.ThelossofintracellularLDHanditsreleaseintothe culturemediumisanindicatorofirreversiblecelldeathduetocell membranedamage.A0.1%TritonXsolutionwasusedaspositive controlandDMEM(assaymedium)asnegativecontrol.

Voltammetricmethods:cyclicvoltammetryandsquarewave voltammetry

Electrochemicaldeterminationoftheantioxidativepotentialof theextractsanda mixtureofstandard solutionwasperformed bymeansofsquare-wavevoltammetry(SWV),aftershort electro-chemicalcharacterizationoftheelectrochemicalfeaturesbycyclic voltammetry(CV),ataglassycarbonworkingelectrode. Experi-mentsincyclicvoltammetrywereconductedoverapotentialrange from−0.200to1.000V, withascan rateof

v

=10mV/s.

Experi-mentalconditionsforSWVwere:potentialstepdE=0.001V,pulse height(SWamplitude) Esw=0.050Vand frequency of f=10Hz. Priortoeachelectrochemicalexperimenttheworkingelectrode waspolishedbyusingAlCl3onapolishingcloth,followedbyrinsing oftheelectrodewithwaterandacetoneanddryinginair.

Statistics

VarianceanalyseswereperformedbyusingaGraphPadPrism 6.0.ProcessingoftheresultswasdonebyonewayANOVA,for threegroupsofresults(differenttimeofexposition),inwhicheach treatmentofthecellswascomparedbythetreatmentwith neg-ativecontroland/orpositivecontrol.Allgraphsrepresentmeans andstandarddeviationsfortriplicatesamplesfromeachofthree independentexperiments(n=9).Resultswhicharestatistically sig-nificantareshowedonthegraphs,p<0.01.Theregressionanalysis wasperformedusingtheprogramGraphPadPrism6.0,XYanalyses, linearregression.

Results

Determinationofthecapsaicinconcentration

Spectrophotometricmeasurementoftheconcentrationof cap-saicinintheethanolicoleoresinswasdeterminedonthebasisof standardsolutionsofcapsaicin.Theconcentrationofcapsaicinin

3

2

A

R2=0.999

1

0

0.0 0.1 0.2

c (capsaicin) /mg/ml

0.3

Fig.1.CalibrationcurveconstructedforstandardsolutionsofcapsaicinbyUVVIS.

thesampleswerecalculatedusingtheregression curveand lin-earityequation(Fig.1).Becausetheextractswereobtainedwith 96%ethanol,wedilutedtheextractswithDMEMinratio1:100,in ordertoescapeanycytotoxiceffectsofethanolonthecells. Addi-tionally,thedilutionaffectedthecoloroftheextracts.Thecolor comesfromthehighconcentrationofpigments,anditvanished afterthedilution.Therefore,toobviatetheprobabilityofgiving falseresults,extractsweredilutedtoappropriateconcentrations fortreatmentofthecellsgiveninTable1.Bombonagenotype con-tainedthehighestconcentrationofcapsaicinwith2.10mM,forthis extract.Concentrationsofcapsaicincalculatedasmg/gDWarein linewiththeresultsfoundintheliterature(Tilahunetal.,2013).

Effectofcapsaicinoncellviabilityandcelldeath

Asshown in Fig.2, capsaicin present in low concentrations (0.5–10␮M)didnotsignificantlyinfluencethecellviability.The concentrationrequired to inhibit 50%of the cells viability was foundtobe61.9␮M,whichisinaccordancewiththeprevious find-ingsforcapsaicin(Richeuxetal.,1999).Inconcentrationof100 and250␮Mitinhibited theviabilityofB104cells comparedto theeffectsof0.1%TritonXsolution,usedaspositivecontroland DMEM(assaymedium)asnegativecontrol.Theincubationof1h resultedinamoderateinhibitionfor100␮M(29±11.5% inhibi-tion,p>0.05),whereastheeffectsweremorepronouncedaftera longerincubationperiodorata higherconcentration.Capsaicin (100␮M)incubatedforaperiodof6hresultedin94±1% inhibi-tion,whileafteranincubationperiodof24h,theinhibitionwas 95±0.1%(p<0.01).Higher concentrationof capsaicin(250␮M)

Table1

ConcentrationofcapsaicininstandardsolutionandextractsofCapsicumusedfor

treatmentofB104cellsinMTTandLDHassays,(extractsweredilutedwithDMEM

inratio1/100).

