Improving bioactive compounds extractability of Amorphophallus paeoniifolius (Dennst.) Nicolson

ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j ourna l h o m e pa g e : w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Improving

bioactive

compounds

extractability

of

Amorphophallus

paeoniifolius

(Dennst.)

Nicolson

Anabela

S.G.

Costa

_,

_João

_C.M.

_Barreira

_,

_Adilson

_Ruas

_,

_Ana

_F.

_Vinha

_,

Filipa

B.

Pimentel

_,

_Rita

_C.

_Alves

_,

_Isabel

_C.F.R.

_Ferreira

_,

_M.

_Beatriz

_P.P.

_Oliveira

a_{REQUIMTE,LAQV/DepartamentodeCiênciasQuímicas,FaculdadedeFarmácia,UniversidadedoPorto,RuaJorgeViterboFerreira,228,4050-313Porto,}

Portugal

b_{MountainResearchCenter(CIMO),ESA,PolytechnicInstituteofBraganc¸a,Apartado1172,5301-855Braganc¸a,Portugal} c_{FCS-UFP/FaculdadedeCiênciasdaSaúde,UniversidadeFernandoPessoa,RuaCarlosdaMaia,296,4200-150Porto,Portugal}

d_{REQUIMTE,LAQV/InstitutoSuperiordeEngenhariadoPorto,InstitutoPolitécnicodoPortoRuaDr.AntónioBernardinodeAlmeida,431,4200-072Porto,}

Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received24May2015

Receivedinrevisedform4November2015 Accepted6November2015

Availableonline21November2015 Keywords:

Elephantfootyamextracts Bioactivecompounds Phenolics

Antioxidantactivity

a

b

s

t

r

a

c

t

Elephantfootyam(Amorphophalluspaeoniifolius(Dennst.)Nicolson)isanunderground,unbranched deciduousplantthatproducesalargetubercle(rhizome)withrecognizedhealtheffects.Inthisstudy,the influenceofsolventnature(water,water/etanol(1:1)andabsoluteethanol)andprocessingtype(fresh, lyophilizedandboiled)ontheantioxidantactivityandbioactivecompoundsextractabilityofelephant footyamwasevaluated.Extractswerecomparedfortheircontentsintotalphenolics,flavonoidsand tan-nins.Moreover,theirantioxidantcapacitywasassessedbytheferricreducingantioxidantpower(FRAP) andthe2,2-diphenyl-1-picrylhydrazylradical(DPPH•)scavengingcapacityassays.Phenolics(154mg GAE/L)andtannins(109mgGAE/L)weremaximizedinlyophilizedsamplesextractedwiththe hydroal-coholicsolvent,whichattainedalsothehighestFRAPvalue(711mgFSE/L).Inturn,flavonoidsreached thehighestyieldsinlyophilizedsamples(95mgECE/L)extractedwithpureethanol,aswellasthehighest DPPH•_{scavengingactivity.Thesefindingsmighthavepracticalapplicationstodefinethebestprocessing} methodologyregardingtheenhancementofelephantfootyam,eitherforpromptconsumption,aswell astodevelopfoodsupplementsorpharmaceuticalrelatedproducts.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction

Oxidativestressis involved in theetiology ofvarious disor-dersanddiseases,beingreasonabletoexpectbeneficialeffectsof antioxidantsinmaintainingourhealthandloweringdiseaserisk (Kris-Ethertonetal.,2002;Niki,2010).Someantioxidantscanbe producedinthebody, buttheamountsmaybeinsufficient, par-ticularlyunder conditions where production of free radicals is increased.Plantsarenatural alternativesources ofantioxidants that mightcomplement the production of thesecompounds in livingorganisms.Theantioxidantactivityinplants isoften cor-relatedtotheirphenolic contents(Caiet al.,2004; Razalietal., 2012).Inaddition,therehasbeenalargevolumeofworkaimed

∗ Correspondingauthorat:REQUIMTE,LAQV/DepartamentodeCiênciasQuímicas, FaculdadedeFarmácia,UniversidadedoPorto,RuaJorgeViterboFerreira,228, 4050-313Porto,Portugal.Fax:+351220184958.

