Valorization of Citrus limon residues for the recovery of antioxidants: evaluation and optimization of microwave and ultrasound application to solvent extraction

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ContentslistsavailableatSciVerseScienceDirect

Industrial

Crops

and

Products

j o u r n a l ho me p a 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

Valorization

of

Citrus

limon

residues

for

the

recovery

of

antioxidants:

Evaluation

and

optimization

of

microwave

and

ultrasound

application

to

solvent

extraction

Farid

Dahmoune

a

_,

_Lila

_Boulekbache

a

_,

_Kamal

_Moussi

a

_,

_Omar

_Aoun

a

_,

_Giorgia

_Spigno

b,∗

_,

Khodir

Madani

a

a_Laboratory_of_{Biomathematics,}_{Biochemistry,}_Biophysics_and_{Scientometrics,}_Abderrahmane_Mira_University_of_Bejaia,₀₆₀₀₀_Bejaia,_Algeria b_Institute_of_Oenology_and_Food_Engineering,_Università_Cattolica_del_Sacro_Cuore,_Via_Emilia_Parmense_84,₂₉₁₂₂_Piacenza,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received11March2013

Receivedinrevisedform17June2013 Accepted4July2013

Keywords: Citruslimonpeels Optimization

Microwaveassistedextraction Ultrasoundassistedextraction Phenoliccompounds

a

b

s

t

r

a

c

t

Ultrasoundassistedextraction(UAE)andmicrowave-assistedextraction(MAE) wereoptimized (by responsesurfacemethodology,RSM)andcomparedfortherecoveryoftotalphenoliccompounds(TPC expressedasgallicacidequivalents(GAE))fromCitruslimonpeels.TheoptimizedresultforMAEwas48% ethanolasextractionsolvent,28:1mL/gofsolvent:solidratio,123sand400Wforirradiationtimeand power.TheoptimizedresultforUAEwas63.93%ethanolasextractionsolvent,40mL/gofliquid/solid ratio,15.05minofholdingtimeand77.79%foramplitude.MaximumpredictedTPCrecoveriesunder theoptimizedconditionsforMAEandUAEwere15.74and15.08mgGAE/grespectively,whichwere closetotheexperimentalvaluesof15.78±0.8and15.22±0.88mgGAE/g,indicatingsuitabilityofthe employedmodelandthesuccessofRSMinoptimizingtheextractionconditions.Theantioxidantactivity determinedbytheDPPHandreducingpowertestsconﬁrmedthesuitabilityofMAEforthepreparation ofantioxidant-richplantextracts.

1. Introduction

Citrusisthemostimportantfruitcropintheworld.The

pro-ductionofcitrusfruitshasbeenincreasingenormouslyinthelast

fewdecades,goingfromanaverageof48milliontonsayearinthe

period1970–1979,tomorethan100milliontonsin2004–2005

(DugoandDiGiacomo,2002;González-Molinaetal.,2010).Citrus

fruitsarecultivatedinmorethan100countriesallovertheworld,

mainlyintropicalandsubtropicalareas,wherefavorablesoiland

climaticconditionsprevail(DugoandDiGiacomo,2002).

Amongfruitandvegetables,citrusfruitsarereportedtobea

veryrichsourceofhealthpromotingsubstances(Artés-Hernández

etal.,2007).Althoughtheirhealth-relatedpropertieshavealways

beenassociatedwiththeircontentofvitaminC,ithasrecentlybeen

shownthatﬂavonoidsalsoplayaroleinthisrespect.Inancient

medicineCitruslimon(C.limonBurm)andmelissa(Melissa

ofﬁci-nalisL.)havelongbeenusedasnaturalinsectrepellents(Oshaghi

etal.,2003).

∗ Correspondingauthor.Tel.:+390523599181;fax:+390523599232. E-mailaddresses:farid.dahmoune@yahoo.fr(F.Dahmoune),

lilaboulekbachemakhlouf@yahoo.fr(L.Boulekbache),moussikamal@yahoo.fr

(K.Moussi),aoun.omar@yahoo.fr(O.Aoun),giorgia.spigno@unicatt.it(G.Spigno),

madani28dz2002@yahoo.fr(K.Madani).

Citrusisanimportantcropmainlyusedinfoodindustriesfor

freshjuiceproduction.Peels,themainwastefractionofcitrusfruits,

represent roughly halfof the fruitmass and have been widely

studiedbecausetheycontainnumerousbiologicallyactive

com-poundsincludingnaturalantioxidantssuchasphenolicacidsand

ﬂavonoids(Hayatetal.,2009,2010).

Theﬁrststepforbothanalysisandexploitation ofmedicinal

plant bioactive constituents is their extraction from the

cellu-larmatrix.The“ideal”extractionmethodshouldbequantitative,

non-destructive, and time saving.Besides conventional solvent

extractionprocessescommonlyusedfortherecoveryof

pheno-lic compounds (Proestos et al., 2006), non-conventional, more

rapidandautomatedmethodshavebeenrecentlyused,e.g.

super-criticalﬂuidextraction(SFE),pressurizedliquidextraction(PLE),

microwave-assisted extraction (MAE) and ultrasound assisted

extraction(UAE)(Aybasteretal.,2013;Liazidetal.,2007).

Actu-ally,thestateoftheartintheseﬁeldshasshownthatultrasound

andmicrowaveradiationcouldacceleratetheextractingprocess

improvingbioactivecompoundsextraction,particularlyphenolic

compounds(Garofuli ´cetal.,2013; Mu ˜niz-Márquezetal.,2013;

MorelliandPrado,2012;Lietal.,2011).

MAEisattractivebecauseitallowsforrapidheatingof

aque-oussamplesandpresentsadvantagesoverconventionalextraction

techniques,suchasimprovedefﬁciency,reducedextractiontime,

lower solvent consumption, higher selectivity toward target

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moleculesandhigherlevelofautomation(Hanetal.,2011;Singh etal.,2011;Zhangetal.,2008).Inadditionoftheseadvantages,a

widerrangeofsolventscanbeusedinMAE,asthetechniqueisless

dependentonsolventafﬁnity(YangandZhai,2010).

