Rev. bras. farmacogn. vol.25 número4

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

In

vitro

photoprotective

effects

of

Marcetia

taxifolia

ethanolic

extract

and

its

potential

for

sunscreen

formulations

Sônia

C.C.

Costa,

Cassia

B.

Detoni,

Carla

R.C.

Branco,

Mariana

B.

Botura,

Alexsandro

Branco

∗

DepartamentodeSaúde,UniversidadeEstadualdeFeiradeSantana,FeiradeSantana,BA,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31May2015

Accepted24July2015

Availableonline17August2015

Keywords:

Marcetiataxifolia

Flavonoid Photoprotective

Sunscreenformulation

a

b

s

t

r

a

c

t

ThespeciesMarcetiataxifolia(A.St.-Hil.)DC.,Melastomataceae,whichisendemicoftherupestrianfields ofnortheasternBrazil,containsasignificantamountofflavonoids.Inthiswork,thepotentialofthe ethanolicextractofM.taxifoliaastheactiveprincipleinasunscreenphotoprotection(UV-AandUV-B) formulationwasinvestigated.TheLiquidChromatographyHighPerformance-DiodeArrayDetector quan-tification(quercetin),totalflavonoidcontent,antioxidantactivitythrough2.2-diphenyl-1-picrylhydrazil method,photoprotectiveactivityagainstUV-BandUV-Aradiationinvitro(spectrophotometricmethod) andpotentialforeyeirritationusingthemethodologyoftheheneggtest-chorioallantoicmembranewere performedintheextract.Afterthat,theformulationswerepreparedusingdifferentconcentrationsof activeethanolicextract(5,10,20and30%)andtheevaluationofthesunprotectionfactorwascarriedout usingthesamemethodologyusedforthecrudeextract.ThecrudeextractshowedUV-Aphotoprotection andloweyeirritationintheheneggtest-chorioallantoicmembranetest.AllformulationscontainingM. taxifoliaextracthad≥6sunprotectionfactor.Itsshowsthepossibilitytousethisextractsasasunscreen inpharmaceuticalpreparations.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Theultraviolet radiation(UVR) isdivided intothree distinct

regions: ultraviolet A (UV-A, 315-400nm), ultraviolet B (UV-B,

280-315nm)andultravioletC(UV-C,100-280nm)(Poloninietal.,

2011).Reachingtheskin,withcumulativeaction,UVRcausesa

complexprocessassociatedwithmorphologicalandchemical

reac-tions.DNAisanimportantmacromoleculethatabsorbsUVRand

hencecanmutate,whatinthefuturecanresultinmalignant trans-formationofthecellskincancer(Baloghetal.,2011).Theeffective protectionagainstUVRisavailableaspreparationsfortopicaluse containingsolarfilters,knownassunscreens.Theefficacyofsuch productsisdependentontheircapacitytoabsorbradiantenergy. Theeffectivenessofasunscreenismeasuredasafunctionoftheir sunprotectionfactor(SPF).Thus,thenecessitytoprovidehighSPF andscreeningefficiencyagainstbothultravioletAandultraviolet Bwavelengthsisevident(Vilelaetal.,2011).

Flavonoids compounds are widely distributed in the plant

kingdom and possess a biological action, especially

antimicro-bial, antioxidant and photoprotective activities (Pietta, 2000;

∗ _{Correspondingauthor.}

E-mail:branco@uefs.br(A.Branco).

Lacombeetal.,2010).Thedemandforactiveflavovoid-richextracts

hasbecomean important componentfor the discoveryof new

moleculesactivetohumanphotoprotection.Thatisduetoits struc-turalsimilaritytochemicalfilterswhichmakesitsusceptibleto radiationabsorptionintheultravioletregion(Agatietal.,2013). Plantextractsrichinflavonoidsarecapableofabsorbing ultravio-letlight,usuallytwomaximumpeaksofultravioletabsorptionin theUV-BandUV-Aregions,whatresultsinthepossibilityforthe useoftheseextractsinthedevelopmentofsunscreenformulations (Bobinetal.,1995).