E-mailaddress:rita.c.alves@gmail.com(R.C.Alves).

at scientific validation of the efficacy of herbal drugs used in thetraditionalmedicine.Furthermore,thepreparationofdietary supplements/nutraceuticalsandsomepharmaceuticalproductsis increasinglybasedontheextractionofbioactivecompoundsfrom naturalmatrices(DaiandMumper,2010).

Amorphophallussp.areperennialherbaceousplants,growingin mountainorhillyareasinsubtropicalregions(Ishrudetal.,2001). Elephantfootyam(Amorphophalluspaeoniifolius(Dennst.) Nicol-son)isanunderground,unbranchedplantwithlargestoutmottled leaves.Theleafblade,whichsitsatopathickfleshystem,isdivided intohundredsofleaflets,varyingamong5and12.5cmlong,with highlyridgeovateoroblongshape.Theplantisdeciduous,dying backtoalargetubercle(rhizome),weighingupto8kgand reach-ingupto50cmindiameter(Sahaetal.,2013;UpretyandPoudel, 2010).Analgesic(Deyetal.,2010),antioxidant(Jayaramanetal., 2010),antibacterial,antifungal(Khanetal.,2008)andcytotoxic (Jayaraman etal., 2007;Khanet al., 2008)activities havebeen described.Therefore,itisassumablethatelephantfootyammight beanimportantsourceofbioactivecompoundssinceitisoften http://dx.doi.org/10.1016/j.indcrop.2015.11.019

A.S.G.Costaetal./IndustrialCropsandProducts79(2016)180–187 181

usedforthetreatmentofpiles,dyspnea,splenomegaly,andcough (RastogiandMehrotra,1995),beingalsorecognizedasanalgesic, livertonic,thermogenic,anthelminticanddiuretic(Arya,1994).

The effectiveness of bioactive compounds extraction from plants,as wellastheircorrespondingactivity,is highly depen-dentonfactorssuchasdifferenttypesofsolvent,solvent-to-solid ratios, extraction times and temperatures(Pinelo et al., 2005), and specially the solventpolarity (Razali et al., 2012). Accord-ingly,this workwasdesignedtoverifytheinfluenceofsolvent nature(water,water:ethanol (1:1)and ethanol)and processing type(fresh,lyophilizedandboiled)ontheantioxidantactivityand bioactivecompoundsextractabilityofelephantfootyam.Extracts werecomparedregardingtheirtotalphenolics,flavonoidsand tan-ninscontents.Moreover,theirantioxidantcapacitywasassessed bytwo complementaryprocedures:theferricreducing antioxi-dantpower(FRAP)methodandthe2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging capacity assay. The obtained results mighthavepracticalapplicationswhendecidingthebest process-ingmethodologyregardingtheenhancementofelephantfootyam extracts,eitherforpromptconsumptionaswellastodevelopfood supplementsorpharmaceuticsrelatedproducts.

2. Materialsandmethods 2.1. Reagentsandstandards

Gallic acid, epicatechin, Folin–Ciocalteu’s phenol reagent, DPPH•(2,2-diphenyl-1-picrylhydrazylradical),sodiumnitrite, fer-ricchloride,aluminumchloride,TPTZ(2,4,6-tripyridyl-s-triazine) solution,andferroussulfateheptahydratewereallobtainedfrom Sigma–Aldrich (St. Louis, U.S.A). Anhydrous sodium carbonate, sodium hydroxide and absolute ethanol were purchased from Merck(Darmstadt, Germany).Ultrapure water wastreatedin a Milli-Qwaterpurificationsystem(Millipore,Bedford,MA,USA)and usedtoprepareallaqueoussolutions.