UAE is particularly attractive for its simplicity, low cost of

equipment(Carreraetal.,2012),efﬁciencyinextractinganalytes

fromdifferentmatrices(HerreraanddeCastro,2005),lowenergy

requiredandreducedsolvent-andtime-consumingmethod(Xia

etal.,2011).Theenhancementoftheextractionprocessby

ultra-soundsisattributedtothedisruptionofthecellwalls,reductionof

theparticlesizeandtheincreaseofthemasstransferofthecell

con-tenttothesolventcausedbythecollapseofthebubblesproduced

byacousticcavitation(Xiaetal.,2011;Rodriguesetal.,2008).

Toourbestknowledge,noliteraturereportexistsonthe

opti-mization and comparison of MAE and UAE procedure for the

extractionoftotalphenoliccompounds(TPC)fromC.limonpeels.

Therefore,theobjectivesofthecurrentstudywereto:

• investigatetheeffectsofdifferentparametersontheextraction

efﬁciency(intermsofrecoveryandantioxidantactivityoftotal

phenoliccompounds)byMAEandUAEprocesses;

• optimize the MAE and UAE conditions by response surface

methodology(RSM);

• comparetheoptimizedMAEandUAEresultswithareference

ConventionalSolventExtractionprocedure(CSE).

2. Materialsandmethods

2.1. Plantmaterial

ThefruitsamplesofC.limonwerecollectedintheareaofSidi

Aich(Bejaia,Algeria),washedwithdistilledwaterandpeeledoff

withhands.Peelsweredriedforabout15daysatroomtemperature

inaventilateddarkroomtoprotecttheactivecompoundscontent

fromlightoxidation.Driedpeelsweregroundwithanelectrical

grinder(IKAmodelA11Basic,Germany),thepowderwaspassed

throughstandard125␮msieveandonlythefractionwithparticle

size<125␮mwasused.Thepowderwasstoredinairtightbags

untiluse.Thewateractivity(aw)wasdeterminedbyHygroPalm

AWandwas0.25±0.02at23◦C.

2.2. Reagents

Sodiumcarbonate(Na2Co3),Folin–Ciocalteau’sphenolreagent

and disodium hydrogen phosphate (Na2HPO4) were obtained

from Prolabo (made in CE) and 1,1-diphenyl-2-picryl-hydrazil

(DPPH)fromSigmaAldrich(Germany).Gallicacid,ferricchloride

(FeCl3·6H2O), potassiumferricyanide (C6N6FeK3), trichloroacetic

acidandsodiumdihydrogenphosphate(NaH2PO4)werepurchased

fromBiochem-chemopharma(UK).Allsolventsusedwereof

ana-lyticalgradeandpurchasedfromProlabo(CE).

2.3. Experimentalwork

For optimization of theMAE and UAE procedure, the

inﬂu-encesoftheprocessparameterswereﬁrstlyseparatelyinvestigated

insingle-factorexperimentstolimitthetotalexperimentalwork

(Tables1and2).Whenonevariablewasnotstudied,itwaskept

constant. In the MAE trials, the constant values for irradiation

time,solvent-to-solidratioandethanolconcentrationwere120s,

20mL/gand50%,respectively.Microwavepowerwassetat500W

inthetrialstoinvestigatetheinﬂuenceofsolventtypeandethanol

concentration,andat400Winthetrialstoinvestigatethe

inﬂu-enceofsolvent-to-solidratio.IntheUAEtrials,theconstantvalues

ofsonicationtime,solvent-to-solidratioandethanol

concentra-tionwere10min,40mL/gand50%.Radiationamplitudewassetat

50%inthetrialstoinvestigatetheinﬂuenceofethanol

concentra-tionandsonicationtime,andat60%inthetrialstoinvestigatethe

inﬂuenceofsolvent-to-solidratio.

Onthebasis ofthesingle-factor experimentalresults,major

inﬂuence factors were selected. Then, an RSM based on a

Box–BehnkenDesign(BBD)forMAEandCentralComposite

Rotat-able Design (CCRD) for UAE was conducted to optimize both

processes(Tables3and4)(Vázquezetal.,2012;Wuetal.,2013).

Regressionanalysisofthedatatoﬁtasecond-orderpolynomial

equation(quadraticmodel)wascarriedoutaccordingtothe

fol-lowinggeneralequation(Eq.(1))whichwas,then,usedtopredict

theoptimumconditionsofextractionprocess.

Y=B0+

k i=1BiXi+

k i=1BiiX 2₊

k i>1BiiXiXj+E (1)

whereYrepresentstheresponsefunction(inourcasetheTPC

yield);B0isaconstantcoefﬁcient;Bi,BiiandBijarethecoefﬁcients

ofthelinear,quadraticandinteractiveterms,respectively,andXi

andXjrepresentthecodedindependentvariables.Accordingtothe

analysisofvariance,theregressioncoefﬁcientsofindividual

lin-ear,quadraticandinteractiontermsweredetermined.Inorderto

visualizetherelationshipbetweentheresponseand

experimen-tallevelsofeach factorandtodeducetheoptimumconditions,

theregressioncoefﬁcientswereusedtogenerate3-Dsurfaceplots

fromtheﬁttedpolynomialequation.Thefactorlevelswerecoded

as−1(low),0(centralpointormiddle)and1(high),respectively.

Thevariableswerecodedaccordingtothefollowingequation(Eq.

(2)):

xi=Xi_X−X0 (2)

wherexiisthe(dimensionless)codedvalueofthevariableXi;X0is

thevalueofXatthecenterpointandXisthestepchange.

