Optimization of the recovery of phenolic compounds from Algerian grape by-products

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ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j ou rn a l h o m epa 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

Optimization

of

the

recovery

of

phenolic

compounds

from

Algerian

grape

by-products

Sonia

Medouni-Adrar

a,∗

_,

_Lila

_{Boulekbache-Makhlouf}

a

_,

_Yves

_Cadot

b

_,

Lamia

Medouni-Haroune

c

_,

_Farid

_Dahmoune

a

_,

_Aida

_Makhoukhe

a

_,

_Khodir

_Madani

a

a_Laboratoire_de_{Biomathématiques,}_Biophysique,_Biochimie,_et_{Scientométrie}_(L3BS),_Faculté_des_Sciences_de_la_Nature_et_de_la_Vie,_Université_de_Bejaia,

06000Bejaia,Algeria

b_INRA,_UE₁₁₁₇_Vigne_Vin,₄₂_rue_Georges_Morel,₄₉₀₇₁_Beaucouzé_Cedex,_France

c_Laboratoire_de_{Microbiologie}_Appliquée,_Faculté_des_Sciences_de_la_Nature_et_de_la_Vie,_Université_de_Bejaia,₀₆₀₀₀_Bejaia,_Algeria

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received28April2015

Receivedinrevisedform17August2015 Accepted18August2015 Keywords: Grapeby-products Phenolics Extractionmethods Optimization

Responsesurfacemethodology

a

b

s

t

r

a

c

t

Grapeseedsandskinareby-productsofwinemakingorjuicemakingoperationswhichare consid-eredasagoodsourceofbioactivecompounds.Inthisstudy,thetotalphenoliccontent(TPC)fromVitis viniferaL.cv.AhmarBou-Amarseedsandskinwasoptimizedbyconventionalsolventextraction(CSE) andmicrowave-assistedextraction(MAE)usingresponsesurfacemethodology(RSM),thena compara-tivestudywascarriedout.TheoptimalconditionsforseedsextractedbyCSEwere:74.33%v/vofethanol concentration,65.23minextractiontime,0.1g/70.86mlsolid/liquidratio,andforthoseextractedby MAEwere:59.88sirradiationtimeand373.15Wmicrowavepower.Theextractobtainedunderthese conditionsshowedaTPCof96.56±1.29mgGAE/gand73.15±0.20mgGAE/gDWforCSEandMAE, respectively.Concerningskin,theoptimalconditionsforCSEwere:51.46%v/vofacetone concentra-tion,89.80minextractiontime,0.1g/32.25mlsolid/liquidratioandforMAEwere:113.74sirradiation timeand384.44Wmicrowavepower.TheextractobtainedundertheseconditionsshowedaTPCof 39.57±0.23mgGAE/gand54.84±0.41mgGAE/gDWforCSEandMAE,respectively.TheTPCofseeds extractobtainedwithMAEwas24%lowerthanthatoftheCSEextract;also,theantioxidantactivityof CSEextractisbetterthanthatofMAEextract.While,theTPCofskinextractobtainedwithMAEwas28% higherthanthatoftheCSEextractandtheantioxidantcapacitywassigniﬁcantlyhigherthanthatofthe CSEextract.Theresultsindicatethattheextractsofcv.AhmarBou-Amarseedsandskincontainahigh quantityofpolyphenols;therefore,theycanbeconsideredasagoodsourceofnaturalantioxidants.

1. Introduction

Grapes (Vitis vinifera L.), are the world’s largest fruit crop (Ghafooretal.,2010;Yangetal.,2009),withatotalproductionof approximately75.1milliontonsin2013(O.I.V.,2015).Although, a highpart of grapesis made into wine, another partis dried intoraisins,asigniﬁcantpartisconsumedastablegrapes(Nelson, 1979).Thecv.AhmarBou-AmargrapeisoriginatedinAlgeria,itis widespreadintheKabilyregionwithalowcommercialvalue,and isgenerallyconsumedasafreshfruit.

VitisviniferaL.containslargeamountsofphytochemicalssuchas phenoliccompounds(Yangetal.,2009)whichofferhealthbeneﬁts viatheirimportantantioxidantactivity(Andjelkovicetal.,2013).

∗ Correspondingauthor.Fax:+21334214762.

E-mailaddress:sonia.medouni@yahoo.fr(S.Medouni-Adrar).

Thegrapepolyphenoliccompoundsarefoundessentiallyinseeds andskinswithapproximately75%(Ghafooretal.,2010);these lev-elsareinﬂuencedbythegrapevarietyandenvironmentalfactors (Cadotetal.,2008;Cheynieretal.,1998;Katalinicetal.,2010).

Grapeseedsandskinswhich arealow-value by-productsof wine making or juice making operations are considered to be a cheap and a good source of the high-quality phenolic com-pounds(Buci ć-Koji ćetal.,2007;Spignoetal.,2007),sotheycan beexploitedasnaturalantioxidantagentstoneutralizefree radi-calsinbiologicalsystems(Buci ć-Koji ćetal.,2007;Ghafooretal., 2010).

Extractionisacriticalstepintheisolationofactivecompounds fromplantmaterial(Afoakwahetal.,2012;Dragovi ´c-Uzelacetal., 2012).Differentextractionmethodshavebeeninvestigatedinthis regard;conventionalextractionmethodsaregenerallybasedon choosingtheaccuratesolventinordertoenhancethesolubility ofactivecompounds(Dragovi ´c-Uzelacetal.,2012;Ravaljietal., http://dx.doi.org/10.1016/j.indcrop.2015.08.039

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2012).These methodsnecessitatelongerextractiontime which producesathermaldeteriorationforthemajorityoftheactive com-pounds(Wong-Pazetal.,2014).Recently,environmentalfriendly techniquesarebeinginterestedtodevelopthe“GreenChemistry” conceptsuchasultrasound-assistedextraction(Bimakretal.,2013) andmicrowave-assistedextraction(MAE)(Afoakwahetal.,2012; Raﬁeeetal.,2011;Wong-Pazetal.,2014;Zhengetal.,2011).

