The efficacy of three formulations of Lippia sidoides Cham. essential oilin the reduction of salivary Streptococcus mutans in children withcaries : a randomized, doubleblind, controlled study

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ContentslistsavailableatScienceDirect

Phytomedicine

j o ur na l h o m e p a g e :w w w . e l s e v i e r . d e / p h y m e d

The

efficacy

of

three

formulations

of

Lippia

sidoides

Cham.

essential

oil

in

the

reduction

of

salivary

Streptococcus

mutans

in

children

with

caries:

A

randomized,

double-blind,

controlled

study

Patrícia

Leal

Dantas

Lobo

a

_,

_Cristiane

_Sá

_Roriz

_Fonteles

b,∗

,

Lídia

Audrey

Rocha

Valadas

Marques

c

_,

_Francisco

_Vagnaldo

_Fechine

_Jamacaru

c

_,

Said

Gonc¸

alves

da

Cruz

Fonseca

d

_,

_Cibele

_Barreto

_Mano

_de

_Carvalho

e

_,

Maria

Elisabete

Amaral

de

Moraes

c

a_Department_of_Clinical_Dentistry,_School_of_{Dentistry-Campus}_Sobral,_Federal_University_of_Ceará,_Brazil

b_Postgraduate_Program_in_Dentistry,_Department_of_Clinical_Dentistry,_School_of_Dentistry,_Federal_University_of_Ceará,_Brazil c_Clinical_Pharmacology_Unit,_Department_of_{Pharmacology,}_School_of_Medicine,_Federal_University_of_Ceará,_Brazil d_Laboratory_of_{Pharmaceutical}_Science,_School_of_Pharmacy,_Federal_University_of_Ceará,_Brazil

e_Department_of_Pathology_and_Legal_Medicine,_School_of_Medicine,_Federal_University_of_Ceará,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24October2013

Receivedinrevisedform23February2014 Accepted20April2014

Keywords:

LippiasidoidesCham.essentialoil Dentalcaries

Streptococcusmutans

a

b

s

t

r

a

c

t

Essentialoilsofmanyplantshavebeenpreviouslytestedinthetreatmentoforaldiseasesandother infections.Thisstudywasarandomized,double-blind,inparallelwithanactivecontrolstudy,which aimedtoevaluatetheefficacyofthreeformulationsoftheLippiasidoidesCham.essentialoil(LSO)inthe reductionofsalivaryStreptococcusmutansinchildrenwithcaries.81volunteers,aged6–12years,both genders,withcaries,wererecruitedtoparticipateinthisstudy,andrandomlyassignedtoeitheroneof fivedifferentgroups.Eachgroupreceivedtopicaltreatmentwitheither1.4%LSOtoothpaste,1.4%LSO gel,0.8%LSOmouthwash,1%chlorhexidinegel,or0.12%chlorhexidinemouthwash.A5-mlvolumeof eachgelwasplacedinsidedisposabletrays,andappliedfor1min,every24h,for5consecutivedays. Themouthwashgroupsused5-mlvolumeofamouthwashinsidedisposablesyringes.Inthetoothpaste group,childrenbrushedtheirteethfor1min,onceadayfor5days.Salivawascollectedbeforeand aftertreatment.MScolonieswerecounted,isolatedandconfirmedthroughbiochemicaltests. Differ-encesinMSlevelsmeasuredindifferentdayswithinthesametreatmentgroupwasonlyverifiedwith LSOtoothpaste,chlorhexidinegelandchlorhexidinemouthwash.ComparisonbetweengroupsofLSO mouthwash,toothpasteandgelshowedthatthetoothpastegroupexpressedsignificantlylowerMS lev-elsthanthemouthwashandgelgroupsatday-30.ChlorhexidinesignificantlyreducedMSlevelsafter 5daysoftreatment,buttheselevelsreturnedtobaselineinotherperiodsofthestudy.LSOtoothpaste reducedMSlevelsafter5daysoftreatment,andMSlevelsremainedlowanddidnotreturntobaseline duringsubsequentanalysis.Hence,LSOtoothpastedemonstratedthemostlong-lastingMSreductionin saliva,whereasotherLSOformulationsdidnoteffectivelyreduceMSlevelsinchildrenwithdentalcaries.