Analysisofvariancewasperformedfortheresponsevariable

usingthefullmodelwhereP-values(partitionedintolinearand

interactionfactors)indicatedwhetherthetermsweresigniﬁcant

ornot.Toverifytheadequacyofthemodels,additionalextraction

trialswerecarriedoutattheoptimalconditionspredictedwiththe

RSMand theobtainedexperimentaldatawerecomparedtothe

valuespredictedbytheregressionmodel.

Efﬁciencyofthetwonon-conventionalmethods(MAEandUAE)

wascomparedbasedontheTPCrecoveryandthequalityofthe

recoveredphenolic compoundsin terms of antioxidantactivity

(accordingtoDPPHassayandFereducingability)whichwas

mea-suredonlyontheextractsobtainedundertheoptimumconditions

selectedbyRSM.

OptimizedMAEandUAEconditionswerethencomparedtoa

referenceCSEprocedure.

Finally, in order to investigate the inﬂuence of ultrasound,

microwaveandmacerationonthemicrostructureofthesamples

powder, thepeel powder samples (before and after the

differ-ent extraction processes) were observed by scanning electron

microscopy(SEM).

2.3.1. Microwaveassistedextraction

Adomesticmicrowaveoven(NN-S674MF,Samsung,Malaysia)

with cavity dimensions of 22.5cm×37.5cm×38.6cm and

2450kHz working frequency was used. The apparatus was

equippedwitha digitalcontrol systemfor irradiationtime and

microwave power (the latter linearly adjustable from 200 to

1000W).Theovenwasmodiﬁedinordertocondensateintothe

samplethevaporsgeneratedduringextractiongivingaconstant

samplevolume.

Fortheextraction, onegramofthepeel powderwasplaced

in a 250mLvolumetric ﬂaskcontaining theextractionsolvent.

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Table1

Resultsofsingle-factorexperimentsformicrowaveassistedextraction.Resultsarereportedasmeans±S.D.Samelettersinthesamecolumnrefertomeansnotstatistically differentaccordingtoANOVAandTukey’stest.TPC,totalphenolsyieldreferredtodryweight(dw)ofC.limonpeels;GAE,gallicacidequivalents.

Solvent Ethanolconcentration Irradiationtime Microwavepower Solvent-to-solidratio Type TPCyield (mgGAE/gdw) (%v/v) TPCyield (mgGAE/gdw) (s) TPCyield (mgGAE/gdw) (W) TPCyield (mgGAE/gdw) (mL/g) TPCyield (mgGAE/gdw) Water 7.38±1.19b ₂₀ _10.43 ±1.28bc 90 10.04±0.95c ₃₀₀ _10.65_±_1.30b ₁₅ _12.20_±_0.46c 50%MeOH 8.50±0.89b ₄₀ _12.11_±_0.62ab ₁₂₀ _13.68_±_0.24a ₄₀₀ _13.68_±_0.34a ₂₀ _13.68_±_0.14b 50%EtOH 12.30±0.47a ₅₀ _13.30_±_0.47a ₁₅₀ _12.88_±_0.30ab ₅₀₀ _12.30_±_0.39ab ₂₅ _14.72_±_0.39a 50%Acetone 9.18±0.44b ₆₀ _10.95_±_0.16bc ₁₈₀ _11.86_±_0.81b ₆₀₀ _12.09_±_0.23ab ₃₀ _13.50_±_0.27b 80 9.95±0.81c ₂₁₀ _11.58_±_0.27bc 240 11.44±0.90bc Table2

Resultsofsingle-factorexperimentsforultrasoundassistedextraction.Resultsarereportedasmeans±S.D.Samelettersinthesamecolumnrefertomeansnotstatistically differentaccordingtoANOVAandTukey’stest.TPC:totalphenolsyieldreferredtodryweight(dw)ofC.limonpeels.GAE,gallicacidequivalents.

Ethanolconcentration Sonicationtime Amplituderadiation Solvent-to-solidratio

(%) TPCyield(mgGAE/gdw) (min) TPCyield(mgGAE/gdw) (%) TPCyield(mgGAE/gdw) (mL/g) TPCyield(mgGAE/gdw)

30 12.02±0.53b ₅ _12.30_±_0.60b ₂₀ _12.03_±_1.20c ₂₀ _11.09_±_1.20b

50 14.10±0.21a ₁₀ _14.16_±_0.42a ₄₀ _13.16_±_0.71bc ₃₀ _12.11_±_0.60b

70 13.20±0.41ab ₁₅ _15.11_±_0.31a ₆₀ _14.90_±_0.83a ₄₀ _14.88_±_0.30a

100 12.11±1.12b ₂₀ _12.21_±_0.36b ₈₀ _14.51_±_0.22ab ₅₀ _15.02_±_0.51a

100 14.00±0.33abc

ovenoperation.Dependingonthetrial,adifferentsolvent,

irradi-ationtime,microwavepowerandsolvent-to-solidratiowereused

(Tables1and3).Attheendofmicrowaveirradiation,thevolumetric

ﬂaskwasallowedtocooltoroomtemperature.

Afterextraction, theextractwasrecovered byﬁltrationin a

BüchnerfunnelthroughWhatmanNo.1paper,andcollectedina

volumetricﬂask.Theextractwasstoredat(4◦C)untiluseand

ana-lyzedfortheTPCand,fortheextractobtainedundertheoptimum

conditionsbyRSM,alsofortheantioxidantactivity.

2.3.2. Ultrasoundassistedextraction

Anultrasonicapparatus(SONICSVibracell,VCX130PB,Stepped

microtipsandprobes,No.630-0422)wasusedforUAEwith

work-ingfrequencyﬁxedat20kHz.

For theextraction, onegramof thepeel powderwasplaced

ina250mLamberglassbottlecontainingtheextractionsolvent.

Thesuspensionwasexposedtoacousticwavesundervariations

ofethanolconcentration,irradiationtime,amplitudeand

liquid-to-solid ratio(Tables 2 and4).The temperaturewascontrolled

Table3

Box–Behnkendesignwiththeobservedresponsesandpredictedvaluesforyieldoftotalphenoliccompounds(TPC)referredtodryweight(dw)ofC.limonpeelsusing microwaveassistedextraction.GAE,gallicacidequivalents.

