Rev. bras. farmacogn. vol.27 número3

(1)

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

Short

communication

Utilization

of

dynamic

light

scattering

to

evaluate

Pterodon

emarginatus

oleoresin-based

nanoemulsion

formation

by

non-heating

and

solvent-free

method

Anna

E.M.F.M.

Oliveira

a,b

_,

_Jonatas

_L.

_Duarte

a,b

_,

_Rodrigo

_A.S.

_Cruz

a,b

_,

_Edemilson

_C.

_da

_Conceic¸

_ão

c

_,

José

C.T.

Carvalho

a,b

_,

_Caio

_P.

_Fernandes

a,b,∗

a_Laboratório_de_Pesquisa_em_Fármacos,_Universidade_Federal_do_Amapá,_Macapá,_AP,_Brazil

b_Laboratório_de_{Nanobiotecnologia}_{Fitofarmacêutica,}_Universidade_Federal_do_Amapá,_Macapá,_AP,_Brazil

c_Laboratório_de_Pesquisa,_{Desenvolvimento}_e_Inovac¸_ão_em_Bioprodutos,_Universidade_Federal_de_Goiás,_Goiânia,_GO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30June2016

Accepted22November2016

Availableonline9February2017

Keywords:

Lowenergymethod

Sucupira-branca

Colloidalsystem

Vouacapanditerpenes

Sesquiterpenes

Phytopharmaceuticalnanobiotechnology

a

b

s

t

r

a

c

t

PterodonemarginatusVogel,Fabaceae,isagreatsourceofbioactivecompounds.Themostknownand studiedherbalderivativefromthisspeciesisanambar-coloredoleoresinthatcontainsvouacapane diterpenesandvolatileterpenoids,suchas␤-caryophyllene.Somerecentpapersaimedtogenerate nanoemulsionsusingthisoleoresinforbiologicalapplications.However,theyusedhigh-energy meth-odsthatelevatecostsoftheprocessorheatingprocedures,whichofferthedisadvantageofpossible volatilesubstancesloss.Thus,aspartofourongoingstudieswithnanobiotechnologyofnatural prod-ucts,especiallyregardingpreparationofnanoemulsionswithpromisingplant-basedoilsbylowcost andlowenergymethods,wedecidedtoevaluatetheabilityofnon-heatingandsolvent-freemethodto generateP.emarginatusoleoresin-basednanoemulsions.Twonon-ionicsurfactantswereusedto gen-eratethenanoemulsionsbyasimplehomogenizationmethodwithvortexstirrer.Lowmeandroplet size(<180nm)andlowpolydispersityindex(<0.200)wereobservedevenafteronedayofpreparation. Thelowcoefficientofvariationfortheanalyzedparametersofdifferentbatchesandsimilarprofilefor dropletsizedistributionsuggestedreproducibilityofthemethod.After30days,somedegreeofdroplet growthwasobservedonnanoemulsionpreparedwithpolyethyleneglycol400monooleate,whilealmost noalterationwasobservedfornanoemulsionpreparedwithpolysorbate85.Programmedtemperature rampanalysisrevealedthatnomajoreffectsondropletsizeandpolydispersityindexwereobserved, suggestingtherobustnessofformednanoemulsions.Thus,thepresentstudyshowsforthefirsttimethe formationofsucupira-basednanoemulsionsbyasimple,lowcostandecofriendlymethod.Thisstudy opensnewperspectivesforbioactiveevaluationofthisnovelnano-product.

Introduction

PterodonemarginatusVogelbelongstothefamilyFabaceaeand

it is widespread in Midwest regionof Brazil,being also found

in South-Eastern,Northeastand North regions (Lima andLima,

2016).Its fruits, as well as those fromother Pterodon species,

arecommonlyknownassucupira-branca.Thisplantpartisused

forextractionofanoleoresinthathasvouacapanditerpenesand

sesquiterpenes.Sucupira-brancaoleoresinhasseveralbiological

properties, including analgesic,antiinflammatory, antinoceptive

∗ Correspondingauthor.

E-mail:caiofernandes@unifap.br(C.P.Fernandes).

andlarvicidalactivities(Hansenetal.,2010;HoscheidandCardoso,

2015).