Microwave-assistedextractionisconsideredasanovel extrac-tionmethodthatcombinesmicrowaveandconventionalsolvent extraction(Lopez-AvliaandYoung,1994).Itisatechniqueused toextractactivecompoundsfromarangeofrawmaterialsusing theenergyofmicrowaveradiationtoheightenthetemperature ofthesolvents successfullyand rapidly(Afoakwah et al.,2012; Zhengetal.,2011).MAEisalsoabletodecreasejointlythesolvent consumptionandextractiontimeincomparisontoconventional techniques.Moreover,ithasthepossibilitytoimproveextraction quality(HithamaniandRamalakshmi,2013;Ravaljietal.,2012; Wong-Pazetal.,2014).

Consideringthevariabilityofthestructuresofphenolic com-poundspresentindifferentpartsofgrape(Rivera-Dominguezetal., 2010),itisimportanttodevelopanoptimalextractionmethodfor eachmatrix.Astheextractionmethodsareaffectedbydifferent parametersaswellastheinteractionofallthesefactors,theuse ofanoptimizationmodelingisnecessarytofacilitatethe deter-minationoftheoptimumextractionconditions.Responsesurface methodology(RSM)isamathematicaltechniquewhichisableto generatestatisticalmodelsandtakeintoconsiderationthe possi-bleinterrelationshipsbetweenthedifferenttestparameterswhile reducingthenumberofessaysandallowingforconsiderable reduc-tionofoperationcostandtime(Dahmouneetal.,2014).

Many authors investigated Conventional Solvent Extraction procedure(CSE)ofphenoliccompoundsandtheirpropertiesfrom grapeseedsandskinsandfromotherplantmaterials(Bordigaetal., 2011;Katalinicetal.,2010;Pineloetal.,2005a).However,limited informationhasbeenpublishedontheuseofmicrowaveforthe extractionoftotalphenoliccompoundsfromgrapeseedsandskins. Moreover,thereisalittlepublishedinformationabout optimiza-tionmodelingandcomparisonofCSEandMAEextractionprocess fromgrapeby-products.Finally,tothebestofourknowledge,no literaturereportexistsontheextractionofpolyphenolsfromgrape cv.AhmarBou-Amar.Therefore,thereisaneedtodevelopan opti-malmethodfortheextractionofphenoliccompoundsfromthe by-productsofthisvariety.Thus,theobjectivesofthecurrentstudy areto:(1)optimizethephenoliccompoundsextractionfromseeds andskinofgrapeVitisviniferacv.AhmarBou-AmarbyCSEandMAE, studyingtheeffectsofdifferentparametersontheextraction efﬁ-ciency,usingBox-BehnkenDesign(BBD)and CentralComposite Design(CCD)ofRSM.(2)ComparetheoptimizedCSEwithMAE pro-cessbasedonthetotalphenoliccontent(TPC)andtheantioxidant activity.

2. Materialsandmethods 2.1. Chemicals

Folin–Ciocalteau’sphenolreagent,sodiumcarbonate(Na2Co3),

disodium hydrogen phosphate (Na2HPO4), sodium

dihydro-genphosphate (NaH2PO4)and allsolvents used wereobtained

from Prolabo (made in CE). Potassium persulfate (K2S2O8),

triﬂuoroacetic Acid (C2HF302) and ABTS:

2,2-azinobis-3-ethylbenzothiazoline-b6-sulfonic acid (C18H18NaO6S4) were

purchasedfromSigmaAldrich(Germany).Potassiumferricyanide (C6N6FeK3),ferricchloride(FeCl3·6H2O),trichloroaceticacid,gallic

acidandquercetinfromBiochem-chemopharma(UK).

2.2. Biologicalmaterial

ThefruitsamplesofVitisviniferacv.AhmarBou-Amarwere col-lectedintheareaofAmizour(Bejaia,Algeria).Grapeseedsandskin weremanuallyseparatedfromthepulpanddriedat40◦Cinahot airoven.Thedriedsamplesweremilledwithanelectricalgrinder, thepowderwassiftedbyastandardsieveandjusttheportionwith particlesize<500␮mwasused.

2.3. Effectofsolventnature

Inafirststage,theeffectofsolventnaturewasstudied:four solventswereused:50% (v/v)methanol,50% (v/v)ethanol,50% (v/v)acetoneand water. Theextractionwascarriedout witha CSEbyfixingextractiontime (30min)and sample/solventratio (100mg/10ml)atambienttemperature,afterthat,each sample wascentrifugedat10000rpm,thenfilteredthroughWhatmanN0₁

paperbyBüchnerfunnel.Thesupernatantwasusedinorderto determinetotalphenoliccompounds(TPC).

2.4. Preparationofextractsaccordingtotheexperimentaldesign 2.4.1. CSEprocess

Samples were extracted using the best solvent selected in theﬁrst step.In order topredict optimalextractionconditions and to determine the best combinations of three independent parametersforCSE,namely:solventconcentration,extractiontime andsolid/liquidratio,a33_factorial_experimental_design_through

Box–BehnkenwithRSMwasusedontherecoveryoftotalphenolic compounds.Thedifferentlevelsforeachindependentparameter werechosen froma series of preliminaryexperimentswithout usingmathematicalmodels.Thelevelsofindependentvariables werereportedinTable1.

50mgofseedsand100mgofskinpowderswereextractedwith differentvolumesofappropriatesolvent,differentextractiontimes andsolid/liquidratioasshowedinTable2.Afterextraction,each samplewascentrifugedat10,000rpm,thenfilteredthrough What-manNo1paperbyBüchnerfunnel.Theextractsobtainedunder theoptimumconditionsbyRSM,wereconcentratedundervacuum usingarotaryevaporatorandrecuperatedin55%ethanolacidified with0.005%oftrifluoroaceticacid(TFA)andstoredat0◦C. 2.4.2. MAEprocess

Using the best solvent, solvent concentration and sam-ple/solventratioﬁxedatoptimumconditionsofCSEprocess,the sampleswereextractedwithMAE,theindependentparametersof extractiontimeandmicrowavepowerwereanalyzedusinga cen-tralcompositedesigntoassesstheireffectsandinteractionsonthe extractionofphenolicscompounds.ARSMwasusedtooptimize theextractionprocess.Thedifferentlevelsforeachindependent parameterwerechosenfromaseriesofpreliminaryexperiments withoutusing mathematicalmodels. Thelevels ofindependent variableswerereportedinTable1.

