Rev. bras. farmacogn. vol.25 número3

(1)

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

Nectandra

falcifolia

:

potential

phytopharmaceutical

for

skin

damage

protection

designed

by

statistical

approach

and

characterized

by

photoacoustic

spectroscopy

Fernanda

B. Borghi-Pangoni

a,b

_,

_Luana

_M.

_Tunin

b

_,

_Kamilla

_L.

_Bonifácio

c

_,

_Ana

_Cláudia

_Nogueira

d

_,

Luzmarina

Hernandes

e

_,

_Fabio

_Yamashita

f

_,

_Décio

_S.

_Barbosa

c

_,

_Mauro

_L.

_Baesso

d

_,

Maria

da

Conceic¸

ão

T. Truiti

b

_,

_Andréa

_Diniz

a,b,∗

a_Programa_de_{Pós-graduac¸}_ão_em_Ciências_da_Saúde,_Universidade_Estadual_de_Londrina,_Londrina,_PR,_Brazil

b_Programa_de_{Pós-graduac¸}_ão_em_Ciências_{Farmacêuticas,}_Departmento_de_Farmácia,_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil c_Departamento_de_Patologia_e_Análises_Clínicas_e_{Toxicológicas,}_Universidade_Estadual_de_Londrina,_Londrina,_PR,_Brazil

d_Departamento_de_Física,_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil e_Departamento_de_{Morfofisiologia,}_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil

f_Departamento_de_Ciência_e_Tecnologia_de_Alimentos,_Universidade_Estadual_de_Londrina,_Londrina,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19November2014 Accepted10February2015 Availableonline16April2015

Keywords:

Antioxidantactivity

Nectandrafalcifolia

Photoacousticspectroscopy Standardizedextract Skinpermeation

a

b

s

t

r

a

c

t

Phytopharmaceuticalproductsarebeingusedinthetreatmentandpreventionofhealthproblems. Nowadays,thedevelopmentandevaluationofnovelpharmaceuticalproductsisexpensiveandtime con-suming.Astatisticalapproachisagoodtoolforoptimaldevelopmentprocesses.Nectandrafalcifolia(Nees) J.A.Castigl.exMart.Crov.&Piccinini,Lauraceae,aBrazilianspecies,isreportedasanti-inflammatory, anti-leishmanialandanti-microbial.However,thereislittleknownaboutitschemicalcomposition.For otherspeciesofNectandragenus,thepresenceofantioxidantcompoundsisreported.Inorderto opti-mizetheprocessofobtainingextractwithhighantioxidantactivity,differentextractionconditionswere testedfollowingastatisticalapproach.Twosequentialexperimentaldesignswereused–first,afactorial 23_design,_followed_by_central_composite₂2_._The_extracts_manufactured_by_these_experimental_statistical

matrixeshadtheirantioxidantactivityandphenoliccontentsquantifiedandtheresponsesurfaceplots werefittedinquadraticmodelsandtheypredictedthebestextractionconditionforthebestantioxidant activity.Thisstandardizedextractanditsantioxidantactivitywerebetterevaluatedbytwo comple-mentarytests(ABTSandBurstrespiratory).Atopicalformulationcontaining1%(w/w)ofstandardized extractwaspreparedandusedforaninvivoskinpermeationstudyusingatwo-doseapplication.The photoacousticspectroscopywasusedtoanalyzethesamplesfromthepermeationstudyandthe com-positionprofileofstandardizedextract.Inratskinsamples,thedatademonstratedthatforthehigher doseoftopicalformulation(5g/cm2_),_the_standardized_extract_could_cross_skin_and_be_seen_in_epidermis

anddermis.Thiswasnotthecaseforthelowerdose(2g/cm2₎_which_was_only_present_in_the_epidermis.

ThisinformationsuggeststhatthisnovelstandardizedextractofN.falcifoliacouldbeexploredforskin damagepreventionortreatmentfordiseasesdevelopedbyoxidativedamage.

Introduction

Currently,manystudiesareconductedtoobtainmore infor-mationaboutantioxidativeactivityof plantmaterialsandtheir

∗ _{Corresponding}_author.

E-mail:[email protected](A.Diniz).

derivatives.Theseresourceshavebeenfrequentlyusedintopical formulationstopreventcelloxidativedamage(Lonnietal.,2012). In abioprospectingapproach tospecies popularlyknownas “canelinha”(Nectandrafalcifolia(Nees)J.A.Castigl.exMart.Crov. &Piccinini,Lauraceae),studieshaveshownthatextractsfromthis speciesexhibitedanti-inflammatory,anti-leishmanialand antibac-terialactivities(OliveiradeMeloetal.,2006;Truitietal.,2005, 2006).Aboutphytochemicalprofile,noinformationaboutN.

fal-cifoliawasfound,butotherspeciesofthesamegenus(Nectandra

http://dx.doi.org/10.1016/j.bjp.2015.02.012

(2)

grandiflorus)haveshownhighcontentofflavonoids(Ribeiroetal., 2002).Bothspeciesaredistributedinmanytropicalandsubtropical areasoftheplanet,mainlyinSouthwestAsiaandBrazil(Oliveira deMeloetal.,2006).

Flavonoidandotherphenoliccompoundsareknownfordiverse biologicalactivities,includingantioxidantactivitybyscavenging offreeradicalsandalsobytheinhibitionofthelipidperoxidation (Hanasakietal.,1994).Thesebiologicalactivitiesincrease scien-tificandeconomicattentionforherbaldrugscontainingphenolic tobeusedfortopicalformulationinpreventionorrecoveryofskin damage.