Standard solutions

Concentration ofcapsaicin [␮M]

Diluted extractsfor treamentof cells

Concentration ofcapsaicinin extracts[mM]

St.solution1 0.5 Solutionof

Vezena

0.51

St.solution2 1 Solutionof

Feferona

0.78

Bombona BBBombona

2.1

Sivrija

0.65

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120 1h incubation

6h incubation

24h incubation

100

80

60

Vibaility of cells (% of control)

40

20

0

DMEM 0.5 Triton X

#

** ** ** ** **

Concentrations of capsaicin (μm)

1 10 100 250

Fig.2.Effectofdifferentcapsaicinconcentrationsoncellviability(MTTassay)on B104neuroblastomacellsincubatedfor1,6or24h.Thereisasignificantdifference intheresultsfortheeffectof100␮Mcapsaicinfor1hcomparedtotheeffectfor 6h.Significance:#(p<0.05),**(p<0.01)vs.DMEM.

100

80

60

40

20

Viability of cells (% of control) ₀

DMEM 0.5 1

Concentrations of capsaicin (μm)

10 100

1h incubation 6h incubation 24h incubation

250

Fig.3. Capsaicinsuppressescellviabilityinatimeanddosedependentmanner. Therewasasignificantcytotoxicityofhigherconcentrationsofcapsaicin(100and 250␮M),after6and24hincubation.IC5061.9␮Mfor6handIC5061.6␮Mfor24h.

resultedin much higher inhibition ofcell viabilityeven for1h

incubation(93±0.5%,p<0.01)andfor6hand24h(94±1%and

95±0.2%,p<0.01,respectively).

TheresultsfromtheMTT assay(Fig.3)showedthatthereis

significantdifferenceintheviabilityofculturedcellswhenthey weretreatedwithcapsaicinfor1h,and6or24h.Thecalculated IC50 valuefor6h(IC5061.9␮M)wasalmostthesameasfound after 24h incubation period (IC50 61.6␮M) indicating that the effectofcapsaicinwasnotpronouncedwithinalongerincubation period.

TheresultobtainedfromtheLDHassayispresentedinFig.4.and itconfirmedtheresultsobtainedfromtheMTTassay.Capsaicin inlowconcentrations(0.5–10␮M)didnotsignificanlyinfluence theLDHrelease.Inconcentrationof100and250␮Mitresulted inhighLDHreleasefromB104cells,indicatingcelldeath.Results werecomparedtotheeffectsof0.1%TritonX solution,usedas positivecontrolandDMEM(assay medium)asnegativecontrol. Concentrationofcapsaicinof100␮Mincubatedfor1hresultedina moderateLDHrelease(13±6%,p>0.05),whereastheeffectswere morepronouncedafteralongerincubationperiodoratahigher concentration.Thesameconcentrationofcapsaicin(100␮M)fora periodof6hincubationresultedin96±2.3%celldeath,whilefor 24htreatment,thecelldeathwasincresedto58±5.1%,(p<0.01). Higher concentration of capsaicin (250␮M) resulted in much higher cytotoxicity of cells even for 1h incubation (100±2.7%, p<0.01),aswellasfor6hand24hconsequently(90±1.38%and 98±6.9%,p<0.01).

120

100

80

60

% of cell death 40

20

0

DMEM 0.5 1 10 100 250

Concentrations of capsaicin (μm) 1h incubation _{∗ ∗}

∗∗

# ∗∗

∗∗ ∗∗

6h incubation 24h incubation

Fig.4.Effectofcapsaicinindifferentconcentration(0.5,1,10,100,250␮M),

exam-inedbyLDHassayonB104neuroblastomacellsfor1,6or24hofincubation.Treating

oftheneuroblastomacellswithcapsaicin(100and250␮M)for6or24hresulted

inhighincreaseinLDHreleaseindicatingcelldeath.(#p<0.05;*p<0.01).Values

aremeans±SEcomparedwithDMEMasnegativecontrol.n=9foreachgroup.

Ethanolic capsicum extracts

DMEM

120

100

80

60

40

Viability of cells (% of control)

20

0

V

ez

ena

F

ef

erona

Bombona

Sivr

ija

T

riton X

Fig.5. EffectofethanolicCapsicumextracts(obtainedfromCapsicumgenotypes:

Vezena,Feferona,Bombona,Sivrija)oncellviability(MTTassay)treatedfor1,6or

24h.Viabilityofthecellswascomparedtonegativecontrol(DMEM).

