w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Pterodon
pubescens
oil
nanoemulsions:
physiochemical
and
microbiological
characterization
and
in
vivo
anti-inflammatory
efficacy
studies
Jaqueline
Hoscheid,
Priscila
M.
Outuki,
Sirlene
A.
Kleinubing,
Paulo
R.N.
de
Goes,
Marli
M.S.
Lima,
Roberto
K.N.
Cuman,
Mara
L.C.
Cardoso
∗ProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,UniversidadeEstadualdeMaringá,Maringá,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received19May2016 Accepted30August2016 Availableonline16February2017
Keywords:
Microbiologicalstability Physicochemicalproperty Peritonitis
Sucupira Shelflifestudy
a
b
s
t
r
a
c
t
Pterodonpubescens(Benth.)Benth.,Fabaceae,fruitshavebeeninvestigatedfortheiranti-inflammatory
andantinociceptiveactivities,andhavedemonstratedeffectivenessininflammatoryconditions.A
phys-iochemicalandmicrobiologicalstabilitystudywasconductedtoinvestigatetwonanoemulsion-based
deliverysystemsoftwodifferenthydrophilicsurfactants(polyethyleneglycol-40Hcastoroilor
polyethy-leneglycol-40castoroil).Thenanoemulsions,containingP.pubescensoil,lecithin,hydrophilicsurfactant
andwater,wereanalyzedfordropletsizedistribution,polydispersityindex,pH,consistencyindex,
stabilityagainstcentrifugalforce,andactivecontent/vouacapanderivatives.Thephysicochemical
char-acteristicswerefollowedfor365days.Thenanoemulsionsystemwasevaluatedforanti-inflammatory
activitybyusingwithaperitonitismodel,immediatelyafterpreparationandafter365daysofstorage
at25◦C.Thestabilitystudydemonstratedthatproperstorage(25◦C)preservedthecharacteristicsofthe
nanoemulsioncontaining7.5%polyethyleneglycol-40Hcastoroil,5%lecithin,and5%P.pubescensoil.
Fur-ther,itensuredashelflifeof365daysasaphytotherapeuticformulation.Intheperitonitisassayinduced
bycarrageenan,nanoemulsionpreparedwithpolyethyleneglycol-40Hcastoroil(125mg/kg)reduced
leukocytemigration,evenafter365daysofstorage(25◦C),highlightingitspotentialforthetreatment
ofinflammatorydiseases.However,furtherstudiesareneededtoconfirmitsclinicaleffectiveness.
©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Several phytotherapeutic formulations have been used as effectivealternativesforthetreatmentofdiseases(Parveenetal., 2015).Theanti-inflammatory(Coelhoetal.,2005;Hoscheidetal., 2013; Pascoa et al., 2015)and antinociceptive activities(Nucci etal.,2012;Servatetal.,2012)oftheoilfromPterodonsp.fruits havebeendemonstrated.However,todate,drugscontainingthis oilarenotavailableonthemarket(HoscheidandCardoso,2015). Similarto severaldrugs, drugs containingPterodon sp. fruit oil are associated with low bioavailability (due to instability, low permeability,andlow solubility)(Parveenetal.,2015).Thus,in recentyears,pharmaceuticalindustrieshavefocusedon improv-ingthepermeability andbioavailability ofpoorlywater-soluble compounds(Alamet al.,2012).Nanoemulsions(NE) havebeen
∗ Correspondingauthor.
E-mail:[email protected](M.L.Cardoso).
shown toincrease thetherapeuticefficacyofseveraldrugsand improvephysicalandchemicalstability(Parveenetal.,2015).
