Photoprotective activity and increase of SPF in sunscreen formulation using lyophilized red propolis extracts from Alagoas.

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Photoprotective

activity

and

increase

of

SPF

in

sunscreen

formulation

using

lyophilized

red

propolis

extracts

from

Alagoas

Wanessa

A. da

S. Almeida

a

_,

_Amanda

_dos

_Santos

_Antunes

a

_,

_Ricardo

_G.

_Penido

a

_,

Helen

S. da

G. Correa

a

_,

_Andrea

_M.

_do

_Nascimento

a

_,

_Ângela

_L.

_Andrade

a

_,

Vagner

R. Santos

b

_,

_Thiago

_Cazati

c

_,

_Tatiane

_Roquete

_Amparo

d

_,

Gustavo

Henrique

Bianco

de

Souza

d

_,

_Kátia

_Michelle

_Freitas

d

_,

Orlando

David

Henrique

dos

Santos

d

_,

_Lucas

_Resende

_Dutra

_Sousa

a

_,

Viviane

M.

R. dos

Santos

a,∗

a_Departamento_de_Química,_Instituto_de_Ciências_Biológicas,_Universidade_Federal_de_Ouro_Preto_,_Campus_Morro_do_Cruzeiro,_Ouro_Preto,_MG,_Brazil b_Laboratório_de_{Microbiologia}_e_{Biomateriais,}_Escola_de_Odontologia,_Universidade_Federal_de_Minas_Gerais_,_Belo_Horizonte,_MG,_Brazil

c_Departamento_de_Física,_Instituto_de_Ciências_Exatas_e_Biológicas,_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_MG,_Brazil

d_Laboratório_de_{Fitotecnologia,}_Escola_de_Farmácia,_Universidade_Federal_de_Ouro_Preto,_Campus_Morro_do_Cruzeiro,_Ouro_Preto,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2018 Accepted7March2019 Availableonline20March2019

Keywords:

Sunprotectionfactor Redpropolis FilterUVA-UVB Totalphenolics HET-CAMtest Antioxidantactivity

a

b

s

t

r

a

c

t

Theexcessivesunexposure,coupledwithlackofsunprotectionrepresentsoneofthebiggestrisksto

theoccurrenceofskincancerandphotoaging.Recentstrategiesforphotoprotectionhaveincludedthe

incorporationofnaturalantioxidantandanti-inﬂammatorycompounds,intosunscreens,andtheoral

administrationofnaturalantioxidantextracts.Inthiswork,weuseBrazilianredpropolisextractbecause

itsantioxidantandanti-inﬂammatoryactivities.Theaimofthisworkwastoevaluatethesunprotection

factorandantioxidantactivityofdifferenthydroalcoholicextractsofredpropolis(70%and75%)prepared

fromlyophilizedredpropolisatroomandhightemperatures.Thesunprotectionfactorinvitrowas

determinedbyaspectrophotometricmethoddevelopedbyMansur.Thehydroalcoholicextractsofred

propolisincorporatedwithFilterUVA-UVB5%GelPermulemTR-1presentedabsorptionintheUVB

region.Also,theinvitrocapacityofthehydroalcoholicextractsofredpropolistoincreasephotoprotective

activityofFilterUVA-UVB5%Gelwasevaluated.Thehydroalcoholicextractsofredpropolisincorporated

presentedhighervaluesofsunprotectionfactorandshowedsynergisminthephotoprotectiveactivityof

FilterUVA-UVB5%GelPermulemTR-1.Theantioxidantactivityandsunprotectionfactorarecorrelated

withtotalphenolicscontentoftheextractsandthehydroalcoholicextractofredpropolis75%atroom

temperaturewaschoosen.TheformulationdevelopedwithFilterUVA-UVB5%GelPermulemTR-1with

thishydroalcoholicextractofredpropolisshowedsafetobeappliedontheskinaccordingHET-CAM

test.Suggestsindicationofhydroalcoholicextractofredpropolis(75%–roomtemperature)associated

tophotoprotectiveformulationsforuseinphotoprotectiveproducts.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Thesearchforsunprotectionhasintensiﬁedinrecentdecades

asthesun’sharmfuleffectshavebecomemoreknownand

publi-cized.Itisawell-knownfactthatanoverexposureofhumanskin byultravioletlightincludedsunburncells,acceleratedskinaging andinductionofskincancer.Sunscreensareproductsthatprovide

∗ Correspondingauthor.

E-mail:vivianesantos@ufop.edu.br(V.M.Santos).

protectionagainsttheeffectsofthesun,buttheycanaccumulate

in humans and frequent exposure may induce adverse effects.

