RevistaBrasileiradeFarmacognosia25(2015)189–192
w w w . s b f g n o s i a . o r g . b r / r e v i s t a
Short
communication
Evaluation
of
larvicidal
activity
of
a
nanoemulsion
of
Rosmarinus
officinalis
essential
oil
Jonatas
L.
Duarte
a,b,
Jesús
R.R.
Amado
a,b,
Anna
E.M.F.M.
Oliveira
a,
Rodrigo
A.S.
Cruz
b,
Adriana
M.
Ferreira
a,
Raimundo
N.P.
Souto
c,
Deborah
Q.
Falcão
d,
José
C.T.
Carvalho
a,
Caio
P.
Fernandes
a,b,∗aLaboratóriodePesquisaemFármacos,UniversidadeFederaldoAmapá,Macapá,AP,Brazil
bLaboratóriodeNanobiotecnologiaFitofarmacêutica,UniversidadeFederaldoAmapá,Macapá,AP,Brazil cLaboratóriodeArtrópodes,UniversidadeFederaldoAmapá,Macapá,AP,Brazil
dLaboratóriodeTecnologiaFarmacêutica,UniversidadeFederalFluminense,Niteroi,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received4December2014 Accepted2February2015 Availableonline31March2015
Keywords: Aedesaegypti Larvicidal Nanoemulsion Rosmarinusofficinalis
a
b
s
t
r
a
c
t
Nanotechnologyhasemergedasapromisingareaforinnovativeproducts,includinginsecticides.Dengue isatropicaldiseasewhichisconsideredacriticalhealthproblemindevelopingcountries,duetonegative impactstotheenvironmentcausedbysyntheticchemicalsusedforvectorcontrol(Aedesaegypti).Thus, developingofnaturalproductsbasedinsecticidalareconsideredverypromising.Onthiscontext,theaim ofthepresentstudywastoobtainanO/WnanoemulsioncontainingRosmarinusofficinalisL.,Lamiaceae, essentialoilandevaluateitslarvicidalactivityagainstA.aegypti.Lowenergymethodwasemployed, allowingachievementofsmalldroplets.Thenanoemulsionalsopresentedlowpolydispersityandmean dropletbelow200nm,evenafter30daysofstorage.Potentialmortalitylevelswereobservedafter24h (80±10%)and48h(90±10%)inA.aegyptilarvaeatfinalconcentrationof250ppm,relatedtoR.officinalis
essentialoil.Thisstudycontributestonanobiotechnologyofnaturalproducts,presentingapotential larvicidalnanoemulsionpreparedwithR.officinalisessentialoil.Moreover,nanoemulsionproduction involvedanon-heatingprocedure,describingeasytechniquewhichmaybeusefulforintegrativecontrol programs.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Nanotechnologyisamultidisciplinaryapproachwhichinvolves creationandutilizationofdifferentsystemsonananometricscale (DeVilliersetal.,2009).Severaltypesofnanoformulationshave beenreported,includingnanoemulsions,whicharedispersed sys-temsconstitutedbyimmiscibleliquidsandoneormorestabilizers (McClements, 2012). Nanoemulsions are characterized by their thermodynamicallystabilityandsmalldroplets,rangingfrom20 to200nm(Ostertagetal.,2012).
Dengue is an endemic illness on South America and other countries. Recently,it wasobservedanincrease inthe morbid-ityofthispathology, beingconsideredacriticalhealth problem (WHO,2014).Manysubstanceshavebeentestedtocontrolthe vec-torAedesaegypti.Severalsubstanceshavebeentestedtocontrolde vectorAedesaegypti(Hirataetal.,2014).However,manyofthem aresyntheticchemicals,includingtheorganophosphatetemephos and thepyrethroid deltamethrin, which maylead resistancein
∗ Correspondingauthor.
E-mail:caiofernandes@unifap.br(C.P.Fernandes).
themosquitoes and even negative impactsto theenvironment (Marcombeetal.,2009).
Onthiscontext,ecofriendlyalternativeintegratedcontrol pro-grams have emergedas promising alternatives(Sugumar etal., 2014)andessentialoilbasednanoemulsionshavebeenrecognized as valuable productsfor mosquito control(Ghosh et al.,2013). RosmarinusofficinalisL.,Lamiaceae,essentialoilhasdemonstrated larvicidalproperties(Prajapatietal.,2005;Freitasetal.,2010;Amer andMehlhorn,2006a)andrepellentactivity(Prajapatietal.,2005; AmerandMehlhorn,2006b).However,intrinsicpoorwater solu-bilityofessentialoilsisatechnologicalchallenge.Theaimofthe presentstudywastoobtainanO/WnanoemulsioncontainingR. officinalisessentialoilandevaluateitslarvicidalactivityagainstA. aegypti.