In the meantime, there are several ways to obtain herbal extracts.Distinctextractivesystemscangeneratedifferent prod-uctsintermsofphenoliccontents,evenwhenmadefromthesame herbalspecies(Audietal.,2001;Laporniketal.,2005).

Becausetheseuncountablenumbersofvariationsin produc-tiveandanalyticprocessesofaplantextract,thefirststepforthe developmentofnewherbalextractshouldbetooptimizeand stan-dardizethemanufactureprocedures.Forthispurpose,theresponse surfacemethodology(RSM)hasbeenaveryusefultoolin improv-ingtheprocess.Itiswidelyusedtoassesstherelationshipbetween chemical contentsand biological activities.RSMis alsousedto understandinteractionsbetweenvariablestoobtainabetter activ-ity(Onsekizogluetal.,2010).

Presently, characterization of extract composition has been reported using different analytical techniques, from traditional spectroscopy (Berbicz et al., 2011) to modern methods like UPLC–MS/MS(Oszmia ´nskietal.,2014).Newmethodsarebeing appliedin scientificscenarios every dayandthe choiceforthe bettertechniqueiscase-dependent.

Inthisway,photoacousticspectroscopy(PAS)wasdescribedas atechniquethatcanbeusedtobiological/pharmaceutical analy-sisfordifferentsamplesmatrix.ThePAScanbeveryefficientto evaluatethepermeationanddistributionofsubstancesthrough

theskininvitro,ex vivoandinvivo(Truiteet al.,2007).PASis

basedonopticalabsorptionspectradeterminedbyphotoacoustic signalgeneratedbytheinteractionofmatterwitharadiationof knownwavelength(Berbiczetal.,2011;Truiteetal.,2007).This canbeused toanalyzethedepthofskinpermeationprofileof formulations,includingherbalextractformulations(Truiteetal., 2007).

Materialsandmethods

Materials

Allreagentsandsolvents usedwereanalyticalgrade, includ-ing gallic acid (Sigma®_), _ethyl _alcohol _P.A. _(Synth®_), _methanol

P.A.(FMaia®_),_{butylatedhydroxy}_toluene_(BHT)_(Aldrich®_),

anhy-drous sodium carbonate P.A. (Neon®

), aluminum chloride P.A. (Synth), 2,2-diphenyl-1-picryl-hydrazyl (Aldrich®_), _Folin

Ciocal-teau(Dinâmica®₎_and_quercetin_(Sigma®_).

Rawmaterial

Leavesof theNectandrafalcifolia(Nees)J.A.Castigl. exMart. Crov. & Piccinini, Lauraceae, were collected in Taquarac¸u, MS, Brazil. The material was herborized and deposited in Nupélia herbarium/UniversidadeEstadualdeMaringá,PR,Brazilwithwide voucherspecimennumberHNUPn◦_1421._The_material_was_dried

at40◦_C_in_a_hot-air_oven,_and_ground_by_blade_mill₍_d₌_1.6_mm)._The

herbaldrugcharacterization(waterandashcontainingand gran-ulometricanalysis)wasmade(WHO,2011).Themethodusedfor theextractsproductionwasmaceration.

Experimentalstatisticaldesign

Twosequentialexperimentalmatriceswereused.Thefirstone wasafactorialdesign23 _(Table_1)._In_order_to_produce_extracts,

thevariableswere:extractiontime(ET)(h),ethanolconcentration (EC)(%)andplantproportions(PP)(w/v)whichwereappliedin twolevelsandacentralpoint(n=4).Thetotalphenoliccontents andantioxidantactivitywerequantifiedforeachextractproduced. Afterstatisticalanalysisofthefirstdataset,theethanol concen-trationwasfixedin100%andtheothervariableswereoptimized following the second experimentalmatrix, a central composite design22_(Table_1).

Forbothdesigns,allextractswereproducedatrandom(n=3). The alcohol was evaporated under reduced pressure at 40◦_C,

lyophilizedandstoredinamberbottlesprotectedfromlight,heat andmoisture,whilewaitingforanalysis.ThesoftwareStatistica 7.0®_(StatSoft,_USA)_was_used_to_compare_the_results.

RadicalscavengingactivityDPPHassay

Theantioxidantactivityofthelyophilizedextractswas quanti-fiedbythephotocolorimetricmethodofthefreeradicalDPPH(Prior etal.,2005).Thismethodwasadaptedandvalidated.Torunthe analysis,stocksolutionwereproducedbydissolvingtheextracts inmethanol(5ml).Analyseswereperformedintriplicate.

Fromstocksolutions,sixconcentrationswereprepared(20,24, 28,32, 36,40␮g/ml).Twomillilitersofthereactingsolutionof DPPH(0.06mg/ml)wereaddedto1.0mlofeachofthese concentra-tions.Thetimeofreactionwas30min.TheUVspectrometerwas calibratedusingablanksolution(3mlofmethanolP.A.)andthe negativesolution(noreaction)wascomposedof1mlofmethanol, addedto2mlofDPPHreactingsolution.Thewavelengthusedwas 516nm.

ThepercentageofinhibitionwascalculatedaccordingtoEq.(1),

IC (%)=

_A

0−A1

A0

×100 (1)

where A0 is the absorbanceof negative solution and A1 is the

absorbanceofsamplesreactions.

ThevaluesoftheIC50wereestimatedbylinearregressionfrom

theIC(%)curveversusconcentrations.