∗ Correspondingauthorat:UnidadedePesquisasClınicas,UniversidadeFederal doCeará,LaboratóriodeFarmacologiaMetabolicaeFisiologiaCelular,AvenidaJosé Bastos3390,Sala106,CaixaPostal3229,CEP60.436-160,Fortaleza-Ce,Brazil.Tel.: +558533668232;fax:+558533668232.

E-mailaddress:[email protected](C.S.R.Fonteles).

Introduction

Theriseofherbalmedicinehasstirredinterestintheeffects

ofplantextractsforthecontrolofplaqueandotheroraldiseases

(Buffonetal.2001).Plaqueisconsideredaprimaryfactorindental

caries, thus justifyingtheuse ofmeasures forits control.

Den-tal caries canprogress rapidlyresulting in mass destructionof

primarydentition,compromisingoralfunctionandthechild’s

well-being(DenBestenandBerkowitz2003).Sinceearlycontamination

withmutansstreptococci(MS)isamajorissueinthispopulation

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(Kohleretal.1988)withthepotentialforsignificantlyincreasing

thepossibilityofcaries,strategiesfortreatingthisdiseasein

chil-drenmustfocusoncontrollinggrowthofthesepathogenicbacteria

(ThibodeauandO’Sullivan1999).

Theessentialoilsofmanydifferentplantshavebeenpreviously

testedinbothinvitroandinvivostudies,aspromisingagentsinthe

treatmentoforaldiseasesandotherinfections(Nostroetal.2007;

Paietal.2004).LippiasidoidesCham.,aplantoftheverbenaceae

family,popularlyknownas“Alecrim-Pimenta”,isabushwitha

brittlestemandodoriferousleaves,typicallyfoundinNortheastern

Brazil.ThechemicalcompositionofLippiasidoidesCham.

essen-tialoil(LSO)hasbeenpreviouslydescribed(Botelhoetal.2007;

Fontenelleetal.2007;Sousaetal.2002).Theoilitselfhasproven

topossesssignificantantifungalactivity,andbroadantimicrobial

actionagainstmanydifferentbacteria(Fontenelleetal.2007).The

twomajorconstituentsofLSOarethymol(50–59%)andcarvacrol

(7–16%)(Botelhoetal.2007;Fontenelleetal.2007;Sousaetal.

2002).Phenolic compoundssuchas carvacrol and thymol have

hadtheirwidespectrumantimicrobialactionagainstyeastsand

bacteriaestablished,beingalsoconstituentsofotheressentialoils

(Nostroetal.2007).Inspiteofalimitednumberofclinicalstudies

demonstratingtheantimicrobialefficacyofLSOondentalcaries

andperiodontaldisease(FernandesFilhoetal.1998;Giraoetal.

2003;Botelhoetal.2007),nopreviousworkhasinvestigatedits

effectinchildrenwithdentalcaries.

Weconductedapilotstudy,whichdemonstratedthat Lippia

sidoides(LSO) wassafeand had agood acceptanceby children.

Thiswasarandomized,double-blind,inparallelwithactivecontrol

study,whichaimedtoevaluatetheefficacyofthreedifferent

for-mulationsofLSOinthereductionofsalivaryStreptococcusmutans

inchildrenwithcaries.

Materialsandmethods

ExtractionandchemicalanalysisofLSO

SamplesofLippiasidoidesCham.wereoriginallyobtainedfrom

themain gardenof the Laboratoryof Natural Products atUFC.

Botanicalidentificationoftheplant’sspecieswasobtainedatthe

Department of Biology.The collected leaveswere dried under

shadow,ground,keptinvacuum-sealedplasticbagsand

identi-fiedforfutureuse.Theessentialoilwasextractedapproximately9

monthslaterbythesteamdistillationmethodinaClevenger

appa-ratus(Craveiroetal.1976)andstoredinglasscontainers,under

refrigerationuntilthemomenttobeused.Chemicalconstituents

wereidentifiedbyspecialistsattheDepartmentofChemistry,inthe

sameuniversitybyusingagaschromatographercoupledtoamass

spectrometersystem(GC–MS,Shimadzu,modelQP5050,Japan).