As regards the interface of the NE, lecithin uses has been described, because have a unique set of properties including biocompatibility, biodegradability and low to absent toxicity (Washington, 1996). Systems stabilized by phospholipids have appropriate carriers as drug delivery systems (Baspinar and Borchert, 2012; Schuh et al., 2014). Polyethylene glycol cas-tor oil(PEG)derived are nonionic surfactants, which are being usedincreasingly inoral,topicaland parenteralpharmaceutical formulations; particularly suitable for the production of liquid preparationscontainingvolatileoils,vitaminsandother hydropho-bic substances (Ran et al., 2001). O/W emulsions’ are often stabilizedusingionicandnonionicethoxylatedsurfactants.Asa rule,suchdispersesystemsarestableagainstflocculationdueto stericandelectrostaticstabilization(KorolevaandYurtov,2012). Productstability referstothephysicalandchemicalintegrityof thedosageunitandwhereappropriate,thedrug’sabilityto main-tain protection against microbiologicalcontamination. An ideal productshouldbefullycharacterized(physically,chemically,and
http://dx.doi.org/10.1016/j.bjp.2016.08.012
microbiologically)atbaselineandoverthedesiredshelflife(Alam etal.,2012).
Dropletsizeisanimportantfactorwhenassessingthe stabil-ityofemulsionsystems.Changesindropletsizearegenerallydue toaggregationandcoalescencethoroughofmicelles(Petsevetal., 1995).Coalescenceandmoleculardiffusion(alsocalledOstwald ripening)inNEsystemsareresponsibleforcontrollingtheaging process.Ostwald ripening is oftenthe dominantaging process, whichoccurswhen amolecularormacromolecularstabilizeris presentinsufficientamountintheinterfaceandconsistsof spon-taneousdiffusionsofsmalldropletstoformlargerdroplets(Kong andPark,2011).
Rheologicalbehaviorisessentialinstabilitystudies.Areduction inviscosityduringthestorageofakineticallyunstableemulsion leadsto the displacement of the droplets, which subsequently collidewitheachother,resultingincoalescencethorough.Thus, changesinflowbehaviorwithrespecttotimeareimportantandcan provideessentialinformationregardingthestabilityofthesystem (Badolatoetal.,2008).
Chemically,achangeinthepHofformulationsmayindicate degradationand/orionizationofoneormoreoftheconstituentsof theformulation.Moreover,thesechemicaltransformationsreflect thesystem incompatibility and can lead to toxic effects when administeredtopatients(Alametal.,2015).
Takingadvantageofthelipidcharacteristicsoftheextractof
Pubescens pubescensfruits,ourgrouppreviously developed and characterizedNEcontainingP.pubescensoil(Hoscheidetal.,2015). Followingourpreviousstudy,weevaluatedthephysical, chemi-cal,andmicrobialstability,andanti-inflammatorypotentialofNE based-formulationofP.pubescensoil.
Materialsandmethods
Chemicals
Polyethylene glycol (PEG)-40 castor oil/sorbitan oleate and PEG-40hydrogenatedcastoroil/sorbitanoleate(PEG-40H)were kindly provided by Oxiteno (São Paulo, Brazil). Purified soy-bean lecithin (Phospholipon 90G) was obtained from Lipoid (Ludwigshafen, Germany). Ultra-purified water (Milli-Q® Plus, MilliporeCorporation,Billerica,MA,USA)wasusedforpreparation ofallaqueoussolutions.SabourauddextroseagarandTrypticsoy agar (TSA) were purchased from BD® (DifcoTM, Dickinson and
Company,Bedford,Massachusetts,USA).-Carrageenan,usedas a phlogistic agent to induce inflammation, was obtained from Sigma-Aldrich(Auckland,NewZealand).
Plantmaterialandoilextraction
Pterodon pubescens (Benth.) Benth., Fabaceae, fruits were obtainedfromNossaSenhoradoLivramento,MatoGrosso,Brazil (15◦89′ S;56◦41′ W). Thetaxonomicidentitywasconfirmedby
Dr.GermanoGuarimNeto,andavoucherspecimen(no.20502) wasdepositedintheHerbariumoftheUniversidadeEstadualde Maringá.
Oilextractionwasperformedaspreviouslyreported(Hoscheid etal.,2012).Briefly,P.pubescensoilwereextractedwithethanolby turboextraction(Ultra-TurraxUTC115KT,IKAWorks,Wilmington, NC,USA)and partitionedwithhexane.Theorganicsolventwas evaporatedinavacuumrotaryevaporator(Büchi®R-210,Flawil, Switzerland)at40◦Cuntilthesolventevaporatedcompletely.