TheMelastomataceaefamilycontainsaround170generaand

4,600speciesdistributedintropicalandsubtropicalareasofthe world(Reisetal.,2005).TheyareespeciallyabundantinBrazil, occurringinforests,savannahsandrupestrianfields.Several popu-lationsoftheMarcetiagenusarefoundinrockyfieldsintheMinas Gerais(SerradoEspinhac¸o)andBahia(ChapadaDiamantina)states. Thesefieldsareasetofelevationsaround1,200mwithhigh

irra-diation,highwinds,droughtandarockysoilquartznaturewith

small depthand low fertility (Giuliettiet al., 1987).The genus Marcetiacurrentlycomprehends29species,withapproximately 90%inhabitingBahia(Brazil).ThespeciesMarcetiataxifolia(A.St.

Hil.)DC.is representedbyshrubandsubshrubbetween15and

300cminheight,smallleaves,oppositeandsessileorshortly peti-ole(Martins,1989).

http://dx.doi.org/10.1016/j.bjp.2015.07.013

Thepreviousinvestigationmadeinourlaboratoryinthe Marce-tiataxifoliashowtheantimicrobialactivityandthepresence of flavonoidsusingHPLC-DADanalysis(Leiteetal.,2012).Inthiswork thepotentialofphotoprotectiveactivityoftheethanolicextract fromthesameplantinasunscreenformulationisinvestigated.

MaterialsAndMethods

Plantmaterial

Marcetiataxifolia (A.St.-Hil.) DC., Melastomataceae, was

col-lected in Rio-de-Contas (Chapada Diamantina, state of Bahia,

Brazil).VoucherspecimensweredepositedintheHerbariumofthe DepartmentofBiologyoftheStateUniversityofFeiradeSantana

(HUEFS)withthefollowingnumber:191053.

Preparationofplantextracts

Thedried powderedleavesofM.taxifolia(1500g)were

sub-jectedtothoroughmacerationin95%ethanol.Theextractionswere carriedoutatintervalsof72h.Theethanolicextractwasfiltered throughtavacuumfilterandthefiltratewasdefattedusinghexane. Theethanolicportionsweredriedandusedforthestudies.

HPLC-DADquantification

APurospher100RP-18(250mm×4.6mmi.d.,5␮m)column

(Merck)wasused.Themobilephasewascomposedbysolvent(A)

H2O/H3PO40.1%andsolvent(B)MeOH.Thesolventgradientwas

programmedasA(75-0%)andB(25-100%)for20min,then100%B

for4min,then75%Aand25%Bfor10min.Aflowrateof1.0␮l/min wasusedina30◦_{Coven,and20␮lofeachsamplewasinjected.}

Theeluatewasmonitoredatadetectionwavelengthof360nm.

Precisely,weighedsampleweredissolvedinmethanol(10mg/␮l).

Themethodwasvalidatedbyanexternalcalibrationcurveusing

standardsolutionsofquercetin,preparedinmethanolinfive dif-ferentconcentrations,rangingfrom0.1to1.5mg/␮l.Thequercetin

standardsolutionswereinjectedthreetimes,andthecurvewas

constructedonMicrosoftOfficeExcel© _{usingtheaverageofthe}

area.Theaccuracywasexpressedastheagreementbetweenthe

experimentallymeasuredvalueandthesetreferencevalue.The

precisionandaccuracywerecalculatedaccordingtotheformula,

respectively:RSD(%)=(SD×100)/C,whereRSD(%)isthe preci-sion,SDisthestandarddeviationandCisthemeanconcentrations calculated;Accuracy(%)=(Cexp×100)/TC,whereCexpisthetotal concentrationofquercetinfromtheextractandTCisthetheoretical concentrationofthestandardreference.Thedetectionlimit(LOD) andquantificationlimit(LOQ)wereestimatedbytheslopeandthe

meanstandarddeviationoftheconcentrationsusedtoconstruct

theanalyticalcurve.

Totalflavonoidcontent(TFC)

TheTFCwasdeterminedbyusingacolorimetricmethodas

pre-viouslydescribedwithafewmodifications(Gursoy,2009).Thedata wereexpressedas␮gofquercetinequivalents(Que)per10mgof ethanolicextractweight.

DPPHFreeradicalscavengingassay

Thefreeradicalscavengingactivitywasmeasuredusingthe

2.2-diphenyl-1-picrylhydrazil(DPPH)assay(Mensoretal.,2001).