Run X1–ethanolconcentration(%v/v) X2–time(s) X3–power(W) X4–ratio(mL/g) TPCyield(mgGAE/gdw)

Experimental Predicted 1 50 120 400 25 15.00±0.83 14.97 2 60 150 400 25 13.49±1.48 13.48 3 50 90 400 30 14.19±0.90 14.23 4 50 120 300 20 13.72±0.88 13.76 5 60 120 300 25 12.49±0.99 12.70 6 50 120 500 20 14.05±1.49 14.17 7 50 150 400 20 14.05±1.41 14.22 8 50 120 400 25 14.98±0.93 14.97 9 50 150 500 25 13.44±0.99 13.57 10 40 120 400 20 14.33±0.93 14.49 11 60 120 400 20 13.65±0.95 13.75 12 50 90 500 25 13.49±1.79 13.59 13 50 120 300 30 13.74±1.11 13.98 14 40 90 400 25 14.30±1.06 13.97 15 50 150 400 30 14.51±0.99 14.76 16 60 90 400 25 12.91±0.88 12.93 17 50 90 400 20 14.23±1.32 14.36 18 50 90 300 25 12.92±0.95 12.99 19 60 120 400 30 13.84±1.25 14.01 20 50 150 300 25 13.25±0.83 13.40 21 40 150 400 25 13.93±1.25 13.82 22 40 120 400 30 14.40±1.14 14.64 23 50 120 500 30 14.61±1.11 14.35 24 40 120 500 25 13.58±0.83 13.77 25 50 120 400 25 14.93±0.88 14.97 26 40 120 300 25 13.40±0.97 13.34 27 60 120 500 25 13.38±1.20 12.99

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Table4

CentralCompositeRotatableDesignwiththeobservedresponsesandpredictedvaluesforyieldoftotalphenoliccompounds(TPC)referredtodryweight(dw)ofC.limon peelsusingultrasoundassistedextraction.GAE,gallicacidequivalents.

Run X1–time X2–amplitude X3–solvent TPCyield(mgGAE/gdw)

Experimental Predicted 1 5 30 30 12.92±1.20 13.21 2 15 30 30 11.09±0.32 11.27 3 5 70 30 14.15±0.53 14.18 4 15 70 30 12.49±0.45 12.71 5 5 30 70 13.01±0.15 12.72 6 15 30 70 13.69±1.02 13.59 7 5 70 70 14.38±0.36 14.13 8 15 70 70 15.82±0.96 15.47 9 10 50 50 15.01±0.89 14.91 10 10 50 50 14.51±0.16 14.91 11 10 50 50 15.01±1.60 14.91 12 10 50 50 14.91±0.99 15.01 13 10 50 50 15.92±1.23 14.57 14 17.35 50 50 14.58±0.52 13.57 15 2.65 50 50 14.91±0.86 15.67 16 10 79.4 50 15.48±0.36 12.28 17 10 20.6 50 13.67±0.45 13.95 18 10 50 79.4 13.32±0.12 13.26 19 10 50 20.6 12.82±1.22 13.25

continuouslyandkeptatroomtemperaturebycirculatingexternal

coldwaterbath.Aftertheextraction,theextractwasrecoveredand

analyzedasreportedforMAEinSection2.3.1.

2.3.3. Conventionalsolventextraction

Fortheconventionalextraction,onegramsofsamplepowder

wereplacedinaconicalﬂask,and50mLof50%(v/v)ethanolwere

added.Themixturewaskeptinathermostaticwaterbath(mod.

WNB22Memmert,Germany)withshakingspeedof110strokesper

minute,at60◦Cfor2h,accordingtothemethodrecommendedby

Spignoetal.(2007).Theextractwasthenrecoveredandanalyzed

asreportedforMAEinSection2.3.1.

2.4. Analyticaldeterminations

2.4.1. Totalphenoliccontent(TPC)

Totalphenoliccontentoftheextractswasdeterminedaccording

totheFolin–Ciocalteuassay(Jaramillo-Floresetal.,2003).Brieﬂy,

100␮Loftheextract(opportunelydilutedindistilledwater)was

mixedwith750␮Lof10-folddilutedFolin–Ciocalteaureagent.The

solutionwasmixedandincubatedatroomtemperaturefor5min.

After5min,750␮Lof7.5%sodiumcarbonate(Na2CO3)wereadded.

Afterincubationat25◦Cfor90min,theabsorbanceofthe

sam-plewasmeasuredat725nmagainstablank(madeasreportedfor

thesamplebutwith100␮Lofdistilledwater)byusingaUV–vis

Spectrophotometer(SpectroScan50,UK).

Gallicacidhydratewasusedasstandardforthecalibrationcurve

toexpresstheTPCconcentrationofthesampleasmg/Lofgallic

acidequivalents(GAE).TPCyieldwasthencalculatedbasedonthe

sampleconcentration,extractvolumeandpeelpowderdryweight,

accordingtothefollowingequation(Eq.(3)):

TPCYield=mgGAE/L·Lextract

gpeelpowder

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2.4.2. AntioxidantactivitybyDPPHassay

Theelectrondonationabilityoftheextracts(CSEsamplesand

MAEandUAEsamplesobtainedunderoptimizedcondition)was

measured by bleaching of the purple-colored solution of

1,1-diphenyl-2-picrylhydrazylradical(DPPH)accordingtothemethod

ofDudonnéetal.(2009).

DPPHradicalshaveanabsorptionmaximumat515nm,which

disappearswithreductionbyanantioxidantcompound.ADPPH•

solutioninabsolutemethanol(60␮M)wasprepared,and3mLof

thissolutionweremixedwith0.1mLofpeelsextractdilutedin

distilledwater(dilutionswerebasedontheTPCinordertohave

100,200,300,400and500_␮g/mL).Thesampleswereincubatedfor

20minat37◦Cinawaterbathand,then,thedecreaseinabsorbance

at515nmwasmeasured.