Becauseofthis,researchhasbeendonetoﬁndanaturalprotector thatisexpectedtohavefewersideeffects.Duringourresearchfor naturalsunscreenagents,wehavefocusedonpropolis.Propolisor beeglueisaresinousstickysubstancethatApismelliferaL.collects fromvariousplants,mixedwithwaxandothersecretions.Ithas beenusedsinceancienttimesbecauseofitsbiologicalproperties

as an antioxidant, anti-microbial, antibiotic, anti-inﬂammatory

and sun photoprotective. These characteristics have attracted

theresearchers’attention.Propolisisoneofthenaturalproducts

https://doi.org/10.1016/j.bjp.2019.02.003

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arisingfromthe Brazilian ﬂorathat hasbeen usedfor the

dis-coveryanddevelopmentofnewtherapeuticagents.Recently,the

Brazilianredpropolistypehasbeenhighlightedduetoitsfeatures.

Thechemicalcompositionand pharmacologicalactivitiesofthis

speciﬁcredpropolis,havebeenintenselyexploredsincethe90s, whichisevidencedbythepublicationofover100papersbetween scientiﬁcarticlesandpatents(Mouraetal.,2017).Themain com-poundsfoundinpropolisaredistributedinseveralmajorclasses:

phenolicacidsandtheiresters,flavonoids(flavones,flavonones,

ﬂavonols, dihydroﬂavonols, chalcones), terpenes, steroids,

aro-maticaldehydes,alcohols,sesquiterpenes,naphthalene,stilbene

derivativesofbenzopyran,benzophenone,caffeicacid, cinnamic

acidderivatives,andbenzoicacid(Castroetal.,2009).Flavonoids

representthemostcommonandwidelydistributedgroupof

phe-nolicsinredpropolis.Theseareamongthemostactivecompounds inthisresin, whichactindifferentphysiologicalprocesses, and performvariousfunctions,includingsunprotection.

Thepurposeofthisstudywastodeterminethe

photoprotec-tivepropertiesofpurelyophilizedBrazilianredpropolisandthe lyophilizedBrazilianredpropolisincorporatedintotheformulation sunscreen(Coiffardetal.,2014).Thelyophilizedpropoliswasused

tomaskorreduceundesiredsmellofthehydroalcoholicextract

ofpropolis.Thephotoprotectiveactionofthepropolisextractwas

measuredbytheMansurmethod.Thephotoprotectiveactionof

asunscreenismeasureduniversallybythesunprotectionfactor (SPF)whichestablishestheincreaseofthedoseofsunexposure

withthephotoprotectiveproductappliedwithouttheoccurrence

oferythema(Gonc¸alvesetal.,2018).

Materialsandmethods

Generalconsiderations

SolventswerepurchasedfromNeonandusedwithoutfurther

puriﬁcation.Thein vitroSolarProtection Factor(SPF)and

Pho-tostabilityweredeterminedby theSpectrophotometricmethod

developed by Mansur. The UV–Vis Ultraviolet readings were

performedonthe BelEngineering UV-M51 Spectrophotometer.

Flavonoidconcentrationsweredeterminedbyultraviolet–visible

absorption spectroscopy (UV–Vis) at 420nm in a Thermo

Sci-entiﬁc – Genesys 840 spectrophotometer. Filter UVA-UVB 5%

GelPermulemTR-1with2-phenyl-benzimidazole-5-sulfonicacid

and2-hydroxy-4-methoxybenzophenoneassunscreenagentswas

obtainedbytheBioFarma.Thelyophilizedredpropolissamples

wereboughtinPharmaNéctar.

Redpropolissamples

ThelyophilizedredpropolissampleswereboughtinPharma

Néctarandthecrude samplesofred propoliswereobtainedin

MarechalDeodoro,stateofAlagoas,locatedintheNortheastern

RegionofBrazil(SL094237andWL355342).

Preparationofethanolicextractsofredpropolis(EEP-70%or75%) heated

Thelyophilizedredpropolissample(20g)wasextractedusing ethanol70%or75%(Neon),15mlofethanolforeach2g,inwater bathat70◦C for30min.Afterthat, thesamplewasﬁlteredon

ﬁlterpaperand 100mlof ethanol (75%or 70%)were addedto

theresidue,andanotheralcoholicextractionwasperformed.The solutionobtainedfromthetwoextractionsweredriedandstored (Borgesetal.,2014).

Preparationofethanolicextractsofredpropolis(EEP-70%or75% theroomtemperature)

Thelyophilizedredpropolissample(20g)wasextractedwith 70%or75%ethanol(15mlofethanolforeach2g)for48hatroom

temperature and the resulting alcoholicsextracts wereﬁltered

undervacuumonﬁlterpaperand100mlofethanol(75%or70%)

wereaddedtotheresidue,andanotheralcoholicextractionwas

made.Thesolutionobtainedfromthetwoextractionsweredried

andstored(Nascimentoetal.,2009).

Incorporationofredpropolisextracts(EPP)intheﬁlterUVA-UVB

5%GelPermulemTR-1

FilterUVA-UVB5%GelPermulemTR-1(1g)and1mlofEEP

solu-tion(1mg/ml)wereaddedtoa100mlbeaker.Themixturewas

maintainedunderstirringfor30minatroomtemperature.After

that,thesamplewasstored(Nascimentoetal.,2009).