Natural products from plant origin have been subjected to

severalstudiesaimingto generatenovelnanotechnology-based

bioactive systems.Regarding severaltypes of nano-size

formu-lations,herbaloils(includingoleoresins)areveryinterestingfor

nanoemulsionpreparation(Zorzietal.,2015).Nanoemulsionsare

kineticstabledispersesystemsconstitutedbytwoimmiscible

liq-uids, oftenstabilized by surfactant (s).The dropletdiameter is

onthenanometerrange, oftenrangingfrom20nm todifferent

upperlimits(e.g.500,300,200and100nm),thatvaries

accord-ingtoauthorcriteria(SolansandSolé,2012).High-energymethods

involvedonnanoemulsionformationoftenmakeuseofhighenergy

devices thatelevate costsoftheprocess,suchashighpressure

homogenizers or ultrasonicators. Spontaneous energy methods

http://dx.doi.org/10.1016/j.bjp.2016.11.005

(2)

involveutilizationofvolatileorganicsolventsthatmaynotbe

con-sideredecofriendly,consideringtheirpotentialtoxicity.Moreover,

itisnecessarytoremovethiscomponentofthenanoemulsification

process. Low-energy methods make use of intrinsic

character-istics of the system,often involving phase inversion (Sugumar

etal.,2016).Mostcommonphaseinversionmethodsare

associ-atedtophasetransitionsinducedbychangeintemperature(phase

inversiontemperature–PIT)orcomposition(phaseinversion

com-position–PIC)(SolansandSolé,2012).

Growinginterest is observed for nanobiotechnology studies

witholeoresinfromsucupira-branca(Hoscheidetal.,2015;Pascoa

etal., 2015; Oliveiraet al., 2016).The oleoresinobtainedfrom

thefruitsof P.emarginatusthat wasusedin thepresent study

have been previously submitted to chemical characterization.

ChromatographicanalysiscarriedoutusingHPLC-DADand

com-parison to authentic external standards revealed the presence

ofmethyl6␣,7␤-dihydroxyvouacapan-17-␤-oate,geranylgeraniol

and␤-caryophyllene,beinginaccordancewithphytochemical

pro-fileofthegenus(Oliveiraetal.,2016).Thus,itisworthmentioning

thatutilizationofaphaseinversiontemperaturemayleadsome

lostofvolatiles,even ifit isabletogeneratefinedroplets. The

presentstudyaimstoinvestigatethepossibilityofgeneratingP.

emarginatusoilinwaternanoemulsionsusingasimplelowenergy,

non-heatingandsolvent-freeapproachthatcanbeconsideredvery

promising for development of viable, low cost and ecofriendly

nanoproductswiththisnaturalrawmaterial.

Materialandmethods

PterodonemarginatusVogel,Fabaceae,fruitswereobtainedat

theCentralMarketofGoiânia,GO,Brazil,andwereidentifiedby

Dr. José Realinode Paula. Voucher specimen was deposited at

theHerbariumofGoiásFederalUniversity(GO,Brazil)underthe

register number 41714. The oleoresin waspreviously obtained

fromfruits by cold pressing, beingstored in amberglass flask

andkeptat−20◦_C₍_Oliveira_et_al.,₂₀₁₆_)._Prior_to_utilization,_the

oleoresinwasleft underroomtemperaturefortwo daysand it

wascentrifugedtoremovenon-solublecompounds.Oilinwater

nanoemulsionswereprepared byblending non-ionicsurfactant

(polysorbate85,Sigma–Aldrich,StLouis,MO,or

polyethylenegly-col400monooleate,Praid,SP,Brazil)andP.emarginatusoleoresin

atafixedsurfactanttooilratio(9:1).Aftercomplete

homogeniza-tionoftheoilyphase(surfactant+oleoresin),distilledwaterwas

slowlyaddeddropwisetothis mixtureundervigorousagitation

usingavortexstirrer.Eachemulsionpresentedfinalmassof10g

and90%(w/w)ofwater.Dynamiclightscattering(DLS)analysiswas

carriedoutusingaZetasizerNanoZS,Malvern,UK)equippedwith

a10mW“red”laser(=632.8nm)andsamplesweremeasuredata

90◦_scattering_detector_angle_for_size_{measurements.}_The_effects_of

dilutiononmeandropletsize,polydispersityindex(pdi)andzeta

potentialwereperformedusingdifferentdilutionsfactors(1:10,

1:25,1:50and 1:100).Reproducibilityofthenanoemulsion

for-mationwascarriedoutbypreparatingofthreedifferentbatchesof

thenanoemulsions.Nanoemulsionswerealsosubjectedtoanalysis

usingaprogrammedlinearrampoftemperaturestartingfrom25◦_C

to80◦_C_at₅◦_C_intervals._Results_are_expressed_as_mean_±_standard

deviation.