ThemaincomponentsoftheLippiasidoidesCham.essentialoil

usedinthepresentstudywere:cycloheptatriene(0.98%),benzene

(2.07%),caryophyllene(3.59%), thymol/carvacrol(93.36%).Three

differentformulationsofLippiasidoidesCham.essentialoil(LSO)

werepreparedforthisclinicaltrial:(1)toothpaste,(2)geland(3)

mouthwash.Thetoothpasteandgelpreparationscontaineda1.4%

LSOconcentration,whichrenderedatotalof1.3%thymol/carvacrol,

whereasthemouthwashformulationconsistedof0.8%LSO,

render-ing0.74%oftheThy/Carmixture.

Patients

ThestudyprotocolwasapprovedbytheMedicalSchool’sEthics

Committee of the Federal University of Ceará, Brazil (Protocol

#182/07).ItcomplieswiththecurrentBrazilianlaws.After

writ-teninformed consentwasgiven by parents or legal guardians,

81volunteers,aged6–12years,frombothgenders,withatleast

onecariouscavitatedornon-cavitatedlesion,wererecruitedto

participate in thestudy.The volunteers wererecruited by two

graduateandonepostgraduatestudentoutofapopulationof400

childrensearchingfordentalcareatthePediatricDentalClinicof

theFederalUniversityofCeará.Patientswithahistoryofallergies

orallergicdiseases,e.g.asthma,urticaria,rhinitis,sinusitis,or

intra-oralsofttissuelesions,wereexcludedfromthestudy.Noneofthe

participantsunderwentantibiotictreatmentduringthecourseof

thisclinicaltrial.

Treatmentapplication

Participantswererandomlyassignedtoeitheroneoffive

dif-ferent groups. Each group receivedtopical treatment that was

formulated bythe Laboratory of PharmaceuticalScience atthe

FederalUniversity ofCeará,Fortaleza, Brazil,witheithera 1.4%

LSOtoothpaste,or1.4%LSOgel,or0.8%LSOmouthwash,or1%

chlorhexidinegel,or0.12%chlorhexidinemouthwash.Thegeland

mouthwashofLSOandchlorhexidinewereformulatedwith

sim-ilarcolorandtasteandtheidentificationofeachsubstancewas

concealedfrom thepostgraduate studentin charge ofapplying

thetreatment,andfromthestudyparticipants,untiltheclinical

trialwasconcluded.Therefore,thisclinicaltrialwiththeexception

ofthetoothpastegroupconsistedofadouble-blind,randomized

study.GelandmouthwashtreatmentswereappliedinthePediatric

DentalClinicattheFederalUniversityofCearáunderthe

supervi-sionofthestudy’sprincipalinvestigatorandwiththeassistanceof

apostgraduatestudent,whereasthetoothpastegroupwastreated

athomebyparents,whowerepreviouslyinstructedtobrushtheir

child’steethwithapea-sizeamountoftoothpaste,during1min,

onceaday,for5consecutivedays.

Beforethestartoftreatment,aclinicalexaminationwas

per-formedby only oneexaminer, using a visual/tactilemethodto

calculatethenumber of decayed,missing andfilled surfacesof

thesepatients. Allofthepatientsreceivedthesametoothpaste,

toothbrushandrecommendationsfororalhygieneanddiettobe

followedthroughoutthestudy.Thegelgroupshadthegelplaced

insidedisposabletrays,asa5-mlvolume,andappliedfor1min,

every24h,for5consecutivedays.Themouthwashgroupsused

mouthwashplacedinsidedisposablesyringes,asa5-mlvolume.