Determinationofthetotalphenoliccontent

The total phenolic content was quantified according to the method of Sousa et al. (2007) which was adapted and vali-dated.Twentymilligramsofeachdriedextractweredissolvedin methanolPAandtheexperimentwasperformed.Theblank solu-tion(methanolandtheotherreagents,excepttheextract)wasalso analyzedandallsampleswerereadat760nmatroomtemperature. Thecalibrationcurvewasbuiltforgallicacid(astandard phe-nolic compound) usingthe same reactionconditions. The total phenoliccontentwasdeterminedbyinterpolatingtheabsorbance ofthesamplesagainstacalibrationcurveforgallicacid.Thetotal phenoliccontentacidwasexpressedinequivalentmilligramsper gramofdryextract(mgGAE/gofextract).Allassayswereperformed intriplicate.

Determinationofthetotalflavonoidcontent

Thetotalflavonoidcontentinthedriedextractswasdetermined accordingtotheBrazilianpharmacopoeia(monographofthe

Cal-endulaofficinalisL.,Asteraceae)(Anvisa,2010),againadaptedand

validated.

(3)

Table1

FactorialDesignMatrix23_and_Central_Composite_Design_Matrix₂2_for_the_optimization_of_the_total_phenolic_contents_and_antioxidant_activity_of_extracts_of_Nectandra_facifolia leaves.

FactorialDesignMatrix23 _Central_Composite_Design_Matrix₂2

X1* X2* X3* X1PP(w/v) X2ET(h) X3EC(%) X1* X2* X1ET(h) X2PP(w/v)

1.00 1.00 1.00 1:20 72 100 −1.00 −1.00 24 1:10

1.00 1.00 −1.00 1:20 72 50 −1.00 1.00 24 1:20

1.00 −1.00 1.00 1:20 24 100 1.00 −1.00 72 1:10

1.00 −1.00 −1.00 1:20 24 50 1.00 1.00 72 1:20

−1.00 1.00 1.00 1:10 72 100 −1.4142 0.00 14 1:15

−1.00 1.00 −1.00 1:10 72 50 1.4142 0.00 82 1:15

−1.00 −1.00 1.00 1:10 24 100 0.00 −1.4142 48 1:08

−1.00 −1.00 −1.00 1:10 24 50 0.00 1.4142 48 1:22

0.00 0.00 0.00 1:15 48 75 0.00 0.00 48 1:15

X1*,X2*,X3*:codedvariable;X1,X2,X3:decodedvariable.

total flavonoid contents wasdetermined applying the samples

absorbancetothequercetincurveequationandexpressedas

mil-ligramsofquercetinequivalentbygramofdriedextract(mgEQ/g

ofextract).Assayswereperformedintriplicate.

Complementaryantioxidativeactivityassaysforstandardized

extract.

Freeradicalsscavengeractivity(ABTS)assays

Theantioxidativecapacityofthestandardizedextractwas

mea-suredincomparisontothefreeradicalABTS+•₍_{Sánchez-González}

etal.,2005).TheABTS+•_cation_was_produced_by_the_reaction_of

7mMofthestocksolutionABTSwith2.45mMofpotassium per-sulfate.TheionABTS+•_solution_was_diluted_with_the_phosphate

buffer(pH7.4)and readforabsorbance(0.7–730nm).Themass extracts(20,30and40␮g)weresolubilizedindimethylsulfoxide (DMSO).Thereactionwasmadebyadding10␮lofthesamples to4mloftheABTS+•_solution_diluted._The_samples_were_read_at

730nmafter6minofreaction.Theantioxidantcapacitywas cal-culatedbythepercentageofinhibitionoftheradicalABTS(IA%), accordingtoEq.(2):

IA (%)=100−

_A

1

A0

×100 (2)

whereA1 isthevalueofabsorbanceofthesampleandA0 isthe

valueoftheabsorbanceofthecontrolsolutioninabsenceofthe sample(Sánchez-Gonzálezetal.,2005).

RespiratoryBurstmethodology

Therespiratoryburstwasperformedusinghumanneutrophils toevaluatethekineticproductionofoxygenreactivespeciesafter stimulation by phorbol-myristate (PMA). Chemioluminescence wasmonitoredina microplatereaderVictorX3(PerkinElmer®

, EUA)(Wymannetal.,1987).Theresultswereexpressedincounts perminute(CPM).ForsamplesofstandardizedN.falcifoliaextract, theconcentrationstestedwere:20,30and40␮g/ml.Resultswere shownaccordingtothevaluesofthepeaksfromthekineticcurves andpresentedintheformofquartiles(1stand3rd).Testswere performedin14repetitions.

Topicalformulationcontainingthestandardizedextract(TPSE)

Anon-ionic emulsion base formulation wasdeveloped with the following composition: distilled water, propyleneglycol, ceteareth-20; glyceryl stearate; cetearyl alcohol; decyloleate; caprylic/caprictriglyceride; methylparaben; propylparaben. The

emulsionwasproducedbyaconventionaltechniqueinalaboratory setting.Subsequently,thestandardizedN.falcifoliaextract(SNFE) wasincorporatedin1%ofthebaseformulation.

Photoacousticspectroscopy(PAS)

TheSNFE,baseformulationandTPSEwereanalyzedby photoa-cousticspectroscopy.Thiswasdonetoverifythespectralresponse ofthecomponentsinultravioletandvisiblespectraineach sam-ple.Themeasurementwasperformedusinganexperimentalsetup (Baessoetal.,1994;Berbiczetal.,2011;OliveiraDeMeloetal., 2011).Themonochromaticlight(xenonarclamp–Oriel,Model 68820)withapowerof103_watts_(W)_and_an_emission_in_the_range

180to4×103_nm,_and_a_{monochromator}_(Oriel,_Model_77250)._The

frequencyofthelightmodulationwascontrolledbya mechan-ical modulator (Stanford Research Systems, Model SR540). The microphone(Brüel&Kjaer,BKModel2669)wasattachedtothe photoacousticcellandconnectedtoapowersourceand preampli-fier.ThesignalfromthemicrophonewastransferredtotheLock-in (tunedamplifier)(EG&GInstrumentsModel5110).TheLock-in providestheintensityandphaseofthephotoacousticsignalwhich istransferredtoamicrocomputerviaGPIBinterface.The photoa-cousticspectrawereobtainedbymodulationfrequenciesof25Hz, powerof700Wandwererecordedbetween200and800nm.

