Rev. bras. farmacogn. vol.25 número3

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Improved

green

coffee

oil

antioxidant

activity

for

cosmetical

purpose

by

spray

drying

microencapsulation

Anna

B.F.L.

Nosari

a

_,

_Juliana

_F.

_Lima

b

_,

_Osvaldo

_A.

_Serra

b

_,

_Luis

_Alexandre

_P.

_Freitas

a,∗

a_Faculdade_de_Ciências_{Farmacêuticas}_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil b_Faculdade_de_Filosofia_Ciências_e_Letras_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10March2015 Accepted23April2015 Availableonline12June2015

Keywords:

Arabicgum Heat Light Conductivity Cosmetic

a

b

s

t

r

a

c

t

Theoilextractedbycoldpressingunroastedcoffeebeans,knownasgreencoffeeoil,hasbeenwidely usedforcosmeticpurposes.Theobjectiveofthisworkwastoprepareandcharacterizemicrocapsules containinggreencoffeeoilandtoverifyitsantioxidantactivityundertheeffectoflight,heatandoxygen. Theencapsulatingmaterialwasarabicgumandthemicrocapsuleswereobtainedbyspraydryingan oil-in-wateremulsioncontaininggreencoffeeoil.Thecharacterizationofthemicrocapsuleswasperformed bylaserdiffraction,scanningelectronmicroscopy,differentialscanningcalorimetryandtheantioxidant activity.Theantioxidantactivitywasdeterminedbyamodifiedactiveoxygenmethodwithlight irradia-tion,heatingandoxygenflux.Themicroparticleswereeffectivelyproducedbytheproposedspraydrying method,whichresultedingreencoffeeoilloadsof10and30%.Themorphologicalevaluationof micro-capsulesshowedsphericalshapewithsmoothandnon-poroussurfaces,demonstratingtheadequacyof arabicgumasencapsulatingmaterial.Calorimetricanalysisofindividualcomponentsandmicrocapsules with10and30%greencoffeeoilshoweddiminisheddegradationtemperaturesandenthalpy, suggest-ingapossibleinteractionbetweenarabicgumandgreencoffeeoil.Theantioxidantactivitiesforpure greencoffeeoilanditsmicrocapsuleswithloadsof10and30%showedhighactivitywhencompared tothereferenceantioxidantalfa-tocopherol.Microcapsulescontaining10and30%ofoilshowed7-fold and3-foldincreaseinantioxidantactivitywhencomparedtopuregreencoffeeoil.Thenewmethodfor antioxidantactivitydeterminationproposedhere,whichappliesheat,lightandoxygensimultaneously, suggestsahighimprovementinencapsulatedgreencoffeeoilwhencomparedtothisactivealone.The resultsshowedhereinindicateapromisingindustrialapplicationofthismicroencapsulatedgreencoffee oil.

Introduction

Productsderivedfromcoffee(CoffeaarabicaL.,Rubiaceae)have beenlongusedbymankindasbeverages,foodsandcosmetics.Most recently,theoilextractedbycoldpressingtheunroastedbeansof coffeewasintroducedtothecosmeticmarketwithgreatimpact. Thissocalledgreencoffeeoil,GCO,hasbeenstudiedforitsactivity ontheskinhealth(Pereda,2009;Peredaetal.,2009;Savianetal.,

2011;Wagemakeretal.,2012;Chiarietal.,2014).Thisvegetable

oilpresentsauniquecompositionandpreviousstudiesshowedan expressiveantioxidantactivityagainstlipidperoxidation(Kroyer

etal.,1989).

∗ Correspondingauthor.

E-mail:lapdfrei@usp.br(L.A.P.Freitas).

The GCO showeda dose dependentstimulation of collagen, elastin and glycosamineglycans synthesis byfibroblasts invitro

(Peredaetal.,2009)besidesanincreasedreleaseofgrowthfactors,

TGF-b1andGM-CSF.Peredaetal.(2009)alsofoundAQP-3mRNA expression6.6foldhigherinthepresenceofGCO,indicatinga pro-tectiveeffectofthisoilonphysiologicalbalanceoftheskin.Pereda

etal.(2009)alsoconcludedthattheGCOiseffectiveagainst

celluli-tis.AlthoughcosmeticformulationscontainingtheGCOshowed lowantioxidantandantimicrobialactivitiesinvitro(Wagemaker etal.,2012)therewasalsoobservedlackoftoxicityinvitroandin clinicalevaluation(Wagemakeretal.,2013).ThoseeffectsofGCO ontheskinhealthmayprobablyberelatedtoitslipidfractionrich intriacylglycerols,sterolsandtocopherols,aswellasditerpenes ofthekaurenefamily(SpeerandKolling-Speer,2006),whichhave beenpreviouslyconnectedtobeneficactionstotheskin(Nakayama etal.,2003).However,themoststudieddermatological applica-tionof GCO iscertainly as a photoprotectionaid(Savian etal.,