Salivacollectionandmicrobiologicalanalysis

During saliva collection patients were asked to chew on a

3cm×3cmpieceofParafilm®during60sinordertostimulate

sali-varysecretionandreleaseplaqueintothesalivaryfluid.Salivawas

thencollectedwithadisposableplasticcannulaandstoredinsterile

ependorfs®_for_subsequent_analysis._Samples_were_transported_to

thelaboratoryformicrobiologicalanalysisinahermeticallysealed

casecontainingice,andanalyzednolongerthan2haftercollection

(Lopezetal.2002).

Avolumeof0.1mlofeachsamplewasasepticallydrawnand

transferredintoonesteriletesttubecontaining0.9mlofsaline.

Procedurewasrepeatedtwice,establishingdilutionsof1:10and

1:100.Acorrespondingvolumeof10␮lofeachdilutionwasplated

ontoMitisSalivarius-Bacitracin(MSB)agarmedium(18)in

trip-licates.The plateswerethenincubatedat 37◦_C, _during₄₈_h, _in

jarsundermicroaerofilicconditions.Representativecolonieswith

morphologicalcharacteristicsof MSwere counted,isolated and

biochemically confirmed to be MS utilizing mannitol, sorbitol,

lactose, raffinose, melibiose and esculin. Bacterial counts were

expressedascolonyformingunits(CFU)/mlofsaliva.

Statisticalanalysis

DataonthenumberofCFUwereinitiallyprocessedinorder

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normality.Tothisend,weusedalogarithmictransformationasthe

followingequation:y=log10(x)=log(x).Becausetherewerezero

values,itwasadded1tox,sincethelogarithmicfunctionisdefined

onlyforx>0.Quantitativevariables,continuousanddiscrete,were

initiallyanalyzedbytheKolmogorov–Smirnovtest,inorderto

ver-ifythenormalityofthedistribution.Fordescriptivestatistics,we

calculatedthemeanandstandarddeviation(parametricdata)or

median,interquartilerangeandminimumandmaximumvalues

(nonparametricdata).Comparisonsbetweentwotreatmentgroups

ateachtimeweremadebyusingtheunpairedttest(parametric

data)orMann–Whitneytest(nonparametricvariables).To

com-parethree ormore groups(between groups analysis), weused

analysis of variance (ANOVA) associated withTukey’s multiple

comparisonstest,tocheckdifferencesbetweeneverytwogroups

(parametricdata),orKruskal–WallistestcomplementedbyDunn’s

multiplecomparisontest(nonparametricvariables).Comparisons

betweendifferenttimesinthesamegroup(withingroupanalysis)

werecarriedoutbyrepeatedmeasuresanalysisofvariance

associ-atedwiththeTukey’smultiplecomparisontest(parametricdata)

orbyFriedmantestcomplementedbyDunn’smultiple

compar-isonstest(nonparametricvariables).Inallcases,thesignificance

levelwassetat0.05(5%),beingconsideredstatisticallysignificant

apvaluelessthan0.05.Thedatawereanalyzedusingthesoftware

GraphPadPrism5.00(GraphPadSoftware,SanDiego,California,

USA,2007).

Results

Noimportantsideeffectsweredescribedbychildren,parentsor

guardians.Parentsandguardiansofchildrenwhousedtoothpaste

LSO,reportedanimprovementinthebreathofthechildrenafter5

daysoftreatment.

Fig.1.Streptococcusmutanslevelsmeasuredatdays1,5,30,60,180and365, expressedinlog(CFU/ml),aftera5-daytreatmentwith(A)chlorhexidine mouth-washor(B)gelformulations.FriedmanandDunn’stestsusedforcomparisons (p<0.05).

Withingroupanalysis

A difference in salivary MS levels measured in different

days within the same treatment group was only verified with

LSOtoothpaste,chlorhexidinegelandchlorhexidinemouthwash.

Chlorhexidinegelandmouthwashtreatmentdemonstrateda

sig-nificantlyhigherefficacyinMSreductionafter5daysoftreatment

(p<0.001), but theamountof Streptococcusmutans returned to

baseline onday30 andremainedsountil theend of thestudy

(Fig.1).ThetemporalprogressionoftheamountofsalivarySMis

showedinFig.2.TheuseofLSOgeldidnotcauseastatistically

sig-nificantvariationinSMlevelsthroughoutthedurationofthestudy.