NEpreparation
NEwaspreparedattheoptimalconditionsthathavebeen pre-viouslydescribed(Hoscheidetal.,2015).Theoilphaseconsisting
ofP.pubescensoil(5%,w/w)andalipophilicemulsifier(soybean lecithin5%,w/w)waspreviouslyhomogenizedandinjectedinto theaqueousphaseconsistingofwaterandahydrophilicemulsifier (PEG-40HorPEG-40,7.5%,w/w),inahigh-speedshearapparatus (IKA®T10basic,Wilmington,DE,USA)at14,500rpmfor15min. ThepHwasadjustedto7.4withNaOHsolution(1M).
Stabilitystudy
ThestudywasperformedaccordingtotheCosmeticStability Guide(Anvisa,2004).NE(10ml)waspackedinglasscontainers (with15ml capacity)withpressurecap,andkeptinincubation chambers(BINDER®)atdifferentstoragetemperaturesand con-ditions:roomtemperature(25±1◦C),intermediatetemperature
withhumidity(30±1◦C with75% relative humidity), and high
temperaturewithhumidity(40±1◦Cwith75%relativehumidity).
Atpre-determinedintervals(30,60,90,120,150and180daysfor NEstoredat30and40◦C;and30,60,90,120,150,180,270and
365days,forNEstoredat25◦C),sampleswereremovedfromthe
storageandphysicochemicalcharacteristicswereevaluated.The freeP.pubescensoilwasstoredunderthesameconditions and temperaturestocomparethechemicalstabilityofthefreeoilto thatoftheNE.Samplesremovedfromtheincubationchambers were maintained at room temperature (25◦C) and analyzed
immediately.Thewithdrawaltimetableofthesampleswasstrictly followed. Droplet size distribution, polydispersity index (PDI), morphology, pH, consistency index, physical stability against centrifugalforce,andvouacapancontentoftheNEwereevaluated.
Assessmentofstabilitybyphysicochemicalcharacterization
Dropletsizedistributionandmorphology
TheaveragedropletsizeandPDIweredeterminedbydynamic lightscattering(DLS)inaparticleanalyzer(Nanoplusnano/zeta particleanalyzer,Georgia, USA).NE wasdilutedwithultrapure water(pH7.4)intheratioof1:10,tominimizethemultiple scat-teringeffectspriortoeachexperiment.Theanalyseswerecarried out intriplicate todeterminetheaveragevalues. The size was expressedinnm.
Themorphologyandformationofaggregateswereassessedby transmissionelectronmicroscopy (TEM)(JEOLJEM 1400 Trans-missionElectronMicroscope,Peabody,MA,USA).NEwasplaced onformvar/Carbon400meshcoppergrid(TedPella,Redding,CA, USA)andnegativelystainedwith2%phosphotungsticacid.After 24hwereobservedat120kV(magnification10k).
pH
Acalibratedpotentiometer(TECNAL,SãoPaulo,Brazil)wasused tomeasurethepHoftheNEsamplesat25±1◦C.
Consistencyindex
RheometrywasconductedusingaMARSII(Haake®)controlled stressrheometer(ThermoFisherScientificInc.,Newington, Ger-many)inflowmodewithcontrolledshearrate(CR),at25±1◦C,
andinconjunctionwithparallelsteelcone-plategeometry(35mm, 2◦ angle,and separatedbya fixeddistanceof 0.052mm).Flow
curves weremeasured over a range of shear rates(0–250s−1).
Theshearratewasincreasedoveraperiodof150s,maintained at the upper limit of 10s, and then decreased over a period of 150s. The consistency index was analyzed according tothe Ostwald-de-Waeleequation(powerlaw).