Theabsorbancevaluesweremeasuredat518nmandconverted

into the percent antioxidant activity (AA) using the following

formula: AA%=[(absorbance of the control−absorbance of the

sample)/absorbanceof the control]×100. TheIC50 values were

calculatedfromalinearregressionofthedatausingtheGraphPad Prism®_5.0program.

Sunprotectionfactor(SPF)ofthecrudeextract

Theethanolicextractwasdissolvedinethanoltoafinal concen-trationof12.5,25,50,125and250␮g/␮l.TheSPFmodelusedin

thisstudywasaccordingtothemethodologydescribedbyMansur

et al. (1986). The sample absorbanceswere measuredin UV-B

wavelengthrange(290-320nm),with5-nmincrementsandthree

determinationsweremadeateachpoint.TheSPFwascalculatedby applyingtheMansurequation:SPF=CFx290

320290EE(␭)xI(␭) xabs(␭),where:CF(correctionfactor)=10;EE(␭)isthe erythe-malefficiencyspectrum;I(␭)isthesolarintensityspectrum;abs (␭)istheabsorbanceofthesolution.ThevaluesofEE(␭)xI(␭)are constantaccordingSayreetal.(1979).

UV-Ablockingactivityofthecrudeextract

A1mg/mlhydroalcoholicsolutionoftrans-resveratrolwas pre-pared.Petridishesof4.5cmdiameterwerefilledwith5␮lofthis solution.EachPetridishwascoveredwitha0.04-gevenlyspread layerofdryethanolicextractofM.taxifoliatobetestedfortheirUV blockingactivity.Petridisheswithcleanlidswereusedascontrol.

ThepetridisheswereplacedinsidetheUV-Achamberandwere

irradiatedwitharadiationintensityofabout830W/m2_(between 320and400nm)foraperiodoftime(0-120min).Inpredetermined

intervals (20min) a sample was collected and diluted at 1:10.

Theabsorbancewasmeasuredspectrophotometrically(306nm)

usingaUV-VISVarian®

(Cary100BIO)(Kerrileeetal.,2009).The absorbanceofasampletotallyprotectedfromlightwasalso

mea-suredinpredeterminedintervalstoassurethatthedegradation

wasinducedbyUV-Alight;transisomer,whenexposedtolight,

becomesthecismakingconstantabsorbance(Kerrileeetal.,2009). Thisexperimentwasperformedintriplicate.

Invitroeyeirritationtests(HET-CAM)

Theirritatingpotentialofthecrudeextractwasperformedusing

HET-CAM(hen’seggtest-chorioallantoicmembrane)test

accord-ingtoICCVAM(ICCVAM,2010).The assayemploystheCAM of

a 10-day-oldfertilizedhen’s egg.TheCAM,a membrane which

surrounds the developing chick embryo, is highly vascularized

(Bagleyetal.,1991),andisregardedasbeinginsensitivetopain (Dannhardtetal.,1996).Forthistest,theethanolicextractofM taxifoliawasdissolvedindistilledwateratconcentrationsof250, 125,62.5␮g/␮l.Thefertilizedchicken eggswereobtainedfrom commercialsources.Onthe10th_{dayofincubation,theeggshell}

wasremovedaroundthechamberair,showingtheshell

mem-brane.AfterCAMwasexposed,300␮loftheextractswasapplied.

Thepositivecontrols(NaOH 0.1M),andanegativecontrol

(dis-tilledwater)wereperformedtodemonstratethevalidityofthe

test.Foreachconcentrationandcontrols,threeeggswereused.

Aftertheapplicationoftheextracts,themembraneandblood ves-selswereexaminedfor5min.Thetimeofappearance,measuredin seconds,ofeachirritanteffect(haemorrhage,lysisandcoagulation)

wasrecorded.

Thefollowingformulaisusedtogenerateanirritationscore(IS):

IS=(301−H)×5

(300) +

(301−L)×7

300 +

(301−C)×9 300

WhereH=thetime takentostart thehemorrhagereactions;

Table1

PolowaxlotioncompositionfortheincorporationofMarcetiaextract.

Phase Rawmaterial Amount Properties

1 Methylparaben 0,15% Preservative

Glycerine 5% Moist

Deionizedwater q.s.p.100g Carrier

2 Propylparaben 0,05% Preservative

LiquidVaseline 3% Lubricant

Octylstearate 4% Emollient

Polowax 5% BasisandAuto-Emulsifier

3 Imidazolidinylurea 0,1% Preservative

Table2

Compositionofthesunscreenformulation.