2.2. Samplesandsamplespreparation

Elephantfootyam(A.paeoniifolius(Dennst.)Nicholsonwas col-lectedfromBaucau,adistrictofEastTimor,ontheNortherncoastin theEasternpartofthecountry.Voucherspecimenswerenumbered anddepositedinthelocalherbarium.Samplesweresubmittedto differentconservationprocesses(fresh,boiledandlyophilized).For thepreparationofthecookedsample,cubesofpeeledtuberswere boiledat100◦Cforabout40min,simulatingthedomesticcooking process.Afterwards,allvisiblewaterwasdrainedout.Toobtain lyophilizedsamples,fine-cutpeeledtuberswerefrozen(−20◦_C) and lyophilized (Telstar Cryodos-80 Terrassa, Barcelona). Sam-plesobtainedfromdifferentconservationprocesseswereground (GrindomixGM200,Retsch,Haan,Germany)andusedtoprepare theextractsdescribedinthenextsection.

2.3. Extractspreparation

Extracts were prepared using three different solvents: (i) ethanol,(ii)waterand(iii)ethanol:water50:50(v/v).Three indi-vidualsamples of fresh,boiled and lyophilizedfoot yam were testedwitheachsolvent.Eachsample(≈1g)wasextractedby stir-ringwith50mlofthecorrespondingsolvent,at40◦C, 600rpm, for1handfilteredthroughWhatmanNo.4paper.Theresidues werethenextractedwithadditionalportionsofthecorresponding solvents.Thecombinedextractsofeachsolventwereevaporated (ethanol)underreducedpressure(Rotavapor®_{R-210,Büchi,Flawil,} Switzerland)orfrozenand lyophilized(water)and re-dissolved inthecorrespondingextractatanadequateconcentration.Stock solutionswerestoredat4◦Cforfurtheruse;alltheassayswere

carriedoutintriplicateandtheresultswereexpressedasmean values±standarddeviations(SD).

2.4. Totalphenolics

Totalphenoliccontentsofdilutedextracts(1:10v/v)were deter-minedaccordingtoAlvesetal.(2010).Briefly,500␮lofeachextract weremixedwith2.5mloftheFolin–Ciocalteaureagent(1:10)and 2mlofasodiumcarbonatesolution(7.5%m/v).Themixturewas firstincubatedat45◦C,during15min,followedby30min incuba-tionatroomtemperaturebeforeabsorbancereadingsat765nm. Totalphenoliccontentswerecalculatedfromacalibrationcurve preparedwithgallicacid(10–100mg/L;r=0.9997)andexpressed asmgofgallicacidequivalents(GAE)/Lofextract.

2.5. Totalflavonoids

TotalflavonoidcontentsweredeterminedaccordingtoSoares et al. (2013). Aliquotsof 1ml of extract weremixed with4ml ofdistilledwaterand300␮lof5%sodiumnitrite.After5minat roomtemperature,300␮lof10%AlCl3wereadded,followed(after 1min)by2mlofsodiumhydroxide(1M)and2.4mlofultrapure water. Theabsorbancewasrecorded at510nm. Totalflavonoid contentswerecalculatedthroughacalibrationcurveof epicate-chin(50–450mg/L;r=0.9998)andexpressedasmgofepicatechin equivalents(ECE)/Lofextract.

2.6. Totaltannins

Total tannins contents were determined according to Shad etal.(2012),withslightmodifications.Briefly,500␮lofextract (dilutedat1:10whennecessary)weremixedwith2.5mlofthe Folin–Ciocalteureagent(1:10).After3min,2mlofsodium carbon-ate(7.5%m/v)wereadded.Themixturewaskeptinthedarkfor 2h.Absorbancereadingswerecarriedoutat725nm.Tannins con-tentwascalculatedfromacalibrationcurvepreparedwithgallic acid(10–100mg/L;r=0.9997)andexpressedasmgofgallicacid equivalents(GAE)/Lofextract.