Sedimentationbehavior
Thisanalyzersimulatesphase separationprocesses under grav-itationalforces (Caddeo etal.,2014)bymeansof infraredlight emittingintheinfraredlengthofthecells,duringcentrifugation andtransmissionthroughthesample;hence,separationkinetics canbestudiedunderacceleratedconditions.Scansarerepeated overtime,eachproducingacurve,andallcurvesaresuperimposed onagraphtoevaluatestabilityovertime.Migrationofparticles,due tocentrifugalforce,leadstovariationinthelocalconcentrationof particlesandtherefore,variationsinthetransmissionwilloccur, indicatingsysteminstabilitysincetheoverlapoftransmission pro-filesovertimehighlightsthequalityofthecolloidaldispersion(Ng etal.,2013).TheNEwerecentrifugedat4000×gforcefor13h and44minat20◦C,conditionsestablishedbytheequipmentfor
evaluatingtheequivalenttothreeyearsofphysicalstability.
Vouacapancontent
Therecoveryandchemical stabilityofvouacapanderivatives wereinvestigatedusinga previousvalidatedmethod(Hoscheid etal.,2015)ofgaschromatographycoupledtomassspectrometry intheselected-ionmonitoring(SIM)mode(GC–MS-SIM)(Thermo ElectronCorporationDSQII;TLC,ThermoFisher,Boston,MA,USA). AliquotsofNEforeachtimeandtemperatureanalysiswerefrozen and lyophilized.Totheoily residue,1500lof chloroformwas addedand1lwasinjected.Vouacapanswerequantifiedusing thecalibrationcurve.Therecoverywascalculatedbydividingthe experimentalconcentrationbythetheoreticalconcentrationand thenmultiplyingby100.
Statisticalanalysis
Alldeterminationswereperformedintriplicatewithtwo sam-ples for each time and temperature, and represented as the means±standarddeviations.Theeffectsofstoragedurationand temperaturewerestatisticallyanalyzedusingtwo-wayanalysisof variance(ANOVA)withtheR-3.2.2software.
Microbiologicalstability
ThenumberofviablepathogensinNEwasdetermined accord-ing to the standards set by the Brazilian Pharmacopoeia V (Farmacopéia Brasileira, 2010). The evaluation was performed usingthepourplatetechnique.Briefly,freshNEwasdilutedwith sterilesalineintheratioof1:10and then1mlwastransferred toPetridishes.Thesolutionwashomogenizedinculturemedium (15ml), allowedto solidify, and then incubated in refrigerated BODincubators(BiogenicOxygenDemand,TE-390model,TECNAL, Brazil).Theculturemediaemployedwereasfollow:Sabouraud dextroseagarforfungiandyeasts,andTSAAgarforbacteria. Stor-ageconditionswereasfollow:7daysat28◦C todeterminethe
presenceoffungiand48hat35◦Cfortotalheterotrophicbacteria.
Todeterminemicrobiologicalstability,NEwasreevaluatedafter 365daysofstorageat25◦C.Allanalyseswereperformedin
tripli-cate.
Stabilityassessedbytheanti-inflammatoryeffectoftheNE
Animals
TheexperimentalprocedureswereapprovedbytheEthics Com-mitteeoftheUniversidadeEstadualdeMaringá(protocolnumber 3968190615). The peritonitis experiment was conducted with Swissmice(weighing30–40g).Micewerehousedundercontrolled conditions (22±2◦C witha 12h light/12hdark cycle and free
accesstofoodandwater).
Anti-inflammatoryactivityoftheNE
Theanti-inflammatoryactivitywasevaluatedby carrageenan-inducedperitonitis.Pharmacologicalexperimentswereconducted withtheNE thatshowedthebest performanceduring the sta-bilitystudy,i.e.,NEcontaining7.5%ofPEG-40Hcastoroilasthe hydrophilicsurfactant.
Groupsofmice(n=5)wereadministeredintramuscularlywith different doses of NE (31.25, 62.50, 125, and 250mg/kg). Ster-ilesalinewasusedasthecontrol.After1h,animalsreceivedan intraperitoneal carrageenaninjection(500g/animal).After4h, theanimalswereanesthetizedwithxylazine(10mg/kg)and pleu-ral exudates werecollectedand thenumber of leukocytes was determinedusingtheNeubauerchamber.
Theanti-inflammatoryactivityofNEwasevaluatedafter365 daysofstorageat25◦C,atadoseof125mg/kg,correspondingtoa
decreaseof60%inleukocytemigration.