Materialsandmethods
Chemicals
Polysorbate20waspurchased fromPraidProdutosQuímicos Ltda(SP,Brazil).
http://dx.doi.org/10.1016/j.bjp.2015.02.010
190 J.L.Duarteetal./RevistaBrasileiradeFarmacognosia25(2015)189–192
Essentialoil
EssentialoilextractionfromleavesofR.officinalisL.,Lamiaceae, wasperformedbyhydrodistillationusingaClevengerapparatus. Experimentalprotocolforextractionandchemicalcharacterization oftheessentialoilusedinthisworkwerepreviouslydescribed (Fernandesetal.,2013).
Nanoemulsionpreparation
Nanoemulsionwasobtainedbyalowenergymethod(Ostertag etal.,2012)using90%(w/w)ofwater,5%(w/w)ofessentialoiland 5%(w/w)ofpolysorbate20atatotalmassof50g.Theessential oilandpolysorbate20werestirredat800rpmusingmagnetic stir-rer(Fisatom,Brazil)for30min.Then,waterwasaddeddropwise ataflowrateof3.5ml/min.Themixturewasstirredat800rpm for 60min.Nanoemulsion was stored under roomtemperature (20±2◦C)andevaluatedafter1,7,21and30daysofpreparation.
Dropletsizeanalysis
Dropletsizeandpolydispersityofthenanoemulsionwas deter-minedbyphotoncorrelationspectroscopy(ZetasizerZS,Malvern, UK). Nanoemulsionwas dilutedwithwater for injection(1:25) (Fernandesetal.,2013).Measurementsweremadeintriplicate. Theaveragedropletsizewasexpressedasthemeandiameter.
Larvicidalassay
A.aegyptilarvaewereobtainedfromtheArthropoda Labora-tory(UniversidadeFederaldoAmapá,Brazil).Biologicalassaywas performedundercontrolledconditions,beingfourth-instarlarvae keptat25±2◦C,relativehumidityof75±5%anda12hlight:dark
cycle.Experimental protocolwasperformedaccording toWHO (2005)withsomemodifications.Allexperimentswereperformed intriplicatewith10forth-instarlarvaeineachsample,usingthe nanoemulsiondilutedindistilledwaterat250ppm(relatedtoR. officinalisessentialoil).Negativecontrolwasperformedwith sur-factantatsameconcentrationoftestedsamples.Mortalitylevels wererecordedafter24and48hofexposure.
Statisticalanalysis
Analysisofvariance (ANOVA)followed byDuncan’stestwas conductedusingStatGraphicsPlussoftwarev.5.1(StatEasyCo., Minneapolis,USA). Difference wasconsidered significant when p≤0.05.
Resultsanddiscussion
Essentialoilsarevolatilecomplexmixtureswithawiderangeof biologicalactivities,includingrepellent,insecticidalandlarvicidal properties(Contietal.,2010).R.officinalisessentialoilusedinthis studyhas1,8-cineole(44.0%),camphor(16.1%),-myrcene(11.1),
␣-pinene(9.4%);verbenone(4.1%),borneol(3.5%)andcamphene
(3.3%)asmajorsubstances(Fernandesetal.,2013),beingessential oilswiththesesubstancesaredescribedaslarvicidalagents(Conti etal.,2010).
However,essentialoilshavepoorwatersolubilityandthisisa technologicalproblemfortheirapplicationaslarvicidalproducts.A. aegyptidevelopmentoccursinwater,thus,activesubstancesmust bedispersedorsolubilizedinthismedium.Onthiscontext,anO/W nanoemulsionofR.officinalisessentialoilcouldsolvetheproblem ofwatersolubility.
Nanoemulsioncontaining5%(w/w)ofessentialoilfromR. offici-nalis,5%(w/w)ofpolysorbate20presentedafineappearanceand
Fig.1.O/WnanoemulsionofRosmarinusofficinalis.
bluishaspect,whichisinaccordancewiththis typeof formula-tion(Fig.1).Itwasnotobservedanysignalofinstability,including creamingorphaseseparation.
Fig.2showsresultsconcerningmeandropletsizeand polydis-persity,during4weeks.Lowmeandiameter(<200nm),whichisin accordancewiththeconceptofnanoemulsions(Solansetal.,2005; Solèetal.,2012)wereobservedinallmeasurements.Particlesize distributionafteronedaypresentedapolimodalprofile,indicating thepresenceofdifferentparticlesizepopulations.