http://dx.doi.org/10.1016/j.bjp.2015.04.006

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2011;Chiarietal.,2014).Anon-ionicO/Wemulsioncontaining3% (w/w)GCOwasproposedasatopicformulationfor

photoprotec-tion(Savianetal.,2011).Recently,astudyofGCOasanadditiveto

sunscreenformulation containing ethylhexylmethoxycinnamate showedasynergisticeffectofthisoilbyincreasingthesun protec-tionfactor,SPF,by20%ascomparedtosyntheticsunscreenalone

(Chiarietal.,2014).

Oneofthedrawbacksforthecosmeticapplicationofvegetable oilsorfatsistheirlipidoxidativestability(RamalhoandJorge,2006) sincetheunsaturatedwaxyacidsmayundergophotooxidation, thermaloxidation,autooxidationandenzymaticoxidation.GCO photooxidationmaybealimitingfactorespeciallyforsunscreen applicationsandtheuseofsyntheticantioxidantsaresubjectto manyformulationandregulatoryaspectsoftopicaladministration. Microencapsulationis aneffectivewaytoprotectthese materi-als,aswellasothercomponents,likethediterpenes,againstlipid oxidation(Puetal.,2011; Jimenezetal.,2006)andother envi-ronmentalfactors.AccordingtoCostaetal.(2007)manystudies havedemonstratedtheuseofmicroparticlestoreducetoxicityand increasetheefficiencyofactivesubstances.

Therearemanytechniquesthatcanbeappliedforthe produc-tionofmicroparticles,includingthespraydrying,spraycoolingand fluidizedbed(Puetal.,2011).Amongthemanytechniques,the spraydryinghascaughtattentionforplantextractsandoils(Jafari

etal.,2008;Coutoetal.,2013a,b;PeixotoandFreitas,2013;Porto

etal.,2013)duetoitsmanyadvantages(OliveiraandPetrovick, 2010).Therearealsomanymaterialsthatcanbeusedas encap-sulatingagentssuchasgums,waxesandpolymers(Oliveiraand

Petrovick,2010;Coutoetal.,2013a,b;PeixotoandFreitas,2013;

Portoetal.,2013).Arabicgumis notedforpresentingexcellent

emulsifyingpropertiesandis widelyusedfor theretention and protectionofoil(Jimenezetal.,2006;Jafarietal.,2008).Theyare widelyusedforcontrolledrelease ofactive andhave good sta-bilityinvariationsofpHandmoisturelevelsinadditiontobeing biocompatible(Jafarietal.,2008;Ranjhaetal.,2010)

Thus,theobjectiveofthisstudywastopreparemicroparticles bythetechniqueofspraydryingcontainingGCOandusingAGas thewallformingmaterial.Inadditionimportantcharacteristicsof microparticlessuchasthemorphology,thermalbehaviorand pho-tocatalyticactivitywerestudied.Possibleinteractionsbetweenthe GCOandAGwereevaluatedbydifferentialscanningcalorimetry, DSC.

Materialandmethods

GCOmicrocapsulespreparation

Materials

Arabicgumpowderanalyticalgradebatchnumber144617was suppliedby Labsynth Ltda(SãoPaulo,Brazil). The green coffee oilbrand name ‘Melscreen Coffee’ (Chemyunion Química Ltda, Sorocaba,Brazil)batchnumberCN102-0811waspurchasedfrom Distriol Comércio de Insumos Ltda (São Paulo, SP). The DL-␣

-tocopherolacetatecosmeticgrade(ZhejiangMedicineCo,China) batchnumber20120615with99.6%puritywassuppliedby Via-farma Ltda (São Paulo, Brazil). The castor oil fatty acid Acros OrganicsBVBA containing85% ricinoleicacid(12-hydroxy-oleic acid)waspurchasedfromJanssenPharmaceuticalaan(Geel, Bel-gium).

