Rev. bras. farmacogn. vol.25 número3

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

Development

of

standardized

extractive

solution

from

Lippia

sidoides

by

factorial

design

and

their

redox

active

profile

Bruno

S.

Lima

_,

_Cledison

_S.

_Ramos

_,

_João

_P.A.

_Santos

_,

_Thallita

_K.

_Rabelo

_,

_Mairim

_R.

_Serafini

_,

Carlos

A.S.

Souza

_,

_Luiz

_A.L.

_Soares

_,

_Lucindo

_J.

_Quintans

_Júnior

_,

_José

_C.F.

_Moreira

_,

_Daniel

_P.

_Gelain

_,

Adriano

A.S.

Araújo

_,

_Francilene

_A.

_Silva

a_{DepartamentodeFarmácia,UniversidadeFederaldeSergipe,SãoCristóvão,SE,Brazil} b_{DepartamentodeBioquímica,UniversidadeFederaldeRioGrandedoSul,PortoAlegre,RS,Brazil} c_{DepartamentodeFarmácia,UniversidadeFederaldePernambuco,Recife,PE,Brazil}

d_{DepartamentodeFisiologia,UniversidadeFederaldeSergipe,SãoCristóvão,SE,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16December2014 Accepted17December2014 Availableonline24April2015

Keywords: Lippiasidoides Factorialdesign Antioxidant Flavonoids

a

b

s

t

r

a

c

t

Theaimofthisstudywastoevaluatetheinfluencesofvariablesofpreparationontotalflavonoidscontent fromextractivesolutionofLippiasidoidesCham.,Verbenaceae.Thusa23_{factorialdesignwasusedtostudy}

theimportanceofplantproportion,theextractionmethodandsolventontheextractionofflavonoid.The methodologyofdeterminationofchemicalsinfactorialdesignwasvalidatedaccordingtotheparameters requiredbyBrazilianHealthAgency.Theextractionsolutionwasselectedthroughafullfactorialdesign wherethebestconditionstoachievethehighestcontentofflavonoidswere:7.5%(w/v)ofplantwith ethanol50%(v/v)assolvent.ThepolyphenolscontentwasdeterminedbyLCmethodanditsrelationship withtheantioxidantandfreeradicalscavengingactivitieswasevaluated.Thefreeradicalscavenging activitiesandantioxidantpotentialsweredeterminedfordifferentconcentrationsusingvariousinvitro

models.Ourresultsindicatethatextractsexhibitedasignificantdose-dependentantioxidanteffectas evaluatedbyTRAP/TARassays.Besides,weobservedanantioxidantactivityagainsthydroxylradicalsand nitricoxide,andprotectionagainstlipidperoxidationinvitro.Ourresultssuggestthattheextractpresents significantinvitroantioxidantpotentialindicatingpromisingperspectivesforitsuseaspharmaceutical/or foodadditive.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Lippia sidoides Cham., Verbenaceae, popularly known as

“alecrim-pimenta”, isa typicalshrubcommonlygrowinginthe

NortheastofBrazil.Thisspeciesproducesanessentialoilrichin thy-molandcarvacrol,whichhasapotentantimicrobialactivityagainst fungiandbacteria(Lemosetal.,1990;Lacosteetal.,1996).The hydro-alcoholicextractsofthisplantarelargelyusedfortreatment ofskinwounds,asamouthantisepticandinliquidsoap prepa-rationstotreatandpreventgeneralfungalinfectionsofthebody (Matos,2000).

Inthisway,thosegroupsofsubstancescanbesuccessfullyused aschemicalsmarkerswhichcanassistinthestudiesof standard-izationofextractivesolutionsfrommedicinalplants.

∗ Correspondingauthor.

E-mail:francilene.silva@pq.cnpq.br(F.A.Silva).

However,validatedqualitycontrolmethodsneedtobe

devel-oped inorder tocomplywithregulatoryrequirements, ifsome

plantistobeusedasrawmaterialbythepharmaceutical indus-try;theabsenceofthosestudieshampersthereproducibilityofthe extractivesqualities,whichcouldaffecttheefficiencyandsafety. Thus,thestandardizationofplantextractivesolutionsshouldbe thefirststepduringthetechnologicaldevelopmentof phytophar-maceuticals.Theinfluenceofseveralparameterssuchasextractive methodandtechnology,typeandconcentrationofsolvent,aswell asplantconcentration,andtheirinfluenceinthephysical-chemical properties of theextractive solutions should be evaluated and quantified(Audietal.,2001;Cunhaetal.,2009).

