w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Amburana
cearensis
seed
extracts
protect
PC-12
cells
against
toxicity
induced
by
glutamate
Erica
P.L.
Pereira
a,
Suzana
Braga-de-Souza
a,
Cleonice
C.
Santos
a,
Leticia
O.
Santos
a,
Martins
D.
Cerqueira
b,
Paulo
R.
Ribeiro
b,c,
Luzimar
G.
Fernandez
b,
Victor
D.A.
Silva
a,
Silvia
L.
Costa
a,∗aLaboratóriodeNeuroquímicaeBiologiaCelular,InstitutodeCiênciasdaSaúde,UniversidadeFederaldaBahia,Salvador,BA,Brazil bLaboratóriodeBioquímica,BiotecnologiaeBioprodutos,InstitutodeCiênciasdaSaúde,UniversidadeFederaldaBahia,Salvador,BA,Brazil cDepartamentodeQuímicaOrgânica,InstitutodeQuímica,UniversidadeFederaldaBahia,Salvador,BA,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received16May2016 Accepted31August2016 Availableonline22November2016
Keywords: Amburanacearensis Glutamate PC12cells Excitotoxicity Neuroprotection
a
b
s
t
r
a
c
t
Amburanacearensis(Allemão)A.C.Sm.,Fabaceae,hasbeenwidelystudiedforitsmedicinalactivities. Manyneurodegenerativedisordersarecausedbyoxidativestress,mitochondrialdysfunction, excito-toxicityinducedbyglutamateandultimatelycelldeath.Thisstudydescribesthechemicalprofileof theethanolic,hexane,dichloromethane,andethylacetateextractsobtainedfromseedsofA.cearensis. Theobjectiveofthisstudywastoinvestigatethechemicalprofileofextractsobtainedfromseedsof
A.cearensis,aswellastheircytotoxicityandneuroprotectiveeffectsinculturesofneuralPC12cells. MetaboliteprofilewasperformedbyGC–MS.PC12cellsweretreatedwithincreasingconcentrationsof theextracts(0.01–2000g/ml)andthecellviabilitywasanalyzedafter24and72husinganMTTtest. Fortheexcitotoxicityassay,PC12cellswerepre-treatedwithglutamate(1mM)for6handtreatedwith increasingconcentrations(0.1–1000g/ml)oftheextracts.Thechromatographicanalysisoftheextracts detectedvariouscompoundswithantioxidantproperties,withthemajorityofpeakscorrespondingto theisoflavonecoumarin.Onlythehexaneextractshowedtoxicityafter72hexposureatthehighest con-centration(1000g/ml).Bycontrast,allextractsincreasedthecellularviabilityofPC12cellsagainstthe toxicitycausedbyglutamate.Therefore,theextractsfromtheseedsofA.cearensisshowednotoxicityand haveneuroprotectivepotentialagainstneuronaldamageinducedbyglutamate,whichmayberelatedto theirantioxidantproperties.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Neurodegenerativediseasesareamongthemostserioushealth problemsfacingmodernsocietyandarecharacterizedbyneuronal celllossinthebrainandspinalcordleadingtofunctionalorsensory impairment(Uttaraetal.,2009).Manyofthesedisorders,including Parkinson’sdisease(PD),Alzheimer’sdisease,multiplesclerosis, stroke,amyotrophiclateralsclerosisandmanyothershavebecome morecommonwiththeaging ofthepopulationand havebeen associatedwithoxidativestress,mitochondrialdysfunction,and excitotoxicity. Finally,cell deathis causedby apoptosis(Uttara etal.,2009).Excitotoxicityiscaused bytheexcessivereleaseof glutamate, the mostexcitatory neurotransmitter in thecentral nervous system(CNS) of mammals (Fujikawa, 2015).Neuronal
∗ Correspondingauthor.