Evaluationofpercutaneouspermeationprofileoftopical

formulationsbyphotoacousticspectroscopy(PAS)

Thepenetrationprofilesoftopicalformulations(baseandTPSE) wereevaluatedbyPAS(Baessoetal.,1994;OliveiraDeMeloetal., 2011).Theexperimentalprocedureinvolvingtheuseofanimals wasapprovedbytheEthicsCommitteeonAnimalUsein Experi-mentation,UniversidadeEstadualdeMaringá(n◦_100/2012).

MaleWistarrats,weighing150–200g,weredividedintothree groupsandexposedto:(G1)baseformulation(doseappliedwas 2mg/cm2 ₍_n₌_3));_(G2)_base_formulation_(dose₅_mg/cm2₍_n₌_4));

(G3andG4)TPSEintwoapplications(2mg/cm2_and₅_mg/cm2_),

respectively.

(4)

Table2

Levelsofphenolictotalcontent(TP)(mgGAE/gextract)andIC50oftheantioxidantactivity(AA)fromtheNectandrafalcifoliaextractsobtainedbythefactorial23_and_Central Composite22_designs.

FactorialDesign23 _Central_Composite_Design₂2

X1 X2 X3 TP AA(DPPH) X1 X3 TP AA(DPPH)

ET EC PP Y1 Y2 ET PP Y1 Y2

24 50 01:10 141.9±18.4 27.7±1.2 24 1:10 765.4±42.6 24.7±1.1 24 50 01:20 231.8±28.5 19.4±0.7 24 1:20 529.8±2.5 27.1±0.3 24 100 01:10 765.4±42.7 24.7±1.1 72 1:10 864.8±1.7 19.8±0.6 24 100 01:20 529.8±2.5 27.1±0.3 72 1:20 567.2±46.9 23.2±0.5 72 50 01:10 278.3±26.6 21.5±1.6 14 1:15 136.9±1.17 32.9±0.4 72 50 01:20 385.5±33.3 26.8±0.7 82 1:15 757.1±10.2 21.4±0.1 72 100 01:10 864.8±1.72 19.8±0.6 48 1:08 903.2±24.7 22.8±0.4 72 100 01:20 567.2±46.9 23.2±0.5 48 1:22 520.5±49.1 30.9±0.5 48 75 01:15 74.5±19.5 22.3±0.1 48 1:15 655.5±13.2 23.2±0.8 48 75 01:15 104.5±25.0 22.4±0.2 48 1:15 648.3±1.1 24.2±0.4 48 75 01:15 94.95±1.0 22.9±0.4 48 1:15 641.2±26.7 22.8±0.7 48 75 01:15 72.25±1.8 23.1±0.1 48 1:15 647.7±1.8 23.9±0.7 Dataaremean±SD,n=3.

X1:ET–extractiontime;X2:EC–ethanolicconcentration;X3:PP–plantproportions;X1,X2,X3:decodedvariable;Y1,Y2:average±sd;TP:totalphenolic(mgGAE/gext); AA:DPPH-IC50(␮g/ml).

Allabsorptionspectrawereobtainedusingamodulation

fre-quency of 25Hz in the range of 200–800nm region of the

ultravioletspectrum(UV)andvisible(VIS).

Resultsanddiscussion

Thefirst matrix projected wasthefactorial 23 _experimental

design.Resultsfortotalphenoliccontentsandantioxidant

activ-itybyDPPHmethodoftheextractsarepresentedinTable2.Total

phenoliccontentsareexpressedintermsofmilligramsofgallic acidpergramofdriedextract(mgGAE/gextract)andantioxidant activityisexpressedinaconcentrationoffiftypercentofinhibition (IC50).ANOVAwasperformedtothemodelsgeneratedbythe

fac-torial23_for_both_dependent_variables_and_the_results_are_presented

inTable3.

The performance of the extracts designed by this matrix wasfitted in a linear model for dependent variables. For total phenolic content, the adjusted model presented the overall mean470.63±_5.31_mgGAE/g_extract._The_main_effects _were:_ET −84.04±10.62mgGAE/gextract,EC422.41±10.62mgGAE/gand PP106.74±10.62mgGAE/g.Significantinteractioneffectsbetween ET×_EC_was−_182.55±_10.62_mgGAE/g_extract_and_for_EC×_PP_was −38.31±10.62mgGAE/g extract. The R2 _was _0.99 _and _the _R2

Adjustedwas0.99.

Theseresultsshowthatethanolconcentration(EC)wasthemost importantvariableforbetterextractiveefficiencyoftotal pheno-lic.Thehigherconcentrationofethanolpresentedbetterphenolic levelsintheextract.Ontheotherhand,theinteractionbetween ET×ECshowednegativeeffect,asdidtheinteractionETandPP.

Thesenegativesignsindicateanegativecontributiondecreasing extractiveefficiencywhenthevariablesECandPPareincreased.