Thefactorialdesignisastatistictoolusedtoscreeningand/or

optimization process,with rapid and economic way,and

max-imization the quality of final products. Besides, the statistical analysisawardresultsarereliable(MyersandMontgomery,1995; Montgomery,1997).Thus,itispossibletorankeachindependent variable according toits significance on thestudied responses. Therefore,withreducedtimeandexperimentaleffort,itmaybe

http://dx.doi.org/10.1016/j.bjp.2014.12.004

possibletochoicetheextractiveconditionsthatareabletoproduce

themaximumexperimentalresponse (MyersandMontgomery,

1995; Montgomery,1997; Canteri-Scheminet al., 2005; Soares etal.,2005).

Theaimofthisworkwastousethefactorialdesigntoevaluate theeffectoftheplantproportion,solventtype,concentrationand theextractionmethodonthetotalflavonoidscontentinextractives solutionsfromL.sidoides.Additionally,theaimwasalsotoevaluate theinvitroantioxidantactivityoftheoptimizedextractivesolution.

Materialsandmethods

Plantmaterial

Aerialpartsof LippiasidoidesCham., Verbenaceae,were col-lectedinthegardenofmedicinalplantsoftheFederalUniversity ofSergipe(SãoCristóvão,Brazil)inMay2009andwasidentified byProf.AnaPaulaN.Pratawhoisa planttaxonomistfromthe

DepartmentofBiology.Avoucherspecimen(ASE2626)hasbeen

depositedintheHerbariumofDepartmentofBiology.Theplant materialafterharvestingwassubjectedtodryingprocessina circu-latingairatatemperatureof40±2◦_{Cuntilstabilizationofresidual} moisture.Afterdryingtheplantmaterialwasmanuallyselected, subjectedtogrindinginagrinderofknives,weighed,sampledand wascharacterizedthroughanalysisof moisturecontent, granu-lometricanalysisusingsieveof150,250,355,500,600,710and 1000␮m,lossofdryingbygravimetricmethodanddetermination

ofextractivecontent.

Evaluationofanalyticmethodologybyspectrophotometry

Throughthetechniqueofdeterminationwithoutacid hydroly-sis(Petryetal.,1998;Silvaetal.,2009;Marquesetal.,2012),the sampleofrawmaterialwassubjectedtoextractionunderreflux (7.5%,w/v)andwasheldinadilutionof3:50mlofstocksolutionin waterdistilled;then,inflasksof20mlaliquotswereaddedto3.2ml ofthedilutedsolutionand1.6mlofAlCl3(2.5%and7.5%)inorderto assesswhatwouldbetheidealconcentrationofAlCl3necessaryfor chelatingflavonoidslikequercetinfoundintheextractivesolution ofL.sidoides.Then,theballoonswerefilledwithethanol(40%)and

30minafterthereadingswereperformedina

spectrophotome-terat423nm(maximumofAlCl3-quercetincomplexed).Giventhe optimalconcentrationofAlCl3 tocomplexationwasperformeda readingofscanningtheextractivesolutionbeforeandafter

com-plexationwhich comprised a range of200–800nm inwhich it

wasintended to strengthen the displacement of bathochromic

flavonoidsolution.

Validationmethodology

Amethodologytodetermineandquantifytheflavonoidsinraw materialmustbevalidatedconsideringthefollowingparameters: specificity,linearity,precision,accuracy androbustness(Anvisa, 2003).

Thestatisticanalysisandexperimentaldesign

Theexperimentalmatrixwasa23_{factorialdesignanditwas} usedtoevaluatetheinfluenceofplantproportions(5.0/7.0%;w/v), solventtype(water/ethanol50%)andtheextractionmethod (infu-sion/decoction)ontheefficiencyoftheextractionofflavonoids.The experimentswereperformedintriplicate,andthetotalflavonoids content(TFC)wasusedasresponses.Thestatisticalanalysisofthe factorialdesignwasperformedusingthesoftwareStatistica®

6.0

(StatSoft,USA).TheexperimentaldatawereanalyzedbyANOVA

andt-testforstandardizedeffects(MyersandMontgomery,1995).