Statisticalanalysis
Datawereexpressedasthemean±standarderrorofthemean (SEM).ResultswerestatisticallyanalyzedusingtheGraphPad soft-ware(GraphPadSoftware,Inc.,SanDiego,CA).Analysisofvariance (ANOVA)posthocTukey’stestwasusedtocomparethecompare.
p<0.05wasconsideredstatisticallysignificant.
Resultsanddiscussion
Physicochemicalstabilityevaluation
Thepurposeofstabilitytestingistoensurethequalityofthe activecompoundorthepharmaceuticalproduct,overtime,under theinfluenceofseveralfactors,suchasenvironment,temperature, humidity,andlight.Further,stabilitystudyestablishesaproduct shelf lifeand recommendsstorageconditions.Anidealproduct shouldbephysicallyandchemicallycharacterized(Alietal.,2014). OuroptimizedNEwascharacterizedbydropletsize,PDI, morphol-ogy,pH,consistencyindex,physical stabilityagainstcentrifugal force,andvouacapancontent,atdifferenttimesandtemperatures. Fromapracticalstandpoint,itisimportantthatNEremain phys-icallystableduringstorage.Therefore,wedeterminedthechanges indropletsizeovertime,atdifferenttemperatures(Fig.1).In gen-eral,aunimodaldistributionprofilewasobservedforallNE,with slightincreaseintheaveragedropletsizeandPDIasafunctionof time,andtheincreaseindropletsizewasthehighestin formula-tionsstoredat30and40◦C(Fig.1CandF).Theincreaseindroplet
sizeandPDIwerelowerinformulationscontaining7.5%PEG-40H castoroil(Fig.1B),whichshowedPDIlessthan0.3throughoutthe testperiod.Takingintoconsiderationthelongdurationofthestudy, thisresultindicatesaconsistentsystemandreflectstheoverall sta-bilityofthisformulation(Junyaprasertetal.,2009;Galindo-Alvarez etal.,2011;LiandGe,2012).
Fig.2showsthemorphologyofthedropletsbyTEMand indi-cates thatthe droplets werenot perfectlyspherical,i.e.exhibit anisotropy,whichisacommonfeatureofsoftwaits.Furthermore, thephotomicrographsdemonstratedthataggregation(Fig.2F)and coalescencethoroughwerepronounced(Fig.2GandH)intheNE preparedwithPEG-40castoroil.Ontheotherhand,NEprepared withPEG-40Hcastoroilshowedsignsofaggregationjustafter365 daysofstorage(Fig.2D).
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Initial time 25 ºC after 180 d 30 ºC after 180 d 40 ºC after 180 d 25 ºC after 12 months
Fig.1.Averagedropletsize,PDI,andnormalizedintensitydistributionofNEpreparedwith7.5%PEG-40H(A,B,andC)and7.5%PEG-40(D,E,andF)respectively,atdifferent timeandtemperatureofstorage.
1 µm 1 µm 1 µm 2 µm
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Fig.2. TEMimage,at120kV(magnification10k)ofNEpreparedwith7.5%ofPEG-40H(A,B,C,andD)andPEG-40(E,F,G,andH)atthestartoftheexperiment,after180 daysat25◦Cand40◦C,andafter365daysstorageat25◦C,respectively.
fromeachother (Han etal.,2004).Aggregationdoesnot imply anincreaseindropletssize,butisacoalescencethorough precur-sor,whichtogetherwithOstwaldmaturationinducesincreasein dropletsize(KongandPark,2011).Furthermore,accordingtoLi andGe(2012),thesolubilityofnonionicsurfactantchangesasthe temperatureincrease,andhightemperaturescanleadtothe break-ageofhydrogenbondsatthesurfactantsurface,resultinginloss ofstabilityandfacilitatesaggregationandcoalescencethorough (Fig.2G).
It is important to monitor pH to evaluate the stability of emulsionssincepHchangeindicatestheoccurrenceofchemical reactionsandmaycompromisethefinalproductquality.Itcanbe seenthatregardlessofthesurfactant,thereductioninpHshowed similarkinetics(Fig.3),withsignificantdifferencesobservedonly
among 0,30, and60 days, andstatisticallysimilarvalues were observedafter60days(25◦C).AccordingtotheCosmeticStability
Guide (Anvisa, 2004), it is important that during formulation packagingofstabilitystudy,thevolumeoftheformulationisnot at themaximum volume of thebottle and maintains a gap of approximatelyonethirdofthecontainercapacityforpossiblegas exchange.