Regardingantioxidant activity,theoverallmean ofIC50 was

23.86±0.16␮g/ml.ThemaineffectswereforETwas0.71±0.32, EC was −0.13±0.32 and for PP −1.93±0.32␮g/ml. Significant interactioneffectswerealsofound.Theywere2.17±0.32␮g/ml (ET×_EC),_3.67±_0.32_␮g/ml_(ET×_PP),−_2.48±_0.32_␮g/ml_(EC×_PP) and−3.14±0.32␮g/ml (ET×EC×PP).Thus,themostimportant factor for thebest result for decreasingIC50 wasPP. However,

thebestresponsefordecreasingIC50 valueswastheinteraction

betweenPP×ECandET×EC×PP.Theimprovementinantioxidant capacityisrelatedtotheincreasingofEC,ETandPPlevelsbecause theypresentednegativeinteractioneffects.TheR2_was_0.94_and

theR2_Adjusted_was_0.92.

ResultsshowthattheoptimalextractswereobtainedusingEC 100%.Additionally,instatisticalanalysisthecurvatureeffectwas significantandindicatedthatthenextstepneededtobethe rota-tiononthematrixplanetocharacterizetheexistenceofquadratic effect.Whereastherotationaxisledtotheestablishmentofaxial points(−_1.4142_and_+1.4142)_to_this_model_(Table_2),_the_upper axialpoint ofexecution wasnot possibleforECbecauseit was alreadytestedinamaximumconcentration(100%)level+1.

Since this point,thevariable ECwasfixedin100% and was designedthesecondexperimentalmatrix,acentralcomposite22_.

ThenewmatrixispresentedinTable1andtheresultsforthese extractsarepresentedinTable2.ANOVAwasperformedtothe modelsgeneratedbycentralcomposite22_for_both_dependent

vari-ablesandtheresultsarepresentedinTable4.Themodelsadjusted fortheseconddatasetarepresentedinTable5.

Table3

ANOVAresultstothemodelgeneratedbythefactorial23_for_total_phenolic_content_and_for_total_antioxidant_activity. Factors Totalphenoliccontent Antioxidantactivity

SS df MS F p SS df MS F p

Curve 1,179,925 1 1,179,925 1741.9 0.00 10.32 1 10.32 16.4 0.00

ET 42,384 1 42,384 62.5 0.00 3.10 1 3.10 4.9 0.03

EC 1,070,591 1 1,070,591 1580.5 0.00 0.10 1 0.10 0.2 0.67

PP 68,369 1 68,369 100.9 0.00 22.57 1 22.57 35.8 0.00

ET×EC 199,966 1 199,966 295.2 0.00 28.39 1 28.39 45.0 0.00

ET×PP 746 1 746 1.10 0.30 81.12 1 81.12 128.5 0.00

EC×PP 8807 1 8807 13.0 0.00 36.97 1 36.97 58.6 0.00

ET×EC×PP 2366 1 2366 3.49 0.07 59.31 1 59.31 94.0 0.00

Pureerror 18,289 27 677 17.04 27 0.63

TotalSS 2,591,443 35 258.9 35

(5)

Table4

ANOVAresultstothemodelgeneratedbycentralcomposite22_for_total_phenolic_and_for_total_antioxidant_activity.

Factors Totalphenoliccontent Antioxidantactivity

SS df MS F p SS df MS F p

ET(L) 385,439 1 385,439 633.42 0.00 235.40 1 235.4 469.42 0.00 ET(Q) 108,006 1 108,006 177.49 0.00 18.45 1 7.37 18.45 0.00 PP(L) 432,859 1 432,859 711.35 0.01 111.61 1 111.6 222.56 0.00 PP(Q) 63,384 1 63,384 104.16 0.00 14.02 1 14.0 27.96 0.00 ET(L)×PP(L) 2884 1 2884 4.74 0.10 0.85 1 0.8 1.69 0.20 Lackoffit 268,302 3 89,434 146.97 0.03 97.84 3 32.6 65.03 0.00

Pureerror 16,429 27 608.5 13.53 27 0.5

TotalSS 1,318,920 35 486.38 35

ET(L)×PP(L):interactionET(L)andPP(L);SS:sumsquare;df:degreeoffreedom;MS:meansquare;ET:extractiontime;PP:plantproportion;L:linearterm;Q:quadratic term.

In the second set of experiments, data of total phenolic

content of the overall mean value was 648.19±7.12mgGAE/g

extract,andsignificantmaineffectswere253.45±_10.07_mgGAE/g

extractforETlinear(L),−150.00±11.25mgGAE/gextractforET

quadratic(Q),−268.59±10.07mgGAE/gextractforthePP(L)and

114.91±_11.25_mgGAE/g_extract_for_PP(Q)._None_meaningful

inter-actioneffectwasobserved.TheR2_was_0.79_and_the_R2_Adjusted

was0.75.

For the antioxidant values in the overall average IC50

was 23.58±0.20␮g/ml, and the significant variable effects

were−6.26±0.28␮g/ml for ET (L),1.96±0.32␮g/ml for ET(Q),

4.31±0.28␮g/mlforPP(L),1.70±0.32␮g/mlforPP(Q).Asfortotal

phenoliccontent,nonesignificantinteractioneffectwasobserved.

TheR2_was_0.78_and_the_R2_Adjusted_was_0.73.

Responsesurfacesgraphsfromcentralcomposite22_for_the_total

phenoliccontent(A)andantioxidantactivity(IC50)(B)oftheN.

falcifoliaextractsareshowninFig.1.

Thecombinedoptimalvalueforthetotalphenoliccontentand antioxidativeactivityoftheN.falcifoliaextractswaspredictedfor extractpreparedwithET100%,ET72handPP1:10(w/w).

Theextractproducedundertheseconditionswascalled stan-dardizedN.falcifoliaextract (SNFE).It wasproduced,dried and storedunderthesameconditionspreviouslydescribed.Thisextract haditsantioxidantactivityevaluatedbytwomoremethods(ABTS andrespiratoryburst)inordertoexplorebetterthisproperty.