Themicrowaveapparatususedinthisstudyisadomesticone (NN-S674MF,Samsung,Malaysia),itwasmodifiedinorderto con-densatethevaporsgeneratedduringextractionprocessgivinga stableextractvolume.Concerningtheextraction,50mgofseeds and100mgofskinpowderswereplacedina250mlvolumetric flaskcontainingtheextractionsolvent.Thesampleswereextracted atdifferentirradiationtimesandmicrowavepowers(Table2).After extraction,eachsamplewascentrifugedat10,000rpm,then fil-teredthroughWhatmanNo1paperbyBüchnerfunnel.Theextracts obtainedundertheoptimum conditions byRSM,were concen-tratedundervacuumusingarotaryevaporatorandrecuperated in55%ethanolacidifiedwith0.005%oftrifluoroaceticacid(TFA) andstoredat0◦C.

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Table1

LevelsofdifferentindependentvariablesinRSMfortheoptimizationofextractsbyCSEandMAE.

Extractionprocess Independentvariables Levels

Seeds Skin

(-1) (0) (+1) (-1) (0) (+1)

CSE X1-solventconcentration

(%v/v) X2-time(min) X3–ratio(mg/ml) 50 30 1 65 60 1.75 80 90 2.5 40 60 2.5 55 90 3.75 70 120 5 MAE X1-Power(W) X2-time(S) 200 30 400 75 600 120 200 60 400 120 600 180 Table2

PreparationconditonsofextractsaccordingtotheexperimentaldesignwithRMSbyCSEandMAE.

Essay CSEFactors Essay MAEfactors

X1-solvent

concentration (%v/v)

X2-time(min) X3–ratio

(mg/ml) X1-time(s) X2-power(W) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 −1 +1 −1 0 0 −1 0 +1 +1 0 0 −1 +1 0 0 +1 0 0 −1 0 0 +1 −1 0 −1 +1 −1 +1 0 0 0 −1 −1 −1 0 +1 +1 0 +1 +1 −1 0 0 0 0 1 2 3 4 5 6 7 8 9 10 11 0 0 −1 91 +1 0 −1 0 0 +1 +1 0 0 0 −1 −1 +1 +1 0 −1 0 +1

To obtaintheoptimum operating conditions and to explain theperformanceofthemethod,asecond-ordermodelwasused accordingtothefollowingequation:

Y= ˇo+SˇiXi+SˇiiXi2+SˇijXiXj (1)

WhereYisthepredictedresponse(TPCmgGAE/gDW),␤o,ˇi,ˇii, and␤ijareregressioncoefﬁcientsforintercept,linear,quadratic andinteractionterms, respectivelyand XiandXjare theactual levelsoftheindependentvariables.

2.5. Analysisoftotalphenoliccompounds

Totalphenoliccontentwasmeasuredaccordingtothemethod described byVelioglu etal. (1998).200␮lof theextractswere mixedwith1500␮lofFolin–Ciocalteaureagent(dilutedtenfold). After3min,1500␮lof6%sodiumcarbonatewereadded.Thetest tubecontentsweremixedandpreservedfor1hatroom temper-atureinthedark.Then,theabsorbancewasmeasuredat765nm versusablankpreparedwithoutextract.Gallicacidwasusedas astandardandtheresultswereexpressedasmilligramgallicacid equivalentspergramofdryweight(GAE/gofDW).

2.6. Evaluationoftheantioxidantactivity 2.6.1. ABTS+_method

TheABTS+ _radical_scavenging_activity_of _extracts_was

deter-minedaccordingtothemethodofAwikaetal.(2003).TheABTS+

radicalwaspreparedbycombiningavolumeof8mMABTS+_with

thesamevolume of3mM potassium persulfateand incubating for16hinthedarkatroomtemperature.Theworkingsolution wasobtainedbydilutingwith50%methanoltoanabsorbanceof 0.70±0.02at734nm.TheresultswereexpressedasQuercetinmg

equivalentspergramofdryweight(mgQE/gDW).Thepercentage inhibitionwascalculatedaccordingtothefollowingformula: %Inhibition =[(Acontrol−Asample)/control]×100 (2) WhereAistheabsorbanceofthecontrolorofthesample. 2.6.2. Ferricreducingpower(FRP)

Thereducing poweroftheextractswasevaluatedusing the method described by Oyaizu (1986); 1ml of phosphate buffer (0.2M,pH6.6)and1mlof1%potassiumferricyanidewereaddedto 1mlofthesample.Themixturewasincubatedat50◦Cfor20min. Afterthat,1mlof10%trichloroaceticacidwasaddedtothemixture andcentrifugedfor10minat1700g.Then0.2mlof0.1%ferric chlo-ridewasaddedtothemixtureof1mlofsupernatantand1mlof distilledwater.Theabsorbancewasmeasuredat700nm.Quercetin was usedas a standard and reducing power wasexpressedas quercetinmgequivalentspergramofdryweight(mgQE/gDW). 2.7. Statisticalanalysis

Alltheessayswererepeatedtreetimesandtheresultswere expressedas means±standard deviation (SD).Statistical anal-ysiswasdonewithJMP software (Version7.0, SAS)tofindthe responsesurfacesandcontourplotsoftheresponsemodel.Data wereanalyzedbyanalysisofvariance(ANOVA)andthefittingof themathematicalmodelswasdeterminedbyevaluatingthelack offitandcoefficientofdetermination(R2_)._While_the_effect_of_type

solventontheTPCinthefirststageandtheresultsof compari-sonbetweenCSEandMAE(ABTS,FRPandTPC)werestatistically assessedbyANOVAusingSTATISTICA5.5 tofindsignificant dif-ferencesamongthedifferentresults.Thestatisticalsignificanceof eachresultwasdeterminedat5%probabilitylevel(p<0.05).