HPLCanalysisofextractivesolution

PreparationofL.sidoidesextracts

Dryextract(100ml)obtainedbydecoction(7.5%,w/vin15min) wasdilutedwithmethanol/milli-Qwater(50:50,v/v)toachievea concentrationof10␮g/ml.ForinjectionintheHPLC,thesolutions

werefilteredina0.44␮m(regeneratedcellulose)membrane.

AnalyticalandpreparativeHPLC

TheHPLCanalysiswasperformedonaShimadzusystem

con-sisting of a degasser DGU-20A3, a SIL-20A autosampler, two

LC-20AD pumps and a SPDM20Avp photodiode array detector

(DAD),coupledwithaCBM20Ainterface.Beforeinjectionintothe HPLC,thesolutionswerefilteredina0.44␮m(regenerated

cellu-lose)membrane.Theanalyticalmethodemployedalineargradient systemwhich consisted of (A) acetic acid:water 1.0%(v/v); (B)

methanol.Thechromatographic separation hasbeenperformed

usingaPhenomenexLunaC18analyticalcolumn250mm×4.6mm

(5mmparticlesize).Theflowratewas0.6ml/minandtheinjection volumewas20␮l.Themobilephaseconsistedofgradientofwater

andmethanolstartingwith10%Bduring20min;40–45%Bduring 5min;45–60%Bduring10min;60–75%Bduring25mintotaltime of60min.Photodiodearraydetectorwassetat254nmfor acquir-ingchromatograms.Theanalysisofpeakswasbasedinaccordance withtheretentiontimeofthestandardsubstanceandmass

spec-trum(MS/MS).Massspectrometricanalysiswasperformedona

Bruckermassspectrometerfittedwithiontrapionizationsource. Thenegativeionmode[m/zM−H]wasusedforallcompounds.Ion trapionizationwasoperatedinMRMandconditionswere: nebu-lizerpressure:40psi;drygasflow:9l/min;dryinggastemperature: 300◦_{C;flowrate:200␮l/min.}

Redoxactiveprofile

Totalantioxidantpotential(TRAP)andtotalantioxidantreactivity (TAR)

TRAP/TAR was determined by measuring the

chemi-luminescence (CL) intensity of luminol induced by 2,2′_-azobis (2-amidinopropan) dihydrochloride (AAPH) (Lissi et al., 1995).

ThebackgroundCLwasmeasuredbyaddingAAPHandluminol.

Then,thesamples(OELSfrom1ngml−1_to1mgml−1_)wereadded,

and the CL was measured in a liquid scintillator counter. The

lastcountbeforetheadditionsampleswasconsideredas100%. Graphswereobtainedbyplottingpercentageofcountspermin(% cpm)versustime(s).TheAUC(TRAPassay)wascalculatedusing GraphPadPrismsoftware.TheTARwascalculatedastheratioof lightintensityinabsenceofsamples(Io)/lightintensityrightafter OELSaddition(I).

TBARS(ThiobarbituricAcidReactiveSpecies)

TBARSassaywasemployedtoquantifylipidperoxidationand

anadaptedTBARSmethodwasusedtomeasuretheantioxidant

at1200gfor10min.Analiquotof0.5mlfromthesupernatantwas mixedwith0.5mlTBAandheatedat95◦_{Cfor30min.After} cool-ing,samplesabsorbancewasmeasuredusingaspectrophotometer at532nm.TheresultswereexpressedasapercentageofTBARS formedbyAAPHalone(inducedcontrol).

Hydroxylradical(•_OH−_{)scavengingassay}

HydroxylradicalsweregeneratedbyaFentonsystem(FeSO2 -H2O2).Whenexposedtohydroxylradicals,thesugardeoxyribose isdegradedtomalonaldehyde(MDA),whichgeneratesapink chro-mogenonheatingwithTBAatlowpH.Themethodfordetermining thescavengingonhydroxylradicalswasperformedaccordingtoa previouslydescribedprocedure.