TheABTS methodhas a differentantioxidant mechanism in comparisontoDPPH.Percentageofinhibition(%)fortheSNFEwas presentedtocompareresults.Themassesofextractusedforboth methodswere20,30and40␮g.For40␮gtheDPPHinhibition(%) was88.13andtheABTSinhibition(%)was93.50,for30␮gwasthe DPPH67.92andtheABTS93.10andfor20␮gtheDPPH47.71and theABTS77.40inhibition(%).

TheseresultsshowthattheSNFEhadanexpressiveantioxidant activityinbothmethodstested.Theywereperformedina sim-plereactionenvironment,asdescribed above.Theseresultswill notnecessarily bethesame ina biologicallycomplex medium. To investigate the antioxidative capacity of the SNFE in com-plexenvironment, the burst respiratory method was used and the results are shown in Fig. 2. This graph shows the activa-tionof humanneutrophilsby PMAin theabsence(neutrophils control)or in the presence of neutrophils added to SNFE. The

data were analyzed by theANOVA test and complemented by theTurkeytest.Forneutrophilscontrol,theaverage(n=14)was 32,497.35±_4469.41_{cintillations}_per_minute_(CPM),_for_the_mass_of 20␮gofSNFEwas13,408.35±1939.23CPMand30␮gofSNFEwas 11,403.28±2177.58CPMand40␮gofSNFEwas9716.57±1658.43 CPM.

Therespiratory burstshowedthatthehigher masshad bet-terantioxidant activity(9716.57CPM)comparedtothecontrol (32,497.35CPM),similartotheABTSandDPPHmethods,indicating thateveninacomplexreactionmediumasthecellular environ-ment,theextractwasabletomaintainhighantioxidantcapacity.

As discussed previously, other species of Nectandra present theflavonoidsasaclassofsecondarymetabolites.Onthatbasis, flavonoids contents characterizationand quantification in SNFE wasevaluatedbyphotoacousticspectroscopy(PAS)andflavonoid test,respectively.

TheopticalabsorptionspectrumbyPASofSNFEisshownin Fig.3(A)andtheresultoftheGaussiandeconvolutionusedto char-acterizethemainwavelengthsfor thespectrum canbeseenin Fig.3(B).

GaussiandeconvolutionofSNFE(Fig.3(B)),suggeststhatthe maximumabsorptionpeakat234nmand390nmarethebands II(ringA)andI(ringB)offlavonoids,respectively.Thissuggests thepresenceofflavonoidsintheextract,asseeninotherspecies (Ribeiroetal.,2002).

ThecontentofflavonoidsinSNFEwasdeterminedandshowed thevalueof41.72±1.92␮QE/mgextract,confirmingthepresence ofthisclassofcompoundswithknownantioxidantactivity.

Thegoalofthisworkwastodevelopanovelproductfor treat-mentand/orpreventionofskindamage.Toaccomplishthis,the permeabilityacrossskinneededtobeevaluated.Therefore,the SNFEwasincorporatedina basetopical formulationpreviously described,containing1%ofSNFE.Thefinal formulationandthe base(withoutSNFE)wereanalyzedbyPASinordertoconfirmtheir absorptionprofiles.

ResultsarepresentedinFig.3(AandB).TheyshowthatSNFE hadawiderangeofabsorption,fromultraviolettovisibleregion (200–800nm).Fig.3(B)presentsthecentersabsorbersobtainedby Gaussiandeconvolution.Theysuggestthepossiblespecies respon-sibleforsuchabsorption.Thetopicalformulationwith1%ofSNFE presentsbandsinpositionssimilartotheSNFEspectrum,butwith

Table5

AdjustedmodelsforphenoliccontentsandantioxidantactivityofNectantrafalcifoliaextractsproducedaccordingacentralcomposite22_design.

Dependentvariables Models R2 _p_value _Equation

Totalphenoliccontent(PC) PC

=(648.19±7.12)+(253.45±10.7)·ETL−(150.00±11.25)· ETQ−(268.59±10.07)·PPL+(114.91±11.25)·PPQ

0.79 <0.05 (3)

Antioxidantactivity(AA) AA

=(23.58±0.20)−6.26±0.28)·ETL+(1.96±0.32)·ETQ+ (4.31±0.28)·PPL+(1.70±0.32)·PPQ

0.78 <0.05 (4)

(6)

1400 1200 1000 800 600 400 200 –200 2.0 2.0 1.5 1.0 0.5 0. 0 –0.₅ –1.0 –1 .5 –1_.0 1.5 1.0 –1.0 –1.5 –2.0 0.5 –0.5 0.0 0

Extraction time (h ) Plant pr oportion (w/v ) To tal phenolic conten

t (mgGAE/g ext) 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 2.0 2.₀ 1.5 1.0 0.₅ 0.0 –0.5 –1_.0 –1.5 –1.₀ 1.5 1.0 –1.0 –1.5 –2.0 0.5 –0.5 0.0 Extraction time (h ) Plant pr oportion (w /v) DPPH IC50 ( µ g/ml ) > 1200

B

A

< 1200 < 1000

< 800 < 600 < 400 < 200 < 0

> 38 < 38 < 34 < 30 < 26 < 22 < 18

Fig.1.Responsesurfaceplotfromcentralcomposite22_for_the_total_phenolic_content_(A)_and_for_antioxidant_activity_(B)_in_Nectandra_falcifolia_extracts

50000 40000 30000 20000 10000 0

Neutrophils Nect 20

µg Nect 30 µg Nect 40 µg Neutrophils Nect 20 µg Nect 30 µg Nect 40 µg

Peak value on the kinetic curve

(C.P

.M.)