EffectofethanolicCapsicumextractsoncellviabilityandcell death

Incontrasttocapsaicin,theethanolicCapsicumextractsdidnot influencesignificantlyneitherthecellviabilitynorthecelldeath.In theMTTassay(Fig.5)therewasnosignificantinhibitionofthecell viabilityaftertreatmentofthecellswiththeextracts.The incu-bationtimeperiod hadnot effectonthecellviability.TheLDH assay(Fig.6)confirmedtheresultsofMTTassay.Noeffectsonthe LDHreleasewasfound,indicatingthattheextractdidnothaveany cytotoxiceffects.

Antioxidativepotentialofcapsaicinandotherco-extracted bioactivecompoundspresentintheethanolicCapsicumextracts

Abriefelectrochemicalcharacterizationofcapsaicin,vitaminE, ascorbicacidandquercetinhasbeenperformedbymeansofcyclic voltammetry(resultsnotshown),whiletheirpotentialsynergistic antioxidativeeffecthasbeenanalyzedbymeansofsquare-wave voltammetry.TypicalnetSWvoltammogramsofcapsaicin, vita-min E, quercetin, and ascorbic acid (each at concentration of 10␮mol/l)recordedataglassycarbonelectrodeinabuffer solu-tionatpH=7.0aregiveninFig.7.ThenetSWpeaksofvitaminE andquercetinarecloselypositionedatpotentialsofEp,net=0.146V

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Ethanolic capsicum extracts

DMEM

120

100

80

60

40

% of cell death

20

0

Vezena _Feferona

Bombona

Sivrij

a

T

riton

X

Fig.6.EffectofethanolicCapsicumextractsoncelldeath(LDHassay)treatedfor

1,6or24h.TherewasnosignificantdifferencewithethanolicCapsicumextracts

comparedtothenegativecontrol(DMEM).

5.0

4.5

4.0

3.5

3.0

I

/

μ

A

E/V (v.s Ag/AgCI) c

d b

a e

2.5

2.0

1.5

–0.20 –0.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Fig.7.Square-wavevoltammogramof:(A)vitaminE;(B)quercetin,(C)ascorbicacid

and(D)capsaicin(10␮M),(E)equimolarmixturecontainingvitaminE,quercetin,

ascorbicacidandcapsaicinatconcentrationof10␮M,recordedataglassy

car-bonelectrodeinabuffersolutionatpH=7.1.Instrumentalparameterswere:step

potentialdE=0.001V,square-waveamplitudeEsw=0.05Vandfrequencyof10Hz.

locatednearbythecapsaicinpotential(atEp,net=0.409Vfor

ascor-bicacidand Ep,net=0.352V forcapsaicin).VitaminEelevatesto

thehighestnetpeak current(Ip,net=1.894␮A) comparedtothe maximalpeakcurrentsofquercetin,ascorbicacidandcapsaicin (0.580␮Aforquercetin,0.193␮Aforascorbicacidand0.086␮A forcapsaicin).Theobtainedsquare-wavevoltammogramsforthe equimolar mixture of 10␮mol/l of each compound show that thevoltammetricresponseconsistsofasingleSWVpeakatthe potentialabout0.128V,whichisbetweenthetypicalpeakof Vita-minE and quercetin. Themeasured netpeak current obtained for the mixture of all four compounds was Ip,net=3.313␮A, Fig.7(e).

Discussion

It is well known that capsaicin has different carcinogenic effects onneuronal and non-neuronalcells (Chouet al., 2009). However, capsaicin induced cytotoxicity on pancreatic neu-roendocrine tumor cells Skrzypski et al. (2014), human skin fibroblasts Kim et al. (2004), human gastric adenocarcinoma cell line (Yi-Ching et al., 2005). Ethanolic extracts of several spices,inwhichchillipepperwasincluded,inhibitedcellgrowth at concentrations of 0.2–1mg/ml in vitro (Unnikrishnan and

4

3.5 y=0.304x+0.368

R2=0.987 3

2.5

2

1 1.5

I

pa/

μ

A

0.5

0

0 2 4

Equimolar concentration of all compounds in mixture μmol/I

6 8 10 12

Fig.8. Dependenceontheanodicpeakcurrentandtheconcentrationofequimolar

mixturescontainingvitaminE,quercetin,ascorbicacidandcapsaicinat

increas-ingconcentrationfrom1to10␮M,inaphosphatebufferatpH=7.1(dE=0.001V,

Esw=0.050Vandfrequencyof10Hz).

Ramadasan,1988).Otherauthorsconfirmedthatethanolic Cap-sicum extracts (0.01–1000␮g/ml) didnot alter endothelial cell survival(Chularojmontrietal.,2010).