DeterminationofmaximumabsorbanceofEEP,ﬁlterUVA-UVB5%

GelPermulemTriandﬁlterUVA-UVB5%GelPermulemTR-1

incorporatedwithEEP

TheabsorptionreadingsoftheultravioletUV–Visbythe

sam-pleswereperformedusingtheUVSpectrophotometerFemto800

Xi.FordeterminationofabsorbanceintheultravioletregionsUVA andUVB10mgofsamplesweredilutedin10mlethanol70%, pro-ducingaconcentrationof1mg/mlthatwasdilutedinethanol70% andproducingdilutedconcentrationsof0.010,0.020,0.030,0.050,

0.070and0.1mg/ml.Thescanningwasperformedforeach

con-centration betweenthewavelengthsof 200and 600nm inthe

UVspectrophotometer,usingquartzbucketwithanopticalway

of1cm.Ethanol70%wasusedaswhiteandtheexperimentwas

carriedoutintriplicate(Gonc¸alvesetal.,2018). InvitrodeterminationoftheSunProtectionFactor(SPF)

UVA-UVB5%GelPermulemTR-1Filter(1g)incorporatedwith

EEPwasweighedandthedilutionswereperformedinethanol70%

(triplicate),untilobtainingaconcentrationof0.2␮l/ml.Also,

sep-arately,1mg/mlofeachEEPand1mg/mlFilterUVA-UVB5%Gel

PermulemTR-1werepreparedandthedilutionswereinethanol

70% (triplicate), until obtaining concentrations of 0.010, 0.020, 0.030,0.050,0.070and 0.1mg/ml.The invitro SolarProtection

Factor was determined to each concentration by the

spectro-photometricmethoddevelopedbyMansur(1984)usingEq.(1).

SPF=FC

320₂₉₀EE()·I()·Abs() (1)

whereFC=10(constant),EE=erythemogeniceffect,I=intensityof

thesunandAbs=absorbanceofthesample.Absorptionreadings

wereperformedintherangeof290–320nmwithintervalsof5nm andaddedinEq.(1).TheconstantsEEandIwerepre-deﬁnedby Mansur(1984),accordingtoTable1.

Evaluationofphotostability

Thephotostabilitytestwasperformedusinga lightchamber

withaUVlampat365nmwavelength.Solutionsof0.1mg/mlEPP,

FilterUVA-UVB5%GelPermulemTR-1andFilterUVA-UVB5%Gel

PermulemTriincorporatedwithEEPwerepreparedin

volumet-ricﬂasks,whichwereexposedtoradiationfor2h,evaluatingthe

effectiveconcentrationevery30minuponexposuretoUV

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Table1

EEandIconstantsforthecalculationofinvitroSPF.

(nm) EE()×I() 290 0.0150 295 0.0817 300 0.2874 305 0.3278 310 0.1864 315 0.0839 320 0.0180

1.0000

EE(),erythemogeniceffectofwavelengthradiation;I(),sunintensityat wave-length();,wavelength(Mansur,1984).

Evaluationofantioxidantactivity

Theantioxidantactivitywasevaluatedbyphoto-colorimetryin vitromethodsusingthefreeradicals2,2-diphenyl-1-picrylhydrazyl

(DPPH) (Sigma) and 2,2

-azinobis-3-ethylbenzotiazoline-6-sulfonicacid(ABTS)(Sigma).

FortheDPPHmethod,theEEPandthequercetin(standard)were solubilizedinethanoltoobtainstocksolutionsof320.0␮g/ml. Dif-ferentaliquotswerepipettedtogiveﬁnalsolutionsfrom0.5to

64␮g/ml.TheFilterUVA-UVB5%GelPermulemTR-1andFilter

UVA-UVB5%GelPermulemTriincorporatedwithEEPwere

sol-ubilizedtogiveﬁnalsolutions of×␮l/ml.Then100␮l ofDPPH

solutionat0.008% w/vin ethanol wereadded toeach ofthese

samples.Theﬁnal volumewasadjustedto240␮lwithethanol.

Thenegativecontrolwasobtainedfrom100␮loftheDPPHand

140␮loftheethanol,whichwasusedtocalculatetheinhibition percentageoffreeradical.Then,allthesampleswereincubatedfor 30minatroomtemperature(25±2◦C)protectedfromlightand

theabsorbance(Abs)werereadata wavelengthof490nmin a

plate-reader.Thetestwasperformedintriplicateandtheabilityto scavengefreeradicalswasevaluatedbythescavengingpercentage offreeradical(%I),calculatedusingtheformula(Sousaetal.,2007):

%I=

Abscontrol−Abssample

Abscontrol

×100

Theconcentrationrequiredtoobtaina50%antioxidanteffect (EC50)wascalculatedbylinearregressionforEEPandquercetin.