Fig.2.Graph–Boxplotofthepeakvaluesonthekineticcurves.Datawereanalyzed byANOVAcomplementedbyTurkey’s*p<0.001comparedtocontrolneutrophils; **p<0.001comparedtocontrolneutrophilsand0.01comparedtoNectandra20␮g. C.P.M.:countperminute.

alowerintensity.Baseformulationspectrumisalsopresentedin

Fig.3(A);itdidnotshowabsorption.

Forevaluatingskinpenetration,thefrequencyofmodulation usedandthethermaldiffusivityoftheskinwasd=4×10−4_cm2_/s.

Thethermal diffusionlength,in whichthephotoacousticsignal generated, wasapproximately23␮m.Considering that samples hadanaveragethicknessof770microns, thickerthanthe pen-etrationcapacity ofthesignal,thesampleswere excitedat the epidermalanddermalsideandevaluated.

Toillustrate,inFig.4(A)and (B)arepresentedtheresultsof thePASspectrumofskinsamplesofoneindividualofG3andG4, respectively.Fig.4(A)showsdataforthelowerdose(2mg/cm2_).

ItindicatesthepresenceoftheemulsionwithSNFEinthe epider-mis,asevidencedbyincreasesinopticalabsorptionobservedin treatedskincomparedtonontreated(290and320nm,450nm and470and650and665nm).The650and665nmoptical absorp-tionassignedtothechlorophyllspresentedinSNFE(Cunhaetal.,

A

0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5 SNFE Fit 234 nm 326 nm 390 nm 444 nm 490 nm 548 nm 611 nm 629 nm 742 nm

Photoacoustic signal (a.u.) Photoacoustic signal (a.u.)

300 400 500 200 300 400 500

Wavelength (nm) Wavelength (nm)

700 700

600 600

B

800 800

(7)

2 mg/cm2

A

5 mg/cm2

B

300 400 500 600 700 800

0.0 0.2 0.4 0.6

A B C D E F G H I

Photoacoustic signal (a.u.)

Wavelength (nm)

A ( Epidermis - control ) B (Dermis -control) C (base)

D (TPSE-rat 1 – Epidermis) E (TPSE-rat 1 - Dermis) F (TPSE-rat 2 – Epidermis) G (TPSE-rat 2 - Dermis ) H (TPSE-rat 2 – Epidermis) I (TPSE-rat 3 - Dermis)

250 300 350 400 450 500 550 600

0.0 0.1 0.2

B C E G I H

Wavelength (nm)

B (Dermis - control) C (base)

E (TPSE-rat 1 - Dermis) G (TPSE-rat 2 - Dermis) I (TPSE-rat 3 - Dermis)

H (TPSE-rat 4 - Dermis)

Fig.4. OpticalabsorptionspectrumobtainedbyPASofTPSAintheepidermalanddermalsideoftheskinsampleswithoutapplicationofproducts(epidermiscontroland dermiscontrol)andfacestheepidermalanddermalsamplesofskinwithTPSAdoses(A)2mg/cm2_and_(B)₅_mg/cm2_.

2009).Regardingthedermis,thepresentdataindicatesthatlittle ornopermeationoccurred,sincetheonlynotabledifferenceisa slightincreaseintheopticalabsorptionnear250nmregion.

Fig.4(B)(higherdose−₅_mg/cm2₎_shows_the_presence_of_the topicalformulationcontainingSNFEinthedermisandepidermis,as indicatedbytheincreasedopticalabsorptioninallregionbetween 240and410nm.

Thecomponentsofbaseformulationdidnotcontributetothe increasedabsorptionbetween240and300nm,sinceitwasnot observedinthedermis.Theseresultsshowthatthefilmthickness oftheproductappliedtotheskinisanotherfactortobeconsidered intermsoftheproductpenetration.

Considering the capacity of SNFE across skin and anti-leishmanial,antimicrobialandantiinflammatoryactivities previ-ouslystated (Oliveira de Melo et al., 2006; Truiti et al., 2005, 2006),canbepostulatednewstudiesfortegumentarleishmaniaor

bacterialinfectionsonskin,aswellinflammatoryprocessthatused tobemediatedbyoxidativeprocess.

Insummary,thedatashowsthatthestatisticalexperimental designcouldcorrectlyguidethestandardizationofextractofN.

fal-cifolia,whenhighantioxidantactivityisdesired.Thisextractcould

crosstheskinbarrierandisthereforeapotentialnovelproductfor skindiseasesandprotection.However,moreexperimentsare nec-essarytoconfirmthisprobableprofileinhumansandintoxicity studies.

Authors’contributions

(8)

contributedtotheexperimentaldesignanalysis.ACNandMLB con-tributedtothephotoacousticspectroscopyanalysis.LHcontributed tothepermeationanalysis.KLBandDSBcontributedtothe com-plementaryantioxidativeactivityassays.Alltheauthorshaveread thefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorswouldliketoexpresstheirgratitudeforfinancial supporttoCAPESandCNPq,toLEPEMC-UEMforUV-spectrometer andtoCOMCAP-UEMforthephotoacousticspectrometer availabil-ities.

References

Anvisa,2010.AgênciaNacionaldeVigilânciaSanitária.FarmacopeiaBrasileira,5th ed.

Audi,E.A.,Campos,E.J.V.,Rufino,M.,GarcíaCortez,D.A.,Amado,C.A.B.,Soares, L.A.L.,Petrovick,P.R.,Mello,J.C.P.,2001.PetiveriaalliaceaL.:plantdrug qual-itycontrol,hydroalcoholicextractstandardizationandpharmacologicalassay oflyophilizedextract.ActaFarm.Bonaer.20,225–232.