E-mails:[email protected],[email protected](S.L.Costa).
damage associated with excitotoxicity is a type of cell death triggeredbytheoveractivationofglutamatereceptorsandtheloss ofcalciumhomeostasisduetoexcessivecalciuminflux(Camacho and Massieu, 2006). Many studies have evaluated compounds that inhibitcascadesthattriggerneuronal damagetominimize theprimarycausesofneuronalinsults.Medicinalherbsthathave beenusedinmanycultures forthousandsofyears haveplayed a vital role in the discovery of new drugs (Silva et al., 2012). Amburanacearensis(Allemão)A.C.Sm.,isaspeciesofthefamily of Fabaceae(Papilionoideae,Leguminoseae), naturallyoccurring innortheasternBrazil,specificallyinCaatinga.Differentpartsof theplants,leaves, stemand seedsareusedinpopularmedicine andhavemanymedicinalapplications.A.cearensisisan antiox-idant(Pereiraetal.,2014),bronchodilator(Lealetal.,2005)and affectsrespiratorytractdiseases(Agraetal.,2007;Roque,2009) amongothers.Thisplantislistedamongendangeredplants(IPEF, 2008), and despite its great therapeutic potential,very little is knownabout theaction of its compoundsin the CNS.Because
http://dx.doi.org/10.1016/j.bjp.2016.08.010
glutamatergictransmissionis related tonumerous neurological andpsychiatricdisorders(ValliandSobrinho,2014),theobjective ofthisstudywastoevaluatetheantioxidantandneuroprotective potentialofextractsfromA.cearensisseedsinPC12cellcultures subjectedtoexcitotoxicitymodelsinducedbyglutamate.
Materialsandmethods
Amburanacearensisseedextracts
TheseedsofAmburanacearensis(Allemão)A.C.Sm.,Fabaceae, wereobtainedinpopulartradeinthecityofFeiradeSantana,Bahia, Brazil,andhaditsauthenticityrecognizedwhencomparedwiththe identificationaccordingtoGuedesetal.(2007)13734,theIDare depositedintheHerbariumoftheBiologyInstituteoftheFederal UniversityofBahia,withthenumber13734.Thepreparationof extractswasperformedbythesoakingmethod.Theseeds(1kg) weregroundinaknifemillanddriedforaperiodof24hinan exhausthood.Then,thematerialwasplacedinaglasscontainer, coveredwithethylalcoholfor72handsuccessivelyshakenevery 24h.Afterthisperiod,thesupernatantwascollectedandfiltered. Additionalethanolwasaddedtotheobtainedpellet.Thisprocess wasrepeatedthreetimes.Theresultingsupernatantfromthethree macerationswascollectedandplaced onarotaryevaporatorat 40–50◦C.Then,theextractwasdriedinanovenat40◦Cforthree days.Thefinalproductwasnamedthecrudeethanolicextractof theseedsofA.cearensis(ETAC).
Thepreparationofthefractionsoccurredusingthelowest sol-vent polaritytothehighest polarity. Fractionationof theother extracts using the partition method was performed using the ethanolicextract.Thedrycrudeethanolicextract(ETAC,2g)was solubilizedin100mlofethanol(PA)witha20%volumeofdistilled water(20mlH2O).Avolumeof50mlhexanesolventwasaddedto
thismixture.Themixturewasstirredandplacedatrestuntilthe phasesseparated.Thehexanicphasewasthencollected.This pro-cesswasrepeatedthreetimestoobtainafinalvolumeof150mlfor thehexanepartition.Thesametechnicalprocedurewasperformed forthesolventsmethylenechlorideandethylacetate.Theresulting extractswereplacedonarotaryevaporatoranddriedinanovenat 40◦Cforthreedays.Theresultingextractswerenamedthehexane extractoftheseedsofA. cearensis(EHAC),thedichloromethane extractoftheseedsofA. cearensis(EDAC)andtheethylacetate extractoftheseedsofA.cearensis(EAAC).