Microencapsulation

AGwasdissolvedinMilli-Q(EMDMillipore,Billerica,MA,USA) water(1:2w/w)undermagneticstirringat250rpmandatroom temperature(25±2◦_C)₂₄_h_before_the_preparation_of_the

emul-sionfordrying.Theemulsionswerepreparedfromtheaqueous

14 000 rpm

GCO

Emulsion Pump

Hot air

Powder

24 h H₂O AG

S P R A Y

Fig.1.SequenceofstepsduringGCOmicrocapsulespreparation.

solutionofAGbyincorporatingGCOinconcentrationsof10and 30%(w/w)relativetoAG.Theemulsionwasthenpreparedusinga highshearhomogenizerTurratecTe-102(TecnalLtda,Piracicaba, Brazil)wasusedat14,000rpmfor5minatroomtemperature.After thepreparationtheenulsionswerereadilyspraydried.

Thedryingprocessoftheemulsionwasperformedusinga lab-oratoryscalespraydryermodelMSD0.5(LabmaqLtda,Ribeirão Preto,Brazil).Theemulsionwasatomizedbyapneumaticspray nozzleinthedryingchamberandthemicroparticleswere sepa-ratedbyacycloneandcollectedinaflask.Thefollowingdrying conditionswerekeptconstantduringtheexperiments:emulsion feedrate6ml/min;dryingairflowrate1.25m3_/min;_atomization pressure6bar,atomizingairflowrate50ml/min;inletandoutlet dryingairtemperature140and100◦_C,_{respectively.}_Fig.₁_depicts

thesequenceofstepsduringGCOmicrocapsulespreparation.

Morfology

The microcapsules were poured on a stub and coated with

goldinaBal-Tecsputtercoater.Microparticlesmorphologywas observedbyScanningElectronMicroscopy,SEM,usinga micro-scopeXL30-TMPNOandFEGXL30(PhillipsCo.,Netherland).

Thermalanalysis

Samples(5mg)wereplacedinaluminumpansandheatedto 420◦_C_at_a_rate_of₁₀◦_C/min_under_a_nitrogen_flux_of₁₀_ml/min._DSC

measurementswereperformedusingaDSC-50(ShimadzuCorp., Kyoto,Japan).

Photocatalyticactivity

ThesampleswereanalyzedbyanActiveOxygenMethod,AOM, adaptedfromtheconductometrictechniqueRancimat®

(Limaetal.,

2009; Nosari, 2012; Lima and Serra, 2013). The experimental

assemblyisshowninFig.2.Castoroil(3ml)wasplacedintheflask 2A(Fig.2)and40mgsampleofalpha-tocopherol,GCOor microcap-suleswasadded.Thismixturewassubmittedtoconstantstirring at120◦_C_and_irradiation_of_light_by_a_xenon_lamp_Xenarc_D-H4R

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Heating Oil +

sample Air inlet

Water Conductivity

cell Light

Fig.2. Theexperimentalassemblyforphotoxidationassay.

Resultsanddiscussion

Physicalcharacterization

Thepreparationofmicroparticlesbyspraydryerwas success-ful,resultinginapowderwithsizesrangingfrom4to11␮m.The

yieldafterdryingwasabout60%,whichmaybeconsideredgood foralabscalespraydryer.Fig.3showsSEMphotomicrographs. TheyindicatethatGCOwasencapsulatedbytheAGwithinatypical morphologyforspraydriedmicroparticles,withasphericalshape, smoothsolidsurface,displayingnocracks,fissuresorpores,which allowsforgreaterprotectionofoil(Santosetal.,2005;Trindade

andGrosso,2000;Bertolinietal.,2001).Someflatorconcave

sur-facesobservedareprobablycausedbyshrinkageoftheliquiddrop duetorapidlymoisturelossduringtheearlystagesofspraydrying

(Santosetal.,2005).Themorphologyofthemicroparticlesshown

inFig.3wassimilarfordifferentconcentrationsofGCO(10%Fig.3a; 30%Fig.3b).

Thermalanalysis

Thermalanalysisofspraydryingmicroparticles(Fig.4)showed endothermicand exothermic effects, where the first endother-micpeakislikelyassociatedwiththewaterlossfromfunctional groupsofthepolymer(ZohuriaanandShokrolahi,2004).The sec-ondpeakischaracterizedbyanexothermiceventfeaturingthe degradation of AG, which showeda shift in peak temperature whencomparingAGtothemicroparticles.Thepeakdegradation ofAGoccursat308◦_C_while_the_thermogram_of_the_{microparticles}

DSC

Exo

Endo

100.00

Temp [C]

200.00 300.00 400.00 AG 10% 30% 5.00

–0.00

mW

Fig.4.Differentialscanningcalorimetryofspraydriedmicroparticles.

showeddegradationpeaksat303and297◦_C_for_GCO_contents_of

10and30%,respectively.Theseshiftsindegradationtemperatures arepossiblyduetochangesinmolecularweightandpolarityofthe polysaccharidegroups(Lossetal.,2006).