Resultsanddiscussion

Pterodonemarginatusoleoresin-basednanoemulsionsprepared

withpolysorbate85 orpolyethyleneglycol400monooleate

pre-sented a fine aspectwith bluish reflect (Fig.1A and B) that is

characteristicforthistypeofcolloidalsystem.Basedonour

ongo-ingstudieswithnaturaloilsandoleoresins,wehaveobservedthat

Fig.1. NanoemulsionspreparedwitholeoresinextractedfromPterodon

emargina-tusfruitsandnon-ionicsurfactantsatday0:(A)polyethyleneglycol400monooleate

or(B)polysorbate85andday30:(C)polyethyleneglycol400monooleateor(D)

polysorbate85.

itiseasiertoformoilinwaternanoemulsionsusingasystem

con-stitutedbylargeamountsofwater(90–95%).Moreover,itiswell

establishedthatincreasingsurfactanttooilratiooftenenhances

kineticstabilitybyreducingthedropletsize(Ostertagetal.,2012;

KomaikoandMcClements,2015).Sinceweaimedtoevaluateifitis

possibletogeneratenanoemulsionsusinglowenergy,non-heating

andsolvent-freemethod,weoptedtousethisratioasabackground

composition.

Severaldilutionswereperformedjust afterwardsthe

prepa-ration of P. emarginatus oleoresin-based nanoemulsions with

polysorbate85 orpolyethyleneglycol400monooleate(Table1).

Higher mean droplet size and polydispersity index (pdi) were

observed at the dilution ratio of 1:10, for nanoemulsions

pre-pared with both surfactants, which also presented lower zeta

potential (in module). On another hand, no major differences

wereobservedfor meandropletsizeontheremainingdilution

ratios(1:25, 1:50and 1:100),which were around 160–170nm

forbothnanoemulsions.Slightlylowerpolydispersityindexwas

observedfornanoemulsionpreparedwithpolysorbate85,which

wasaround0.111–0.122,whilepdiofdropletsfromnanoemulsion

preparedwithpolyethyleneglycol400monooleatewerearound

0.149–1.152.Zetapotentialofthesenanoemulsionsrangedfrom

−21.9±0.5mV (dilution ratio 1:25 for nanoemulsion prepared

withpolysorbate85)to−28.7±0.7mV(dilutionratioof1:100of

(3)

Table1

Effectofdilutionondropletsize,polydispersityindex(pdi)andzetapotentialofnanoemulsionspreparedwitholeoresinextractedfromPterodonemarginatusfruitsand

non-ionicsurfactants(polyethyleneglycol400monooleateorpolysorbate85).Eachmeasurementrepresentsmean±standarddeviation.Eachanalysiswasperformedin

triplicate.

Dilution Polysorbate85 Polyethyleneglycol400monooleate Size(nm) Pdi Zeta(mV) Size(nm) Pdi Zeta(mV) 1:10 224.6±0.6 0.265±0.008 −16.1±0.3 176.5±1.8 0.188±0.014 −20.1±0.4 1:25 166.7±1.8 0.113±0.011 −21.9±0.5 167.0±1.9 0.149±0.005 −25.2±0.8 1:50 164.4±0.5 0.122±0.001 −24.9±0.1 160.8±0.2 0.152±0.021 −23.3±0.3 1:100 163.5±0.2 0.111±0.015 −26.2±1.2 159.2±0.9 0.150±0.006 −28.7±0.7

Dilutionpriortodropletsizedistributionanalysisisconsidereda criticalparameterandoptimizeddilutionfactorshouldbe deter-mined,in order toavoid multiplescattering effects. Moreover, dilutionofpreviouslypreparednanoemulsionshasbeen consid-ered an important parameter that affects stability and droplet growthofnanoemulsions(Saberietal.,2013a,b,2014).Considering

thatmoreconcentratedstocknanoemulsionshouldbepreferred

for further dilutions in practical applicable productsand since

nomajordifferencewasobservedformostofthem,wedecided

tofurtherinvestigateeffectofstorageortemperatureusingthe

nanoemulsionat1:25dilution.