Nitricoxide(NO)scavengingassay

Nitricoxidewasgenerated fromspontaneousdecomposition

ofsodiumnitroprusside(SNP)inthephosphatebuffer(pH7.4). OncegeneratedNOinteractswithoxygentoproducenitriteions, whichweremeasuredbytheGriessreaction.Thereactionmixture containingSNPinphosphatebufferandOELSatdifferent concen-trationswereincubatedat37◦_{Cfor1h.Analiquotwastakenand} homogenizedwithGriessreagent.Theabsorbanceofchromophore wasmeasuredat540nm.Percentinhibitionofnitricoxide gener-atedwasmeasuredbycomparingtheabsorbancevaluesofnegative controls(SNPandvehicle)andassaypreparations.

Resultsanddiscussion

Characterizationoftheplantmaterial

TheplantmaterialofL.sidoidesusedinthisresearchpresented particlemeandiameterof325␮m,lossondryingof11.25%and

extractivecontentof19.83%.Theseproprietiesareimportantfor standardizationofextractiveprocess,sinceparticlesmean diam-eterinfluencestheextractionefficiencyandthelossondryingis importanttoconservationofrawmaterial(ListandSchimdt,1989).

Developmentofanalyticmethodologybyspectrophotometry

Thealuminumcationformsstablecomplexeswithflavonoids, whichleadstoadeviationofthepeakabsorptiontolonger wave-length.Thusitispossibletodeterminetheamountofflavonoids, avoidingtheinterferenceofotherphenolicsubstances,mainly phe-nolicacids,whichinvariably accompanytheflavonoidsinplant tissues.Thisreadingisdoneinspectrophotometerat425nm,using aluminumchlorideto2%.Therewasnostatisticallysignificant dif-ferenceinamplitudeofthepeakabsorptionoratthetimethatthis pointhasbeenreachedregardingtheconcentrationofAlCl3was used.

Validationofthemethodology

AccordingtoBrazilianHealthAgency(Anvisa,2003),the pur-poseofvalidationistodemonstratethatthemethodissuitable for their intended purpose, and to identify qualitative, semi-quantitativeand/orquantitiesof drugsand othersubstances in

pharmaceuticals. Thus, the method is validated when has the

specificity,linearity,precision,accuracyandrobustnesswithinthe parametersrequired.

Thespecificityofmethodwasdemonstratedacrossthe spec-trumscanningoftheextractivesolutionfromL.sidoides(7.5%,w/v) andthestandardquercetin(50.0␮g/ml),bothcomplexedwith

alu-minumchloride(2.5%),onlyonepeakbeingshownofmaximum

4.500

4.000

2.000

–2.000

–4.000

–4.500

200.00 400.00 600.00

L.sidoides

Quercetin

nm.

Abs.

800.00 000

Fig.1. Spectrumscanningof theextractivesolution fromL. sidoidesandthe quercetinstandard.

absorptionat423nmfor quercetinandotherat405nmfor the extractivesolution(Fig.1).

Thelinearitywasobtainedthroughofthecalibrationcurveof quercetindilutedinethanol(40%).Thiscurvewasmadewithfive differentconcentrations:10,20,30,40and50␮g/ml.The

param-eterlinearityisveryimportantbecauseitdemonstratesthatthe resultsobtainedaredirectlyproportionaltotheconcentrationof analyteinthesample,withinaspecifiedrange.

Thetestwasmadeintriplicateandtheresultsofthestatistical analysisareshowedinTable1.Theequationofthelineobtained wasy=0.01467x+0.3062,theaccuracyofthedeterminationswas between102.10and101.11%(Table1)andthedetermination coef-ficientobtainedwasgreaterthan0.99(r2_{=0.9992)whichiswithin} theparametersofexistinglegislation(Anvisa,2003).

Theprecisionwasobtainedattwolevels:repeatability,which

evaluated the concordance betweenthe results within a short

periodoftimewiththesameanalystandsameinstrumentation. Thislevelwascarriedintriplicatewithsixdeterminationsof100% oftheconcentrationofthetest;andthesecondlevelwasthe

inter-mediate precision,which analyzes the correlationbetween the

resultsfromthesamelaboratory,obtainedindifferentdayswith differentanalystsperformedinduplicatewithsixdeterminations. Theresultsofthestatisticalanalysisoftheprecisiontestareshown inTable2.