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Fig.3. pHvaluesofNEatdifferenttimeandtemperatureofstorage.(A)FormulationspreparedwithPEG-40H.(B)FormulationspreparedwithPEG-40.*p<0.05compared totheinitialtimewithTukey’stest.
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Fig.4.Consistencyindex(K)ofNEatdifferenttimeandtemperatureofstorage.(A)FormulationspreparedwithPEG-40H.(B)FormulationspreparedwithPEG-40. *p<0.05comparedtotheinitialtimewithTukey’stest.
asthetemperatureincreasebutthisdidnotaffecttheoverall qual-ityoftheNEcontainingPEG-40Hcastoroil,sinceatidealstorage conditions(25◦C),pHremainedabove6.0,whichisanacceptable
levelforparenteralformulations(GhoshandJasti,2004).
Fig.4showstheconsistencyindexoftheNE.Increasingshear gradientswereappliedtotheformulationstodeterminetheflow parametersandensurethequalityoftheformulationsunderstress conditions.Areductionintheconsistencyindexwasobservedin theNEthatweresubjectedtohightemperaturesandaninverse proportionalrelationshipwasdemonstrated.Inmostemulsions, theconsistencyindexdecreased athightemperatureduetothe activation energy generated through heat, which is necessary tobreak thehydrogenbondsresponsible for theassociationof moleculesandfacilitatedmovement(Sinko,2008).
Moreover,itwasfoundthattheviscositydecreasedovertime inallformulations.Thisfactcanbeexplained,inpart,bythePDI ofthedroplets.Thewiderdistributionofdropletsize,thelower theviscosity,comparedtosystemscontainingnarrowerPDI(Sinko, 2008).Inthis study,weobservedthatthePDIincreasedduring storage,whichmaybeattributedtothereductioninconsistency index.
Itisbelievedthatviscousemulsionsaremorestablethanless viscousemulsions,duetothedelayinflocculationandcoalescence thorough.However,the stabilityof emulsion dependsonother factors,whichcanbeassessedbytheflowbehavioroftheNEas a function of time. For all samples,thixotropic behaviorswere observed,witha smallareaofhysteresis. Asthesamplesaged, allNEcontainingPEG-40Hcastoroilshowedthesamebehavior, whileNEwithPEG-40castoroilshowedrheopecticbehavior(data notshown).Thus,topromotestability,whichismoreimportant
thandevelopingahighviscosityformulation,istoachieve ideal characteristicsfor thesystem.Viscosity alonedoesnotindicate stableemulsions;itisonlya smallinfluenceoftheoverallO/W emulsions’stability(Sinko,2008).TheNEpreparedwithPEG-40H castoroil(Fig.4A)showedalowerinitialconsistencyindex,due tohydrogenationonPEGchains,andlowerrateofchangeofthe consistencyindexcomparedtothoseoftheNEwithPEG-40castor oil,whichisindicativeofgreaterstabilityofthissystem.
Thekineticdemixingwasanalyzedbyatransmissionprofile sequenceusingamultisamplinganalyticalcentrifuge(Fig.5),which allowsopticalcharacterizationofanytypeofdispersioninamore rapidandsensitivemannerthanwiththenakedeye.
After180days ofstorage atdifferenttemperaturesand 365 daysat25◦C,aseriesofsmoothcurvesinacentrifugalfieldwere
observedandshowedminimalchanges,whichconfirmedthe phys-icalstabilityofcolloidaldispersions.
DegradationoffreeoilandoilcontainedintheNEareshownin
Fig.6.Rapiddegradationofvouacapanswasobservedwiththefree oilatalltestedtemperatures, resultingin yieldsof19.16% after 30daysat40◦Cand18.70%after120daysofstorageat25◦C.In
addition,NEpreparedwith7.5%PEG-40castoroilsufferedmore accelerated degradationcomparedtothose preparedwith PEG-40Hcastoroil.Thekineticmodelofdegradationwasnon-linear andsuggeststhatcomplexreactionsoccurredowingtothe vari-abilityofthesesquiterpenes,andlinearandcyclicditerpenes.Thus, amoredetailedandthoroughanalysisofthedegradationmodelis required.