FortheABTSmethod,theworkingsolutionwasobtainedby

mixingABTS(7.4mmol/l)withpotassiumpersulfate(2.6mmol/l) andkeptfor16hatroomtemperatureprotectedfromlight.On thedayofanalysis,this solutionwasdilutedwithethanoltoan absorbanceof0.70(±0.02)at650nm.TheconcentrationsfromEEP, quercetinandgelswereobtainedinthesamemannerasthe pre-vioustest.Then120␮lofABTSwereaddedtothesesamples.The ﬁnalvolumewasadjustedto150␮lwithadditionofethanol.The negativecontrolwasobtainedfrom120␮loftheABTSand30␮l oftheethanol.Then,allthesampleswereincubatedfor6minat roomtemperature(25±2◦C)protectedfromlight(Lietal.,2009).

Thereadingswereperformedat650nmandthe%scavengingof

thesampleandEC50wascalculatedlikepreviouslydescribed.

Determinationoftotalphenolicandﬂavonoidcontent

The measurement of total phenolic was made by the

Folin-Ciocauteu method according Bonoli et al. (2004), with

modiﬁcations. The extracts (80␮l) and fractions (5mg/ml in

ethanol95%)weretransferredtoa96-wellplateandwereadded 60␮lofwaterand10␮lofFolin-Ciocauteu(Cromoline).Following theplatewasagitatedfor1minandwereadded40_␮lofsodium carbonatesolution(15%w/v).Theplatewasagitatedfor30s,were added10␮lofwaterandafterincubationfor2htheabsorbance

wasreadat650nminmicroplatereader(MolecularDevices).The totalphenolicwerequantiﬁedbyusingastandardcalibrationcurve ofgallicacid(10–320␮g/ml;r2₌_0.9983;_y₌_0.0046x₊_0.0886)._The

experimentwasmadeintriplicateandtheresultswereexpressed asmgofgallicacidequivalents(GAE)pergofsample(mgGAE/g) andaspercentage(w/w).

The measurement of total ﬂavonoids was madeby the

alu-minumchloride(AlCl3)colorimetricmethodaccordingChanget

al.(2002),withmodiﬁcations.Theextracts(100_␮l)andfractions (5mg/mlinethanol95%)weretransferredtoa96-wellplateand wereadded40␮lofethanol95%,4␮lofAlCl3 (10%w/v),4␮lof

potassiumacetate(1mol/l)and52␮lofwater.Theabsorbancewas readafterincubationfor40mininmicroplatereader(Molecular Devices)at405nm.Thetotalﬂavonoidswerequantiﬁedbyusing astandardcalibrationcurveofquercetin(2–64␮g/ml;r2₌_0.9982;

y=0.0017x+0.0418).Theexperimentwasmadeintriplicateand theresultswereexpressedasmgofquercetinequivalents(QE)per gofsample(mgQE/g)andaspercentage(w/w).

HET-CAMtest

HET-CAMtestwasperformedaccordingtoaprotocolsuggested byLuepke,1985forUVA-UVB5%GelPermulemTR-1Filter incorpo-ratedornotwithEEP75%(roomtemperature).Theassaybasesthe analysisoftheappearanceofirritativereactionsinthe

chorioal-lantoic membrane ofthe fertilizedchicken eggsin response to

exposureof tested substances.For the procedurewasobtained

commerciallyfertileWhiteLeghornchickeneggs(GranjaTomolei, RJ,Brazil)withoutmycoplasmsandusedafterthe9thdayof incu-bation.Afterhavingbeencontrolledforembryoviabilityareopened neartheaircellusingapairofsurgicalscissorstorevealthehighly

vascularizedchorioallantoicmembrane(CAM).Aftertheproduct

application(0.1mg),theCAMsurfacewasobservedoveraperiod of300sandthetimeoftheappearanceofhemorrhage,lysisand coagulationoccurringonthevascularsystemandthealbuminwas recorded.Thepositiveirritantcontrolwas0.1Nsodiumhydroxide andthenegativecontrolwas0.9%sodiumchloride.Foreachsample threeeggswereused.

Theirritanteffects wereclassiﬁedbyscoresaccordingtothe

time they were observed: less than 30s (hyperemia: 5;

hem-orrhage: 7; clot formation/opacity: 9); between 30 and 120s

(hyperemia: 3; hemorrhage: 5; clot formation/opacity: 7); or

between120and 300s(hyperemia:1;hemorrhage: 3;

clotfor-mation/opacity:5). Ifan effectwas notobserved after300s, it

wasscoredaszero.Eachformulationwasclassiﬁedaccordingto

the scores mean value of three eggs: 0–4.99 corresponding to

non-irritant/slightly irritant (NI/SI); 5.00–8.99 corresponding to moderatelyirritant(MI);and9.00–21.00correspondingtoseverely irritant(SVI)(Mansuretal.,2016).