Phase Rawmaterial Amount Properties

1 Methylparaben 0,15% Preservative

Glycerine 5% Moist

Deionizedwater q.s.p.100g Carrier

2 Propylparaben 0,05% Preservative

Vaselineliquid 3% Lubricant

Octylstearate 4% Emollient

Polowax 5% BasisAutoEmulsifier

Benzophenone-3 3% SunscreenUVA/UVB

3 Imidazolidinylurea 0,1% Preservative

The average score was calculated for each extract, and the extractswereclassifiedintofourcategories:non-irritant(IS>1), lowirritant(1≤IS<5),Moderateirritant(5≤IS<9)andirritant(IS

≥9)(Debbaschetal.,2005).

Preparationofsunscreenformulations

ThepharmaceuticalbaseforincorporatingMarcetiaextractand

sunscreenformulawereadaptedfromthePharmaceutical

Medi-calForm(Batistuzzoetal.,2006)asdescribedinTables1and2, respectively.

Thevehicle,polowaxlotion,usedfortheincorporationofherbal extracts,waspreparedbyheatingthephase1and2at75◦_C, sep-arately(Proenc¸aetal.,2009).Afterthat,phase2waspouredinto phase1underconstantagitation,endingwiththeadditionofphase 3andthemixingtemperaturebelow40◦_{C.Theformulaconsistedof}

astandardsunscreenemulsionofO/Wcontaining

benzophenone-3as chemical filter,inaccording toTable2. Theemulsionwas

preparedwiththesamemethoddescribedforpreparingtheO/W

emulsionwithoutbenzophenone-3.Theextractwasincorporated

tothelotionpolowaxvaryingintheirconcentrationsof5,10,20 and30%.Onceformulated,thepHwasmeasuredandtunedwith tri-ethanolaminetopH6.0to7.0,whichpHisdesirableforsunscreen accordingtoRibeiro(2010).

DeterminationoftheSPFofsunscreens

ForthedeterminationoftheSPFoftheformulationsextracts(5, 10,20and30%),theyweredissolvedinethanoltoafinal concen-trationof0.2,2.0,5.0,10,15,20,30and50mg/␮ltoevaluatethe

profileofthesunscreenformulationsprepared.Themethodology

usedforSPFevaluationoftheseformulationswasthesameused

forthecrudeextract.Theabsorbancereadingswereperformedin

triplicate.

Statisticalanalysis

The analyses were performed in triplicate, and the results

expressed as mean±standard deviation (SD).Differences were

considered significant when p<0.05. Multiple comparisons

betweenmore thantwo groups wereperformedwithone-way

5 10 15 20

Minutes

Quercetin standard

A

B

Que

25

Fig. 1.HPLCchromatogram of theethanolicextract ofMarcetia taxifolia (A):

flavonoidglycosylatedregion(Rt:10–15min),flavonoidnon-glycosilatedregion

(Rt:16-20min);HPLCchromatogram(B)ofthequercetinstandard(Rt:14.35min).

ANOVAsupplementedwithTukey’stest.Thedataobtainedwere

analyzedusingtheGraphPadPrism®_version5.0.

ResultsandDiscussion

HPLCquantification

Thecrude extractof Marcetiataxifoliawasanalyzedthrough

HPLC-DAD (Fig. 1A), what shows the presence of the

non-glycosylated (peaks betweenRt 10 to15min) and glycosylated

(peaksbetweenRt16to20min)flavonoids.TheUV-spectraofthe peakatTr14.34minwassimilartotheflavonolquercetin.Thus,the

quercetinwasidentifiedfromtheretentiontime(Rt)comparison

usingstandard(Fig.1B).Duetothegoodphotoprotectiveactionof quercetin,thiscompoundwasHPLC-quantifiedintheM.taxifolia.

Thelinearitywasconfirmedbypreparing standardsolutions

of quercetin solutions in methanolat five concentrations.