Ourresultsshowedthatthecytotoxicactivityofcapsaicinon neuroblastomaB104cellswaspronouncedatconcentrationsof100 and200␮M.TheIC50valueswerefoundtobe61.9␮Mfor incuba-tiontimeperiodof6hand61.6␮Mfor24h,respectively,indicating thattheefficiencyunderthegivenconditionsdidnotchangewhen theincubationtimeperiodwasextendedform6hto12h. Previ-ousfindingsofcapsaicincytotoxicityhaveshowna similarIC50 valuebutit wasobtainedfor5daystreatmentofthecells with capsaicin(Richeuxetal.,1999).Lowerconcentrationsofcapsaicin (Figs.2and4)didnotshowanycytotoxicactivity.Interestingly,the extractsobtainedfromdifferentgenotypes(Figs.5and6)which containedevenhigherconcentrationofcapsaicindidnotinduce theexpectedcytotoxicity.

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we could report that vitamin E and quercetin can eventually neutralizethe ROS produced bycapsaicin atthe mitochondrial membrane.

Huetal.(2008)haveshownthatTRPV1receptorisincluded incapsaicininducedCa2+_influx_by_generation_of_reactive_oxygen species(ROS),depolarizationofthemitochondrialmembrane,and ultimatelycelldeathonthesynovialfibroblastsinrats.Huangetal. (2009)havedemonstratedthattheapoptoticprocesson hepatocel-lularcancerwasalsoaccompaniedbyincreasingoftheintracellular Ca2+_level,_increased_production_of_ROS,_and_disruption_of mito-chondrialmembranepotential.Thisapoptoticmechanismwasalso confirmedformanyothertypesofcancer.

Therefore,weassumethathighantioxidativepotentialofthese co-extractedcompoundspresentintheethanolicextractscould haveanantagonisticeffecttocapsaicincytotoxicmechanism.This hypothesis enforced us toconsider that thesynergistic antiox-idativeeffectofthecomplexcompositionofhotpepperfruitsis responsiblethatethanolicCapsicumextractshavenotshown cyto-toxicactivity,besideitshighconcentrationofcapsaicin.

Conclusions

Thisstudyexhibitedthatcapsaicincanactascytotoxicagent inneruoblastomacellsinadosedependentmanner.Knowingthat capsaicincanbeeasilyextractedandisolatedfromCapsicumfruits offersthechance for discovering a phytochemicalagent which possessesastrongpharmacologicalactivityinantitumortherapy. Incontrast,Capsicumextractsdidnotshowanyanti-proliferative activity.Therefore,additionalelectrochemicalexperimentswere performedtoexplainthesynergisticeffectsbetweencapsaicinand vitaminE,quercetinandascorbicacid,presenttogetherina com-plexmixture.Asshownintheliterature,acommonmechanismof capsaicincytotoxicityisachievedthroughproductionofreactive oxygenspeciesoncellularlevel.Thisleadstodisruptionof mito-chondrialmembranepotential,activationofcaspase-3activityand successiveapoptosis.Weassumedthatthisphenomenonof syner-gismbetweenthestudiedcompoundscouldbeapossiblereasonfor antagonisticeffectsoftheotherco-extractedphytochemicalsfrom thehotpepperfruitsonthecytotoxicityofcapsaicin.Inorderto ensureinthishypothesisfurtherexperimentsareneededtoobtain moredetailedresultsofthemechanismofcytotoxicityofcapsaicin whenitisinamixturewithotherbioactivecompoundsfoundin peppers.

Ethicaldisclosures

Protection of human subjects and animals in research.The authorsdeclarethatnoexperimentswereperformedonhumans oranimalsforthisstudy.

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authorcontributions

VM(PhDstudent)contributedincollectionandidentification ofthepepperfruits,preparationoftheextracts,runningthe lab-oratory experiments in (spectrophotometry, cytotoxic analyses and voltammetry), analysesof the dataand preparation ofthe manuscript.LKGcontributedintheUVanalysesandsupervised theextractionprocedures.RGdesignedthestudyofthe

antioxida-tiveanalysesinvoltammetryandsupervisedallthevoltammetric experimentandresults.KNdesignedthestudyofcytotoxicity anal-yses and supervised allthe experimentsperformed onthecell cultures.Allcoauthorscontributedwiththeircritical readingof themanuscript.Alltheauthorshavereadthefinalmanuscriptand approveditforpublication.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethanktoDAADorganizationforcollaborationbetweenGoce DelcevUniversity,Stip,RepublicofMacedoniaandUniversityof Leipzig,Leipzig, Germany,throughtheprojectMatCatNet, 2013. Someexperiments were performedin theframe of studystay, whichwasfinanciallysupportedwithascholarshipbyDAAD foun-dation.

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