Statisticalanalysis

TheresultswerestatisticallyanalyzedbytheOne-WayANOVA

testandmultiplecomparisonbyTukeyconsideringasigniﬁcance

level of0.05 (p<0.05).Pearson test wasusedtocorrelate phe-nolicandﬂavonoidcontentwithSPFandantioxidantresults.All statisticalanalysisweremadeusingGraphPadPrism5.0software.

Resultsanddiscussion

DeterminationofabsorbancemaximumofEEP,UVA-UVB5%Gel

PermulemTR-1FilterandUVA-UVB5%GelPermulemTriFilter incorporatedwithEEP

TheEPPincorporatedintheFilterUVA-UVB5%GelPermulem

TR-1causedabathocromicdisplacement.TheUVbandsofthe

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200 300 400 500 600 0.0 1.0 2.0 3.0 4.0 Ab so rba nc e / a.u . Wavelength / nm EEP 70%

Permulen TR-1 with EEP 70% Permulen TR-1 Room Temperature 200 300 400 500 600 0.0 1.0 2.0 3.0 4.0 Ab so rba nc e / a.u . Wavelength / nm EEP 75%

Permulen TR-1 with EEP 75% Permulen TR-1 Room Temperature

Fig.1. UV/VisabsorptionspectraofEEP(70%and75%),EEP(70%and75%)incorporatedinPermulenTR-1andPermulenTR-1Filter,preparedatroomtemperature.

200 300 400 500 600 0.0 1.0 2.0 3.0 4.0 Ab sor ban ce / a.u. Wavelength / nm EEP 70%

Permulen TR-1 with EEP 70% Permulen TR-1 Heated 200 300 400 500 600 0.0 1.0 2.0 3.0 4.0 Ab s o rb an c e / a. u . Wavelength / nm EEP 75%

Permulen TR-1 with EEP 75% Permulen TR-1

Heated

Fig.2.UV/VisabsorptionspectraofheatedEEP(70%and75%),EEP(70%and75%)incorporatedinPermulenTR-1andPermulenTR-1Filter.

Table2

SPFvalues(mean±SD)calculatedfromEEPandPositiveControl(UVA-UVB5%GelPermulemFilter)indifferentconcentrations.

Concentration SPF

70%(roomtemperature) 70%(hot) 75%(roomtemperature) 75%(hot) Positivecontrol

0.02 2.29±0.70a _2.25_±_0.68a _2.47_±_0.75a _2.41_±_0.73a _4.16_±_1.28a

0.03 3.66±1.11a _3.49_±_1.06a _3.90_±_1.19a _3.54_±_1.07a _5.42_±_1.67a

0.05 6.04±1.84a _5.66_±_1.72a _6.30_±_1.91a _6.03_±_1.83a _10.36_±_3.19a

0.07 8.48±2.58a _8.00_±_2.43a _9.04_±_2.75a _8.49_±_2.58a _13.86_±_4.27a

0.1 12.30±3.74a _11.2₁_±_3.41a _12.68_±_3.85a _12.24_±_3.7a _19.75_±_6.07a

Resultsexpressedasmean±standarddeviation.Differentlettersindicatep<0.05atthesamelinebyOne-WayANOVAtest.

Filtermovedtoalongerwavelenght,conﬁrmingtheincorporation (Figs.1and2).

InvitrodeterminationoftheSunProtectionFactor(SPF)

The SPF was evaluated by the methodology developed by

Mansurmethod.Theanalysiswascarriedoutonanultraviolet spec-trophotometer,wherethevaluesoftheobtainedabsorbanceswere placedinEq.(1),providingthedataofSPFvalues, presentedin theTable2.UVA-UVB5%GelPermulemTR-1Filterhasbeenused aspositivecontrolinpreviousstudies.AccordingtoTable2,the EEPsshowedsimilarSPFthanpositivecontrol,withoutstatistical difference.

TheUVA-UVB5%GelPermulemTR-1Filterataconcentration

of0.20␮l/mlpresentedanSPFof10.22(Table3).TheEEP70%and

75%(roomtemperature)withFilterUVA-UVB5%GelPermulem

TR-1showedthattherewasasynergismbetweentheformulations.

Extractsofredpropoliswhenincorporatedwiththeﬁltershowed

moresigniﬁcantUVabsorptionvalues.Theresultsobtainedwith

theincorporationofEPP(roomtemperature)showthatthese mix-turescauseaintensificationintheSPFthroughthesynergisticeffect oftheFilter(Table3).Ingeneral,theincorporationoftheEPP70% and75%(roomtemperature)onthefiltershowthatthesemixtures promoteanintensificationoftheSPFthroughthesynergisticeffect oftheextractswiththesyntheticfilteremployed,ensuringgreater sunprotection.

Table3

SPFvaluescalculatedfromFilterUVA-UVB5%GelPermulemTR-1incorporatedwith EEP.