Theantioxidantcapacitywasexpressedaspercentageof

inhi-bitionofDPPHradical(%DPPHinhibition)calculatedaccordingto

thefollowingequation(Eq.(4)):

%DPPHinhibition=Abscontrol−Abssample

Abscontrol ×

100 (4)

whereAbscontrolistheabsorbanceofDPPHradical+distilledwater;

Abssample is the absorbance of DPPH radical+sample extract at

20min.Fromdataelaboration (%inhibition plotted versustotal

phenolsconcentration),theconcentrationofTPCrequiredtoreach

50%radicalinhibition(IC50)wascalculated.

2.4.3. Antioxidantactivityasironreducingpower

Thereducingpoweroftheextracts(CSEsamplesandMAEand

UAEsamplesobtainedunderoptimizedcondition)wasevaluated

asthecapacityofreducingFe3+_to_Fe2+_according_to_the_method

ofShon(2003).Todeterminethereducingpower,four

concentra-tionsofpeelextract(50,100,150and200␮gGAE/mLobtained

dilutingwithdistilledwater)wereaddedseparatelyto2.5mLof

phosphatebuffer(0.2M,pH6.6)and2.5mLof1%potassium

fer-ricyanide.The mixturewasincubatedat50◦C for20min.After

thistime, thereactionwasterminatedbyadding 2.5mLof10%

trichloroaceticacidandthemixturewascentrifugedat650×gfor

10min.Thesupernatantwasmixedwith5mLofdistilledwater

and1mLof0.1%ferricchloride(FeCl3),andthenabsorbancewas

measuredat700nm.Highabsorbancevalueshowshighreducing

activity(Panetal.,2010).

2.4.4. Scanningelectronmicroscopy(SEM)analysis

ThepowderbeforeandafterextractionwasobservedunderSEM

(Quanta200,FEIcompany)formorphologicalcharacterization(Lou

etal.,2010).Foursamplesofpowders(untreatedandresiduesafter

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werecollectedanddrieduntilconstantmassinovenat60◦Cbefore

SEManalysis.

Sampleparticleswerefixedonthespecificcarbonfilmsupport,

andtheirshape and surfacecharacterswere observedbyusing

GSEDdetectorwithenvironmentalmode(ESEM).

2.5. Statisticalanalysis

Eachextractiontrialand alltheanalyseswerecarriedoutin

triplicate and allthe data in this paper have been reportedas

means±S.D.InﬂuenceofeachfactorontheTPCyieldinthe

single-factor experiment for both the MAE and UAE was statistically

assessedbyANOVAandTukey’sposthoctestwith95%conﬁdence

level.

DataobtainedfromtheBBDandCCRDtrialsfortheMAEand

UAE,respectively,werestatisticallyanalyzedusingANOVAforthe

responsevariableinordertotestthemodelsigniﬁcanceand

suit-ability.P<0.05 andP<0.01weretakenassigniﬁcantandhighly

signiﬁcantlevel,respectively.

TheJMP (Version 7.0, SAS)and Design-Expert (Trialversion

8.0.7.1)softwarewereusedtoconstructtheBBDand CCRDand

toanalyzealltheresults.

3. Resultsanddiscussion

3.1. Microwaveassistedextraction

3.1.1. Single-factorexperiments

Table1showstheresultsofthesingle-factorexperiments

car-riedoutforpreliminaryoptimizationofMAE.

Atthebeginningofthis study,aneffectofsolventtype was

investigated. Theinvestigated solvents arethe mostcommonly

employedsolventsinphenolicextractionfrombotanicalmaterials

(Inglettetal.,2010;Spignoetal.,2007).Typeofsolvent

signiﬁ-cantlyinﬂuencedtheTPCyield,withaqueousethanolbeingthe

bestoneandwater,acetoneandaqueousmethanolgiving

compa-rableresultswhichmaybeattributedtothedifferenceindielectric

propertiesofthesolvent.Waterhasthehighestdielectricconstant

(ε₌_80.4) _and _the _lowest _dissipation _factor _(tan₌_1570), _hence,

therateatwhichwaterabsorbsmicrowaveenergyishigherthan

therateatwhich thesystemcandissipatetheheat,these

phe-nomenaaccountforthe“superheating”effectwhichoccurswhen

waterisused(Proestos andKomaitis,2008).Furthermore,since

theFolinassayusedtoquantifyTPCactuallymeasuresthe

reduc-ingpowerofthecompounds,theFolinresultsareinﬂuencedby

theoxidative status ofthe sample,therefore lowervalues may

suggestthatintenseheatinghascauseddegradationofthe

bioac-tivecompounds.However,mixturesofhighandlowmicrowave

absorbingsolventscanbeexploitedtoproduceoptimumresults.

Forthetestedaqueoussolvents,theethanol/watersystemhasthe

highestboilingpoint.Thisallowedcarryingoutextractionathigher

temperaturesresultinginimprovedextractionefﬁcienciesdueto

increasedmasstransfercoefﬁcients ofTPC andreduced surface

tensionandsolventviscositywhichimprovesamplewettingand

matrixpenetration.Thisisinagreementwithresultsreportedby

Panetal.(2003).Aqueousethanolwasthenselectedasthesolvent

fortheRSMtrialsandforthenextsingle-factortrials.

Inagreementwithotherliteratureresults(Spignoetal.,2007;

Pan et al., 2003), TPC yield increased with increasing ethanol

concentrationupto50%(v/v)andthendecreasedforhigher

con-centrations.ThemaximumTPCyieldwasobtainedwithethanol

50%(eventhoughstatisticallyequaltoethanol40%)whichwas

ﬁxedforthenextsingle-factorexperiments,while,basedonthe

statisticalanalysis reportedin Table1,the concentrationrange

40–60%wasselectedfortheRSMtrials.