After 30 days of storage, the nanoemulsion prepared with

polyethyleneglycol 400 monooleate (1:25 dilution factor)

pre-sentedaslightcreaming(Fig.1C),whilethenanoemulsionprepared

withpolysorbate85(1:25dilutionfactor)maintainedthevisual

appearance (Fig. 1D). It was observed that mean droplet size

and polydispersity index of the nanoemulsion prepared with

polyethyleneglycol 400 monooleate presented major increase

(Fig.2AandB).Onanotherhand,lowerincreasedonmeandroplet

size(181.5±0.9nm)andpolydispersityindex(0.111±0.012)was

observedfornanoemulsionpreparedwithpolysorbate85,

show-ingasimilarprofileofdropletsizedistribution,whencomparedto

day0(Fig.2CandD).Thesurfactanttypeisamajorparameteron

nanoemulsionformationandstabilization.Onthiscontext,

inves-tigationsregarding surfactant concentrationarevery important

fordevelopmentofoptimalnanoemulsions(Guttoffetal.,2015).

Consideringthatweoptedonthisstudyforafixedsurfactanttooil

ratio,itisworthmentioningthatdifferencesbetweenthe

surfac-tantsmayberesponsiblebydifferencesondropletsizegrowthand

nanoemulsionstabilization.

Fig.3showstheinfluenceoftemperatureondropletsize

dis-tribution and zeta potential. Analysis of mean droplet size of

nanoemulsionpreparedwithpolysorbate85asfunctionof

tem-perature revealedthat it remained almostconstant from25 to

35◦_C _(around₁₇₀_nm)._Then, _it_rapidly_decreased, _being_around

140nm from 40 to 80◦_C. _On _another _hand, _droplet _diameter

reduction whichwasobservedfornanoemulsionpreparedwith

polyethyleneglycol400monooleateoccurredmoregradually,also

reachinglowvaluesathighertemperatures.Probablythecoating

filmthatthissurfactantformthroughthedropletsismorerigid

duetotheplasticizercharacterofpolyethyleneglycolsurfactants.

Thus,rearrangementoftheinternalphasemaynotbesodrastic

asitwasobservedforpolysorbate85.Polydispersityindexofboth

nanoemulsionsstartedbelow0.200,beingthevalueassociatedto

nanoemulsionpreparedwithpolysorbate85slightlylowerthanthe

valueobservedfornanoemulsionpreparedwith

polyethylenegly-col400monooleate.Then,theyreachedamaximumaround0.250

after35◦_C_and_decreased_to_minimum_values_below_0.100_at₈₀◦_C.

Zetapotentialslightchangedbelow40◦_C,_however,_overall_gradual

increasewasobservedasfunctionoftemperature.

Non-ionic surfactants stabilizes nanoemulsions by reducing

interfacialtensionandtheyalsoformalayerthatpromotessteric

repulsionbetweendroplets(Wangetal.,2009).Themain

mech-anismfornanoemulsiondestabilizationisassociatedtoOstwald

ripening,whichinducesformationoflargerdroplets(Solansand

Solé, 2012). Interestingly, increased temperatures induced

for-mation ofsmaller droplets whileno increase onpolydispersity

indexwasobservedabove0.300.Moreover,pdivaluesreturned

tobasalvaluesaround0.100.Thisparameterreflectshomogeneity

ofdropletsizedistributionandgradualformationoflargerdroplets

fromastartingmonomodaldistributionwouldresultonincreased

polydispersityindex.

Migration of acid compounds from copaiba oleoresin was

associatedtonegativezetapotentialvaluesonnanoemulsions

pre-paredwiththismaterial(Diasetal.,2014).Vouacapanditerpenes,

includingsomecompoundswithacidmoiety, arecharacteristic

ofsucupiraoleoresinandmayexplainthesestartingzeta

poten-tial values. Further increase of temperature may have induced

growingpartitionofadditionalsubstancesthatdonoteasily

disso-ciate,thereforereducingthiszetapotential.Moreover,deposition

20

15

10

5

0

0.1 1 10 100 1000 10 000

20

15

10

5

0

Size (d.nm) Size (d.nm)

0.1 1 10 100 1000 10 000

20

15

10

5

0 15

10

5

0

Size (d.nm)

0.1 1 10 100 1000 10 000

Size (d.nm)

0.1 1 10 100 1000 10 000

A

B

C

D

Fig.2.DropletsizedistributionofnanoemulsionspreparedwitholeoresinextractedfromPterodonemarginatusfruitsandpolyethyleneglycol400monooleateat(A)day