Formulationsintheconcentrationsof0.20␮l/ml SPF FilterUVA-UVB5%GelPermulemTR-1 10.22±3.16a

EEP70%(roomTemperature)withFilterUVA-UVB5%Gel PermulemTR-1

18.75±5.77b

EEP70%(hot)withFilterUVA-UVB5%GelPermulemTR-1 3.78±0.80c

EEP75%(roomTemperature)withFilterUVA-UVB5%Gel PermulemTR-1

17.29±5.33b

EEP75%(hot)withFilterUVA-UVB5%GelPermulemTR-1 9.39±2.89a

Resultsexpressedasmean±standarddeviation.Differentlettersindicatep<0.05 atthesamecolumnbyOne-WayANOVAtest.

Evaluationofphotostability

EEP,UVA-UVB5%GelPermulemTR-1FilterandEEPwith

UVA-UVB5%GelPermulemTR-1Filterwereanalyzedseparatelyinthe photostabilityassay.Thesamplesweredissolvedinethanol, form-ingsolutionsofconcentrationequalto0.10mg/ml.AccordingFig.3, thegraphsshowhowthesesolutionsbehave,intermsof photosta-bility,afterexposuretoUVradiationat365nmwavelength.The

sampleswerephotostablein theUVradiationat365nm

wave-lengthinthetimeof2h. Evaluationofantioxidantactivity

Results of antioxidant activity showed that EPP had similar

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200 300 400 500 600 0.0 1.0 2.0 3.0 4.0 0 20 40 60 80 1.5 2.0 2.5 3.0 A b so rb a n ce In te n si ty a t 2 80 nm Time / min A b so rb a n ce / a .u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 70% (heated)

200 300 400 500 600 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 0.0 0.5 1.0 Ab so rban ce I nt en si ty a t 30 0 nm Time / min Ab so rban ce / a. u. Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 70% incorparated in Permulem TR-1 (heated) 200 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0 20 40 60 80 1.5 2.0 2.5 3.0 3.5 A b so rban ce I n ten si ty a t 280 nm Time / min Ab s o rba nc e / a. u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm Photostability EEP 75% (heated) 200 300 400 500 600 0.0 0.4 0.8 1.2 1.6 2.0 0 20 40 60 80 0.4 0.8 1.2 1.6 2.0 Ab s o rb an ce I n te n si ty a t 300 nm Time / min Ab s o rba nc e / a. u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm Photostability EEP 75% incorparated in Permulem TR-1 (heated) 200 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 20 40 60 80 1.8 2.0 2.2 2.4 2.6 Ab so rb an ce I n ten si ty a t 280 nm Time / min A b so rban ce / a .u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 75% (room Temperature)

200 300 400 500 600 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 0 20 40 60 80 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Ab s o rb an ce I nt e n s it y at 3 00 nm Time / min Ab so rban ce / a. u. Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 75% incorparated in Permulem TR-1 (room Temperature)

200 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0 20 40 60 80 1.6 1.8 2.0 2.2 2.4 2.6 2.8 Ab so rban ce I nt en s ity a t 280 nm Time / min A b so rb a n ce / a .u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 70% (room Temperature)

200 300 400 500 600 0.0 0.4 0.8 1.2 1.6 2.0 0 20 40 60 80 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 Ab s o rb an ce In ten si ty a t 300 nm Time / min Ab s o rba nc e / a. u . Wavelength / nm 0 min 20 min 40 min 60 min 80 nm

Photostability EEP 70% incorparated in Permulem TR-1 (room Temperature)

Fig.3.GraphsofphotostabilityoftheEEP(70%or75%)andPermulemTR-1incorporatedwithEEP(70%or75%),preparedatroomtemperatureandheated.Theinsertsshow theintensityofabsorptionversusexposuretimetoUVradiation.

GelPermulemTR-1showedaweakantioxidantactivity(low

inhi-bitionpercentage%I)(Table5).Thisresultcanbeduetothelack ofantioxidantactivityofoneofthebothsunscreenagentspresent inthisﬁltergel,the2-phenyl-1H-benzimidazole-5-sulfonicacid

(Binoetal.,2017).However,theresultsobtainedwiththe

incorpo-rationofEPPwithFilterUVA-UVB5%GelPermulemTR-1showed

thattheseextractsinducedintensiﬁcationintheantioxidant activ-ityofthisformulation(Table5).

(6)

Table4

Antioxidantactivityofthepropolisextracts(EEP).

Ethanolicextract(EEP) EC50(␮g/ml)

DPPH ABTS 70%(roomtemperature) 51.15±3.11a _9.96_±_0.35a 70%hot 47.66±1.30a _9.86_±_0.50a 75%(roomtemperature) 52.01±4.85a _10.07_±_0.26a 75%hot 43.45±0.70a _9.83_±_0.39a Quercetin 2.96±0.08c _2.00_±_0.07c

EC50:concentrationrequiredtoobtaina50%antioxidanteffect.Resultsexpressed

asmean±standarddeviation.Differentlettersindicatep<0.05atthesamecolumn byOne-WayANOVAtest.