Oxidativeactivity

Theoxidativeactivitywasevaluatedbythechangesinwater solutionconductivityinthepresenceofthevolatilecompounds whichareformedduringthedegradationofcastoroilsubjected toair,heatingandlightirradiation(Limaetal.,2009;Limaand

Serra,2013).Thehighdegradationofcastoroilincreasesunder

thisoxidativeenvironmentandconsequentlyincreasesthe con-ductivityofwaterincollectionvessel.Thus,theoxidativeactivity isdirectlyproportionaltothemeasuredconductivity(Limaetal.,

2009;Nosari,2012;LimaandSerra,2013).Fig.5showstheresults

obtainedforthewaterconductivityinthecollectionflaskversus

timefortheoxidativereferenceCsOilandfortheCsOiltogether withtheGCO,microparticlescontaining10and30%ofGCO(SD10 and SD30) and also with␣-tocopherol,␣-TOH. Ascan beseen

inFig.5,thehighestconductivityvaluesarefoundfortheCsOil alone,showingvaluesashighas117␮Scm−1after7hof

irradia-tion.ThelowerconductivitiesfoundfortherunsofCsOilwiththe samplesofGCO,␣-TOHandthemicroencapsulatedGCOproves

thatallsamplesshowsomeantioxidantactivity.The␣-TOHisa

commonreferenceasantioxidantformanyfoodand pharmaceu-ticalproducts,especiallyforoilsandwaxesandasshowninFig.5

itdecreasedsubstantiallythewaterconductivitymeasuredinthe collectionflask.ThismeansthattheformationofCsOildegradation productswasinhibitedbythepresenceof␣-TOH.Intheincreasing

orderofinhibitorycapacityforCsOildegradationtherearethe␣

-TOH,SD10,GCOandSD30.Ingeneral,itwasobservedaconstant conductivityincreaseforallsamplesstudied,startingwitha con-ductivityverycloseto100␮Scm−1,whichisusuallyreportedfor

20μm

EHT = 20.00 kV Mag = 2.00 K X Detector = SE1 10μm EHT = 2000 kV Mag = 2.00 K X Detector = SE1

a

b

(4)

0 95 100 105 110 115 120

CsOil CsOil+GCO CsOil+SD10 CsOil+SD30 CsOil+a -TOH

1 2 3 4 5 6 7

Time (h)

Conductivity (

μ

S

, cm

–1

)

Fig.5. Conductivityinaqueoussolutionasafunctionoftimeforsamplesofcastor oilaloneandaddedwithgreencoffeeoil,vitaminEandmicroencapsulatedgreen coffeeoil.

freshwater.FortheCsOilsample,thereisasignificantburstinthe conductivityintheinitialcurveandthenaconstantincrease.For CsOiladdedwith␣-TOH,GCOandSD10thereisaminorburst

dur-ingthefirsthourandthentheconductivityreachesaplateau,with littleorinsignificantincreaseuntiltheendoftheexperiment. How-ever,thebehavioroftheSD30sampleisquitedifferentfromthe others,sincethereisnoincreaseintheconductivityduringthefirst hourbutanincreaseinconductivityintheperiodbetween1and 2h,followedbyaplateaufrom2.5to4.5handconstantdecrease inconductivityuntiltheendoftheexperiment.Thisisprobably explainedbythefasterreleaseofGCOfromSD30ascomparedto SD10duetoloweramountofencapsulatingmaterial.Theeffect oflongerreleasetimesforhigherratiosofencapsulatingagentto activehasbeenlargelyreportedinliterature(Martinsetal.,2014). Furtheranalysisofthisresultcouldbeattainedbymeasuring theglobalreductionoftheCsOildegradation,orbyintegratingthe conductivityasafunctionoftimetogivetheareaunderthecurves, AUC,andcomparingtheareasfortheseveralexperiments.TheAUC isgivenbyequation1.Theantioxidantactivitycanbecalculated bythereductionintheAUCforeachexperimentusingtheAUC ofCsOilasthereference.Thepercentantioxidantactivity,AOA,is definedbyEq.(2).

AUC=

t=tf

t=0

C(t)dt (1)

AOA(%)= AUCCsOil_AUC−AUCSample

CsOil ×

100 (2)

where,C(t)istheconductivityasafunctionoftime,tf=7h,andthe

areasunderdecurvesaregivenin␮Scm−1s.

TheAOAresultsforthesamplesstudiedareshowninFig.6A. Thepercentreductionoftheareasunderthecurveare5.02%,6.77%, 8.10%and9.77%forthe␣-TOH,SD10,GCOandSD30respectively.