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3. Resultsanddiscussion

3.1. Effectsofthesolventonthetotalphenoliccontent

Preliminaryexperiments wereconductedtodetermine opti-mumextractionsolventforeachmatrix.Theefficiencyofethanol, methanol,acetoneandwaterontheextractionofthetotalphenolic contentfromgrapeseedsandskin,wascompared.Significant dif-ferences(p<0.05)solventextractionforskinsamplesasshownin Table3,thehighercontentofTPCwasdetectedinacetoneextract (27.90±0.39mgGAE/g DW)followed bytheethanol, methanol andwaterextracts.Whileforseeds,thehighercontentofTPCwas detectedinethanolextract(86.51±5.36mgGAE/gDW)followed bythemethanol,water thenacetoneextracts.Therefore,inthe followingstudy,acetonewaschosentobethebestsolvent extrac-tionforskinmatrixandethanolwaschosentobethebestsolvent extractionforseeds.Theseresultsareinagreementwiththe previ-ouspublishedresults.Indeed,aqueousethanolwasusedbyseveral authorsfortheextractionofphenoliccompoundsfromgrape,grape seeds,skinsand/orpomace(Buci ć-Koji ćetal.,2007;Carreraetal., 2012;Katalinicetal.,2010;Pouralietal.,2014;Spignoetal.,2007) andYilmazandToledo(2006)haveusedaqueousacetone.Kennedy etal.(2002)extractedthephenoliccompoundsfromgrapeskins with66% aqueous acetone.Cadot et al. (2011, 2006) used dif-ferent solvents for skins and seeds:acetone/methanol/H2O/TFA

(50:15:35:0.05)forskinsandacetone/H20(60:40)forseeds.

3.2. OptimizationoftheCSEconditions

Theexperimentaldesignandcorrespondingresponsesforthe obtainingofgrapecv.AhmarBou-Amarseedsandskinextractsare presentedinTable4.Modelpresentedthetotalof15experiments. Treereplicationsatthecentralpointswereincludedinorderto evaluateexperimentalerrormeasurement(GoupyandCreighton, 2006).

Followingtheresultsobtainedondifferentsolventsextraction, seedsandskinswereextractedwiththeselectedappropriate sol-vent(ethanolandacetoneforseedsandskinsrespectively).The inﬂuenceofthevariables:solventconcentrations,solid/liquidratio, and extractiontime onthe extraction of TPC was investigated througha33_factorial_experimental_design_with_response_surface

methodology(RSM)andBox–Behnkendesign.Theexperimental andpredictedvaluesofTPCatvariousexperimentalconditionsare giveninTable4

Asshown,TPCofseedsrangedfrom87.99to95.97mgGAE/g DW,while the bestresult wasobtained when solvent concen-tration, extracting time and solid/solventratio were 65% (v/v), 60minand1.75(m/v),respectively. Ontheotherhand,TPC for skinrangefrom33.19to39.14mgGAE/gDW,wherethecentral pointpresentsthebestextractionconditions.Theobtainedresults werehigherthanthoseobtainedbyCasazzaetal.(2010).Indeed, theseauthorshavefoundvaluesof55.98±0.58and5.60±0.20mg GAE/gin ethanolic extractsfrom grapeseeds and skin, respec-tively.Buci ´c-Koji ´cetal.(2007)foundtheconcentrationrangedfrom 14.72mgGAE/gDWto66.81mgGAE/gDWin50%ethanolicextract ofgrapeseeds.Krishnaswamyetal.(2012),haveobtainedavalue of13.5±0.48mgGAE/gDWin30%ethanolicextractfromgrape seeds.

The second order polynomial equation was generated to describetheempiricalrelationshipbetweentheTPCofseeds(Y1)

andskin(Y2)andoperationalconditions(solventconcentration,

solid/extractiontimeandsolid/liquidratio)intermsofcoded val-ues: the models were simpliﬁed by elimination of statistically insigniﬁcantterms.

Y1 = 95.65+0.46X1+0.35X2–1.78X3–0.87X2X3

–0.54X12–2.68X22–2.41X32 (3)

Y2= 38.85–0.43X1–0.55X2–1.21X3–0.87X2X3–1.07X12

–1.71X22–1.19X32 (4)

Theresultsoftheanalysisofvariance(ANOVA)fortheadequacy oftheselectedmathematicalmodelsarereportedinTable5.The significanceofeveryfactorandtheirinteractionsareverifiedby p-values,wherethevalueslessthan0.05indicatesignificanceand lessthan0.001indicatehighsignificance.

verylowp-values(p<0.0001andp=0.0022forseedsandskin, respectively)indicatedthateachgeneratedmodelwasstatistically signiﬁcantandsuggeststhattheCSEofgrapeseedsandskincould bewelldescribed withthose appropriatemodels.Thevaluesof R-squaredarecloseto1foreachmodel(R2₌_0.9924_and_0.9722

forseedsandskin,respectively),whichisveryhighandindicates agood correlationbetweentheexperimentaland thepredicted values,andindicatedthateachmodelcouldexplain99.24%and 97.22%ofthevariationintheTPCingrapeseedsandskin, respec-tively.Also,thevalueofF-valueforthelackoffit,ofbothmodels, wasnotsignificant(p>0.05),thusconfirmingthevalidityofthe model.Allresultsindicatedthatbothmodelscouldworkwellfor thepredictionofTPCextractionfromgrapeseedsandskin.

Theregressionanalysisofthedatashowedthatallthethree parameters(solventconcentration(X1),solid/liquidratio(X2)and

extractiontime(X3))have asigniﬁcanteffect(p<0.05)onboth

seeds and skin extracts. Meanwhile, solid/liquid ratio has the dominanteffect onTPC,followed bysolvent concentrationand extraction time. However, interactions X1X2 and X1X3 did not

exhibitanysigniﬁcanteffectinanycases.Whereas,theeffectsof X2X3,X12,X22 andX32ontheTPCofseedsandskinextractwere

signiﬁcant(p<0.05).