However,inrelationtobaseline,thetreatmentwithLSOtoothpaste

significantlyreducedtheamountofsalivarySMondays5(p<0.05),

180(p<0.01)and365(p<0.001),whiletheLSOmouthwashcaused

asignificantdecreaseonlyinthesecondhalfofthestudy,ondays

180(p<0.05)and365(p<0.001).

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Fig.3.ComparisonofStreptococcusmutans(logCFU/ml)levelsbetweengroupstreatedwithLippiasidoidesCham.(LSO)mouthwash,gel,ortoothpasteformulations,atdays 1,5,30,60,180and365.Kruskal–WallisandDunn’stestsusedforcomparisons(p<0.05).

Betweengroupanalysis

DuringcomparisonbetweengroupsofLSOmouthwash,

tooth-pasteandgelitwasfoundthattheamountofMSmattermeasured

inthetoothpastegroupwassignificantlylowerthanthatofthe

mouthwashandgelgroupsondays30(p<0.05comparedtogroups

mouthwashand gel), 60 (p<0.01 compared tomouthwash and

p<0.05 comparedtothegel), 180(p<0.05compared togroups

mouthwashandgel)and365(p<0.01comparedtogelandp<0.01

comparedtomouthwash)(Fig.3).

In thecomparison betweengroups of chlorhexidine

mouth-wash,chlorhexidinegelandLSOtoothpasteattheendofa5day

period,MSmattermeasuredinthechlorhexidinegelgroupwas

sig-nificantlylower(***p<0.001)thanthatofthegroupschlorhexidine

mouthwash and LSOtoothpaste. Further,chlorhexidine

mouth-washwassignificantly lower(*p<0.05) thanLSO-dentifrice. On

day30,theamountofMSmattermeasuredintheLSO-toothpaste

group was significantly lower (**p<0.01) than that observed

in group-Chlorhexidine mouthwash. Moreover, in 60 days, the

quantitiesofsalivarySMmeasuredinthegroupsLSO-dentifrice

(**p<0.01) and chlorhexidine gel (**p<0.01) were significantly

lower than that observed in group-Chlorhexidine mouthwash.

It was further observed that in 180 days, the SM quantities

measuredingroupsLSO-dentifrice(**p<0.01)andchlorhexidine

gel(*p<0.05)weresignificantlylowerthanthatobservedinthe

chlorhexidinemouthwashgroup.Finally,itwasfoundthatin365

days,theamountofMSmattermeasuredintheLSO-toothpaste

groupwassignificantlylowerthan thatobservedin thegroups

chlorhexidine mouthwash (***p<0.001) and chlorhexidine gel

(**p<0.01)(Fig.4).Itwasalsoobservedatendencyofdecreasingin

theamountofsalivarySMintheLSOtoothpastegroupovertime.

Discussion

Studies have shown the success of antimicrobial treatment

against dental caries (Lopez et al. 2002; Zhan et al. 2006).

Chlorhexidineasanantiplaque andantigingivitisagentremains

a gold standard, but in dental caries itseffectiveness hasbeen

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controversial(Twetman2004).Chlorhexidineresultsinthepresent

study confirmed those from previous studies reporting that it

stronglyreducesMSbutitstillallowsrecolonization(Emilson1981,

1994;Loboet al.2008).Theaction of chlorhexidineis perhaps

dueitsfavorablechemicalproperties.Inadditiontobindingtothe

negativelychargedbacterialcellwall,exertingabacteriostaticor

bactericidaleffect,chlorhexidinebindstotheoralsurfaces,dental

pellicleandsaliva(Oliveiraetal.2007),butrecolonizationremains

aproblem.

In this study, LSO toothpaste demonstrated a reduction of

salivaryMSafter 5days of treatment, and lowMS levelswere

maintainedthroughoutthestudyandtheselevelsdidnotreturn

tobaselineduringsubsequentanalysis(days30,60,180and365).

Allpatientsreceivedthesamebrushandtoothpastethroughout

thestudy,andnewtoothbrusheswereprovidedevery3months.