Cali-brationcurve wasplotted and determinedusing thestandards

data:forquercetin,y=30000000x+14619andR2_{=0.9987,which}

werelinearforspecifiedconcentrationranges.Thedetectionlimit was 0.0145mg/␮land quantification limit was 0.048mg/␮lfor

quercetin.The quantification resultsshow that theamounts of

quercetinpresentintheethanolicextractwashighabovethe detec-tionandquantificationlimits,furtheremphasizingthereliability

ofthemethod.Theprecisionandaccuracyofthemeasurements

werewithinallowablevalues;theaccuracydidnotallowvalues

that exceeded2.09%. Theconcentrationof quercetinpresent in

ethanolicextractfromM.taxifoliawas1.12mg/␮l.

Totalflavonoidcontentandantioxidantactivity

TheflavonoidcontentofM.taxifoliaextractcalculatedasthe quercetinequivalenceshows168±0.35␮g/␮lofquercetin,which isequivalentto10mgofeachethanolicextractweight.The antirad-icalactivityoftheantioxidantisshowintheTable3.Theseresults

showassociationbetweenantioxidativeactivitiesand flavonoid

Table3

AntioxidantactivityoftheethanolicextractobtainedfromMarcetiataxifolia.

250* _{125 50 25 10 5}

Extract 95.31 93.97 93.43 74.19 35.52 20.25

BHT* _{94.64 94.07 86.58 57.16 30.20 18.38}

AA** _{97.03 96.62 96.98 96.57 74.32 24.60}

* _␮g/ml

** _{Butylatedhydroxytoluene}

***_Ascorbicacid

Sunprotectionfactor(SPF)ofthecrudeextract

TheethanolicextractofM.taxifoliahadthesunscreenactivity

evaluatedwiththemethoddevelopedbyMansuretal.,1986.The

resultsofthedeterminationsofinvitroSPFvaluesareshownin Fig.2.AccordingtotheBrazilianlaw,RDC30fromJune1,2012 (Anvisa,2012a),onlySPFgreaterthanorequalto6issuitablefor useincosmeticproductswithphotoprotectiveactivity.TheM. tax-ifoliaextractswithdifferentconcentrations(250and125␮g/␮l) hadsatisfactorysunscreenactivity(15.52and8.35,respectively), higherthantheminimumrequiredbyAnvisa.TheSPFvaluesofthe extractstestedwereconcentration-dependent,theincreasein con-centrationresultedinanincrementoftheSPF.Thisactivitycanbe attributedtotheflavonoidsfoundinspeciesoftheMarcetiafamily.

The plant extracts rich in flavonoids which are efficient in

absorbing ultraviolet light, usually show two maximum peaks

ofultravioletabsorption,one between240-280nmandanother

300-550nm(Bobinetal.,1995).Severalclassesofnatural

com-poundswereexaminedfortheirantioxidantandphotoprotective

activity.Recentstudies(Poloninietal.,2011)reviewedthe

impor-tanceof natural sunscreens in thecompositions ofcommercial

sunscreens and therefore analyzedtheir role in theprevention

of skincancer. Photoprotective propertieshave been evaluated

inextractsof agreat varietyof plants,extractsChrysanthemum

ramosumandCrocussativusL.provedtobegoodweather

reduc-ingagent-inducederythemaascomparedtobenzophenone4(Hu

andWang,1998).Violanteetal.(2009)havedevelopedstudieson thespeciesMacrosiphoniavelame,OxalishirsutissimaandLafoensia pacari,whichthesespeciesshowedabsorptionintheUVin␭max.

318nm,324nmand356nm.Thephytochemicalstudiesdescribed

thepresenceofflavonoids,andotherconstituentsastanninsand

alkaloids,whicharesubstancesdescribedwithaphotoprotective

action.TheCalophylluminophyllumextractisrichinvariousnatural sunscreencompounds,suchasbiflavonoids,etocotrienols tocofer-ols,andbroadabsorbanceintheUV,aswellasantioxidantactivity (Saidetal.,2007).Studiesusingthedry extractfromtheleaves ofEncholiriumspectabile,Bromeliaceae,describedapotent antiox-idantactivityand significantphotoprotective activitycorrelated withflavonoidcompoundspresent(Oliveiraetal.,2013).

0 5 10 15 20

250 200

150 100

50 0

SPF

Concentration (μg/ml)

Fig.2.SunProtectionFactor(SPF)oftheethanolicextractofMarcetiataxifolia.