TheEq. (3)shows thatethanol concentrationand extraction time havesignificantpositive lineareffects onTPC grape seeds extract,whereas,solid/liquidratioandtheirquadraticinteraction havesignificantnegativeeffects.Concerningskin,Eq.(4),indicates thatallparametersandtheirquadraticinteractionhavesignificant negativeeffectsonTPC.Theinfluenceofthethreeparameterson TPCofgrapeseedsandskinisdepictedinFig.1.Resultsindicate that,yieldofTPCincreasedwithincreasingethanolconcentration upto74%andthenitremainedsignificantlyconstant.Whilethe TPCinthegrapeskinextractsincreasedwithdecreasingofacetone concentration,sothemaximumTPCwasobtainedwithminimal valueofacetoneconcentration.Similarresultshavebeenobtained byChewetal.,(2011);the40%ethanolextractofCentella asiat-icacontainedmorephenoliccompoundsthan60%,80%and100% ethanolextracts.Also,Al-FarsiandLee(2008)reportedthat50% acetonewasthemostefficientsolventforphenolicextractionfrom dateseeds.

FromFig.1AtoF,itcanbeconcludedthattheTPC, indepen-dentlyofsolventconcentrationand solid/liquidratio,increased rapidlyforgrapeseedsandslightlyforskinextracts,respectively, withtheincreaseoftheextractiontimefrom30to60andfrom 60to90minforgrapeseedsandskinextracts,respectively;then slightlydecreasedfrom65to90andrapidlyfrom91to120minfor grapeseedsandskinextracts,respectively.Ingeneral,the high-estvalue ofTPC was achievedat anextractiontime of 65 and 90min for seeds and skin extracts,respectively. But, after this point,theTPCwasdecreased.Similarresultswerereportedinthe literature(Spignoetal., 2007;Wissametal., 2012;Yang etal., 2013).Pilenoetal.(2005b)showedthattheTPCofgrapeextracts decreasewiththeincreaseoftheextractiontime.Thisphenomenon

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Table3

EffectofsolventnatureontheextractionofTPCfromseedsandskin.

Solvents TPCofseedsextracts(mg

GAE/gDW) TPCofskinextracts(mg GAE/gDW) Water Ethanol50% Methanol50% Acetone50% 57.56±0.23b 86.51±5.36a 69.18±1.41b 38.92±0.68c 07.58±0.83d 20.61±1.64b 13.09±0.79c 27.90±0.39a

Resultsarereportedasmeans±S.D.DifferentlettersinthesamecolumnrefertomeansstatisticallydifferentaccordingtoANOVAandTukey’stest.

Table4

Box–BehnkendesignwiththeobservedresponsesandpredictedvaluesofTPCreferredtoDWofseedsandskinusingCSE.

Essay Factors Reponses

X1-solvent

concentration (%v/v)

X2-time(min) X3–ratio

(mg/ml)

Seeds

TPCmgGAE/gDW

Skin

TPCmgGAE/gDW

Experimental predicted Experimental predicted 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 −1 +1 −1 0 0 −1 0 +1 +1 0 0 −1 +1 0 0 +1 0 0 −1 0 0 +1 −1 0 −1 +1 −1 +1 0 0 0 −1 −1 −1 0 +1 +1 0 +1 +1 −1 0 0 0 0 92.06±0.84 95.13±1.63 93.61±1.36 91.38±1.29 95.74±1.09 91.01±0.90 87.99±0.54 92.16±0.78 91.01±0.38 89.33±1.10 93.54±1.48 91.71±0.48 93.76±0.82 95.97±0.67 95.24±0.78 92.00 95.31 93.64 91.13 95.65 90.83 88.24 92.22 90.98 89.30 93.57 91.93 93.54 95.65 95.65 36.06±0.70 36.89±1.05 38.11±0.37 36.95±1.70 39.14±0.36 35.98±1.17 33.19±0.37 36.68±0.55 35.32±0.31 35.70±0.69 37.94±1.38 36.88±1.13 34.63±1.02 38.83±1.84 38.58±0.66 36.26 37.21 38.37 36.83 38.85 35.66 33.31 36.48 35.07 36.16 37.48 36.74 34.77 38.85 38.85 Table5

Analysisofvariance(ANOVA)ofthequadraticmodelobtainedwithCSE.df.degreesoffreedom.

Source Seeds Skin

Df Sumofsquares F-value P-valueProb>F Df Sumofsquares F-value P-valueProb>F Model X1–Solvent X2-Time X3-Ratio X1X2 X1X3 X2X3 X12 X22 X32 Residual Lackofﬁt Pureerror Cortotal 9 1 1 1 1 1 1 1 1 1 5 3 2 14 76.9125 1.6718 0.9583 25.5628 0.3906 0.5779 3.0648 1.1126 26.5220 21.4407 0.5855 0.3060 0.2794 77.4980 72.9768 14.2766 8.1834 218.2930 3.3360 4.9356 26.1724 9.5012 226.4838 183.0925 0.7301 <0.0001 0.0129 0.0353 2.56815e-5 0.1273 0.0769 0.0037 0.0273 2.34629e-5 3.94951e-5 0.6221 9 1 1 1 1 1 1 1 1 1 5 3 2 14 37.0018 1.5287 2.4088 11.7540 0.3791 0.0788 3.0548 4.2876 10.7707 5.2694 1.0576 0.9022 0.1554 38.0595 19.4359 7.2272 11.3874 55.5664 1.7926 0.3727 14.4414 20.2693 50.9180 24.9108 3.8692 0.0022 0.0433 0.0197 0.0006 0.2382 0.5681 0.0126 0.0063 0.0008 0.0041 0.2122 R2₌_0.9924 _R2₌_0.9722

couldbeexplained byFick’s secondlawof diffusion,whenthe solventsaturatestheextractedcompound,theconcentration gradi-entbecomesnullafteraparticulardurationandthephenomenon stops(Rodriguesetal.,2008;Yangetal.,2013).However,itwas foundthataugmentationoftheextractiontimeprolonged expo-suretooxygenandlight(Wissametal.,2012),thus,increasethe chancesforformationoffreeradicalswhichcanbescavengedby phenoliccompounds(NaczkandShahidi,2004).Alsoplantcells containenzymescapableforalteringthephenoliccompounds,in particularpolyphenoloxidaseviareactionsenzymatic browning (Tomãs-BarberãnandEspin,2001),thelongerextractiontimemay favorthosereactions(Garcia-Salasetal.,2010).Hence,anexcessive extractiontimewasnotusefultoextractmorephenolic antioxi-dants(Chaalaletal.,2012).