TheuseofLSOwasbasedonpreviousworkbyNunesetal.(2006)

thathasstandardizedLippia sidoidesrawmaterialandits

vege-talextract,andhasdetermineditsantimicrobialactivityagainst

Streptococcusmutansinaninvitrostudy.Theauthorsconcluded

thatLippiasidoidesrawmaterialandextractcontainedvolatile

con-stituents,whichinplantsrelatetoanintrinsicbiologicalaction,and

thuscouldbeemployedintheproductionofclinicalformulations

fordentaluse,sinceitseffectivenessagainstStreptococcusmutans

wasdemonstrated.In1990,Lemosetal.analyzedthe

antimicro-bialactionofdifferentessentialoils,andverifiedactivityofLSO

againstE.coli,S.aureus,P.aeruginosa,B.subtilisandM.smegmatis.

Limitedantimicrobialactivitywasobserved againstgram

nega-tivebacteria,suchasP.aeruginosaandE.coli.Alaterstudyofthe

antibacterialeffectsofessentialoilsofplantsfromNortheastern

Brazil,demonstratedsensitivityofS.aureusandE.colitoLSO;

how-ever,P.aeruginosashowedresistancetotheactionofthisessential

oil(Bertinietal.2005).TheseresultshaveconfirmedLSOas

hav-ingmainlyagrampositiveantibacterialspectrum,inadditionto

alimitedactionagainstgramnegativebacteria.However,toour

knowledge,onlytwoclinicaltrialstestingtheeffectivenessofLSO

formulationshavebeenreportedintheliteratureandnonehave

testedLSOinagelortoothpasteformulation.Inaddition,thisstudy

isthefirsttoprovideclinicaldataontheefficacyofLSOinreducing

salivaryMSinchildrenwithdentalcaries.

LSOmouthwashand geldidnot demonstratesignificantMS

reductions,probablyduetothelackofmechanicalbrushing.

Fur-thermorethecombinationofnaturalproductswithchemicals,like

fluoride,mightimprovetheperformanceofantimicrobialagents,as

asynergismbetweenfluorideandLSOmayimproveMSreduction

andthereforethecontrolofcaries.Thesefindingswereobserved

previously(Ditterichetal.2007;Oliveiraetal.2007;Roselletal.

2004).Somestudieshavethwartedtheantimicrobialactivityof

natural substancesin toothpastes. Ditterichetal. (2007)

evalu-atedtheinvitroantimicrobialactionof7toothpastescomposed

of natural substances where the formation of inhibition zones

wereobserved.Theauthorsconcludedthat theadditionof

nat-uralantimicrobialagentstodentifricescanactasanadjuvantin

themechanicalcontrolofthedentalbiofilm.Anotherinvitrostudy

evaluatedtheantimicrobialactivityof9toothpastesagainstMSin

individualswithhighbacteriallevelsinsaliva,andobservedthe

inhibitionofMS,concludingthatthecombinationofnatural

ingre-dientscanhelpmaximizetheperformanceoftoothpastesagainst

themainbacteriumthatcausestoothdecay(Roselletal.2004).

Inthepresentstudy,alldifferentformulationsofchlorhexidine

significantlyreducedMSlevelsinthesalivaofchildrenwithdental

cariesafter5daysoftreatment,butMSlevelsreturnedtobaseline

onday30andremainedsountiltheendofthestudy.LSO

mouth-washandgelformulationsdidnotsignificantlyreducesalivaryMS

levelsinchildrenwithdentalcaries.However,thepresentlytested

5-dayLSOtoothpastetreatmentsuccessfullyreducedMSlevels,

andmostimportantly,MSlevelsremainedlowanddidnotreturnto

baselineduringsubsequentanalysis,suggestingasynergisteffect

betweenfluorideandtheantibacterialconstituentspresentin

Lip-piasidoidesCham.essentialoil,andemphasizingtheimportanceof

themechanicalremovalofthedentalbiofilminlong-term

antibac-terialeffectagainstStreptococcusmutans.

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