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20 30 40 50 60 70 80 90 120

140 160 180 200

Temperature (°C)

20 30 40 50 60 70 80 90

Temperature (°C)

20 30 40 50 60 70 80 90

Temperature (°C)

Size (nm)

0.0 0.1 0.2 0.3

Polydispersity index

–30 –20 –10 0 10

Tween 85 MP 400

Zeta potential (mV)

Fig.3.Influenceoftemperatureondropletsize,polydispersityindex(pdi)and

zetapotentialofnanoemulsionspreparedwitholeoresinextractedfromPterodon

emarginatusfruitsandnon-ionicsurfactants(polyethyleneglycol400monooleate

orpolysorbate85).Eachmeasurementrepresentsmean±standarddeviation.Each

analysiswasperformedintriplicate.

ofsomesubstancesontheinterfacemayformmorecompactfilms

andtherefore,enhancingthestabilityofthesystem(Wangetal.,

2009).

Wedecidedtoinvestigatethereproducibilityofthemethod

on nanoemulsion formation and low coefficient of variation

values of three individual batches were observed for droplet

size (CVMP400=0.00499; CVT85=0.0113), pdi (CVMP400=0.0534;

CVT85=0.0732) and zeta potential (CVMP400=0.0416;

CVT85=0.0741). Fig. 4 reflects the technical feasibility of the

methodtoinduceformationofnanoemulsionswithlowdroplet

sizeand lowpolydispersity index(<0.300),beingin accordance

with satisfactory results for size distribution of aqueous

nan-odispersions (Tan et al., 2016). A study aiming to optimize

thepreparation of nanoemulsions with copaiba oleoresinfrom

Copaiferamultijuga,whichbelongs tothesamebotanicalfamily

(Fabaceae)ofP.emarginatus,performedatriplicateofcenterpoint

fromafactorialdesignexperimentandalsoobservedlow

coeffi-cientofvariationforanalyzedparameters(Diasetal.,2014).Thus,

overallourresultssuggestthereproducibilityofthemethodfor

thepreparationofnanoemulsionsusingP.emarginatusoleoresin

withahighprevalenceofdropletsbelow200nm.

Dropletsizedistributionofnanoemulsionsfromthethree

dif-ferentbatchesalsorevealalessabundantdropletpopulationbelow

100nm, for those prepared withboth surfactants. Moreover, a

dropletpopulationwithsizeabove1000nm(microdroplets)was

observedonnanoemulsionpreparedwithpolysorbate85.Sincethe

firststudieswithP.emarginatusoleoresin,it wasobservedthat

diterpenesgradually precipitate onthis viscousoily raw

mate-rial(MahajanandMonteiro,1973).Consideringthehighviscosity

of theP.emarginatus oleoresin,somemicroparticles (or

micro-crystals) of the diterpenes on suspension may remain on the

supernatant that wasused for nanoemulsification,even after a

centrifugingstep,beingresponsiblebythismicro-sizepopulation.

Furtherstudiesaimingtobetterinvestigatethishypothesisor

con-comitantformation ofanother systems(e.g. multiphaseregion,

microdroplets/coarsedroplets)arerequiredtosolvethese

ques-tions.

Alarvicidalnanoemulsionpreparedwiththesameoleoresin

thatwasusedinthepresentstudy,amixtureofpolysorbate

80/sor-bitanmonooleate(HLBmixture=11)and waterpresented mean

droplet sizearound 130nm, polydispersity index around 0.200

andzetapotentialaround−30mV.Thenanoemulsificationmethod

involvedhomogenizationofoilyphase(P.emarginatusoleoresin

andsurfactants)at80◦_C_for₃₀_min₍_Oliveira_et_al.,₂₀₁₆_)._A

pro-cedurethatalsoinvolvedheatingprocesswasperformedaiming

toprepareP.emarginatusbasednanoemulsionsduetothe

antiin-flammatorypotential(Pascoaetal.,2015).Itisworthmentioning

thatvolatilesubstancesfromtheoleoresinmaybelostduringheat

oftheoilyphase.Forthisreason,utilizationofeffective

nanoemul-sificationmethodsthat donotinvolvea heatingstepshouldbe

consideredanadvantage,consideringthattheywouldprotectthe

undesiredlossofvolatiles,includingsometerpenoids.