0 0 0.2 0.4 0.6 0.8 1.2 1.4 1.6

1

20 40 60

Time (min)

Absorbance (306 nm)

Control

M. taxifolia

80 100 120 140

Fig.3.EvaluationofthephotoprotectivepotentialagainstUVAradiationbythe

Marcetiaethanolicextract.

UV-Ablockingactivityofthecrudeextract

The plant extracts shown had the ability to protect a

pho-tolabile solution (trans-resveratrol) from UVA radiation. When

trans-resveratrolisexposedtoUVAradiation,itundergoes

degra-dationwhichcanbeseenbytheabsorbancedecreasesduringthe

timeintervals(Kerrilee,2009).Itwasfoundthatduringthecontrol

boardofexpositiontoUVA radiation,there wasphotobleaching

of50.73%trans-resveratrol.TheplatewascoatedwithM. taxifo-liaextractandphotobleachingwaslesspronounced;theendofthe 120minexposureconversionoftrans-resveratroltocis-resveratrol was9.48%.ThisstudyshowedthattheextractfromtheM. taxifo-liaiscapableofpreventingUV-Aradiationfrompermeatingacross aPetridishlid(Fig.3).ThisstudyofM.taxifoliashoweditsgood abilityofphotoprotectionagainstUV-A.

Invitroeyeirritationtests(HET-CAM)

TheHET-CAMtestshowedthatM.taxifolia(250and125␮g/␮l) contendlowirritationpotential:1.93.Theseresultsindicatedthat theethanolextractofM.taxifoliaissuitableforuseincosmetics (Table4).Reportsonthetoxicologicalevaluationsofspeciesofthe Marcetiagenerawerenotfoundintheliterature.

In addition to the protection against UV-B radiation,

prod-uctsbeingcurrentlyresearchedalsohavephotoprotectiveactivity againstUV-Arays.Initially,itwasbelievedthatUV-Aradiationdid

notcausedamagetotheskin.However,studieshaveshowedthat

thisprocesscontributestoradiationandpromotestheappearance

ofphoto-agingskintumors.TheUV-Aradiationpenetratesmore

deeplyintothedermallayersoftheskin,unlikeUV-Bwhich is

absorbedintotheskinepidermis.UV-Ageneratesmoreoxidative

stressthandoesUV-B,andatlevelsfoundinsunlight,itistentimes moreefficientthanisUV-Bincausinglipidperoxidationleading

toplasmamembranedamage(Damiani,2006).Somestudieshave

demonstratedthatsunscreenphoto-instabilityisprimarilya prob-lemconcerningtheUV-Aregion(Maieretal.,2001;Hojerovaetal., 2011).

Table4

Classificationofextractsaccordingtothescoreofthephenomena.

Concentration (␮g/ml)

IrritationScore (IS)

Classification Irritancy

M.taxifolia 250 1.93 lowirritant

125 1.08 lowirritant

62,5 0.14 non-irritant

NaOH0,1M* _{0.1M 12.6 irritant}

Distilledwater** _{0.0 non-irritant}

* _{Positivecontrol}

Table5

Dilution(mg/␮l)andSPFfortheformulationswiththeMarcetiataxifoliaethanolic.

MT5% MT10% MT20% MT30% Benzophenone5%

0.2mg/ml

1.241±0.119d _{1.796±0.0800}c _{2.231±0.0494}c _{2.860±0.0655}b _{4.823±0.0907}a

2.0mg/ml

6.397±0.0626e _{7.336±0.2748}d _{13.360±0.0625}c _{14.530±0.0598}b _{38.530±0.3256}a

5.0mg/ml

10.200 ± 0.3160e _{15.810± 0.2379}d _{17.170± 0.0511}c _{27.460 ± 01150}b _{38.800 ± 0.01464}a

10.0mg/ml

25.430±0.1806e _{28.480±0.0265}d _{30.54±0.0505}c _33.780±0754b _{38.730±0.01201}a

15.0mg/ml

32.620±0.1931e _{33.150±0.0775}d _{34.590±0.3471}c _35.340±0987b _{39.100±0.0808}a

20.0mg/ml

32.730± 0.1561d _{33.30± 0.1103}c _{33.730± 0.0657}b _{33.890 ± 0887}b _{39.20 ± 0.0624}a

30.0mg/ml

33.250±0.1402e _{34.140±0.0340}d _{24.610±0.0209}c _36.550±0417b _{42.370±0.2960}a

50.0mg/ml

37.310±0.1708c _{38.430±0.0377}b _{42.550±0.2215}a _{43.110±0.0800}a _{39.840±0.2343}a

Thelevelofsignificativewasp<0.05.Theequallettersrepresentstatisticallyequalvaluesforthesamedilution.