Theaccuracyobtainedasindicatedbytheresolutionforthis testwasthusappliedintheanalyticalmethodologyproposedin theanalysisofasubstanceofknownpurity;inlow(22.5␮g/ml),

medium(45␮g/ml)andhigh(65␮g/ml)concentrationaccording

totheminimumandmaximumvaluesonthecurvealltheresults areshowninTable3.

The robustness was evaluated by variation of temperature

between25and35◦_{Candthesolutionconcentrationofaluminum}

Table1

Resultsofthestatisticalanalysisofthelinearitytest.

TC C SD RSD A

10 10.2152 1.723 1.68 102.10 20 20.4642 2.351 1.15 102.32 30 30.6136 2.093 0.68 101.65 40 40.6821 1.254 0.28 101.90 50 50.5461 2.372 0.46 101.11 TC(␮g/ml),theoreticalconcentration;C(␮g/ml),meanconcentrationofthree

Table2

Resultsofthestatisticalanalysisoftheprecisiontest.

Test TC C SD RSD A

Repeatability 50.0 50.47 2.45 1.09 99.59 50.0 50.91 1.75 0.38 100.32 50.0 49.97 2.12 0.32 100.21 Intermediate

precision

Analyst1 44.0 44.05 2.11 0.47 100.76 Analyst2 44.0 44.16 2.09 0.47 100.32 TC(␮g/ml),theoreticalconcentration;C(␮g/ml),meanconcentrationofthree

deter-minations;SD,standarddeviation;RSD(%),relativestandarddeviation;A(%), accuracy.

Table3

Resultofthestatisticalanalysisoftheaccuracytest.

Test CL TC C SD RSD A

Accuracy Low 22.5 22.475 2.45 1.09 99.89 Medium 45.0 44.912 1.75 0.38 99.80 High 65.0 64.978 2.12 0.32 99.95 CL,concentrationlevel;TC(␮g/ml),theoreticalconcentration;C(␮g/ml),mean

concentrationofthreedeterminations;SD,standarddeviation;RSD(%),relative standarddeviation;A(%),accuracy.

chlorideinmethodbetween2.5and7.5%.Theanalysesweredonein triplicate.Themethodwasrobustforthevariationoftemperature andsolutionconcentrationofaluminumchloride.

Experimentaldesign

Theresultsforthetotalflavonoidscontentofeachextractive solutionarepresentedinTable4.Thestandardizedeffectsofeach main factor as wellas respectiveinteractions are presented in Table5.Regarding thestatisticalanalysisof experimentaldata, themostimportanteffectcouldbeattributedtothefactorSolvent, whichprovidesmoreefficientextractiveprocedurewhenethanol 50%(v/v)wasusedassolvent.Accordingtot-test,thesecondmain factorwasthedrugproportion.Nostatisticallysignificanteffect couldbeimputedtoextractionmethods(infusion/decoction).On theotherhand,theinteractionsofextractionmethodwereableto provideimprovementsontheextractiveefficiencyeitherwith Sol-ventorPlantproportion.Theexpectedimprovementsintheyield ofTFCduetoincreasingdrugamountaswellasthepositiveeffect ofethanol:watermixtureundergoextraenhancedeffectderived frommethodofextraction.Althoughnosignificanteffectswere observedfortheMethodofExtraction,theinfluenceof maintain-ingthetemperaturebydecoctionseemstobeessentialtopromote betterwettabilityoftherawmaterialandhigherdiffusivityofthe solvent.

SeparationpolyphenolsbyHPLC

Photodiodearraydetectorwassetat254nmforacquiring

chro-matograms.ThechromatogramL.sidoidesextract showedthree

Table5

Statisticalanalysisofexperimentaldata:standardizedeffects(mainfactorsand interactions).