Withregardtosolid/liquidratio,whenthisparameterdecreases from2.5to1forseedsandfrom5to2.5forskinanincreaseonTPC wasobserved.OurresultsagreewiththosereportedbyChaalaletal. (2012),whoconﬁrmedanincreaseofTPCwhenthesolid/liquid ratiowasdecreasedfrom0.08(0.8g/10ml)to0.02(0.2g/10ml). Oneoftheprobableexplanationforthisphenomenonisthat regu-larlyusageoflargervolumeofsolventcouldobtainlargeramountof phenoliccompounds(Inceetal.,2014);accordingtomasstransfer principles,thedrivingforceduringmasstransferisthe concentra-tiongradientbetweenthesampleandthesolvent,whichisgreatest whenasmallsample/solventratioisused(Pineloetal.,2005b).

Theobtainedresultsshowthat theoptimumextraction con-ditionsfortherecoveryoftheTPCusingCSEfromseeds(1)are: 74.33%v/vofethanolconcentration,65.23minextractiontimeand 0.1g/70.86mlsolid/liquidratiowithapredictedyieldof96.23mg

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Fig.1.Three-dimensionalpresentationofthedevelopedresponsesurfacemodelfortheTPCfromgrapeseedsandskinwithCSEwithrespecttoextractiontimeandsolvent percentage(A:seeds;B:skin);solid/liquidratioandsolventpercentage(C:seeds;D:skin);extractiontimewithsolid/liquidratio(E:seeds;F:skin).

GAE/g.Averiﬁcationexperimentattheoptimumconditionswas performedandthepracticalrecoveryof96.56±1.29mgGAE/gwas obtained.Whilethosefromtheskin(2)are:at51.46%v/vof ace-toneconcentration,89.80minextractiontimeand0.1g/32.25ml solid/liquidratiowithapredictedyieldof39.22mgGAE/g.A veriﬁ-cationexperimentattheoptimumconditionswasperformedand thepracticalrecoveryof39.57±0.23mgGAE/gwasobtained.

3.3. OptimizationoftheMAEconditions

RSMbasedonaCentralCompositeDesign(CCD),througha23

factorialexperimentaldesign,tooptimizetheMicrowave-Assisted Extraction (MAE) of total phenol content fromseeds and skin, wasinvestigated.TheexperimentalvaluesofTPCextractsat vari-ousexperimentalconditionsarepresentedinTable6.Inthisnew design,onlytheirradiationtimeandmicrowavepowerwere stud-ied.Thesolventconcentrationandsolid/liquidratiowereﬁxedat optimumthatwasfoundinCSEstudy.Eachfactor,containingthree levels,waschosenfromaseriesofpreliminaryexperiments with-outusingmathematicalmodels.Themodelpresentedthetotalof11

experiments.Threereplicationsatthecentralpointswereincluded inordertoevaluateexperimentalerrormeasurement(Goupyand Creighton,2006).Theobtainedresultsshowedthatthemaximum TPCofthegrapeseedsandskinextractswere72.92and55.82mg GAE/gDW,respectively.

Eq.(5)and(6),showtherelationshipbetweenirradiationtime andmicrowavepowerfortheextractionof totalphenolic com-poundsofgrapeseeds(Y1)andskin(Y2),respectively.

Y1= 72.33–3.47X1–4.98X2–6.88X1X2–3.51X12–9.22X22 (5)

Y2= 54.31–0.82X1–0.72X2–2.30X1X2–3.08X12–3.12X22 (6)

The resultsof ANOVA,goodness of ﬁt and the adequacy of themodelsareshowedinTable7.TheANOVAresponsesurface quadraticregressionmodelshowedthatthemodelswerehighly signiﬁcantforbothmatrixes(p<0.001)withhighlyF-value(129.22 and69.87forseedsand skin,respectively),atthesametime, a highproportionofvariabilitywasexplainedbytheRSMmodels forTPCasindicatedbyR2_(R2₌_0.99_and_0.98_for_seeds_and_skin,

respectively)therefore,themodelswereadequateandexplained mostofthevariabilityforeachmatrix.Also,thelack-ofﬁtstatistics

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Table6

CentralCompositeDesignwiththeobservedresponsesandpredictedvaluesofTPCreferredtoDWofseedsandskinusingMAE.

Essay Factors Reponses

X1–time(s) X2–power(W) Seeds Skin

TPCmgGAE/gDW TPCmgGAE/gDW

Experimental predicted Experimental predicted

1 0 0 71.83±0.79 72.32 54.92±0.84 55.06 2 0 0 72.44±0.46 72.32 55.82±0.82 55.06 3 −1 0 72.00±0.60 72.29 52.60±0.98 52.79 4 −1 −1 60.68±0.95 61.17 47.76±0.34 47.78 5 1 −1 67.30±1.08 67.97 51.28±1.02 51.21 6 0 1 56.74±1.23 58.12 50.58±0.72 51.09 7 −1 1 65.74±0.73 64.97 51.51±0.64 51.3 8 0 0 72.92±0.79 72.32 55.01±0.52 55.06 9 0 −1 69.23±1.00 68.08 52.48±0.57 52.53 10 1 0 65.39±1.19 65.33 50.90±0.69 51.27 11 1 1 44.86±1.90 44.25 45.12±0.61 44.82 Table7

Analysisofvariance(ANOVA)ofthequadraticmodelobtainedwithMAE.df.degreesoffreedom.