2.7. Antioxidantactivity 2.7.1. DPPHscavengingactivity

Theradicalscavengingabilityofextractswasanalyzed accord-ing tothemethoddescribed byHariniet al.(2012) withsome modifications.Briefly,14␮lofdilutedextract(1:10v/v)weremixed with186␮lofafreshlypreparedDPPH•solution(6.0×10−5mol/L inethanol).Theabsorbancedecreaseat525minwasmeasuredin timeintervalsof2min,inordertoobservethereactionkinetics. Thereactionendpointwasattainedin40min.Theradical scaveng-ingactivity(RSA)wasexpressedaspercentageofinhibitionand calculatedusingthefollowingequation:

%RSA=Acontrol−Asample Acontrol ×100

2.7.2. Ferricreducingantioxidantpower(FRAP)assay

TheFRAPassaywasperformedaccordingtoBenzieandStrain (1996)withslightmodifications.Briefly,90␮lofdilutedextract (1:10v/v)weremixedwith270␮lof distilledwaterand 2.7ml oftheFRAPsolution(containing0.3Macetatebuffer,10mMTPTZ solution, and 20mM of ferric chloride). After homogenization, themixture waskeptfor 30minat 37◦C protected fromlight. Absorbance was measured at 595nm. A calibration curve was preparedwithferroussulfate(50–450mg/L,r=0.9998)andferric

Fig.1.Interactionsbetweensolventtype(ST)andprocessingtype(PT)effectsonthebioactivecompoundsofA.paeoniifoliussamples.Totalphenolics(A),totaltannins(B), totalflavonoids(C).

A.S.G.Costaetal./IndustrialCropsandProducts79(2016)180–187 183

Table1

Bioactivecompoundscontentsobtainedforthepolarextractsoffootyamsubmittedtodifferentprocessingtypesa_.

Totalphenolics(mgGAE/L) Totaltannins(mgGAE/L) Totalflavonoids(mgECE/L)

Solvent type (ST) Water 43±17 41±16 18±7 Water:ethanol(1:1) 132±79 129±78 58±32 Ethanol 69±71 29±9 41±39 pvalue(n=54) <0.001 <0.001 <0.001 Processing type (PT) Fresh 24±11 26±7 10±5 Lyophilized 154±68 109±83 70±33 Boiled 67±47 64±46 37±23 pvalue(n=54) <0.001 <0.001 <0.001 ST×PT pvalue(n=162) <0.001 <0.001 <0.001

a_{Theresultsarepresentedasmean±SD.GAE,gallicacidequivalents;ECE,epicatechinequivalents.}

reducingantioxidantpowerwasexpressedasmgofferroussulfate

equivalents(FSE)/Lofextract.

2.8. Statisticalanalysis

Allstatisticaltestswereperformedata5%significancelevel,

usingSPSSv.22.0program(IBMCorp.,Armonk,NY,USA).Foreach

processingtype(PT)andsolventtype(ST),threesampleswere

ana-lyzed,withalltheassaysbeingalsocarriedoutintriplicate.The

resultsareexpressedasmeanvalue±standarddeviation(SD).

Ananalysisofvariance(ANOVA)withtypeIIIsumsofsquares

wasperformedusingtheGLM(GeneralLinearModel)procedure

oftheSPSSsoftware.Thedependentvariableswereanalyzedusing

2-wayANOVA,withthefactorsPTandST.Inthiscase,whena

sta-tisticallysignificantinteraction(PT×ST)isdetected,themultiple

comparisonsclassificationresultscannotbeconsidered,andthe

twofactorsshouldbeevaluatedsimultaneouslybytheestimated

marginal meansplots for alllevels of each singlefactor.

Alter-natively,ifnostatisticalsignificantinteractionisverified,means

mightbecomparedusingTukey’shonestlysignificantdifference

(HSD)multiplecomparisontest.

Further,alineardiscriminantanalysis(LDA)wasusedto

com-paretheeffectofthePTandSTonantioxidantactivityandextracted

bioactivecompounds. Astepwisetechnique, usingtheWilks’

methodwiththeusualprobabilitiesofF(3.84toenterand2.71

to remove), was applied for variable selection. This procedure

usesacombinationof forwardselection andbackward

elimina-tionprocesses,wheretheinclusionofanewvariableispreceded

byensuringthatallvariablesselectedpreviouslyremain

signifi-cant(Maroco,2003;Lópezetal.,2008).Withthisapproach,itis possibletoidentifythesignificantvariablesobtainedforeach sam-ple.Toverifythesignificanceofcanonicaldiscriminantfunctions, theWilks’testwasapplied.Aleaving-one-outcross-validation procedurewascarriedouttoassessthemodelperformance.