Standardizedeffect t-Test ANOVA(F) Mean/interc. 14.64 53.11* _–

(1)Method 1.014 1.84 3.38

(2)Plant(%) 2.69 4.88* _23.85*

(3)Solvent 4.37 7.92* _62.67*

1by2 1.33 2.42* _5.83*

1by3 1.41 2.57* _6.58*

2by3 1.00 1.82 6.30

*_˛=0.05.

majorpeakswellseparated:P1(R.T.21min),P2(R.T.34min)and P3(R.T.41min).P1wasidentifiedaschlorogenicacid(Fig.2).P2 andP3 werenot identifiedbut exhibitedUV spectrapattern of caffeoylquinicacidderivatives,indicatingthatthisclassof polyphe-nolsare themajorconstituents inthe extract.P1 had itsmass

spectrum analysis and Showed MS 353 m/z and MS2 _{191 m/z}

(chlorogenicacidmolecule).

Redoxactiveprofile

TheTRAPandTARmethodsarewidelyemployedtoestimatethe generalantioxidantcapacityofsamplesinvitro.Thus,thegeneral antioxidantpotentialofextractfromL.sidoideswasfirstevaluated bytheTRAP/TARassays.TheTRAP/TARassaysindicatethat opti-mizedextractivesolutionofL.sidoides(OELS)presentsasignificant antioxidantactivityattheallconcentrationsstudied(Fig.3).

Lipidperoxidation(LPO)hasbeendefinedasthebiological dam-agecausedbyfreeradicalsthatareformedunderoxidativestress (Zinetal.,2002).Severalplantextractshavebeenshowntoinhibit LPOasmeasuredbythelevelsofTBARS.Thelipidsinmembrane arecontinuouslysubjectedtooxidantchallenges.Oxidantinduced abstractionofahydrogenatomfromanunsaturatedfattyacylchain ofmembranelipidsinitiatestheprocessofLPO,whichpropagates asachainreaction.Intheprocess,cyclicperoxides,lipid peroxi-desandcyclicendperoxidesaregenerated,whichultimatelyare fragmentedintoaldehydeslikeMDA.Similarly,theOELSwereable topreventlipoperoxidationinducedbyAAPHinvitroina lipid-enriched system(Fig.4).TheOELSat 100ngml−1 _to1mgml−1 showedantioxidantactivityagainsthydroxylradicals(Fig.5).To determinetheabilityofextracttoactasareactivenitrogenspecies (RNS)scavenger,weevaluatedtheNO-scavengingactivitybythe Griessmethodand OELSat100ngml−1 _to1mgml−1 _showeda significant(p<0.05)NO-scavengingactivity(Fig.6).

The resultsfoundin this studyare in agreementwith both observationsandsuggestadirectcorrelationbetweenantioxidant activityandpolyphenolcontent.Itisprobablethattheactive prin-cipleresponsiblebytheredoxactivityinthisworkisthepolyphenol content.Severalstudieshaveshownthattheredoxactivity asso-ciatedwithnaturalantioxidantsisattributedtothetotalcontent

Table4

Matrixofthefactorialdesign23_.

Exp Codedvariable Naturalvariable TFT(␮g/ml)

Method Plant Solvent Method Plant(w/v) Solvent

1 +1 +1 −1 Decoction 7.5 Water 13.043

2 +1 +1 +1 Decoction 7.5 EtOH50% 21.280

3 +1 −1 +1 Decoction 5.0 EtOH50% 14.798

4 +1 −1 −1 Decoction 5.0 Water 11.476

5 −1 +1 −1 Infusion 7.5 Water 13.568

6 −1 +1 +1 Infusion 7.5 EtOH50% 16.064

7 −1 −1 +1 Infusion 5.0 EtOH50% 15.157

8 −1 −1 −1 Infusion 5.0 Water 11.752

65.0

254nm, 4nm (1.00)

A

62.0 60.0 57.5 55.0 52.5 50.0 47.5 45.0 42.5 40.0 37.5 35.0 32.5 30.0 27.5 25.0 22.5 20.5 17.5 15.0 12.5 10.0 7.5 5.0 2.5 0.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 P1

P3 P2

35.0 40.0 45.0 50.0 55.0 min mAU

950 900 850 800 750

650

550

450

350

250

150 100 50 0 200 300 400 500 600 700

254nm, 4nm (1.00)

B

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min mAU

Fig.2.(A)ChromatogramofextractivesolutionofL.sidoides.(B)Chromatogramofthereferencesubstance(chlorogenicacid).