Thestocksolutionsforthebioassayswerepreparedby dissolv-ingeachextractinwatertoafinalconcentrationof100mg/ml.The solutionswerethenstoredat4◦C.
Chemicalanalyses
MetaboliteprofilingwasperformedwithanAgilent7809Agas chromatograph(AgilentTechnologies,California,USA)coupledto aTriple-Axisdetector(Agilent5975C)usingaZB-5(Phenomenex; 30m×0.25mm)capillarycolumn(0.25mmfilmthickness)with heliumasthecarriergasataflowrateof1ml/minasdescribed byRibeiroetal.(2014).Theinlettemperatureoftheinjectorwas setto250◦C.Theinitialoventemperaturewas45◦Cfor1minand wasincreasedto300◦Cafter1minatarateof10◦C/minandheld at300◦Cfor5min.Asolventdelayof420swasset.Stock solu-tionsoftheextractswerepreparedinamixtureofchloroform: acetone(1:1).Analiquot(50l)wastransferredtoa 2mlglass vialand a glassinsertof a200lwith150lofdiazomethane solutioninether wasadded.Octadecanewasusedasthe inter-nalstandard,andthesampleswereinjectedinasplitlessmode. Allanalyseswereperformedthreetimes.Metaboliteswere puta-tivelyannotated(e.g.,withoutchemicalreferencestandards,based
upon the physicochemical properties and/or spectral similarity withpublic/commercialspectrallibraries)accordingtoSumnerand colleagues(2007).
PC12cellcultureandtreatments
ThePC12ratpheochromocytomacelllinewaspurchasedfrom theATCCCellBank.ThesecellswereculturedinRPMImedium (Cul-tilab,SP,Brazil),supplementedwithl-glutamine,10%inactivated
fetalbovineserum(Cultilab,SP,Brazil),5%inactivatedhorseserum (Cultilab,SP,Brazil),1%penicillinand1%streptomycin(Cultilab, SP,Brazil).PC12cells werecultureduntilconfluencein100mm polystyreneplates(TPP,Trasadingen,Switzerland),trypsinizedand replatedin96-well(7.5×103cells/cm2)polystyreneculturedishes (TPP,Trasadingen,Switzerland).Thecellsweremaintainedinan incubatorwithahumidifiedatmosphereof95%airand5%CO2at
37◦C.
After24h,themediumwaschangedandextractsand/or glu-tamatedilutedinmediumwithoutserumwereaddeddirectlyin PC12cellcultures.Negativecontrolgroupwasexposedtomedium withoutserum.
Cellviabilityassay
The cytotoxicity of the A. cearensis extracts on the PC12 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT;Sigma,St.Louis,MO).TheMTT testis basedontheprincipleof convertinga yellowcolor sub-strateofformazancrystalstoavioletcolorbythemitochondrial dehydrogenasesoflivingcells(Mosmann,1983).PC12cellswere cultivatedfor24hin 96-wellplates(TPP, Trasadingen, Switzer-land).Theculturemediumwereremovedandthecellsweretreated with increasing concentrations (0.001, 0.01, 0.1, 1, 10, 100 or 2000g/ml)oftheextractsETAC,EAAC,EDAC,orEHAC.Negative
controlgroupwasculturemaintainedinmediumwithoutserum. TheMTTassaywasperformed24or72hafterthetreatments.The MTTsolution(100l)dilutedinRPMImediumwasaddedtoeach wellatafinalconcentrationof1g/ml.Theplateswereincubated for2hinahumidifiedatmospherewith5%CO2 at37◦C.
There-after,tocompletethedissolutionoftheformazancrystals,thecells werelysedwith100l/welllysisbuffer,containing20%sodium dodecylsulfate(SDS)and50%dimethylformamide(DMF),pH4.7. After12hatroomtemperature, theopticalabsorbanceof each samplewasmeasuredusingaspectrophotometerat595nm.To determinewhethertheextractshaveabsorbanceorreactwithMTT, theopticdensityoftheextractswasmeasuredaloneandinthe presenceofMTTat595nminwellswithoutcells.Allexperiments wereperformedineightwellsandwererepeatedatleastthree times.