3. Resultsanddiscussion

Inwhatregardstoantioxidantactivity,twoassayswere per-formedtoevaluatedifferentmechanismsofaction:ferricreducing antioxidantpower(FRAP),anelectrontransfermethod,which can-notdetectcompoundsthatactbyradicalquenching,butdetects compounds with redox potentials lower than 0.7V (the redox potentialofFe3+_{-TPTZ);andtheDPPH}•_{scavengingassay,wherethe} radicalsmaybeneutralizedeitherbydirectreductionviaelectron transfers,orbyradicalquenchingviaHatomtransfer(Prioretal., 2005).Also,threegroupsofcompoundswerequantified,namely totalphenolics,totaltanninsandtotalflavonoids.Besidesstudying theeffectsofphysicalvariablesrelatedwithmasstransfer kinet-ics(specificallythesolventtype),itwasalsointendedtoverifyif thewayinwhichthestudiedmatriceswereprocessedexerteda significanteffect.

Theeffects ofsolventand processingwereevaluatedby fix-ingoneofthefactors,i.e.,theresultsarepresentedasthemean valueofeachST,includingallthePT,andasthemeanvalueofeach PT,withthecontributionofallST.Hence,thestandarddeviation valuesshouldnotbelookedupasameasureofassays repeatabil-ity.AsitcanbeconcludedfromTables1andTable2,eachfactor showedasignificanteffectpersi,buttheinteractionamong fac-tors(ST×PT)wasalsoasignificant(p<0.001)sourceofvariation forallparameters, indicatinga strongdependence betweenthe solventusedandthewayinwhichsampleswereprocessed.This significantinteractionmightbeeasilyobservedintheestimated marginalmeans(EMM),wherethevariationintotalphenols, tan-ninsandflavonoids(Fig.1A–C)amongfresh,lyophilizedandboiled sampleswereclearlydependentonthesolventtype.Forinstance, tanninlevelsaresimilarforfresh,lyophilizedandboiledsamples whenextractedwithwaterorethanol, butcompletelydifferent when extractedwiththe hydroalcoholicsolvent(Fig.1B). Like-wise, whileDPPH• wasmaximal in boiledsampleswhenusing water orwater:ethanol,thehighestactivityinlyophilized sam-pleswasreachedwhenthesampleswereextractedwithethanol

Table2

Antioxidantpropertiesobtainedfortheextractsoffootyamsubmittedtodifferentprocessingtypesa_.

DPPH•_{scavengingactivity(%ofinhibition)}b _{FRAPassay(mgFSE/L)}

Solvent type (ST) Water 26±9 116±20 Water:etanol(1:1) 57±21 505±248 Ethanol 49±35 287±273 pvalue(n=54) <0.001 <0.001 Processing type (PT) Fresh 18±8 97±53 Lyophilized 61±29 506±259 Boiled 53±18 306±244 pvalue(n=54) <0.001 <0.001 ST×PT pvalue(n=162) <0.001 <0.001

a_{Theresultsarepresentedasmean±SD.FSE,ferroussulphateequivalents.} b_{Dilutedextracts(1:10)weretestedagainstaDPPH}•_{solutionof6×10}−5_mol/L.

Fig.2.Interactionsbetweensolventtype(ST)andprocessingtype(PT)effectsontheantioxidantactivityofA.paeoniifoliussamples.DPPH•_{scavengingassay(A),FRAPassay}

(B).