AlsoirradiationtimesigniﬁcantlyinﬂuencedtheTPCyield,with

120–150s as the best values, with apparentthermal

degrada-tionofphenoliccompoundsatlongerirradiationtimes(Proestos

andKomaitis,2008;Proestosetal.,2006).Basedontheseresults,

120swasselectedforthenextsingle-factortrials,whiletherange

90–150swasselectedfortheRSMtrials.

MicrowavepowersigniﬁcantlyinﬂuencedtheTPCyieldinthe

tested conditions (Table 1). However, the yield increased with

increasing microwave power from 300 to 400W and then it

remainedconstantorslightlydecreasedforhigherpowers(average

valuesat500and600Wwerestatisticallynotdifferentfromboth

valuesat300and400W).Itmustbeunderlinedthattheoperating

temperaturecouldnotberegulatedintheusedequipmentandthat

foraconstantsamplesizetemperatureincreaseswithmicrowave

power(SpignoandDeFaveri,2009).Sinceithasbeenreportedthat

themaineffectofmicrowaves,andinmanycasestheonly,isthe

heatingeffect(Kappeetal.,2013),theobtainedresultswere

prob-ablyduetoanenhancementofphenolsrecoveryduetoaheating

effect,withconsequentincreaseofmasstransferphenomena,upto

acertainmicrowavepowervalueand,then,tothermaldegradation

ofbioactivecompoundsathigherpowers.

Therange300–500WwasselectedfortheRSMstudy,whilethe

400Wpowerwasusedforthelastsingle-factortrials.

Thesolvent-to-solidratiosigniﬁcantlyinﬂuencedtheTPCyield,

showingthesametrendasreportedbySpignoandDeFaveri(2009).

Infact,inthepresentstudy,theratiowasincreasedkeeping

con-stantthesolidweight,therefore,thepositiveeffectofanincreased

volumeduetoalargerconcentrationgradienttodrivethemass

transfer,is,atacertainpoint,compensatedbyalowerheatingdue

toreducedmicrowavepenetrationinthesample.Furthermore,the

solventvolumemustbesufﬁcienttoensurethattheentiresample

isimmersed,especiallywhenhavingasamplethatwillswell

dur-ingtheextractionprocess(EskilssonandBj ¨orklund,2000).Based

onstatisticalanalysis,therange20–30mL/gwasselectedforthe

RSMoptimization.

3.1.2. OptimizationbyRSM

TheTPCyieldsobtainedinthetrialsoftheBBDarereportedin

Table3,whileTable5reportsthestatisticalanalysisofthe

regres-sionmodel(Eq.(1)).Neglectingthenon-signiﬁcantterms(P>0.01)

thefollowingpredictiveequationwasobtained:

Y =14.91−0.35X1−0.23X3+0.13X4+0.23X1X2+0.22X3X4

−0.72X12₋₀_.6X22₋₀_.97X32 ₍₅₎

Itcanbeseenthatirradiationtime(X2)didnotinﬂuencethe

extractionyieldbutitsinteractionwithethanoldidit.The

inter-actionpowerandsolvent-to-solidratiowashighlysigniﬁcantsuch

asthequadratictermsforethanol,timeandmicrowavepower.

Eq.(5)wasusedtoobtaintheresponsesurfacesforallthe

pos-siblevariablesinteractions(Fig.1),wheremaximalTPCyieldcould

beobtainedat optimal-valuesas combination oftwo variables,

withthethird and fourthvariables keptconstantat zerolevel.

Consequently,thequadraticexperimentalmodelhadastationary

point,andthepredictiveTPCyieldwasthemaximalvalueinthe

stationarypoint(Panetal.,2012).Conﬁrming theresultsofthe

single-factortrials,theTPCyieldreachedamaximumlevelwhen

irradiationtime,ethanolconcentrationandextractionpowerwere

setatmediumlevels(0codedvalues),whilethesolvent-to-solid

ratioshowedaverylimitedinﬂuencewhichwasapparentlyin

con-trastwithsingle-factorexperiments.However,inthe20–30mL/g

solvent-to-solidratiorangeselectedfortheRSMoptimization,the

single-factorresultshadshownasigniﬁcantincreaseinTPCyield

fromthe20to25ratioand,then,asigniﬁcantreductionfromthe

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Fig.1.ResponsesurfaceanalysisforthetotalphenolicyieldfromC.limonpeelswithmicrowaveassistedextractionwithrespecttoirradiationtimeandethanolpercentage (A);solvent-to-solidratioandethanolpercentage(B);microwavepowerandethanolpercentage(C);solvent-to-solidratioandmicrowavepower(D);solvent-to-solidratio andirradiationtime(E);microwavepowerandirradiationtime(F).

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Table5

Analysisofmeansquaredeviationofthequadraticmodelterms(Eq.(1))appliedtotheexperimentalvaluesoftotalphenolicyieldsobtainedwithmicrowaveassisted extraction.df,degreesoffreedom.

Source Sumofsquares df F-value P-valueProb>F

Model 0.0550 14 39.7721 <0.0001 Signiﬁcant X1–ethanol 0.0079 1 79.8241 <0.0001 X2–time 0.0001 1 2.0073 0.1820 X3–power 0.0034 1 34.0340 <0.0001 X4–ratio 0.0010 1 10.6458 0.0068 X1X2 0.0012 1 12.4428 0.0042 X1X3 0.0008 1 7.7829 0.0164 X1X4 2.0028E−05 1 0.2025 0.6607 X2X3 0.0002 1 2.1162 0.1714 X2X4 0.0003 1 3.1018 0.1036 X3X4 0.0009 1 9.5278 0.0094 X12 _0.0153 ₁ _154.6386 _<0.0001 X22 _0.0104 ₁ _105.3051 _<0.0001 X32 _0.0271 ₁ _274.5798 _<0.0001 X42 _0.0001 ₁ _1.3568 _0.2667 Residual 0.0012 12

Lackofﬁt 0.0012 10 20.2406 0.0580 Notsigniﬁcant

Pureerror 1.16E−05 2

Cortotal 0.0550 26

obtainedwiththe20ratio,whichcanexplainthelimitedinﬂuence

observedintheRSMstudy.Optimal conditionsresultedin

irra-diationtime120s,extractionpower400W,solvent-to-solidratio

28:1and48%ethanolconcentrationwithapredictedTPCyieldof

15.74mgGAE/g.MAEwascarriedoutattheseoptimalconditions

obtainingaTPCyieldof15.78±0.80mgGAE/g,veryclosetothe

valuepredictedbythemodel.