Thelowestreductioninthedegradationofcastoroilwasobserved forthe␣-TOH,whichiswellknownforitsantioxidantproperties

andalsorecommendedforthisapplicationinfoodsand pharma-ceuticals.Considering that ␣-TOH is a lipid-soluble antioxidant

widelystudiedandusedincosmetics,theresultsherein demon-stratesthatthegreencoffeeoilhasastrongantioxidantactivity sinceitsAOAinthisphotocatalyticassayisconsiderablyhigher than the vitamin E. This result is important because there are contradictoryconclusionsintheliterature,sinceapreviouswork

(Wagemakeretal.,2012)reportedapoorantioxidantactivityfor

GCOwhentheDPPHmethodwasusedwhileagoodantioxidant

activityagainstlipidperoxidationwasalsoreported(Kroyeretal., 1989).AccordingtoFig.6A,allsamplescontaininggreencoffeeoil hadahigherantioxidativeactivitythanvitamineE,suggestingthat undertheseconditions,thestudiedsamplesshowedabetter pro-tectionagainstcastoroildegradation.Fig.6Ashowsgrossvaluesof AOA%whenusing40mgofeachsamplestudied.Itisworth remem-beringthattheamountofgreencoffeeoilvariedinthesamples from10%(SD10),30%(SD30)and100%(GCO).Atleastforthe com-parisonamongthesamplesthatcontainingGCO,whichisbelieved tobethemostimportantantioxidantcompound,itisinterestingto correcttheAOA%valuestakingintoconsiderationthe concentra-tionofGCO.TheresultingcorrectedAOA%areshowninFig.6Band demonstrateanexpressiveincreaseinantioxidantactivityofSD10 sample.Usingthis base,GCOmaintainsits8.10%inAUC reduc-tion,butSD10nowhas67.7%andSD30presentsnow29.3%ofAUC reduction.Thisbaseofcalculationrepresentsbettertheincreasein AOA%afterGCOencapsulation,sinceismeasuredtakingthemass ofGCOineachsample,anddemonstratesthegreatprotectionthat thematrixofGAgivestogreencoffeeoilwhenitis microencap-sulated.SD10andSD30representa7-foldanda3-foldincreasein AOA%,respectively.OnemightexpectahigherAOA%forSD30then SD10,basedintheirGCOcontents.However,theexplanationisthe fasterreleasefromSD30,asobservedanddiscussedinFig.5,where SD30sampleshowednoburstintheconductivityduringthefirst hoursofexperiment.ThisshowsthatSD10hasaslowerreleaseof GCOandthenalongerlastingeffectwhichreflectsinitsAOA%.

Theresultsstimulatesfurtherstudiestoevaluateother impor-tantparametersofspraydrying,suchasdryingtemperatureand flowrateofdispersionatomization,aswellasdetailedanalysisto verifytheencapsulationefficiencyandGCOstability.

TOH 0 2 4 6 8 10

0 20 40 60 80

SD10

Sample

A B

Sample

A

O

A (%)

A

O

A (%)

GCO SD30 TOH SD10 GCO SD30

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Conclusion

Theresultshereinconfirmthehighantioxidantactivityofgreen coffeeoil,whichisworldwideacceptedinthecosmeticindustry. Thisshowsthattheantioxidanttestproposedhere,which com-binesheat,lightandoxygen,shouldbeadoptedforotherstudies forAOA%ofnaturaloilsandsunscreenproducts.Anotherimportant resultisthatGCOantioxidantactivityinthisworkwassuperiorto alfa-tocopherol,awidelyusedproductasantioxidantincosmetic industry.Furthermore,themicroencapsulatedsystemsdeveloped hereinprovidedbetterstabilityandlongreleaseofGCO,thus giv-inghigherprotectiontoCsOilthanthenon-encapsulatedGCO.The increaseinantioxidantactivityformicrocapsulescontaining10and 30%GCOwereveryexpressive,indicatingfutureindustrial appli-cationincosmeticmarket.

Authorscontribution

ABFLNpreparedthemicrocapsulesandcarriedoutthe acqui-sitionandanalysisofphotooxidationdatatogetherwithJFL.OAS andLAPFcontributedfortheanalysisandinterpretationofdata.All authorsparticipatedindraftingthearticleandrevisingitcritically.

Conflictofinterest

Theauthorsdeclarenoconflictofinterest.

Acknowledgements

The financial support from FAPESP (2011/20872-7 and

2012/04071-7)andCNPq(PQ2)aregratefullyacknowledged.

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