Aftersevendays ofpreparation it wasobservedan increase inmeandropletsize.However,itwasmaintainedbelow200nm (Fig.2)andpolydispersitywasreduced(Fig.3B).Fig.3CandDshow thatmeandropletsizeafter21and30daysalsoremainedunder 200nm.Moreover,nosignificantdifferencewasobservedbetween polydispersity (t=−0.6351; p=0.5599) in the interval of 21–30 days.Micellesarecontinuouslydisintegratingandreassembling, beingindynamicequilibriumwithindividualsurfactantmolecules
200
180
160
140
120
100
Particle size (nm)
Polidispersion index
80
60
40
0 7 14
0.136 0.147
0.281
50.15 115.5 0.506
Particle size Polidispersion index
174.1
180.0
Time (days)
21 28 35
0,1 0,2 0,3 0,4 0,5 0,6
J.L.Duarteetal./RevistaBrasileiradeFarmacognosia25(2015)189–192 191
15
10
5
0
0.1 1 10 100
Size (d.nm)
Size distribution by intensity
Intensity (%)
1000 10000
25
20
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5
0
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1000 10000
15 20
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0.1 1 10 100
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Intensity (%)
15 20
10
5
0
Intensity (%)
1000 10000 0.1 1 10 100
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Size distribution by intensity
1000 10000
A
B
C
D
Fig.3.ParticlesizedistributionofR.officinalisnanoemulsionafter(A)1day:meandroplet–50.15±1.306nm;polydispersity–0.506±0.036nm.(B)7days:mean droplet–115.5±5.147nm;polydispersity–0.281±0.089.(C)21days:meandroplet–174.1±2.536nm;polydispersity–0.136±0.026nm.(D)30days:meandroplet –184.0±4.133nm;polydispersity–0.147±0.016nm.
(Patistetal.,2002).Thus,itcouldbesuggestedthatmicellesreached dynamicequilibriuminthisperiod,showingakineticstabilityof thenanoemulsion.
Previousstudy withR.officinalis essential oilallowed deter-mination of required HLB value and achievement of an O/W nanoemulsion.Emulsificationmethodinvolvedintheprocedure usedheatingoftheoilphase,constitutedbyessentialoiland sur-factant,inordertoobtainsmalldroplets(Fernandesetal.,2013). Essential oils are complex mixtures of volatile substances and heatingstepwouldleadlossofsubstances.Aspartofour ongo-ingstudiesconcerningnanobiotechnologyofR.officinalisessential oil,wedecidedtotestamethodwithoutheating,whichproved to successfully generate a nanoemulsion. Titration low energy methodusedinthisstudyisbasedinacatastrophicphaseinversion (Ostertagetal.,2012).
Itwasobservedthatthenanoemulsioncontainingessentialoil ofR.officinaliscaused80±10%ofmortalityafter24hand90±10% ofmortalityafter48h(Fig.4).Nomortalitywasobservedfor con-trolgroup.Afterone dayofpreparation, it wasobserved some particlesaround10nm,whichwereresponsiblebythepolimodal profile.Furthercharacterizationrevealedthatnarrowdistribution wasachieved, probably due to disintegration and regeneration ofmicelles.Penetrationthroughcuticleiscrucialforinsecticidal activityandrecognizedasoneofpossiblemechanismsof insec-tides(Kasaietal.,2014).Consideringthatparticlesizeofdroplets remainedinananometricrange,penetrationandpotential larvi-cidalactivitymaynotbeaffected.Furtherinvestigationswouldbe necessarytoconfirmthesefindings.
Potential larvicidal application of natural products can be obtainedconsideringmortalitylevelsoflarvaeafter48hof treat-ment with samples at 250ppm as follows: promising (>75%), partiallypromising(>50%and<75%),weaklypromising(>25%and <50%)andinactive(<25%)(Montenegroetal.,2006).Onthis con-text, our results suggest that the nanoemulsion containing 5% (w/w)ofR.officinalisessentialoil,5%(w/w)ofpolysorbate20(w/w) and90%(w/w)ofwatercanbeconsideredapromisinglarvicidal agent.
100
80
a
a
60
40
20
0
24 48
Time (h)
Mortality (%)
Fig.4. MortalitylevelsofAedesaegyptilarvaeaftertreatmentwith nanoemul-sioncontainingRosmarinusofficinalisessentialoil.Testedconcentration–250ppm (relatedtoR.officinalisessentialoil).Columnswiththesamesuperscriptdonothave significantdifference.
Conclusion
192 J.L.Duarteetal./RevistaBrasileiradeFarmacognosia25(2015)189–192
Authors’contributions
JLD(undergraduate student) and AEMFM (master’s student) contributedrunningthelaboratorywork,analysisofthedataand draftingthe paper. AMF contributed in nanoemulsions charac-terization.RASC and DQF contributed tocritical reading of the manuscript.JRRA and RNPS contributedto biologicalassay and statisticalanalysis.JCTCandCPFdesigned thestudy,supervised the laboratorywork and contributed to critical reading of the manuscript.Alltheauthorshave readthefinal manuscript and approvedthesubmission.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
AuthorswouldliketothankCNPQandFAPEAPforthefinancial support.
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