Results arein agreementwith otherliterature works which

underlined the inﬂuence of ethanol concentration, microwave

powerand irradiationtime on theextractionof phenolic

com-poundsfromvegetabletissues(Songetal.,2011;Sutivisedsaketal.,

2010).

3.2. Ultrasoundassistedextraction

3.2.1. Single-factorexperiments

Table2showstheresultsofthesingle-factorexperiments

car-riedoutforpreliminaryoptimizationofUAE.

Theeffectofethanolconcentrationwassigniﬁcantwitha

max-imumTPCyieldat50%ethanol/water.Thisisinagreementwith

Wangetal.(2008)andwiththeresultsobtainedwithMAE.

Theethanolconcentrationrangeof30–70%wasselectedforthe

RSMtrials,whilethe50%forthenextsingle-factortrials.

Withregardtoextractiontime,theTPCyieldincreased

signiﬁ-cantlyupto10–15min.Resultsindicatedthatextractionofactive

compoundsfromthepeelsextractwereaccomplishedafter10min.

Overmuchsonicationtimewould increaseenergysupply which

is needed torelease thetarget compounds but can also

accel-eratedegradationof phenoliccompounds (Carreraet al.,2012).

Similarobservationswerereportedby Hossainetal. (2012)for

theoptimizationofUAEofantioxidantcompoundsfrom

marjo-ram(Origanummajorana).Sonicationtimerangeof5–15minwas

selectedfortheRSMtrials,whiletimeof10minwasusedforthe

nextsingle-factortrials.

Asitconcernstheradiationamplitude,TPCyieldwasconstantat

20and40%amplitudeand,then,increasedandremainedconstant

foramplitudebetween60and100%,asalsoreportedbyHossain

etal.(2012).Increasingradiationamplitudemayfavorthe

disrup-tionoftheplantcellwallduetocavitationphenomena,thereby

increasingthecontactsurfaceareabetweensolidandliquidphase,

enhancingsolventpenetrationintotheplantmaterialand

facili-tatingthereleaseofsolutes(Jermanetal.,2010;Maetal.,2009).

However,all reactions, including theformation of free radicals

which canbescavenged byphenoliccompounds (Jermanetal.,

2010),arepromotedwhenhighamplitudesareused(Stanisavljevi ´c

etal.,2009).

In this study, operating temperature during UAE was kept

constantatroomtemperature;thereforeanyheatingeffectwas

excluded.

A30–70%amplituderangewasselectedfortheRSMtrials,while

the60%wasusedforthelastsingle-factortrials.

Finally,theTPCyieldaugmentedsigniﬁcantlywiththeincrease

ofliquid/solidratioupto40mL/gwhichwas,then,ﬁxedforthe

RSMtrials.

3.2.2. OptimizationbyRSM

TheTPCyieldsobtainedinthetrialsoftheCCRDarereportedin

Table4.

Regression coefﬁcients and analysisof theregression model

are summarized inTable 6withindicationthat themodel had

adequatelyrepresentedtherealrelationshipbetweenthechosen

parameters(LiyanapathiranaandShahidi,2005).

Neglectingthenon-signiﬁcantterms(P>0.01),theﬁnal

predic-tiveequationforUAEwas(Eq.(6)):

Y(TPC)₌14_.75₋0_.71X2₊0_.57X3₊0_.7X1X3₋1_.02X32 ₍₆₎

Toinvestigatetheinteractiveeffectsofoperationalparameters

anddetermineoptimallevelsofthevariables,three-dimensional

surface plots wereconstructedaccording toEq.(6) as for MAE

(Fig.2).Theplots showclearly thegreat effectof ethanol

con-centrationonTPCyield,asalsoobservedbyotherauthorswho

investigatedandoptimizedbyRSMtheUAEofphenolic

antiox-idants fromdifferent plantmaterials(Morelli and Prado,2012;

Rodriguesetal.,2008).

Summarizing,ethanolconcentrationandextractionamplitude

weretwocrucialfactorsinUAE,withsigniﬁcanteffectoftheir

cor-respondinglineartermsandalsoofthequadratictermforethanol

concentration.TimedidnotinﬂuencetheTPCyieldaslinearterm,

butitdidasinteractivefactorwithethanolconcentration.Thiswas

apparentlyincontrastwiththesingle-factorexperiments,

accord-ingtowhichtimewasaninﬂuentfactor.However,inthe5–15min

time range selectedfor theRSMoptimization,thesingle-factor

resultshadshownasigniﬁcantincreaseinTPCyieldonlyfrom5

to10min,whichcanexplainthelimitedinﬂuenceobservedinthe

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Fig.2.ResponsesurfaceanalysisforthetotalphenolicyieldfromC.limonpeelswithultrasoundassistedextractionwithrespecttoultrasoundamplitudeandextraction time(A);ethanolpercentageandextractiontime(B);ethanolpercentageandultrasoundamplitude(C).

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Table6

Analysisofmeansquaredeviationofthequadraticmodelterms(Eq.(1))appliedtotheexperimentalvaluesoftotalphenolicyieldsobtainedwithultrasoundassisted extraction.df,degreesoffreedom.