Table5

AntioxidantactivityofFilterUVA-UVB5%GelPermulemTriincorporatedwithEEP. Formulationsintheconcentrationsof

160␮l/ml

%I

DPPH ABTS

FilterUVA-UVB5%GelPermulemTR-1 4.78±1.65a _66.40_±_1.04a

EEP70%(roomtemperature)withFilter UVA-UVB5%Gel

27.12±2.07b _86.34_±_0.21b

EEP70%(hot)withFilterUVA-UVB5%Gel 36.44±0.90c _86.13_±_0.20b

EEP75%(roomtemperature)withFilter UVA-UVB5%Gel

33.81±0.41c _89.72_±_8.90b

EEP75%(hot)withFilterUVA-UVB5%Gel 27.84±1.49b _86.68_±_3.50b

%I,inhibitionpercentageofradicals.Resultsexpressedasmean±standard devi-ation.Differentlettersindicatep<0.05atthesamecolumnbyOne-WayANOVA test.

Theadditionofantioxidantstocommercialsunscreenscan pre-ventthedamagescausedbytheoxidativestressinducedbyUVA radiationthroughreactiveoxygenspecies(ROS)thatleadstoDNA lesions(Matsuietal.,2009).Extractsfrommedicinalplantsdueits antioxidantactivityandSPFmayprovidenewpossibilitiesforthe

treatmentandpreventionofUV-mediateddiseases(Ebrahimzadeh

etal.,2014).

Determinationoftotalphenolicandﬂavonoidcontent

InBrazilian red propolissamples,phenolic compounds have

already been identiﬁed as tannins, catechins, ﬂavonones and

flavonols(Mendonçaetal.,2015).Severalstudiesdemonstrated thatphenolicacidsandflavonoidsderivedfrompropolis,tea,grape,

fern,andmilkthistleprovidephotoprotection.Thesecompounds

canprevent penetration of radiationinto the skin,resulting in

thereductionofinﬂammation,oxidativestress,andDNA

damag-ingeffects(NicholsandKatiyar,2010).Phenoliccompoundsalso

areimportant antioxidantsand theelimination of free radicals

isamongthemechanismsrelatedtothispharmacologicalaction

(Soobratteeetal.,2005).Becauseofthis,itisreasonableto com-paretotalphenolicandﬂavonoidcontentofthedifferentextracts studied.Therefore,thecontentofbothgroupsofphenolicswasalso determinedintheextracts(Table6).

Table7

CoefﬁcientofPearsoncorrelation(r2₎_between_the_results.

Totalphenolics Totalﬂavonoids

SPF 0.5 −0.2

DPPH 0.9 0.8

ABTS 0.9 0.8

Table8

Timeforreaction(s)inHET-CAMtestofFilterUVA-UVB5%GelPermulemTri incor-poratedornotwithEEP.

Formulation tH1 tH2 tC Irritant

index

Classiﬁcation

FilterUVA-UVB5%Gel PermulemTR-1

0 0 0 0 NI/SI

EEP75%(roomtemperature) withFilterGel

120 0 0 3 NI/SI

Positivecontrol(NaOH1.0N) 30 120 120 19 SVI

Negativecontrol(NaCl0.9%) 0 0 0 0 NI/SI

H1,hyperemia;H2,hemorrhage;C,coagulation;NI/SI,non-irritant/slightlyirritant; SVI,severelyirritant.

EEP 75% hot presented a lower quantity of phenolic and

ﬂavonoidsinrelationtotheothersextracts.Theminimum require-mentsaccordingtotheBrazilianregulationofidentifyandquality ofpropolisare5%(w/w)forphenoliccompoundsand0.5%(w/w)for ﬂavonoids(MinistériodaAgricultura,2001).Therefore, between theextractsonlyEEP75%isbelowthatrequiredbyBrazilian regu-lation.

The total phenolic content have strong correlation

(0.7≤r2_≤_1.0) _with _the _antioxidant _results _and _moderate

cor-relation (0.5≤r2_<_0.7) _with _the _SPF _results ₍_Table ₇_). _In _the

other way, the total ﬂavonoid content have weak correlation

(0.0≤r2_<_0.5)_with_the_SPF_results₍_Table₇_)._Thus,_the_activities

arebetterrelatedtototalphenoliccontentthatﬂavonoid.