0 5 10 15 20 25 30 35 40 45 50

50 45 40 35 30 25 20 15 10 5 0

SPF

Diluition (mg/ml)

5% 10% 20% 30%

Benzophenone 5%

Fig.4. ProfileofsunscreenformulationswiththeMarcetiataxifoliaethanolicextract.

Currently,thepharmaceuticalandcosmeticindustries, along withgovernmentregulatoryandqualitycontrolofproducts,have usedalternativemethodologiesforassessingthepotentialforeye irritation. In this perspective, the National Health Surveillance Agency(Anvisa)hasadoptedtheGuidelinefortheSafetyEvaluation ofCosmeticProducts(Anvisa,2012b).TheinvitroHET-CAM,which providesinformationontheeye-irritatingproperties,isan essen-tialpartofthehazardidentificationofchemicalsandproducts.The HET-CAMbasicallyassessesvascularchangesandgeneratesresults which,inturn,comparedtotheanimalmodel,haveagreat relation-shipwithphenomenaobservedinconjunctiva,eveniftheyare,as arule,lighterweightthanchangesdetectedinothersystemssuch as,forexample,thoseinducedinisolatedorgans(Nóbregaetal., 2008)

DeterminationoftheSPFofsunscreens

Tukey’smultiple-comparisontestwasusedtoevaluatewhether

theSFPinthedilutionswasthesame,thereby formingaUV-B

sunscreenprofileforformulationsofM.taxifoliaextract(Fig.4).

TheSFPevaluationshowgoodresultsforformulationscontent

M.taxifoliaextracts(5,10,20and30%)whichwereobservedSFP≥6 dilutionfrom2mg/␮l(Table5).Forthespeciesinvestigated,itwas observedthatthedilutionof50mg/␮lwasclosetotheSFPstandard ofbenzophenone5%,inthefixedprotectivefactorof30-50,which

indicatesthat,accordingtotheRDC30/12,theformulationsmay

besuitableforverysensitiveskinsunburn,becauseoftheirhigh sunprotection,asdemonstratedintheinvitromethodology.

AnalyzingstatisticallytheSPFvaluesispossiblenotedthatthe formulationswithM.taxifoliaextract10%and20%(Table5)showed thesameSPFvaluesforthedilutionof0.2mg/␮l.Theformulations at20%and30%ataconcentrationof20mg/␮lwerealsostatistically equal.Thesameistruefortheconcentrationof50mg/␮landthe

standardformulationof5%ofbenzophenone.

Conclusion

TheethanolicextractofM.taxifoliashowgoodantioxidantand

photoprotectiveactivityagainstUV-BandUV-Aradiation.These

effects canbeattributedtoflavonoidderivatives presentin the

plant including quercetin.Sunscreens were developed and SPF

wasevaluated.TheformulationscontainingM.taxifoliaethanolic extractshowedSPFvaluesnearachemicalfilterfrequentlyusedin thepharmaceuticalindustry,benzophenone-3.TheM.taxifoliahas greatapotentialforuseasanactiveingredientforsunscreens.

Authors’Contributions

SCCChelpedwiththemeasurementoftheinvitroantioxidant

activity,totalflavonoidandHPLCanalysis.Shealsoparticipatedof thepreparationoftheformulations,interpretationofdataand sta-tisticalanalysis,evaluationoftheinvitrophotoprotectiveactivityof sunscreensandtheevaluationstabilitytests.CBDwasinchargeof theevaluationoftheinvitrophotoprotectiveactivityofsunscreens.

MBBhelpedwiththemeasurement oftheinvitroEye Irritation

Tests(HET-CAM)and carriedouttheinterpretationofdataand

statisticalanalysis.CRCBconductedstudiesoftheHPLCandthe

developmentofthesunscreenformulation.ABparticipatedinthe

coordinationoftheproject.Allauthorshavereadandapprovedthe finalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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