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Fig.5.PhysicalstabilityagainstcentrifugationforceofNEpreparedwithPEG-40HandPEG-40castoroil,attheinitialtime(AandBrespectively),after180daysofstorageat 25◦C(CandD,respectively),after180daysstorageat30◦C(EandF,respectively)and40◦C(GandH,respectively),andafter365daysstorageat25◦C(IandJ,respectively).
wereinfluencedbystoragetemperatureandthechemical stabil-ityoftheformulationsat40◦Cwaslowerthanthoseat25 and
30◦C were.However,NEcontainingPEG-40Hcastoroilshowed
slowdegradationandmaintainedvouacapanscontentgreaterthan
90%after180daysat30◦C(content:92.01%)and365daysat25◦C
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40 ºC 17.82 19.16
62.22
Fig.6.Vouacapancontent(%)inNEatdifferenttimeandstoragetemperatures.(A)Formulationspreparedwith7.5%ofPEG-40H(solidlines)andfreePterodonpubescensoil (dashedlines).(B)Formulationspreparedwith7.5%ofPEG-40(solidlines)andfreeP.pubescensoil(dashedlines).
TheinstabilityofNEisprimarilyduetotheinteractionsbetween activecontentsandexcipientsviaoxidation,hydrolysis,photolysis, andthermolysis(Alametal.,2015).Thelowdegradationof voua-capansinNEcontainingPEG-40Hcastoroilindicateslowriskof chemicalinstabilityandhighlightstheimportanceofpreparation, storage,andtransportationatappropriateconditionstopreserve stabilityandbioactivity.
Microbiologicalstability
Parenteraldrugsneedtobesteriletoensurethestabilityofthe productandpreventdetrimentaleffectstothepatient.Microbial contaminationisassociatedwiththelossoftherapeuticefficacy duetodegradationoftheactiveingredients,orchangesin phys-icalandchemicalparameters(Baumeretal.,2011).Therefore,to confirmmicrobiologicalstability,freshNE(24hafterpreparation) and NEstored for 365days at 25◦C, were evaluatedfor viable
pathogens.AllNEweresubjectedtomicrobiologicalanalysisand showedabsenceofbacterialand/orfungal.Theseresults demon-stratethattheNEwerewellprepared.
Themicrobialqualityof thefinal productcanbeaffectedby direct sources of contamination (water, raw materials, packag-ingmaterial,andproductionenvironment)andindirectsources, resultingfromcleaningequipmentandsurgicalscruband/or train-ingoperator(Eguchi,2011).
Previousstudieshaveconfirmedtheantimicrobialactivityofoil extractedfromPterodongenus(Menna-Barretoetal.,2008;Dutra etal.,2009;Bustamanteetal.,2010)suggestingthat microbiolog-icalstabilityisrelatedtothepresenceandantimicrobialproperty ofP.pubescensoil.
Anti-inflammatoryevaluation
During an inflammatory response, leukocytes circulating in thebloodleavetheintravascularcompartmentandmigrateinto theinflamedsite viachemotaxis,toremovetheinjuriousagent and promote tissue repair (Noursharghand Alon, 2014). How-ever,uncontrolledinflammatoryresponseaccompaniedbysevere leukocyte migration can lead to tissue damage. Therefore, the developmentanduseofdrugsthatinhibitchemotaxismaybean interestingtherapeuticstrategyforthetreatmentofinflammation (Kummeretal.,2015).Thus,theanti-inflammatorypropertiesofP. pubescensoilNE,preparedwithPEG-40Hcastoroil,wereevaluated usingtheperitonitistestinducedbycarrageenaninSwissmice.