Neuralexcitotoxicityassay
Toevaluatetheneuroprotective effectof theextractsofthe seedsof A.cearensis, PC12cells werecultivated for 24hin 96-wellplates.Then,theculturemediumwasremovedandthecells weretreatedwith1mMl-glutamicacidmonosodiumsalthydrate
(SigmaRES5063G)(Maetal.,2013)dilutedintheRPMImedium. After6h,thecultureswerepost-treatedwiththeextracts(ETAC, EAAC,EDACandEHAC)atfinalconcentrationsof0.1,1,10,100 and1000g/mlwith1mMglutamate.Thecellsweremaintained inahumidifiedatmosphereof95%airand5%CO2at37◦Cfor24h.
Table1
CompoundsidentifiedbyGC-MSinextractsETAC,EAAC,EDACandEHACfrom Amburanacearensisseeds.
ETAC EDAC EAAC EHAC
Coumarin 1.1±0.1 14.9±1.0 1.0±0.0 1.1±0.0 (b) (a,c,d) (b) (a,b) Methylhexadecanoate 1.8±0.0 5.4±0.2 1.0±0.1 4.1±0.1
(b,c,d) (a,c,d) (a,b,d) (a,b,c) Methyl
9-cis-11-trans-Octadecanoate
1.0±0.0 6.9±0.3 Nd 2.9±0.2
(b,c,d) (a,c,d) (a,c,b) Methyl13-trans-octadecanoate 1.0±0.1 11.0±0.7 1.0±0.1 6.3±0.2
(b,c,d) (a,c,d) (a,b,d) (a,b,c) Methyloctadecanoate 2.3±0.1 3.9±0.7 1.0±0.1 2.8±0.1
(b,d) (c,d) (a,b,d) (a,b,c) ␥-Sitosterol 1.5±0.0 1.1±0.3 Nd 6.1±0.4
(b,c,d) (c,d) (a,b,c) Ethylhexadecanoate Nd 56.8±2.4 Nd 1.0±0.0
(a,c,d) (b)
Ethanolic(ETAC),hexane(EHAC),dichloromethane(EDAC)andethylacetate(EAAC) extractsobtainedfromseedsofAmburanacearensis.Datarepresentmeans±s.e.m. DifferentlettersindicatesignificantdifferencesbetweensamplesandETAC(a), EDAC (b), EAAC (c), EHAC(d) by one-way analysisof variance followed by Tukey–Kramerposthoctest(p<0.001).Nd,notdetected.
Table2
MetaboliteprofilingofAmburanacearensisseedextracts.
Compounds TR IR NI
Coumarin 10.32 1445.38 2
Methylhexadecanoate 13.85 1969.20 1
Methyl9-cis-11-trans-octadecadienoate 14.96 2155.41 2 Methyl13-trans-octadecenoate 14.99 2161.73 2
Methyloctadecanoate 15.14 2187.26 1
␥-Sitosterol 23.61 3493.82 2
Methyl3-coumarin 10.85 1514.87 2
Ethylhexadecanoate 14.30 2043.52 1
Ethyl9,12-octadecadienoate 15.37 2225.60 2
Ethyl9-cis-octadecenoate 15.41 2232.67 1
Ethyloctadecanoate 15.54 2256.47 2
Campesterol 22.57 3373.30 2
Stigmasterol 22.85 3407.43 1
-amyrin 24.31 3563.39 1
TR,retentiontime;IR,retentionindex;NI,identificationlevel.