Source Seeds Skin

df Sumofsquares F-value P-valueProb>F df Sumofsquares F-value P-valueProb>F Model X1–Time X2-Power X1X2 X12 X22 Residual Lackofﬁt Pureerror Cortotal 5 1 1 1 1 1 5 3 2 10 723.1177 207.6950 480.6025 496.4884 85.2034 539.8073 5.5956 4.9984 0.49984 728.7133 129.2295 72.5813 148.7268 189.1988 31.2173 215.4265 5.5803 2.78383e-5 0.0004 8.61256e-5 <0.0001 0.0032 3.49442e-5 0.1557 5 1 1 1 1 1 5 3 2 10 94.8697 4.0520 3.1520 21.1829 24.1343 24.6201 1.3560 0.9293 0.4266 96.2257 69.9606 14.9408 11.6220 78.1057 88.9880 90.7793 1.4520 0.0001 0.0118 0.0190 0.0003 0.0002 0.0002 0.4326 R2₌_0.9923 _R2₌_0.9859

(p=0.1557and0.4326forseedsandskin,respectively),whichwas usedtotesttheadequacyofthemodelwasnotsigniﬁcant.Thus, itcanbeconcludedthatthemodelswerewellﬁttedandcouldbe usedtopredicttheTPCfromgrapeseedsandskinextractswith MAE.

AsshowninTable7,ANOVAresultsindicatedthateffectof irra-diationtime(X1)andmicrowavepower(X2)onTPCofseedsand

skinextractsishighlysigniﬁcant(p<0.0001)suchastheir inter-actionandtheirquadraticterms.AccordingtoEq.(5)and(6)the negativecoefﬁcientsforX1andX2indicatelineareffectsthatmay

decreasetheresponses(TPC).TherelationshipbetweenTPCand dependentvariablesisgraphicallypresentedinthreedimensional responsesurfaceplots(Fig.2).Fromthoseﬁgures,itcanbe con-cludedthattheTPCslightlyincreases,ataconstantirradiationtime, withanincreaseofmicrowavepowerfrom200to380and410W forseedsandskin,respectively,thenrapidlydecreasesfrom460 and480to600Wforseedsandskin,respectively.

Itwasalsoobservedthattheincreaseintheirradiationtimeat lowermicrowavepower,leadstotheincreaseoftheresponse.At lowermicrowavepower,theimpactofextractiontimehasa pos-itiveeffectontheTPCuptotheextractiontimeof84sand146 sforseedsandskinextractsrespectively,andthenithasa nega-tiveimpact.ItisveryinterestingtonotethattheTPC(1)decreases withtheincreasingof themicrowavepower forlonger extrac-tiontimesand(2)increasesatlowmicrowavepowerwithlong extractiontimeand highpowerwithshortexposure.Thereisa stronginteractionbetweenthesetwoparameters,whichisbased onthefunctionshape.Theseresultsconcludethatthemost efﬁ-cientmeantoextractphenolsfromgrapeseedsandskinsbyMAE istheapplicationofamoderatemicrowavepowerforashort expo-sure.Therefore,thelongerextractiontimewithhighermicrowave powercanaffectthequalityofthephenoliccompounds.Afoakwah

etal.(2012)reportedthat,theextractionprocessofMAEiswidely inﬂuenced bytime exposuretoheat. Thus,the quantityof tar-getcompoundsextractedcanbeincreasedwithanincreaseinthe extractiontime,butthereisanassociatedriskofdeteriorationof thermolabileones.Theresultsobtainedcanbeexplainedbythe factthattheincreaseofthemicrowavepowerinshortextraction timeleadstotheincreaseofthecontentofphenoliccompounds byincreasing thesolubilityofthetarget analytes,thediffusion rate, and themass transfer (Afoakwah et al.,2012; Dahmoune etal.,2013;Garcia-Salasetal.,2010).However,itwasnotedthat increasingthepoweruptoacertainvaluesmaypromote possi-bleconcurrentdecompositionofphenoliccompounds(Afoakwah etal.,2012;Muthuselvietal.,2012).Additionally,highpowermay encourage solventlossthrough evaporation(Garcia-Salas etal., 2010;Thabitetal.,2014).

Optimal conditions forseedsextract are: irradiationtime of 58.63sandmicrowavepowerof373.15WwithapredictedTPC yieldof73.29mgGAE/gDW.Forskinextract:irradiationtimeof 113.74sandmicrowavepower384.44WwithapredictedTPCyield of 54.38mg GAE/gDW. Thevalidity ofthose predictedoptimal valuesofTPCforeachmatrixextracts,wasalsoexperimentally con-ﬁrmed:73.15±0.20and54.84±0.41mgGAE/gDWforseedsand skin,respectively,veryclosetothevaluespredictedbythemodels. 3.4. ComparisonbetweenCSEandMAE

Table8showstheconcentrationsofphenoliccompoundsand the antioxidant activity (ABTS radical scavenging activity and reducing power) of the extracts obtained under the best con-ditions.The resultsindicate that TPC of seedsextract obtained withMAE wassigniﬁcantlylower (73.15±0.20mg/g)than that obtainedwithCSE (96.56±1.29mg/g).The sameobservationis

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Fig.2.ResponsesurfacemodelfortheTPCfromgrapeseedsandskinswithMAEtoirradiationtimeandmicrowavepower(A:seeds;B:skin).

Table8

Totalphenoliccontentandantioxidantactivityofdifferentextracts.

Matrixes TPCmgGAE/gDW ABTSmgQE/gDW FRPmgQE/gDW

CSE MAE CSE MAE CSE MAE

Seeds 96.56±1.29a _73.15_±_0.20b _182.24_±_5.44a _134.62_±_1.99b _181.90_±_1.09a _122.₉₄_±_1.10b

Skin 39.57±0.23b _54.84_±_0.41a _104.94_±_0.54b _108.36_±_1.14a _90.₉₈_±_0.94b _93.52_±_1.11a

Resultsarereportedasmeans±S.D.DifferentlettersinthesamelineandintwoconsecutivecolumnsrefertomeansstatisticallydifferentaccordingtoANOVAandTukey’s test.

notedfortheantioxidantactivity,indeedtheABTSandtheFRP values(182.24±5.44and181.90±1.09mgEQ/gDW,respectively) ofextractobtainedbyCSE weresigniﬁcantlyhigherthan those obtainedbyMAE(134.62±1.99and122.94±1.10mgEQ/gDW, respectively).

The lower TPC of seeds extracts obtained with microwave extractionascomparedtoconventionalextractioncanduetotheir heatsensibility.Theeffectoftemperaturecannotbegeneralized sinceitstronglydependsontypologyofcompounds(Spignoetal., 2007).