HET-CAMtest

Theresultsoftotalphenoliccontentshowedgoodcorrelation withantioxidantandSPFresults,thusthetoxicitytestHET-CAM

wasperformedtoFilterUVA-UVB5%GelPermulemTR-1

incorpo-ratedornotwithEEP75%(roomtemperature),whichpresented

thebiggesttotalphenoliccontentamongEEP(Table6).HET-CAM

resultsshowednonealterationsinthemembranewhenincontact

withFilter UVA-UVB5% GelPermulem TR-1, butthe FilterGel

incorporatedwithEEP75%(roomtemperature)inducedapontual

slighthyperemia(Fig.4).Accordingtotheclassiﬁcationbyscores, bothformulationswereclassiﬁedasnon-irritant/slightlyirritant (NI/SI),suggesting that theyare safetobe appliedontheskin (Table8).HET-CAMalterationsisbaseduponvasculareffectson

theCAM,thenonobservationoftheseeffectsmaysuggestthat

formulation possessalso a lowerskin irritationpotential,since skinirritationstartswithvascularalterationsandtheresultscan

Table6

Totalphenoliccompoundsandtotalﬂavonoidscontentinthepropolisextracts(EEP).

Ethanolicextract(EEP) Totalphenolics Totalﬂavonoids

mgGAE/ga _%_(w/w) _mgQE/gb _%_(w/w)

70%(roomtemperature) 160.76±0.77a _1.60_±_0.08a _19.11_±_0.33a _1.91_±_0.03a

70%hot 113.48±9.22b _11.30_±_0.92b _20.28_±_3.99a _2.03_±_0.40a

75%(roomtemperature) 224.89±3.84c _22.49_±_0.38c _19.18_±_0.83a _1.92_±_0.08a

75%hot 38.48±6.15d _3.85_±_0.61d _3.22_±_1.50b _0.32_±_0.15b

a_mg_of_galic_acid_equivalents_per_g_of_sample. b _mg_of_quercetin_equivalents_(QE)_per_g_of_sample.

(7)

Fig.4. PhotographsofHET-CAMtestresultsofPermulemTR-1incorporatedornotwithEEP75%,preparedatroomtemperature.Thearrowsindicatethealterations.

beassociatedtocosmeticapplicationontheface,neartotheocular mucousmembrane(Mansuretal.,2016).

Conclusion

Thepresentstudydemonstratedtheimportanceandinterestof usingredpropolisextractsinsunscreenpreparationswith incorpo-ratedUVA-UVB5%GelTR-1Filter,sinceitwasobservedarelative

increase ofSPFand antioxidantactivity,leadingtogreater skin

protection.TheresultsobtainedbyUVspectrophotometryallow

abetterevaluationoftheefﬁcacyofEPP75%(roomtemperature) assunscreenswhenassociatedwithaphotoprotectiveformulation. TheantioxidantactivityandSPFarecorrelatedwithtotal pheno-licscontentoftheextractsandtheEEP75%atroomtemperature

waschosen.TheformulationdevelopedwithFilterUVA-UVB5%

(8)

accordingHET-CAMtest.Itcanbeconcludedintheexperimental conditionsofthisstudythatEPP(75%-roomtemperature) associ-atedtothephotoprotectiveformulationshowedanincreaseinthe activityoftheformulation,whichsuggestsitsindicationforusein photoprotectiveproducts.

Authorscontributions

WASA(undergraduate studentt)contributed incorporationof

redpropolisextractsintheﬁlter,contributedSunProtectionFactor

studies,running the laboratorywork. AAS (undergraduate

stu-dent)contributedevaluationofphotostability.RGP(undergraduate student)contributedSunProtectionFactorstudies.HSGC

(under-graduatestudent) contributed preparation ofethanolic extracts

of red propolis. AMN contributed chemical studies, supervised

thelaboratoryworkand criticalreadingofthemanuscript.ALA

designed the study and contributed to critical reading of the

manuscript.VRScontributedincollectingredpropolissampleand identiﬁcation.TCcontributedSunProtectionFactorstudies, con-tributedphotostabilitstudiesanddesignedthegraphs.TRA(post gradstudent)contributedtoevaluatetheantioxidantactivity, con-tributeddeterminationoftotalphenolicandﬂavonoidcontentand

Statistical analysis.GHBS supervised the laboratory work,

con-tributedBiologicalstudiesandcontributedtocriticalreading of

themanuscript.KMF(post-docfellow)contributedHETCAMtest.

ODHSsupervisedthelaboratorywork,contributedbiological

stud-iesandcontributedHETCAMstudies.LRScontributedantioxidant

activitystudies.VMRSdesignedthestudy,supervisedthe exper-iments,wrotethemanuscriptandcontributedtocritical,reading ofthemanuscript.Alltheauthorshavereadtheﬁnalmanuscript,

approvedthesubmissionandcontributedbiologicalstudiesand

chemicalstudies.Alltheauthorshaverealcontributiontotheﬁnal manuscriptwriting.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

TheauthorsthankChemistryDepartment,InstituteofExactand BiologicalSciences,FederalUniversityofOuroPreto.Theauthors

alsothankstheﬁnancialsupportfromFAPEMIGandPROPP/UFOP

(#23109.003517/2018-85).

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