Statisticalanalysis
Statisticalanalyseswereperformedusingthesoftware Graph-pad Prism 5.0. The cytotoxicityassay was analyzed byANOVA followed by the Tukey test. The neuronal excitotoxicity assay inducedbyglutamatewasanalyzedbyANOVAfollowedby Stu-dent’st-test. The resultsare expressed as themean±standard deviationrelativetothenegativecontrol,considered100%.Values ofp<0.05wereconsideredsignificant.
Resultsanddiscussion
MetaboliteprofilingofAmburanacearensisseedextracts
Fourteenmetaboliteswereidentifiedintheextracts,including theisoflavonescoumarinand3-methyl-coumarin,eightfattyacids (methylhexadecanoate,methyl9-cis-11-trans-octadecadienoate, methyl 13-trans-methyl-octadecanoate, methyl hexadecanoate, ethyl hexadecanoate, octadecanoate acetate, 9,12-cis-9-ethyl octadecanoate, and ethyl octadecanoate), three steroids (␥-sitosterol, campesterol, and stigmasterol) and the triterpenoid -amyrin(Tables1and2).Theisoflavonecoumarinwasdetected inallfourextracts.However,itsconcentrationwas14.9-foldhigher attheEDACthanattheotherthreeextracts.
Four methyl esters were detectedin theextracts. The first, methyl hexadecanoate, was detected in the four extracts and
wasin differentconcentrationsin theEDAC(44%±1.6%),EHAC (32%±1.2%),ETAC (14%±0.5%),and EAAC(8%±0.3%).The sec-ond,9-cis-11-trans-methyloctadecadienoate,wasdetectedonly in three extracts, with different concentrations in the EDAC (63%±2.5%),EHAC(26%±1.5%),andETAC(9%±0.3%).Thethird, 13-trans-methyl-octadecanoate,wasdetectedinthefourextracts andexhibiteddifferentconcentrationsintheEDAC(53%±3.3%), EHAC(31%±0.7%),ETAC(11%±0.2%)andEAAC(5%±0.3%).The lastmethylester,ethylhexadecanoate,waspresentatsimilar con-centrationstheinEDAC(42%±7.8%)andinEHAC(30%±1.4%),but waspresentatlowconcentrationsintheETAC(16%±0.3%)andin EAAC(10%±1.5%).
The compound of the phytosterol class, ␥-sitosterol, was detectedinthreeextractsatdifferentconcentrationsintheEHAC (69%±5%), EDAC (12%±3%)and ETAC(18%±5%). Furthermore, theethylhexadecanoatefromtheethylatedcompoundsclasswas detectedintwoextracts,withdifferentconcentrationsintheEDAC (98%±4%)andEHAC(2%±0.02%).Severalstudiesshowedvarious biologicalactionsofsomeofthesecompoundsintheA. cearen-sisextracts,butmanywereisolatedfromthestembarkorleaves. Our resultsarepromising and identified thecompoundsin the extractsoftheA.cearensisseeds.Therearefewstudiesthatusing theseedsofA.cearensistoidentifycompounds,suchasNegrietal. (2004),CanutoandSilveira(2006),andBezerraetal.(2006),who isolated6-hydroxycoumarin,o-coumaricacid,aderivativeofthe esterifiedamburoside,acid-p-hydroxy-benzoicacid,glycosylated (E)-o-coumaric acidand glycosylated (Z)-o-coumaric. The com-poundcoumarinwasidentifiedbyNegrietal.(2004)inthestem barkofA.cearensis.Additionally,severalmethylatedcompounds wereidentifiedinthestembarkofA.cearensis;however,theywere notpreviouslyidentifiedintheseeds.