Theheattreatmentcausesirreversiblechangesintannins struc-ture,indeed,Padmaja(1989),hasreportedthedecreaseoftannin contentsincassavaleavesdriedat60◦C.Severalstudiesshowed that extractionoftannins and proantocyanidins,which arethe majorphenolic compoundsingrapeseeds(Bordigaetal.,2011; Cadotetal.,2006;Shietal.,2003),decreasewiththeincreaseof temperature(Makkarand Becker, 1996;Padmaja,1989; Spigno etal.,2007).Liazidetal.,(2007)alsoshowedthatthemicrowave radiationcaneasilydegradeunstablephenolicsduetotheir struc-tural properties. Phenolic compounds with higher number of hydroxyl substituent in their aromatic rings are unstable and caneasilybedegradedunder hightemperature conditions.The degradationofphenoliccompoundscangenerateadeclineonthe antioxidantactivity,whichcanexplainthelowerantioxidant activ-ityofextractsobtainedbyMAEincomparisontothoseobtainedby CSEmethod.Indeed,severalauthorsfoundasigniﬁcant correla-tionbetweenTPCandantioxidantactivity(Slusarczyketal.,2009; Veliogluetal.,1998).

Intheotherhand,TPC ofskinextractsobtainedbydifferent methodsshowedsigniﬁcantdifference(Table6).TheTPCobtained byMAEmethod(54.84±0.41)wassigniﬁcantly(p<0.05)higher thanthatobtainedbyCSEmethod(39.57±0.23mg/g).Thesame observationisnotedfortheantioxidantactivity.IndeedtheABTS andtheFRPvalues(104.94_±0.54and90.98_±0.94mgEQ/gDW, respectively)ofextract obtainedby CSEwerelowerthan those obtainedby MAE (108.36±1.14and 93.52±1.11mg EQ/g DW, respectively).

For extracting phenolic compounds, Hithamani and Ramalakshmi,(2013)andRafieeetal.(2011)haveshownthatMAE wasmoreeffectivethantheconventionalextractionmethodswith reducinginextractiontime.Thereductionoftheextractiontime wasduetotheheatingmechanismofthemicrowaves(Rafieeetal., 2011;Yangetal.,2013).Anenhanceintemperatureaddstothe efficacyoftheprocess,indeedheat(1)decreasestheviscosityof thesolvent,whichmakeseasyitspassagethroughthesolidmatrix sotheefficiencyoftheextraction.Intheotherhand,it(2)increases thepermeabilityofcellwallswhichpromotesthesolubilityand thediffusioncoefficientsofthebiologicalactivecompoundstobe extracted(Afoakwahetal.,2012;Muthuselvietal.,2012;Wissam etal.,2012);Asaresult,microwaveincomparisontoconventional extractioncanextractpolyphenolsinshortertimes(Bayramoglu etal.,2008).

Theshorterextractiontimeinmicrowaveprocessingcanreduce thedegradationofbiologicalactivecompounds(Inceetal.,2014; Ravaljietal.,2012),whichexplainstheresultoftheantioxidant activityofgrapeskinextracts.In fact,theloweractivityofCSE extractcouldbearesultoftheextensionintheextractiontime; thelongexposureoftheextractstounfavorableconditionssuch aslight,togetherwithoxygenandtemperature,isthemost impor-tantfactorthatfacilitatesdegradationreactions(Garcia-Salasetal., 2010).

4. Conclusion

Theaimofthisstudywas,ontheonehand,toobtaina bet-tersurveyintooptimizationofextractionconditionsofphenolic compoundsfrom Algerian grapecv. AhmarBou-Amar withtwo extractionmethods(CSEandMAE)usingRSM,andontheother handtocomparethoseextractionmethodsbasedonthemaximum TPCandantioxidantactivitymeasuredwithABTSandFRP.

Theresultsindicatedthatthemathematicalmodelsusedinthe presentinvestigationworkwellforthepredictionofTPCextraction fromgrapecv.AhmarBou-Amarseedsandskin.Thus,thetwo stud-iedextractionmethodsweresuccessfullyoptimizedbyBBDand CCDofRSM.Ontheotherhand,theTPCofseedsextractobtained

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withMAEwas24%lowerthanthatoftheCSEextract;also,the antioxidantactivityofCSEextract wasbetterthan thatofMAE extract.While,theTPCofskinextractobtainedwithMAEwas28% higherthanthatoftheCSEextractandtheantioxidantcapacitywas signiﬁcantlyhigherthanthatoftheCSEextract.Therefore,(1)itcan beconcludedthat,MAEisagoodalternativefortheextractionof phenoliccompoundsfromgrapeskinofthestudiedvariety,sinceit providesahighquantityandahighantioxidantqualitycompared toCSEbydrasticallyreducingthetimeoftheprocess(98%).(2)In ordertomaximizerecoveryofTPCfromgrapeseedsofthestudied variety,itisbettertorealizetheextractionwithCSEmethodwith longerextractiontimecomparedtoMAE,butitisnoteworthyto pointthat,MAEpermitstoextractinonly58.63sabout76%ofTPC whichwereextractedbyCSEduring65.23min.

Thus,fromthepointofviewofindustrialapplication,MAEcan berecommendedforextractingantioxidantcompoundsfromgrape cv.AhmarBou-Amarbyproductswithareductioninextractiontime ofabout98%comparedtoCSE.Additionally,fortheirhigh con-tentsofphenoliccompoundsincomparisontoothersvarieties,the extractsofgrapecv.AhmarBou-Amarseedsandskincanbe consid-eredasagoodsourceofnaturalantioxidantsandmaybeexploited asnaturalantioxidantagentsforhealthfoodassubstituted antiox-idanttothesyntheticantioxidantswithgreatcommercialinterest inthefoodandphyto-pharmaceuticalmarket.

Acknowledgements

WewouldliketothankallmembersofUVVlaboratoryatthe INRAofAngers(France)fortheirprecioushelp,especiallyGérard Barbeau,Marie-HélèneBouvetandAnneMège.Aswellasaspecial thanktoMrsWahibaAzegaghforherprovidingEnglishlanguage help.

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