(Fig.2A).Ontheotherhand,theferricreducingpowerremained nearlythesamefor fresh,lyophilizedandboiledsampleswhen thesewereextractedwithwater,butthesamesamplesgave sig-nificantdifferencesifextractedwithethanolorthewater:ethanol (Fig.2B).Besidestheindividualvariations,somegeneral conclu-sionsmightalsobedrawnfromtheEMM:forinstance,thehighest amountsoftotalphenolics(132mgGAE/Lextract)andtotaltannins (129mgGAE/Lextract),aswellasthemostpowerfulFRAP(505mg FSE/L)wereachieved withthehydroalcoholic solvent;aqueous extractsrevealedthe lowestcontents in total phenolics (43mg GAE/Lextract),totalflavonoids(18mgECE/Lextract)andalsothe weakestDPPH•scavengingactivity(26%)andFRAP(116mgFSE/L extract);total tanninscontentpresented theleast value(29mg GAE/Lextract) in ethanol extracts.In previousstudies,ethanol extractswerereportedashavingthehighestantioxidantefficiency alongwiththehighcontentofphenoliccompounds(Angayarkanni etal., 2010; Jayaprakashaet al., 2008). Herein,thebestresults wereobtainedwithwater:ethanol(1:1),potentiallyindicatingthe presenceofphenolicacids(whicharereadilysolubleinwater)or ahighpercentageof glycosylatedphenolics(it isawell-known factthatglycosylationincreasesthewatersolubilityofphenolic compounds).Besidesthepolarityofextractionsolventsandthe sol-ubilityofphenoliccompounds,differencesmightalsobeexplained bychangesintherateofmasstransfer(Bietal.,2009).Regarding thetypeofprocessing,lyophilizedsamplesallowedthebestresults forallassays,exceptforDPPH•scavengingactivity,whereitwasnot possibletodifferentiatefromlyophilizedandboiledsamples.The

detectedamountsofbioactivecompoundsaregenerallyin agree-mentwithpreviousresultsinalcoholicextractsofelephantfoot yam(Angayarkannietal.,2010;Natarajetal.,2009).

Sincetheantioxidantactivityisoftencorrelatedwiththe con-tents in total phenolics of a determined matrix (Razali et al., 2012),thecorrelationcoefficientsamongbioactivecompoundsand antioxidantactivitywerealsocalculated.Despitethefactthatthe DPPHassayisrepresentativeofthecapacity oftestcompounds toscavengefreeradicalsindependentlyfromanyenzymatic activ-ity,thedetectedcorrelationswerehigherforFRAPassay,especially withtotalphenolics(y=18.01+3.49x,R2_{=0.91)andtotalflavonoids} (y=3.71+7.69x,R2_=0.95).

Inthefollowingsectiontheresultsobtainedfromthe conju-gatedanalysisofallparametersarecomprehensivelyanalyzed.This approachwasfollowedusinglineardiscriminantanalysisinorder tohaveanintegratedperspectiveabouttheeffectofsolventand processingontheantioxidantactivityandbioactivecompounds amounts.Thesignificantindependentvariables(evaluated param-eters) were selectedusing the stepwise procedureof the LDA, accordingtotheWilks’test.Onlyvariableswithastatistically significantclassificationperformance (p<0.05)werekeptinthe analysis.

Starting with the ST effect, two significant functions were defined(plottedinFig.3),whichintegrated100.0%oftheobserved variance(first,80.3%;second,19.7%).Asitcanbeobserved,the naturally occurring groups (each used solvent) were not com-pletely individualized. However, the classification performance

A.S.G.Costaetal./IndustrialCropsandProducts79(2016)180–187 185

water

water:etanol (1:1) ethanol group centroid

Fig.3.Discriminantscoresscatterplotofthecanonicalfunctionsdefinedforbioactivecompoundscontentsandantioxidantactivityresultsaccordingwithsolventtype(ST).

fresh lyophilized boiled group centroid

Fig.4. Discriminantscoresscatterplotofthecanonicalfunctionsdefinedforbioactivecompoundscontentsandantioxidantactivityresultsaccordingwiththeprocessing type(PT).

Table3

ContingencymatrixobtainedusingLDAbasedonantioxidantactivityandbioactive compoundscontentsofelephantyamextracts.