30 0000

A

20 0000

10 0000

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

TRAP

(AUC arbitary units)

0

System Trolox

100 ng/ml 1 1 mg/ml µg/ml

10 µ g/ml

100 µg/ml

100

80

60

40

20

B

∗∗∗

∗∗∗

∗∗∗

∗∗∗

T

AR (Io/I)

0

System Trolox

100 ng/ml 1 1 mg/ml µg/ml

10 µ g/ml

100 µg/ml

Fig.3.(A)TotalRadical-TrappingAntioxidantParameter(TRAP)atdifferent con-centrations.Valuesrepresentmean±S.E.D.,experimentsintriplicate,***p<0.001 differentfromsystemandtrolox(ANOVAfollowedbyTukey).(B)Thetotal antiox-idantreactivity(TAR)wascalculatedastheratiooflightintensityinabsenceof samplesexpressedaspercentofinhibition(I0/I).Valuesrepresentmean±S.E.D.,

experimentsintriplicate,***p<0.001differentfromsystemandtrolox(ANOVA followedbyTukey).

100

50

0 150

System Trolox

100 ng/ml 1 1 mg/ml µg/ml

10 µ g/ml

100 µg/ml

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗

TBARS

(% AAPH-induced

damage)

Fig.4. ThiobarbituricAcidReactiveSpecies(TBARS)wasevaluatedfromL.sidoides

(100ngml−1_to1mgml−1_{).Valuesrepresentmean}

±S.E.D.,experimentsin tripli-cate,ANOVAfollowedbyTukey,*p<0.05;***p<0.001differentfromsystem.

100

50

0 150

System Trolox

100 ng/ml 1 1 mg/ml µg/ml

10 µg/ml

100 µg/ml

∗∗∗

∗∗

_∗∗

_∗∗∗

∗∗∗

∗∗

% of hydroxyl

Fig. 5.Hydroxyl radical-scavenging activity from L. sidoides (100ngml−1 _to

1mgml−1_{).Valuesrepresentmean}

100

50

0 150

System Trolox

100 ng/ml1 1 mg/ml µg/ml

10 µg/ml

100 µg/ml

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

% of nitrite formation

Fig.6. Nitricoxide (NO) scavenging activity from L.sidoides (100ngml−1 _to

1mgml−1_{).Valuesrepresentmean±S.E.D.,experimentsintriplicate,ANOVA}

fol-lowedbyTukey*p<0.05;***p<0.001differentfromsystem(SNP).

ofphenoliccompounds(Halliwell,2008;Rice-Evansetal.,1997; Scalbertetal.,2005;Serafinietal.,2011).

Conclusions

Theanalysisof datafromthis studyconfirmedtheability of

thedeveloped methodforevaluatingthepolyphenolcontentin

extractsofL.sidoidesandtheanalysisoffactorialdesignshowed thattheoptimumconditiontopreparetheextractivesolutionswith maximumtotalflavonoidcontent,foundextractionbydecoction, using7.5%(w/v)ofplantandethanol50%(v/v)astheextractive solvent.Concluding,thedatepresentedhereinindicatesthattheL. sidoidesextracthaveinvitroantioxidantactivityandshouldbe con-sideredasnewsourcesofnaturalantioxidantsjointlywithother phenolicrichplants.Furtherstudiesareneededtoexaminethe potentialuseoftheseextractsinthepreventionortreatmentof

pathologies where oxidativestress seemstoplay an important

role.

Authors’contributions

BSLandCSRcontributedwithchromatographicanalysis, fac-torialdesignandwritingofthemanuscript.JPAS,TKRandCASS contributedwiththeredoxactiveprofile.MRS,LALS,LJQJ,JCFM, DPG,AASAandFASdesignedthestudy,supervisedthelaboratory work,contributedtocriticalreadingofthemanuscript,writingof themanuscriptandfinaleditingofthemanuscript.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors are grateful to CAPES, CNPq, FACEPE/PE and

FAPITEC/SEforfinancialsupportandfellowships.

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