Rosmarinusofficinalisessentialoilwasformerlyusedfor

devel-opmentof anoilin waternanoemulsionusing phase inversion

temperature (PIT) method and droplets with mean diameter

around 100nm were obtained (Fernandes et al., 2013). Latter,

nanoemulsion phase inversion method using a titration

pro-cesswasperformedforachievementofbioactiverosemary-based

nanoemulsionsusingthesamenaturalrawmaterial.Afterseven

daysofstorage,meandropletsizearound115.0nmandlow

poly-dispersityindex(<0.300)wereobserved(Duarteetal.,2015).

The nanoemulsions prepared with oleoresin from Copaifera

duckei were prepared by using phase inversion temperature

method.Lowmeandropletsize(145.2nm)andrelativelybroad

droplet size distribution (pdi=0.378) were observed for this

nanoproductafteronedayofpreparation(Rodriguesetal.,2014).

The oleoresin from another copaiba species (C. multijuga) was

nanoemulsifiedusingahigh-pressurehomogenizeranddroplets

sizearound120–140nmandzetapotentialaround−20mVwere

obtained (Dias et al., 2012). On another study, nanoemulsions

preparedwithC.multijugaoleoresinwerepreparedusing

high-pressurehomogenizationmethodorspontaneousemulsification

usingorganicsolvents.Mostofnanoemulsionspreparedbyhigh

energymethodpresentedmeandropletsizearound160–200nm

andmonomodaldistribution,beingconsideredmoreefficientthan

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20

15

10

5

0

15

10

5

0

0.1 1 10 100 1000 10 000

Size (d.nm)

MP400

T85

Size (d.nm)

Fig.4.DropletsizedistributionofPterodonemarginatusoleoresin-basednanoemulsionspreparedwithpolyethyleneglycol400monooleate(MP400)orpolysorbate85(T85).

Eachmeasurement(intriplicate)representsthreedifferentbatchesofnanoemulsions.

TheoleoresinfromP.pubescensmixedwithphospholipon90G

wasaddedthroughaqueousdispersionsofseveralsurfactantsand

thennanoemulsifiedusinghigh-speedshearhomogenizer.Mean

diameterofdropletsrangedfrom199to860nmandlowestsize

wereobservedfornanoemulsionspreparedwithpolyethylene

gly-col(PEG-40)castoroil/sorbitanoleateandPEG-40hydrogenated

castoroil/sorbitanoleate(PEG-40H)(Hoscheidetal.,2015).The

andiroba(Carapaguianensis)oilwassubjectedtonanoemulsion

preparationby aself-nanoemulsifyingmethodusingacetoneas

organicphaseconstituent.Onthissamestudy,anaroeira(Schinus

molle)-basednanoemulsionwasprepared usingahigh-pressure

homogenizer(Baldisseraetal.,2013).

Conclusion

The present study shows that it is possible to generate P.

emarginatus nanoemulsions by a non-heating and solvent free

approach,withoutusinghighenergyequipment.Thisecofriendly

concepthasmajoradvantagesduetopossiblereducingcostsand

even less impairment to environment and health. It is worth

mentioningthat furtherstudiesshouldbenecessarytodevelop

optimalP.emarginatusnanoemulsionsusingthismethod.

How-ever,webelievethatitopensgreatperspectivesforfastandsimple

preparationofnaturalproduct-basednanoemulsionsforbiological

evaluation,beingcomparableorevenpotentiallymoreeffective

thanmostusedmethods. It alsoprovidesgreat information for

studiesaimingtodevelopthistypeofcolloidalsystem,sincethis

informationarestillscarceforcomplexBraziliannaturaloils.

Authors’contributions

AEMFMO(PhDstudent),RASC(PhDstudent)andJLD

(under-graduate student) contributed running the laboratory work,

analysisof the data and draftedthe paper. ECCcontributed to

oleoresinextraction,characterization andcriticalreading ofthe

manuscript.JCTCandCPFdesignedthestudy,supervisedthe

labo-ratoryworkandcontributedtocriticalreadingofthemanuscript.

Alltheauthorshavereadthefinalmanuscriptandapprovedthe

submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors thank CNPq (Rede Amazônica de Pesquisa em

Biofármacos–407768/2013-0),Fapeap(Prodetec Araguari–

Pro-cess n◦ _{250.203.035/2013)} _and _PROPESPG _(PROPUBLIC _and

PAPESQ)forfinancialsupport.

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