15000
10000
5000
0
Control
31.25 mg/kg of NE62.50 mg/kg of NE125.00 mg/kg of NE250.00 mg/kg of NE
125.00 mg/kg of NE after 12 months
Number of leuk
ocytes (cell/mm
3)
* *
* *
# * #
Fig.7.EffectoftheNE-basedformulationofPterodonpubescensoil,preparedwith PEG-40Hcastoroil,oncarrageenan-inducedperitonitis(500g/i.p.)inSwissmice. Valuesexpressedasthemean±standarderrorofthemean(n=5animals/group). *p<0.0001comparedtothecontrolgroup(ANOVA,Tukey’stest).#Donotshow
statisticaldifferencebetweenthem.
TheNEshowedsignificantanti-inflammatoryeffectand drasti-callyreducedinflammationinadose-dependentmannercompared tothatofthecontrolgroup(Fig.7).
The resultsfor theNE-based formulation of P. pubescensoil wereinagreementwiththefindingsofstudiesthatdemonstrated theanti-inflammatoryactivityoffreshoil(Cardosoetal.,2008; Hoscheidetal.,2015;Pascoaetal.,2015)andisolatedcompounds (Galceranetal.,2011)ofPterodonsp.fruits.
Sincechangesinevaluatedphysicochemicalparametersarenot necessarilyrelatedtothelossoftherapeuticefficacy(Eguchi,2011), evaluationofanti-inflammatoryactivitywasperformedto inves-tigatethetherapeuticefficiencyversusthestability ofNE.Thus, anti-inflammatoryactivitywasreassessedafter365daysofstorage at25◦C,atadoseof125mg/kg.
suggestingarelationbetweenvouacapansderivativescontentand therapeuticefficacy.
Thisstudywasthefirsttoreporttheanti-inflammatoryactivity ofaNE-basedsystemofP.pubescensoilandtheresultsencourage theprogress of the research,since thesystem showed signifi-cantinhibitoryeffectsonthemigrationofleukocytesandappears promising for the treatment of inflammatory diseases. Further studiesshouldbeconductedtocompletetheanti-inflammatory efficacyofthisNE,andtoevaluateitsmechanismofaction, possi-bletoxiceffects,andperformpre-clinicalandclinicaltrials,inthe nearfuture.
Conclusion
Thisstudydemonstratedthatproperstorage(at25◦C)during
transport andstorage canpreserve thecontents presentin the dropletsoftheNE,ensuringaphysical,chemical,and microbial stabilityof365days,andpotentialanti-inflammatoryactivityof formulationpreparedwith7.5%PEG-40Hcastoroil,5%lecithinand 5%P.pubescensoil.Ontheotherhand,thefreeoilshowedrapid anddrasticdegradation.Furthermore,thesystemsolved inconve-niencesrelatedtolowwatersolubility.Notethatco-adjuvantswere absentintheNE,suchasantioxidantsandpreservatives,andthe additionofthesecouldextendtheshelflifeofthesystem.Finally, asignificantinhibitoryeffectonleukocytemigrationwas demon-stratedwiththeNE,bycarrageenan-inducedperitonitisinmice, whichisanimportantprocessoftheinflammatoryresponse.Thus, thissystemmaybeusefulintreatinginflammatorydiseaseswith excessiveleukocytemigration.However,furtherinvestigationsare neededtoconfirmthis.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare
thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).
Confidentialityofdata. Theauthorsdeclarethatnopatientdata
appearinthisarticle.
Right to privacy and informed consent. The authors have
obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.
Authors’contributions
JH,PMOandSAK(PhDstudent)contributedinrunningthe lab-oratorywork,analysisofthedataand draftedthepaper.PRNG and RKNC contributed tobiological studies. MMSL contributed torheometryanalisysand contributedtocriticalreading ofthe manuscript.JHandMLCCdesignedthestudy,supervisedthe labo-ratoryworkandcontributedtocriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorsaregratefultoFinanciadoradeEstudoseProjetos, Fundac¸ãoAraucária,CAPES,andCNPqfortheirfinancialsupport. TheyalsoacknowledgetheEdsonMarquesdosReisfor GC–MS analysis,Dr.NoboruHiokaforDLSanalysis,andComplexode Cen-traisdeApoioaPesquisa(COMCAP-UEM).
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