Source Sumofsquares df F-value P-valueProb>F

Model 24.7117 9 11.3956 0.0011 Signiﬁcant X1–time 0.2792 1 1.1589 0.3131 X2–amplitude 6.2710 1 26.0264 0.0009 X3–ethanol 3.9580 1 16.4271 0.0037 X1X2 0.1081 1 0.4486 0.5218 X1X3 3.9340 1 16.4271 0.0037 X2X3 0.0946 1 0.3926 0.5484 X12 _0.4289 ₁ _1.7804 _0.2188 X22 _0.7978 ₁ _3.3114 _0.1063 X32 _9.0160 ₁ _37.4189 _0.0003 Residual 1.9275 8

Lackofﬁt 1.2508 5 1.1090 0.4978 Notsigniﬁcant

Pureerror 0.6767 3

Cortotal 28.4531 18

FromthemodeltheoptimalconditionsofUAEwere:63.93%

ethanol concentration,15.05minat 77.79% amplituderadiation

withapredictedyieldof15.08mgGAE/g.UAEwascarriedoutunder

theseconditionsgivingarealrecoveryof15.22±0.88mgGAE/g

(notstatisticallydifferentfromtheMAEyield).

3.3. ComparisonbetweenMAE,UAEandCSE

Thetwoalternativeextractiontechnologies,MAEandUAE,were

comparedwitheachotherandwithCSEconsideringtheTPCyield

alongwiththeantioxidantactivityoftheextracts.Selectionofan

extractionmethodwouldmainlydependontheadvantagesand

disadvantagesoftheprocessessuchasextractionyield,complexity,

productioncost,environmentalfriendlinessand safety(Lietal.,

2010).

TheCSEgaveaTPCyieldof15.03±0.12mgGAE/gdwwhichwas

notstatisticallydifferentthanMAEandUAEyields.MAEshould

bepreferredbasedonthelowersolventconsumptionand

extrac-tiontime(SpignoandDeFaveri,2009;Lietal.,2011;Upadhyay

et al., 2012).However, in this study, operating temperature in

theUAEwaskeptconstantatroomtemperature,excludingany

heatingeffectwichmightpositevelyornegativelyinﬂuecethe

phe-nolsrecoverydependingontheappliedamplitude.Furthermore,a

punctualenergycostanalysisshouldberequiredtocomparethe

twotechniquesfromtheenergyconsumptionpointofview.

0 10 20 30 40 50 60 70 80 90 100 0 100 200 300 400 500 600 700 800 % D P PH Inhibitio n µg GAE/mL

MAE UAE CSE

Fig.3.DPPHactivity(as%DPPHinhibition)asafunctionoftotalphenol concentra-tion(asgallicacidequivalents(GAE))ofmicrowaveassisted(MAE)andultrasound assisted(UAE)extracts(obtainedunderRSMoptimizedconditions)comparedto conventionalsolventCSEextract.Errorbarsindicate±S.D.

Fromthepointofviewofthequalityoftheextracts,theDPPH

test showed again MAE as the best technology(Fig.3)due to

thesigniﬁcantlylowerIC50(203.59±5.59␮gGAE/mL)thanUAE

(268.24±10.62␮gGAE/mL)whichwas,onitsturn,signiﬁcantly

lowerthanCSE(298.82±8.60␮gGAE/mL).

AlsotheFRAPtestsshowedahigheractivityfortheMAEextract

(Fig.4).

TheloweractivityofUAEandCSEextractcouldberesultedfrom

extendedextractiontime,henceexposuretounfavorable

condi-tionssuchaslightandoxygen.Moreover,itiscommonlyknown

thatultrasonicationcouldinducefree radicalsformation within

theliquidmedium,thuscausingoxidationanddegradationofthe

activecompounds(Hayatetal.,2009).

SEMobservationoftheresidueafterMAE,UAEandCSE(Fig.5)

showedthat,incomparison withtheuntreatedsample, solvent

extractionproducedcellchangesinallsamples,althoughtheextent

ofdamagediffered(AspéandFernández,2011).

Itiswellknownthatultrasoundsdisruptplantcellsvia

cavi-tationphenomena(Kongetal.,2010),butUAEdidnotseriously

affectthesampleinthepresent studyifcompared toCSE.This

suggeststhatthedriedC.limonpeelsparticlesarehighlyresistant

toultrasound energy(Aspéand Fernández,2011).Onthe

oppo-site,microwaveheatingcausedahighercellulardamagehelping

therapidrelease ofsolutesintothesolventsandenhancingthe

well-knownmainheatingeffectofmicrowaves.

0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 150 200 250 Absorbance at 7 00nm µg GAE/mL

MAE UAE CSE

Fig.4. Ironreducingpower(absorbanceat700nm)asafunctionoftotal phe-nolconcentration(asgallicacidequivalents(GAE))ofmicrowaveassisted(MAE) andultrasoundassisted(UAE)extracts(obtainedunderRSMoptimizedconditions) comparedtoconventionalsolvent(CSE)extract.Errorbarsindicate±S.D.

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Fig.5.ScanningelectronmicroscopeimagesofC.limonpeelspowderbefore(A)andafterextractionbyconventionalsolventextraction(B),microwaveassistedextraction (C)andultrasoundassistedextraction(D).

4. Conclusions

Theresponsesurfacemethodologywassuccessfullyemployed

tooptimizetotalphenolicyieldfromdriedC.limonpeels,according

todifferentnon-conventionalsolventextractionprocesses(MAE

andUAE).Theappliedsecond-orderpolynomialmodelgavea

sat-isfactorydescriptionoftheexperimentaldatashowingthatthe

TPCyieldwasmostaffectedbyethanolconcentration,extraction

powerandliquid–solidratioforMAE,and bysonication

ampli-tudeandethanolconcentrationforUAE.TheproposedMAEmethod

appearedtobebetterthanbothUAEandCSEallowingforhigher

recovery yield and speciﬁc antioxidant activity with a shorter

workingtimeandalowersolventconsumption.SEMobservation

suggestedthattheseresultswereduetomoreintensetissue

degra-dationundermicrowaveirradiation.

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