Predictedgroupmembership Total Sensitivity(%)

Water Water:etanol(1:1) Ethanol

Water 54 0 0 54 100

Water:etanol(1:1) 18 36 0 54 67

Ethanol 18 0 36 54 67

Total 90 36 36 162 78

Specificity(%) 60 100 100 87

wassatisfactory,resultingin78% ofcorrectlyclassifiedsamples

(sensitivity)and87%ofoverallspecificitywithintheleave-one-out

cross-validationprocedure(Table3).Theanalysiskeptallvariables

inthefinaldiscriminantmodel,beingverifiedthattotaltanninsand DPPH•scavengingactivity(bothhigherinhydroalcoholicsamples) werethevariableswiththehighestcorrelationwithfunction1and 2,respectively.Thediscriminantpowerproportionofthenth func-tionmaybeestimatedbytheratioamongitsownvalueandthe sumofalldiscriminantfunctionsvalues.Thecanonical discrimi-nantfunctioncoefficientsallowedobtainingthefollowingmodel:

D1 =−1.20+0.003×phenolics+0.02×tannins−0.14 ×flavonoids−0.002×DPPH+0.02×FRAP

Regarding classification function coefficients, the following functionswereobtained:

water=−5.08+0.14×phenolics+0.04×tannins−0.32 ×flavonoids+0.42×DPPH−0.04×FRAP

water;ethanol=−9.76+0.06×phenolics+0.10×tannins −0.52×fla

v

ethanol=−4.70−0.01×phenolics+0.04×tannins−0.05 ×flavonoids+0.31×DPPH−0.04×FRAP

ForPT,thediscriminantmodelselectedalso2significant func-tions(Fig. 4), which included 100.0% of the observed variance (function1:75.1%,function2:24.9%).Intheobtainedmodel,all sampleswerecorrectlyclassified(sensitivityandspecificitywere obviously100.0%).Allvariableswereincludedinthefinalmodel, withDPPH•scavengingactivityandtotalphenolics(bothhigherin lyophilizedsamples)asthevariableswiththehighestcorrelation withfunction1and2,respectively.

The canonical discriminant function coefficients allowed obtainingthefollowingmodel:

D1 =−6.63−0.14×phenolics+0.11×tannins−0.12×flavonoids +0.36×DPPH−0.03×FRAP

Regarding classification function coefficients, the following functionswereobtained:

fresh=−10.59−0.40×phenolics+0.44×tannins+0.27 ×flavonoids+1.66×DPPH−0.16×FRAP

4. Conclusions

Thestatisticalinteractionamongthetypeofsolventandtype ofprocessingwassignificantinallcases,showingthattheeffects causedbyeachselectedsolventmightdependontheforminwhich thesamplewasprocessed.Inaddition,theresultsobtainedwith LDA indicatethat type ofprocessing had a higherinfluence on theantioxidantactivityandbioactivecompoundscontentofthe extractsthantheextractionsolvent.Phenolsandtannins concen-trationsweremaximizedinhydroalcoholicextractsoflyophilized samples(which presentedalso thehighest FRAP values),while flavonoids reachthe highest yieldsin ethanolic extracts ofthe lyophilizedsamples(whichalsoshowedthehighestDPPH scav-engingactivity).Thesefindingsmighthavepracticalapplications todefinethebestprocessingmethodologyregardingthe enhance-mentofelephantfootyam,eitherforpromptconsumptionaswell astodevelopfoodsupplementsorpharmaceuticsrelatedproducts.

Acknowledgments

J.C.M.BarreiraandR.C.AlvesthanktoFCT,POPH-QRENandFSE fortheirgrants(SFRH/BD/76019/2011andSFRH/BPD/68883/2010, respectively).Thisworkreceivedfinancialsupportfromthe Euro-peanUnion(FEDERfundsthroughCOMPETE)andNationalFunds (FCT)throughprojectLAQVUID/QUI/50006/2013.Thisworkalso receivedfinancialsupportfromtheEuropeanUnion(FEDERfunds) undertheframeworkofQRENthroughProject NORTE-07-0124-FEDER-000069.

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