CytotoxicityassayoftheextractsfromtheseedsofAmburana cearensis
ToevaluatethecytotoxicityofETAC,EAAC,EDACand EHAC, we performed in vitro assays using an MTT test onPC12 cells exposedtotheseextractsatdifferentconcentrationsandperiods of time. All fourextracts did not decrease the cell viability of PC12cell culturesexposed toconcentrations ofextract ranging from0.1to2000g/mlafter24hofexposure(Fig.1).TheETAC extractat2000g/ml increasedthemitochondrialactivity, sug-gesting an increase of celldensity of theculture visualizedby optical microscopy(datanot shown).After72hof exposureto 1000g/ml of ETAC and EHAC, a decrease of cell viability of 10%and 70%,respectively,wasobservedforthePC12cells cul-tures. EAAC and EDAC were not toxic under some conditions (0.1–1000g/ml,after72h)(Fig.2).Lealetal.(2003)evaluatedthe hydro-alcoholicextractobtainedfromthestembarkofA.cearensis anddemonstratedthatitwasnottoxictorats.Moreover,several compoundsfromA.cearensis,suchaskaempferol,isokaempferide, amburosideA andprotocatechuic acidwereevaluated toverify theircytotoxicityontumorcelllines,andtheresultsshowedan inhibitionofviabilitybykaempferolandisokaempferide( Costa-Lantufo et al., 2003).We demonstrated that extracts obtained from the seeds of A. cearensis did not toxic to the PC12 cell line.
NeuroprotectiveeffectoftheAmburanacearensisseedextracts againstexcitotoxicityinducedbyglutamate
Fig.1. CytotoxicityassaysoftheextractsfromAmburanacearensisseedsinPC12cellculture.TheETAC,EAAC,EDACandEHACextracts(0.001,2000g/ml)wereaddedto theculturemedium.ThecellviabilitywasdeterminedusingMTTassay24hafterthetreatments.Theresultsareexpressedasapercentageofthecontrol,considered100% (***p<0.05,ANOVAfollowedbyTukeytest).
Fig.2. CytotoxicityassaysofextractsfromAmburanacearensisseedsinPC12cellcultures.TheETAC,EAAC,EDACandEHACextracts(0.1–1000g/ml)wereaddedtothe culturemedium.ThecellviabilitywasdeterminedusingtheMTTassay72hafterthetreatments.Theresultsareexpressedasapercentageofthecontrol,considered100% (***p<0.05,ANOVAfollowedbyTukeytest).
treatedwithETACandEHACextracts,reduceddeathinducedby glutamate (1mM) neuronal PC12 cells, occurred at concentra-tionsof10mg/ml,andthisreductionby27%and29%deathcell respectively.InthegroupofcellstreatedwithEDACextractonly observed front protective effect on the damage induced by
glutamate (1mM), at the highest concentration tested in 1000g/ml,areductionofupto17%celldeath(Fig.3).
Fig.3. NeuroprotectiveeffectofextractsfromseedsofAmburanacearensisinPC12cellsculturesagainstglutamate-inducedtoxicity.Thecellswerepre-treatedwithglutamate (1mM),andafter4htheextractsETAC,EAAC,EDACorEHAC(0.1–1000g/ml)wereaddedtotheculturemedium.CellviabilitywasdeterminedusingtheMTTassay24h afterthetreatments.Theresultsarestatisticallysignificantcomparedtothecontrol(*)andglutamate(#)(p<0.05,ANOVA).
biological activityof A. cearensis and wasobserved in previous studiesevaluatingthemethanolextractfromseedsofA.cearensis (Pereiraetal.,2014).Theresultsofthepresentstudysuggesta ther-apeuticpotentialformoleculesintheEAAC,whichhasfewer com-poundscomparedtotheotherthreeextractsbecauseitcontains onlycoumarinastheprimarycomponent,hexadecanoatemethyl, trans-13-methyl-octadecanoateandmethyloctadecanoate.