Baesso,M.L.,Shen,J.,Snook,R.D.,1994.Laser-inducedphotoacousticsignalphase studyofstratumcorneumandepidermis.Analyst119,561–562.

Berbicz,F.,Nogueira,A.C.,Neto,A.M.,Natali,M.R.M.,Baesso,M.L.,Matioli, G., 2011.Useofphotoacousticspectroscopyinthecharacterizationofinclusion complexesofbenzophenone-3-hydroxypropyl-␤-cyclodextrinandexvivo eval-uationofthepercutaneouspenetrationofsunscreen.Eur.J.Pharm.Biopharm. 79,449–457.

Cunha, F.P.,daCosta, L.J.L.,da Fernandes, A.J.D.,Souza, T.P.,de Soares,L.A.L., 2009.DevelopmentandoptimizationofextractivesfromAstroniumurundeuva (allemão)Engl. by factorialdesign.Braz.Arch.Biol. Technol.52, 647–652, http://dx.doi.org/10.1590/S1516-89132009000300017.

Hanasaki,Y.,Ogawa,S.,Fukui,S.,1994.Thecorrelationbetweenactiveoxygens scavengingandantioxidativeeffectsofflavonoids.FreeRadic.Biol.Med.16, 845–850.

Lapornik,B.,Proˇsek,M.,Wondra,A.G.,2005.Comparisonofextractspreparedfrom plantby-productsusingdifferentsolventsandextractiontime.J.FoodEng.71, 214–222.

Lonni,A.A.S.G.,Longhini,R.,Lopes,G.C.,deMello,J.C.P.,Scarminio,I.S.,2012. Sta-tisticalmixturedesignselectiveextractionofcompoundswith antioxidant activityandtotalpolyphenolcontentfromTrichiliacatigua.Anal.Chim.Acta719, 57–60.

OliveiradeMelo,J.,daConceic¸ãoTorradoTruiti,M.,Muscará,M.N.,Bolonheis,S.M., Dantas,J.A.,Caparroz-Assef,S.M.,Cuman,R.K.N.,Bersani-Amado,C.A.,2006. Anti-inflammatoryactivityofcrudeextractandfractionsofNectandrafalcifolia leaves.Biol.Pharm.Bull.29,2241–2245.

Oliveira De Melo, J., Pedrochi, F., Baesso, M.L., Hernandes, L., Truiti, M.C.T., Baroni,S.,Bersani-Amado,C.A.,2011.Evidenceofdeeppercutaneous penetra-tionassociatedwithanti-inflammatoryactivityoftopicallyappliedHelicteres gardneriana extract A photoacoustic spectroscopy study. Pharm. Res. 28, 331–336.

Onsekizoglu,P.,SavasBahceci,K.,Acar,J.,2010.Theuseoffactorialdesignfor mod-elingmembranedistillation.J.Membr.Sci.349,225–230.

Oszmia ´nski,J.,Kolniak-Ostek,J.,Wojdyło,A.,2014.Characterizationofphenolic compoundsandantioxidantactivityofSolanumscabrumandSolanumburbankii berries.J.Agric.FoodChem.62,1512–1519.

Prior,R.L.,Wu,X.,Schaich,K.,2005.Standardizedmethodsforthedeterminationof antioxidantcapacityandphenolicsinfoodsanddietarysupplements.J.Agric. FoodChem.53,4290–4302,http://dx.doi.org/10.1021/jf0502698.

Ribeiro, A.B., Silva,D.H.S., Bolzani, V.da S., 2002. Antioxidant flavonol glyco-sides from Nectandragrandiflora (Lauraceae). Eclética Química 27,35–44, http://dx.doi.org/10.1590/S0100-46702002000200004.

Sánchez-González,I.,Jiménez-Escrig,A.,Saura-Calixto,F.,2005.Invitroantioxidant activityofcoffeesbrewedusingdifferentprocedures(Italianespressoandfilter). FoodChem.90,133–139.

Sousa, C.M.de M., Silva, H.R.E., Vieira-Jr., G.M., Ayres, M.C.C.,Costa, C.L.S.da, Araújo, D.S., Cavalcante, L.C.D., Barros,E.D.S., Araújo, P.B.de M., Brandão, M.S.,Chaves, M.H.,2007. Fenóis totaise atividade antioxidante de cinco plantas medicinais. Quim. Nova 30, 351–355, http://dx.doi.org/10.1590/ S0100-40422007000200021.

Truite,C.V.R.,Philippsen,G.S.,Ueda-Nakamura,T.,Natali,M.R.M.,Filho,B.P.D.,Bento, A.C.,Baesso,M.L.,Nakamura,C.V.,2007.Percutaneouspenetration,melanin activationandtoxicityevaluationofaphytotherapicformulationforvitiligo therapeutic.Photochem.Photobiol.83,1529–1536.

Truiti,M.daC.T.,Bersani-Amado,C.A.,DiasFilho,B.P.,Sarragiotto,M.H.,deSouza, M.C.,2006.ScreeningoffiveBrazilianplantsforanti-inflammatoryand antimi-crobialactivities.Pharm.Biol.44,516–521.

Truiti,M.C.T.,Ferreira,I.C.P.,Zamuner,M.L.M.,Nakamura,C.V.,Sarragiotto,M.H., Souza,M.C.,2005.AntiprotozoalandmolluscicidalactivitiesoffiveBrazilian plants.Braz.J.Med.Biol.Res.38,1873–1878.

WHO,2011.QualityControlMethodsforHerbalMaterials,Switzerland.

Wymann,M.,Vontscharner,V.,Deranleau,D.,Baggiolini,M.,1987.Theonsetof therespiratoryburstinhuman-neutrophilsrealtimestudiesofH2O2