Fig.4shows themorphologicalanalysisresultsfor neuronal PC12 cells when analyzed by phase contrast microscopy. PC12 neuronalcells incontrolconditions haveabipolarmorphology, refractile,showingcellular viability.In groups treatedwiththe extract(ETAC,EAAC,EDACandEHAC)cellmorphologywas sim-ilartothecontrolgroupcells.Incellculturestreatedwith1mM glutamatecanobservecellswithcellbodyandretractedrefractile bitindicatinglossofviability,and reducedcellularity.However, culturesofcellsexposedtoglutamateandtreatedwithA. cearen-sisextractscanbeverifiedcellswithsimilarmorphologytothose incontrolconditionsandwithfewcellsandatypicalmorphology refractilebit,whichindicatesdamageprotection.
AmongthecompoundsisolatedfromdifferentpartsofA. cearen-siswithproven biologicalactivitywhich maybe relatedtothe neuroprotective effect observed in the present study, we can highlightaction phenolic compoundssuchas coumarin.Invivo studiesconductedbyLealetal.(2006)associatedwithantioxidant activityantinociceptive effectof phenolic compoundsextracted from the bark and stem A. cearensis. Lopes, 2010 associated antioxidantactivitywithanti-inflammatoryeffectofafrormosina, phenoliccompoundextractedfromthebarkandstemA. cearen-sis. Oliveira et al. (2014) observed antioxidant activity in vitro 8-methoxypsoralen, synthetic coumarin in astrocytic lineages. In addition, Pereira et al. (2014) observed in free antioxidant systemcells ofthetotal methanolextract of A.cearensis seeds.
The combined findings, it concluded that extracts of seeds of A. cearensis containcompounds withneuroprotective potential, particularly theextract in ethyl acetate. Additional studies are needed tocharacterize theeffects of thecompounds coumarin methyl ester alone or in combination in cultured neuronal cells.
Ourresultsdemonstratethat24hafterexposuretoEAAC,all concentrations tested reduced from 12% to 30% the PC12 cell deathinducedby1mM glutamate.Moreover,fortheETACand EAACextracts,concentrations between10 and 1000g/ml also reducedtheglutamateinduceddeathofPC12cells,reducingby 17%andby17%thecelldeath,respectively.EDAConlyinduced neuroprotectionatthehighestconcentrationtested(1000g/ml), reducing by 17% the cell death induced by glutamate (Fig. 3). Phase contrast microscopy (Fig. 4) shows that treatment with 1mMglutamateinducedretractionofthecellbodyinthemajority of PC12cells. However, PC12cells post-treated withA. cearen-sis extracts at 100g/ml showed viable adherent cells with different phenotypes and with similar morphology to control cells,Indicatingprotectionagainstglutamateexcitotoxicity.Cell protectionwas alsoevidenced in cultures treatment100g/ml EAAC.
Fig.4. MicrographyanalysisofPC-12cellsundercontrolconditions(A)orafter24h exposureto1mMglutamate(B),0.1mg/mlEAACextract(C)orboth1mMglutamate and0.1mg/mlEAAC.Objective×40.Scale=100m.
Conclusion
The present investigation shows that the extracts obtained toA. cearensis seeds contained ethyl esters, methyl esters and coumarins.Moreoverethanolicextract(ETAC)andfourextracts derivedfromitdonotpresenttoxiceffecttoPC12neuronalcells. Furthermore,apotentialneuroprotectioneffectagainstglutamate toxicitywasdeterminedinthiswork.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Authorcontributions
EPP,CCS,LOS,VDAS,SBS,andSLC,participatedinstudyconcept anddesign,acquisitionofdata,analysisandinterpretationofdata, andcriticalrevisionofthemanuscriptforimportantintellectual content.LGFandMDCparticipatedinstudyconcept.EPP,SBS,VDAS andSLC,draftedthemanuscript.EPP,MDCandPRcarriedoutthe extraction,fractionationandGC-MSanalysis,andparticipatedin draftingthemanuscript.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Wegratefullyacknowledgetheresearchsupportprovidedby CNPq,FAPESB,CAPES,andbyProgramadePós-graduac¸ãoem Ciên-ciaAnimalnosTrópicos,UniversidadeFederaldaBahia.
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